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Sample records for vitro biofilm reveals

  1. Biofilms of vaginal Lactobacillus in vitro test.

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    Wei, Xiao-Yu; Zhang, Rui; Xiao, Bing-Bing; Liao, Qin-Ping

    2017-01-01

    This paper focuses on biofilms of Lactobacillus spp. - a type of normal flora isolated from healthy human vaginas of women of childbearing age; thereupon, it broadens the research scope of investigation of vaginal normal flora. The static slide culture method was adopted to foster biofilms, marked by specific fluorescence staining. Laser scanning confocal and scanning electron microscopy were used to observe the microstructure of the biofilms. Photographs taken from the microstructure were analysed to calculate the density of the biofilms. The body of Lactobacillus spp., though red, turned yellow when interacting with the green extracellular polysaccharides. The structure of the biofilm and aquaporin within the biofilm were imaged. Lactobacillus density increases over time. This study provides convincing evidence that Lactobacillus can form biofilms and grow over time in vitro. This finding establishes an important and necessary condition for selecting proper strains for the pharmaceutics of vaginal ecology.

  2. In vitro phenotypic differentiation towards commensal and pathogenic oral biofilms

    NARCIS (Netherlands)

    Janus, M.M.; Keijser, B.J.F.; Bikker, F.J.; Exterkate, R.A.M.; Crielaard, W.; Krom, B.P.

    2015-01-01

    Commensal oral biofilms, defined by the absence of pathology-related phenotypes, are ubiquitously present. In contrast to pathological biofilms commensal biofilms are rarely studied. Here, the effect of the initial inoculum and subsequent growth conditions on in vitro oral biofilms was studied.

  3. Characterization of Biofilm Formation by Borrelia burgdorferi In Vitro

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    Sapi, Eva; Bastian, Scott L.; Mpoy, Cedric M.; Scott, Shernea; Rattelle, Amy; Pabbati, Namrata; Poruri, Akhila; Burugu, Divya; Theophilus, Priyanka A. S.; Pham, Truc V.; Datar, Akshita; Dhaliwal, Navroop K.; MacDonald, Alan; Rossi, Michael J.; Sinha, Saion K.; Luecke, David F.

    2012-01-01

    Borrelia burgdorferi, the causative agent of Lyme disease, has long been known to be capable of forming aggregates and colonies. It was recently demonstrated that Borrelia burgdorferi aggregate formation dramatically changes the in vitro response to hostile environments by this pathogen. In this study, we investigated the hypothesis that these aggregates are indeed biofilms, structures whose resistance to unfavorable conditions are well documented. We studied Borrelia burgdorferi for several known hallmark features of biofilm, including structural rearrangements in the aggregates, variations in development on various substrate matrices and secretion of a protective extracellular polymeric substance (EPS) matrix using several modes of microscopic, cell and molecular biology techniques. The atomic force microscopic results provided evidence that multilevel rearrangements take place at different stages of aggregate development, producing a complex, continuously rearranging structure. Our results also demonstrated that Borrelia burgdorferi is capable of developing aggregates on different abiotic and biotic substrates, and is also capable of forming floating aggregates. Analyzing the extracellular substance of the aggregates for potential exopolysaccharides revealed the existence of both sulfated and non-sulfated/carboxylated substrates, predominately composed of an alginate with calcium and extracellular DNA present. In summary, we have found substantial evidence that Borrelia burgdorferi is capable of forming biofilm in vitro. Biofilm formation by Borrelia species might play an important role in their survival in diverse environmental conditions by providing refuge to individual cells. PMID:23110225

  4. Characterization of biofilm formation by Borrelia burgdorferi in vitro.

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    Eva Sapi

    Full Text Available Borrelia burgdorferi, the causative agent of Lyme disease, has long been known to be capable of forming aggregates and colonies. It was recently demonstrated that Borrelia burgdorferi aggregate formation dramatically changes the in vitro response to hostile environments by this pathogen. In this study, we investigated the hypothesis that these aggregates are indeed biofilms, structures whose resistance to unfavorable conditions are well documented. We studied Borrelia burgdorferi for several known hallmark features of biofilm, including structural rearrangements in the aggregates, variations in development on various substrate matrices and secretion of a protective extracellular polymeric substance (EPS matrix using several modes of microscopic, cell and molecular biology techniques. The atomic force microscopic results provided evidence that multilevel rearrangements take place at different stages of aggregate development, producing a complex, continuously rearranging structure. Our results also demonstrated that Borrelia burgdorferi is capable of developing aggregates on different abiotic and biotic substrates, and is also capable of forming floating aggregates. Analyzing the extracellular substance of the aggregates for potential exopolysaccharides revealed the existence of both sulfated and non-sulfated/carboxylated substrates, predominately composed of an alginate with calcium and extracellular DNA present. In summary, we have found substantial evidence that Borrelia burgdorferi is capable of forming biofilm in vitro. Biofilm formation by Borrelia species might play an important role in their survival in diverse environmental conditions by providing refuge to individual cells.

  5. Characterizing the in vitro biofilm phenotype of Staphylococcus epidermidis isolates from central venous catheters.

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    Van Kerckhoven, Marian; Hotterbeekx, An; Lanckacker, Ellen; Moons, Pieter; Lammens, Christine; Kerstens, Monique; Ieven, Margareta; Delputte, Peter; Jorens, Philippe G; Malhotra-Kumar, Surbhi; Goossens, Herman; Maes, Louis; Cos, Paul

    2016-08-01

    Central venous catheter (CVC)-related infections are commonly caused by Staphylococcus epidermidis that is able to form a biofilm on the catheter surface. Many studies involving biofilm formation by Staphylococcus have been published each adopting an own in vitro model. Since the capacity to form a biofilm depends on multiple environmental factors, direct comparison of results obtained in different studies remains challenging. This study characterized the phenotype (strong versus weak biofilm-producers) of S. epidermidis from CVCs in four different in vitro biofilm models, covering differences in material type (glass versus polymer) and nutrient presentation (static versus continuous flow). A good correlation in phenotype was obtained between glass and polymeric surfaces independent of nutrient flow, with 85% correspondence under static growth conditions and 80% under dynamic conditions. A 80% correspondence between static and dynamic conditions on polymeric surfaces could be demonstrated as well. Incubation time had a significant influence on the biofilm phenotype with only 55% correspondence between the dynamic models at different incubation times (48h versus 17h). Screening for the presence of biofilm-related genes only revealed that ica A was correlated with biofilm formation under static but not under dynamic conditions. In conclusion, this study highlights that a high level of standardization is necessary to interpret and compare results of different in vitro biofilm models. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Pseudomonas aeruginosa Cystic Fibrosis isolates of similar RAPD genotype exhibit diversity in biofilm forming ability in vitro

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    Elborn Stuart J

    2010-02-01

    Full Text Available Abstract Background Pseudomonas aeruginosa is considered to grow in a biofilm in cystic fibrosis (CF chronic lung infections. Bacterial cell motility is one of the main factors that have been connected with P. aeruginosa adherence to both biotic and abiotic surfaces. In this investigation, we employed molecular and microscopic methods to determine the presence or absence of motility in P. aeruginosa CF isolates, and statistically correlated this with their biofilm forming ability in vitro. Results Our investigations revealed a wide diversity in the production, architecture and control of biofilm formation. Of 96 isolates, 49% possessed swimming motility, 27% twitching and 52% swarming motility, while 47% were non-motile. Microtitre plate assays for biofilm formation showed a range of biofilm formation ability from biofilm deficient phenotypes to those that formed very thick biofilms. A comparison of the motility and adherence properties of individual strains demonstrated that the presence of swimming and twitching motility positively affected biofilm biomass. Crucially, however, motility was not an absolute requirement for biofilm formation, as 30 non-motile isolates actually formed thick biofilms, and three motile isolates that had both flagella and type IV pili attached only weakly. In addition, CLSM analysis showed that biofilm-forming strains of P. aeruginosa were in fact capable of entrapping non-biofilm forming strains, such that these 'non-biofilm forming' cells could be observed as part of the mature biofilm architecture. Conclusions Clinical isolates that do not produce biofilms in the laboratory must have the ability to survive in the patient lung. We propose that a synergy exists between isolates in vivo, which allows "non biofilm-forming" isolates to be incorporated into the biofilm. Therefore, there is the potential for strains that are apparently non-biofilm forming in vitro to participate in biofilm-mediated pathogenesis in the CF

  7. Optimization of conditions for in vitro development of Trichoderma viride-based biofilms as potential inoculants.

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    Triveni, Sodimalla; Prasanna, Radha; Saxena, Anil Kumar

    2012-09-01

    Biofilms represent mixed communities present in a diverse range of environments; however, their utility as inoculants is less investigated. Our investigation was aimed towards in vitro development of biofilms using fungal mycelia (Trichoderma viride) as matrices and nitrogen-fixing and P-solubilizing bacteria as partners, as a prelude to their use as biofertilizers (biofilmed biofertilizers, BBs) and biocontrol agents for different crops. The most suitable media in terms of population counts, fresh mass and dry biomass for Trichoderma and Bacillus subtilis/Pseudomonas fluorescens was found to be Pikovskaya broth ± 1 % CaCO(3), while for Trichoderma and Azotobacter chroococcum, Jensen's medium was most optimal. The respective media were then used for optimization of the inoculation rate of the partners in terms of sequence of addition of partners, fresh/dry mass of biofilms and population counts of partners for efficient film formation. Microscopic observations revealed significant differences in the progress of growth of biofilms and dual cultures. In the biofilms, the bacteria were observed growing intermingled within the fungal mycelia mat. Further, biofilm formation was compared under static and shaking conditions and the fresh mass of biofilms was higher in the former. Such biofilms are being further characterized under in vitro conditions, before using them as inoculants with crops.

  8. The characterization of functions involved in the establishment and maturation of Klebsiella pneumoniae in vitro biofilm reveals dual roles for surface exopolysaccharides

    DEFF Research Database (Denmark)

    Balestrino, D.; Ghigo, J.M.; Charbonnel, N.

    2008-01-01

    The ability to form biofilm is seen as an increasingly important colonization strategy among both pathogenic and environmental Klebsiella pneumoniae strains. The aim of the present study was to identify abiotic surface colonization factors of K. pneumoniae using different models at different phases...

  9. In Vitro Biofilm Models for Device-Related Infections.

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    Buhmann, Matthias T; Stiefel, Philipp; Maniura-Weber, Katharina; Ren, Qun

    2016-12-01

    Many promising antimicrobial materials fail to translate from bench to bedside, in part owing to a lack of in vitro biofilm models that can be used to predict their long-term in vivo antimicrobial and anti-biofilm activity. Various factors need to be considered for predictive modeling to mimic the conditions in vivo. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Development and pyrosequencing analysis of an in-vitro oral biofilm model.

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    Kistler, James O; Pesaro, Manuel; Wade, William G

    2015-02-10

    Dental caries and periodontal disease are the commonest bacterial diseases of man and can result in tooth loss. The principal method of prevention is the mechanical removal of dental plaque augmented by active agents incorporated into toothpastes and mouthrinses. In-vitro assays that include complex oral bacterial biofilms are required to accurately predict the efficacy of novel active agents in vivo. The aim of this study was to develop an oral biofilm model using the Calgary biofilm device (CBD) seeded with a natural saliva inoculum and analysed by next generation sequencing. The specific objectives were to determine the reproducibility and stability of the model by comparing the composition of the biofilms over time derived from (i) the same volunteers at different time points, and (ii) different panels of volunteers. Pyrosequencing yielded 280,093 sequences with a mean length of 432 bases after filtering. A mean of 320 and 250 OTUs were detected in pooled saliva and biofilm samples, respectively. Principal coordinates analysis (PCoA) plots based on community membership and structure showed that replicate biofilm samples were highly similar and clustered together. In addition, there were no significant differences between biofilms derived from the same panel at different times using analysis of molecular variance (AMOVA). There were significant differences between biofilms from different panels (AMOVA, P < 0.002). PCoA revealed that there was a shift in biofilm composition between seven and 14 days (AMOVA, P < 0.001). Veillonella parvula, Veillonella atypica/dispar/parvula and Peptostreptococcus stomatis were the predominant OTUs detected in seven-day biofilms, whilst Prevotella oralis, V. parvula and Streptococcus constellatus were predominant in 14-day biofilms. Diverse oral biofilms were successfully grown and maintained using the CBD. Biofilms derived from the same panel of volunteers were highly reproducible. This model could be used to screen both

  11. In VitroStreptococcus pneumoniae Biofilm Formation and In Vivo Middle Ear Mucosal Biofilm in a Rat Model of Acute Otitis Induced by S. pneumoniae.

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    Yadav, Mukesh Kumar; Chae, Sung-Won; Song, Jae-Jun

    2012-09-01

    Streptococcus pneumoniae is one of the most common pathogens of otitis media (OM) that exists in biofilm, which enhances the resistance of bacteria against antibiotic killing and diagnosis, compared to the free-floating (planktonic) form. This study evaluated biofilm formation by S. pneumoniae on an abiotic surface and in the middle ear cavity in a rat model of OM. In vitro biofilm formation was evaluated by inoculation of a 1:100 diluted S. pneumoniae cell suspension in a 96-well microplate. Adherent cells were quantified spectrophotometrically following staining with crystal violet by measurement of optical density at 570 nm. The ultrastructure of pneumococcal biofilm was assessed by scanning electron microscopy (SEM). For in vitro biofilm study, S. pneumoniae cell suspensions containing 1×10(7) colony forming units were injected through transtympanic membrane into the middle ear cavity of Sprague Dawley rats. The ultrastructure of middle ear mucus was observed by SEM 1 and 2 weeks post-inoculation. The in vitro study revealed robust biofilm formation by S. pneumoniae after 12-18 hours of incubation in high glucose medium, independent of exogenously supplied competence stimulating peptide and medium replacement. Adherent cells formed three-dimensional structures approximately 20-30 µm thick. The in vivo study revealed that ciliated epithelium was relatively resistant to biofilm formation and that biofilm formation occurred mainly on non-ciliated epithelium of the middle ear cavity. One week after inoculation, biofilm formation was high in 50% of the treated rats and low in 25% of the rats. After 2 weeks, biofilm formation was high and low in 25% and 37.5% of rats, respectively. The results imply that glucose level is important for the S. pneumoniae biofilm formation and S. pneumoniae biofilm formation may play important role in the pathophysiology of OM.

  12. The in vitro effect of xylitol on chronic rhinosinusitis biofilms.

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    Jain, R; Lee, T; Hardcastle, T; Biswas, K; Radcliff, F; Douglas, R

    2016-12-01

    Biofilms have been implicated in chronic rhinosinusitis (CRS) and may explain the limited efficacy of antibiotics. There is a need to find more effective, non-antibiotic based therapies for CRS. This study examines the effects of xylitol on CRS biofilms and planktonic bacteria. Crystal violet assay and spectrophotometry were used to quantify the effects of xylitol (5% and 10% solutions) against Staphylococcus epidermidis, Pseudomonas aeruginosa, and Staphylococcus aureus. The disruption of established biofilms, inhibition of biofilm formation and effects on planktonic bacteria growth were investigated and compared to saline and no treatment. Xylitol 5% and 10% significantly reduced biofilm biomass (S. epidermidis), inhibited biofilm formation (S. aureus and P. aeruginosa) and reduced growth of planktonic bacteria (S. epidermidis, S. aureus, and P. aeruginosa). Xylitol 5% inhibited formation of S. epidermidis biofilms more effectively than xylitol 10%. Xylitol 10% reduced S. epidermidis planktonic bacteria more effectively than xylitol 5%. Saline, xylitol 5% and 10% disrupted established biofilms of S. aureus when compared with no treatment. No solution was effective against established P. aeruginosa biofilm. Xylitol has variable activity against biofilms and planktonic bacteria in vitro and may have therapeutic efficacy in the management of CRS.

  13. The Small Molecule DAM Inhibitor, Pyrimidinedione, Disrupts Streptococcus pneumoniae Biofilm Growth In Vitro.

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    Yadav, Mukesh Kumar; Go, Yoon Young; Chae, Sung-Won; Song, Jae-Jun

    2015-01-01

    Streptococcus pneumoniae persist in the human nasopharynx within organized biofilms. However, expansion to other tissues may cause severe infections such as pneumonia, otitis media, bacteremia, and meningitis, especially in children and the elderly. Bacteria within biofilms possess increased tolerance to antibiotics and are able to resist host defense systems. Bacteria within biofilms exhibit different physiology, metabolism, and gene expression profiles than planktonic cells. These differences underscore the need to identify alternative therapeutic targets and novel antimicrobial compounds that are effective against pneumococcal biofilms. In bacteria, DNA adenine methyltransferase (Dam) alters pathogenic gene expression and catalyzes the methylation of adenine in the DNA duplex and of macromolecules during the activated methyl cycle (AMC). In pneumococci, AMC is involved in the biosynthesis of quorum sensing molecules that regulate competence and biofilm formation. In this study, we examine the effect of a small molecule Dam inhibitor, pyrimidinedione, on Streptococcus pneumoniae biofilm formation and evaluate the changes in global gene expression within biofilms via microarray analysis. The effects of pyrimidinedione on in vitro biofilms were studied using a static microtiter plate assay, and the architecture of the biofilms was viewed using confocal and scanning electron microscopy. The cytotoxicity of pyrimidinedione was tested on a human middle ear epithelium cell line by CCK-8. In situ oligonucleotide microarray was used to compare the global gene expression of Streptococcus pneumoniae D39 within biofilms grown in the presence and absence of pyrimidinedione. Real-time RT-PCR was used to study gene expression. Pyrimidinedione inhibits pneumococcal biofilm growth in vitro in a concentration-dependent manner, but it does not inhibit planktonic cell growth. Confocal microscopy analysis revealed the absence of organized biofilms, where cell-clumps were scattered

  14. The Small Molecule DAM Inhibitor, Pyrimidinedione, Disrupts Streptococcus pneumoniae Biofilm Growth In Vitro.

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    Mukesh Kumar Yadav

    Full Text Available Streptococcus pneumoniae persist in the human nasopharynx within organized biofilms. However, expansion to other tissues may cause severe infections such as pneumonia, otitis media, bacteremia, and meningitis, especially in children and the elderly. Bacteria within biofilms possess increased tolerance to antibiotics and are able to resist host defense systems. Bacteria within biofilms exhibit different physiology, metabolism, and gene expression profiles than planktonic cells. These differences underscore the need to identify alternative therapeutic targets and novel antimicrobial compounds that are effective against pneumococcal biofilms. In bacteria, DNA adenine methyltransferase (Dam alters pathogenic gene expression and catalyzes the methylation of adenine in the DNA duplex and of macromolecules during the activated methyl cycle (AMC. In pneumococci, AMC is involved in the biosynthesis of quorum sensing molecules that regulate competence and biofilm formation. In this study, we examine the effect of a small molecule Dam inhibitor, pyrimidinedione, on Streptococcus pneumoniae biofilm formation and evaluate the changes in global gene expression within biofilms via microarray analysis. The effects of pyrimidinedione on in vitro biofilms were studied using a static microtiter plate assay, and the architecture of the biofilms was viewed using confocal and scanning electron microscopy. The cytotoxicity of pyrimidinedione was tested on a human middle ear epithelium cell line by CCK-8. In situ oligonucleotide microarray was used to compare the global gene expression of Streptococcus pneumoniae D39 within biofilms grown in the presence and absence of pyrimidinedione. Real-time RT-PCR was used to study gene expression. Pyrimidinedione inhibits pneumococcal biofilm growth in vitro in a concentration-dependent manner, but it does not inhibit planktonic cell growth. Confocal microscopy analysis revealed the absence of organized biofilms, where cell

  15. Calcium fluoride nanoparticles induced suppression of Streptococcus mutans biofilm: an in vitro and in vivo approach.

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    Kulshrestha, Shatavari; Khan, Shakir; Hasan, Sadaf; Khan, M Ehtisham; Misba, Lama; Khan, Asad U

    2016-02-01

    Biofilm formation on the tooth surface is the root cause of dental caries and periodontal diseases. Streptococcus mutans is known to produce biofilm which is one of the primary causes of dental caries. Acid production and acid tolerance along with exopolysaccharide (EPS) formation are major virulence factors of S. mutans biofilm. In the current study, calcium fluoride nanoparticles (CaF2-NPs) were evaluated for their effect on the biofilm forming ability of S. mutans in vivo and in vitro. The in vitro studies revealed 89 % and 90 % reduction in biofilm formation and EPS production, respectively. Moreover, acid production and acid tolerance abilities of S. mutans were also reduced considerably in the presence of CaF2-NPs. Confocal laser scanning microscopy and transmission electron microscopy images were in accordance with the other results indicating inhibition of biofilm without affecting bacterial viability. The qRT-PCR gene expression analysis showed significant downregulation of various virulence genes (vicR, gtfC, ftf, spaP, comDE) associated with biofilm formation. Furthermore, CaF2-NPs were found to substantially decrease the caries in treated rat groups as compared to the untreated groups in in vivo studies. Scanning electron micrographs of rat's teeth further validated our results. These findings suggest that the CaF2-NPs may be used as a potential antibiofilm applicant against S. mutans and may be applied as a topical agent to reduce dental caries.

  16. Patterned biofilm formation reveals a mechanism for structural heterogeneity in bacterial biofilms.

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    Gu, Huan; Hou, Shuyu; Yongyat, Chanokpon; De Tore, Suzanne; Ren, Dacheng

    2013-09-03

    Bacterial biofilms are ubiquitous and are the major cause of chronic infections in humans and persistent biofouling in industry. Despite the significance of bacterial biofilms, the mechanism of biofilm formation and associated drug tolerance is still not fully understood. A major challenge in biofilm research is the intrinsic heterogeneity in the biofilm structure, which leads to temporal and spatial variation in cell density and gene expression. To understand and control such structural heterogeneity, surfaces with patterned functional alkanthiols were used in this study to obtain Escherichia coli cell clusters with systematically varied cluster size and distance between clusters. The results from quantitative imaging analysis revealed an interesting phenomenon in which multicellular connections can be formed between cell clusters depending on the size of interacting clusters and the distance between them. In addition, significant differences in patterned biofilm formation were observed between wild-type E. coli RP437 and some of its isogenic mutants, indicating that certain cellular and genetic factors are involved in interactions among cell clusters. In particular, autoinducer-2-mediated quorum sensing was found to be important. Collectively, these results provide missing information that links cell-to-cell signaling and interaction among cell clusters to the structural organization of bacterial biofilms.

  17. Baicalein Inhibits Staphylococcus aureus Biofilm Formation and the Quorum Sensing System In Vitro.

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    Yan Chen

    Full Text Available Biofilm formed by Staphylococcus aureus significantly enhances antibiotic resistance by inhibiting the penetration of antibiotics, resulting in an increasingly serious situation. This study aimed to assess whether baicalein can prevent Staphylococcus aureus biofilm formation and whether it may have synergistic bactericidal effects with antibiotics in vitro. To do this, we used a clinically isolated strain of Staphylococcus aureus 17546 (t037 for biofilm formation. Virulence factors were detected following treatment with baicalein, and the molecular mechanism of its antibiofilm activity was studied. Plate counting, crystal violet staining, and fluorescence microscopy revealed that 32 μg/mL and 64 μg/mL baicalein clearly inhibited 3- and 7-day biofilm formation in vitro. Moreover, colony forming unit count, confocal laser scanning microscopy, and scanning electron microscopy showed that vancomycin (VCM and baicalein generally enhanced destruction of biofilms, while VCM alone did not. Western blotting and real-time quantitative polymerase chain reaction analyses (RTQ-PCR confirmed that baicalein treatment reduced staphylococcal enterotoxin A (SEA and α-hemolysin (hla levels. Most strikingly, real-time qualitative polymerase chain reaction data demonstrated that 32 μg/mL and 64 μg/mL baicalein downregulated the quorum-sensing system regulators agrA, RNAIII, and sarA, and gene expression of ica, but 16 μg/mL baicalein had no effect. In summary, baicalein inhibited Staphylococcus aureus biofilm formation, destroyed biofilms, increased the permeability of vancomycin, reduced the production of staphylococcal enterotoxin A and α-hemolysin, and inhibited the quorum sensing system. These results support baicalein as a novel drug candidate and an effective treatment strategy for Staphylococcus aureus biofilm-associated infections.

  18. Enzymatic degradation of in vitro Staphylococcus aureus biofilms supplemented with human plasma.

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    Watters, Chase M; Burton, Tarea; Kirui, Dickson K; Millenbaugh, Nancy J

    2016-01-01

    Enzymatic debridement is a therapeutic strategy used clinically to remove necrotic tissue from wounds. Some of the enzymes utilized for debridement have been tested against bacterial pathogens, but the effectiveness of these agents in dispersing clinically relevant biofilms has not been fully characterized. Here, we developed an in vitro Staphylococcus aureus biofilm model that mimics wound-like conditions and employed this model to investigate the antibiofilm activity of four enzymatic compounds. Human plasma at concentrations of 0%-50% was supplemented into growth media and used to evaluate biofilm biomass accumulation over 24 hours and 48 hours in one methicillin-sensitive and five methicillin-resistant strains of S. aureus. Supplementation of media with 10% human plasma resulted in the most robust biofilms in all six strains. The enzymes α-amylase, bromelain, lysostaphin, and papain were then tested against S. aureus biofilms cultured in 10% human plasma. Quantification of biofilms after 2 hours and 24 hours of treatment using the crystal violet assay revealed that lysostaphin decreased biomass by up to 76%, whereas α-amylase, bromelain, and papain reduced biomass by up to 97%, 98%, and 98%, respectively. Scanning electron microscopy confirmed that the dispersal agents detached the biofilm exopolysaccharide matrix and bacteria from the growth surface. Lysostaphin caused less visible dispersal of the biofilms, but unlike the other enzymes, induced morphological changes indicative of bacterial cell damage. Overall, our results indicate that use of enzymes may be an effective means of eradicating biofilms and a promising strategy to improve treatment of multidrug-resistant bacterial infections.

  19. Enzymatic degradation of in vitro Staphylococcus aureus biofilms supplemented with human plasma

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    Watters CM

    2016-04-01

    Full Text Available Chase M Watters,1,2 Tarea Burton,1 Dickson K Kirui,1 Nancy J Millenbaugh1 1Maxillofacial Injury and Disease Department, Naval Medical Research Unit San Antonio, Joint Base San Antonio-Fort Sam Houston, TX, USA; 2Wound Infections Department, Naval Medical Research Center, Silver Spring, MD, USA Abstract: Enzymatic debridement is a therapeutic strategy used clinically to remove necrotic tissue from wounds. Some of the enzymes utilized for debridement have been tested against bacterial pathogens, but the effectiveness of these agents in dispersing clinically relevant biofilms has not been fully characterized. Here, we developed an in vitro Staphylococcus aureus biofilm model that mimics wound-like conditions and employed this model to investigate the antibiofilm activity of four enzymatic compounds. Human plasma at concentrations of 0%–50% was supplemented into growth media and used to evaluate biofilm biomass accumulation over 24 hours and 48 hours in one methicillin-sensitive and five methicillin-resistant strains of S. aureus. Supplementation of media with 10% human plasma resulted in the most robust biofilms in all six strains. The enzymes α-amylase, bromelain, lysostaphin, and papain were then tested against S. aureus biofilms cultured in 10% human plasma. Quantification of biofilms after 2 hours and 24 hours of treatment using the crystal violet assay revealed that lysostaphin decreased biomass by up to 76%, whereas a-amylase, bromelain, and papain reduced biomass by up to 97%, 98%, and 98%, respectively. Scanning electron microscopy confirmed that the dispersal agents detached the biofilm exopolysaccharide matrix and bacteria from the growth surface. Lysostaphin caused less visible dispersal of the biofilms, but unlike the other enzymes, induced morphological changes indicative of bacterial cell damage. Overall, our results indicate that use of enzymes may be an effective means of eradicating biofilms and a promising strategy to improve

  20. In vitro management of hospital Pseudomonas aeruginosa biofilm using indigenous T7-like lytic phage.

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    Ahiwale, Sangeeta; Tamboli, Nilofer; Thorat, Kiran; Kulkarni, Rajendra; Ackermann, Hans; Kapadnis, Balasaheb

    2011-02-01

    Pseudomonas aeruginosa, a human pathogen capable of forming biofilm and contaminating medical settings, is responsible for 65% mortality in the hospitals all over the world. This study was undertaken to isolate lytic phages against biofilm forming Ps. aeruginosa hospital isolates and to use them for in vitro management of biofilms in the microtiter plate. Multidrug resistant strains of Ps. aeruginosa were isolated from the hospital environment in and around Pimpri-Chinchwad, Maharashtra by standard microbiological methods. Lytic phages against these strains were isolated from the Pavana river water by double agar layer plaque assay method. A wide host range phage bacterial virus Ps. aeruginosa phage (BVPaP-3) was selected. Electron microscopy revealed that BVPaP-3 phage is a T7-like phage and is a relative of phage species gh-1. A phage at MOI-0.001 could prevent biofilm formation by Ps. aeruginosa hospital strain-6(HS6) on the pegs within 24 h. It could also disperse pre-formed biofilms of all hospital isolates (HS1-HS6) on the pegs within 24 h. Dispersion of biofilm was studied by monitoring log percent reduction in cfu and log percent increase in pfu of respective bacterium and phage on the peg as well as in the well. Scanning electron microscopy confirmed that phage BVPaP-3 indeed causes biofilm reduction and bacterial cell killing. Laboratory studies prove that BVPaP-3 is a highly efficient phage in preventing and dispersing biofilms of Ps. aeruginosa. Phage BVPaP-3 can be used as biological disinfectant to control biofilm problem in medical devices.

  1. Efficacy of Ethanol against Trichosporon asahii Biofilm in vitro.

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    Liao, Yong; Zhao, Hui; Lu, Xuelian; Yang, Suteng; Zhou, Jianfeng; Yang, Rongya

    2015-05-01

    Trichosporon asahii (T. asahii) can cause invasive infections, particularly catheter-related bloodstream infections (CR-BSIs). T. asahii biofilm, which is resistant to the most common clinical antifungal agents, may play an important role in these life-threatening infections. This study focused on the effects of ethanol on the different phases of T. asahii biofilm formation. At the concentrations clinically used, ethanol killed T. asahii planktonic cells (MIC90 = 15% and m-MIC90 = 15%) and biofilm (SMIC90 = 50%), and exposure to 25% ethanol for 12 h or to 50% ethanol for 8 h completely inhibited biofilm development and eradicated mature T. asahii biofilm. Thus, our results showed that ethanol effectively inhibited the main phases of T. asahii biofilm formation. This study reveals a new potential strategy to prevent and treat T. asahii biofilm-related CR-BSIs. © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. A novel assay of biofilm antifungal activity reveals that amphotericin B and caspofungin lyse Candida albicans cells in biofilms.

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    DiDone, Louis; Oga, Duana; Krysan, Damian J

    2011-08-01

    The ability of Candida albicans to form drug-resistant biofilms is an important factor in its contribution to human disease. Assays to identify and characterize molecules with activity against fungal biofilms are crucial for the development of drugs with improved anti-biofilm activity. Here we report the application of an adenylate kinase (AK)-based cytotoxicity assay of fungal cell lysis to the characterization of agents active against C. albicans biofilms. We have developed three protocols for the AK assay. The first measures AK activity in the supernatants of biofilms treated with antifungal drugs and can be performed in parallel with a standard 2,3-bis-(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-caboxanilide-based biofilm susceptibility assay; a second, more sensitive protocol measures the AK activity present within the biofilm matrix; and a third procedure allows the direct visualization of lytic activity toward biofilms formed on catheter material. Amphotericin B and caspofungin, the two most effective anti-biofilm drugs currently used to treat fungal infections, both directly lyse planktonic C. albicans cells in vitro, leading to the release of AK into the culture medium. These studies serve to validate the AK-based lysis assay as a useful addition to the methods for the characterization of antifungal agents active toward biofilms and provide insights into the mode of action of amphotericin B and caspofungin against C. albicans biofilms. Copyright © 2011 John Wiley & Sons, Ltd.

  3. Inefficacy of vancomycin and teicoplanin in eradicating and killing Staphylococcus epidermidis biofilms in vitro.

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    Claessens, J; Roriz, M; Merckx, R; Baatsen, P; Van Mellaert, L; Van Eldere, J

    2015-04-01

    Biofilm-associated bacteria display a decreased susceptibility towards antibiotics. Routine assessment of antibiotic susceptibility of planktonic bacteria therefore offers an insufficient prediction of the biofilm response. In this study, in vitro biofilms of eight clinical Staphylococcus epidermidis strains were subjected to treatment with vancomycin, teicoplanin, oxacillin, rifampicin and gentamicin. In addition, the biofilms were subjected to combinations of an antibiotic with rifampicin. The effects on the biofilms were assessed by crystal violet staining to determine the total biofilm biomass, staining with XTT to determine bacterial cell viability, and microscopy. Combining these methods showed that treatment of S. epidermidis biofilms with glycopeptides increased the total biofilm biomass and that these antibiotics were not effective in killing bacteria embedded in biofilms. The decreased killing efficacy was more pronounced in biofilms produced by strains that were classified as 'strong' biofilm producers. Rifampicin, oxacillin and gentamicin effectively killed biofilm-associated bacteria of all tested strains. Combining antibiotics with rifampicin increased the killing efficacy without influencing the total biofilm biomass. When vancomycin or teicoplanin were combined with rifampicin, the increase in biofilm biomass was neutralised and also the killing efficacy was influenced in a positive way. We conclude that the combined methodology used in this study showed that glycopeptides were not effective in eradicating S. epidermidis biofilms but that combination with rifampicin improved the killing efficacy in vitro. Copyright © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

  4. A novel technique using potassium permanganate and reflectance confocal microscopy to image biofilm extracellular polymeric matrix reveals non-eDNA networks in Pseudomonas aeruginosa biofilms.

    Science.gov (United States)

    Swearingen, Matthew C; Mehta, Ajeet; Mehta, Amar; Nistico, Laura; Hill, Preston J; Falzarano, Anthony R; Wozniak, Daniel J; Hall-Stoodley, Luanne; Stoodley, Paul

    2016-02-01

    Biofilms are etiologically important in the development of chronic medical and dental infections. The biofilm extracellular polymeric substance (EPS) determines biofilm structure and allows bacteria in biofilms to adapt to changes in mechanical loads such as fluid shear. However, EPS components are difficult to visualize microscopically because of their low density and molecular complexity. Here, we tested potassium permanganate, KMnO4, for use as a non-specific EPS contrast-enhancing stain using confocal laser scanning microscopy in reflectance mode. We demonstrate that KMnO4 reacted with EPS components of various strains of Pseudomonas, Staphylococcus and Streptococcus, yielding brown MnO2 precipitate deposition on the EPS, which was quantifiable using data from the laser reflection detector. Furthermore, the MnO2 signal could be quantified in combination with fluorescent nucleic acid staining. COMSTAT image analysis indicated that KMnO4 staining increased the estimated biovolume over that determined by nucleic acid staining alone for all strains tested, and revealed non-eDNA EPS networks in Pseudomonas aeruginosa biofilm. In vitro and in vivo testing indicated that KMnO4 reacted with poly-N-acetylglucosamine and Pseudomonas Pel polysaccharide, but did not react strongly with DNA or alginate. KMnO4 staining may have application as a research tool and for diagnostic potential for biofilms in clinical samples. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. In Vitro Evaluation of Bacteriocins Activity Against Listeria monocytogenes Biofilm Formation.

    Science.gov (United States)

    Camargo, Anderson Carlos; de Paula, Otávio Almeida Lino; Todorov, Svetoslav Dimitrov; Nero, Luís Augusto

    2016-03-01

    The present study aimed to assess the activity of cell-free supernatant (CFS) containing bacteriocins on the formation and maintenance of biofilms developed by Listeria monocytogenes, and the associated effect of bacteriocins and ethylene-diamine-tetra-acetic acid (EDTA) on the formed biofilm. CFS from 9 lactic acid bacteria (LAB) strains was tested for inhibitory activity against 85 L. monocytogenes isolates and 21 LAB strains. Then, 12 L. monocytogenes strains were selected based on genetic profiles and sensitivity to CFS and were subjected to an in vitro assay to assess biofilm formation in microtiter plates, considering different culture media and incubation conditions. Based on these results, 6 L. monocytogenes strains were subjected to the same in vitro procedure to assess biofilm formation, being co-inoculated with CFS. In addition, these strains were subjected to the same in vitro procedure, modified by adding the CFS after biofilm formation. Relevant decrease in biofilm formation was observed in the first experiment, but CFS added after biofilm formation did not eliminate them. CFS from Lactobacillus curvatus ET31 were selected due to its anti-biofilm activity, being associated to EDTA at different concentrations and tested for biofilm control of three strains of L. monocytogenes, using the same in vitro procedure described previously. Concentrated bacteriocin presented poor performance in eliminating formed biofilms, and EDTA concentration presented no evident interference on biofilm elimination. Twelve selected L. monocytogenes strains were positive for investigated virulence makers and negative for luxS gene, recognized as being involved in biofilm formation. Selected L. monocytogenes strains were able to produce biofilms under different conditions. CFSs have the potential to prevent biofilm formation, but they were not able to destroy already formed biofilms. Nevertheless, low concentrations of CFS combined with EDTA caused a relevant reduction in

  6. Acute and substantive action of antimicrobial toothpastes and mouthrinses on oral biofilm in vitro

    NARCIS (Netherlands)

    Otten, Marieke P. T.; Busscher, Henk J.; van der Mei, Henny C.; van Hoogmoed, Chris G.; Abbas, Frank; Hoogmoed, G.G. van

    The aim of this study was to compare acute action by killing or disrupting oral biofilms through the use of antimicrobial toothpastes and mouthrinses in vitro and to investigate substantive action by absorption of antimicrobials in a biofilm. Biofilms from freshly collected human saliva were grown

  7. Characteristics of Aspergillus fumigatus in Association with Stenotrophomonas maltophilia in an In Vitro Model of Mixed Biofilm.

    Directory of Open Access Journals (Sweden)

    Elise Melloul

    Full Text Available Biofilms are communal structures of microorganisms that have long been associated with a variety of persistent infections poorly responding to conventional antibiotic or antifungal therapy. Aspergillus fumigatus fungus and Stenotrophomonas maltophilia bacteria are examples of the microorganisms that can coexist to form a biofilm especially in the respiratory tract of immunocompromised patients or cystic fibrosis patients. The aim of the present study was to develop and assess an in vitro model of a mixed biofilm associating S. maltophilia and A. fumigatus by using analytical and quantitative approaches.An A. fumigatus strain (ATCC 13073 expressing a Green Fluorescent Protein (GFP and an S. maltophilia strain (ATCC 13637 were used. Fungal and bacterial inocula (105 conidia/mL and 106 cells/mL, respectively were simultaneously deposited to initiate the development of an in vitro mixed biofilm on polystyrene supports at 37°C for 24 h. The structure of the biofilm was analysed via qualitative microscopic techniques like scanning electron and transmission electron microscopy, and fluorescence microscopy, and by quantitative techniques including qPCR and crystal violet staining.Analytic methods revealed typical structures of biofilm with production of an extracellular matrix (ECM enclosing fungal hyphae and bacteria. Quantitative methods showed a decrease of A. fumigatus growth and ECM production in the mixed biofilm with antibiosis effect of the bacteria on the fungi seen as abortive hyphae, limited hyphal growth, fewer conidia, and thicker fungal cell walls.For the first time, a mixed A. fumigatus-S. maltophilia biofilm was validated by various analytical and quantitative approaches and the bacterial antibiosis effect on the fungus was demonstrated. The mixed biofilm model is an interesting experimentation field to evaluate efficiency of antimicrobial agents and to analyse the interactions between the biofilm and the airways epithelium.

  8. Synergistic Effects of Nonthermal Plasma and Disinfecting Agents against Dental Biofilms In Vitro

    OpenAIRE

    Koban, Ina; Geisel, Marie Henrike; Holtfreter, Birte; Jablonowski, Lukasz; H?bner, Nils-Olaf; Matthes, Rutger; Masur, Kai; Weltmann, Klaus-Dieter; Kramer, Axel; Kocher, Thomas

    2013-01-01

    Aim. Dental biofilms play a major role in the pathogenesis of many dental diseases. In this study, we evaluated the synergistic effect of atmospheric pressure plasma and different agents in dentistry on the reduction of biofilms. Methods and Results. We used monospecies (S. mutans) and multispecies dental biofilm models grown on titanium discs in vitro. After treatment with one of the agents, the biofilms were treated with plasma. Efficacy of treatment was determined by the number of colony f...

  9. Synergy of ambroxol with vancomycin in elimination of catheter-related Staphylococcus epidermidis biofilm in vitro and in vivo.

    Science.gov (United States)

    Zhang, Yunhui; Fu, Yakun; Yu, Jialin; Ai, Qing; Li, Junshuai; Peng, Ningning; Song, Sijie; He, Yu; Wang, Zhengli

    2015-11-01

    Central venous catheters are widely used in neonatal intensive care units (NICUs) nowadays. The commonest cause of catheter-related bloodstream infections (CRBSIs) is coagulase-negative staphylococci (CoNS). Ambroxol, an active metabolite of bromhexine, exhibits antimicrobial activity against strains producing biofilm and enhances the bactericidal effect of some antibiotic by breaking the structure of biofilm. In this study, we aimed to determine the effect of ambroxol with vancomycin on the biofilm of Staphylococcus epidermidis (S. epidermidis) in vitro and in vivo. In the in vitro study, the biofilm of S. epidermidis was assessed by XTT reduction assay and analysed by confocal laser scanning microscopy (CLSM). In the in vivo study, a rabbit model of CRBSIs was created by intravenous intubation with a tube covered with S. epidermidis biofilm. The rabbits received one of the following four treatments by means of antibiotic lock therapy: normal heparin, ambroxol, vancomycin, or vancomycin plus ambroxol each for 3 days. The microstructure of the biofilm was assessed by scanning electron microscopy (SEM). The number of bacterial colonies in the organs (liver, heart, and kidney) and on the intravenous tubes was measured on agar plates. Pathological changes in the organs (liver, heart, and kidney) were observed with Hematoxylin-Eosin staining. The ambroxol exhibits significant efficacy to potentiate the bactericidal effect of vancomycin on S. epidermidis biofilm both in vitro and in vivo. The antibiotic lock therapy using a combination of ambroxol and vancomycin reveals a high ability to eradicate S. epidermidis biofilms in vivo. These results provide the basis of a useful anti-infection strategy for the treatment of CRBSIs. Copyright © 2015 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  10. An in vitro biofilm model associated to dental implants: structural and quantitative analysis of in vitro biofilm formation on different dental implant surfaces.

    Science.gov (United States)

    Sánchez, M C; Llama-Palacios, A; Fernández, E; Figuero, E; Marín, M J; León, R; Blanc, V; Herrera, D; Sanz, M

    2014-10-01

    The impact of implant surfaces in dental biofilm development is presently unknown. The aim of this investigation was to assess in vitro the development of a complex biofilm model on titanium and zirconium implant surfaces, and to compare it with the same biofilm formed on hydroxyapatite surface. Six standard reference strains were used to develop an in vitro biofilm over sterile titanium, zirconium and hydroxyapatite discs, coated with saliva within the wells of pre-sterilized polystyrene tissue culture plates. The selected species used represent initial (Streptococcus oralis and Actinomyces naeslundii), early (Veillonella parvula), secondary (Fusobacterium nucleatum) and late colonizers (Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans). The developed biofilms (growth time 1 to 120h) were studied with confocal laser scanning microscopy using a vital fluorescence technique and with low-temperature scanning electron microscopy. The number (colony forming units/biofilm) and kinetics of the bacteria within the biofilm were studied with quantitative PCR (qPCR). As outcome variables, the biofilm thickness, the percentage of cell vitality and the number of bacteria were compared using the analysis of variance. The bacteria adhered and matured within the biofilm over the three surfaces with similar dynamics. Different surfaces, however, demonstrated differences both in the thickness, deposition of the extracellular polysaccharide matrix as well as in the organization of the bacterial cells. While the formation and dynamics of an in vitro biofilm model was similar irrespective of the surface of inoculation (hydroxyapatite, titanium or zirconium), there were significant differences in regards to the biofilm thickness and three-dimensional structure. Copyright © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

  11. Standardization and Classification of In vitro Biofilm Formation by Clinical Isolates of Staphylococcus aureus.

    Science.gov (United States)

    Singh, Ashish Kumar; Prakash, Pradyot; Achra, Arvind; Singh, Gyan Prakash; Das, Arghya; Singh, Rakesh Kumar

    2017-01-01

    Staphylococcus aureus is Gram-positive bacterium commonly associated with nosocomial infections. The development of biofilm exhibiting drug resistance especially in foreign body associated infections has enabled the bacterium to draw considerable attention. However, till date, consensus guidelines for in vitro biofilm quantitation and categorization criterion for the bacterial isolates based on biofilm-forming capacity are lacking. Therefore, it was intended to standardize in vitro biofilm formation by clinical isolates of S. aureus and then to classify them on the basis of their biofilm-forming capacity. A study was conducted for biofilm quantitation by tissue culture plate (TCP) assay employing 61 strains of S. aureus isolated from clinical samples during May 2015- December 2015 wherein several factors influencing the biofilm formation were optimized. Therefore, it was intended to propose a biofilm classification criteria based on the standard deviation multiples of the control differentiating them into non, low, medium, and high biofilm formers. Brain-heart infusion broth was found to be more effective in biofilm formation compared to trypticase soy broth. Heat fixation was more effective than chemical fixation. Although, individually, glucose, sucrose, and sodium chloride (NaCl) had no significant effect on biofilm formation, a statistically significant increase in absorbance was observed after using the supplement mix consisting of 222.2 mM glucose, 116.9 mM sucrose, and 1000 mM NaCl (P= 0.037). The present study puts forth a standardized in vitro TCP assay for biofilm biomass quantitation and categorization criteria for clinical isolates of S. aureus based on their biofilm-forming capacity. The proposed in vitro technique may be further evaluated for its usefulness in the management of persistent infections caused by the bacterium.

  12. Standardization and classification of In vitro biofilm formation by clinical isolates of Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Ashish Kumar Singh

    2017-01-01

    Full Text Available Background: Staphylococcus aureus is Gram-positive bacterium commonly associated with nosocomial infections. The development of biofilm exhibiting drug resistance especially in foreign body associated infections has enabled the bacterium to draw considerable attention. However, till date, consensus guidelines for in vitro biofilm quantitation and categorization criterion for the bacterial isolates based on biofilm-forming capacity are lacking. Therefore, it was intended to standardize in vitro biofilm formation by clinical isolates of S. aureus and then to classify them on the basis of their biofilm-forming capacity. Materials and Methods: A study was conducted for biofilm quantitation by tissue culture plate (TCP assay employing 61 strains of S. aureus isolated from clinical samples during May 2015– December 2015 wherein several factors influencing the biofilm formation were optimized. Therefore, it was intended to propose a biofilm classification criteria based on the standard deviation multiples of the control differentiating them into non, low, medium, and high biofilm formers. Results: Brain-heart infusion broth was found to be more effective in biofilm formation compared to trypticase soy broth. Heat fixation was more effective than chemical fixation. Although, individually, glucose, sucrose, and sodium chloride (NaCl had no significant effect on biofilm formation, a statistically significant increase in absorbance was observed after using the supplement mix consisting of 222.2 mM glucose, 116.9 mM sucrose, and 1000 mM NaCl (P = 0.037. Conclusions: The present study puts forth a standardized in vitro TCP assay for biofilm biomass quantitation and categorization criteria for clinical isolates of S. aureus based on their biofilm-forming capacity. The proposed in vitro technique may be further evaluated for its usefulness in the management of persistent infections caused by the bacterium.

  13. Effect of 5-aminolevulinic acid photodynamic therapy on Candida albicans biofilms: An in vitro study.

    Science.gov (United States)

    Shi, Hang; Li, Jiyang; Zhang, Hui; Zhang, Jie; Sun, Hongying

    2016-09-01

    In this study, the photoinactivation of 5-aminolevulinic acid (ALA) has been investigated on Candida albicans biofilms in vitro. After culture and proliferation of Candida albicans biofilms in vitro, the metabolic activity was confirmed using XTT reduction assay. Then, the suitable incubation time and concentration of ALA were determined by measuring PpIX accumulation quantities. Photosensitivity of the biofilms treated with ALA solution was studied in optical doses of 50, 100, 200 and 300J/cm(2) while light irradiation was applied by a red light semiconductor. Finally, rapid immunofluorescence staining method using the LIVE/DEAD FungaLight Yeast Viability Kit and XTT assay were conducted to visualize and quantify the antifungal effect of ALA-PDT on Candida albicans biofilms. A 5h incubation time and 15mM ALA concentration were determined for this study. Photoinactivation of ALA-PDT on Candida albicans biofilms showed a significant increase of protoporphyrin IX (PpIX) in the biofilms. The metabolic activity of Candida albicans biofilms tread with ALA-PDT confirmed the inhibition efficacy compared with control groups. Upon radiation at 300J/cm(2), cells in Candida albicans biofilms were 74.45% inhibited. PpIX can be absorbed in biofilm-grown Candida albicans in vitro and under appropriate parameters, photochemistry can be triggered by light in combination with ALA and inhibits Candida albicans biofilms effectively. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. How can in vitro models best reflect in vivo Staphylococcus biofilms?

    DEFF Research Database (Denmark)

    Meyer, Rikke Louise

    In vitro biofilm models are the basis for most studies of biofilm biology because they enable high-throughput analyses without the expenditure of animals. But how do we ensure that what we learn from in vitro studies is relevant in vivo? Biofilms grown in standard laboratory media do not interact...... fold in different S. aureus strains, and increasing the calcium and magnesium concentration to physiological levels further doubled biofilm formation for MRSA. When grown in plasma, S. aureus biofilms consisted of fibrinogen-coated cell clusters interspersed in a matrix of fibrin fibers....... This was in stark contrast to the homogenous biofilms formed in the laboratory media. While vancomycin susceptibility was similar for all biofilms, daptomycin more effectively eradicated biofilms grown in plasma. S. epidermidis biofilms were also strongly affected by human plasma. Strains that did not produce...... polysaccharides could not form biofilms in the absence of plasma, while in the presence of plasma, the structure of polysaccharide-producers and non-producers were indistinguishable. Extracellular polysaccharides may thus be of little consequence to S. epidermidis biofilms in vivo. Access to host factors...

  15. In vitro interactions between anidulafungin and nonsteroidal anti-inflammatory drugs on biofilms of Candida spp.

    Science.gov (United States)

    Rosato, Antonio; Catalano, Alessia; Carocci, Alessia; Carrieri, Antonio; Carone, Addolorata; Caggiano, Giuseppina; Franchini, Carlo; Corbo, Filomena; Montagna, Maria Teresa

    2016-03-01

    Candida spp. are responsible for many biomaterial-related infections; they give rise to infective pathologies typically associated with biofilm formation. We recently reported that the echinocandin anidulafungin (ANF) showed a strong in vitro activity against both planktonic and biofilms cells. Herein, we report the antifungal activities of ANF alone and in association with some non-steroidal anti-inflammatory drugs (NSAIDs) against nine Candida strain biofilms: four Candida albicans, two Candida glabrata and three Candida guilliermondii. The activity of ANF was assessed using an in vitro microbiological model relevant for clinical practice. ANF proved oneself to be active against biofilms cells, and a clear-cut synergism was found against Candida species biofilms when ANF was used in combination with three NSAIDs: aspirin, diclofenac, ibuprofen. The positive synergism against Candida spp. of ANF in association with aspirin or the other NSAIDs proved to be a very effective antifungal treatment (FICICandida biofilm pathologies. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. From in vitro to in vivo Models of Bacterial Biofilm-Related Infections

    OpenAIRE

    Christophe Beloin; Olaya Rendueles; David Lebeaux; Ashwini Chauhan

    2013-01-01

    International audience; The influence of microorganisms growing as sessile communities in a large number of human infections has been extensively studied and recognized for 30-40 years, therefore warranting intense scientific and medical research. Nonetheless, mimicking the biofilm-life style of bacteria and biofilm-related infections has been an arduous task. Models used to study biofilms range from simple in vitro to complex in vivo models of tissues or device-related infections. These diff...

  17. A review of telavancin activity in in vitro biofilms and animal models of biofilm-associated infections.

    Science.gov (United States)

    Chan, Cynthia; Hardin, Thomas C; Smart, Jennifer I

    2015-01-01

    Tissue- and device-associated biofilm infections are important medical problems. These infections are difficult to treat due to a high-level of tolerance to antibiotics. Telavancin has been studied in several in vitro biofilm models and has demonstrated efficacy against staphylococcal and enterococcal-associated biofilm infections, including those formed by methicillin-resistant Staphylococcus aureus. Telavancin was effective against the difficult-to-treat vancomycin- and glycopeptide-intermediate strains of S. aureus in these models. Furthermore, the efficacy of telavancin has been evaluated in several biofilm-related in vivo models, including osteomyelitis, endocarditis and device-associated infections in rabbits. Overall, telavancin exhibited similar or greater efficacy than vancomycin and other comparators in these animal models and maintained activity against vancomycin-intermediate and daptomycin nonsusceptible strains of S. aureus.

  18. Potential applications of nonthermal plasmas against biofilm-associated micro-organisms in vitro.

    Science.gov (United States)

    Puligundla, P; Mok, C

    2017-05-01

    Biofilms as complex microbial communities attached to surfaces pose several challenges in different sectors, ranging from food and healthcare to desalination and power generation. The biofilm mode of growth allows microorganisms to survive in hostile environments and biofilm cells exhibit distinct physiology and behaviour in comparison with their planktonic counterparts. They are ubiquitous, resilient and difficult to eradicate due to their resistant phenotype. Several chemical-based cleaning and disinfection regimens are conventionally used against biofilm-dwelling micro-organisms in vitro. Although such approaches are generally considered to be effective, they may contribute to the dissemination of antimicrobial resistance and environmental pollution. Consequently, advanced green technologies for biofilm control are constantly emerging. Disinfection using nonthermal plasmas (NTPs) is one of the novel strategies having a great potential for control of biofilms of a broad spectrum of micro-organisms. This review discusses several aspects related to the inactivation of biofilm-associated bacteria and fungi by different types of NTPs under in vitro conditions. A brief introduction summarizes prevailing methods in biofilm inactivation, followed by introduction to gas discharge plasmas, active plasma species and their inactivating mechanism. Subsequently, significance and aspects of NTP inactivation of biofilm-associated bacteria, especially those of medical importance, including opportunistic pathogens, oral pathogenic bacteria, foodborne pathogens and implant bacteria, are discussed. The remainder of the review discusses majorly about the synergistic effect of NTPs and their activity against biofilm-associated fungi, especially Candida species. © 2017 The Society for Applied Microbiology.

  19. Physical disruption of oral biofilms by sodium bicarbonate: an in vitro study.

    Science.gov (United States)

    Pratten, J; Wiecek, J; Mordan, N; Lomax, A; Patel, N; Spratt, D; Middleton, A M

    2016-08-01

    Sodium bicarbonate has been shown clinically to be efficacious at removing dental plaque; however, its effect of mechanism against biofilms has not been evaluated in vitro. Here, we used a well-established in vitro plaque biofilm model to investigate the disruption of dental plaque biofilms. Biofilms were grown in a constant depth film fermentor for up to 14 days. The fermentor was inoculated with pooled human saliva and growth maintained with artificial saliva. After various time points, replicate biofilms were removed and subjected to treatment at varying concentrations of sodium bicarbonate. Disruption of the plaque was assessed by viable counts and microscopy. The viable count results showed that younger biofilms were less susceptible to the action of sodium bicarbonate; however, biofilms of 7 days and older were increasingly susceptible to the material with the oldest biofilms being the most susceptible. Sixty-seven percentage of sodium bicarbonate slurry was able to reduce the number of organisms present by approx. 3 log10 . These quantitative data were corroborated qualitatively with both confocal and electron microscopy, which both showed substantial qualitative removal of mature biofilms. The results from this study have shown that sodium bicarbonate is able to disrupt mature dental plaque grown in vitro and that its reported efficacy in maintaining oral hygiene may be related to this key factor. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. Development and antimicrobial susceptibility studies of in vitro monomicrobial and polymicrobial biofilm models with Aspergillus fumigatus and Pseudomonas aeruginosa

    National Research Council Canada - National Science Library

    Manavathu, Elias K; Vager, Dora L; Vazquez, Jose A

    2014-01-01

    .... The primary objective of this study was to develop an in vitro model for P. aeruginosa and A. fumigatus polymicrobial biofilm to study the efficacy of various antimicrobial drugs alone and in combinations against biofilm-embedded cells...

  1. Development of an in vitro periodontal biofilm model for assessing antimicrobial and host modulatory effects of bioactive molecules

    National Research Council Canada - National Science Library

    Millhouse, Emma; Jose, Anto; Sherry, Leighann; Lappin, David F; Patel, Nisha; Middleton, Andrew M; Pratten, Jonathan; Culshaw, Shauna; Ramage, Gordon

    2014-01-01

    .... An in vitro multi-species biofilm containing S. mitis, F. nucleatum, P. gingivalis and A. actinomycetemcomitans was created to represent a disease-associated biofilm and the oral epithelial cell in OKF6-TERT2...

  2. In Vitro Effects of Sports and Energy Drinks on Streptococcus mutans Biofilm Formation and Metabolic Activity.

    Science.gov (United States)

    Vinson, LaQuia A; Goodlett, Amy K; Huang, Ruijie; Eckert, George J; Gregory, Richard L

    2017-09-15

    Sports and energy drinks are being increasingly consumed and contain large amounts of sugars, which are known to increase Streptococcus mutans biofilm formation and metabolic activity. The purpose of this in vitro study was to investigate the effects of sports and energy drinks on S. mutans biofilm formation and metabolic activity. S. mutans UA159 was cultured with and without a dilution (1:3 ratio) of a variety of sports and energy drinks in bacterial media for 24 hours. The biofilm was washed, fixed, and stained. Biofilm growth was evaluated by reading absorbance of the crystal violet. Biofilm metabolic activity was measured by the biofilm-reducing XTT to a water-soluble orange compound. Gatorade Protein Recovery Shake and Starbucks Doubleshot Espresso Energy were found to significantly increase biofilm (30-fold and 22-fold, respectively) and metabolic activity (2-fold and 3-fold, respectively). However, most of the remaining drinks significantly inhibited biofilm growth and metabolic activity. Several sports and energy drinks, with sugars or sugar substitutes as their main ingredients inhibited S. mutans biofilm formation. Among the drinks evaluated, Gatorade Protein Recovery Chocolate Shake and Starbucks Doubleshot Energy appear to have cariogenic potential since they increased the biofilm formation and metabolic activity of S. mutans.

  3. In vitro studies evaluating the effects of biofilms on wound-healing cells: a review.

    Science.gov (United States)

    Kirker, Kelly R; James, Garth A

    2017-04-01

    Chronic wounds are characterized as wounds that have failed to proceed through the well-orchestrated healing process and have remained open for months to years. Open wounds are at risk for colonization by opportunistic pathogens. Bacteria that colonize the open wound bed form surface-attached, multicellular communities called biofilms, and chronic wound biofilms can contain a diverse microbiota. Investigators are just beginning to elucidate the role of biofilms in chronic wound pathogenesis, and have simplified the complex wound environment using in vitro models to obtain a fundamental understanding of the impact of biofilms on wound-healing cell types. The intent of this review is to describe current in vitro methodologies and their results. Investigations started with one host cell-type and single species biofilms and demonstrated that biofilms, or their secretions, had deleterious effects on wound-healing cells. More complex systems involved the use of multiple host cell/tissue types and single species biofilms. Using human skin-equivalent tissues, investigators demonstrated that a number of different species can grow on the tissue and elicit an inflammatory response from the tissue. A full understanding of how biofilms impact wound-healing cells and host tissues will have a profound effect on how chronic wounds are treated. © 2017 APMIS. Published by John Wiley & Sons Ltd.

  4. DNase inhibits Gardnerella vaginalis biofilms in vitro and in vivo

    National Research Council Canada - National Science Library

    Hymes, Saul R; Randis, Tara M; Sun, Thomas Yang; Ratner, Adam J

    2013-01-01

    .... Gardnerella vaginalis predominates in bacterial vaginosis. Biofilms of G. vaginalis are present in human infections and are implicated in persistent disease, treatment failure, and transmission...

  5. In vitro enterococcus faecalis biofilm formation on five adhesive systems

    Science.gov (United States)

    Baca, Pilar; Furtado-Antunes de Freitas, Márcia; Ferrer-Luque, Carmen M.; González-Rodríguez, María P.

    2012-01-01

    Objective: To determine the E. faecalis biofilm formation on the surface of five adhesive systems (AS) and its relationship with roughness. Study Design: The formation of E. faecalis biofilms was tested on the surface of four dual-cure AS: AdheSE DC, Clearfil DC Bond, Futurabond DC and Excite DSC and one light-cure antimicrobial AS, Clearfil Protect Bond, after 24 hours of incubation, using the MBEC high-throughput device. Results: E. faecalis biofilms grew on all the adhesives. The least growth of biofilm was on Excite DSC, Clearfil Protect Bond, and the control. Futurabond DC resulted in the greatest roughness and biofilm amount. There was a close relationship between the quantity of biofilm and roughness, except for Clearfil Protect Bond, which showed little biofilm but high roughness. Conclusion: None of the tested AS prevented E. faecalis biofilm formation, although the least quantity was found on the surface of Clearfil Protect Bond. Key words:Adhesive systems, biofilm, Enterococcus faecalis, roughness. PMID:22143728

  6. Pharmacokinetics and pharmacodynamics of antibiotics in biofilm infections of Pseudomonas aeruginosa in vitro and in vivo

    DEFF Research Database (Denmark)

    Hengzhuang, Wang; Høiby, Niels; Ciofu, Oana

    2014-01-01

    Although progress on biofilm research has been obtained during the past decades, the treatment of biofilm infections with antibiotics remains a riddle. The pharmacokinetic (PK) and pharmacodynamic (PD) profiles of an antimicrobial agent provide important information helping to establish an effici......Although progress on biofilm research has been obtained during the past decades, the treatment of biofilm infections with antibiotics remains a riddle. The pharmacokinetic (PK) and pharmacodynamic (PD) profiles of an antimicrobial agent provide important information helping to establish...... an efficient dosing regimen and to minimize the development of antimicrobial tolerance and resistance in biofilm infections. Unfortunately, most previous PK/PD studies of antibiotics have been done on planktonic cells, and extrapolation of the results on biofilms is problematic as bacterial biofilms differ...... from planktonic grown cells in the growth rate, gene expression, and metabolism. Here, we set up several protocols for the studies of PK/PD of antibiotics in biofilm infections of P. aeruginosa in vitro and in vivo. It should be underlined that none of the protocols in biofilms have yet been...

  7. Strawberry Extract’s Effects on Enterococcus faecalis and Porphyromonas gingivalis Biofilms in vitro

    Directory of Open Access Journals (Sweden)

    Armelia Sari Widyarman

    2017-09-01

    Full Text Available Background: Enterococcus faecalis (E. faecalis and Porphyromonas gingivalis (P. gingivalis are oral bacteria related to root canal infection and periodontal disease pathogenesis. Strawberries (Fragaria x ananassa fruit are rich in vitamins and minerals, have antibacterial and antioxidant effects. Objective: This study investigated the inhibition effect of strawberry extract on monospecies and multispecies E. faecalis and P. gingivalis bacteria grown as biofilms in vitro. Methods: This study used E. faecalis ATCC 29212 and P. gingivalis ATCC 33277. It analyzed the effect of strawberry extract on bacteria biofilm formation using a biofilm assay on microplate wells. Five concentrations of strawberry extracts were used (100%, 50%, 25%, 12.5%, and 6.25%, and the inhibition effect was observed after a 1h, 3h, 6h, and 24h incubation period. Biofilms without the strawberry extract were used as the negative controls, and crystal violet and safranin (0.5%w/v were used to count the biofilm mass. The biofilms grown on microplates were counted using an ELISA reader at 450 nm after 200 mL of 90% ethanol was added to attract the absorbed stain. The strawberry extract inhibition effectiveness on the biofilm formation of each bacterium tested was analyzed using one-way Anova, where p<0.05 was defined as a significant difference. Result: The strawberry extract inhibited the tested monospecies and multispecies bacteria biofilm formation. The optimal strawberry extract concentration for the inhibition of either monospecies biofilms was 100%. However, the optimal incubation time for the strawberry extract to inhibit the multispecies biofilm formation was 24h, which was the study’s biofilm maturity phase. Conclusions: The 100% strawberry extract concentration inhibited the formation of both the monospecies and multispecies E. faecalis and P. gingivalis biofilms. Future studies are needed to evaluate the potential of strawberry extract as an alternative dental

  8. A novel antifungal is active against Candida albicans biofilms and inhibits mutagenic acetaldehyde production in vitro.

    Directory of Open Access Journals (Sweden)

    Mikko T Nieminen

    Full Text Available The ability of C. albicans to form biofilms is a major virulence factor and a challenge for management. This is evident in biofilm-associated chronic oral-oesophageal candidosis, which has been shown to be potentially carcinogenic in vivo. We have previously shown that most Candida spp. can produce significant levels of mutagenic acetaldehyde (ACH. ACH is also an important mediator of candidal biofilm formation. We have also reported that D,L-2-hydroxyisocaproic acid (HICA significantly inhibits planktonic growth of C. albicans. The aim of the present study was to investigate the effect of HICA on C. albicans biofilm formation and ACH production in vitro. Inhibition of biofilm formation by HICA, analogous control compounds or caspofungin was measured using XTT to measure biofilm metabolic activity and PicoGreen as a marker of biomass. Biofilms were visualised by scanning electron microscopy (SEM. ACH levels were measured by gas chromatography. Transcriptional changes in the genes involved in ACH metabolism were measured using RT-qPCR. The mean metabolic activity and biomass of all pre-grown (4, 24, 48 h biofilms were significantly reduced after exposure to HICA (p40 µM of ACH were detected in 24 and 48 h biofilms at both pHs. Interestingly, no ACH production was detected from D-glucose in the presence of HICA at acidic pH (p<0.05. Expression of genes responsible for ACH catabolism was up-regulated by HICA but down-regulated by caspofungin. SEM showed aberrant hyphae and collapsed hyphal structures during incubation with HICA at acidic pH. We conclude that HICA has potential as an antifungal agent with ability to inhibit C. albicans cell growth and biofilm formation. HICA also significantly reduces the mutagenic potential of C. albicans biofilms, which may be important when treating bacterial-fungal biofilm infections.

  9. A novel antifungal is active against Candida albicans biofilms and inhibits mutagenic acetaldehyde production in vitro.

    Science.gov (United States)

    Nieminen, Mikko T; Novak-Frazer, Lily; Rautemaa, Wilma; Rajendran, Ranjith; Sorsa, Timo; Ramage, Gordon; Bowyer, Paul; Rautemaa, Riina

    2014-01-01

    The ability of C. albicans to form biofilms is a major virulence factor and a challenge for management. This is evident in biofilm-associated chronic oral-oesophageal candidosis, which has been shown to be potentially carcinogenic in vivo. We have previously shown that most Candida spp. can produce significant levels of mutagenic acetaldehyde (ACH). ACH is also an important mediator of candidal biofilm formation. We have also reported that D,L-2-hydroxyisocaproic acid (HICA) significantly inhibits planktonic growth of C. albicans. The aim of the present study was to investigate the effect of HICA on C. albicans biofilm formation and ACH production in vitro. Inhibition of biofilm formation by HICA, analogous control compounds or caspofungin was measured using XTT to measure biofilm metabolic activity and PicoGreen as a marker of biomass. Biofilms were visualised by scanning electron microscopy (SEM). ACH levels were measured by gas chromatography. Transcriptional changes in the genes involved in ACH metabolism were measured using RT-qPCR. The mean metabolic activity and biomass of all pre-grown (4, 24, 48 h) biofilms were significantly reduced after exposure to HICA (pMutagenic levels (>40 μM) of ACH were detected in 24 and 48 h biofilms at both pHs. Interestingly, no ACH production was detected from D-glucose in the presence of HICA at acidic pH (pagent with ability to inhibit C. albicans cell growth and biofilm formation. HICA also significantly reduces the mutagenic potential of C. albicans biofilms, which may be important when treating bacterial-fungal biofilm infections.

  10. DNase inhibits Gardnerella vaginalis biofilms in vitro and in vivo.

    Science.gov (United States)

    Hymes, Saul R; Randis, Tara M; Sun, Thomas Yang; Ratner, Adam J

    2013-05-15

    Bacterial vaginosis is a highly prevalent and poorly understood polymicrobial disorder of the vaginal microbiota, with significant adverse sequelae. Gardnerella vaginalis predominates in bacterial vaginosis. Biofilms of G. vaginalis are present in human infections and are implicated in persistent disease, treatment failure, and transmission. Here we demonstrate that G. vaginalis biofilms contain extracellular DNA, which is essential to their structural integrity. Enzymatic disruption of this DNA specifically inhibits biofilms, acting on both newly forming and established biofilms. DNase liberates bacteria from the biofilm to supernatant fractions and potentiates the activity of metronidazole, an antimicrobial agent used in the treatment of bacterial vaginosis. Using a new murine vaginal colonization model for G. vaginalis, we demonstrate >10-fold inhibition of G. vaginalis colonization by DNase. We conclude that DNase merits investigation as a potential nonantibiotic adjunct to existing bacterial vaginosis therapies in order to decrease the risk of chronic infection, recurrence, and associated morbidities.

  11. Impact of early colonizers on in vitro subgingival biofilm formation.

    Directory of Open Access Journals (Sweden)

    Thomas W Ammann

    Full Text Available The aim of this study was to investigate the impact of early colonizing species on the structure and the composition of the bacterial community developing in a subgingival 10-species biofilm model system. The model included Streptococcus oralis, Streptococcus anginosus, Actinomycesoris, Fusobacterium nucleatum subsp. nucleatum, Veillonella dispar, Campylobacter rectus, Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. Based on literature, we considered Streptococcus oralis, Streptococcus anginosus, and Actinomyces oris as early colonizers and examined their role in the biofilms by either a delayed addition to the consortium, or by not inoculating at all the biofilms with these species. We quantitatively evaluated the resulting biofilms by real-time quantitative PCR and further compared the structures using confocal laser scanning microscopy following fluorescence in situ hybridisation. The absence of the early colonizers did not hinder biofilm formation. The biofilms reached the same total counts and developed to normal thickness. However, quantitative shifts in the abundances of individual species were observed. In the absence of streptococci, the overall biofilm structure appeared looser and more dispersed. Moreover, besides a significant increase of P. intermedia and a decrease of P. gingivalis , P. intermedia appeared to form filamented long chains that resembled streptococci. A. oris, although growing to significantly higher abundance in absence of streptococci, did not have a visible impact on the biofilms. Hence, in the absence of the early colonizers, there is a pronounced effect on P. intermedia and P. gingivalis that may cause distinct shifts in the structure of the biofilm. Streptococci possibly facilitate the establishment of P. gingivalis into subgingival biofilms, while in their absence P. intermedia became more dominant and forms elongated chains.

  12. Restoration materials and secondary caries using an in vitro biofilm model

    NARCIS (Netherlands)

    Kuper, N.K.; van de Sande, F.H.; Opdam, N.J.M.; Bronkhorst, E.M.; de Soet, J.J.; Cenci, M.S.; Huysmans, M.C.D.N.J.M.

    2015-01-01

    This in vitro study investigated whether restoration materials and adhesives influence secondary caries formation in gaps using a short-term in vitro biofilm model. Sixty enamel-dentin blocks were restored with 6 different restoration materials with or without adhesives (n = 10 per group) with a

  13. In Vitro Streptococcus pneumoniae Biofilm Formation and In Vivo Middle Ear Mucosal Biofilm in a Rat Model of Acute Otitis Induced by S. pneumoniae

    OpenAIRE

    Yadav, Mukesh Kumar; Chae, Sung-Won; Song, Jae-Jun

    2012-01-01

    Objectives Streptococcus pneumoniae is one of the most common pathogens of otitis media (OM) that exists in biofilm, which enhances the resistance of bacteria against antibiotic killing and diagnosis, compared to the free-floating (planktonic) form. This study evaluated biofilm formation by S. pneumoniae on an abiotic surface and in the middle ear cavity in a rat model of OM. Methods In vitro biofilm formation was evaluated by inoculation of a 1:100 diluted S. pneumoniae cell suspension in a ...

  14. Microbial dynamics during conversion from supragingival to subgingival biofilms in an in vitro model.

    Science.gov (United States)

    Thurnheer, T; Bostanci, N; Belibasakis, G N

    2016-04-01

    The development of dental caries and periodontal diseases result from distinct shifts in the microbiota of the tooth-associated biofilm. This in vitro study aimed to investigate changes in biofilm composition and structure, during the shift from a 'supragingival' aerobic profile to a 'subgingival' anaerobic profile. Biofilms consisting of Actinomyces oris, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus mutans and Veillonella dispar were aerobically grown in saliva-containing medium on hydroxyapatite disks. After 64 h, Campylobacter rectus, Prevotella intermedia and Streptococcus anginosus were further added along with human serum, while culture conditions were shifted to microaerophilic. After 96 h, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola were finally added and the biofilm was grown anaerobically for another 64 h. At the end of each phase, biofilms were harvested for species-specific quantification and localization. Apart from C. albicans, all other species gradually increased during aerobic and microaerophilic conditions, but remained steady during anaerobic conditions. Biofilm thickness was doubled during the microaerophilic phase, but remained steady throughout the anaerobic phase. Extracellular polysaccharide presence was gradually reduced throughout the growth period. Biofilm viability was reduced during the microaerophilic conversion, but was recovered during the anaerobic phase. This in vitro study has characterized the dynamic structural shifts occurring in an oral biofilm model during the switch from aerobic to anaerobic conditions, potentially modeling the conversion of supragingival to subgingival biofilms. Within the limitations of this experimental model, the findings may provide novel insights into the ecology of oral biofilms. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. In Vitro Antimicrobial Susceptibility of Pathogenic Leptospira Biofilm.

    Science.gov (United States)

    Vinod Kumar, Kirubakaran; Lall, Chandan; Raj, Ratchagadasse Vimal; Vedhagiri, Kumaresan; Sunish, Ittoop Pulikkottil; Vijayachari, Paluru

    2016-10-01

    Pathogenic Leptospira spp. are the causative agent of leptospirosis. Biofilm formation in leptospires is a new area of study, and its role in pathogenesis is not fully explored. As in other biofilm-forming bacteria, Leptospira biofilm may play a significant role in antibiotic resistance. In this study, the antimicrobial susceptibility of Leptospira biofilm was investigated by 96-well plate assay using Alamar Blue. Leptospira biofilm showed five to sixfold increase in resistance in all the strains used. The range of minimal bactericidal concentrations for penicillin G, ampicillin, tetracycline, and doxycycline was 1,600 U/ml, 800-1,600 μg/ml, 800-1,600 μg/ml, and 800-1,600 μg/ml, respectively. In agar substrate, the biofilm showed six- to sevenfold increase in resistance to antibiotics compared to planktonic cell. The present study emphasizes the importance of biofilm formation and its antibiotic susceptibility patterns. This could pave the way for devising appropriate strategy to prevent the occurrence of potential chronic leptospirosis in endemic areas and also during an outbreak situation.

  16. In vitro characterization of Trichophyton rubrum and T. mentagrophytes biofilms.

    Science.gov (United States)

    Costa-Orlandi, C B; Sardi, J C O; Santos, C T; Fusco-Almeida, A M; Mendes-Giannini, M J S

    2014-01-01

    Dermatophytes are fungi responsible for a disease known as dermatophytosis. Biofilms are sessile microbial communities surrounded by extracellular polymeric substances (EPS) with increased resistance to antimicrobial agents and host defenses. This paper describes, for the first time, the characteristics of Trichophyton rubrum and T. mentagrophytes biofilms. Biofilm formation was analyzed by light microscopy, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) as well as by staining with crystal violet and safranin. Metabolic activity was determined using the XTT reduction assay. Both species were able to form mature biofilms in 72 h. T. rubrum biofilm produced more biomass and EPS and was denser than T. mentagrophytes biofilm. The SEM results demonstrated a coordinated network of hyphae in all directions, embedded within EPS in some areas. Research and characterization of biofilms formed by dermatophytes may contribute to the search of new drugs for the treatment of these mycoses and might inform future revisions with respect to the dose and duration of treatment of currently available antifungals.

  17. Synergistic Effects of Nonthermal Plasma and Disinfecting Agents against Dental Biofilms In Vitro

    Science.gov (United States)

    Koban, Ina; Geisel, Marie Henrike; Holtfreter, Birte; Jablonowski, Lukasz; Hübner, Nils-Olaf; Matthes, Rutger; Masur, Kai; Weltmann, Klaus-Dieter; Kramer, Axel; Kocher, Thomas

    2013-01-01

    Aim. Dental biofilms play a major role in the pathogenesis of many dental diseases. In this study, we evaluated the synergistic effect of atmospheric pressure plasma and different agents in dentistry on the reduction of biofilms. Methods and Results. We used monospecies (S. mutans) and multispecies dental biofilm models grown on titanium discs in vitro. After treatment with one of the agents, the biofilms were treated with plasma. Efficacy of treatment was determined by the number of colony forming units (CFU) and by live-dead staining. For S. mutans biofilms no colonies could be detected after treatment with NaOCl or H2O2. For multispecies biofilms the combination with plasma achieved a higher CFU reduction than each agent alone. We found an additive antimicrobial effect between argon plasma and agents irrespective of the treatment order with cultivation technique. For EDTA and octenidine, antimicrobial efficacy assessed by live-dead staining differed significantly between the two treatment orders (P effective treatment of dental biofilms on titanium discs with atmospheric pressure plasma could be increased by adding agents in vitro. PMID:24159388

  18. Synergistic Effects of Nonthermal Plasma and Disinfecting Agents against Dental Biofilms In Vitro.

    Science.gov (United States)

    Koban, Ina; Geisel, Marie Henrike; Holtfreter, Birte; Jablonowski, Lukasz; Hübner, Nils-Olaf; Matthes, Rutger; Masur, Kai; Weltmann, Klaus-Dieter; Kramer, Axel; Kocher, Thomas

    2013-01-01

    Aim. Dental biofilms play a major role in the pathogenesis of many dental diseases. In this study, we evaluated the synergistic effect of atmospheric pressure plasma and different agents in dentistry on the reduction of biofilms. Methods and Results. We used monospecies (S. mutans) and multispecies dental biofilm models grown on titanium discs in vitro. After treatment with one of the agents, the biofilms were treated with plasma. Efficacy of treatment was determined by the number of colony forming units (CFU) and by live-dead staining. For S. mutans biofilms no colonies could be detected after treatment with NaOCl or H2O2. For multispecies biofilms the combination with plasma achieved a higher CFU reduction than each agent alone. We found an additive antimicrobial effect between argon plasma and agents irrespective of the treatment order with cultivation technique. For EDTA and octenidine, antimicrobial efficacy assessed by live-dead staining differed significantly between the two treatment orders (P effective treatment of dental biofilms on titanium discs with atmospheric pressure plasma could be increased by adding agents in vitro.

  19. Development and pyrosequencing analysis of an in-vitro oral biofilm model

    OpenAIRE

    Kistler, James O.; Pesaro, Manuel; Wade, William G.

    2015-01-01

    Background Dental caries and periodontal disease are the commonest bacterial diseases of man and can result in tooth loss. The principal method of prevention is the mechanical removal of dental plaque augmented by active agents incorporated into toothpastes and mouthrinses. In-vitro assays that include complex oral bacterial biofilms are required to accurately predict the efficacy of novel active agents in vivo. The aim of this study was to develop an oral biofilm model using the Calgary biof...

  20. In Vitro Antimicrobial Effect of a Cold Plasma Jet against Enterococcus faecalis Biofilms

    OpenAIRE

    Chunqi Jiang; Christoph Schaudinn; Jaramillo, David E.; Paul Webster; J. William Costerton

    2012-01-01

    The hypothesis that a cold plasma jet has the antimicrobial effect against Enterococcus faecalis biofilms was tested in vitro. 27 hydroxyapatite discs were incubated with E. faecalis for six days to form a monoculture biofilm on the disc surface. The prepared substrata were divided into three groups: the negative control, the positive control (5.25% NaOCl solution), and the plasma treatment group. Resultant colony-forming unit counts were associated with observations of bacterial cell morphol...

  1. A Novel Antifungal Is Active against Candida albicans Biofilms and Inhibits Mutagenic Acetaldehyde Production In Vitro

    Science.gov (United States)

    Nieminen, Mikko T.; Novak-Frazer, Lily; Rautemaa, Vilma; Rajendran, Ranjith; Sorsa, Timo; Ramage, Gordon; Bowyer, Paul; Rautemaa, Riina

    2014-01-01

    The ability of C. albicans to form biofilms is a major virulence factor and a challenge for management. This is evident in biofilm-associated chronic oral-oesophageal candidosis, which has been shown to be potentially carcinogenic in vivo. We have previously shown that most Candida spp. can produce significant levels of mutagenic acetaldehyde (ACH). ACH is also an important mediator of candidal biofilm formation. We have also reported that D,L-2-hydroxyisocaproic acid (HICA) significantly inhibits planktonic growth of C. albicans. The aim of the present study was to investigate the effect of HICA on C. albicans biofilm formation and ACH production in vitro. Inhibition of biofilm formation by HICA, analogous control compounds or caspofungin was measured using XTT to measure biofilm metabolic activity and PicoGreen as a marker of biomass. Biofilms were visualised by scanning electron microscopy (SEM). ACH levels were measured by gas chromatography. Transcriptional changes in the genes involved in ACH metabolism were measured using RT-qPCR. The mean metabolic activity and biomass of all pre-grown (4, 24, 48 h) biofilms were significantly reduced after exposure to HICA (pbiofilms pre-grown for 4 h at neutral pH. Mutagenic levels (>40 µM) of ACH were detected in 24 and 48 h biofilms at both pHs. Interestingly, no ACH production was detected from D-glucose in the presence of HICA at acidic pH (pbiofilm formation. HICA also significantly reduces the mutagenic potential of C. albicans biofilms, which may be important when treating bacterial-fungal biofilm infections. PMID:24867320

  2. Microbial biofilms are able to destroy hydroxyapatite in the absence of host immunity in vitro

    Science.gov (United States)

    Junka, Adam Feliks; Szymczyk, Patrycja; Smutnicka, Danuta; Kos, Marcin; Smolina, Iryna; Bartoszewicz, Marzenna; Chlebus, Edward; Turniak, Michal; Sedghizadeh, Parish P.

    2014-01-01

    Introduction It is widely thought that inflammation and osteoclastogenesis result in hydroxyapatite (HA) resorption and sequestra formation during osseous infections, and microbial biofilm pathogens induce the inflammatory destruction of HA. We hypothesized that biofilms associated with infectious bone disease can directly resorb HA in the absence of host inflammation or osteoclastogenesis. Therefore, we developed an in vitro model to test this hypothesis. Materials and Methods Customized HA discs were manufactured as a substrate for growing clinically relevant biofilm pathogens. Single-species biofilms of S.mutans, S.aureus, P.aeruginosa and C.albicans, and mixed-species biofilms of C.albicans + S.mutans were incubated on HA discs for 72 hours to grow mature biofilms. Three different non-biofilm control groups were also established for testing. HA discs were then evaluated by means of scanning electron microscopy, micro-CT metrotomography, x-ray spectroscopy and confocal microscopy with planimetric analysis. Additionally, quantitative cultures and pH assessment were performed. ANOVA was used to test for significance between treatment and control groups. Results All investigated biofilms were able to cause significant (P<0.05) and morphologically characteristic alterations in HA structure as compared to controls. The highest number of alterations observed was caused by mixed biofilms of C.albicans + S.mutans. S. mutans biofilm incubated in medium with additional sucrose content was the most detrimental to HA surfaces among single-species biofilms. Conclusion These findings suggest that direct microbial resorption of bone is possible in addition to immune-mediated destruction, which has important translational implications for the pathogenesis of chronic bone infections and for targeted antimicrobial therapeutics. PMID:25544303

  3. The synthetic killer peptide KP impairs Candida albicans biofilm in vitro.

    Science.gov (United States)

    Paulone, Simona; Ardizzoni, Andrea; Tavanti, Arianna; Piccinelli, Serena; Rizzato, Cosmeri; Lupetti, Antonella; Colombari, Bruna; Pericolini, Eva; Polonelli, Luciano; Magliani, Walter; Conti, Stefania; Posteraro, Brunella; Cermelli, Claudio; Blasi, Elisabetta; Peppoloni, Samuele

    2017-01-01

    Candida albicans is a commensal organism, commonly inhabiting mucosal surfaces of healthy individuals, as a part of the resident microbiota. However, in susceptible hosts, especially hospitalized and/or immunocompromised patients, it may cause a wide range of infections. The presence of abiotic substrates, such as central venous or urinary catheters, provides an additional niche for Candida attachment and persistence, particularly via biofilm development. Furthermore, Candida biofilm is poorly susceptible to most antifungals, including azoles. Here we investigated the effects of a synthetic killer peptide (KP), known to be active in vitro, ex vivo and/or in vivo against different pathogens, on C. albicans biofilm. Together with a scrambled peptide used as a negative control, KP was tested against Candida biofilm at different stages of development. A reference strain, two fluconazole-resistant and two fluconazole-susceptible C. albicans clinical isolates were used. KP-induced C. albicans oxidative stress response and membrane permeability were also analysed. Moreover, the effect of KP on transcriptional profiles of C. albicans genes involved in different stages of biofilm development, such as cell adhesion, hyphal development and extracellular matrix production, was evaluated. Our results clearly show that the treatment with KP strongly affected the capacity of C. albicans to form biofilm and significantly impairs preformed mature biofilm. KP treatment resulted in an increase in C. albicans oxidative stress response and membrane permeability; also, biofilm-related genes expression was significantly reduced. Comparable inhibitory effects were observed in all the strains employed, irrespective of their resistance or susceptibility to fluconazole. Finally, KP-mediated inhibitory effects were observed also against a catheter-associated C. albicans biofilm. This study provides the first evidence on the KP effectiveness against C. albicans biofilm, suggesting that KP may

  4. Haemophilus parainfluenzae Strain ATCC 33392 Forms Biofilms In Vitro and during Experimental Otitis Media Infections.

    Science.gov (United States)

    Pang, Bing; Swords, W Edward

    2017-09-01

    Haemophilus parainfluenzae is a nutritionally fastidious, Gram-negative bacterium with an oropharyngeal/nasopharyngeal carriage niche that is associated with a range of opportunistic infections, including infectious endocarditis and otitis media (OM). These infections are often chronic/recurrent in nature and typically involve bacterial persistence within biofilm communities that are highly resistant to host clearance. This study addresses the primary hypothesis that H. parainfluenzae forms biofilm communities that are important determinants of persistence in vivo The results from in vitro biofilm studies confirmed that H. parainfluenzae formed biofilm communities within which the polymeric matrix was mainly composed of extracellular DNA and proteins. Using a chinchilla OM infection model, we demonstrated that H. parainfluenzae formed surface-associated biofilm communities containing bacterial and host components that included neutrophil extracellular trap (NET) structures and that the bacteria mainly persisted in these biofilm communities. We also used this model to examine the possible interaction between H. parainfluenzae and its close relative Haemophilus influenzae, which is also commonly carried within the same host environments and can cause OM. The results showed that coinfection with H. influenzae promoted clearance of H. parainfluenzae from biofilm communities during OM infection. The underlying mechanisms for bacterial persistence and biofilm formation by H. parainfluenzae and knowledge about the survival defects of H. parainfluenzae during coinfection with H. influenzae are topics for future work. Copyright © 2017 American Society for Microbiology.

  5. (+-Dehydroabietic Acid, an Abietane-Type Diterpene, Inhibits Staphylococcus aureus Biofilms in Vitro

    Directory of Open Access Journals (Sweden)

    Pia Vuorela

    2013-06-01

    Full Text Available Potent drugs are desperately needed to counteract bacterial biofilm infections, especially those caused by gram-positive organisms, such as Staphylococcus aureus. Moreover, anti-biofilm compounds/agents that can be used as chemical tools are also needed for basic in vitro or in vivo studies aimed at exploring biofilms behavior and functionability. In this contribution, a collection of naturally-occurring abietane-type diterpenes and their derivatives was tested against S. aureus biofilms using a platform consisting of two phenotypic assays that have been previously published by our group. Three active compounds were identified: nordehydroabietylamine (1, (+-dehydroabietic acid (2 and (+-dehydroabietylamine (3 that prevented biofilm formation in the low micromolar range, and unlike typical antibiotics, only 2 to 4-fold higher concentrations were needed to significantly reduce viability and biomass of existing biofilms. Compound 2, (+-dehydroabietic acid, was the most selective towards biofilm bacteria, achieving high killing efficacy (based on log Reduction values and it was best tolerated by three different mammalian cell lines. Since (+-dehydroabietic acid is an easily available compound, it holds great potential to be used as a molecular probe in biofilms-related studies as well as to serve as inspirational chemical model for the development of potent drug candidates.

  6. 'Get in Early'; Biofilm and Wax Moth (Galleria mellonella) Models Reveal New Insights into the Therapeutic Potential of Clostridium difficile Bacteriophages.

    Science.gov (United States)

    Nale, Janet Y; Chutia, Mahananda; Carr, Philippa; Hickenbotham, Peter T; Clokie, Martha R J

    2016-01-01

    Clostridium difficile infection (CDI) is a global health threat associated with high rates of morbidity and mortality. Conventional antibiotic CDI therapy can result in treatment failure and recurrent infection. C. difficile produces biofilms which contribute to its virulence and impair antimicrobial activity. Some bacteriophages (phages) can penetrate biofilms and thus could be developed to either replace or supplement antibiotics. Here, we determined the impact of a previously optimized 4-phage cocktail on C. difficile ribotype 014/020 biofilms, and additionally as adjunct to vancomycin treatment in Galleria mellonella larva CDI model. The phages were applied before or after biofilm establishment in vitro, and the impact was analyzed according to turbidity, viability counts and topography as observed using scanning electron and confocal microscopy. The infectivity profiles and efficacies of orally administered phages and/or vancomycin were ascertained by monitoring colonization levels and larval survival rates. Phages prevented biofilm formation, and penetrated established biofilms. A single phage application reduced colonization causing extended longevity in the remedial treatment and prevented disease in the prophylaxis group. Multiple phage doses significantly improved the larval remedial regimen, and this treatment is comparable to vancomycin and the combined treatments. Taken together, our data suggest that the phages significantly reduce C. difficile biofilms, and prevent colonization in the G. mellonella model when used alone or in combination with vancomycin. The phages appear to be highly promising therapeutics in the targeted eradication of CDI and the use of these models has revealed that prophylactic use could be a propitious therapeutic option.

  7. Biofilm Forming Ability Of Salmonella Enteritidis In Vitro

    National Research Council Canada - National Science Library

    Čabarkapa Ivana; Škrinjar Marija; Lević Jovanka; Kokić Bojana; Blagojev Nevena; Milanov Dubravka; Suvajdžić Ljiljana

    2015-01-01

    ... out by Cristal violet assay on microtiter plates. A total of 14 isolates of S. Enteritidis were tested for biofilm forming ability, while Salmonella Enteritidis ATTC 13076 was used as the reference strain...

  8. Dps promotes survival of nontypeable Haemophilus influenzae in biofilm communities in vitro and resistance to clearance in vivo

    Directory of Open Access Journals (Sweden)

    Bing ePang

    2012-05-01

    Full Text Available Nontypeable Haemophilus influenzae (NTHi is a common airway commensal and opportunistic pathogen that persists within surface-attached biofilm communities. In this study, we tested the hypothesis that bacterial stress-responses are activated within biofilms. Transcripts for several factors associated with bacterial resistance to environmental stress were increased in biofilm cultures as compared to planktonic cultures. Among these, a homolog of the DNA-binding protein from starved cells (dps was chosen for further study. An isogenic NTHi 86-028NP dps mutant was generated and tested for resistance to environmental stress, revealing a significant survival defects in high-iron conditions, which was mediated by oxidative stress and was restored by genetic complementation. As expected, NTHi 86-028NP dps had a general stress-response defect, exhibiting decreased resistance to many types of environmental stress. While no differences were observed in density and structure of NTHi 86-028NP and NTHi 86-028NP dps biofilms, bacterial survival was decreased in NTHi 86-028NP dps biofilms as compared to the parental strain. The role of dps persistence in vivo was tested in animal infection studies. NTHi 86-028NP dps had decreased resistance to clearance after pulmonary infection of elastase-treated mice as compared to NTHi 86-028NP, whereas minimal differences were observed in clearance from mock-treated mice. Similarly, lower numbers of NTHi 86-028NP dps were recovered from middle-ear effusions and bullar homogenates in the chinchilla model for otitis media. Therefore, we conclude that Dps promotes bacterial survival within NTHi biofilm communities both in vitro and in chronic infections in vivo.

  9. Human pathogenic viruses are retained in and released by Candida albicans biofilm in vitro.

    Science.gov (United States)

    Mazaheritehrani, Elham; Sala, Arianna; Orsi, Carlotta Francesca; Neglia, Rachele Giovanna; Morace, Giulia; Blasi, Elisabetta; Cermelli, Claudio

    2014-01-22

    Candida albicans is the most prevalent human fungal pathogen associated with biofilm formation on indwelling medical devices. Under this form, Candida represents an infectious reservoir difficult to eradicate and possibly responsible for systemic, often lethal infections. Currently, no information is available on the occurrence and persistence of pathogenic viruses within C. albicans biofilm. Therefore, the aim of this study was to investigate whether Herpes Simplex Virus type 1 (HSV-1) and Coxsackievirus type B5 (CVB5) can be encompassed in Candida biofilm, retain their infectivity and then be released. Thus, cell-free virus inocula or HSV-1-infected cells were added to 24h-old fungal biofilm in tissue culture plates; 48 h later, the biofilm was detached by washing and energetic scratching and the presence of virus in the rescued material was end-point titrated on VERO cells. Planktonic Candida cultures and samples containing only medium were run in parallel as controls. We found that both HSV-1 and CVB5 free virus particles, as well as HSV-1 infected cells remain embedded in the biofilm retaining their infectivity. As a second step, the influence of biofilm on virus sensitivity to sodium hypochlorite and to specific neutralizing antibodies was investigated. The results showed that virus encompassment in fungal biofilm reduces virus sensitivity to chemical inactivation but does not affect antibody neutralization. Overall, these data provide the first in vitro evidence that viruses can be encompassed within Candida biofilm and then be released. Thus, it may be speculated that Candida biofilm can be a reservoir of viruses too, posing a further health risk. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Inhibition of Streptococcus mutans Growth and Biofilm Formation by Probiotics in vitro.

    Science.gov (United States)

    Schwendicke, Falk; Korte, Franziska; Dörfer, Christof E; Kneist, Susanne; Fawzy El-Sayed, Karim; Paris, Sebastian

    2017-01-01

    To exert anticaries effects, probiotics are described to inhibit growth and biofilm formation of cariogenic bacteria such as Streptococcus mutans (SM). We screened 8 probiotics and assessed how SM growth or biofilm formation inhibition affects cariogenicity of probiotic-SM mixed-species biofilms in vitro. Growth inhibition was assessed by cocultivating probiotics and 2 SM strains (ATCC 20532/25175) on agar. Probiotics were either precultured before SM cultivation (exclusion), or SM precultured prior to probiotic cultivation (displacement). Inhibition of SM culture growth was assessed visually. Inhibition of SM biofilm formation on bovine enamel was assessed using a continuous-flow short-term biofilm model, again in exclusion or displacement mode. The cariogenicity of mixed-species biofilms of SM with the most promising growth and biofilm formation inhibiting probiotic strains was assessed using an artificial mouth model, and enamel mineral loss (ΔZ) was measured microradiographically. We found limited differences in SM growth inhibition in exclusion versus displacement mode, and in inhibition of SM 20532 versus 25175. Results were therefore pooled. Lactobacillus acidophilus LA-5 inhibited significantly more SM culture growth than most other probiotics. L. casei LC-11 inhibited SM biofilm formation similarly to other alternatives but showed the highest retention of probiotics in the biofilms (p < 0.05). Mineral loss from SM monospecies biofilms (ΔZ = 9,772, 25th/75th percentiles: 6,277/13,558 vol% × µm) was significantly lower than from mixed-species SM × LA-5 biofilms (ΔZ = 24,578, 25th/75th percentiles: 19,081/28,768 vol% × µm; p < 0.01) but significantly higher than from SM × LC-11 biofilms (ΔZ = 4,835, 25th/75th percentiles: 263/7,865 vol% × µm; p < 0.05). Probiotics inhibiting SM culture growth do not necessarily reduce the cariogenicity of SM-probiotic biofilms. Nevertheless, SM biofilm formation inhibition may be relevant in the reduction of

  11. In vitro antimicrobial effects and mechanism of atmospheric-pressure He/O2 plasma jet on Staphylococcus aureus biofilm

    Science.gov (United States)

    Xu, Zimu; Shen, Jie; Cheng, Cheng; Hu, Shuheng; Lan, Yan; Chu, Paul K.

    2017-03-01

    The antimicrobial effects and associated mechanism of inactivation of Staphylococcus aureus (S. aureus) NCTC-8325 biofilms induced by a He/O2 atmospheric-pressure plasma jet (APPJ) are investigated in vitro. According to CFU (colony forming units) counting and the resazurin-based assay, the 10 min He/O2 (0.5%) APPJ treatment produces the optimal inactivation efficacy (>5 log10 ml-1) against the S. aureus biofilm and 5% of the bacteria enter a viable but non-culturable (VBNC) state. Meanwhile, 94% of the bacteria suffer from membrane damage according to SYTO 9/PI counterstaining. Scanning electron microscopy (SEM) reveals that plasma exposure erodes the extracellular polymeric substances (EPS) and then the cellular structure. The H2DCFDA-stained biofilms show larger concentrations of intracellular reactive oxygen species (ROS) in membrane-intact bacteria with increasing plasma dose. The admixture of oxygen in the working gas highly contributes to the deactivation efficacy of the APPJ against S. aureus and the plasma-induced endogenous ROS may work together with the discharge-generated ROS to continuously damage the bacterial membrane structure leading to deactivation of the biofilm microbes.

  12. Emergent Bacteria in Cystic Fibrosis: In Vitro Biofilm Formation and Resilience under Variable Oxygen Conditions

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    Susana P. Lopes

    2014-01-01

    Full Text Available Concurrent to conventional bacterial pathogens, unusual microbes are emerging from cystic fibrosis (CF airways. Nonetheless, little is known about the contribution of these newly microbes to the resilience of CF-associated biofilms, particularly under variable-oxygen concentrations that are known to occur in vivo in the mucus of CF patients. Two CF-emergent bacterial species, Inquilinus limosus and Dolosigranulum pigrum, and the major pathogen Pseudomonas aeruginosa were studied in terms of biofilm development and antibiotic susceptibilities under in vitro atmospheres with different oxygen availabilities. All species were able to develop in vitro biofilms under different oxygen-available environments, with D. pigrum accumulating high amounts of biomass and respiratory activities. When established, biofilms were of difficult eradication, with antibiotics losing their effectiveness in comparison with the corresponding planktonic populations. Surprisingly, biofilms of each emergent organism displayed multidrug resistance under aerobic environments, enduring even in low-oxygen atmospheres. This study suggests a potential prospect on the impact of nonconventional organisms I. limosus and D. pigrum on CF lung infections, demonstrating capacity to adapt to biofilm mode of life under restricted-oxygen atmospheres resembling CF airways, which may ultimately endanger the efficacy of currently used antibiotic regimens.

  13. Candida albicans survival, growth and biofilm formation are differently affected by mouthwashes: an in vitro study.

    Science.gov (United States)

    Paulone, Simona; Malavasi, Giulia; Ardizzoni, Andrea; Orsi, Carlotta Francesca; Peppoloni, Samuele; Neglia, Rachele Giovanna; Blasi, Elisabetta

    2017-01-01

    Candida albicans is the most common cause of oral mycoses. The aim of the present study was to investigate in vitro the susceptibility of C. albicans to mouthwashes, in terms of growth, survival and biofilm formation. Candida albicans, laboratory strain SC5314, and 7 commercial mouthwashes were employed: 3 with 0.2% chlorhexidine digluconate; 1 with 0.06% chlorhexidine digluconate and 250 ppm F- sodium fluoride; 3 with fluorine-containing molecules. None of the mouthwashes contained ethanol in their formulations. The anti-Candida effects of the mouthwashes were assessed by disk diffusion, crystal violet and XTT assays. By using five protocols combining different dilutions and contact times the mouthwashes were tested against: 1) C. albicans growth; 2) biofilm formation; 3) survival of fungal cells in early, developing and mature Candida biofilm. Chlorhexidine digluconate-containing mouthwashes consistently exhibited the highest anti-Candida activity, irrespective of the protocols employed. Fungal growth, biofilm formation and survival of Candida cells within biofilm were impaired, the effects strictly depending on both the dilution employed and the time of contact. These in vitro studies provide evidence that mouthwashes exert anti-Candida activity against both planktonic and biofilm fungal structures, but to a different extent depending on their composition. This suggests special caution in the choice of mouthwashes for oral hygiene, whether aimed at prevention or treatment of oral candidiasis.

  14. From in vitro to in vivo Models of Bacterial Biofilm-Related Infections

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    Christophe Beloin

    2013-05-01

    Full Text Available The influence of microorganisms growing as sessile communities in a large number of human infections has been extensively studied and recognized for 30–40 years, therefore warranting intense scientific and medical research. Nonetheless, mimicking the biofilm-life style of bacteria and biofilm-related infections has been an arduous task. Models used to study biofilms range from simple in vitro to complex in vivo models of tissues or device-related infections. These different models have progressively contributed to the current knowledge of biofilm physiology within the host context. While far from a complete understanding of the multiple elements controlling the dynamic interactions between the host and biofilms, we are nowadays witnessing the emergence of promising preventive or curative strategies to fight biofilm-related infections. This review undertakes a comprehensive analysis of the literature from a historic perspective commenting on the contribution of the different models and discussing future venues and new approaches that can be merged with more traditional techniques in order to model biofilm-infections and efficiently fight them.

  15. Bordetella adenylate cyclase toxin interacts with filamentous haemagglutinin to inhibit biofilm formation in vitro.

    Science.gov (United States)

    Hoffman, Casandra; Eby, Joshua; Gray, Mary; Heath Damron, F; Melvin, Jeffrey; Cotter, Peggy; Hewlett, Erik

    2017-01-01

    Bordetella pertussis, the causative agent of whooping cough, secretes and releases adenylate cyclase toxin (ACT), which is a protein bacterial toxin that targets host cells and disarms immune defenses. ACT binds filamentous haemagglutinin (FHA), a surface-displayed adhesin, and until now, the consequences of this interaction were unknown. A B. bronchiseptica mutant lacking ACT produced more biofilm than the parental strain; leading Irie et al. to propose the ACT-FHA interaction could be responsible for biofilm inhibition. Here we characterize the physical interaction of ACT with FHA and provide evidence linking that interaction to inhibition of biofilm in vitro. Exogenous ACT inhibits biofilm formation in a concentration-dependent manner and the N-terminal catalytic domain of ACT (AC domain) is necessary and sufficient for this inhibitory effect. AC Domain interacts with the C-terminal segment of FHA with ∼650 nM affinity. ACT does not inhibit biofilm formation by Bordetella lacking the mature C-terminal domain (MCD), suggesting the direct interaction between AC domain and the MCD is required for the inhibitory effect. Additionally, AC domain disrupts preformed biofilm on abiotic surfaces. The demonstrated inhibition of biofilm formation by a host-directed protein bacterial toxin represents a novel regulatory mechanism and identifies an unprecedented role for ACT. © 2016 John Wiley & Sons Ltd.

  16. From in vitro to in vivo Models of Bacterial Biofilm-Related Infections

    Science.gov (United States)

    Lebeaux, David; Chauhan, Ashwini; Rendueles, Olaya; Beloin, Christophe

    2013-01-01

    The influence of microorganisms growing as sessile communities in a large number of human infections has been extensively studied and recognized for 30–40 years, therefore warranting intense scientific and medical research. Nonetheless, mimicking the biofilm-life style of bacteria and biofilm-related infections has been an arduous task. Models used to study biofilms range from simple in vitro to complex in vivo models of tissues or device-related infections. These different models have progressively contributed to the current knowledge of biofilm physiology within the host context. While far from a complete understanding of the multiple elements controlling the dynamic interactions between the host and biofilms, we are nowadays witnessing the emergence of promising preventive or curative strategies to fight biofilm-related infections. This review undertakes a comprehensive analysis of the literature from a historic perspective commenting on the contribution of the different models and discussing future venues and new approaches that can be merged with more traditional techniques in order to model biofilm-infections and efficiently fight them. PMID:25437038

  17. In Vitro Effect of Porphyromonas gingivalis Methionine Gamma Lyase on Biofilm Composition and Oral Inflammatory Response.

    Science.gov (United States)

    Stephen, Abish S; Millhouse, Emma; Sherry, Leighann; Aduse-Opoku, Joseph; Culshaw, Shauna; Ramage, Gordon; Bradshaw, David J; Burnett, Gary R; Allaker, Robert P

    2016-01-01

    Methanethiol (methyl mercaptan) is an important contributor to oral malodour and periodontal tissue destruction. Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum are key oral microbial species that produce methanethiol via methionine gamma lyase (mgl) activity. The aim of this study was to compare an mgl knockout strain of P. gingivalis with its wild type using a 10-species biofilm co-culture model with oral keratinocytes and its effect on biofilm composition and inflammatory cytokine production. A P. gingivalis mgl knockout strain was constructed using insertion mutagenesis from wild type W50 with gas chromatographic head space analysis confirming lack of methanethiol production. 10-species biofilms consisting of Streptococcus mitis, Streptococcus oralis, Streptococcus intermedius, Fusobacterium nucleatum ssp polymorphum, Fusobacterium nucleatum ssp vincentii, Veillonella dispar, Actinomyces naeslundii, Prevotella intermedia and Aggregatibacter actinomycetemcomitans with either the wild type or mutant P. gingivalis were grown on Thermanox cover slips and used to stimulate oral keratinocytes (OKF6-TERT2), under anaerobic conditions for 4 and 24 hours. Biofilms were analysed by quantitative PCR with SYBR Green for changes in microbial ecology. Keratinocyte culture supernatants were analysed using a multiplex bead immunoassay for cytokines. Significant population differences were observed between mutant and wild type biofilms; V. dispar proportions increased (pgingivalis has been shown to affect microbial ecology in vitro, giving rise to a markedly different biofilm composition, with a more pro-inflammatory cytokine response from the keratinocytes observed. A possible role for methanethiol in biofilm formation and cytokine response with subsequent effects on oral malodor and periodontitis is suggested.

  18. The effect of inoculum source and fluid shear force on the development of in vitro oral multispecies biofilms.

    Science.gov (United States)

    Fernández, C E; Aspiras, M B; Dodds, M W; González-Cabezas, C; Rickard, A H

    2017-03-01

    Saliva has been previously used as an inoculum for in vitro oral biofilm studies. However, the microbial community profile of saliva is markedly different from hard- and soft-tissue-associated oral biofilms. Here, we investigated the changes in the biofilm architecture and microbial diversity of in vitro oral biofilms developed from saliva, tongue or plaque-derived inocula under different salivary shear forces. Four inoculum types (saliva, bacteria harvested from the tongue, toothbrush and curette-harvested plaque) were collected and pooled. Biofilms (n ≥ 15) were grown for 20 h in cell-free human saliva flowing at three different shear forces. Stained biofilms were imaged using a confocal laser scanning microscope. Biomass, thickness and roughness were determined by image analysis and bacterial community composition analysed using Ion Torrent. All developed biofilms showed a significant reduction in observed diversity compared with their respective original inoculum. Shear force altered biofilm architecture of saliva and curette-collected plaque and community composition of saliva, tongue and curette-harvested plaque. Different intraoral inocula served as precursors of in vitro oral polymicrobial biofilms which can be influenced by shear. Inoculum selection and shear force are key factors to consider when developing multispecies biofilms within in vitro models. © 2016 The Society for Applied Microbiology.

  19. Candida albicans biofilm development in vitro for photodynamic therapy study; Desenvolvimento de biofilme formado por Candida albicans in vitro para estudo da terapia fotodinamica

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Luis Claudio

    2009-07-01

    Photodynamic therapy (PDT) is a phototherapy based on the use of a photo sensitizer (PS) in the presence of low intensity light with resonant wavelength of absorption of the PS and biological systems that can raise awareness, generating reactive oxygen species. Studies show that PDT has a lethal effect on Candida albicans. The biofilm formed by C. albicans is the cause of infections associated with medical devices such as catheters, with a proven resistance to antifungal agents, and the removal of the catheter colonized almost always is necessary. However, few studies in literature report the behavior and response of biofilm organized by C. albicans against PDT. The aims of this study were to develop a methodology for in vitro biofilm formation of C. albicans, evaluate the sensitivity of the biofilm of C. albicans to antimicrobial photodynamic therapy using PS as the methylene blue (MB) and hypocrellin B: La{sup +3} (HBL{sup a+3}) and analyze the biofilm by Optical Coherence Tomography (OCT). For biofilm formation, discs were made from elastomeric silicone catheters. The PS were dissolved in solution of PBS, and the MB had two different concentrations tested in the biofilm: 100{mu}M and 1mM; HBLa{sup +3} only one of 10{mu}M. The irradiation of both dyes with the microorganism was done by two different LEDs, one with red emission at {lambda} = 630nm {+-} 20nm and the other one blue emission at {lambda} = 460nm {+-} 30nm. We performed a curve of survival fraction versus time of irradiation of each sample with biofilm and suspension of the microorganism in the yeast form to verify the susceptibility of the front PDT. The yeast showed 100% reduction using both PS, but at different times of irradiation (30s to HBLa{sup +3} and 6 min for the MB at 100{mu}M). When the therapy was applied in biofilm, the MB 100{mu}M did not show any significant reduction, while at concentration of 1mM was reduced by 100% after 6 min of irradiation. The HBLa{sup +3} biofilm group showed a

  20. From Mouth to Model: Combining in vivo and in vitro oral biofilm growth

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    Barbara Klug

    2016-09-01

    Full Text Available Oral biofilm studies based on simplified experimental setups are difficult to interpret. Models are limited mostly by the number of bacterial species observed and the insufficiency of artificial media. Few studies have attempted to overcome these limitations and to cultivate native oral biofilm. This study aimed to grow oral biofilm in vivo before transfer to a biofilm reactor for ex-situ incubation. The in-vitro survival of this oral biofilm and the changes in bacterial composition over time were observed. Six human enamel-dentin slabs embedded buccally in dental splints were used as biofilm carriers. Fitted individually to the upper jaw of 25 non-smoking male volunteers, the splints were worn continuously for 48 hours. During this time, tooth-brushing and alcohol-consumption were not permitted. The biofilm was then transferred on slabs into a biofilm reactor and incubated there for 48 hours while being nourished in BHI medium. Live/dead staining and confocal laser scanning microscopy were used to observe bacterial survival over four points in time: directly after removal (T0 and after 1h (T1, 24h (T2 and 48h (T3 of incubation. Bacterial diversity at T0 and T3 was compared with 454-pyrosequencing. Fluorescence in situ hybridization was performed to show specific taxa. Survival curves were calculated with a specially designed MATLAB script. Acacia and QIIME 1.9.1 were used to process pyrosequencing data. SPSS 21.0 and R 3.3.1 were used for statistical analysis.After initial fluctuations at T1, survival curves mostly showed approximation of the bacterial numbers to the initial level at T3. Pyrosequencing analysis resulted in 117 OTUs common to all samples. The genera Streptococcus and Veillonella (both Firmicutes dominated at T0 and T3. They make up two thirds of the biofilm. Genera with lower relative abundance had grown significantly at T3. FISH analysis confirmed the pyrosequencing results, i.e. the predominant staining of Firmicutes. We

  1. Dexamethasone diffusion across contact lenses is inhibited by Staphylococcus epidermidis biofilms in vitro

    Science.gov (United States)

    Brothers, Kimberly M.; Nau, Amy C.; Romanowski, Eric G.; Shanks, Robert M. Q.

    2014-01-01

    Purpose This study was designed to measure the impact of bacterial biofilms on diffusion of an ocular therapeutic through silicone hydrogel bandage lenses in vitro. Methods An assay was designed to study the passage of a commonly used steroid dexamethasone through the silicone hydrogel soft contact lenses. Diffused dexamethasone was measured using a spectrophotometer over a period of 18 hours and quantified using a standard curve. This assay was performed with control and Staphylococcus epidermidis biofilm-coated contact lenses composed of lotrafilcon A and methafilcon. Biofilms were formed in brain heart infusion broth supplemented with D-glucose. Results The presented data validate a simple in vitro model that can be used to measure penetration of a topical therapeutic through silicone hydrogel soft contact lenses. Using this model we measured a reduction of dexamethasone diffusion by up to 88% through S. epidermidis biofilm-coated silicon hydrogel lenses compared to control lenses. Conclusions The results of this in vitro study demonstrate that bacterial biofilms impede dexamethasone diffusion through silicon hydrogel contact lenses, and warrant future studies regarding the clinical benefit of using ocular therapeutics in the setting of bandage contact lens use for corneal epithelial defects. PMID:25090165

  2. In vitro biofilm models to study dental caries: a systematic review.

    Science.gov (United States)

    Maske, T T; van de Sande, F H; Arthur, R A; Huysmans, M C D N J M; Cenci, M S

    2017-09-01

    The aim of this systematic review is to characterize and discuss key methodological aspects of in vitro biofilm models for caries-related research and to verify the reproducibility and dose-response of models considering the response to anti-caries and/or antimicrobial substances. Inclusion criteria were divided into Part I (PI): an in vitro biofilm model that produces a cariogenic biofilm and/or caries-like lesions and allows pH fluctuations; and Part II (PII): models showing an effect of anti-caries and/or antimicrobial substances. Within PI, 72.9% consisted of dynamic biofilm models, while 27.1% consisted of batch models. Within PII, 75.5% corresponded to dynamic models, whereas 24.5% corresponded to batch models. Respectively, 20.4 and 14.3% of the studies reported dose-response validations and reproducibility, and 32.7% were classified as having a high risk of bias. Several in vitro biofilm models are available for caries-related research; however, most models lack validation by dose-response and reproducibility experiments for each proposed protocol.

  3. Dexamethasone diffusion across contact lenses is inhibited by Staphylococcus epidermidis biofilms in vitro.

    Science.gov (United States)

    Brothers, Kimberly M; Nau, Amy C; Romanowski, Eric G; Shanks, Robert M Q

    2014-10-01

    This study was designed to measure the impact of bacterial biofilms on diffusion of an ocular therapeutic through silicone hydrogel bandage lenses in vitro. An assay was designed to study the passage of a commonly used steroid, dexamethasone, through silicone hydrogel soft contact lenses. Diffused dexamethasone was measured using a spectrophotometer over a period of 18 hours and quantified using a standard curve. This assay was performed with control and Staphylococcus epidermidis biofilm-coated contact lenses comprised of lotrafilcon A and methafilcon. Biofilms were formed in brain heart infusion broth supplemented with D-glucose. The presented data validate a simple in vitro model that can be used to measure the penetration of a topical therapeutic through silicone hydrogel soft contact lenses. Using this model, we measured a reduction in dexamethasone diffusion up to 88% through S. epidermidis biofilm-coated silicone hydrogel lenses compared with control lenses. The results of this in vitro study demonstrate that bacterial biofilms impede dexamethasone diffusion through silicone hydrogel contact lenses and warrant future studies regarding the clinical benefit of using ocular therapeutics in the setting of bandage contact lens use for corneal epithelial defects.

  4. Effects of CO2 laser irradiation on matrix-rich biofilm development formation–an in vitro study

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    Bruna Raquel Zancopé

    2016-11-01

    Full Text Available Background A carbon dioxide (CO2 laser has been used to morphologically and chemically modify the dental enamel surface as well as to make it more resistant to demineralization. Despite a variety of experiments demonstrating the inhibitory effect of a CO2 laser in reduce enamel demineralization, little is known about the effect of surface irradiated on bacterial growth. Thus, this in vitro study was preformed to evaluate the biofilm formation on enamel previously irradiated with a CO2 laser (λ = 10.6 µM. Methods For this in vitro study, 96 specimens of bovine enamel were employed, which were divided into two groups (n = 48: 1 Control-non-irradiated surface and 2 Irradiated enamel surface. Biofilms were grown on the enamel specimens by one, three and five days under intermittent cariogenic condition in the irradiated and non-irradiated surface. In each assessment time, the biofilm were evaluated by dry weigh, counting the number of viable colonies and, in fifth day, were evaluated by polysaccharides analysis, quantitative real time Polymerase Chain Reaction (PCR as well as by contact angle. In addition, the morphology of biofilms was characterized by fluorescence microscopy and field emission scanning electron microscopy (FESEM. Initially, the assumptions of equal variances and normal distribution of errors were conferred and the results are analyzed statistically by t-test and Mann Whitney test. Results The mean of log CFU/mL obtained for the one-day biofilm evaluation showed that there is statistical difference between the experimental groups. When biofilms were exposed to the CO2 laser, CFU/mL and CFU/dry weight in three day was reduced significantly compared with control group. The difference in the genes expression (Glucosyltransferases (gtfB and Glucan-binding protein (gbpB and polysaccharides was not statically significant. Contact angle was increased relative to control when the surface was irradiated with the CO2 laser. Similar

  5. In vitro analysis of finasteride activity against Candida albicans urinary biofilm formation and filamentation.

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    Chavez-Dozal, Alba A; Lown, Livia; Jahng, Maximillian; Walraven, Carla J; Lee, Samuel A

    2014-10-01

    Candida albicans is the 3rd most common cause of catheter-associated urinary tract infections, with a strong propensity to form drug-resistant catheter-related biofilms. Due to the limited efficacy of available antifungals against biofilms, drug repurposing has been investigated in order to identify novel agents with activities against fungal biofilms. Finasteride is a 5-α-reductase inhibitor commonly used for the treatment of benign prostatic hyperplasia, with activity against human type II and III isoenzymes. We analyzed the Candida Genome Database and identified a C. albicans homolog of type III 5-α-reductase, Dfg10p, which shares 27% sequence identity and 41% similarity to the human type III 5-α-reductase. Thus, we investigated finasteride for activity against C. albicans urinary biofilms, alone and in combination with amphotericin B or fluconazole. Finasteride alone was highly effective in the prevention of C. albicans biofilm formation at doses of ≥16 mg/liter and the treatment of preformed biofilms at doses of ≥128 mg/liter. In biofilm checkerboard analyses, finasteride exhibited synergistic activity in the prevention of biofilm formation in a combination of 4 mg/liter finasteride with 2 mg/liter fluconazole. Finasteride inhibited filamentation, thus suggesting a potential mechanism of action. These results indicate that finasteride alone is highly active in the prevention of C. albicans urinary biofilms in vitro and has synergistic activity in combination with fluconazole. Further investigation of the clinical utility of finasteride in the prevention of urinary candidiasis is warranted. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  6. In vitro biofilm forming capacity on abiotic contact surfaces by outbreak-associated Vibrio harveyi strains

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    Pallaval Veera Bramha Chari

    2014-02-01

    Full Text Available Objective: To evaluate the in vitro biofilm forming capacity on abiotic food contact surfaces by Vibrio harveyi (V. harveyi strains. Methods: Thirty six Gram-negative V. harveyi strains were isolated from various street vended seafood outlets in a food processing line and evaluated for their ability to produce mucoid biofilms on food contact surfaces using a microplate assay. Phenotypic characterization of mucoid biofilm producing V. harveyi strains were screened on Congo red agar, thiosulfate-citrate-bile salts-sucrose agar and tryptic soy agar, respectively. Results: Only five V. harveyi strains (14% were mucoid biofilm producers characterized by formation of black colonies, whereas the remaining 31 strains (86% were not capable of producing biofilm characterized by formation of red colonies or pinkish-red colonies with darkening at the centre. The morphological, physiological and biochemical characteristics of these isolates were studied using standard protocols. Strain identification was confirmed by polymerase chain reaction targeted to species-specific polymerase chain reaction primers VH-1 and VH-2 corresponding to variable regions of V. harveyi 16S rRNA sequence. All the biofilm-forming strains showed resistance to at least three antimicrobial compounds tested. V. harveyi strains isolated from various seafood were able to form biofilms of different capacity, and the strains VB267, VB238 and VB166 isolated from cat fish, shrimp and eel fish exhibited significantly greater biofilm forming ability compared to other isolates. Conclusions: It can be concluded from the present study that the strain VB166 was able to better attach and form subsequent biofilms on glass and stainless steel compared to high density polyethylene. These properties allow these bacteria to survive, proliferate and persist in street vended seafood outlets.

  7. A three-phase in-vitro system for studying Pseudomonas aeruginosa adhesion and biofilm formation upon hydrogel contact lenses

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    Kohlmann Thomas

    2010-11-01

    Full Text Available Abstract Background Pseudomonas aeruginosa is commonly associated with contact lens (CL -related eye infections, for which bacterial adhesion and biofilm formation upon hydrogel CLs is a specific risk factor. Whilst P. aeruginosa has been widely used as a model organism for initial biofilm formation on CLs, in-vitro models that closely reproduce in-vivo conditions have rarely been presented. Results In the current investigation, a novel in-vitro biofilm model for studying the adherence of P. aeruginosa to hydrogel CLs was established. Nutritional and interfacial conditions similar to those in the eye of a CL wearer were created through the involvement of a solid:liquid and a solid:air interface, shear forces and a complex artificial tear fluid. Bioburdens varied depending on the CL material and biofilm maturation occurred after 72 h incubation. Whilst a range of biofilm morphologies were visualised including dispersed and adherent bacterial cells, aggregates and colonies embedded in extracellular polymer substances (EPS, EPS fibres, mushroom-like formations, and crystalline structures, a compact and heterogeneous biofilm morphology predominated on all CL materials. Conclusions In order to better understand the process of biofilm formation on CLs and to test the efficacy of CL care solutions, representative in-vitro biofilm models are required. Here, we present a three-phase biofilm model that simulates the environment in the eye of a CL wearer and thus generates biofilms which resemble those commonly observed in-situ.

  8. Development of an in vitro Assay, Based on the BioFilm Ring Test®, for Rapid Profiling of Biofilm-Growing Bacteria.

    Science.gov (United States)

    Di Domenico, Enea G; Toma, Luigi; Provot, Christian; Ascenzioni, Fiorentina; Sperduti, Isabella; Prignano, Grazia; Gallo, Maria T; Pimpinelli, Fulvia; Bordignon, Valentina; Bernardi, Thierry; Ensoli, Fabrizio

    2016-01-01

    Microbial biofilm represents a major virulence factor associated with chronic and recurrent infections. Pathogenic bacteria embedded in biofilms are highly resistant to environmental and chemical agents, including antibiotics and therefore difficult to eradicate. Thus, reliable tests to assess biofilm formation by bacterial strains as well as the impact of chemicals or antibiotics on biofilm formation represent desirable tools for a most effective therapeutic management and microbiological risk control. Current methods to evaluate biofilm formation are usually time-consuming, costly, and hardly applicable in the clinical setting. The aim of the present study was to develop and assess a simple and reliable in vitro procedure for the characterization of biofilm-producing bacterial strains for future clinical applications based on the BioFilm Ring Test® (BRT) technology. The procedure developed for clinical testing (cBRT) can provide an accurate and timely (5 h) measurement of biofilm formation for the most common pathogenic bacteria seen in clinical practice. The results gathered by the cBRT assay were in agreement with the traditional crystal violet (CV) staining test, according to the κ coefficient test (κ = 0.623). However, the cBRT assay showed higher levels of specificity (92.2%) and accuracy (88.1%) as compared to CV. The results indicate that this procedure offers an easy, rapid and robust assay to test microbial biofilm and a promising tool for clinical microbiology.

  9. In vitro biofilm models to study dental caries: a systematic review

    NARCIS (Netherlands)

    Maske, T.T.; Sande, F.H. van de; Arthur, R.A.; Huysmans, M.C.; Cenci, M.S.

    2017-01-01

    The aim of this systematic review is to characterize and discuss key methodological aspects of in vitro biofilm models for caries-related research and to verify the reproducibility and dose-response of models considering the response to anti-caries and/or antimicrobial substances. Inclusion criteria

  10. Transcriptome Sequencing Reveals Wide Expression Reprogramming of Basal and Unknown Genes in Leptospira biflexa Biofilms.

    Science.gov (United States)

    Iraola, Gregorio; Spangenberg, Lucía; Lopes Bastos, Bruno; Graña, Martín; Vasconcelos, Larissa; Almeida, Áurea; Greif, Gonzalo; Robello, Carlos; Ristow, Paula; Naya, Hugo

    2016-01-01

    The genus Leptospira is composed of pathogenic and saprophytic spirochetes. Pathogenic Leptospira is the etiological agent of leptospirosis, a globally spread neglected disease. A key ecological feature of some pathogenic species is their ability to survive both within and outside the host. For most leptospires, the ability to persist outside the host is associated with biofilm formation, a most important bacterial strategy to face and overcome hostile environmental conditions. The architecture and biochemistry of leptospiral biofilms are rather well understood; however, the genetic program underpinning biofilm formation remains mostly unknown. In this work, we used the saprophyte Leptospira biflexa as a model organism to assess over- and underrepresented transcripts during the biofilm state, using transcriptome sequencing (RNA-seq) technology. Our results showed that some basal biological processes like DNA replication and cell division are downregulated in the mature biofilm. Additionally, we identified significant expression reprogramming for genes involved in motility, sugar/lipid metabolism, and iron scavenging, as well as for outer membrane-encoding genes. A careful manual annotation process allowed us to assign molecular functions to many previously uncharacterized genes that are probably involved in biofilm metabolism. We also provided evidence for the presence of small regulatory RNAs in this species. Finally, coexpression networks were reconstructed to pinpoint functionally related gene clusters that may explain how biofilm maintenance is regulated. Beyond elucidating some genetic aspects of biofilm formation, this work reveals a number of pathways whose functional dissection may impact our understanding of leptospiral biology, in particular how these organisms adapt to environmental changes. IMPORTANCE In this work, we describe the first transcriptome based on RNA-seq technology focused on studying transcriptional changes associated with biofilm growth

  11. In vitro activity of xanthorrhizol isolated from the rhizome of Javanese turmeric (Curcuma xanthorrhiza Roxb.) against Candida albicans biofilms.

    Science.gov (United States)

    Rukayadi, Yaya; Hwang, Jae-Kwan

    2013-07-01

    The purpose of this study was to investigate the activity of xanthorrhizol isolated from Curcuma xanthorrhiza Roxb. on Candida albicans biofilms at adherent, intermediate, and mature phase of growth. C. albicans biofilms were formed in flat-bottom 96-well microtiter plates. The biofilms of C. albicans at different phases of development were exposed to xanthorrhizol at different concentrations (0.5 µg/mL-256 µg/mL) for 24 h. The metabolic activity of cells within the biofilms was quantified using the XTT reduction assay. Sessile minimum inhibitory concentrations (SMICs) were determined at 50% and 80% reduction in the biofilm OD₄₉₀ compared to the control wells. The SMIC₅₀ and SMIC₈₀ of xanthorrhizol against 18 C. albicans biofilms were 4--16 µg/mL and 8--32 µg/mL, respectively. The results demonstrated that the activity of xanthorrhizol in reducing C. albicans biofilms OD₄₉₀ was dependent on the concentration and the phase of growth of biofilm. Xanthorrhizol at concentration of 8 µg/mL completely reduced in biofilm referring to XTT-colorimetric readings at adherent phase, whereas 32 µg/mL of xanthorrhizol reduced 87.95% and 67.48 % of biofilm referring to XTT-colorimetric readings at intermediate and mature phases, respectively. Xanthorrhizol displayed potent activity against C. albicans biofilms in vitro and therefore might have potential therapeutic implication for biofilm-associated candidal infections. Copyright © 2012 John Wiley & Sons, Ltd.

  12. Morin inhibits biofilm production and reduces the virulence of Listeria monocytogenes - An in vitro and in vivo approach.

    Science.gov (United States)

    Sivaranjani, Murugesan; Gowrishankar, Shanmugaraj; Kamaladevi, Arumugam; Pandian, Shunmugiah Karutha; Balamurugan, Kirshnaswamy; Ravi, Arumugam Veera

    2016-11-21

    The current study explores the in vitro and in vivo antibiofilm efficacy of morin against a leading foodborne pathogen-Listeria monocytogenes (LM). Minimum inhibitory concentration (MIC) of morin against LM strains was found to be 100μg/ml. The non-antibacterial effect of morin at its sub-MICs (6.25, 12.5 and 25μg/ml) was determined through growth curve and XTT assay. Morin at its sub-MICs demonstrated a significant dose dependent inhibitory efficacy against LM biofilm formation which was also evidenced through light, confocal and scanning electron microscopic analyses. However, morin failed to disperse the mature biofilm of LM even at its MIC. Our data also revealed the anti-virulence efficacy of morin, as it significantly inhibited the production of hemolysin and motility of LM. Concentration-dependent susceptibility of morin treated LM cells to normal human serum was observed. In vivo studies revealed that morin extended the lifespan of LM infected Caenorhabditis elegans by about 85%. Furthermore, the non-toxic nature and in vivo anti-adherence efficacy of morin were also ascertained through C. elegans-LM infection model. Overall, the data of the current study identifies morin as a promising antibiofilm agent and its suitability to formulate protective strategies against biofilm associated infections caused by LM. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Molecular Techniques Revealed Highly Diverse Microbial Communities in Natural Marine Biofilms on Polystyrene Dishes for Invertebrate Larval Settlement

    KAUST Repository

    Lee, On On

    2014-01-09

    Biofilm microbial communities play an important role in the larval settlement response of marine invertebrates. However, the underlying mechanism has yet to be resolved, mainly because of the uncertainties in characterizing members in the communities using traditional 16S rRNA gene-based molecular methods and in identifying the chemical signals involved. In this study, pyrosequencing was used to characterize the bacterial communities in intertidal and subtidal marine biofilms developed during two seasons. We revealed highly diverse biofilm bacterial communities that varied with season and tidal level. Over 3,000 operational taxonomic units with estimates of up to 8,000 species were recovered in a biofilm sample, which is by far the highest number recorded in subtropical marine biofilms. Nineteen phyla were found, of which Cyanobacteria and Proteobacteria were the most dominant one in the intertidal and subtidal biofilms, respectively. Apart from these, Actinobacteria, Bacteroidetes, and Planctomycetes were the major groups recovered in both intertidal and subtidal biofilms, although their relative abundance varied among samples. Full-length 16S rRNA gene clone libraries were constructed for the four biofilm samples and showed similar bacterial compositions at the phylum level to those revealed by pyrosequencing. Laboratory assays confirmed that cyrids of the barnacle Balanus amphitrite preferred to settle on the intertidal rather than subtidal biofilms. This preference was independent of the biofilm bacterial density or biomass but was probably related to the biofilm community structure, particularly, the Proteobacterial and Cyanobacterial groups. © 2014 Springer Science+Business Media New York.

  14. Cold plasma therapy of a tooth root canal infected with enterococcus faecalis biofilms in vitro.

    Science.gov (United States)

    Pan, Jie; Sun, Ke; Liang, Yongdong; Sun, Peng; Yang, Xiaohui; Wang, Jing; Zhang, Jue; Zhu, Weidong; Fang, Jing; Becker, Kurt H

    2013-01-01

    Complete sterilization of an infected root canal is an important challenge in endodontic treatment. Traditional methods often cannot achieve high-efficiency sterilization because of the complexity of the root canal system. The objective of the study was to investigate in vitro the feasibility of using a cold plasma treatment of a root canal infected with Enterococcus faecalis biofilms. Seventy single-root teeth infected with E. faecalis biofilms were divided into 7 groups. Group 1 served as the negative control group (no treatment), and group 7 was the positive control group with teeth treated with calcium hydroxide intracanal medication for 7 days. Groups 2 to 6 included teeth treated by cold plasma for 2, 4, 6, 8, and 10 minutes, respectively. The disinfection of the E. faecalis biofilm was evaluated by colony-forming unit (CFU) counting. Scanning electron microscopy was used to evaluate the structural changes of the E. faecalis biofilm before and after plasma treatment. Confocal scanning laser microscopy was used to investigate the vitality of the microorganisms in the biofilm before and after plasma treatment. A significant decrease in the number of CFUs was observed after prolonged cold plasma treatment (based on the statistical analysis of the teeth in groups 2-6). Compared with the positive control group, cold plasma treatment of 8 or 10 minutes (groups 5 and 6) had a significantly higher antimicrobial efficacy (P faecalis death and destruction of the biofilm. The cold plasma had a high efficiency in disinfecting the E. faecalis biofilms in in vitro dental root canal treatment. Copyright © 2013 American Association of Endodontists. All rights reserved.

  15. Streptococcus uberis: In vitro biofilm production in response to carbohydrates and skim milk.

    Science.gov (United States)

    Dieser, Silvana A; Fessia, Aluminé S; Ferrari, Miriam P; Raspanti, Claudia G; Odierno, Liliana M

    Streptococcus uberis has become one of the most important environmental pathogens associated with clinical and subclinical bovine mastitis. Biofilm confers to bacteria more resistance to physical and chemical agents as well as to different mechanisms of the innate immune system. The aim of this work was to evaluate the ability of in vitro biofilm production in 32 S. uberis isolates from bovine mastitis and identified by biochemical tests and subsequently confirmed by the amplification of the pauA gene. The isolates were cultivated in TMP broth and TMP broth with the addition of 0.5% glucose, 1% sucrose, 1% lactose or 0.5% skim milk in microtiter plates stained with crystal violet. We demonstrated that S. uberis isolated from bovine mastitis are able to produce biofilms in TMP broth and, also that biofilm formation by S. uberis can be significantly enhanced by the addition of 0.5% glucose or 1% sucrose to TMP broth. This may suggest that the carbohydrates in milk or within the ruminant gut might affect the growth mode of S. uberis. In addition, our results showed that in vitro biofilm production under different conditions of supplementation displays variation among the isolates and that each isolate shows a particular profile of biofilm production. This phenotypic heterogeneity in biofilm production exhibited by S. uberis could at least partly explain why this bacterium has the ability to adapt to different niches facilitating survival to diverse and stressful conditions. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  16. Influence of Candida krusei and Candida glabrata on Candida albicans gene expression in in vitro biofilms.

    Science.gov (United States)

    Barros, Patrícia Pimentel; Ribeiro, Felipe Camargo; Rossoni, Rodnei Dennis; Junqueira, Juliana Campos; Jorge, Antonio Olavo Cardoso

    2016-04-01

    The present study aimed to evaluate the interactions between the species Candida albicans, Candida krusei and Candida glabrata in monotypic and mixed biofilm models formed in vitro as well as the relative expression of the ALS1, ALS3, HWP1, BCR1, EFG1, TEC1, SAP5, PLB2 and LIP9 genes. Mixed (C. albicans/C. krusei and C. albicans/C. glabrata) and monotypic biofilms were cultured for 0, 12 and 24h. Gene expression was analyzed in the same biofilm model in which the number of CFU/mL was counted. The C. albicans CFU/mL values were lower at the 12 and 24h time points in the mixed biofilms compared with the monotypic biofilms, and decreases of 56.23% and 64.4% in C. albicans were observed when this species was associated with C. glabrata and C. krusei, respectively. In the presence of C. krusei, the expression of the ALS3, HWP1, BCR1, EFG1 and TEC1 genes of C. albicans was completely inhibited, indicating both transcriptome and the phenotypic antagonism between these two species, but genes related to the secretion of enzymes were stimulated. In the presence of C. glabrata, C. albicans showed a similar gene expression profile to that obtained in association with C. krusei, though it was altered to a lesser degree. We conclude that C. krusei and C. glabrata may alter or inhibit the mechanisms involved in the in vitro adherence and formation of C. albicans biofilms, influencing the pathogenicity of this species and suggesting a competitive interaction with C. krusei and C. glabrata in biofilm formation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Antifungal activity of amphotericin B and voriconazole against the biofilms and biofilm-dispersed cells of Candida albicans employing a newly developed in vitro pharmacokinetic model.

    Science.gov (United States)

    El-Azizi, Mohamed; Farag, Noha; Khardori, Nancy

    2015-04-03

    Candida albicans is a common cause of a variety of superficial and invasive disseminated infections the majority of which are associated with biofilm growth on implanted devices. The aim of the study is to evaluate the activity of amphotericin B and voriconazole against the biofilm and the biofilm-dispersed cells of Candida albicans using a newly developed in vitro pharmacokinetic model which simulates the clinical situation when the antifungal agents are administered intermittently. RPMI medium containing 1-5 X 10(6) CFU/ml of C. albicans was continuously delivered to the device at 30 ml/h for 2 hours. The planktonic cells were removed and biofilms on the catheter were kept under continuous flow of RPMI medium at 10 ml/h. Five doses of amphotericin B or voriconazole were delivered to 2, 5 and 10 day-old biofilms at initial concentrations (2 and 3 μg/ml respectively) that were exponentially diluted. Dispersed cells in effluents from the device were counted and the adherent cells on the catheter were evaluated after 48 h of the last dose. The minimum inhibitory concentration of voriconazole and amphotericin B against the tested isolate was 0.0325 and 0.25 μg/ml respectively. Amphotericin B significantly reduced the dispersion of C. albicans cells from the biofilm. The log10 reduction in the dispersed cells was 2.54-3.54, 2.30-3.55, and 1.94-2.50 following addition of 5 doses of amphotericin B to 2-, 5- and 10-day old biofilms respectively. The number of the viable cells within the biofilm was reduced by 18 (±7.63), 5 and 4% following addition of the 5 doses of amphotericin B to the biofilms respectively. Voriconazole showed no significant effect on the viability of C. albicans within the biofilm. Both antifungal agents failed to eradicate C. albicans biofilm or stop cell dispersion from them and the resistance progressed with maturation of the biofilm. These findings go along with the need for removal of devices in spite of antifungal therapy in patients

  18. Effects of Streptococcus mutans gtfC deficiency on mixed oral biofilms in vitro.

    Science.gov (United States)

    Thurnheer, T; van der Ploeg, J R; Giertsen, E; Guggenheim, B

    2006-01-01

    The aim of this study was to examine the influence of glucosyltransferase-gene-negative (gtf-) Streptococcus mutans strains unable to synthesize water-insoluble or soluble glucan on the structure and macromolecular diffusion properties of in vitro grown mixed oral biofilms. Biofilms modeling supragingival plaque consisted of Actinomyces naeslundii OMZ 745, Candida albicans OMZ 110, Fusobacterium nucleatum KP-F2, Streptococcus oralis SK 248, Veillonella dispar ATCC 17748T and one of the S. mutans strains UA159, OMZ 966, OMZ 937 or OMZ 977. Biofilms were grown anaerobically on sintered hydroxyapatite disks for 64.5 h at 37 degrees C. To perform confocal laser scanning microscopy analyses, microorganisms were stained with Syto 13 and extracellular polysaccharides (EPS) with Calcofluor. Macromolecular diffusion properties were measured following timed biofilm exposure to Texas-Red-labeled 70-kDa dextran. Results showed that replacing wild-type S. mutans by a gtfC- mutant led to an increase in the volume fraction occupied by cells from 29 to 48% and a decrease of the EPS volume fraction from 51 to 33%. No such changes were observed when the S. mutans wild-type strain was replaced by a gtfB- or gtfD- mutant. The diffusion coefficient of 70-kDa dextran in biofilms containing the gtfC- S. mutans was 16-fold higher than in biofilms with the wild-type strain indicating a strong macromolecular sieving effect of GTF C-generated glucans. Our data demonstrate the influence of EPS on the structure and macromolecular diffusion properties of an oral biofilm model and uncover our still limited knowledge of the function of EPS in biofilms and plaque. Copyright (c) 2006 S. Karger AG, Basel.

  19. Real time optical coherence tomography monitoring of Candida albicans biofilm in vitro during photodynamic treatment

    Science.gov (United States)

    Suzuki, Luis Cláudio; Araujo Prates, Renato; Raele, Marcus Paulo; Zanardi di Freitas, Anderson; Simões Ribeiro, Martha

    2010-04-01

    The biofilm formed by Candida albicans is the mainly cause of infections associated to medical devices such as catheters. Studies have shown that photodynamic antimicrobial therapy (PAT) has lethal effect on C. albicans, and it is based on photosensitizer (PS) in the presence of low intensity light to generate reactive oxygen species in biological systems. The aim of this study was to analyze in real time, by Optical Coherence Tomography (OCT), the alterations in C. albicans biofilm in vitro during PAT using methylene blue (MB) as a PS and red light. An OCT system with working at 930nm was used, sequential images of 2000×512 pixels were generated at the frame rate of 2.5frames/sec. The dimension of the analyzed sample was 6000μm wide by 1170μm of depth corrected by refraction index of 1.35. We recorded 1min. before and after the irradiation with LED for PAT, generating 8min. of video. For biofilm formation, discs were made from elastomeric silicone catheters. The PS was dissolved in PBS solution, and a final concentration of 1mM MB was applied on biofilm, followed by a red LED irradiation (λ=630nm+/-20nm) during 6min. We performed a curve of survival fraction versus time of irradiation and it was reduced by 100% following 6min. of irradiation. OCT was performed for measurement of biofilm thickness of 110μm when biofilm was formed. During irradiation, the variation of biofilm thickness was ~70μm. We conclude that OCT system is able to show real time optical changes provided by PAT in yeasts organized in biofilm.

  20. Early staphylococcal biofilm formation on solid orthopaedic implant materials: in vitro study.

    Directory of Open Access Journals (Sweden)

    Hironobu Koseki

    Full Text Available Biofilms forming on the surface of biomaterials can cause intractable implant-related infections. Bacterial adherence and early biofilm formation are influenced by the type of biomaterial used and the physical characteristics of implant surface. In this in vitro research, we evaluated the ability of Staphylococcus epidermidis, the main pathogen in implant-related infections, to form biofilms on the surface of the solid orthopaedic biomaterials, oxidized zirconium-niobium alloy, cobalt-chromium-molybdenum alloy (Co-Cr-Mo, titanium alloy (Ti-6Al-4V, commercially pure titanium (cp-Ti and stainless steel. A bacterial suspension of Staphylococcus epidermidis strain RP62A (ATCC35984 was added to the surface of specimens and incubated. The stained biofilms were imaged with a digital optical microscope and the biofilm coverage rate (BCR was calculated. The total amount of biofilm was determined with the crystal violet assay and the number of viable cells in the biofilm was counted using the plate count method. The BCR of all the biomaterials rose in proportion to culture duration. After culturing for 2-4 hours, the BCR was similar for all materials. However, after culturing for 6 hours, the BCR for Co-Cr-Mo alloy was significantly lower than for Ti-6Al-4V, cp-Ti and stainless steel (P0.05. These results suggest that surface properties, such as hydrophobicity or the low surface free energy of Co-Cr-Mo, may have some influence in inhibiting or delaying the two-dimensional expansion of biofilm on surfaces with a similar degree of smoothness.

  1. Evaluation of the Enterococcus faecalis Biofilm-Associated Virulence Factors AhrC and Eep in Rat Foreign Body Osteomyelitis and In Vitro Biofilm-Associated Antimicrobial Resistance.

    Directory of Open Access Journals (Sweden)

    Kristi L Frank

    Full Text Available Enterococcus faecalis can cause healthcare-associated biofilm infections, including those of orthopedic devices. Treatment of enterococcal prosthetic joint infection is difficult, in part, due to biofilm-associated antimicrobial resistance. We previously showed that the E. faecalis OG1RF genes ahrC and eep are in vitro biofilm determinants and virulence factors in animal models of endocarditis and catheter-associated urinary tract infection. In this study, we evaluated the role of these genes in a rat acute foreign body osteomyelitis model and in in vitro biofilm-associated antimicrobial resistance. Osteomyelitis was established for one week following the implantation of stainless steel orthopedic wires inoculated with E. faecalis strains OG1RF, ΩahrC, and ∆eep into the proximal tibiae of rats. The median bacterial loads recovered from bones and wires did not differ significantly between the strains at multiple inoculum concentrations. We hypothesize that factors present at the infection site that affect biofilm formation, such as the presence or absence of shear force, may account for the differences in attenuation in the various animal models we have used to study the ΩahrC and ∆eep strains. No differences among the three strains were observed in the planktonic and biofilm antimicrobial susceptibilities to ampicillin, vancomycin, daptomycin, linezolid, and tetracycline. These findings suggest that neither ahrC nor eep directly contribute to E. faecalis biofilm-associated antimicrobial resistance. Notably, the experimental evidence that the biofilm attachment mutant ΩahrC displays biofilm-associated antimicrobial resistance suggests that surface colonization alone is sufficient for E. faecalis cells to acquire the biofilm antimicrobial resistance phenotype.

  2. Evaluation of the Enterococcus faecalis Biofilm-Associated Virulence Factors AhrC and Eep in Rat Foreign Body Osteomyelitis and In Vitro Biofilm-Associated Antimicrobial Resistance.

    Science.gov (United States)

    Frank, Kristi L; Vergidis, Paschalis; Brinkman, Cassandra L; Greenwood Quaintance, Kerryl E; Barnes, Aaron M T; Mandrekar, Jayawant N; Schlievert, Patrick M; Dunny, Gary M; Patel, Robin

    2015-01-01

    Enterococcus faecalis can cause healthcare-associated biofilm infections, including those of orthopedic devices. Treatment of enterococcal prosthetic joint infection is difficult, in part, due to biofilm-associated antimicrobial resistance. We previously showed that the E. faecalis OG1RF genes ahrC and eep are in vitro biofilm determinants and virulence factors in animal models of endocarditis and catheter-associated urinary tract infection. In this study, we evaluated the role of these genes in a rat acute foreign body osteomyelitis model and in in vitro biofilm-associated antimicrobial resistance. Osteomyelitis was established for one week following the implantation of stainless steel orthopedic wires inoculated with E. faecalis strains OG1RF, ΩahrC, and ∆eep into the proximal tibiae of rats. The median bacterial loads recovered from bones and wires did not differ significantly between the strains at multiple inoculum concentrations. We hypothesize that factors present at the infection site that affect biofilm formation, such as the presence or absence of shear force, may account for the differences in attenuation in the various animal models we have used to study the ΩahrC and ∆eep strains. No differences among the three strains were observed in the planktonic and biofilm antimicrobial susceptibilities to ampicillin, vancomycin, daptomycin, linezolid, and tetracycline. These findings suggest that neither ahrC nor eep directly contribute to E. faecalis biofilm-associated antimicrobial resistance. Notably, the experimental evidence that the biofilm attachment mutant ΩahrC displays biofilm-associated antimicrobial resistance suggests that surface colonization alone is sufficient for E. faecalis cells to acquire the biofilm antimicrobial resistance phenotype.

  3. A transposon mutant library of Bacillus cereus ATCC 10987 reveals novel genes required for biofilm formation and implicates motility as an important factor for pellicle-biofilm formation.

    Science.gov (United States)

    Okshevsky, Mira; Louw, Matilde Greve; Lamela, Elena Otero; Nilsson, Martin; Tolker-Nielsen, Tim; Meyer, Rikke Louise

    2017-11-22

    Bacillus cereus is one of the most common opportunistic pathogens causing foodborne illness, as well as a common source of contamination in the dairy industry. B. cereus can form robust biofilms on food processing surfaces, resulting in food contamination due to shedding of cells and spores. Despite the medical and industrial relevance of this species, the genetic basis of biofilm formation in B. cereus is not well studied. In order to identify genes required for biofilm formation in this bacterium, we created a library of 5000 +  transposon mutants of the biofilm-forming strain B. cereusATCC 10987, using an unbiased mariner transposon approach. The mutant library was screened for the ability to form a pellicle biofilm at the air-media interface, as well as a submerged biofilm at the solid-media interface. A total of 91 genes were identified as essential for biofilm formation. These genes encode functions such as chemotaxis, amino acid metabolism and cellular repair mechanisms, and include numerous genes not previously known to be required for biofilm formation. Although the majority of disrupted genes are not directly responsible for motility, further investigations revealed that the vast majority of the biofilm-deficient mutants were also motility impaired. This observation implicates motility as a pivotal factor in the formation of a biofilm by B. cereus. These results expand our knowledge of the fundamental molecular mechanisms of biofilm formation by B. cereus. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  4. Cytotoxicity, interaction with dentine and efficacy on multispecies biofilms of a modified salt solution intended for endodontic disinfection in a new in vitro biofilm model.

    Science.gov (United States)

    van der Waal, S V; Scheres, N; de Soet, J J; Wesselink, P R; Crielaard, W

    2015-02-01

    To investigate the cytotoxicity of a modified salt solution (MSS) and evaluate the antimicrobial properties of MSS on in vitro biofilm models. In a metabolic assay, fibroblasts derived from periodontal ligaments (PDL) of human extracted teeth were cultured and challenged with MSS or controls. Then, in active attachment biofilm models, the efficacy of MSS in the presence of dentine powder and in eliminating mature biofilms was investigated. In the dentine assay, a biofilm of Enterococcus faecalis was employed. For the final assay, microorganisms were retrieved from infected root canals and cultured to produce biofilms. After the treatments with MSS or the controls, the biofilms were collected, serially diluted and plated. The colony-forming units were counted. One-way anova was used to analyse the differences between the groups. A P 0.05). In endodontic biofilms, the culturable bacteria were equally reduced by MSS, 2% chlorhexidine (CHX) or 2% sodium hypochlorite (NaOCl) (P > 0.05). Modified salt solution is noncytotoxic in vitro and has good antimicrobial properties equal to CHX and NaOCl. Although the results are promising, ex vivo and in vivo studies are needed before its use as an interappointment root canal dressing can be considered. © 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  5. Formation of In Vitro Mixed-Species Biofilms by Lactobacillus pentosus and Yeasts Isolated from Spanish-Style Green Table Olive Fermentations.

    Science.gov (United States)

    León-Romero, Ángela; Domínguez-Manzano, Jesús; Garrido-Fernández, Antonio; Arroyo-López, Francisco Noé; Jiménez-Díaz, Rufino

    2015-11-13

    The present work details the in vitro interactions between Lactobacillus pentosus and yeast strains isolated from table olive processing to form mixed biofilms. Among the different pairs assayed, the strongest biofilms were obtained from L. pentosus and Candida boidinii strain cocultures. However, biofilm formation was inhibited in the presence of d-(+)-mannose. In addition, biofilm formation by C. boidinii monoculture was stimulated in the absence of cell-cell contact with L. pentosus. Scanning electron microscopy revealed that a sort of "sticky" material formed by the yeasts contributed to substrate adherence. Hence, the data obtained in this work suggest that yeast-lactobacilli biofilms may be favored by the presence of a specific mate of yeast and L. pentosus, and that more than one mechanism might be implicated in the biofilm formation. This knowledge will help in the design of appropriate mixed starter cultures of L. pentosus-yeast species pairs that are able to improve the quality and safety of Spanish-style green table olive processing. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  6. Modeling Reveals the Role of Aging and Glucose Uptake Impairment in L1A1 Listeria monocytogenes Biofilm Life Cycle

    Directory of Open Access Journals (Sweden)

    Eva Balsa-Canto

    2017-11-01

    Full Text Available Listeria monocytogenes is a food-borne pathogen that can persist in food processing plants by forming biofilms on abiotic surfaces. The benefits that bacteria can gain from living in a biofilm, i.e., protection from environmental factors and tolerance to biocides, have been linked to the biofilm structure. Different L. monocytogenes strains build biofilms with diverse structures, and the underlying mechanisms for that diversity are not yet fully known. This work combines quantitative image analysis, cell counts, nutrient uptake data and mathematical modeling to provide a mechanistic insight into the dynamics of the structure of biofilms formed by L. monocytogenes L1A1 (serotype 1/2a strain. Confocal laser scanning microscopy (CLSM and quantitative image analysis were used to characterize the structure of L1A1 biofilms throughout time. L1A1 forms flat, thick structures; damaged or dead cells start appearing early in deep layers of the biofilm and rapidly and massively loss biomass after 4 days. We proposed several reaction-diffusion models to explain the system dynamics. Model candidates describe biomass and nutrients evolution including mechanisms of growth and cell spreading, nutrients diffusion and uptake and biofilm decay. Data fitting was used to estimate unknown model parameters and to choose the most appropriate candidate model. Remarkably, standard reaction-diffusion models could not describe the biofilm dynamics. The selected model reveals that biofilm aging and glucose impaired uptake play a critical role in L1A1 L. monocytogenes biofilm life cycle.

  7. Biofilm inhibitory and eradicating activity of wound care products against Staphylococcus aureus and Staphylococcus epidermidis biofilms in an in vitro chronic wound model.

    Science.gov (United States)

    Brackman, G; De Meyer, L; Nelis, H J; Coenye, T

    2013-06-01

    Although several factors contribute to wound healing, bacterial infections and the presence of biofilm can significantly affect healing. Despite that this clearly indicates that therapies should address biofilm in wounds, only few wound care products have been evaluated for their antibiofilm effect. For this reason, we developed a rapid quantification approach to investigate the efficacy of wound care products on wounds infected with Staphylococcus spp. An in vitro chronic wound infection model was used in which a fluorescent Staph. aureus strain was used to allow the rapid quantification of the bacterial burden after treatment. A good correlation was observed between the fluorescence signal and the bacterial counts. When evaluated in this model, several commonly used wound dressings and wound care products inhibited biofilm formation resulting in a decrease between one and seven log CFU per biofilm compared with biofilm formed in the absence of products. In contrast, most dressings only moderately affected mature biofilms. Our model allowed the rapid quantification of the bacterial burden after treatment. However, the efficacy of treatment varied between the different types of dressings and/or wound care products. Our model can be used to compare the efficacy of wound care products to inhibit biofilm formation and/or eradicate mature biofilms. In addition, the results indicate that treatment of infected wounds should be started as soon as possible and that novel products with more potent antibiofilm activity are needed. © 2013 The Society for Applied Microbiology.

  8. Comparison of the in vitro activity of five antimicrobial drugs on Staphylococcus pseudintermedius and Staphylococcus aureus biofilms

    Directory of Open Access Journals (Sweden)

    Aude A Ferran

    2016-08-01

    Full Text Available Resistance in canine pathogenic staphylococci is necessitating re-evaluation of the current antimicrobial treatments especially for biofilm-associated infections. Long, repeated treatments are often required to control such infections due to the tolerance of bacteria within the biofilm. To comply with the goal of better antibiotic stewardship in veterinary medicine, the efficacies of the available drugs need to be directly assessed on bacterial biofilms.We compared the activities of amoxicillin, cefalexin, clindamycin, doxycycline and marbofloxacin on in vitro biofilms of Staphylococcus pseudintermedius and Staphylococcus aureus. Exposure of biofilms for 15 hours to maximum concentrations of the antibiotics achievable in canine plasma only reduced biofilm bacteria by 0.5 to 2.0 log10 CFU, compared to the control, except for marbofloxacin which reduced S. aureus biofilms by 5.4 log10 CFU. Two-antibiotic combinations did not improve, and even decreased, bacterial killing. In comparison, 5 min-exposure to 2 % chlorhexidine reduced biofilms of the 2 tested strains by 4 log10 CFU. Our results showed that S. pseudintermedius biofilm, unlike S. aureus biofilm, was highly tolerant to all the drugs tested, consistent with the treatment failures observed in practice. Under our conditions, the use of topical chlorhexidine would probably be the best currently available strategy to reduce S. pseudintermedius biofilm.

  9. Reduced ability to detect surface-related biofilm bacteria after antibiotic exposure under in vitro conditions

    DEFF Research Database (Denmark)

    Ravn, Christen; Furustrand Tafin, Ulrika; Bétrisey, Bertrand

    2016-01-01

    Background and purpose - Antibiotic treatment of patients before specimen collection reduces the ability to detect organisms by culture. We investigated the suppressive effect of antibiotics on the growth of non-adherent, planktonic, and surface-related biofilm bacteria in vitro by using sonication...... and microcalorimetry methods. Patients and methods - Biofilms of Staphylococcus aureus, S. epidermidis, Escherichia coli, and Propionibacterium acnes were formed on porous glass beads and exposed for 24 h to antibiotic concentrations from 1 to 1,024 times the minimal inhibitory concentration (MIC) of vancomycin......, daptomycin, rifampin, flucloxacillin, or ciprofloxacin. The beads were then sonicated to dislodge biofilm, followed by culture and measurement of growth-related heat flow by microcalorimetry of the resulting sonication fluid. Results - Vancomycin did not inhibit the heat flow of staphylococci and P. acnes...

  10. Interactions between Lactobacillus rhamnosus GG and oral micro-organisms in an in vitro biofilm model.

    Science.gov (United States)

    Jiang, Qingru; Stamatova, Iva; Kainulainen, Veera; Korpela, Riitta; Meurman, Jukka H

    2016-07-12

    Probiotics have shown favourable properties in maintaining oral health. By interacting with oral microbial communities, these species could contribute to healthier microbial equilibrium. This study aimed to investigate in vitro the ability of probiotic Lactobacillus rhamnosus GG (L.GG) to integrate in oral biofilm and affect its species composition. Five oral strains, Streptococcus mutans, Streptococcus sanguinis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum and Candida albicans were involved. The group setup included 6 mono-species groups, 3 dual-species groups (L.GG + S. mutans/S. sanguinis/C. albicans), and 4 multi-species groups (4/5 species and 4/5 species + L.GG, 4 species were all the tested strains except S. mutans). Cell suspensions of six strains were pooled according to the group setup. Biofilms were grown on saliva-coated hydroxyapatite (HA) discs at 37 °C in anaerobic conditions for 64.5 h. Biofilm medium was added and refreshed at 0, 16.5, and 40.5 h. The pH of spent media was measured. Viable cells of the 16.5 h and 64.5 h biofilms were counted. 64.5 h biofilms were stained and scanned with confocal laser scanning microscopy. Our results showed that L.GG and S. mutans demonstrated stronger adhesion ability than the other strains to saliva-coated HA discs. L.GG, C. albicans, S. mutans and F. nucleatum, with poor ability to grow in mono-species biofilms demonstrated better abilities of adhesion and reproduction in dual- and/or multi-species biofilms. L.GG slightly suppressed the growth of C. albicans in all groups, markedly weakened the growth of S. sanguinis and F. nucleatum in 4sp + L.GG group, and slightly reduced the adhesion of S. mutans in L.GG+ S. mutans group. To conclude, in this in vitro model L.GG successfully integrated in all oral biofilms, and reduced the counts of S. sanguinis and C. albicans and lowered the biofilm-forming ability of F. nucleatum, but only slightly reduced the adhesion of S. mutans

  11. In Vitro and In Vivo Biofilm Characterization of Methicillin-Resistant Staphylococcus aureus from Patients Associated with Pharyngitis Infection

    Directory of Open Access Journals (Sweden)

    Shanmugaraj Gowrishankar

    2016-01-01

    Full Text Available The present investigation was deliberately aimed at evaluating the biofilm-forming ability of 63 clinical MRSA isolates recovered from pharyngitis patients through different phenotypic assays. The molecular detection of adhesion (icaA/icaD/icaB/icaC, adhesins (fnbA/fnbB, clfA, and cna, staphylococcal accessory regulator (sarA, and α-toxin (hla genes was done by employing polymerase chain reaction (PCR. Out of 63 isolates, 49 (77.8% were found slime positive by the Congo red agar (CRA method and 44 (69.8% as biofilm positive by the quantitative microtitre plate assays. The results of MATH assay showed that most of the test pathogens are hydrophilic in nature. The molecular investigation of biofilm-associated genes revealed that 84.13% (n=53 of isolates were found positive for icaADBC genes. The fnbA and fnbB genes were present in 49 (77.8% and 51 (81% MRSA isolates, respectively. In addition, 58.7% (n=37, 73% (n=46, and 69.8% (n=44 of the isolates harboured the clfA, cna, and hla genes, respectively. Further, nearly 81% (n=51 of the isolates were found positive for the gene sarA and all the ica negative isolates were also negative for the gene. Furthermore, the results of in vivo adherence assay unveiled the factual commonness in the in vitro adherence method.

  12. In vitro interference of tigecycline at subinhibitory concentrations on biofilm development by Enterococcus faecalis.

    Science.gov (United States)

    Maestre, Juan Ramón; Aguilar, Lorenzo; Mateo, María; Giménez, María-José; Méndez, María-Luisa; Alou, Luis; Granizo, Juan-José; Prieto, José

    2012-05-01

    Since biofilm formation is the hallmark of Enterococcus faecalis isolates, the aim of this study was to quantify biofilm formation in the presence of subinhibitory concentrations of tigecycline. Interference of tigecycline on biofilm formation was spectrophotometrically quantified using 20 biofilm-producing E. faecalis isolates with tigecycline MICs of 0.12 (8 strains) or 0.25 mg/L (12 strains). Biofilm production was measured in antibiotic-free tryptic soy broth supplemented with 1% glucose and compared with biofilm production in the same medium with tigecycline at subinhibitory concentrations (0.25× or 0.5× MIC, similar to trough concentrations in serum or concentrations in the colon after a standard dose) by reading the optical density at 450 nm (OD(450)) after staining with Crystal Violet. In the presence of subinhibitory tigecycline concentrations, pooled OD(450) values for the 20 strains [median (IQR)] were significantly lower than those for controls: 0.468 (0.379-0.516) for antibiotic-free controls versus 0.295 (0.200-0.395) for 0.25× MIC tigecycline (P < 0.001) and 0.287 (0.245-0.479) for 0.5× MIC tigecycline (P < 0.001), with significant differences between pooled OD(450) values obtained with each concentration of tigecycline (P = 0.022). In 17 out of 20 (85%) strains the OD(450) obtained with 0.25× MIC tigecycline was significantly (P < 0.05) lower than the basal OD(450), while this occurred in 12 out of 20 (60%) strains with 0.5× MIC. In vitro tigecycline subinhibitory concentrations were able to interfere with biofilm formation by E. faecalis.

  13. Porphyromonas gingivalis biofilm formation in different titanium surfaces, an in vitro study.

    Science.gov (United States)

    Di Giulio, Mara; Traini, Tonino; Sinjari, Bruna; Nostro, Antonia; Caputi, Sergio; Cellini, Luigina

    2016-07-01

    The aim of this work was to evaluate the biofilm formation of Porphyromonas gingivalis on disks of titanium (Ti) grade 4 (G4) and Ti-6Al-4V alloy grade 5 (G5) with different surface topographies. Porphyromonas gingivalis ATCC 33277 was used to develop an in vitro mature biofilm on a total of 96 disk-shaped specimens of laser-treated (L), sandblasted (S), and machined (M) surfaces of Ti G4 and Ti G5. Surface roughness (Ra) and the wettability contact angle (WCA) were measured to characterize the surface of the specimens. The bacterial biofilm was evaluated by biomass quantification, bacterial viability, visualization of the biofilm extracellular matrix, and bacterial cell count. Data were analyzed using one-way ANOVA and Holm-Sidak tests and expressed as mean ± standard deviation. The Ra for the L group was 0.10 (±0.07) μm inside the craters and 0.40 (±0.08) μm in the area surrounding the craters resulting the smoothest (P gingivalis bacterial biomass (0.38 ± 0.01 for G4; 0.62 ± 0.02 for G5) that was significant in respect to G4-S (P gingivalis biofilm formation. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. ‘Get in early’; biofilm and wax moth (Galleria mellonella models reveal new insights into the therapeutic potential of Clostridium difficile bacteriophages

    Directory of Open Access Journals (Sweden)

    Janet Yakubu Nale

    2016-08-01

    Full Text Available Clostridium difficile infection (CDI is a global health threat associated with high rates of morbidity and mortality. Conventional antibiotic CDI therapy can result in treatment failure and recurrent infection. C. difficile produces biofilms which contribute to its virulence and impair antimicrobial activity. Some bacteriophages (phages can penetrate biofilms and thus could be developed to either replace or supplement antibiotics. Here, we determined the impact of a previously optimized 4-phage cocktail on C. difficile ribotype 014/020 biofilms, and additionally as adjunct to vancomycin treatment in Galleria mellonella larva CDI model. The phages were applied before or after biofilm establishment in vitro, and the impact was analyzed according to turbidity, viability counts and topography as observed using scanning electron and confocal microscopy. The infectivity profiles and efficacies of orally administered phages and/or vancomycin were ascertained by monitoring colonization levels and larval survival rates. Phages prevented biofilm formation, and penetrated established biofilms. A single phage application reduced colonization causing extended longevity in the remedial treatment and prevented disease in the prophylaxis group. Multiple phage doses significantly improved the larval remedial regimen, and this treatment is comparable to vancomycin and the combined treatments. Taken together, our data suggest that the phages significantly reduce C. difficile biofilms, and prevent colonization in the G. mellonella model when used alone or in combination with vancomycin. The phages appear to be highly promising therapeutics in the targeted eradication of CDI and the use of these models has revealed that prophylactic use could be a propitious therapeutic option.

  15. A novel in vitro wound biofilm model used to evaluate low-frequency ultrasonic-assisted wound debridement

    DEFF Research Database (Denmark)

    Crone, S.; Garde, Christian; Bjarnsholt, T.

    2015-01-01

    disrupts the biofilm leaving bacteria more vulnerable to antiseptic or antibiotic treatment. The objective of this study is to develop a realistic model to elucidate the effect of ultrasound on biofilms. Method: A novel in vitro wound biofilm model was developed. Biofilms of Staphylococcus aureus were......Objective: Bacterial biofilms remain difficult to treat. The biofilm mode of growth enables bacteria to survive antibiotic treatment and the inflammatory reaction. Low-frequency ultrasound has recently been shown to improve healing in a variety of settings. It is hypothesised that ultrasound...... casted in a semi-solid agar gel composed of either tryptic soy broth (TSB) or a wound simulating media (WSM; composed of Bolton broth with blood and plasma), to resemble the non-surface attached aggregates. The model was used to evaluate the antibiofilm effect of an ultrasonic-assisted wound debridement...

  16. In vitro Dynamic Model of a Catheterized Bladder and Biofilm Assay

    OpenAIRE

    Maierl, Mario; Jörger, Michael; Rosker, Patrik; Reisner, Andreas

    2015-01-01

    Biofilm formation on catheters is thought to contribute to persistence of catheter-associated urinary tract infections (CAUTI) which represent the most frequent nosocomial infections. Understanding of factors relevant for CAUTI pathogenesis and evaluation of new therapeutics or interference strategies requires a model system that mirrors the physico-chemical conditions prevailing in a catheterized human bladder. The described in vitro dynamic model of a catheterized bladder enables to emulate...

  17. Activity of Imipenem against Klebsiella pneumoniae Biofilms In Vitro and In Vivo

    Science.gov (United States)

    2014-02-01

    Activity of Imipenem against Klebsiella pneumoniae Biofilms In Vitro and In Vivo Ping Chen,a Akhil K. Seth,b Johnathan J. Abercrombie,a Thomas A...USAa; Division of Plastic Surgery, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USAb Encapsulated Klebsiella pneumoniae has... Klebsiella pneumoniae is a Gram-negative, encapsulated, non-motile, rod-shaped opportunistic pathogen. This organism can cause a wide range of

  18. In vitro interactions between farnesol and fluconazole, amphotericin B or micafungin against Candida albicans biofilms

    Science.gov (United States)

    Katragkou, Aspasia; McCarthy, Matthew; Alexander, Elizabeth L.; Antachopoulos, Charalampos; Meletiadis, Joseph; Jabra-Rizk, Mary Ann; Petraitis, Vidmantas; Roilides, Emmanuel; Walsh, Thomas J.

    2015-01-01

    Objectives Biofilm formation by Candida albicans poses an important therapeutic challenge in human diseases. Typically, conventional antifungal agents encounter difficulty in treating and fully eradicating biofilm-related infections. Novel therapeutic approaches are needed to treat recalcitrant Candida biofilms. Farnesol is a quorum-sensing molecule, which induces apoptosis, inhibits Ras protein pathways and profoundly affects the morphogenesis of C. albicans. We therefore investigated the interactions between farnesol and different classes of antifungal agents. Methods The combined antifungal effects of triazoles (fluconazole), polyenes (amphotericin B) and echinocandins (micafungin) with farnesol against C. albicans biofilms were assessed in vitro. Antifungal activity was determined by the XTT metabolic assay and confocal microscopy. The nature and the intensity of the interactions were assessed using the Loewe additivity model [fractional inhibitory concentration (FIC) index] and the Bliss independence (BI) model. Results Significant synergy was found between each of the three antifungal agents and farnesol, while antagonism was not observed for any of the combinations tested. The greatest synergistic effect was found with the farnesol/micafungin combination, for which the BI-based model showed the observed effects as being 39%–52% higher than expected if the drugs had been acting independently. The FIC indices ranged from 0.49 to 0.79, indicating synergism for farnesol/micafungin and farnesol/fluconazole and no interaction for farnesol/amphotericin B. Structural changes in the biofilm correlated well with the efficacies of these combinations. The maximum combined effect was dependent on the farnesol concentration for micafungin and amphotericin B. Conclusions Farnesol exerts a synergistic or additive interaction with micafungin, fluconazole and amphotericin B against C. albicans biofilms, thus warranting further in vivo study. PMID:25288679

  19. In vitro biofilm distribution on the intraocular lens surface of different biomaterials.

    Science.gov (United States)

    Mazoteras, Paloma; Casaroli-Marano, Ricardo Pedro

    2015-09-01

    To study the disposition of bacterial adhesion to intraocular lens (IOL) biomaterials depending on the material and region of the optic IOL surface: center or peripheral edge. School of Medicine, University of Barcelona, Barcelona, Spain. Experimental study. For the in vivo study, IOLs were explanted from donor ocular globes without clinical symptoms of endophthalmitis. Biofilm formation was qualitatively studied by scanning electron microscopy (SEM). For the in vitro study, 5 IOL biomaterials (hydrophilic acrylic, hydrophobic acrylic, poly[methyl methacrylate] [PMMA], heparinized PMMA, and silicone) were contaminated with a biofilm-producing strain of Staphylococcus epidermidis. Bacterial densities were quantitatively (colony-forming units per area) compared by SEM and direct counting of viable adherent bacteria, according to the biomaterial, region of the IOL optic surface, and time of incubation. For SEM, bacterial adhesion was also qualitatively classified according to the characteristics of biofilm observed: structure, cocci per cluster, homogeneity of cluster distribution, and extracellular matrix production. At 3 hours of incubation, bacterial counts for hydrophilic acrylic and PMMA IOLs were significantly lower, but at 72 hours there were no statistically significant differences among biomaterials. A higher density of bacteria was observed at the periphery of the IOL's optic of assayed biomaterials for in vitro and in vivo studies. Biofilm formation and the presence of extracellular matrix were predominantly restricted to the edges of IOL optic surface. Bacterial adhesion and biofilm development on the IOL optic surface depended on the region and biomaterial of the IOL. Neither author has a financial or proprietary interest in any material or method mentioned. Copyright © 2015 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

  20. Variable and complex food web structures revealed by exploring missing trophic links between birds and biofilm.

    Science.gov (United States)

    Kuwae, Tomohiro; Miyoshi, Eiichi; Hosokawa, Shinya; Ichimi, Kazuhiko; Hosoya, Jun; Amano, Tatsuya; Moriya, Toshifumi; Kondoh, Michio; Ydenberg, Ronald C; Elner, Robert W

    2012-04-01

    Food webs are comprised of a network of trophic interactions and are essential to elucidating ecosystem processes and functions. However, the presence of unknown, but critical networks hampers understanding of complex and dynamic food webs in nature. Here, we empirically demonstrate a missing link, both critical and variable, by revealing that direct predator-prey relationships between shorebirds and biofilm are widespread and mediated by multiple ecological and evolutionary determinants. Food source mixing models and energy budget estimates indicate that the strength of the missing linkage is dependent on predator traits (body mass and foraging action rate) and the environment that determines food density. Morphological analyses, showing that smaller bodied species possess more developed feeding apparatus to consume biofilm, suggest that the linkage is also phylogenetically dependent and affords a compelling re-interpretation of niche differentiation. We contend that exploring missing links is a necessity for revealing true network structure and dynamics. © 2012 Blackwell Publishing Ltd/CNRS.

  1. Shin’iseihaito (Xinyiqingfeitang Suppresses the Biofilm Formation of Streptococcus pneumoniae In Vitro

    Directory of Open Access Journals (Sweden)

    Masaaki Minami

    2017-01-01

    Full Text Available Streptococcus pneumoniae (S. pneumoniae is the important pathogen that causes otolaryngeal diseases such as sinusitis. S. pneumoniae frequently forms the biofilm to prevent severe circumstances such as antimicrobial agents. Shin’iseihaito (xinyiqingfeitang is a formula of Japanese traditional Kampo medicine that has 9 crude drugs and provides the medicinal usage for sinusitis. The objective of the present study is to reveal the mechanism of antibiofilm activity by Shin’iseihaito extract (SSHT. SSHT significantly inhibited the formation of biofilm from S. pneumoniae ATCC 49619 in dose- and time-dependent manners. SSHT also significantly suppressed the biofilm formation by other five different cps types of S. pneumoniae clinical isolates. We found that the extracts of 8 out of 9 components in Shin’iseihaito had the inhibitory effects of biofilm formation, and the extract of the root of Scutellaria baicalensis had the strongest effect among the ingredients of Shin’iseihaito. We found that the capsule of SSHT-treated S. pneumoniae was significantly thinner than that of the untreated group and that SSHT reduced the hydrophobicity of bacterial cell surface. Our results suggest that Shin’iseihaito may be a useful agent for the treatment of S. pneumoniae-induced sinusitis because of the inhibition of biofilm formation of S. pneumoniae.

  2. Chlorhexidine Antiseptic Irrigation Eradicates Staphylococcus epidermidis From Biofilm: An In Vitro Study.

    Science.gov (United States)

    Schmidt, Kenneth; Estes, Chris; McLaren, Alex; Spangehl, Mark J

    2018-03-01

    Antiseptic and antibacterial solutions used for intraoperative irrigation are intended to kill bacteria and thereby decrease the incidence of surgical site infections. It is unknown if the concentrations and exposure times of irrigation solutions commonly used for prophylaxis in clean cases (povidone-iodine 0.35% for 3 minutes) are effective against bacteria in biofilm that are present in implant infections. Currently, povidone-iodine (0.35%), chlorhexidine (0.05%), sodium hypochlorite (0.125%), and triple antibacterial solution are all being used off-label for wound irrigation after surgical débridement for orthopaedic infections. Do commonly used antibacterials and antiseptics kill bacteria in established biofilm at clinically relevant concentrations and exposure times? Staphylococcus epidermidis (ATCC#35984) biofilms were exposed to chlorhexidine (0.025%, 0.05%, and 0.1%), povidone-iodine (0.35%, 1.0%, 3.5%, and 10%), sodium hypochlorite (0.125%, 0.25%, and 0.5%,), and triple antibacterial solution (bacitracin 50,000 U/L, gentamicin 80 mg/L, and polymyxin 500,000 U/L) for 1, 5, and 10 minutes in triplicate. Surviving bacteria were detected by 21-day subculture. Failure to eradicate all bacteria in any of the three replicates was considered to be "not effective" for that respective solution, concentration, and exposure time. Chlorhexidine 0.05% and 0.1% at all three exposure times, povidone-iodine 10% at all three exposure times, and povidone-iodine 3.5% at 10 minutes only were effective at eradicating S epidermidis from biofilm. All concentrations and all exposure times of sodium hypochlorite and triple antibacterial solution were not effective. Chlorhexidine is capable of eradicating S epidermidis from biofilm in vitro in clinically relevant concentrations and exposure times. Povidone-iodine at commonly used concentrations and exposure times, sodium hypochlorite, and triple antibacterial solutions are not. This in vitro study suggests that chlorhexidine may be

  3. Evaluation of biofilm production by Pseudomonas aeruginosa from canine ears and the impact of biofilm on antimicrobial susceptibility in vitro.

    Science.gov (United States)

    Pye, Charlotte C; Yu, Anthony A; Weese, J Scott

    2013-08-01

    Pseudomonas aeruginosa is a common cause of canine otitis; P. aeruginosa biofilm formation has been documented in human medicine, but the role of biofilms in canine disease is not well documented. Bacteria within biofilms can be more resistant to antibiotics compared with their planktonic form; therefore, understanding the biofilm-forming capacity of isolates and their susceptibility to antimicrobials is important when developing treatment regimens. To evaluate the biofilm-forming capacity of canine otic isolates of P. aeruginosa and to compare the minimal inhibitory concentration (MIC) of the planktonic versus biofilm-embedded bacteria. Biofilm-forming ability was assessed using a microtitre plate assay. Broth microdilution was used to assess the MICs of neomycin, polymyxin B, enrofloxacin and gentamicin for the planktonic and biofilm-embedded bacteria. Eighty-three isolates from dogs with otitis were tested; 33 (40%) were classified as biofilm producers. Biofilm MICs for polymyxin B, neomycin, gentamicin and enrofloxacin were significantly higher than for the planktonic form (P otitis isolates of P. aeruginosa is common and may play a role in the pathogenesis of disease. The MICs for biofilm-embedded bacteria differ from their planktonic counterparts, potentially leading to a lack of response to treatment. If polymyxin B, gentamicin, neomycin or enrofloxacin is to be used for topical treatment of a Pseudomonas otitis, the concentration of the medication should be increased, in particular if addressing chronic otitis, because biofilms may have developed. © 2013 The Authors. Veterinary Dermatology © 2013 ESVD and ACVD.

  4. Cytotoxicity, interaction with dentine and efficacy on multispecies biofilms of a modified salt solution intended for endodontic disinfection in a new in vitro biofilm model

    NARCIS (Netherlands)

    van der Waal, S.V.; Scheres, N.; de Soet, J.J.; Wesselink, P.R.; Crielaard, W.

    2014-01-01

    Aim To investigate the cytotoxicity of a modified salt solution (MSS) and evaluate the antimicrobial properties of MSS on in vitro biofilm models. Methodology In a metabolic assay, fibroblasts derived from periodontal ligaments (PDL) of human extracted teeth were cultured and challenged with MSS or

  5. Fluorescence and NMR spectroscopy together with molecular simulations reveal amphiphilic characteristics of a Burkholderia biofilm exopolysaccharide.

    Science.gov (United States)

    Kuttel, Michelle M; Cescutti, Paola; Distefano, Marco; Rizzo, Roberto

    2017-06-30

    Biofilms are a collective mode of bacterial life in which a self-produced matrix confines cells in close proximity to each other. Biofilms confer many advantages, including protection from chemicals (including antibiotics), entrapment of useful extracellular enzymes and nutrients, as well as opportunities for efficient recycling of molecules from dead cells. Biofilm matrices are aqueous gel-like structures composed of polysaccharides, proteins, and DNA stabilized by intermolecular interactions that may include non-polar connections. Recently, polysaccharides extracted from biofilms produced by species of the Burkholderia cepacia complex were shown to possess clusters of rhamnose, a 6-deoxy sugar with non-polar characteristics. Molecular dynamics simulations are well suited to characterizing the structure and dynamics of polysaccharides, but only relatively few such studies exist of their interaction with non-polar molecules. Here we report an investigation into the hydrophobic properties of the exopolysaccharide produced by Burkholderia multivorans strain C1576. Fluorescence experiments with two hydrophobic fluorescent probes established that this polysaccharide complexes hydrophobic species, and NMR experiments confirmed these interactions. Molecular simulations to model the hydrodynamics of the polysaccharide and the interaction with guest species revealed a very flexible, amphiphilic carbohydrate chain that has frequent dynamic interactions with apolar molecules; both hexane and a long-chain fatty acid belonging to the quorum-sensing system of B. multivorans were tested. A possible role of the non-polar domains of the exopolysaccharide in facilitating the diffusion of aliphatic species toward specific targets within the biofilm aqueous matrix is proposed. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Biofilm formation on stainless steel and gold wires for bonded retainers in vitro and in vivo and their susceptibility to oral antimicrobials.

    Science.gov (United States)

    Jongsma, Marije A; Pelser, Floris D H; van der Mei, Henny C; Atema-Smit, Jelly; van de Belt-Gritter, Betsy; Busscher, Henk J; Ren, Yijin

    2013-05-01

    Bonded retainers are used in orthodontics to maintain treatment result. Retention wires are prone to biofilm formation and cause gingival recession, bleeding on probing and increased pocket depths near bonded retainers. In this study, we compare in vitro and in vivo biofilm formation on different wires used for bonded retainers and the susceptibility of in vitro biofilms to oral antimicrobials. Orthodontic wires were exposed to saliva, and in vitro biofilm formation was evaluated using plate counting and live/dead staining, together with effects of exposure to toothpaste slurry alone or followed by antimicrobial mouthrinse application. Wires were also placed intra-orally for 72 h in human volunteers and undisturbed biofilm formation was compared by plate counting and live/dead staining, as well as by denaturing gradient gel electrophoresis for compositional differences in biofilms. Single-strand wires attracted only slightly less biofilm in vitro than multi-strand wires. Biofilms on stainless steel single-strand wires however, were much more susceptible to antimicrobials from toothpaste slurries and mouthrinses than on single-strand gold wires and biofilms on multi-strand wires. Also, in vivo significantly less biofilm was found on single-strand than on multi-strand wires. Microbial composition of biofilms was more dependent on the volunteer involved than on wire type. Biofilms on single-strand stainless steel wires attract less biofilm in vitro and are more susceptible to antimicrobials than on multi-strand wires. Also in vivo, single-strand wires attract less biofilm than multi-strand ones. Use of single-strand wires is preferred over multi-strand wires, not because they attract less biofilm, but because biofilms on single-strand wires are not protected against antimicrobials as in crevices and niches as on multi-strand wires.

  7. In Vitro Analyses of the Effects of Heparin and Parabens on Candida albicans Biofilms and Planktonic Cells

    Science.gov (United States)

    Miceli, Marisa H.; Bernardo, Stella M.; Ku, T. S. Neil; Walraven, Carla

    2012-01-01

    Infections and thromboses are the most common complications associated with central venous catheters. Suggested strategies for prevention and management of these complications include the use of heparin-coated catheters, heparin locks, and antimicrobial lock therapy. However, the effects of heparin on Candida albicans biofilms and planktonic cells have not been previously studied. Therefore, we sought to determine the in vitro effect of a heparin sodium preparation (HP) on biofilms and planktonic cells of C. albicans. Because HP contains two preservatives, methyl paraben (MP) and propyl paraben (PP), these compounds and heparin sodium without preservatives (Pure-H) were also tested individually. The metabolic activity of the mature biofilm after treatment was assessed using XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction and microscopy. Pure-H, MP, and PP caused up to 75, 85, and 60% reductions of metabolic activity of the mature preformed C. albicans biofilms, respectively. Maximal efficacy against the mature biofilm was observed with HP (up to 90%) compared to the individual compounds (P < 0.0001). Pure-H, MP, and PP each inhibited C. albicans biofilm formation up to 90%. A complete inhibition of biofilm formation was observed with HP at 5,000 U/ml and higher. When tested against planktonic cells, each compound inhibited growth in a dose-dependent manner. These data indicated that HP, MP, PP, and Pure-H have in vitro antifungal activity against C. albicans mature biofilms, formation of biofilms, and planktonic cells. Investigation of high-dose heparin-based strategies (e.g., heparin locks) in combination with traditional antifungal agents for the treatment and/or prevention of C. albicans biofilms is warranted. PMID:21986822

  8. The transcriptional programme of Salmonella enterica serovar Typhimurium reveals a key role for tryptophan metabolism in biofilms.

    LENUS (Irish Health Repository)

    Hamilton, Shea

    2009-12-11

    exogenous tryptophan or indole. Conclusions Biofilm growth of S. Typhimurium causes distinct changes in gene and protein expression. Our results show that aromatic amino acids make an important contribution to biofilm formation and reveal a link between SPI2 expression and surface-associated growth in S. Typhimurium.

  9. An Essential Role for Coagulase in Staphylococcus aureus Biofilm Development Reveals New Therapeutic Possibilities for Device-Related Infections.

    Science.gov (United States)

    Zapotoczna, Marta; McCarthy, Hannah; Rudkin, Justine K; O'Gara, James P; O'Neill, Eoghan

    2015-12-15

    High-level resistance to antimicrobial drugs is a major factor in the pathogenesis of chronic Staphylococcus aureus biofilm-associated, medical device-related infections. Antimicrobial susceptibility analysis revealed that biofilms grown for ≤ 24 hours on biomaterials conditioned with human plasma under venous shear in iron-free cell culture medium were significantly more susceptible to antistaphylococcal antibiotics. Biofilms formed under these physiologically relevant conditions were regulated by SaeRS and dependent on coagulase-catalyzed conversion of fibrinogen into fibrin. In contrast, SarA-regulated biofilms formed on uncoated polystyrene in nutrient-rich bacteriological medium were mediated by the previously characterized biofilm factors poly-N-acetyl glucosamine, fibronectin-binding proteins, or autolytic activity and were antibiotic resistant. Coagulase-mediated biofilms exhibited increased antimicrobial resistance over time (>48 hours) but were always susceptible to dispersal by the fibrinolytic enzymes plasmin or nattokinase. Biofilms recovered from infected central venous catheters in a rat model of device-related infection were dispersed by nattokinase, supporting the important role of the biofilm phenotype and identifying a potentially new therapeutic approach with antimicrobials and fibrinolytic drugs, particularly during the early stages of device-related infection. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  10. Increased Effects of Extracorporeal Shock Waves Combined with Gentamicin against Staphylococcus aureus Biofilms In Vitro and In Vivo.

    Science.gov (United States)

    Qi, Xin; Zhao, Yaochao; Zhang, Jieyuan; Han, Dan; Chen, Chunyuan; Huang, Yinjun; Chen, Xiaofeng; Zhang, Xianlong; Wang, Ting; Li, Xiaolin

    2016-09-01

    An implant-associated bacterial infection is one of the most common and costly complications of orthopedic surgery. Once biofilms develop, it is extremely difficult to cure infections with antimicrobial agents. High-energy extracorporeal shock wave (ESW) treatment has been used for orthopedic-related diseases and has been found to be an effective bactericidal agent that is tolerable both in vitro and in vivo. The broad-spectrum antibiotic gentamicin exhibits bactericidal activity against Staphylococcus aureus, and bacterial resistance to gentamicin is lower. We tested the effectiveness of gentamicin in combination with ESW treatment against S. aureus biofilms in vivo and in vitro. The spread plate method, crystal violet staining, confocal laser scanning microscopy, scanning electron microscopy and microbiologic evaluation were used to compare the effects of combined treatment with those of either treatment alone. The results revealed statistically significant differences between the group treated with ESWs combined with gentamicin and all other groups. Our findings indicate that use of the combination of ESWs with gentamicin is more effective against S. aureus biofilms in vitro and in vivo. Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

  11. Biofilm formation by Borrelia burgdorferi sensu lato.

    Science.gov (United States)

    Timmaraju, Venkata Arun; Theophilus, Priyanka A S; Balasubramanian, Kunthavai; Shakih, Shafiq; Luecke, David F; Sapi, Eva

    2015-08-01

    Bacterial biofilms are microbial communities held together by an extracellular polymeric substance matrix predominantly composed of polysaccharides, proteins and nucleic acids. We had previously shown that Borrelia burgdorferi sensu stricto, the causative organism of Lyme disease in the United States is capable of forming biofilms in vitro. Here, we investigated biofilm formation by B. afzelii and B. garinii, which cause Lyme disease in Europe. Using various histochemistry and microscopy techniques, we show that B. afzelii and B. garinii form biofilms, which resemble biofilms formed by B. burgdorferi sensu stricto. High-resolution atomic force microscopy revealed similarities in the ultrastructural organization of the biofilms form by three Borrelia species. Histochemical experiments revealed a heterogeneous organization of exopolysaccharides among the three Borrelia species. These results suggest that biofilm formation might be a common trait of Borrelia genera physiology. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  12. In vitro activity of micafungin against biofilms of Candida albicans, Candida glabrata, and Candida parapsilosis at different stages of maturation.

    Science.gov (United States)

    Prażyńska, Małgorzata; Bogiel, Tomasz; Gospodarek-Komkowska, Eugenia

    2018-03-01

    Candida spp. is able to form a biofilm, which is considered resistant to the majority of antifungals used in medicine. The aim of this study was to evaluate the in vitro activity of micafungin against Candida spp. biofilms at different stages of their maturation (2, 6, and 24 h). We assessed the inhibitory effect of micafungin against 78 clinical isolates of Candida spp., growing as planktonic or sessile cells, by widely recommended broth microdilution method. The in vitro effect on sessile cells viability was evaluated by colorimetric reduction assay. All examined strains were susceptible or intermediate to micafungin when growing as planktonic cells. At the early stages of biofilm maturation, from 11 (39.3%) to 20 (100%), tested strains, depending on the species, exhibited sessile minimal inhibitory concentrations (SMICs) of micafungin at ≤ 2 mg/L. For 24-h-old Candida spp. biofilms, from 3 (10.7%) to 20 (100%) of the tested strains displayed SMICs of micafungin at ≤ 2 mg/L. Our findings confirm that micafungin exhibits high potential anti-Candida-biofilm activity. However, this effect does not comprise all Candida species and strains. All strains were susceptible or intermediate to micafungin when growing as planktonic cells, but for biofilms, micafungin displays species- and strain-specific activity. Paradoxical growth of C. albicans and C. parapsilosis was observed. Antifungal susceptibility testing of Candida spp. biofilms would be the best solution, but to date, no reference method is available. The strongest antibiofilm activity of micafungin is observed at early stages of biofilm formation. Possibly, micafungin could be considered as an effective agent for prevention of biofilm-associated candidiasis, especially catheter-related candidaemia.

  13. Effectiveness of Hypochlorous Acid to Reduce the Biofilms on Titanium Alloy Surfaces in Vitro

    Directory of Open Access Journals (Sweden)

    Chun-Ju Chen

    2016-07-01

    Full Text Available Chemotherapeutic agents have been used as an adjunct to mechanical debridement for peri-implantitis treatment. The present in vitro study evaluated and compared the effectiveness of hypochlorous acid (HOCl, sodium hypochlorite (NaOCl, and chlorhexidine (CHX at eliminating Gram-negative (E. coli and P. gingivalis and Gram-positive (E. faecalis and S. sanguinis bacteria. The effect of irrigating volume and exposure time on the antimicrobial efficacy of HOCl was evaluated, and a durability analysis was completed. Live/dead staining, morphology observation, alamarBlue assay, and lipopolysaccharide (LPS detection were examined on grit-blasted and biofilm-contaminated titanium alloy discs after treatment with the three chemotherapeutic agents. The results indicated that HOCl exhibited better antibacterial efficacy with increasing irrigating volumes. HOCl achieved greater antibacterial efficacy as treatment time was increased. A decrease in antimicrobial effectiveness was observed when HOCl was unsealed and left in contact with the air. All the irrigants showed antibacterial activity and killed the majority of bacteria on the titanium alloy surfaces of biofilm-contaminated implants. Moreover, HOCl significantly lowered the LPS concentration of P. gingivalis when compared with NaOCl and CHX. Thus, a HOCl antiseptic may be effective for cleaning biofilm-contaminated implant surfaces.

  14. Effectiveness of Hypochlorous Acid to Reduce the Biofilms on Titanium Alloy Surfaces in Vitro.

    Science.gov (United States)

    Chen, Chun-Ju; Chen, Chun-Cheng; Ding, Shinn-Jyh

    2016-07-19

    Chemotherapeutic agents have been used as an adjunct to mechanical debridement for peri-implantitis treatment. The present in vitro study evaluated and compared the effectiveness of hypochlorous acid (HOCl), sodium hypochlorite (NaOCl), and chlorhexidine (CHX) at eliminating Gram-negative (E. coli and P. gingivalis) and Gram-positive (E. faecalis and S. sanguinis) bacteria. The effect of irrigating volume and exposure time on the antimicrobial efficacy of HOCl was evaluated, and a durability analysis was completed. Live/dead staining, morphology observation, alamarBlue assay, and lipopolysaccharide (LPS) detection were examined on grit-blasted and biofilm-contaminated titanium alloy discs after treatment with the three chemotherapeutic agents. The results indicated that HOCl exhibited better antibacterial efficacy with increasing irrigating volumes. HOCl achieved greater antibacterial efficacy as treatment time was increased. A decrease in antimicrobial effectiveness was observed when HOCl was unsealed and left in contact with the air. All the irrigants showed antibacterial activity and killed the majority of bacteria on the titanium alloy surfaces of biofilm-contaminated implants. Moreover, HOCl significantly lowered the LPS concentration of P. gingivalis when compared with NaOCl and CHX. Thus, a HOCl antiseptic may be effective for cleaning biofilm-contaminated implant surfaces.

  15. Coagulase-negative staphylococci: pathogenesis, occurrence of antibiotic resistance genes and in vitro effects of antimicrobial agents on biofilm-growing bacteria.

    Science.gov (United States)

    Szczuka, Ewa; Jabłońska, Lucyna; Kaznowski, Adam

    2016-12-01

    Coagulase-negative staphylococci (CoNS) are opportunistic pathogens that particularly cause infections in patients with implanted medical devices. The present research was performed to study the virulence potential of 53 clinical isolates of Staphylococcus capitis, Staphylococcus auricularis, Staphylococcus lugdunensis, Staphylococcus simulans, Staphylococcus cohnii and Staphylococcus caprae. All clinical strains were clonally unrelated. Isolates carried genes encoding resistance to β-lactam (mecA) (15 %), aminoglycoside [aac(6')/aph(2″)(11 %), aph (3')-IIIa (15 %), ant(4')-Ia (19 %)] and macrolide, lincosamide and streptogramin B (MLSB) [erm(A) (4 %), erm(B) (13 %), erm(C) (41 %), msr(A) (11 %)] antibiotics. CoNS isolates (64 %) were able to form biofilms. Confocal laser scanning microscopy revealed that these biofilms formed a three-dimensional structure composed mainly of living cells. All biofilm-positive strains carried the ica operon. In vitro studies demonstrated that a combination treatment with tigecycline and rifampicin was more effective against biofilms than one with ciprofloxacin and rifampicin. The minimum biofilm eradication concentration values were 0.062-0.5 µg ml-1 for tigecycline/rifampicin and 0.250-2 µg ml-1 for ciprofloxacin/rifampicin. All CoNS strains adhered to the human epithelial cell line HeLa, and more than half of the isolates were able to invade the HeLa cells, although most invaded relatively poorly. The virulence of CoNS is also attributed to their cytotoxic effects on HeLa cells. Incubation of HeLa cells with culture supernatant of the CoNS isolates resulted in cell death. The results indicate that the pathogenicity of S. capitis, S. auricularis, S. lugdunensis, S. cohnii and S. caprae is multi-factorial, involving the ability of these bacteria to adhere to human epithelial cells, form biofilms and invade and destroy human cells.

  16. In vitro and in vivo activity of EDTA and antibacterial agents against the biofilm of mucoid Pseudomonas aeruginosa.

    Science.gov (United States)

    Liu, Zhenqiu; Lin, Yaying; Lu, Qi; Li, Fang; Yu, Jialin; Wang, Zhengli; He, Yu; Song, Chao

    2017-02-01

    Refractory infection caused by bacterial biofilm is an important clinical problem. Pseudomonas aeruginosa is a common pathogen responsible for persistent and chronic biofilm infections. We aimed to explore the in vitro and in vivo activity of ethylenediamine tetraacetic acid (EDTA) in combination with antibacterial agents against mucoid P. aeruginosa biofilm. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration of ciprofloxacin, gentamicin, and ampicillin alone or with EDTA against P. aeruginosa were determined in vitro. Extracellular polysaccharides (EPS) and structural parameters of the biofilm were monitored. P. aeruginosa was aerosolized and delivered into the lungs of guinea pigs, which were treated with ciprofloxacin with or without EDTA. The colony-forming units (CFUs) of P. aeruginosa were determined from the lungs. EDTA reduced the MIC of ciprofloxacin and ampicillin by about 30-fold and that of gentamicin by twofold. EDTA reduced the biofilm EPS and the proportion of viable bacteria. The thickness, average diffusion distance, and textural entropy of EDTA-treated biofilm were significantly decreased. EDTA plus antibiotics reduced the colony counting from 107 to 103 CFU/mL. In vivo, EDTA plus ciprofloxacin had a significantly lower mean CFU/g of lung tissue (EDTA + ciprofloxacin 1.3 ± 0.19; EDTA 4.4 ± 0.57; ciprofloxacin 4.2 ± 0.47), and lung lesions were less severe compared with the single treatment groups. EDTA can destroy the biofilm structures of mucoid P. aeruginosa in vitro. Moreover, EDTA and ciprofloxacin had a significant bactericidal effect against biofilm in vivo.

  17. In vitro evaluation of antifungal activity of monolaurin against Candida albicans biofilms

    Directory of Open Access Journals (Sweden)

    Dalia Seleem

    2016-06-01

    Full Text Available Monolaurin (also known as glycerol monolaurate is a natural compound found in coconut oil and is known for its protective biological activities as an antimicrobial agent. The nature of oral candidiasis and the increased antifungal resistance demand the search for novel antifungal therapeutic agents. In this study, we examine the antifungal activity of monolaurin against Candida albicans biofilms (strain ATCC:SC5314/MYA2876 in vitro and investigate whether monolaurin can alter gene expression of host inflammatory cytokines, IL-1α and IL-1β. In a co-culture model, oral fibroblast cells were cultured simultaneously with C. albicans for 24 hrs followed by the exposure to treatments of monolaurin (3.9–2,500 µM, positive control fluconazole (32.2 µM, and vehicle control group (1% ethanol, which was a model used to evaluate the cytotoxicity of monolaurin on fibroblasts as well as to analyze morphological characteristics of biofilms through fluorescence microscopy. In addition, the co-culture model was used for RNA extraction of oral fibroblasts to assess gene expression of host inflammatory cytokines, using quantitative real-time PCR. Our results showed the MIC and MFC of monolaurin were in the range 62.5–125 µM and 125–250 µM, respectively. Biofilm antifungal assay showed significant reduction in Log (CFU/ml of biofilms treated with 1,250 and 2,500 µM of 1-monolaurin when compared to the control groups . There was also a significant down-regulation of IL-1α and IL-1β in the co-culture treated with monolaurin. It can be concluded that monolaurin has a potential antifungal activity against C. albicans and can modulate the pro-inflammatory response of the host.

  18. Combinatorial effects of aromatic 1,3 – disubstituted ureas and fluoride on in vitro inhibition of Streptococcus mutans biofilm formation

    Directory of Open Access Journals (Sweden)

    Gurmeet eKaur

    2016-06-01

    Full Text Available Dental caries occurs as a result of disequilibrium between acid producing pathogenic bacteria and alkali generating commensal bacteria within a dental biofilm (dental plaque. S. mutans has been reported as a primary cariogenic pathogen associated with dental caries. Emergence of multidrug resistant as well as fluoride resistant strains of Streptococcus mutans due to over usage of various antibiotics is a rising problem and prompted the researchers worldwide to search for alternative therapies. In this perspective, the present study was aimed to screen selective inhibitors against ComA, a bacteriocin associated ABC transporter, involved in the quorum sensing of S. mutans. In light of our previous in silico findings, 1,3- disubstituted urea derivatives which had better affinity to ComA were chemically synthesized in the present study for in vitro evaluation of S. mutans biofilm inhibition. The results revealed that 1,3- disubstituted urea derivatives showed good biofilm inhibition. In addition, the synthesized compounds exhibited potent synergy with a very low concentration of fluoride (31.25 ppm to 62.5 ppm in inhibiting the biofilm formation of S. mutans without affecting the bacterial growth. Further, the results were confirmed by confocal laser scanning microscopy. On the whole, from our experimental results we conclude that the combinatorial application of fluoride and disubstituted ureas has a potential synergistic effect which has a promising approach in combating multidrug resistant and fluoride resistant S. mutans in dental caries management.

  19. Candida parapsilosis complex water isolates from a haemodialysis unit: biofilm production and in vitro evaluation of the use of clinical antifungals

    Directory of Open Access Journals (Sweden)

    Regina Helena Pires

    2011-09-01

    Full Text Available Candida parapsilosis, currently divided into three distinct species, proliferates in glucose-rich solutions and has been associated with infections resulting from the use of medical devices made of plastic, an environment common in dialysis centres. The aims of this study were (i to screen for Candida orthopsilosis and Candida metapsilosis (100 environmental isolates previously identified as C. parapsilosis, (ii to test the ability of these isolates to form biofilm and (iii to investigate the in vitro susceptibility of Candida spp biofilms to the antifungal agents, fluconazole (FLC and amphotericin B (AMB. Isolates were obtained from a hydraulic circuit collected from a haemodialysis unit. Based on molecular criteria, 47 strains were re-identified as C. orthopsilosis and 53 as C. parapsilosis. Analyses using a formazan salt reduction assay and total viable count, together with microscopy studies, revealed that 72 strains were able to form biofilm that was structurally similar, but with minor differences in morphology. A microtitre-based colorimetric assay used to test the susceptibility of fungal biofilms to AMB and FLC demonstrated that the C. parapsilosis complex displayed an increased resistance to these antifungal agents. The results from these analyses may provide a basis for implementing quality controls and monitoring to ensure the microbiological purity of dialysis water, including the presence of yeast.

  20. The in vivo biofilm

    DEFF Research Database (Denmark)

    Bjarnsholt, Thomas; Alhede, Maria; Alhede, Morten

    2013-01-01

    Bacteria can grow and proliferate either as single, independent cells or organized in aggregates commonly referred to as biofilms. When bacteria succeed in forming a biofilm within the human host, the infection often becomes very resistant to treatment and can develop into a chronic state. Biofilms...... have been studied for decades using various in vitro models, but it remains debatable whether such in vitro biofilms actually resemble in vivo biofilms in chronic infections. In vivo biofilms share several structural characteristics that differ from most in vitro biofilms. Additionally, the in vivo...... experimental time span and presence of host defenses differ from chronic infections and the chemical microenvironment of both in vivo and in vitro biofilms is seldom taken into account. In this review, we discuss why the current in vitro models of biofilms might be limited for describing infectious biofilms...

  1. Biofilm Formation by Pseudomonas Species Onto Graphene Oxide-TiO2 Nanocomposite-Coated Catheters: In vitro Analysis

    Science.gov (United States)

    Deb, Ananya; Vimala, R.

    The present study focuses on the development of an in vitro model system for biofilm growth by Pseudomonas aerouginosa onto small discs of foley catheter. Catheter disc used for the study was coated with graphene oxide-titanium oxide composite (GO-TiO2) and titanium oxide (TiO2) and characterized through XRD, UV-visible spectroscopy. Morphological analysis was done by scanning electron microscopy (SEM). The biofilm formed on the catheter surface was quantified by crystal violet (CV) staining method and a colorimetric assay (MTT assay) which involves the reduction of tetrazolium salt. The catheter coated with GO-TiO2 showed reduced biofilm growth in comparison to the TiO2-coated and uncoated catheter, thus indicating that it could be successfully used in coating biomedical devices to prevent biofilm formation which is a major cause of nosocomial infection.

  2. Silver-Containing Hydroxyapatite Coating Reduces Biofilm Formation by Methicillin-Resistant Staphylococcus aureus In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Masaya Ueno

    2016-01-01

    Full Text Available Biofilm-producing bacteria are the principal causes of infections associated with orthopaedic implants. We previously reported that silver-containing hydroxyapatite (Ag-HA coatings exhibit high antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA. In the present study, we evaluated the effects of Ag-HA coating of implant surfaces on biofilm formation. Titanium disks (14-mm diameter, 1-mm thickness, one surface of which was coated with HA or 0.5%–3.0% Ag-HA with a thermal spraying technique, were used. In vitro, the disks were inoculated with an MRSA suspension containing 4×105 CFU and incubated for 1-2 weeks. In vivo, MRSA-inoculated HA and 3% Ag-HA disks (8.8–10.0 × 108 CFU were implanted subcutaneously on the back of rats for 1–7 days. All disks were subsequently stained with a biofilm dye and observed under a fluorescence microscope, and biofilm coverage rates (BCRs were calculated. The BCRs on the Ag-HA coating were significantly lower than those on the HA coating at all time points in vitro (p<0.05. Similar results were observed in vivo (p<0.001 without argyria. Ag-HA coating reduced biofilm formation by MRSA in vitro and in vivo; therefore, Ag-HA coating might be effective for reducing implant-associated infections.

  3. Antimicrobial activity of a bioelectric dressing using an in vitro wound pathogen colony drip-flow reactor biofilm model.

    Science.gov (United States)

    Kim, H; Izadjoo, M

    2016-07-01

    We performed in vitro antibiofilm testing of a silver and zinc containing microcurrent generating bioelectric dressing (BED) against clinical wound pathogens to determine its efficacy in preventing biofilm formation under low shear and continuous flow conditions, simulating wound infection environments. We customised an in vitro colony drip-flow reactor (DFR) biofilm model for efficacy evaluation of BED. Each bacterial pathogen was diluted to 10(4)CFU/ml and inoculated on the polycarbonate filter membrane as an abiotic support. BED and controls (no treatment, gauze, and blank polyester with no silver and zinc) were applied directly on the membranes where bacterial cultures were inoculated. Biofilms were continuously developed onto the membranes for 72 hours at room temperature. Biofilm formation was confirmed by crystal violet staining and microscopic observation. Through vigorous shaking and sonication, the released bacteria were serially diluted, plated, and incubated for 24 hours at 37°C to determine the numbers of surviving bacteria. Biofilms were well developed onto blank polyesters, but not the BED after 72 hours incubation. Crystal violet staining from the blank polyesters showed large and fully grown biofilms. We observed an inhibition in bacterial growth on BED treatment. The antibiofilm activity of the BED against each of eight monospecies biofilms showed a 1- or 2 log10 (or 10- or 100-fold) reduction in bacterial numbers compared with those of controls. Our results demonstrated that colony DFR biofilm model was an appropriate for testing the antibiofilm efficacy of BED under low shear and continuous flow conditions for simulating clinical wound environments. The bioelectric currents generated from the silver and zinc active ingredients in the dressing resulted in antibiofilm activity of this wound care device.

  4. Formation of Biofilms by Foodborne Pathogens and Development of Laboratory In Vitro Model for the Study of Campylobacter Genus Bacteria Based on These Biofilms.

    Science.gov (United States)

    Efimochkina, N R; Bykova, I B; Markova, Yu M; Korotkevich, Yu V; Stetsenko, V V; Minaeva, L P; Sheveleva, S A

    2017-02-01

    We analyzed the formation of biofilms by 7 strains of Campylobacter genus bacteria and 18 strains of Enterobacteriaceae genus bacteria that were isolated from plant and animal raw materials, from finished products, and swabs from the equipment of the food industry. Biofilm formation on glass plates, slides and coverslips, microtubes made of polymeric materials and Petri dishes, and polystyrene plates of different profiles were analyzed. When studying the process of films formation, different effects on bacterial populations were simulated, including variation of growth factor composition of culture media, technique of creating of anaerobiosis, and biocide treatment (active chlorine solutions in a concentration of 100 mg/dm(3)). The formation of biofilms by the studied cultures was assessed by the formation of extracellular matrix stained with aniline dyes on glass and polystyrene surfaces after incubation; 0.1% crystal violet solution was used as the dye. The presence and density of biomatrix were assessed by staining intensity of the surfaces of contact with broth cultures or by optical density of the stained inoculum on a spectrophotometer. Biofilms were formed by 57% Campylobacter strains and 44% Enterobacteriaceae strains. The intensity of the film formation depended on culturing conditions and protocols, species and genus of studied isolates, and largely on adhesion properties of abiotic surfaces. In 30% of Enterobacteriaceae strains, the biofilm formation capacity tended to increase under the influence of chlorine-containing biocide solutions. Thus, we developed and tested under laboratory conditions a plate version of in vitro chromogenic model for evaluation of biofilm formation capacity of C. jejuni strains and studied stress responses to negative environmental factors.

  5. Antimicrobial penetration in a dual-species oral biofilm after noncontact brushing : an in vitro study

    NARCIS (Netherlands)

    He, Y.; Peterson, B. W.; Ren, Y.; van der Mei, H. C.; Busscher, H. J.

    Oral biofilm is inevitably left behind, even after powered brushing. As a special feature, powered brushing removes biofilm in a noncontact mode. When the brushing distance becomes too large, biofilm is left behind. We hypothesize that biofilm left behind after brushing has different viscoelastic

  6. In vitro biofilm formation of commensal and pathogenic Escherichia coli strains: impact of environmental and genetic factors

    DEFF Research Database (Denmark)

    Reisner, A.; Krogfelt, Karen; Klein, B.M.

    2006-01-01

    Our understanding of Escherichia coli biofilm formation in vitro is based on studies of laboratory K-12 strains grown in standard media. However, pathogenic E. coli isolates differ substantially in their genetic repertoire from E. coli K-12 and are subject to heterogeneous environmental condition...

  7. Solar Radiation Stress in Natural Acidophilic Biofilms of Euglena mutabilis Revealed by Metatranscriptomics and PAM Fluorometry.

    Science.gov (United States)

    Puente-Sánchez, Fernando; Olsson, Sanna; Gómez-Rodriguez, Manuel; Souza-Egipsy, Virginia; Altamirano-Jeschke, Maria; Amils, Ricardo; Parro, Victor; Aguilera, Angeles

    2016-02-01

    The daily photosynthetic performance of a natural biofilm of the extreme acidophilic Euglena mutabilis from Río Tinto (SW, Spain) under full solar radiation was analyzed by means of pulse amplitude-modulated (PAM) fluorescence measurements and metatrascriptomic analysis. Natural E. mutabilis biofilms undergo large-scale transcriptomic reprogramming during midday due to a dynamic photoinhibition and solar radiation stress. Photoinhibition is due to UV radiation and not to light intensity, as revealed by PAM fluorometry analysis. In order to minimize the negative effects of solar radiation, our data supports the presence of a circadian rhythm in this euglenophyte that increases their opportunity to survive. Differential gene expression throughout the day (at 12:00, 20:00 and night) was monitored by massive Illumina parallel sequencing of metatranscriptomic libraries. The transcription pattern was altered in genes involved in Photosystem II stability and repair, UV damaged DNA repair, non-photochemical quenching and oxidative stress, supporting the photoinhibition detected by PAM fluorometry at midday. Copyright © 2016 Elsevier GmbH. All rights reserved.

  8. An in vitro dynamic microcosm biofilm model for caries lesion development and antimicrobial dose-response studies.

    Science.gov (United States)

    Maske, T T; Brauner, K V; Nakanishi, L; Arthur, R A; van de Sande, F H; Cenci, M S

    2016-01-01

    Some dynamic biofilm models for dental caries development are limited as they require multiple experiments and do not allow independent biofilm growth units, making them expensive and time-consuming. This study aimed to develop and test an in vitro dynamic microcosm biofilm model for caries lesion development and for dose-response to chlorhexidine. Microcosm biofilms were grown under two different protocols from saliva on bovine enamel discs for up to 21 days. The study outcomes were as follows: the percentage of enamel surface hardness change, integrated hardness loss, and the CFU counts from the biofilms formed. The measured outcomes, mineral loss and CFU counts showed dose-response effects as a result of the treatment with chlorhexidine. Overall, the findings suggest that biofilm growth for seven days with 0.06 ml min(-1) salivary flow under exposure to 5% sucrose (3 × daily, 0.25 ml min(-1), 6 min) was suitable as a pre-clinical model for enamel demineralization and antimicrobial studies.

  9. Chitosan nanoparticles enhance the efficiency of methylene blue-mediated antimicrobial photodynamic inactivation of bacterial biofilms: An in vitro study.

    Science.gov (United States)

    Darabpour, Esmaeil; Kashef, Nasim; Mashayekhan, Shohreh

    2016-06-01

    Biodegradable chitosan nanoparticles (CSNPs) with an intrinsic antimicrobial activity may be a good choice to improve the effectiveness of antimicrobial photodynamic inactivation (APDI). The aim of this study was to investigate the effect of CSNPs on the efficiency of methylene blue (MB)-mediated APDI of Staphylococcus aureus and Pseudomonas aeruginosa biofilms. We also assessed the phototoxicity of MB+CSNPs towards human fibroblasts. CSNPs were prepared using ionic gelation method and characterized by dynamic light scattering (DLS) and field-emission scanning electron microscope (FESEM). Biofilms were developed in a 96-well polystyrene plate for 24h. In vitro phototoxic effect of MB+CSNPs (at final concentrations of 50μM MB) at fluence of 22.93J/cm(2)) on biofilms were studied. Appropriate controls were included. Also, in vitro cytotoxicity and phototoxicity of the above mixture was assessed on human dermal fibroblasts. DLS and FESEM measurements confirmed the nanometric size of the prepared CSNPs. APDI mediated by the mixture of MB and CSNPs showed significant anti-biofilm photoinactivation (P3 and >2 log10 CFU reduction in S. aureus and P. aeruginosa biofilms, respectively) while MB-induced APDI led to approximately <1 log10 CFU reduction. At the same experimental conditions, only 25.1% of the fibroblasts were photoinactivated by MB+CSNPs. Our findings showed that CSNPs enhanced the efficacy of MB-APDI; it may be due to the disruption of biofilm structure by polycationic CSNPs and subsequently deeper and higher penetration of MB into the biofilms. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Effect of chlorhexidine/thymol and fluoride varnishes on dental biofilm formation in vitro.

    Science.gov (United States)

    Takeuchi, Yasuo; Guggenheim, Bernhard; Filieri, Anna; Baehni, Pierre

    2007-12-01

    This study evaluated the effect of chlorhexidine/thymol (CHX/T) and fluoride (F) varnishes on biofilm formation in vitro. Hydroxyapatite discs coated with varnish were first immersed in saline for 0, 3, 7 or 14 d, then immersed in pasteurized saliva. The discs were incubated for 20 h with a bacterial suspension containing Actinomyces naeslundii, Fusobacterium nucleatum, Streptococcus oralis, and Veillonella dispar. Uncoated discs were used as controls. Growth of bacteria on the discs was evaluated by culture and by scanning electron microscopy (SEM). Bacterial vitality was examined by fluorescence staining. In the CHX/T-treated group, bacterial accumulation was delayed, and the total number of bacteria was significantly lower than in the controls. In the F-treated group, the total number of bacteria did not differ from the control, although the number of S. oralis was lower. Bacterial vitality in the CHX/T and F groups did not differ from that in the controls. The total number of bacteria on the CHX/T-treated discs immersed in saline was significantly higher than that on the non-immersed discs. Biofilm development was inhibited by the CHX/T varnish but not by the F varnish. The effect of the CHX/T varnish decreased following the immersion of discs in saline.

  11. In vitro activities of amoxicillin-clavulanate, doxycycline, ceftazidime, imipenem, and trimethoprim-sulfamethoxazole against biofilm of Brazilian strains of Burkholderia pseudomallei.

    Science.gov (United States)

    Bandeira, Tereza de Jesus Pinheiro Gomes; Moreira, Camila Alencar; Brilhante, Raimunda Sâmia Nogueira; Castelo-Branco, Débora de Souza Collares Maia; Neto, Manoel Paiva de Araújo; Cordeiro, Rossana de Aguiar; Rodrigues, Terezinha de Jesus Santos; Rocha, Marcos Fábio Gadelha; Sidrim, José Júlio Costa

    2013-11-01

    This study aimed at investigating the in vitro activities of amoxicillin-clavulanate, doxycycline, ceftazidime, imipenem, and trimethoprim-sulfamethoxazole against Burkholderia pseudomallei in planktonic and biofilm forms, through broth microdilution and resazurin-based viability staining, respectively. In planktonic growth, the strains were susceptible to the drugs, while in biofilm growth, significantly higher antimicrobial concentrations were required, especially for ceftazidime and imipenem, surpassing the resistance breakpoints. These results highlight the importance of the routine evaluation of biofilm antimicrobial susceptibility.

  12. Diversity and functions of bacterial community in drinking water biofilms revealed by high-throughput sequencing

    Science.gov (United States)

    Chao, Yuanqing; Mao, Yanping; Wang, Zhiping; Zhang, Tong

    2015-06-01

    The development of biofilms in drinking water (DW) systems may cause various problems to water quality. To investigate the community structure of biofilms on different pipe materials and the global/specific metabolic functions of DW biofilms, PCR-based 454 pyrosequencing data for 16S rRNA genes and Illumina metagenomic data were generated and analysed. Considerable differences in bacterial diversity and taxonomic structure were identified between biofilms formed on stainless steel and biofilms formed on plastics, indicating that the metallic materials facilitate the formation of higher diversity biofilms. Moreover, variations in several dominant genera were observed during biofilm formation. Based on PCA analysis, the global functions in the DW biofilms were similar to other DW metagenomes. Beyond the global functions, the occurrences and abundances of specific protective genes involved in the glutathione metabolism, the SoxRS system, the OxyR system, RpoS regulated genes, and the production/degradation of extracellular polymeric substances were also evaluated. A near-complete and low-contamination draft genome was constructed from the metagenome of the DW biofilm, based on the coverage and tetranucleotide frequencies, and identified as a Bradyrhizobiaceae-like bacterium according to a phylogenetic analysis. Our findings provide new insight into DW biofilms, especially in terms of their metabolic functions.

  13. Biofilm formation on stainless steel and gold wires for bonded retainers in vitro and in vivo and their susceptibility to oral antimicrobials

    NARCIS (Netherlands)

    Jongsma, Marije A.; Pelser, Floris D. H.; van der Mei, Henny C.; Atema-Smit, Jelly; van de Belt-Gritter, Betsy; Busscher, Henk J.; Ren, Yijin

    OBJECTIVE: Bonded retainers are used in orthodontics to maintain treatment result. Retention wires are prone to biofilm formation and cause gingival recession, bleeding on probing and increased pocket depths near bonded retainers. In this study, we compare in vitro and in vivo biofilm formation on

  14. Rapid development in vitro and in vivo of resistance to ceftazidime in biofilm-growing Pseudomonas aeruginosa due to chromosomal beta-lactamase

    DEFF Research Database (Denmark)

    Bagge, N; Ciofu, O; Skovgaard, L T

    2000-01-01

    The aim of this study was to examine the development of resistance of biofilm-growing P. aeruginosa during treatment with ceftazidime. Biofilms were established in vitro using a modified Robbins device (MRD) and in vivo in the rat model of chronic lung infection. Three P. aeruginosa strains...

  15. Achromobacter Species Isolated from Cystic Fibrosis Patients Reveal Distinctly Different Biofilm Morphotypes

    DEFF Research Database (Denmark)

    Nielsen, Signe M; Nørskov-Lauritsen, Niels; Bjarnsholt, Thomas

    2016-01-01

    findings were visualized in sputum samples from cystic fibrosis patients using an Achromobacter specific peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) probe, confirming the presence of Achromobacter biofilms in the CF lung. High antibiotic tolerance was associated with the biofilm......Achromobacter species have attracted attention as emerging pathogens in cystic fibrosis. The clinical significance of Achromobacter infection is not yet fully elucidated; however, their intrinsic resistance to antimicrobials and ability to form biofilms renders them capable of establishing long...

  16. The effect of berberine hydrochloride on Enterococcus faecalis biofilm formation and dispersion in vitro.

    Science.gov (United States)

    Chen, Lihua; Bu, Qianqian; Xu, Huan; Liu, Yuan; She, Pengfei; Tan, Ruichen; Wu, Yong

    2016-01-01

    Enterococcus faecalis (E. faecalis) is one of the major causes of biofilm infections. Berberine hydrochloride (BBH) has diverse pharmacological effects; however, the effects and mechanisms of BBH on E. faecalis biofilm formation and dispersion have not been reported. In this study, 99 clinical isolates from the urine samples of patients with urinary tract infections (UTIs) were collected and identified. Ten strains of E. faecalis with biofilm formation ability were studied. BBH inhibited E. faecalis biofilm formation and promoted the biofilm dispersion of E. faecalis. In addition, sortase A and esp expression levels were elevated during early E. faecalis biofilm development, whereas BBH significantly reduced their expression levels. The results of this study indicated that BBH effectively prevents biofilm formation and promotes biofilm dispersion in E. faecalis, most likely by inhibiting the expressions of sortase A and esp. Copyright © 2016 Elsevier GmbH. All rights reserved.

  17. Pentacyclic triterpenes combined with ciprofloxacin help to eradicate the biofilm formed in vitro by Escherichia coli

    Directory of Open Access Journals (Sweden)

    Dorota Wojnicz

    2015-01-01

    Interpretation & conclusions: Although ciprofloxacin is recommended in the treatment of urinary tract infections caused by UPECs, but its efficacy is arguable. Subinhibitory concentrations of ciprofloxacin did not inhibit the formation of biofilm. Pentacyclic triterpenes used in combination with ciprofloxacin enhanced its anti-biofilm effectiveness. However, this anti-biofilm activity was found to depend on the type of surface on which biofilm was formed.

  18. In Vitro Efficacy of Nonantibiotic Treatments on Biofilm Disruption of Gram-Negative Pathogens and an In Vivo Model of Infectious Endometritis Utilizing Isolates from the Equine Uterus.

    Science.gov (United States)

    Ferris, Ryan A; McCue, Patrick M; Borlee, Grace I; Loncar, Kristen D; Hennet, Margo L; Borlee, Bradley R

    2016-03-01

    In this study, we evaluated the ability of the equine clinical treatments N-acetylcysteine, EDTA, and hydrogen peroxide to disrupt in vitro biofilms and kill equine reproductive pathogens (Escherichia coli, Pseudomonas aeruginosa, or Klebsiella pneumoniae) isolated from clinical cases. N-acetylcysteine (3.3%) decreased biofilm biomass and killed bacteria within the biofilms of E. coli isolates. The CFU of recoverable P. aeruginosa and K. pneumoniae isolates were decreased, but the biofilm biomass was unchanged. Exposure to hydrogen peroxide (1%) decreased the biofilm biomass and reduced the CFU of E. coli isolates, K. pneumoniae isolates were observed to have a reduction in CFU, and minimal effects were observed for P. aeruginosa isolates. Chelating agents (EDTA formulations) reduced E. coli CFU but were ineffective at disrupting preformed biofilms or decreasing the CFU of P. aeruginosa and K. pneumoniae within a biofilm. No single nonantibiotic treatment commonly used in equine veterinary practice was able to reduce the CFU and biofilm biomass of all three Gram-negative species of bacteria evaluated. An in vivo equine model of infectious endometritis was also developed to monitor biofilm formation, utilizing bioluminescence imaging with equine P. aeruginosa isolates from this study. Following infection, the endometrial surface contained focal areas of bacterial growth encased in a strongly adherent "biofilm-like" matrix, suggesting that biofilms are present during clinical cases of infectious equine endometritis. Our results indicate that Gram-negative bacteria isolated from the equine uterus are capable of producing a biofilm in vitro, and P. aeruginosa is capable of producing biofilm-like material in vivo. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  19. In vitro activity of anidulafungin in combination with amphotericin B or voriconazole against biofilms of five Candida species.

    Science.gov (United States)

    Valentín, A; Cantón, E; Pemán, J; Fernandez-Rivero, M E; Tormo-Mas, M A; Martínez, J P

    2016-12-01

    To evaluate the in vitro activity of anidulafungin combined with amphotericin B or voriconazole against Candida spp. biofilms. Four Candida albicans, four Candida tropicalis, four Candida glabrata, two Candida parapsilosis and two Candida orthopsilosis blood isolates were tested by the microdilution chequerboard method combined with the XTT metabolic assay. Biofilm MIC was defined as the lowest concentration producing 50% metabolic inhibition with respect to control (BMIC50). Concentrations in the combinations ranged from 1/8 × BMIC50 to 4 × BMIC50 found for each antifungal tested alone. Anidulafungin plus amphotericin B acted synergistically against C. albicans and C. glabrata biofilms [fractional inhibitory concentration index (FICI): 0.082-0.387], but showed no interaction against C. tropicalis, C. parapsilosis and C. orthopsilosis (FICI: 0.516-2.099). The combination of these antifungals failed to completely remove biofilms of C. albicans and C. glabrata, decreasing the metabolic activity of the biofilms up to 80% and 95%, respectively, which did not occur when each antifungal was used alone. Anidulafungin plus voriconazole showed no interaction against all isolates. Using a less stringent criterion previously proposed to define synergism (FICI  1.25), antagonistic interactions were found against some isolates. Anidulafungin with amphotericin B results in a synergistic effect against C. albicans and C. glabrata biofilms at serum concentrations of the drugs, but showed no interaction against C. tropicalis and C. parapsilosis complex. Anidulafungin plus voriconazole showed no interaction against the five Candida species assayed. Biofilms of C. tropicalis were found to be the most resistant towards the combinations assayed. The results presented may be of potential interest in the clinical setting. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For

  20. Anti-caries DNA vaccine-induced secretory immunoglobulin A antibodies inhibit formation of Streptococcus mutans biofilms in vitro.

    Science.gov (United States)

    Huang, Li; Xu, Qing-an; Liu, Chang; Fan, Ming-wen; Li, Yu-hong

    2013-02-01

    To investigate the effects of anti-caries DNA vaccine-induced salivary secretory immunoglobulin A (S-IgA) antibodies on Streptococcus mutans (S. mutans) adherence and biofilms formation in vitro. Adult female Wistar rats were intranasally immunized with the anti-caries DNA vaccine pGJA-P/VAX. Their saliva samples were collected at different times after the immunization, and S-IgA antibody level in the saliva and its inhibition on S. mutans adherence were examined. The effects of S-IgA in the saliva with the strongest inhibitory effects were examined at 3 different stages, ie acquired pellicles, biofilm formation and production of mature biofilms. The number of viable bacteria and depth of the biofilm at 16 h in each stage were determined using counting colony forming units and using a confocal laser scanning microscopy (CLSM). The participation of S-IgA in acquired pellicles and its aggregation with S. mutans were also observed under CLSM. The S-IgA titer in saliva reached its peak and exhibited the strongest inhibition on S. mutans adhesion at 10 weeks after the immunization. The colonies and depth of the biofilm in the saliva-pretreated group were 41.79% and 41.02%, respectively, less than the control group. The colonies and depth of the biofilm in the co-culture group were 27.4% and 22.81% less than the control group. The assembly of S. mutans and S-IgA was observed under CLSM after co-cultivation. In the mature-stage biofilm, no differences were observed between the different groups. These results demonstrate that the anti-caries DNA vaccine induces the production of specific S-IgA antibodies that may prevent dental caries by inhibiting the initial adherence of S. mutans onto tooth surfaces, thereby reducing the accumulation of S. mutans on the acquired pellicles.

  1. Anti-caries DNA vaccine-induced secretory immunoglobulin A antibodies inhibit formation of Streptococcus mutans biofilms in vitro

    Science.gov (United States)

    Huang, Li; Xu, Qing-an; Liu, Chang; Fan, Ming-wen; Li, Yu-hong

    2013-01-01

    Aim: To investigate the effects of anti-caries DNA vaccine-induced salivary secretory immunoglobulin A (S-IgA) antibodies on Streptococcus mutans (S. mutans) adherence and biofilms formation in vitro. Methods: Adult female Wistar rats were intranasally immunized with the anti-caries DNA vaccine pGJA-P/VAX. Their saliva samples were collected at different times after the immunization, and S-IgA antibody level in the saliva and its inhibition on S. mutans adherence were examined. The effects of S-IgA in the saliva with the strongest inhibitory effects were examined at 3 different stages, ie acquired pellicles, biofilm formation and production of mature biofilms. The number of viable bacteria and depth of the biofilm at 16 h in each stage were determined using counting colony forming units and using a confocal laser scanning microscopy (CLSM). The participation of S-IgA in acquired pellicles and its aggregation with S. mutans were also observed under CLSM. Results: The S-IgA titer in saliva reached its peak and exhibited the strongest inhibition on S. mutans adhesion at 10 weeks after the immunization. The colonies and depth of the biofilm in the saliva-pretreated group were 41.79% and 41.02%, respectively, less than the control group. The colonies and depth of the biofilm in the co-culture group were 27.4% and 22.81% less than the control group. The assembly of S. mutans and S-IgA was observed under CLSM after co-cultivation. In the mature-stage biofilm, no differences were observed between the different groups. Conclusion: These results demonstrate that the anti-caries DNA vaccine induces the production of specific S-IgA antibodies that may prevent dental caries by inhibiting the initial adherence of S. mutans onto tooth surfaces, thereby reducing the accumulation of S. mutans on the acquired pellicles. PMID:23274411

  2. Biofilm Infections

    DEFF Research Database (Denmark)

    Bjarnsholt, Thomas; Jensen, Peter Østrup; Moser, Claus Ernst

    A still increasing interest and emphasis on the sessile bacterial lifestyle biofilms has been seen since it was realized that the vast majority of the total microbial biomass exists as biofilms. Aggregation of bacteria was first described by Leeuwenhoek in 1677, but only recently recognized...... as being important in chronic infection. In 1993 the American Society for Microbiology (ASM) recognized that the biofilm mode of growth was relevant to microbiology. This book covers both the evidence for biofilms in many chronic bacterial infections as well as the problems facing these infections...... such as diagnostics, pathogenesis, treatment regimes and in vitro and in vivo models for studying biofilms. This is the first scientific book on biofilm infections, chapters written by the world leading scientist and clinicians. The intended audience of this book is scientists, teachers at university level as well...

  3. Photosensitization of in vitro biofilms formed on denture base resin.

    Science.gov (United States)

    de Freitas-Pontes, Karina Matthes; Gomes, Carlos Eduardo de Albuquerque; de Carvalho, Bruna Marjorie Dias Frota; Sabóia, Rafael de Sousa Carvalho; Garcia, Bruna Albuquerque

    2014-09-01

    Proper sterilization or disinfection of removable prostheses and surgical guides has been problematic in dental practice because of the absence of simple and low-cost techniques that do not cause damage to acrylic resins. The purpose of this study was to study the effect of photodynamic therapy against Streptococcus mutans, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans biofilms formed on acrylic resin specimens. The specimens were sterilized in ethylene oxide gas and submitted to in vitro biofilm growth. The photodynamic therapy consisted of the application of 0.05% methylene blue (P+) conjugated to irradiation with a light-emitting-diode of 630 nm and 150 mW (L+). The specimens were randomly divided into groups (n=5): negative control (P-L-); stained and irradiated at 10 J/cm(2) (P+L+ 10); stained and irradiated at 30 J/cm(2) (P+L+ 30); stained and not irradiated (P+L-); not stained and irradiated at 10 J/cm(2) (P-L+ 10); not stained and irradiated at 30 J/cm(2) (P-L+ 30); and gold standard (GS), sterilized. Afterward, the specimens were submitted to contact with culture medium agar for 10 minutes in petri plates, which were incubated for 48 hours at 37°C. The number of colony-forming units was obtained, and the data were expressed according to scores (1=0; 2=1-10; 3=11-100; 4=101-1000) and analyzed by the Friedman and Dunn tests (α=.05). Streptococcus mutans was sensitized by (P+L-); P aeruginosa and C albicans were also sensitized by the dye but showed a slight microbial reduction with (P+L+ 30), as did S aureus (P>.05); E coli presented an initial score of 3 and achieved a bacterial reduction to score 2 with (P+L+ 30) (P=.039). Photodynamic therapy was effective in reducing E coli counts on biofilms formed on acrylic resin specimens. The inhibition of microorganism growth tended to be directly proportional to the amount of energy provided by the light-emitting diode. Copyright © 2014 Editorial Council for the

  4. In vitro activity of gentamicin as an adjunct to penicillin against biofilm group B Streptococcus.

    Science.gov (United States)

    Ruppen, Corinne; Hemphill, Andrew; Sendi, Parham

    2017-02-01

    Group B Streptococcus (GBS) increasingly causes invasive disease in non-pregnant adults, particularly in elderly persons and those with underlying diseases. Combination therapy with penicillin plus gentamicin has been suggested for periprosthetic joint infection. The postulated synergism of this combination is based on experiments with planktonic bacteria. We aimed to assess the efficacy of this combination against sessile bacteria. Four different GBS strains were used. We compared results of MICs with those of minimal biofilm eradication concentrations (MBECs), applied chequerboard assays to the MBEC device and calculated the fractional inhibitory concentration index. Synergism was evaluated with time-kill assays against bacteria adherent to cement beads, using penicillin (0.048, 0.2 and 3 mg/L), gentamicin (4 and 12.5 mg/L) and a combination thereof. Results were evaluated via colony counting after sonication of beads and scanning electron microscopy. MBEC/MIC ratios were 2000-4000 for penicillin and 1-4 for gentamicin. In chequerboard assays, synergism was observed in all four isolates. In time-kill assays, penicillin and 12.5 mg/L gentamicin showed synergism in two isolates. In the other two isolates 12.5 mg/L gentamicin alone was as efficient as the combination therapy. These in vitro investigations show activity of 12.5 mg/L gentamicin, alone or as an adjunct to penicillin, against four strains of biofilm GBS. This concentration cannot be achieved in bone with systemic administration, but can be reached if administered locally. The combination of systemic penicillin plus local gentamicin indicates a potential application in orthopaedic-device-associated GBS infections. Studies with a larger number of strains are required to confirm our results. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  5. Efficacy of natural antimicrobials in toothpaste formulations against oral biofilms in vitro.

    Science.gov (United States)

    Verkaik, Martinus J; Busscher, Henk J; Jager, Debbie; Slomp, Anje M; Abbas, Frank; van der Mei, Henny C

    2011-03-01

    To evaluate the antimicrobial efficacies of two toothpaste formulations containing natural antimicrobials (herbal extracts and chitosan) against oral biofilms of different composition and maturational status. Bacteria from a buffer suspension or fresh saliva were adhered for 2h to a salivary conditioning film and subsequently grown for 16h. Dual-species biofilms were prepared from Actinomyces naeslundii T14V-J1 and Streptococcus oralis J22, whilst multi-species biofilms were grown from freshly collected human saliva. Biofilms were exposed to 25wt% toothpaste supernatants. A chlorhexidine-containing mouthrinse and a buffer were used as positive- and negative-controls, respectively. Antibacterial efficacy was concluded from acute killing, bacterial removal, prevention of bacterial re-deposition and continued killing during re-deposition. The herbal- and chitosan-based supernatants showed immediate killing of oral biofilm bacteria, comparable with chlorhexidine. Moreover, exposure of a biofilm to these supernatants or chlorhexidine, yielded ongoing killing of biofilm bacteria after exposure during re-deposition of bacteria to a matured 16h biofilm, but not to a much thinner initial biofilm formed by 2h adhesion only. This suggests that thicker, more matured biofilms can absorb and release oral antimicrobials. Supernatants based on herbal- and chitosan-based toothpastes have comparable immediate and ongoing antibacterial efficacies as chlorhexidine. Natural antimicrobials and chlorhexidine absorb in oral biofilms which contributes to their substantive action. Copyright © 2010 Elsevier Ltd. All rights reserved.

  6. Combination of microscopic techniques reveals a comprehensive visual impression of biofilm structure and composition

    DEFF Research Database (Denmark)

    Alhede, Morten; Qvortrup, Klaus; Liebrechts, Ramon

    2012-01-01

    Bacterial biofilms are imaged by various kinds of microscopy including confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). One limitation of CLSM is its restricted magnification, which is resolved by the use of SEM that provides high-magnification spatial images of how...... the single bacteria are located and interact within the biofilm. However, conventional SEM is limited by the requirement of dehydration of the samples during preparation. As biofilms consist mainly of water, the specimen dehydration might alter its morphology. High magnification yet authentic images...... are important to understand the physiology of biofilms. We compared conventional SEM, Focused Ion Beam (FIB)-SEM and CLSM with SEM techniques [cryo-SEM and environmental-SEM (ESEM)] that do not require dehydration. In the case of cryo-SEM, the biofilm is not dehydrated but kept frozen to obtain high-magnification...

  7. Sound waves effectively assist tobramycin in elimination of Pseudomonas aeruginosa biofilms in vitro.

    Science.gov (United States)

    Bandara, H M H N; Harb, A; Kolacny, D; Martins, P; Smyth, H D C

    2014-12-01

    Microbial biofilms are highly refractory to antimicrobials. The aim of this study was to investigate the use of low-frequency vibration therapy (20-20 kHz) on antibiotic-mediated Pseudomonas aeruginosa biofilm eradication. In screening studies, low-frequency vibrations were applied on model biofilm compositions to identify conditions in which surface standing waves were observed. Alginate surface tension and viscosity were also measured. The effect of vibration on P. aeruginosa biofilms was studied using a standard biofilm assay. Subminimal inhibitory concentrations (sub-MIC) of tobramycin (5 μg/ml) were added to biofilms 3 h prior, during, and immediately after vibration and quantitatively assessed by (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) reduction assay (XTT) and, qualitatively, by confocal laser scanning microscopy (CLSM). The standing waves occurred at frequencies sound waves together with antibiotics are a promising approach in eliminating pathogenic biofilms.

  8. Biofilm-specific extracellular matrix proteins of non-typeable Haemophilus influenzae

    Science.gov (United States)

    Wu, Siva; Baum, Marc M.; Kerwin, James; Guerrero-Given, Debbie; Webster, Simon; Schaudinn, Christoph; VanderVelde, David; Webster, Paul

    2014-01-01

    Non-typeable Haemophilus influenzae (NTHi), a human respiratory tract pathogen can form colony biofilms in vitro. Bacterial cells and the amorphous extracellular matrix (ECM) constituting the biofilm can be separated using sonication. The ECM from 24 hr and 96 hr NTHi biofilms contained polysaccharides and proteinaceous components as detected by NMR and FTIR spectroscopy. More conventional chemical assays on the biofilm ECM confirmed the presence of these components and also DNA. Proteomics revealed eighteen proteins present in biofilm ECM that were not detected in planktonic bacteria. One ECM protein was unique to 24 hr biofilms, two were found only in 96 hr biofilms, and fifteen were present in the ECM of both 24 hr and 96 hr NTHi biofilms. All proteins identified were either associated with bacterial membranes or were cytoplasmic proteins. Immunocytochemistry showed two of the identified proteins, a DNA-directed RNA polymerase and the outer membrane protein OMP P2, associated with bacteria and biofilm ECM. Identification of biofilm-specific proteins present in immature biofilms is an important step in understanding the in vitro process of NTHi biofilm formation. The presence of a cytoplasmic protein and a membrane protein in the biofilm ECM of immature NTHi biofilms suggests that bacterial cell lysis may be a feature of early biofilm formation. PMID:24942343

  9. Unintended Laboratory-Driven Evolution Reveals Genetic Requirements for Biofilm Formation by Desulfovibrio vulgaris Hildenborough

    Directory of Open Access Journals (Sweden)

    Kara B. De León

    2017-10-01

    Full Text Available Biofilms of sulfate-reducing bacteria (SRB are of particular interest as members of this group are culprits in corrosion of industrial metal and concrete pipelines as well as being key players in subsurface metal cycling. Yet the mechanism of biofilm formation by these bacteria has not been determined. Here we show that two supposedly identical wild-type cultures of the SRB Desulfovibrio vulgaris Hildenborough maintained in different laboratories have diverged in biofilm formation. From genome resequencing and subsequent mutant analyses, we discovered that a single nucleotide change within DVU1017, the ABC transporter of a type I secretion system (T1SS, was sufficient to eliminate biofilm formation in D. vulgaris Hildenborough. Two T1SS cargo proteins were identified as likely biofilm structural proteins, and the presence of at least one (with either being sufficient was shown to be required for biofilm formation. Antibodies specific to these biofilm structural proteins confirmed that DVU1017, and thus the T1SS, is essential for localization of these adhesion proteins on the cell surface. We propose that DVU1017 is a member of the lapB category of microbial surface proteins because of its phenotypic similarity to the adhesin export system described for biofilm formation in the environmental pseudomonads. These findings have led to the identification of two functions required for biofilm formation in D. vulgaris Hildenborough and focus attention on the importance of monitoring laboratory-driven evolution, as phenotypes as fundamental as biofilm formation can be altered.

  10. Inhibitory effect of biofilm-forming Lactobacillus kunkeei strains against virulent Pseudomonas aeruginosa in vitro and in honeycomb moth (Galleria mellonella) infection model.

    Science.gov (United States)

    Berríos, P; Fuentes, J A; Salas, D; Carreño, A; Aldea, P; Fernández, F; Trombert, A N

    2017-11-10

    Biofilms correspond to complex communities of microorganisms embedded in an extracellular polymeric matrix. Biofilm lifestyle predominates in Pseudomonas aeruginosa, an opportunistic Gram negative pathogen responsible for a wide spectrum of infections in humans, plants and animals. In this context, anti-biofilm can be considered a key strategy to control P. aeruginosa infections, thereby more research in the field is required. On the other hand, Lactobacillus species have been described as beneficial due to their anti-biofilm properties and their consequent effect against a wide spectrum of pathogens. In fact, biofilm-forming Lactobacilli seem to be more efficient than their planktonic counterpart to antagonise pathogenic bacteria. In this work, we demonstrated that Lactobacillus kunkeei, a novel Lactobacillus species isolated from honeybee guts, can form biofilms in vitro. In addition, the L. kunkeei biofilm can, in turn, inhibit the formation of P. aeruginosa biofilms. Finally, we found that L. kunkeei strains attenuate infection of P. aeruginosa in the Galleria mellonella model, presumably by affecting P. aeruginosa biofilm formation and/or their stability. Since L. kunkeei presents characteristics of a probiotic, this work provides evidence arguing that the use of this Lactobacillus species in both animals (including insects) and humans could contribute to impair P. aeruginosa biofilm formation.

  11. In vitro prevention of Pseudomonas aeruginosa early biofilm formation with antibiotics used in cystic fibrosis patients.

    Science.gov (United States)

    Fernández-Olmos, Ana; García-Castillo, María; Maiz, Luis; Lamas, Adelaida; Baquero, Fernando; Cantón, Rafael

    2012-08-01

    The ability of antibiotics used in bronchopulmonary infections in cystic fibrosis (CF) patients to prevent Pseudomonas aeruginosa early biofilm formation was studied using a biofilm microtitre assay with 57 non-mucoid P. aeruginosa isolates (44 first colonisers and 13 recovered during the initial intermittent colonisation stage) obtained from 35 CF patients. Minimum biofilm inhibitory concentrations (BICs) of levofloxacin, ciprofloxacin, imipenem, ceftazidime, tobramycin, colistin and azithromycin were determined by placing a peg lid with a formed biofilm onto microplates containing antibiotics. A modification of this protocol consisting of antibiotic challenge during biofilm formation was implemented in order to determine the biofilm prevention concentration (BPC), i.e. the minimum concentration able to prevent biofilm formation. The lowest BPCs were for fluoroquinolones, tobramycin and colistin and the highest for ceftazidime and imipenem. The former antibiotics had BPCs identical to or only slightly higher than their minimum inhibitory concentrations (MICs) determined by standard Clinical and Laboratory Standards Institute (CLSI) microdilution and were also active on formed biofilms as reflected by their low BIC values. In contrast, ceftazidime and imipenem were less effective for prevention of biofilm formation and on formed biofilms. In conclusion, the new BPC parameter determined in non-mucoid P. aeruginosa isolates recovered during early colonisation stages in CF patients supports early aggressive antimicrobial treatment guidelines in first P. aeruginosa-colonised CF patients. Copyright © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

  12. Differential control of Yersinia pestis biofilm formation in vitro and in the flea vector by two c-di-GMP diguanylate cyclases.

    Directory of Open Access Journals (Sweden)

    Yi-Cheng Sun

    2011-04-01

    Full Text Available Yersinia pestis forms a biofilm in the foregut of its flea vector that promotes transmission by flea bite. As in many bacteria, biofilm formation in Y. pestis is controlled by intracellular levels of the bacterial second messenger c-di-GMP. Two Y. pestis diguanylate cyclase (DGC enzymes, encoded by hmsT and y3730, and one phosphodiesterase (PDE, encoded by hmsP, have been shown to control biofilm production in vitro via their opposing c-di-GMP synthesis and degradation activities, respectively. In this study, we provide further evidence that hmsT, hmsP, and y3730 are the only three genes involved in c-di-GMP metabolism in Y. pestis and evaluated the two DGCs for their comparative roles in biofilm formation in vitro and in the flea vector. As with HmsT, the DGC activity of Y3730 depended on a catalytic GGDEF domain, but the relative contribution of the two enzymes to the biofilm phenotype was influenced strongly by the environmental niche. Deletion of y3730 had a very minor effect on in vitro biofilm formation, but resulted in greatly reduced biofilm formation in the flea. In contrast, the predominant effect of hmsT was on in vitro biofilm formation. DGC activity was also required for the Hms-independent autoaggregation phenotype of Y. pestis, but was not required for virulence in a mouse model of bubonic plague. Our results confirm that only one PDE (HmsP and two DGCs (HmsT and Y3730 control c-di-GMP levels in Y. pestis, indicate that hmsT and y3730 are regulated post-transcriptionally to differentially control biofilm formation in vitro and in the flea vector, and identify a second c-di-GMP-regulated phenotype in Y. pestis.

  13. D-amino acids reduce Enterococcus faecalis biofilms in vitro and in the presence of antimicrobials used for root canal treatment.

    Directory of Open Access Journals (Sweden)

    Peter S Zilm

    Full Text Available Enterococcus faecalis is the most frequent species present in post-treatment disease and plays a significant role in persistent periapical infections following root canal treatment. Its ability to persist in stressful environments is inter alia, due to its ability to form biofilms. The presence of certain D-amino acids (DAAs has previously been shown to reduce formation of Bacillus subtilis biofilms. The aims of this investigation were to determine if DAAs disrupt biofilms in early and late growth stages for clinical E. faecalis strains and to test their efficacy in disrupting E. faecalis biofilms grown in sub-minimum inhibitory concentrations of commonly used endodontic biocides. From thirty-seven E. faecalis strains, the ten "best" biofilm producers were used to test the ability of a mixture containing D-leucine, D-methionine, D-tyrosine and D-tryptophan to reduce biofilm growth over a period of 24, 72 and 144 hours and when compared to their cognate L-Amino Acids (LAAs. We have previously shown that sub-MIC levels of tetracycline and sodium hypochlorite promotes biofilm growth in clinical strains of E. faecalis. DAAs were therefore tested for their effectiveness to reduce biofilm growth in the presence of sub-minimal concentrations of sodium hypochlorite (NaOCl-0.031% and Odontocide™ (0.25% w/v, and in the presence of Odontopaste™ (0.25% w/v. DAAs significantly reduced biofilm formation for all strains tested in vitro, while DAAs significantly reduced biofilm formation compared to LAAs. The inhibitory effect of DAAs on biofilm formation was concentration dependent. DAAs were also shown to be effective in reducing E. faecalis biofilms in the presence of Odontopaste™ and sub-MIC levels of NaOCl and Odontocide™. The results suggest that the inclusion of DAAs into current endodontic procedures may reduce E. faecalis biofilms.

  14. D-amino acids reduce Enterococcus faecalis biofilms in vitro and in the presence of antimicrobials used for root canal treatment.

    Science.gov (United States)

    Zilm, Peter S; Butnejski, Victor; Rossi-Fedele, Giampiero; Kidd, Stephen P; Edwards, Suzanne; Vasilev, Krasimir

    2017-01-01

    Enterococcus faecalis is the most frequent species present in post-treatment disease and plays a significant role in persistent periapical infections following root canal treatment. Its ability to persist in stressful environments is inter alia, due to its ability to form biofilms. The presence of certain D-amino acids (DAAs) has previously been shown to reduce formation of Bacillus subtilis biofilms. The aims of this investigation were to determine if DAAs disrupt biofilms in early and late growth stages for clinical E. faecalis strains and to test their efficacy in disrupting E. faecalis biofilms grown in sub-minimum inhibitory concentrations of commonly used endodontic biocides. From thirty-seven E. faecalis strains, the ten "best" biofilm producers were used to test the ability of a mixture containing D-leucine, D-methionine, D-tyrosine and D-tryptophan to reduce biofilm growth over a period of 24, 72 and 144 hours and when compared to their cognate L-Amino Acids (LAAs). We have previously shown that sub-MIC levels of tetracycline and sodium hypochlorite promotes biofilm growth in clinical strains of E. faecalis. DAAs were therefore tested for their effectiveness to reduce biofilm growth in the presence of sub-minimal concentrations of sodium hypochlorite (NaOCl-0.031%) and Odontocide™ (0.25% w/v), and in the presence of Odontopaste™ (0.25% w/v). DAAs significantly reduced biofilm formation for all strains tested in vitro, while DAAs significantly reduced biofilm formation compared to LAAs. The inhibitory effect of DAAs on biofilm formation was concentration dependent. DAAs were also shown to be effective in reducing E. faecalis biofilms in the presence of Odontopaste™ and sub-MIC levels of NaOCl and Odontocide™. The results suggest that the inclusion of DAAs into current endodontic procedures may reduce E. faecalis biofilms.

  15. Bad to the Bone: On In Vitro and Ex Vivo Microbial Biofilm Ability to Directly Destroy Colonized Bone Surfaces without Participation of Host Immunity or Osteoclastogenesis.

    Science.gov (United States)

    Junka, Adam; Szymczyk, Patrycja; Ziółkowski, Grzegorz; Karuga-Kuzniewska, Ewa; Smutnicka, Danuta; Bil-Lula, Iwona; Bartoszewicz, Marzenna; Mahabady, Susan; Sedghizadeh, Parish Paymon

    2017-01-01

    Bone infections are a significant public health burden associated with morbidity and mortality in patients. Microbial biofilm pathogens are the causative agents in chronic osteomyelitis. Research on the pathogenesis of osteomyelitis has focused on indirect bone destruction by host immune cells and cytokines secondary to microbial insult. Direct bone resorption by biofilm pathogens has not yet been seriously considered. In this study, common osteomyelitis pathogens (Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and Streptococcus mutans) were grown as biofilms in multiple in vitro and ex vivo experiments to analyze quantitative and qualitative aspects of bone destruction during infection. Pathogens were grown as single or mixed species biofilms on the following substrates: hydroxyapatite, rat jawbone, or polystyrene wells, and in various media. Biofilm growth was evaluated by scanning electron microscopy and pH levels were monitored over time. Histomorphologic and quantitative effects of biofilms on tested substrates were analyzed by microcomputed tomography and quantitative cultures. All tested biofilms demonstrated significant damage to bone. Scanning electron microscopy indicated that all strains formed mature biofilms within 7 days on all substrate surfaces regardless of media. Experimental conditions impacted pH levels, although this had no impact on biofilm growth or bone destruction. Presence of biofilm led to bone dissolution with a decrease of total volume by 20.17±2.93% upon microcomputed tomography analysis, which was statistically significant as compared to controls (p biofilm formation (Kruskall-Wallis test, post-hoc Dunne's test; p biofilms associated with osteomyelitis have the ability to directly resorb bone. These findings should lead to a more complete understanding of the etiopathogenesis of osteomyelitis, where direct bone resorption by biofilm is considered in addition to the well-known osteoclastic and host cell destruction

  16. Bad to the Bone: On In Vitro and Ex Vivo Microbial Biofilm Ability to Directly Destroy Colonized Bone Surfaces without Participation of Host Immunity or Osteoclastogenesis

    Science.gov (United States)

    Junka, Adam; Szymczyk, Patrycja; Ziółkowski, Grzegorz; Karuga-Kuzniewska, Ewa; Smutnicka, Danuta; Bil-Lula, Iwona; Bartoszewicz, Marzenna; Mahabady, Susan; Sedghizadeh, Parish Paymon

    2017-01-01

    Bone infections are a significant public health burden associated with morbidity and mortality in patients. Microbial biofilm pathogens are the causative agents in chronic osteomyelitis. Research on the pathogenesis of osteomyelitis has focused on indirect bone destruction by host immune cells and cytokines secondary to microbial insult. Direct bone resorption by biofilm pathogens has not yet been seriously considered. In this study, common osteomyelitis pathogens (Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and Streptococcus mutans) were grown as biofilms in multiple in vitro and ex vivo experiments to analyze quantitative and qualitative aspects of bone destruction during infection. Pathogens were grown as single or mixed species biofilms on the following substrates: hydroxyapatite, rat jawbone, or polystyrene wells, and in various media. Biofilm growth was evaluated by scanning electron microscopy and pH levels were monitored over time. Histomorphologic and quantitative effects of biofilms on tested substrates were analyzed by microcomputed tomography and quantitative cultures. All tested biofilms demonstrated significant damage to bone. Scanning electron microscopy indicated that all strains formed mature biofilms within 7 days on all substrate surfaces regardless of media. Experimental conditions impacted pH levels, although this had no impact on biofilm growth or bone destruction. Presence of biofilm led to bone dissolution with a decrease of total volume by 20.17±2.93% upon microcomputed tomography analysis, which was statistically significant as compared to controls (p <0.05, ANOVA). Quantitative cultures indicated that media and substrate did not impact biofilm formation (Kruskall-Wallis test, post-hoc Dunne’s test; p <0.05). Overall, these results indicate that biofilms associated with osteomyelitis have the ability to directly resorb bone. These findings should lead to a more complete understanding of the etiopathogenesis of

  17. Metagenomic study of red biofilms from Diamante Lake reveals ancient arsenic bioenergetics in haloarchaea.

    Science.gov (United States)

    Rascovan, Nicolás; Maldonado, Javier; Vazquez, Martín P; Eugenia Farías, María

    2016-02-01

    Arsenic metabolism is proposed to be an ancient mechanism in microbial life. Different bacteria and archaea use detoxification processes to grow under high arsenic concentration. Some of them are also able to use arsenic as a bioenergetic substrate in either anaerobic arsenate respiration or chemolithotrophic growth on arsenite. However, among the archaea, bioenergetic arsenic metabolism has only been found in the Crenarchaeota phylum. Here we report the discovery of haloarchaea (Euryarchaeota phylum) biofilms forming under the extreme environmental conditions such as high salinity, pH and arsenic concentration at 4589 m above sea level inside a volcano crater in Diamante Lake, Argentina. Metagenomic analyses revealed a surprisingly high abundance of genes used for arsenite oxidation (aioBA) and respiratory arsenate reduction (arrCBA) suggesting that these haloarchaea use arsenic compounds as bioenergetics substrates. We showed that several haloarchaea species, not only from this study, have all genes required for these bioenergetic processes. The phylogenetic analysis of aioA showed that haloarchaea sequences cluster in a novel and monophyletic group, suggesting that the origin of arsenic metabolism in haloarchaea is ancient. Our results also suggest that arsenite chemolithotrophy likely emerged within the archaeal lineage. Our results give a broad new perspective on the haloarchaea metabolism and shed light on the evolutionary history of arsenic bioenergetics.

  18. Resistance of non-typeable Haemophilus influenzae biofilms is independent of biofilm size.

    Science.gov (United States)

    Reimche, Jennifer L; Kirse, Daniel J; Whigham, Amy S; Swords, W Edward

    2017-02-01

    The inflammatory middle ear disease known as otitis media can become chronic or recurrent in some cases due to failure of the antibiotic treatment to clear the bacterial etiological agent. Biofilms are known culprits of antibiotic-resistant infections; however, the mechanisms of resistance for non-typeable Haemophilus influenzae biofilms have not been completely elucidated. In this study, we utilized in vitro static biofilm assays to characterize clinical strain biofilms and addressed the hypothesis that biofilms with greater biomass and/or thickness would be more resistant to antimicrobial-mediated eradication than thinner and/or lower biomass biofilms. Consistent with previous studies, antibiotic concentrations required to eliminate biofilm bacteria tended to be drastically higher than concentrations required to kill planktonic bacteria. The size characterizations of the biofilms formed by the clinical isolates were compared to their minimum biofilm eradication concentrations for four antibiotics. This revealed no correlation between biofilm thickness or biomass and the ability to resist eradication by antibiotics. Therefore, we concluded that biofilm size does not play a role in antibiotic resistance, suggesting that reduction of antibiotic penetration may not be a significant mechanism for antibiotic resistance for this bacterial opportunist. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  19. Effets d'agents antimicrobiens sur un modèle de biofilm dentaire "in vitro"

    OpenAIRE

    Takinami, Hiroyuki

    2006-01-01

    L'objectif de cette étude était d'évaluer l'effet d'agents antimicrobiens sur un modèle de biofilm. Des disques d'hydroxypatite sont incubés en présence de 4 espèces bactérienne ("Actinomyces naeslundii, Fusobacterium nucleatum, Streptococcus oralis, Veillonella dispar") en milieu anaérobie pendant 12h (biofilm immature) ou 73h (biofilm mature). Les biofilms ont alors été exposés 3x 1min à la chlorhexidine(CHX), triclosan (TRI), cetylpyridinium chloride (CPC) et isopropyl methylphenol (IPMP)....

  20. In vitro antimicrobial activity of mouth washes and herbal products against dental biofilm-forming bacteria.

    Science.gov (United States)

    Da Silva, Naiana B; Alexandria, Adílis K F; De Lima, Aline L; Claudino, Lígia V; De Oliveira Carneiro, Thiago F; Da Costa, Adalberto C; Valença, Ana M G; Cavalcanti, Alessandro L

    2012-07-01

    To evaluate in vitro, the antimicrobial effect of Cymbopogon citrates (lemon grass), Plectranthusamboinicus (Mexican mint) and Conyzabonariensis (hairy fleabane) tinctures as well as pure and diluted commercial mouth washes (Malvatricin(®), Periogard(®) and Listerine(®)) on wild isolates of Streptococcusmutans and reference strains of S. mutans, Streptococcus salivarius, Streptococcus oralis and Lactobacillus casei by determination of minimum inhibitory dilution (MID). 0.12% chlorhexidine and 70% corn alcohol were used as positive and negative controls, respectively. Saliva samples were collected from 3 volunteers and seeded in MSB broth to obtain Streptococcus isolates after 72-hour incubation. Using the agar diffusion method, susceptibility tests were performed with overnight incubation in microaerophilia at 37°C. All tests were performed in duplicate. The bacterial species were resistant to the tinctures and Listerine(®), but were susceptible to 0.12% chlorhexidine, Malvatricin(®) and Periogard(®), with MIDs ranging from 12.5% to 1.56%. Plectrantusamboinicus, Conyzabonariensis and Cymbopongoncitratus tinctures and Listerine(®) did not show inhibitory action against the tested biofilm-forming bacteria.

  1. In vitro antimicrobial activity of mouth washes and herbal products against dental biofilm-forming bacteria

    Directory of Open Access Journals (Sweden)

    Naiana B Da Silva

    2012-01-01

    Full Text Available Aim: To evaluate in vitro, the antimicrobial effect of Cymbopogon citrates (lemon grass, Plectranthusamboinicus (Mexican mint and Conyzabonariensis (hairy fleabane tinctures as well as pure and diluted commercial mouth washes (Malvatricin® , Periogard® and Listerine® on wild isolates of Streptococcusmutans and reference strains of S. mutans, Streptococcus salivarius, Streptococcus oralis and Lactobacillus casei by determination of minimum inhibitory dilution (MID. Materials and Methods: 0.12% chlorhexidine and 70% corn alcohol were used as positive and negative controls, respectively. Saliva samples were collected from 3 volunteers and seeded in MSB broth to obtain Streptococcus isolates after 72-hour incubation. Using the agar diffusion method, susceptibility tests were performed with overnight incubation in microaerophilia at 37°C. All tests were performed in duplicate. Results: The bacterial species were resistant to the tinctures and Listerine® , but were susceptible to 0.12% chlorhexidine, Malvatricin® and Periogard® , with MIDs ranging from 12.5% to 1.56%. Conclusions: Plectrantusamboinicus, Conyzabonariensis and Cymbopongoncitratus tinctures and Listerine® did not show inhibitory action against the tested biofilm-forming bacteria.

  2. Confocal microscopy evaluation of the effect of irrigants on Enterococcus faecalis biofilm: An in vitro study.

    Science.gov (United States)

    Flach, Nicole; Böttcher, Daiana Elisabeth; Parolo, Clarissa Cavalcanti Fatturi; Firmino, Luciana Bitello; Malt, Marisa; Lammers, Marcelo Lazzaron; Grecca, Fabiana Soares

    2016-01-01

    The purpose of this study was to evaluate in vitro the effectiveness of two endodontic irrigants and their association against Enterococcus faecalis (E. faecalis) by confocal laser scanning microscope (CLSM). Twenty-four bovine incisors were inoculated in a monoculture of E. faecalis for 21 days. After this period, the teeth were divided into three test groups (n = 5) according to the chemical used. Group 1: 2.5% sodium hypochlorite (NaOCl), group 2: 2% chlorhexidine gel (CHX), group 3: 2.5% NaOCl + 2% CHX gel, and two control groups (n = 3): negative control group (NCG)-sterile and without root canals preparation and positive control group (PCG)-saline. Then, the samples were stained with SYTO9 and propidium iodide and subjected to analysis by CLSM. Bacterial viability was quantitatively analyzed by the proportions of dead and live bacteria in the biofilm remnants. Statistical analysis was performed by the One-way ANOVA test (p = 0.05). No statistical differences were observed to bacterial viability. According to CLSM analysis, none of the tested substances could completely eliminate E. faecalis from the root canal space. Until now, there are no irrigant solutions able to completely eliminate E. faecalis from the root canal. In this regard, the search for irrigants able to intensify the antimicrobial action is of paramount importance. © Wiley Periodicals, Inc.

  3. Disruption of Biofilms and Neutralization of Bacteria Using Hypochlorous Acid Solution: An In Vivo and In Vitro Evaluation.

    Science.gov (United States)

    Day, Anna; Alkhalil, Abdulnaser; Carney, Bonnie C; Hoffman, Hilary N; Moffatt, Lauren T; Shupp, Jeffrey W

    2017-12-01

    The aims of this study were to assess the effectiveness of a hypochlorous acid-based wound cleanser (Vashe Wound Solution [VWS], SteadMed Medical, Fort Worth, Texas) in disrupting methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa biofilms relative to other cleansers using an in vitro collagen biofilm model and to evaluate cleansers' cytotoxicity. The bioburden reduction of venous stasis wounds by VWS and another cleanser was evaluated. Plates coated with collagen films incubated with active bacteria cultures to yield biofilm mimics were treated with VWS, 1% and 10% povidone-iodine (PI), 0.05% chlorhexidine wound solution (CWS), or normal saline for 3 or 10 minutes. Biofilms were then analyzed for biomass density using a crystal violet assay, quantitative cultures, and fluorescent microscopy. Cytotoxicity was measured using neutral red uptake by primary human dermal fibroblasts. Pre- and postcleansing exudates and swab samples obtained from venous stasis wounds of patients were processed and plated on a series of selective agar plates for bacteria typing and quantification. All agents tested significantly neutralized methicillin-resistant S aureus and P aeruginosa biofilms compared with saline control as assessed by crystal violet assay and fluorescent microscopy assays. Undiluted VWS was significantly less cytotoxic compared with 1% PI, CWS, and 10% PI (in increasing order of cytotoxicity). There was no significant difference in bacterial reduction in wounds after treatment with VWS or CWS for any type of bacteria examined using selective media. In wounds that were treated with VWS or CWS, there was a similar percentage reduction in bacterial colony-forming units from precleansing levels when plated on tryptic soy agar, MacConkey, streptococcal, and mannitol salt agar plates. Plates treated with CWS trended toward higher bacterial reduction on nonselective and gram-negative agars, whereas VWS trended toward higher bacterial reduction in

  4. Effect of Commonly Prescribed Liquid Medications on Streptococcus mutans Biofilm. An in vitro study.

    Science.gov (United States)

    Clark, Samantha A; Vinson, LaQuia A; Eckert, George; Gregory, Richard L

    This study addressed the effect of pediatric liquid antibiotic medications on Streptococcus mutans UA159. These suspensions commonly contain sugars such as sucrose to make them more palatable for children. The study was designed to evaluate the effects of oral liquid antibiotics on Streptococcus mutans growth and biofilm formation. A 24 hour culture of S. mutans was treated with various concentrations of liquid medications commonly prescribed to children for odontogenic or fungal infections- amoxicillin, penicillin VK, clindamycin, and nystatin. The study was conducted in sterile 96-well flat bottom microtiter plates. The minimum inhibitory and biofilm inhibitory concentrations (MIC/MBIC) of S. mutans were determined for each medication. S. mutans was cultured with and without the test drugs, the amount of total growth measured, the biofilms washed, fixed, and stained with crystal violet. The absorbance was determined to evaluate biofilm formation. Higher concentrations of amoxicillin, penicillin VK and clindamycin had decreased biofilm and overall growth than the control. The MICs were 1:2,560 (1.95 ug/ml), 1:2,560 (1.95 ug/ml) and 1:40 (9.375 ug/ml), while the MBIC were 1:640 (7.8 ug/ml), 1:1,280 (3.9 ug/ml) and 1:20 (18.75 ug/ml), respectively. Lower concentrations provided increased biofilm and overall growth. Nystatin induced significantly more biofilm and overall growth than the control at all concentrations. At high concentrations, approximately at the levels expected to be present in the oral cavity of children, amoxicillin, penicillin, and clindamycin inhibited S. mutans biofilm and overall growth due to their antibiotic activity, while at lower concentrations the three antibiotics demonstrated an increase in biofilm and growth. The increase in S. mutans biofilm and overall growth is most likely attributed to the sugar content in the medications. Nystatin provided an increase in biofilm and growth at each concentration tested.

  5. Comparative genome analysis reveals genetic adaptation to versatile environmental conditions and importance of biofilm lifestyle in Comamonas testosteroni.

    Science.gov (United States)

    Wu, Yichao; Arumugam, Krithika; Tay, Martin Qi Xiang; Seshan, Hari; Mohanty, Anee; Cao, Bin

    2015-04-01

    Comamonas testosteroni is an important environmental bacterium capable of degrading a variety of toxic aromatic pollutants and has been demonstrated to be a promising biocatalyst for environmental decontamination. This organism is often found to be among the primary surface colonizers in various natural and engineered ecosystems, suggesting an extraordinary capability of this organism in environmental adaptation and biofilm formation. The goal of this study was to gain genetic insights into the adaption of C. testosteroni to versatile environments and the importance of a biofilm lifestyle. Specifically, a draft genome of C. testosteroni I2 was obtained. The draft genome is 5,778,710 bp in length and comprises 110 contigs. The average G+C content was 61.88 %. A total of 5365 genes with 5263 protein-coding genes were predicted, whereas 4324 (80.60 % of total genes) protein-encoding genes were associated with predicted functions. The catabolic genes responsible for biodegradation of steroid and other aromatic compounds on draft genome were identified. Plasmid pI2 was found to encode a complete pathway for aniline degradation and a partial catabolic pathway for chloroaniline. This organism was found to be equipped with a sophisticated signaling system which helps it find ideal niches and switch between planktonic and biofilm lifestyles. A large number of putative multi-drug-resistant genes coding for abundant outer membrane transporters, chaperones, and heat shock proteins for the protection of cellular function were identified in the genome of strain I2. In addition, the genome of strain I2 was predicted to encode several proteins involved in producing, secreting, and uptaking siderophores under iron-limiting conditions. The genome of strain I2 contains a number of genes responsible for the synthesis and secretion of exopolysaccharides, an extracellular component essential for biofilm formation. Overall, our results reveal the genomic features underlying the adaption

  6. The Limitations of In Vitro Experimentation in Understanding Biofilms and Chronic Infection

    DEFF Research Database (Denmark)

    Roberts, Aled E. L.; Kragh, Kasper N.; Bjarnsholt, Thomas

    2015-01-01

    We have become increasingly aware that, during infection, pathogenic bacteria often grow in multicellular biofilms that are often highly resistant to antibacterial strategies. In order to understand how biofilms form and contribute to infection, many research groups around the world have heavily...

  7. An in vitro biofilm model for enamel demineralization and antimicrobial dose-response studies

    NARCIS (Netherlands)

    Sande, F.H. van de; Azevedo, M.S.; Lund, R.G.; Huysmans, M.C.D.N.J.M.; Cenci, M.S.

    2011-01-01

    Microcosm biofilms formed in microplates have demonstrated complex community dynamics similar to natural dental biofilm. No simplified microcosm models to evaluate enamel demineralization and dose-response effect to anticariogenic therapies have yet been established, thus this study was designed to

  8. Biofilm formation on voice prostheses : Influence of dairy products in vitro

    NARCIS (Netherlands)

    Free, RH; Van der Mei, HC; Dijk, F; Van Weissenbruch, R; Busscher, HJ; Albers, FWJ

    2001-01-01

    Laryngectomized patients use silicone rubber voice prostheses to regain their speech: however. the lifetime of these devices is limited due to biofilm formation. Following anecdotal evidence. the influence of various dairy products on biofilm formation on voice prostheses was studied, using the

  9. In Vitro Evaluation of Biofilm Dispersal as a Therapeutic Strategy To Restore Antimicrobial Efficacy

    DEFF Research Database (Denmark)

    Roizman, Dan; Vidaillac, Celine; Givskov, Michael

    2017-01-01

    engineered strain PAO1/pBAD-yhjH resulted in increased antimicrobial efficacy and synergy of the imipenem-tobramycin combination. These results support the use of biofilm dispersal to enhance antimicrobial efficacy in the treatment of biofilm-associated infections, representing a promising therapeutic...

  10. Potential of a lytic bacteriophage to disrupt Acinetobacter baumannii biofilms in vitro.

    Science.gov (United States)

    Liu, Yannan; Mi, Zhiqiang; Niu, Wenkai; An, Xiaoping; Yuan, Xin; Liu, Huiying; Wang, Yong; Feng, Yuzhong; Huang, Yong; Zhang, Xianglilan; Zhang, Zhiyi; Fan, Hang; Peng, Fan; Li, Puyuan; Tong, Yigang; Bai, Changqing

    2016-10-01

    The ability of Acinetobacter baumannii to form biofilms and develop antibiotic resistance makes it difficult to control infections caused by this bacterium. In this study, we explored the potential of a lytic bacteriophage to disrupt A. baumannii biofilms. The potential of the lytic bacteriophage to disrupt A. baumannii biofilms was assessed by performing electron microscopy, live/dead bacterial staining, crystal violet staining and by determining adenosine triphosphate release. The bacteriophage inhibited the formation of and disrupted preformed A. baumannii biofilms. Results of disinfection assay showed that the lytic bacteriophage lysed A. baumannii cells suspended in blood or grown on metal surfaces. These results suggest the potential of the lytic bacteriophage to disrupt A. baumannii biofilms.

  11. Gardnerella vaginalis outcompetes 29 other bacterial species isolated from patients with bacterial vaginosis, using in an in vitro biofilm formation model.

    Science.gov (United States)

    Alves, Patrícia; Castro, Joana; Sousa, Cármen; Cereija, Tatiana B; Cerca, Nuno

    2014-08-15

    Despite the worldwide prevalence of bacterial vaginosis (BV), its etiology is still unknown. Although BV has been associated with the presence of biofilm, the ability of BV-associated bacteria to form biofilms is still largely unknown. Here, we isolated 30 BV-associated species and characterized their virulence, using an in vitro biofilm formation model. Our data suggests that Gardnerella vaginalis had the highest virulence potential, as defined by higher initial adhesion and cytotoxicity of epithelial cells, as well as the greater propensity to form a biofilm. Interestingly, we also demonstrated that most of the BV-associated bacteria had a tendency to grow as biofilms. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  12. Preparation and evaluation of lipid polymer nanoparticles for eradicating H. pylori biofilm and impairing antibacterial resistance in vitro.

    Science.gov (United States)

    Cai, Jie; Huang, Huizhi; Song, Weijuan; Hu, Haiyan; Chen, Jiesi; Zhang, Liyan; Li, Pengyu; Wu, Rui; Wu, Chuanbin

    2015-11-30

    The resistance of Helicobacter pylori to classical antimicrobial treatment has become increasingly common, whereupon biofilms are considered to play an important role in the resistance mechanism. Here 10.2% of amoxicillin (AMX) and a novel anti H. pylori adhesion material pectin sulfate (PECS) loaded lipid polymer nanoparticles (LPN) were prepared, with rhamnolipid and phospholipids as the outer mixed lipids layer (RHL-PC-LPN). The size of RHL-PC-LPN was around 200 nm, was negatively-charged, and showed sustained and complete drug release within 24h. In an in vitro study, H. pylori biofilm models were successfully established. RHL-PC-LPN, superior to PC-LPN (employing phospholipids only as the outer lipid layer), PECS+AMX (mixture of PECS and AMX) and AMX only, was proven to significantly eradicate H. pylori in the biofilm form. In accordance to our previous results, the RHL-PC-LPN group, together with the PC-LPN and PECS+AMX group, inhibited H. pylori from adhering to AGS cells. Investigating the underlying mechanisms contributing to the death of H. pylori caused by RHL-PC-LPN, we found that LPN could lower the antibiotic minimal inhibition concentration (MIC) to biofilm form from 125 μg/ml to 15.6 μg/ml. Furthermore, FITC-ConA labeled extracellular polymeric substances (EPS) were decreased in the RHL-PC-LPN group observed by a laser scanning confocal microscope. Therefore, we conclude that employing the mixed lipids of rhamnolipid and phospholipids as the outer layer of nanoparticles and PECS as the inner core produces a system capable of significantly disrupting H. pylori biofilm by eliminating the EPS as well as inhibiting the adherence and colonization of bacteria. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Meningococcal biofilm formation

    DEFF Research Database (Denmark)

    Lappann, M.; Haagensen, Janus Anders Juul; Claus, H.

    2006-01-01

    We show that in a standardized in vitro flow system unencapsulated variants of genetically diverse lineages of Neisseria meningitidis formed biofilms, that could be maintained for more than 96 h. Biofilm cells were resistant to penicillin, but not to rifampin or ciprofloxacin. For some strains, m......X alleles was identified among genetically diverse meningococcal strains. PilX alleles differed in their propensity to support autoaggregation of cells in suspension, but not in their ability to support microcolony formation within biofilms in the continuous flow system......., microcolony formation within biofilms was observed. Microcolony formation in strain MC58 depended on a functional copy of the pilE gene encoding the pilus subunit pilin, and was associated with twitching of cells. Nevertheless, unpiliated pilE mutants formed biofilms showing that attachment and accumulation...... of cells did not depend on pilus expression. Mutation and complementation analysis revealed that the type IV pilus-associated protein PilX, which was recently shown to mediate interbacterial aggregation, indirectly supported microcolony formation by contributing to pilus expression. A large number of Pil...

  14. Bacteriophage-Mediated Control of a Two-Species Biofilm Formed by Microorganisms Causing Catheter-Associated Urinary Tract Infections in an In Vitro Urinary Catheter Model

    OpenAIRE

    Lehman, Susan M.; Donlan, Rodney M.

    2014-01-01

    Microorganisms from a patient or their environment may colonize indwelling urinary catheters, forming biofilm communities on catheter surfaces and increasing patient morbidity and mortality. This study investigated the effect of pretreating hydrogel-coated silicone catheters with mixtures of Pseudomonas aeruginosa and Proteus mirabilis bacteriophages on the development of single- and two-species biofilms in a multiday continuous-flow in vitro model using artificial urine. Novel phages were pu...

  15. Clotrimazole and econazole inhibit Streptococcus mutans biofilm and virulence in vitro.

    Science.gov (United States)

    Qiu, Wei; Ren, Biao; Dai, Huanqin; Zhang, Lixin; Zhang, Qiong; Zhou, Xuedong; Li, Yuqing

    2017-01-01

    The aim of this study was to determine the inhibitory effect of eight antifungal drugs on S. mutans growth, biofilm formation and virulence factors. The actions of antifungal drugs on S. mutans were determined by recovery plates and survival kinetic curves. Biofilms were observed by scanning electron microscopy and the viable cells were recovered on BHI plates, meanwhile biofilms were stained by BacLight live/dead kit to investigate the biofilm viability. Bacteria/extracellular polysaccharides staining assays were performed to determine the EPS production of S. mutans biofilms. Acidogenicity and acidurity of S. mutans were determined using pH drop and acid tolerance assays, and the expression of ldh gene was evaluated using qPCR. We found that clotrimazole (CTR) and econazole (ECO) showed antibacterial activities on S. mutans UA159 and S. mutans clinical isolates at 12.5 and 25mg/L, respectively. CTR and ECO could also inhibit S. mutans biofilm formation and reduce the viability of preformed biofilm. CTR and ECO affected the live/dead ratio and the EPS/bacteria ratio of S. mutans biofilms. CTR and ECO also inhibited the pH drop, lactate acid production, and acid tolerance. The abilities of CTR and ECO to inhibit S. mutans ldh expression were also confirmed. We found that two antifungal azoles, CTR and ECO, had the abilities to inhibit the growth and biofilm formation of S. mutans and more importantly, they could also inhibit the virulence factors of S. mutans. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. In vitro effects of ambroxol on Cryptococcus adherence, planktonic cells, and biofilms.

    Science.gov (United States)

    Kong, Qingtao; Du, Xue; Huang, Suyang; Yang, Rui; Zhang, Chengzhen; Shen, Yongnian; Liu, Weida; Sang, Hong

    2017-07-01

    The antifungal effects of ambroxol (Amb; the metabolite VIII of bromhexine) against Cryptococcus planktonic cells and mature biofilms were investigated in this study. Amb showed antifungal activity against planktonic cells and mature biofilms. Disk diffusion test similarly showed antifungal profile for planktonic cells. Furthermore, Amb was found to be synergetic with fluconazole against planktonic cells and reduced the adherence of cells to polystyrene. Our results suggest that Amb can inhibit cryptococcal cells and biofilms, indicating its potential role in the prevention and treatment of cryptococcosis. © 2017 APMIS. Published by John Wiley & Sons Ltd.

  17. Biofilm on orthodontic retention wires : an in vitro and in vivo study

    NARCIS (Netherlands)

    Jongsma, Marije

    2015-01-01

    Orthodontische behandelingen worden steeds populairder. Om na een orthodontische behandeling het resultaat vast te houden, worden retentiedraden achter de voortanden geplaatst. Deze draden zijn heel effectief, maar hebben als nadeel dat er biofilm (tandplaque) aan de draden hecht, waardoor rondom

  18. Efficacy of natural antimicrobials in toothpaste formulations against oral biofilms in vitro

    NARCIS (Netherlands)

    Verkaik, M.J.; Busscher, H.J.; Jager, D.; Slomp, A.M.; Abbas, F.; Mei, H.C. van der

    Objectives: To evaluate the antimicrobial efficacies of two toothpaste formulations containing natural antimicrobials (herbal extracts and chitosan) against oral biofilms of different composition and maturational status. Methods: Bacteria from a buffer suspension or fresh saliva were adhered for 2 h

  19. Analysis of the Cariogenic Potential of Various Almond Milk Beverages using a Streptococcus mutans Biofilm Model in vitro.

    Science.gov (United States)

    Lee, Janelle; Townsend, Janice A; Thompson, Tatyana; Garitty, Thomas; De, Arpan; Yu, Qingzhao; Peters, Brian M; Wen, Zezhang T

    2017-12-15

    To evaluate the cariogenic properties of almond milk beverages, 6 almond milks, along with soy and whole bovine milk, were analyzed for their abilities to support Streptococcus mutans biofilm formation and acid production, and their capacity to buffer changes in pH. Biofilm formation by S. mutans was analyzed using an in vitro 96-well plate model and measured by crystal violet staining. Acid production by S. mutans was evaluated by a colorimetric L-lactate assay and pH measurement of bacterial cultures. Buffering capacity was assessed by a pH titration assay. Soy milk supported the most biofilm growth, while the least was observed with unsweetened almond milk (both p almond milk yielded the lowest pH (4.56 ± 0.66), followed by soy milk and bovine milk; the highest pH was with unsweetened almond milk (6.48 ± 0.5). When analyzed by pH titration, the unsweetened almond milk displayed the weakest buffering capacity while bovine milk showed the highest (p almond milk beverages, except those that are sweetened with sucrose, possess limited cariogenic properties, while soy milk exhibits the most cariogenic potential. As milk alternatives become increasingly popular, dentists must counsel their patients that almond milks, especially sucrose-sweetened varieties, have cariogenic potential. For patients who are lactose-intolerant or suffer from milk allergy, almond milks may be a better alternative than soy-based products. © 2017 S. Karger AG, Basel.

  20. Bacterial adhesion on direct and indirect dental restorative composite resins: An in vitro study on a natural biofilm.

    Science.gov (United States)

    Derchi, Giacomo; Vano, Michele; Barone, Antonio; Covani, Ugo; Diaspro, Alberto; Salerno, Marco

    2017-05-01

    Both direct and indirect techniques are used for dental restorations. Which technique should be preferred or whether they are equivalent with respect to bacterial adhesion is unclear. The purpose of this in vitro study was to determine the affinity of bacterial biofilm to dental restorative composite resins placed directly and indirectly. Five direct composite resins for restorations (Venus Diamond, Adonis, Optifil, Enamel Plus HRi, Clearfil Majesty Esthetic) and 3 indirect composite resins (Gradia, Estenia, Signum) were selected. The materials were incubated in unstimulated whole saliva for 1 day. The biofilms grown were collected and their bacterial cells counted. In parallel, the composite resin surface morphology was analyzed with atomic force microscopy. Both bacterial cell count and surface topography parameters were subjected to statistical analysis (α=.05). Indirect composite resins showed significantly lower levels than direct composite resins for bacterial cell adhesion, (Presins (P>.05). However, within the indirect composite resins a significantly lower level was found for Gradia than Estenia or Signum (Presin roughness and bacterial adhesion when the second and particularly the third-order statistical moments of the composite resin height distributions were considered. Indirect dental restorative composite resins were found to be less prone to biofilm adhesion than direct composite resins. A correlation of bacterial adhesion to surface morphology exists that is described by kurtosis; thus, advanced data analysis is required to discover possible insights into the biologic effects of morphology. Copyright © 2016 Editorial Council for the Journal of Prosthetic Dentistry. Published by Elsevier Inc. All rights reserved.

  1. Carbohydrate Derived Fulvic Acid: An in vitro Investigation of a Novel Membrane Active Antiseptic Agent Against Candida albicans Biofilms.

    Science.gov (United States)

    Sherry, Leighann; Jose, Anto; Murray, Colin; Williams, Craig; Jones, Brian; Millington, Owain; Bagg, Jeremy; Ramage, Gordon

    2012-01-01

    Carbohydrate derived fulvic acid (CHD-FA) is a heat stable low molecular weight, water soluble, cationic, colloidal material with proposed therapeutic properties. The aim of this study was to evaluate the antifungal activity of CHD-FA against Candida albicans, and to characterize its mode of action. A panel of C. albicans isolates (n = 50) derived from a range of clinical specimens were grown planktonically and as biofilms, and the minimum inhibitory concentrations determined. Scanning electron microscopy was performed to examine ultrastructural changes and different cell membrane assays were used to determine its mode of action. In addition, the role of C. albicans biofilm resistance mechanisms were investigated to determine their effects on CHD-FA activity. CHD-FA was active against planktonic and sessile C. albicans at concentrations 0.125 and 0.25% respectively, and was shown to be fungicidal, acting through disruption of the cell membrane activity. Resistance mechanisms, including matrix, efflux, and stress, had a limited role upon CHD-FA activity. Overall, based on the promising in vitro spectrum of activity and minimal biofilm resistance of the natural and cheap antiseptic CHD-FA, further studies are required to determine its applicability for clinical use.

  2. Gallic acid/hydroxypropyl-β-cyclodextrin complex: Improving solubility for application on in vitro/ in vivo Candida albicans biofilms.

    Directory of Open Access Journals (Sweden)

    Guilherme Rodrigues Teodoro

    Full Text Available The aim of this study was to increase the solubility of gallic acid (GA for the treatment of Candida albicans biofilm, which is very difficult to treat and requires high drug concentrations. Cyclodextrins (CDs were used for this purpose. Complexes were evaluated by phase-solubility studies, prepared by spray drying and characterized by drug loading, scanning electron microscopy (SEM and differential scanning calorimetry (DSC. The complexes were tested on C. albicans biofilm using in vitro and in vivo models. HPβCD formed soluble inclusion complexes with GA. The percentage of GA in GA/HPβCD was 10.8 ± 0.01%. The SEM and DSC analyses confirmed the formation of inclusion complexes. GA/HPβCD maintained the antimicrobial activity of the pure GA. GA/HPβCD was effective on C. albicans biofilms of 24 and 48h. The in vivo results showed an anti-inflammatory activity of GA/HPβCD with no difference in invading hypha counting among the groups. This study encourages the development of new antifungal agents.

  3. Pseudomonas aeruginosa Biofilm Infections

    DEFF Research Database (Denmark)

    Rybtke, Morten; Hultqvist, Louise Dahl; Givskov, Michael

    2015-01-01

    Studies of biopsies from infectious sites, explanted tissue and medical devises have provided evidence that biofilms are the underlying cause of a variety of tissue-associated and implant-associated recalcitrant human infections. With a need for novel anti-biofilm treatment strategies, research...... in biofilm infection microbiology, biofilm formation mechanisms and biofilm-associated antimicrobial tolerance has become an important area in microbiology. Substantial knowledge about biofilm formation mechanisms, biofilm-associated antimicrobial tolerance and immune evasion mechanisms has been obtained...... through work with biofilms grown in in vitro experimental setups, and the relevance of this information in the context of chronic infections is being investigated by the use of animal models of infection. Because our current in vitro experimental setups and animal models have limitations, new advanced...

  4. In vitro activities of nisin and nisin derivatives alone and in combination with antibiotics against Staphylococcus biofilms

    Directory of Open Access Journals (Sweden)

    Des eField

    2016-04-01

    activity of established biofilms treated with nisin V + chloramphenicol and nisin I4V + chloramphenicol combinations revealed a significant decrease in S. aureus SA113 and S. pseudintermedius DSM21284 biofilm viability respectively compared to the nisin A + antibiotic combinations as determined by the rapid colorimetric XTT assay. The results indicate that the activities of the nisin derivative and antibiotic combinations represent a significant improvement over that of the wild-type nisin and antibiotic combination and merit further investigation with a view to their use as anti-biofilm agents.

  5. In Vitro Effects of Polyphosphate against Prevotella intermedia in Planktonic Phase and Biofilm.

    Science.gov (United States)

    Jang, Eun-Young; Kim, Minjung; Noh, Mi Hee; Moon, Ji-Hoi; Lee, Jin-Yong

    2016-02-01

    Polyphosphate (polyP) has gained a wide interest in the food industry due to its potential as a decontaminating agent. In this study, we examined the effect of sodium tripolyphosphate (polyP3; Na5P3O10) against planktonic and biofilm cells of Prevotella intermedia, a major oral pathogen. The MIC of polyP3 against P. intermedia ATCC 49046 determined by agar dilution method was 0.075%, while 0.05% polyP3 was bactericidal against P. intermedia in time-kill analysis performed using liquid medium. A crystal violet binding assay for the assessment of biofilm formation by P. intermedia showed that sub-MICs of polyP3 significantly decreased biofilm formation. Under the scanning electron microscope, decreased numbers of P. intermedia cells forming the biofilms were observed when the bacterial cells were incubated with 0.025% or higher concentrations of polyP3. Assessment of biofilm viability with LIVE/DEAD staining and viable cell count methods showed that 0.05% or higher concentrations of polyP3 significantly decreased the viability of the preformed biofilms in a concentration-dependent manner. The zone sizes of alpha-hemolysis formed on horse blood agar produced by P. intermedia were decreased in the presence of polyP3. The expression of the genes encoding hemolysins and the genes of the hemin uptake (hmu) locus was downregulated by polyP3. Collectively, our results show that polyP is an effective antimicrobial agent against P. intermedia in biofilms as well as planktonic phase, interfering with the process of hemin acquisition by the bacterium. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  6. Metabolic differentiation of surface and invasive cells of yeast colony biofilms revealed by gene expression profiling

    Czech Academy of Sciences Publication Activity Database

    Maršíková, J.; Wilkinson, D.; Hlaváček, Otakar; Gilfillan, G.D.; Mizeranschi, A.; Hughes, T.; Begany, Markéta; Rešetárová, Stanislava; Váchová, Libuše; Palková, Z.

    2017-01-01

    Roč. 18, OCT 23 (2017), s. 814 ISSN 1471-2164 R&D Projects: GA MŠk(CZ) 7F14083; GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : Saccharomyces cerevisiae * Colony biofilms * Cell differentiation Subject RIV: EE - Microbiology, Virology Impact factor: 3.729, year: 2016

  7. Osteopontin reduces biofilm formation in a multi-species model of dental biofilm.

    Directory of Open Access Journals (Sweden)

    Sebastian Schlafer

    Full Text Available BACKGROUND: Combating dental biofilm formation is the most effective means for the prevention of caries, one of the most widespread human diseases. Among the chemical supplements to mechanical tooth cleaning procedures, non-bactericidal adjuncts that target the mechanisms of bacterial biofilm formation have gained increasing interest in recent years. Milk proteins, such as lactoferrin, have been shown to interfere with bacterial colonization of saliva-coated surfaces. We here study the effect of bovine milk osteopontin (OPN, a highly phosphorylated whey glycoprotein, on a multispecies in vitro model of dental biofilm. While considerable research effort focuses on the interaction of OPN with mammalian cells, there are no data investigating the influence of OPN on bacterial biofilms. METHODOLOGY/PRINCIPAL FINDINGS: Biofilms consisting of Streptococcus oralis, Actinomyces naeslundii, Streptococcus mitis, Streptococcus downei and Streptococcus sanguinis were grown in a flow cell system that permitted in situ microscopic analysis. Crystal violet staining showed significantly less biofilm formation in the presence of OPN, as compared to biofilms grown without OPN or biofilms grown in the presence of caseinoglycomacropeptide, another phosphorylated milk protein. Confocal microscopy revealed that OPN bound to the surface of bacterial cells and reduced mechanical stability of the biofilms without affecting cell viability. The bacterial composition of the biofilms, determined by fluorescence in situ hybridization, changed considerably in the presence of OPN. In particular, colonization of S. mitis, the best biofilm former in the model, was reduced dramatically. CONCLUSIONS/SIGNIFICANCE: OPN strongly reduces the amount of biofilm formed in a well-defined laboratory model of acidogenic dental biofilm. If a similar effect can be observed in vivo, OPN might serve as a valuable adjunct to mechanical tooth cleaning procedures.

  8. Osteopontin reduces biofilm formation in a multi-species model of dental biofilm.

    Science.gov (United States)

    Schlafer, Sebastian; Raarup, Merete K; Wejse, Peter L; Nyvad, Bente; Städler, Brigitte M; Sutherland, Duncan S; Birkedal, Henrik; Meyer, Rikke L

    2012-01-01

    Combating dental biofilm formation is the most effective means for the prevention of caries, one of the most widespread human diseases. Among the chemical supplements to mechanical tooth cleaning procedures, non-bactericidal adjuncts that target the mechanisms of bacterial biofilm formation have gained increasing interest in recent years. Milk proteins, such as lactoferrin, have been shown to interfere with bacterial colonization of saliva-coated surfaces. We here study the effect of bovine milk osteopontin (OPN), a highly phosphorylated whey glycoprotein, on a multispecies in vitro model of dental biofilm. While considerable research effort focuses on the interaction of OPN with mammalian cells, there are no data investigating the influence of OPN on bacterial biofilms. Biofilms consisting of Streptococcus oralis, Actinomyces naeslundii, Streptococcus mitis, Streptococcus downei and Streptococcus sanguinis were grown in a flow cell system that permitted in situ microscopic analysis. Crystal violet staining showed significantly less biofilm formation in the presence of OPN, as compared to biofilms grown without OPN or biofilms grown in the presence of caseinoglycomacropeptide, another phosphorylated milk protein. Confocal microscopy revealed that OPN bound to the surface of bacterial cells and reduced mechanical stability of the biofilms without affecting cell viability. The bacterial composition of the biofilms, determined by fluorescence in situ hybridization, changed considerably in the presence of OPN. In particular, colonization of S. mitis, the best biofilm former in the model, was reduced dramatically. OPN strongly reduces the amount of biofilm formed in a well-defined laboratory model of acidogenic dental biofilm. If a similar effect can be observed in vivo, OPN might serve as a valuable adjunct to mechanical tooth cleaning procedures.

  9. Set potential regulation reveals additional oxidation peaks of Geobacter sulfurreducens anodic biofilms

    KAUST Repository

    Zhu, Xiuping

    2012-08-01

    Higher current densities produced in microbial fuel cells and other bioelectrochemical systems are associated with the presence of various Geobacter species. A number of electron transfer components are involved in extracellular electron transfer by the model exoelectrogen, Geobacter sulfurreducens. It has previously been shown that 5 main oxidation peaks can be identified in cyclic voltammetry scans. It is shown here that 7 separate oxidation peaks emerged over relatively long periods of time when a larger range of set potentials was used to acclimate electroactive biofilms. The potentials of oxidation peaks obtained with G. sulfurreducens biofilms acclimated at 0.60 V (vs. Ag/AgCl) were different from those that developed at - 0.46 V, and both of their peaks were different from those obtained for biofilms incubated at - 0.30 V, 0 V, and 0.30 V. These results expand the known range of potentials for which G. sulfurreducens produces identifiable oxidation peaks that could be important for extracellular electron transfer. © 2012 Elsevier B.V.

  10. Glass ionomer cement inhibits secondary caries in an in vitro biofilm model.

    Science.gov (United States)

    Krämer, Norbert; Schmidt, Miriam; Lücker, Susanne; Domann, Eugen; Frankenberger, Roland

    2017-07-24

    The objective of this study was to investigate the effect of different glass ionomer cements on secondary caries inhibition in a fully automated in vitro biofilm model. One hundred and twenty-four extracted third molars received class V cavities and were filled with one conventional (Ketac Molar/KM), and two resin-modified glass ionomer cements (Photac Fil/PF, Ketac N100/KN, 3M Espe). A bonded resin composite (Single Bond Plus/Filtek Supreme XTE) served as control. After 14 days water storage at 37 °C, specimens were thermocycled (10,000 × 5/55 °C). Over a period of 10 days, specimens were subjected to cariogenic challenge for 3/4/6 h/day. Demineralization was caused by Streptococcus mutans (DSM 20523) alternatingly being rinsed over specimens using artificial saliva. After biological loading, teeth were cut longitudinally and demineralization depths were evaluated at the margins and at a distance of 0.5 mm from the margins using fluorescence microscopy. Marginal quality was investigated under a SEM at ×200 magnification. Four-hour demineralization depths were for enamel margins (EM), enamel (E), dentin margin (DM), and dentin (D) (μm ± SD): KM: EM 12 ± 8, E 33 ± 7, DM 56 ± 11, D 79 ± 6; PF: EM 19 ± 13, E 34 ± 13, DM 53 ± 10, D 77 ± 12; and KN: EM 26 ± 5, E 38 ± 6, DM 57 ± 11, D 71 ± 7. For all glass ionomer cements (GICs), demineralization depth at the margins was less compared to 0.5 mm distance, with demineralization depth having been correlated to duration of cariogenic challenge (ANOVA [mod. LSD, p Glass ionomer cements have a higher secondary caries inhibiting effect than resin composites.

  11. Klebsiella pneumoniae Planktonic and Biofilm Reduction by Different Plant Extracts: In Vitro Study

    Directory of Open Access Journals (Sweden)

    Lucas De Paula Ramos

    2016-01-01

    Full Text Available This study evaluated the action of Pfaffia paniculata K., Juglans regia L., and Rosmarius officinalis L. extracts against planktonic form and biofilm of Klebsiella pneumoniae (ATCC 4352. Minimum inhibitory concentration (MIC and minimum microbicidal concentration (MMC values were determined for each extract by microdilution broth method, according to Clinical and Laboratory Standards Institute. Next, antimicrobial activity of the extracts on biofilm was analyzed. For this, standardized suspension at 107 UFC/mL of K. pneumoniae was distributed into 96-well microplates (n=10 and after 48 h at 37°C and biofilm was subjected to treatment for 5 min with the extracts at a concentration of 200 mg/mL. ANOVA and Tukey tests (5% were used to verify statistical significant reduction (p<0.05 of planktonic form and biofilm. P paniculata K., R. officinalis L., and J. regia L. showed reductions in biomass of 55.6, 58.1, and 18.65% and cell viability reduction of 72.4, 65.1, and 31.5%, respectively. The reduction obtained with P. paniculata and R. officinalis extracts was similar to the reduction obtained with chlorhexidine digluconate 2%. In conclusion, all extracts have microbicidal action on the planktonic form but only P. paniculata K. and R. officinalis L. were effective against biofilm.

  12. Inside-out Ultraviolet-C Sterilization of Pseudomonas aeruginosa Biofilm In Vitro.

    Science.gov (United States)

    Jones, Cameron C; Valdeig, Steffi; Sova, Raymond M; Weiss, Clifford R

    2016-11-01

    Biofilms are difficult to eradicate due to a protective architecture and create major challenges in patient care by diminishing both host immune response and therapeutic approaches. This study investigated a new strategy for treating surface-attached biofilms by delivering germicidal UV through a material surface in a process referred to as "inside-out sterilization" (IOS). Mature Pseudomonas aeruginosa (ATCC® 27853™ ) biofilms were irradiated with up to 1400 mJ cm-2 of germicidal UV from both ambient and IOS configurations. The lethal dose for the ambient exposure group was 461 mJ cm-2 95% CI [292, 728] compared to the IOS treatment group of 247 mJ cm-2 95% CI [187, 325], corresponding to 47% less UV dosage for the IOS group (P < 0.05). This study demonstrated that with IOS, a lower quantal dosage of UV energy is required to eradicate biofilm than with ambient exposure by leveraging the organizational structure of the biofilm. © 2016 The American Society of Photobiology.

  13. Characterization and evolution of natural aquatic biofilm communities exposed in vitro to herbicides.

    Science.gov (United States)

    Bricheux, Geneviève; Le Moal, Gwenaël; Hennequin, Claire; Coffe, Gérard; Donnadieu, Florence; Portelli, Christophe; Bohatier, Jacques; Forestier, Christiane

    2013-02-01

    River biofilms are assemblies of autotrophic and heterotrophic microorganisms that can be affected by pollutants such as those found in watersheds and wastewater treatment plants. In the laboratory, experimental biofilms were formed from river water, and their overall composition was investigated. Denaturing gradient gel electrophoresis (DGGE) and cytometry were used to assess the richness and diversity of these communities. The software Cytostack (available on request) was developed to treat and analyze the cytometric data. Measurements of chlorophyll-a and carotenoids were used to assess the global composition of the photoautotrophic community, whereas proteins, polysaccharides (PS) content, and esterase activities were used to assess overall changes in the mixed communities. We evaluated the effects that 3 weeks of treatment with the herbicides diuron and glyphosate (10 μg L(-1)) had on these biofilms. Exposed to diuron, bacterial communities adapted, changing their composition. Glyphosate inhibited growth of one autotrophic community but caused no chlorophyll deficit. As a whole, the biofilm acted as a micro-ecosystem, able to regulate and maintain a constant level of photosynthetic pigment through the structural adaptation of the autotrophic community. These results are one more proof that microbial diversity of aquatic biofilms is influenced by chemical stresses, potentially leading to disturbances within the ecosystems. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. The Biofilm Challenge

    DEFF Research Database (Denmark)

    Alhede, Maria; Alhede, Morten

    2014-01-01

    The concept of biofilms has emerged in the clinical setting during the last decade. Infections involving biofilms have been documented in all parts of the human body, and it is currently believed that the presence of biofilm-forming bacteria is equivalent to chronic infection. A quick Pubmed search...... reveals the significance of biofilms, as evidenced by a dramatic increase in scientific publications on the topic, as well as in publications concerning wounds with biofilms, which reached 600 publications in 2013. Judged from the number of publications, it appears that biofilms play a significant role...... in wounds. However, the impact of biofilms is often debated, because infected wounds were also treated before the concept of biofilms was coined. In this short review, we will address the significance of biofilms and their role in wounds, and discuss the future tasks of the biofilm challenge....

  15. In vitro anti-biofilm activity of Quercus brantii subsp. persica on human pathogenic bacteria

    Directory of Open Access Journals (Sweden)

    Z. Bahar

    2017-01-01

    Full Text Available Background and objectives: Quercus brantii subsp. persica is used in folk medicine to treat infections in Iran. There is not available report on the anti-biofilm activity of Quercus brantii subsp.  persica. The aim of the present study was to investigate the effects of Quercus brantii subsp. persica against bacterial biofilms. Methods: Eighty biofilm producing strains of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Pseudomonas aeruginosa were collected. Quercus brantii subsp. persica fruits aqueous extraction (QBAE was prepared though maceration method. Chemical analysis to distinguish the main components of the QBAE was carried out using thin-layer chromatography. The antibacterial effects of QBAE on bacterial isolates were determined by the Kirby-Bauer and broth microdilution methods. The antibiofilm effects of QBAE on bacterial isolates were determined using a microtiter assay. Results: The Quercus brantii subsp. persica exhibited bacterial growth inhibition and bactericidal activity on the majority of the strains at concentrations between 0.2 and 1.2 mg/mL. The average of biofilm formation inhibition by Quercus brantii subsp. persica at a minimum inhibitory concentration MIC50 in Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis and Staphylococcus aureus strains were 35%, 45%, 57% and 61%, respectively. coumarins, phenols, terpenes and steroids were found in the QBAE by TLC. Conclusion: The results showed that Quercus brantii subsp. persica aqueous extraction was effective against the tested microorganisms and showed anti-biofilm activity which can be a basis for future studies to investigate for new anti-biofilm drugs.

  16. In Vitro Efficacy of Continuous Mild High Temperature on the Biofilm Formation of Aspergillus Niger.

    Science.gov (United States)

    Zeng, Rong; Tong, Jian Bo; Liu, Yu Zhen; Chen, Qing; Lin, Tong; Li, Min; Lü, Gui Xia

    2017-12-20

    Objective To investigate whether continuous mild high temperature (increased temperature without causing significant damage to host cells) can inhibit the biofilm formation of Aspergillus niger (A.niger) and its vitality.Methods A.niger biofilms were formed on a coverslip in 24-well tissue culture plate and were checked at the time points 4,8,10,16,24,48 and 72 hours.Confocal laser scanning microscopy (CLSM) was used to image and quantify A.niger biofilm formation under three different continuous mild high temperatures at 37℃,39℃,and 41℃.Furthermore,2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay was used to quantify the dynamic growth of A.niger biofilm under the above conditions.Results Compared with the culture condition 37℃,CLSM analysis at 39℃ or 41℃ showed that higher temperature induced later germination at 4 hours (t=8.603,P=0.047;t=14.550,P=0.008),poorer hyphal elongation at 8 hours(t=35.118,P=0.039;t=63.450,P=0.006),poorer polar growth,and reduced biofilm thickness from 10 to 24 hours.The XTT assay showed that higher temperature (39℃ or 41℃) lead to lower vitality at 10 hours,higher vitality at 16 hours,but finally lower vitality from 24 to 72 hours (t=24.262,P=0.038;t=7.556,P=0.031).Conclusion Continuous mild high temperature may have a negative regulatory effect on biofilm formation of A.niger and its vitality.

  17. The European Confederation of Medical Mycology (ECMM) survey of candidaemia in Italy: in vitro susceptibility of 375 Candida albicans isolates and biofilm production.

    Science.gov (United States)

    Tortorano, Anna Maria; Prigitano, Anna; Biraghi, Emanuela; Viviani, Maria Anna

    2005-10-01

    To investigate the in vitro antifungal susceptibility pattern of 375 Candida albicans bloodstream isolates recovered during the European Confederation of Medical Mycology survey of candidaemia performed in Lombardia, Italy and to test the ability to form biofilm. In vitro susceptibility to flucytosine, fluconazole, itraconazole, posaconazole, voriconazole and caspofungin was performed by broth microdilution following the NCCLS guidelines. Biofilm production was measured using the XTT reduction assay in 59 isolates selected as representative of different patterns of susceptibility to flucytosine and azoles. MICs (mg/L) at which 90% of the strains were inhibited were < or =0.25 for flucytosine, 0.25 for caspofungin, 4 for fluconazole and 0.06 for itraconazole, voriconazole and posaconazole. Flucytosine resistance was detected in five isolates and was associated with serotype B in 2/29 and serotype A in 3/346. Resistance to fluconazole was detected in 10 isolates; nine of these exhibited reduced susceptibility to the other azoles. Among the 10 patients with fluconazole-resistant C. albicans bloodstream infection, only one, an AIDS patient, had been previously treated with fluconazole. Biofilm production was observed in 23 isolates (39%) and was significantly associated with serotype B. No relationship was detected with the pattern of antifungal susceptibility. Resistance is uncommon in C. albicans isolates recovered from blood cultures, while biofilm production is a relatively frequent event. Periodic surveillance is warranted to monitor the incidence of in vitro antifungal resistance as well as of biofilm production.

  18. Properties of silver and copper nanoparticle-containing aqueous solutions and evaluation of their in vitro activity against Candida albicans and Staphylococcus aureus biofilms

    Science.gov (United States)

    Montes Aguirre, Melissa Mariluz

    Most microorganisms grow on surfaces as biofilms rather than as individual planktonic cells, and cells within biofilms show high levels of resistance against antimicrobial drugs. Thereby biofilm formation complicates treatment and contributes to high morbidity and mortality rates associated with infections. This study explores the physical, optical, and nano-structural properties of selected nanoparticles dispersed in aqueous solutions (nanoparticulate colloidal water or nanofluids) and examines their in vitro activity against microbial biofilms. Silver and copper nanofluids of various concentrations were prepared and studied. Their surface energies, surface charge and surface plasmonic resonance properties were obtained using contact angle measurement, zeta potential and optical spectrometer, respectively. The temperature dependence of the surface plasmon resonance behavior was also determined for the selected nanoparticulate aqueous solutions. A model of biofilm formation on the wells of microtiter plates was used to determine the in vitro activity of the nanoparticle preparations against both fungal (Candida albicans) and bacterial (Staphylococcus aureus) biofilms. Scanning electron microscopy (SEM) was used to observe the nanoparticle interactions with microbial cells. Results show that silver nanofluid has higher surface energy than that of the copper, the surface energy increases as the concentration of silver nanoparticles increases; and both nanoparticles in liquid are positively charged. The interaction between silver nanoparticles and water molecules produces notable changes on the usual temperature properties of water. Altogether, effectiveness of silver nanoparticle-containing liquids in controlling biofilm formation is observed and reported. For a given size of silver nanoparticles studied, it is found that the effective concentrations of silver nanoparticles against microbial biofilms are far lower than their cytotoxic concentrations, indicating an

  19. Biofilm formation of Pasteurella multocida on bentonite clay.

    Science.gov (United States)

    Rajagopal, Ramachandranpillai; Nair, Govindapillai Krishnan; Mini, Mangattumuruppel; Joseph, Leo; Saseendranath, Mapranath Raghavan; John, Koshy

    2013-06-01

    Biofilms are structural communities of bacterial cells enshrined in a self produced polymeric matrix. The studies on biofilm formation of Pasteurella multocida have become imperative since it is a respiratory pathogen and its biofilm mode could possibly be one of its virulence factors for survival inside a host. The present study describes a biofilm assay for P. multocida on inert hydrophilic material called bentonite clay. The potential of the organism to form in vitro biofilm was assessed by growing the organism under nutrient restriction along with the inert substrate bentonite clay, which will provide a surface for attachment. For quantification of biofilm, plate count by the spread plate method was employed. Capsule production of the attached bacteria was demonstrated by light microscopic examination following Maneval staining and capsular polysaccharide estimation was done using standard procedures. The biofilm formation peaked on the third day of incubation (1.54 ×10(6) cfu/g of bentonite clay) while the planktonic cells were found to be at a maximum on day one post inoculation (8.10 ×10(8) cfu/ml of the broth). Maneval staining of late logarithmic phase biofilm cultures revealed large aggregates of bacterial cells, bacteria appearing as chains or as a meshwork. The capsular polysaccharide estimation of biofilm cells revealed a 3.25 times increase over the planktonic bacteria. The biofilm cells cultured on solid media also produced some exclusive colony morphotypes.

  20. Bacteriophage-mediated control of a two-species biofilm formed by microorganisms causing catheter-associated urinary tract infections in an in vitro urinary catheter model.

    Science.gov (United States)

    Lehman, Susan M; Donlan, Rodney M

    2015-02-01

    Microorganisms from a patient or their environment may colonize indwelling urinary catheters, forming biofilm communities on catheter surfaces and increasing patient morbidity and mortality. This study investigated the effect of pretreating hydrogel-coated silicone catheters with mixtures of Pseudomonas aeruginosa and Proteus mirabilis bacteriophages on the development of single- and two-species biofilms in a multiday continuous-flow in vitro model using artificial urine. Novel phages were purified from sewage, characterized, and screened for their abilities to reduce biofilm development by clinical isolates of their respective hosts. Our screening data showed that artificial urine medium (AUM) is a valid substitute for human urine for the purpose of evaluating uropathogen biofilm control by these bacteriophages. Defined phage cocktails targeting P. aeruginosa and P. mirabilis were designed based on the biofilm inhibition screens. Hydrogel-coated catheters were pretreated with one or both cocktails and challenged with approximately 1×10(3) CFU/ml of the corresponding pathogen(s). The biofilm growth on the catheter surfaces in AUM was monitored over 72 to 96 h. Phage pretreatment reduced P. aeruginosa biofilm counts by 4 log10 CFU/cm2 (P≤0.01) and P. mirabilis biofilm counts by >2 log10 CFU/cm2 (P≤0.01) over 48 h. The presence of P. mirabilis was always associated with an increase in lumen pH from 7.5 to 9.5 and with eventual blockage of the reactor lines. The results of this study suggest that pretreatment of a hydrogel urinary catheter with a phage cocktail can significantly reduce mixed-species biofilm formation by clinically relevant bacteria. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  1. In vitro and In vivo efficacy of an LpxC inhibitor, CHIR-090, alone or combined with colistin against Pseudomonas aeruginosa biofilm

    DEFF Research Database (Denmark)

    Tan, Jun Hou; Vidaillac, Celine; Yam, Joey Kuok Hoong

    2017-01-01

    With the rapid spread of antimicrobial resistance in Gram-negative pathogens, biofilm-associated infections are increasingly harder to treat and combination therapy with colistin has become one of the most efficient therapeutic strategies. Our study aimed to evaluate the potential for the synergy...... of colistin combined with CHIR-090, a potent LpxC inhibitor, against in vitro and in vivo Pseudomonas aeruginosa biofilms. Four P. aeruginosa isolates with various colistin susceptibilities were chosen for evaluation. The tested isolates of P. aeruginosa exhibited MIC values ranging from 1 to 64 and 0...... highlights the in vivo and in vitro synergistic activity of the colistin and CHIR-090 combination against both colistin-susceptible and -nonsusceptible P. aeruginosa biofilms. Further studies are warranted to investigate the clinical relevance of the combination of these two antimicrobials and outline...

  2. Sodium chloride and potassium sorbate : a synergistic combination against Enterococcus faecalis biofilms: an in vitro study

    NARCIS (Netherlands)

    van der Waal, Suzette V.; Jiang, Lei-Meng; de Soet, Johannes J.; van der Sluis, Lucas W. M.; Wesselink, Paul R.; Crielaard, Wim

    2012-01-01

    Incomplete disinfection of the root canal system is a major cause of post-treatment disease. This study aimed to investigate the disinfecting property of organic acid salts and sodium chloride (NaCl), in a double-hurdle strategy, on Enterococcus faecalis biofilms. First of all, the high-throughput

  3. Sodium chloride and potassium sorbate: a synergistic combination against Enterococcus faecalis biofilms: an in vitro study

    NARCIS (Netherlands)

    van der Waal, S.V.; Jiang, L.M.; de Soet, J.J.; van der Sluis, L.W.M.; Wesselink, P.R.; Crielaard, W.

    2012-01-01

    Incomplete disinfection of the root canal system is a major cause of post-treatment disease. This study aimed to investigate the disinfecting property of organic acid salts and sodium chloride (NaCl), in a double-hurdle strategy, on Enterococcus faecalis biofilms. First of all, the high-throughput

  4. Developing an in vitro artificial sebum model to study Propionibacterium acnes biofilms.

    Science.gov (United States)

    Spittaels, Karl-Jan; Coenye, Tom

    2017-11-22

    The aim of the present study was to develop a new model system to study Propionibacterium acnes biofilms. This model should be representative for the conditions encountered in the pilosebaceous unit. The new model, consists of an artificial sebum pellet supported by a silicone disc. Sebum pellets were inoculated with various P. acnes strains isolated from both normal and acneic skin. Growth and biofilm formation was verified by conventional plating at different time points, as well as by resazurin assays and fluorescence microscopy after LIVE/DEAD staining. The artificial sebum pellets were also used in assays to measure the production of certain virulence factors implicated in the pathogenesis of acne, including lipase, protease and the presence of CAMP factors. The artificial sebum model can sustain biofilm growth of P. acnes, as was determined by increasing CFU counts for up to 1 week after inoculation. Metabolic activity and biofilm formation were confirmed using resazurin staining and fluorescence microscopy respectively. The production of virulence factors in this model was demonstrated as well. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Oral biofilm models for mechanical plaque removal

    NARCIS (Netherlands)

    Verkaik, Martinus J.; Busscher, Henk J.; Rustema-Abbing, Minie; Slomp, Anje M.; Abbas, Frank; van der Mei, Henny C.

    In vitro plaque removal studies require biofilm models that resemble in vivo dental plaque. Here, we compare contact and non-contact removal of single and dual-species biofilms as well as of biofilms grown from human whole saliva in vitro using different biofilm models. Bacteria were adhered to a

  6. The effects of hyperosmosis or high pH on a dual-species biofilm of Enterococcus faecalis and Pseudomonas aeruginosa : an in vitro study

    NARCIS (Netherlands)

    van der Waal, S. V.; van der Sluis, L. W. M.; Ozok, A. R.; Exterkate, R. A. M.; van Marle, J.; Wesselink, P. R.; de Soet, J. J.

    2011-01-01

    van der Waal SV, van der Sluis LWM, Ozok AR, Exterkate RAM, van Marle J, Wesselink PR, de Soet JJ. The effects of hyperosmosis or high pH on a dual-species biofilm of Enterococcus faecalis and Pseudomonas aeruginosa: an in vitro study. International Endodontic Journal, 44, 11101117, 2011. Aim To

  7. In Vitro Activity of Miltefosine against Candida albicans under Planktonic and Biofilm Growth Conditions and In Vivo Efficacy in a Murine Model of Oral Candidiasis.

    Science.gov (United States)

    Vila, Taissa Vieira Machado; Chaturvedi, Ashok K; Rozental, Sonia; Lopez-Ribot, Jose L

    2015-12-01

    The generation of a new antifungal against Candida albicans biofilms has become a major priority, since biofilm formation by this opportunistic pathogenic fungus is usually associated with an increased resistance to azole antifungal drugs and treatment failures. Miltefosine is an alkyl phospholipid with promising antifungal activity. Here, we report that, when tested under planktonic conditions, miltefosine displays potent in vitro activity against multiple fluconazole-susceptible and -resistant C. albicans clinical isolates, including isolates overexpressing efflux pumps and/or with well-characterized Erg11 mutations. Moreover, miltefosine inhibits C. albicans biofilm formation and displays activity against preformed biofilms. Serial passage experiments confirmed that miltefosine has a reduced potential to elicit resistance, and screening of a library of C. albicans transcription factor mutants provided additional insight into the activity of miltefosine against C. albicans growing under planktonic and biofilm conditions. Finally, we demonstrate the in vivo efficacy of topical treatment with miltefosine in the murine model of oropharyngeal candidiasis. Overall, our results confirm the potential of miltefosine as a promising antifungal drug candidate, in particular for the treatment of azole-resistant and biofilm-associated superficial candidiasis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  8. Role of interspecies interactions in dual-species biofilms developed in vitro by uropathogens isolated from polymicrobial urinary catheter-associated bacteriuria.

    Science.gov (United States)

    Galván, E M; Mateyca, C; Ielpi, L

    2016-10-01

    Most catheter-associated urinary tract infections are polymicrobial. Here, uropathogen interactions in dual-species biofilms were studied. The dual-species associations selected based on their prevalence in clinical settings were Klebsiella pneumoniae-Escherichia coli, E. coli-Enterococcus faecalis, K. pneumoniae-E. faecalis, and K. pneumoniae-Proteus mirabilis. All species developed single-species biofilms in artificial urine. The ability of K. pneumoniae to form biofilms was not affected by E. coli or E. faecalis co-inoculation, but was impaired by P. mirabilis. Conversely, P. mirabilis established a biofilm when co-inoculated with K. pneumoniae. Additionally, E. coli persistence in biofilms was hampered by K. pneumoniae but not by E. faecalis. Interestingly, E. coli, but not K. pneumoniae, partially inhibited E. faecalis attachment to the surface and retarded biofilm development. The findings reveal bacterial interactions between uropathogens in dual-species biofilms ranged from affecting initial adhesion to outcompeting one bacterial species, depending on the identity of the partners involved.

  9. Inhibitory effect on in vitro Streptococcus oralis biofilm of a soda-lime glass containing silver nanoparticles coating on titanium alloy.

    Directory of Open Access Journals (Sweden)

    Belén Cabal

    Full Text Available This paper reports the effect of soda-lime-glass-nAg coating on the viability of an in vitro biofilm of Streptococcus oralis. Three strains (ATCC 35037 and two clinical isolates from periodontitis patients were grown on coated with glass, glass containing silver nanoparticles, and uncoated titanium alloy disks. Two different methods were used to quantify biofilm formation abilities: crystal violet staining and determination of viable counts. The influence of the surface morphology on the cell attachment was studied. The surface morphology was characterized by scanning electron microscopy (SEM and using a profilometer. SEM was also used to study the formation and the development of biofilm on the coated and uncoated disks. At least a >99.7% inocula reduction of biofilm respect to titanium disks and also to glass coated disks was observed in the glass-nAg coated disks for all the studied strains. A quantitative evaluation of the release of silver was conducted in vitro to test whether and to what extend the biocidal agent (silver could leach from the coating. These findings suggest that the biofilm formation of S. oralis strains is highly inhibited by the glass-nAg and may be useful for materials which require durable antibacterial effect on their surfaces, as it is the case of dental implants.

  10. Development of In Vitro Denture Biofilm Models for Halitosis Related Bacteria and their Application in Testing the Efficacy of Antimicrobial Agents

    Science.gov (United States)

    Wu, Tingxi; He, Xuesong; Lu, Hongyang; Bradshaw, David J; Axe, Alyson; Loewy, Zvi; Liu, Honghu; Shi, Wenyuan; Lux, Renate

    2015-01-01

    Objective : Since dentures can serve as a reservoir for halitosis-causing oral bacteria, halitosis development is a concern for denture wearers. In this study, we surveyed the prevalence of four selected halitosis-related species (Fusobacterium nucleatum, Tannerella forsythia, Veillonella atypica and Klebsiella pneumoniae) in clinical denture plaque samples, and developed denture biofilm models for these species in vitro to facilitate assessment of antimicrobial treatment efficacy. Design : Denture plaque from ten healthy and ten denture stomatitis patients was screened for the presence of aforementioned four species by PCR. Biofilm formation by these halitosis-associated species on the surfaces of denture base resin (DBR) discs was evaluated by crystal violet staining and confocal laser scanning microscopy. The efficacy of denture cleanser treatment on these mono-species biofilms was evaluated by colony counting. Results : 80% of the subjects in the denture stomatitis group and 60% in the healthy group contained at least one of the targeted halitosis-related species in their denture plaque. All halitosis species tested were able to form biofilms on DBR disc surfaces to varying degrees. These in vitro mono-species resin biofilm models were used to evaluate the efficacy of denture cleansers, which exhibited differential efficacies. When forming biofilms on resin surfaces, the halitosis-related species displayed enhanced resistance to denture cleansers compared with their planktonic counterparts. Conclusion : The four selected halitosis-related bacterial species examined in this study are present on the majority of dentures. The mono-species biofilm models established on DBR discs for these species are an efficient screening tool for dental product evaluation. PMID:25926895

  11. Next generation in vitro systems for biofilm studies - a cystic fibrosis patient airway perspective

    DEFF Research Database (Denmark)

    Weiss Nielsen, Martin

    Bacterial infections have a large impact on the health of the general public, however individuals with cystic fibrosis (CF) are immensely susceptible to acquire pulmonary infections with environmental bacteria, viruses and fungal species. Ultimately pulmonary infections are the major cause......, while the ongoing immune response gradually leads to pulmonary deterioration. Physiologically different oxygen environments are present in the different compartments of the CF airways and this has a profound impact on bacterial growth and the effect of chemotherapeutics to eradicate the bacteria...... was to develop an accurate tool for growing biofilms that can mimic the cystic fibrosis airways, emulating one of the most important characteristics of the human airways, the oxygen environments. Microfluidic approaches that allow biofilm formation under controllable oxygen concentrations, and furthermore enable...

  12. In vitro antimicrobial activity of sodium hypochlorite and chlorhexidine against selected single-species biofilms.

    Science.gov (United States)

    Sena, N T; Gomes, B P F A; Vianna, M E; Berber, V B; Zaia, A A; Ferraz, C C R; Souza-Filho, F J

    2006-11-01

    To investigate the antimicrobial activity of 2.5% and 5.25% sodium hypochlorite and 2.0% chlorhexidine gel and liquid as endodontic-irrigating substances against selected single-species biofilms. Single-species biofilms of Enterococcus faecalis, Staphylococcus aureus, Candida albicans, Prevotella intermedia, Porphyromonas gingivalis, Porphyromonas endodontalis and Fusobacterium nucleatum were generated on a cellulose nitrate membrane placed on agar medium. The biofilms were then immersed in the endodontic-irrigating substances for 30 s and also for 5, 10, 15, 30 and 60 min, with and without mechanical agitation. Sterile saline was used as control. After each time period, the membrane filters were then transferred to tubes containing 2 mL of fresh broth medium plus neutralizers (in order to prevent the residual action of the tested substances). The micro-organisms were suspended using a vortex, and the inoculum was serially diluted 10-fold. Aliquots of the dilutions were plated on 5% sheep blood agar medium, and incubated under adequate gaseous conditions. Colony-forming units were calculated. The samples were compared using the Friedman and Tukey test, when necessary, at a significance level of P chlorhexidine, killed the tested micro-organisms more rapidly. Saline did not inhibit the growth of any of the tested micro-organisms, with or without agitation, being statistically different (P chlorhexidine. P. intermedia, P. gingivalis, P. endodontalis and F. nucleatum were eliminated in 30 s by all antimicrobial agents, with our without agitation, in contrast with the facultative and aerobe strains. Mechanical agitation improved the antimicrobial properties of the chemical substances tested using a biofilm model, favouring the agents in liquid presentation, especially 5.25% NaOCl and 2% chlorhexidine.

  13. Allicin from garlic inhibits the biofilm formation and urease activity of Proteus mirabilis in vitro.

    Science.gov (United States)

    Ranjbar-Omid, Mahsa; Arzanlou, Mohsen; Amani, Mojtaba; Shokri Al-Hashem, Seyyedeh Khadijeh; Amir Mozafari, Nour; Peeri Doghaheh, Hadi

    2015-05-01

    Several virulence factors contribute to the pathogenesis of Proteus mirabilis. This study determined the inhibitory effects of allicin on urease, hemolysin and biofilm of P. mirabilis ATCC 12453 and its antimicrobial activity against 20 clinical isolates of P. mirabilis. Allicin did not inhibit hemolysin, whereas it did inhibit relative urease activity in both pre-lysed (half-maximum inhibitory concentration, IC50 = 4.15 μg) and intact cells (IC50 = 21 μg) in a concentration-dependent manner. Allicin at sub-minimum inhibitory concentrations (2-32 μg mL(-1)) showed no significant effects on the growth of the bacteria (P > 0.05), but it reduced biofilm development in a concentration-dependent manner (P allicin was needed to inhibit the established biofilms. Using the microdilution technique, the MIC90 and MBC90 values of allicin against P. mirabilis isolates were determined to be 128 and 512 μg mL(-1), respectively. The results suggest that allicin could have clinical applications in controlling P. mirabilis infections. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  14. Genome-wide mutagenesis of Xanthomonas axonopodis pv. citri reveals novel genetic determinants and regulation mechanisms of biofilm formation.

    Directory of Open Access Journals (Sweden)

    Jinyun Li

    Full Text Available Xanthomonas axonopodis pv. citri (Xac causes citrus canker disease, a major threat to citrus production worldwide. Accumulating evidence suggests that the formation of biofilms on citrus leaves plays an important role in the epiphytic survival of this pathogen prior to the development of canker disease. However, the process of Xac biofilm formation is poorly understood. Here, we report a genome-scale study of Xac biofilm formation in which we identified 92 genes, including 33 novel genes involved in biofilm formation and 7 previously characterized genes, colR, fhaB, fliC, galU, gumD, wxacO, and rbfC, known to be important for Xac biofilm formation. In addition, 52 other genes with defined or putative functions in biofilm formation were identified, even though they had not previously reported been to be associated with biofilm formation. The 92 genes were isolated from 292 biofilm-defective mutants following a screen of a transposon insertion library containing 22,000 Xac strain 306 mutants. Further analyses indicated that 16 of the novel genes are involved in the production of extracellular polysaccharide (EPS and/or lipopolysaccharide (LPS, 7 genes are involved in signaling and regulatory pathways, and 5 genes have unknown roles in biofilm formation. Furthermore, two novel genes, XAC0482, encoding a haloacid dehalogenase-like phosphatase, and XAC0494 (designated as rbfS, encoding a two-component sensor protein, were confirmed to be biofilm-related genes through complementation assays. Our data demonstrate that the formation of mature biofilm requires EPS, LPS, both flagellum-dependent and flagellum-independent cell motility, secreted proteins and extracellular DNA. Additionally, multiple signaling pathways are involved in Xac biofilm formation. This work is the first report on a genome-wide scale of the genetic processes of biofilm formation in plant pathogenic bacteria. The report provides significant new information about the genetic

  15. Extracellular DNases of Ralstonia solanacearum modulate biofilms and facilitate bacterial wilt virulence.

    Science.gov (United States)

    Minh Tran, Tuan; MacIntyre, April; Khokhani, Devanshi; Hawes, Martha; Allen, Caitilyn

    2016-11-01

    Ralstonia solanacearum is a soil-borne vascular pathogen that colonizes plant xylem vessels, a flowing, low-nutrient habitat where biofilms could be adaptive. Ralstonia solanacearum forms biofilm in vitro, but it was not known if the pathogen benefits from biofilms during infection. Scanning electron microscopy revealed that during tomato infection, R. solanacearum forms biofilm-like masses in xylem vessels. These aggregates contain bacteria embedded in a matrix including chromatin-like fibres commonly observed in other bacterial biofilms. Chemical and enzymatic assays demonstrated that the bacterium releases extracellular DNA in culture and that DNA is an integral component of the biofilm matrix. An R. solanacearum mutant lacking the pathogen's two extracellular nucleases (exDNases) formed non-spreading colonies and abnormally thick biofilms in vitro. The biofilms formed by the exDNase mutant in planta contained more and thicker fibres. This mutant was also reduced in virulence on tomato plants and did not spread in tomato stems as well as the wild-type strain, suggesting that these exDNases facilitate biofilm maturation and bacterial dispersal. To our knowledge, this is the first demonstration that R. solanacearum forms biofilms in plant xylem vessels, and the first documentation that plant pathogens use DNases to modulate their biofilm structure for systemic spread and virulence. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

  16. Pyrosequencing Reveals Bacterial Communities in Unchlorinated Drinking Water Distribution System: An Integral Study of Bulk Water, Suspended Solids, Loose Deposits, and Pipe Wall Biofilm

    KAUST Repository

    Liu, G.

    2014-05-20

    The current understanding of drinking water distribution system (DWDS) microbiology is limited to pipe wall biofilm and bulk water; the contributions of particle-associated bacteria (from suspended solids and loose deposits) have long been neglected. Analyzing the composition and correlation of bacterial communities from different phases helped us to locate where most of the bacteria are and understand the interactions among these phases. In the present study, the bacteria from four critical phases of an unchlorinated DWDS, including bulk water, pipe wall biofilm, suspended solids, and loose deposits, were quantified and identified by adenosine triphosphate analysis and pyrosequencing, respectively. The results showed that the bulk water bacteria (including the contribution of suspended solids) contributed less than 2% of the total bacteria. The bacteria associated with loose deposits and pipe wall biofilm that accumulated in the DWDS accounted for over 98% of the total bacteria, and the contributions of bacteria in loose deposits and pipe wall biofilm were comparable. Depending on the amount of loose deposits, its contribution can be 7-fold higher than the pipe wall biofilm. Pyrosequencing revealed relatively stable bacterial communities in bulk water, pipe wall biofilm, and suspended solids throughout the distribution system; however, the communities present in loose deposits were dependent on the amount of loose deposits locally. Bacteria within the phases of suspended solids, loose deposits, and pipe wall biofilm were similar in phylogenetic composition. The bulk water bacteria (dominated by Polaromonas spp.) were clearly different from the bacteria from the other three phases (dominated by Sphingomonas spp.). This study highlighted that the integral DWDS ecology should include contributions from all of the four phases, especially the bacteria harbored by loose deposits. The accumulation of loose deposits and the aging process create variable microenvironments

  17. Pyrosequencing reveals bacterial communities in unchlorinated drinking water distribution system: an integral study of bulk water, suspended solids, loose deposits, and pipe wall biofilm.

    Science.gov (United States)

    Liu, G; Bakker, G L; Li, S; Vreeburg, J H G; Verberk, J Q J C; Medema, G J; Liu, W T; Van Dijk, J C

    2014-05-20

    The current understanding of drinking water distribution system (DWDS) microbiology is limited to pipe wall biofilm and bulk water; the contributions of particle-associated bacteria (from suspended solids and loose deposits) have long been neglected. Analyzing the composition and correlation of bacterial communities from different phases helped us to locate where most of the bacteria are and understand the interactions among these phases. In the present study, the bacteria from four critical phases of an unchlorinated DWDS, including bulk water, pipe wall biofilm, suspended solids, and loose deposits, were quantified and identified by adenosine triphosphate analysis and pyrosequencing, respectively. The results showed that the bulk water bacteria (including the contribution of suspended solids) contributed less than 2% of the total bacteria. The bacteria associated with loose deposits and pipe wall biofilm that accumulated in the DWDS accounted for over 98% of the total bacteria, and the contributions of bacteria in loose deposits and pipe wall biofilm were comparable. Depending on the amount of loose deposits, its contribution can be 7-fold higher than the pipe wall biofilm. Pyrosequencing revealed relatively stable bacterial communities in bulk water, pipe wall biofilm, and suspended solids throughout the distribution system; however, the communities present in loose deposits were dependent on the amount of loose deposits locally. Bacteria within the phases of suspended solids, loose deposits, and pipe wall biofilm were similar in phylogenetic composition. The bulk water bacteria (dominated by Polaromonas spp.) were clearly different from the bacteria from the other three phases (dominated by Sphingomonas spp.). This study highlighted that the integral DWDS ecology should include contributions from all of the four phases, especially the bacteria harbored by loose deposits. The accumulation of loose deposits and the aging process create variable microenvironments

  18. In Vitro Antifungal Activity of Sertraline and Synergistic Effects in Combination with Antifungal Drugs against Planktonic Forms and Biofilms of Clinical Trichosporon asahii Isolates.

    Science.gov (United States)

    Cong, Lin; Liao, Yong; Yang, Suteng; Yang, Rongya

    2016-01-01

    Trichosporon asahii (T. asahii) is the major pathogen of invasive trichosporonosis which occurred mostly in immunocompromised patients. The biofilms formation ability of T. asahii may account for resistance to antifungal drugs and results a high mortality rate. Sertraline, a commonly prescribed antidepressant, has been demonstrated to show in vitro and in vivo antifungal activities against many kinds of pathogenic fungi, especially Cryptococcus species. In the present study, the in vitro activities of sertraline alone or combined with fluconazole, voriconazole, itraconazole, caspofungin and amphotericin B against planktonic forms and biofilms of 21 clinical T. asahii isolates were evaluated using broth microdilution checkerboard method and XTT reduction assay, respectively. The fractional inhibitory concentration index (FICI) was used to interpret drug interactions. Sertraline alone exhibited antifungal activities against both T. asahii planktonic cells (MICs, 4-8 μg/ml) and T. asahii biofilms (SMICs, 16-32 μg/ml). Furthermore, SRT exhibited synergistic effects against T. asahii planktonic cells in combination with amphotericin B, caspofungin or fluconazole (FICI≤0.5) and exhibited synergistic effects against T. asahii biofilms in combination with amphotericin B (FICI≤0.5). SRT exhibited mostly indifferent interactions against T. asahii biofilms in combination with three azoles in this study. Sertraline-amphotericin B combination showed the highest percentage of synergistic effects against both T. asahii planktonic cells (90.5%) and T. asahii biofilms (81.0%). No antagonistic interaction was observed. Our study suggests the therapeutic potential of sertraline against invasive T. asahii infection, especially catheter-related T. asahii infection. Further in vivo studies are needed to validate our findings.

  19. Genome-Wide Mutagenesis of Xanthomonas axonopodis pv. citri Reveals Novel Genetic Determinants and Regulation Mechanisms of Biofilm Formation

    OpenAIRE

    Li, Jinyun; Wang, Nian

    2011-01-01

    Xanthomonas axonopodis pv. citri (Xac) causes citrus canker disease, a major threat to citrus production worldwide. Accumulating evidence suggests that the formation of biofilms on citrus leaves plays an important role in the epiphytic survival of this pathogen prior to the development of canker disease. However, the process of Xac biofilm formation is poorly understood. Here, we report a genome-scale study of Xac biofilm formation in which we identified 92 genes, including 33 novel genes inv...

  20. Effect of gaseous ozone on Enterococcus faecalis biofilm-an in vitro study.

    Science.gov (United States)

    Boch, Tanja; Tennert, Christian; Vach, Kirstin; Al-Ahmad, Ali; Hellwig, Elmar; Polydorou, Olga

    2016-09-01

    The aim of this study was to evaluate the antimicrobial effect of gaseous ozone compared to conventional methods against Enterococcus faecalis. One hundred twenty-five teeth were infected by E. faecalis and were incubated for 72 h to form biofilm. Teeth were distributed among five groups. In the first group, ozone was used; in the second group, teeth were rinsed with 20 % ethylenediaminetetraacetic acid (EDTA); in the third group, with 3 % sodium hypochlorite (NaOCl). Group 4 combined 20 % EDTA with ozone. NaOCl and ozone were combined in group 5. After treatment, the samples with paper points were taken, followed by dentin samples taken with K-file, and cultured for 24 h. Then bacterial colonies were counted. All treatments reduced significantly (p faecalis up to 91.33 %. The dentin chips showed the following: the highest CFU counts were observed in EDTA group, followed by ozone and NaOCl group. The lowest CFU counts were found in NaOCl-ozone group and EDTA-ozone group. Ozone reduced E. faecalis, even organised in a biofilm, however, lower than NaOCl. No treatment reduced totally the bacteria. Used as an adjuvant, ozone can increase the efficacy of conventional rinsing like EDTA and presents an alternative treatment when NaOCl cannot be used e.g. in teeth with a wide-open apical foramen.

  1. High in vitro antibacterial activity of Pac-525 against Porphyromonas gingivalis biofilms cultured on titanium.

    Science.gov (United States)

    Li, Ji-yin; Wang, Xue-jin; Wang, Li-na; Ying, Xiao-xia; Ren, Xiang; Liu, Hui-ying; Xu, Li; Ma, Guo-wu

    2015-01-01

    In order to investigate the potential of short antimicrobial peptides (AMPs) as alternative antibacterial agents during the treatment of peri-implantitis, the cytotoxic activity of three short AMPs, that is, Pac-525, KSL-W, and KSL, was determined using the MTT assay. The antimicrobial activity of these AMPs, ranging in concentration from 0.0039 mg/mL to 0.5 mg/mL, against the predominant planktonic pathogens, including Streptococcus sanguis, Fusobacterium nucleatum, and Porphyromonas gingivalis, involved in peri-implantitis was investigated. Furthermore, 2-day-old P. gingivalis biofilms cultured on titanium surfaces were treated with Pac-525 and subsequently observed and analysed using confocal laser scanning microscopy (CLSM). The average cell proliferation curve indicated that there was no cytotoxicity due to the three short AMPs. The minimum inhibitory concentration and minimum bactericidal concentration values of Pac-525 were 0.0625 mg/mL and 0.125 mg/mL, respectively, for P. gingivalis and 0.0078 mg/mL and 0.0156 mg/mL, respectively, for F. nucleatum. Using CLSM, we confirmed that compared to 0.1% chlorhexidine, 0.5 mg/mL of Pac-525 caused a significant decrease in biofilm thickness and a decline in the percentage of live bacteria. These data indicate that Pac-525 has unique properties that might make it suitable for the inhibition the growth of pathogenic bacteria around dental implants.

  2. A transposon mutant library of Bacillus cereus ATCC 10987 reveals novel genes required for biofilm formation and implicates motility as an important factor for pellicle-biofilm formation

    DEFF Research Database (Denmark)

    Okshevsky, Mira; Louw, Matilde Greve; Lamela, Elena Otero

    2018-01-01

    Bacillus cereus is one of the most common opportunistic pathogens causing foodborne illness, as well as a common source of contamination in the dairy industry. B. cereus can form robust biofilms on food processing surfaces, resulting in food contamination due to shedding of cells and spores. Desp...

  3. In Vitro Interactions between Non-Steroidal Anti-Inflammatory Drugs and Antifungal Agents against Planktonic and Biofilm Forms of Trichosporon asahii.

    Directory of Open Access Journals (Sweden)

    Suteng Yang

    Full Text Available Increasing drug resistance has brought enormous challenges to the management of Trichosporon spp. infections. The in vitro antifungal activities of non-steroidal anti-inflammatory drugs (NSAIDs against Candida spp. and Cryptococcus spp. were recently discovered. In the present study, the in vitro interactions between three NSAIDs (aspirin, ibuprofen and diclofenac sodium and commonly used antifungal agents (fluconazole, itraconazole, voriconazole, caspofungin and amphotericin B against planktonic and biofilm cells of T. asahii were evaluated using the checkerboard microdilution method. The spectrophotometric method and the XTT reduction assay were used to generate data on biofilm cells. The fractional inhibitory concentration index (FICI and the ΔE model were compared to interpret drug interactions. Using the FICI, the highest percentages of synergistic effects against planktonic cells (86.67% and biofilm cells (73.33% were found for amphotericin B/ibuprofen, and caspofungin/ibuprofen showed appreciable percentages (73.33% for planktonic form and 60.00% for biofilm as well. We did not observe antagonism. The ΔE model gave consistent results with FICI (86.67%. Our findings suggest that amphotericin B/ibuprofen and caspofungin/ibuprofen combinations have potential effects against T. asahii. Further in vivo and animal studies to investigate associated mechanisms need to be conducted.

  4. Unintended Laboratory-Driven Evolution Reveals Genetic Requirements for Biofilm Formation byDesulfovibrio vulgarisHildenborough.

    Science.gov (United States)

    De León, Kara B; Zane, Grant M; Trotter, Valentine V; Krantz, Gregory P; Arkin, Adam P; Butland, Gareth P; Walian, Peter J; Fields, Matthew W; Wall, Judy D

    2017-10-17

    Biofilms of sulfate-reducing bacteria (SRB) are of particular interest as members of this group are culprits in corrosion of industrial metal and concrete pipelines as well as being key players in subsurface metal cycling. Yet the mechanism of biofilm formation by these bacteria has not been determined. Here we show that two supposedly identical wild-type cultures of the SRB Desulfovibrio vulgaris Hildenborough maintained in different laboratories have diverged in biofilm formation. From genome resequencing and subsequent mutant analyses, we discovered that a single nucleotide change within DVU1017, the ABC transporter of a type I secretion system (T1SS), was sufficient to eliminate biofilm formation in D. vulgaris Hildenborough. Two T1SS cargo proteins were identified as likely biofilm structural proteins, and the presence of at least one (with either being sufficient) was shown to be required for biofilm formation. Antibodies specific to these biofilm structural proteins confirmed that DVU1017, and thus the T1SS, is essential for localization of these adhesion proteins on the cell surface. We propose that DVU1017 is a member of the lapB category of microbial surface proteins because of its phenotypic similarity to the adhesin export system described for biofilm formation in the environmental pseudomonads. These findings have led to the identification of two functions required for biofilm formation in D. vulgaris Hildenborough and focus attention on the importance of monitoring laboratory-driven evolution, as phenotypes as fundamental as biofilm formation can be altered. IMPORTANCE The growth of bacteria attached to a surface (i.e., biofilm), specifically biofilms of sulfate-reducing bacteria, has a profound impact on the economy of developed nations due to steel and concrete corrosion in industrial pipelines and processing facilities. Furthermore, the presence of sulfate-reducing bacteria in oil wells causes oil souring from sulfide production, resulting in

  5. In vitro inhibitory effects of farnesol and interactions between farnesol and antifungals against biofilms of Candida albicans resistant strains.

    Science.gov (United States)

    Xia, Jinping; Qian, Fang; Xu, Wenqian; Zhang, Zhenzhen; Wei, Xin

    2017-04-01

    Antifungal resistance is a serious problem in clinical infections. Farnesol, which is a potential antifungal agent against biofilms formed by Candida albicans resistant strains (a fluconazole-resistant isolate derived from SC5314 and two clinical Candida resistant isolates), was investigated in this study. The inhibitory effects of farnesol on biofilms were examined by XTT assay. The morphological changes and biofilm thicknesses were analyzed by scanning electron microscopy and confocal laser scanning microscopy, respectively. Additionally, the checkerboard microdilution method was used to investigate the interactions between farnesol and antifungals (fluconazole, amphotericin B, caspofungin, itraconazole, terbinafine and 5-flurocytosine) against biofilms. The results showed decreased SMICs of farnesol and thinner biofilms in the farnesol-treated groups, indicating that farnesol inhibited the development of biofilms formed by the resistant strain. Furthermore, there were synergistic effects between farnesol and fluconazole/5-flurocytosine, while there were antagonistic effects between farnesol and terbinafine/itraconazole, respectively, on the biofilms formed by the resistant strains.

  6. In vitro oxidant effects of D-glucosamine reduce adhesión and biofilm formation of Staphylococcus epidermidis Los efectos oxidantes de la D-glucosamina in vitro reducen la adhesión y formación de biofilms de Staphylococcus epidermidis

    Directory of Open Access Journals (Sweden)

    Virginia Aiassa

    2012-03-01

    Full Text Available Staphylococcus epidermidis is a common pathogen in medical device-associated infections. Its major pathogenic factor is the ability to form adherent biofilms. In this work, three S. epidermidis strains isolated from infected catheters were chosen with the objective of investigating the effect of D-glucosamine (D-Glu on reactive oxygen species (ROS production, adhesion and biofilm formation. The chemiluminescence and nitroblue tetrazolium reduction assays were used to determine ROS production by planktonic S. epidermidis and the microtiter plate assay to quantify in vitro biofilm formation. D-Glu generated a dose-dependent increase in ROS in planktonic cells with maximum stimuli at a concentration of 0.05 mM, and reduced adhesion and biofilm formation. On the other hand, glucose showed an antioxidative stress action and promoted biofilm adhesion and growth. This study suggests a potential application of D-Glu against infections associated with indwelling medical devices, since the oxidative stress caused by this hexosamine in planktonic S. epidermidis contributed to reducing biofilm formation.Staphylococcus epidermidis es un patógeno común en infecciones asociadas a dispositivos médicos. Su factor de patogenicidad más importante es la capacidad para formar biofilms. Se trabajó con tres cepas de S. epidermidis aisladas de catéteres, con las que se efectuaron ensayos de quimioluminiscencia y de reducción de azul de nitrotetrazolio, para determinar la producción de especies reactivas del oxígeno (ERO en S. epidermidis planctónico, y ensayos dirigidos a cuantificar la formación de biofilm in vitro, empleando placas multipocillos. La D-glucosamina generó un aumento dependiente de la dosis en la producción de ERO en las células planctónicas, con un estímulo máximo a una concentración de 0,05 mM. Este aumento condμlo a la reducción de la adhesión y de la formación de biofilm. La adición de glucosa, en cambio, mostró un efecto

  7. Silver against Pseudomonas aeruginosa biofilms

    DEFF Research Database (Denmark)

    Bjarnsholt, Thomas; Kirketerp-Møller, K.; Kristiansen, S.

    2007-01-01

    bacteria in both the planktonic and biofilm modes of growth. The action of silver on mature in vitro biofilms of Pseudomonas aeruginosa, a primary pathogen of chronic infected wounds, was investigated. The results show that silver is very effective against mature biofilms of P. aeruginosa...

  8. High In Vitro Antibacterial Activity of Pac-525 against Porphyromonas gingivalis Biofilms Cultured on Titanium

    Directory of Open Access Journals (Sweden)

    Ji-yin Li

    2015-01-01

    Full Text Available In order to investigate the potential of short antimicrobial peptides (AMPs as alternative antibacterial agents during the treatment of peri-implantitis, the cytotoxic activity of three short AMPs, that is, Pac-525, KSL-W, and KSL, was determined using the MTT assay. The antimicrobial activity of these AMPs, ranging in concentration from 0.0039 mg/mL to 0.5 mg/mL, against the predominant planktonic pathogens, including Streptococcus sanguis, Fusobacterium nucleatum, and Porphyromonas gingivalis, involved in peri-implantitis was investigated. Furthermore, 2-day-old P. gingivalis biofilms cultured on titanium surfaces were treated with Pac-525 and subsequently observed and analysed using confocal laser scanning microscopy (CLSM. The average cell proliferation curve indicated that there was no cytotoxicity due to the three short AMPs. The minimum inhibitory concentration and minimum bactericidal concentration values of Pac-525 were 0.0625 mg/mL and 0.125 mg/mL, respectively, for P. gingivalis and 0.0078 mg/mL and 0.0156 mg/mL, respectively, for F. nucleatum. Using CLSM, we confirmed that compared to 0.1% chlorhexidine, 0.5 mg/mL of Pac-525 caused a significant decrease in biofilm thickness and a decline in the percentage of live bacteria. These data indicate that Pac-525 has unique properties that might make it suitable for the inhibition the growth of pathogenic bacteria around dental implants.

  9. Advancement of the 10-species subgingival Zurich biofilm model by examining different nutritional conditions and defining the structure of the in vitro biofilms.

    Science.gov (United States)

    Ammann, Thomas W; Gmür, Rudolf; Thurnheer, Thomas

    2012-10-05

    Periodontitis is caused by a highly complex consortium of bacteria that establishes as biofilms in subgingival pockets. It is a disease that occurs worldwide and its consequences are a major health concern. Investigations in situ are not possible and the bacterial community varies greatly between patients and even within different loci. Due to the high complexity of the consortium and the availability of samples, a clear definition of the pathogenic bacteria and their mechanisms of pathogenicity are still not available. In the current study we addressed the need of a defined model system by advancing our previously described subgingival biofilm model towards a bacterial composition that reflects the one observed in diseased sites of patients and analysed the structure of these biofilms. We further developed the growth media by systematic variation of key components resulting in improved stability and the firm establishment of spirochetes in the 10-species subgingival Zurich biofilm model. A high concentration of heat-inactivated human serum allowed the best proliferation of the used species. Therefore we further investigated these biofilms by analysing their structure by confocal laser scanning microscopy following fluorescence in situ hybridisation. The species showed mutual interactions as expected from other studies. The abundances of all organisms present in this model were determined by microscopic counting following species-specific identification by both fluorescence in situ hybridisation and immunofluorescence. The newly integrated treponemes were the most abundant organisms. The use of 50% of heat-inactivated human serum used in the improved growth medium resulted in significantly thicker and more stable biofilms, and the quantitative representation of the used species represents the in vivo community of periodontitis patients much closer than in biofilms grown in the two media with less or no human serum. The appearance of T. denticola, P. gingivalis, and

  10. Paired methods to measure biofilm killing and removal: a case study with Penicillin G treatment of Staphylococcus aureus biofilm.

    Science.gov (United States)

    Ausbacher, D; Lorenz, L; Pitts, B; Stewart, P S; Goeres, D M

    2017-12-30

    Biofilms are microbial aggregates that show high tolerance to antibiotic treatments in vitro and in vivo. Killing and removal are both important in biofilm control, therefore methods that measure these two mechanisms were evaluated in a parallel experimental design. Kill was measured using the single tube method (ASTM method E2871) and removal was determined by video microscopy and image analysis using a new treatment flow cell. The advantage of the parallel test design is that both methods used biofilm covered coupons harvested from a CDC biofilm reactor, a well-established and standardized biofilm growth method. The control Staphylococcus aureus biofilms treated with growth medium increased by 0·6 logs during a 3-h contact time. Efficacy testing showed biofilms exposed to 400 μmol l-1 penicillin G decreased by only 0·3 logs. Interestingly, time-lapse confocal scanning laser microscopy revealed that penicillin G treatment dispersed the biofilm despite being an ineffective killing agent. In addition, no biofilm removal was detected when assays were performed in 96-well plates. These results illustrate that biofilm behaviour and impact of treatments can vary substantially when assayed by different methods. Measuring both killing and removal with well-characterized methods will be crucial for the discovery of new anti-biofilm strategies. Biofilms are tolerant to antimicrobial treatments and can lead to persistent infections. Finding new anti-biofilm strategies and understanding their mode-of-action is therefore of high importance. Historically, antimicrobial testing has focused on measuring the decrease in viability. While kill data are undeniably important, measuring biofilm disruption provides equally useful information. Starting with biofilm grown in the same reactor, we paired assessment of biofilm removal using a new treatment-flow-cell and real-time microscopy with kill data collected using the single tube method (ASTM E2871). Pairing these two methods

  11. Loss of viability and induction of apoptosis in human keratinocytes exposed to Staphylococcus aureus biofilms in vitro.

    Science.gov (United States)

    Kirker, Kelly R; Secor, Patrick R; James, Garth A; Fleckman, Philip; Olerud, John E; Stewart, Philip S

    2009-01-01

    Bacteria colonizing chronic wounds are believed to exist as polymicrobial, biofilm communities; however, there are few studies demonstrating the role of biofilms in chronic wound pathogenesis. This study establishes a novel method for studying the effect of biofilms on the cell types involved in wound healing. Cocultures of Staphylococcus aureus biofilms and human keratinocytes (HK) were created by initially growing S. aureus biofilms on tissue culture inserts then transferring the inserts to existing HK cultures. Biofilm-conditioned medium (BCM) was prepared by culturing the insert-supported biofilm in cell culture medium. As a control planktonic-conditioned medium (PCM) was also prepared. Biofilm, BCM, and PCM were used in migration, cell viability, and apoptosis assays. Changes in HK morphology were followed by brightfield and confocal microscopy. After only 3 hours exposure to BCM, but not PCM, HK formed dendrite-like extensions and displayed reduced viability. After 9 hours, there was an increase in apoptosis (pPCM-exposed HK all exhibited reduced scratch closure (p< or =0.0001). The results demonstrated that soluble products of both S. aureus planktonic cells and biofilms inhibit scratch closure. Furthermore, S. aureus biofilms significantly reduced HK viability and significantly increased HK apoptosis compared with planktonic S. aureus.

  12. In vivo Candida glabrata biofilm development on foreign bodies in a rat subcutaneous model.

    Science.gov (United States)

    Kucharíková, Soňa; Neirinck, Bram; Sharma, Nidhi; Vleugels, Jef; Lagrou, Katrien; Van Dijck, Patrick

    2015-03-01

    Biofilm studies have been mostly dedicated to the major human fungal pathogen Candida albicans, whereas much less is known about this virulence factor in Candida glabrata, certainly under in vivo conditions. This study provides a deeper understanding of the biofilm development of C. glabrata, its architecture and susceptibility profile to fluconazole and echinocandins. In vitro and in vivo C. glabrata biofilms were developed inside serum-coated triple-lumen catheters placed in 24-well polystyrene plates or implanted subcutaneously in the back of a rat, respectively. Scanning electron microscopy and confocal scanning laser microscopy were used to visualize the biofilm architecture. Quantitative real-time PCR was used to demonstrate the expression profile of EPA1, EPA3, EPA6 and AWP1-AWP7 during in vivo biofilm formation. Mature biofilms were observed within the first 48 h and the amount of biofilm reached its maximum by 6 days. Architecturally, mature C. glabrata biofilms consisted of a thick network of yeast cells embedded in an extracellular matrix. Moreover, in vivo biofilms were susceptible to echinocandin drugs, whereas fluconazole remained ineffective. Gene expression profiling revealed that EPA3, EPA6, AWP2, AWP3 and AWP5 were up-regulated in in vivo biofilms compared with in vitro biofilms. C. glabrata is a unique microorganism, which, despite the lack of transition to the hyphal form, formed thick biofilms inside foreign bodies in vivo. To our knowledge, this is the first study that has described in vivo C. glabrata biofilm development and its architectural changes in detail and provides an insight into the susceptibility profile, as well as the gene expression machinery, of biofilm-associated infections. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  13. RNA-Seq Reveals Enhanced Sugar Metabolism in Streptococcus mutans Co-cultured with Candida albicans within Mixed-Species Biofilms

    Directory of Open Access Journals (Sweden)

    Jinzhi He

    2017-06-01

    Full Text Available Early childhood caries (ECC, which can lead to rampant tooth-decay that is painful and costly to treat, is one of the most prevalent infectious diseases affecting children worldwide. Previous studies support that interactions between Streptococcus mutans and Candida albicans are associated with the pathogenesis of ECC. The presence of Candida enhances S. mutans growth, fitness and accumulation within biofilms in vitro, although the molecular basis for these behaviors is undefined. Using an established co-cultivation biofilm model and RNA-Seq, we investigated how C. albicans influences the transcriptome of S. mutans. The presence of C. albicans dramatically altered gene expression in S. mutans in the dual-species biofilm, resulting in 393 genes differentially expressed, compared to mono-species biofilms of S. mutans. By Gene Ontology analysis, the majority of up-regulated genes were related to carbohydrate transport and metabolic/catabolic processes. KEGG pathway impact analysis showed elevated pyruvate and galactose metabolism, suggesting that co-cultivation with C. albicans influences carbohydrate utilization by S. mutans. Analysis of metabolites confirmed the increases in carbohydrate metabolism, with elevated amounts of formate in the culture medium of co-cultured biofilms. Moreover, co-cultivation with C. albicans altered transcription of S. mutans signal transduction (comC and ciaRH genes associated with fitness and virulence. Interestingly, the expression of genes for mutacins (bacteriocins and CRISPR were down-regulated. Collectively, the data provide a comprehensive insight into S. mutans transcriptomic changes induced by C. albicans, and offer novel insights into how bacterial–fungal interactions may enhance the severity of dental caries.

  14. RNA-Seq Reveals Enhanced Sugar Metabolism in Streptococcus mutans Co-cultured with Candida albicans within Mixed-Species Biofilms

    Science.gov (United States)

    He, Jinzhi; Kim, Dongyeop; Zhou, Xuedong; Ahn, Sang-Joon; Burne, Robert A.; Richards, Vincent P.; Koo, Hyun

    2017-01-01

    Early childhood caries (ECC), which can lead to rampant tooth-decay that is painful and costly to treat, is one of the most prevalent infectious diseases affecting children worldwide. Previous studies support that interactions between Streptococcus mutans and Candida albicans are associated with the pathogenesis of ECC. The presence of Candida enhances S. mutans growth, fitness and accumulation within biofilms in vitro, although the molecular basis for these behaviors is undefined. Using an established co-cultivation biofilm model and RNA-Seq, we investigated how C. albicans influences the transcriptome of S. mutans. The presence of C. albicans dramatically altered gene expression in S. mutans in the dual-species biofilm, resulting in 393 genes differentially expressed, compared to mono-species biofilms of S. mutans. By Gene Ontology analysis, the majority of up-regulated genes were related to carbohydrate transport and metabolic/catabolic processes. KEGG pathway impact analysis showed elevated pyruvate and galactose metabolism, suggesting that co-cultivation with C. albicans influences carbohydrate utilization by S. mutans. Analysis of metabolites confirmed the increases in carbohydrate metabolism, with elevated amounts of formate in the culture medium of co-cultured biofilms. Moreover, co-cultivation with C. albicans altered transcription of S. mutans signal transduction (comC and ciaRH) genes associated with fitness and virulence. Interestingly, the expression of genes for mutacins (bacteriocins) and CRISPR were down-regulated. Collectively, the data provide a comprehensive insight into S. mutans transcriptomic changes induced by C. albicans, and offer novel insights into how bacterial–fungal interactions may enhance the severity of dental caries. PMID:28642749

  15. In vitro interference of beta-lactams with biofilm development by prevalent community respiratory tract isolates.

    Science.gov (United States)

    Maestre, J R; Mateo, M; Méndez, M L; Aguilar, L; Gimenez, M J; Alou, L; Coronel, P; Granizo, J J; Prieto, J

    2010-03-01

    Interference of cefditoren (CDN) and amoxicillin/clavulanic acid (AMC) with biofilm production was studied using 11 Streptococcus pneumoniae isolates with minimum inhibitory concentrations (MICs) ranging from 0.015microg/mL to 0.5microg/mL for CDN and from 0.06microg/mL to 2microg/mL for AMC (except for one isolate with an AMC MIC of 8microg/mL) and 5 Haemophilus influenzae isolates with MICs of 0.03-0.06microg/mL for CDN and 0.5-16microg/mL for AMC. Slime production was assessed in antibiotic-free medium and with 0.03microg/mL CDN or 1/0.5microg/mL AMC by measuring the optical density at 450nm (OD(450)). Significantly lower mean OD(450) values were obtained for S. pneumoniae with antibiotics compared with controls (CDN, 0.088 vs. 0.118, P=0.003; and AMC, 0.095 vs. 0.112, P=0.003), with significant correlation between both antibiotics (r=0.752; P=0.008). Percent reduction in OD(450) values was higher for CDN compared with AMC (24.02% vs. 15.92%; P=0.008). For H. influenzae, significantly lower mean OD(450) values were obtained with CDN compared with controls (0.083 vs. 0.096; P=0.043) but not with AMC (0.086 vs. 0.095; P=0.08). Comparing percent reductions in S. pneumoniae versus H. influenzae for each antibiotic, no differences were found for AMC (15.92% vs. 9.40%; P=0.36), with a tendency for CDN (24.02% vs. 13.79%; P=0.069). Different beta-lactams may have different capabilities of interfering with S. pneumoniae biofilm development when tested under the same experimental conditions. Copyright 2009 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

  16. Virulence of Cryptococcus sp. Biofilms In Vitro and In Vivo using Galleria mellonella as an Alternative Model.

    Science.gov (United States)

    Benaducci, Tatiane; Sardi, Janaina de C O; Lourencetti, Natalia M S; Scorzoni, Liliana; Gullo, Fernanda P; Rossi, Suélen A; Derissi, Jaqueline B; de Azevedo Prata, Márcia C; Fusco-Almeida, Ana M; Mendes-Giannini, Maria J S

    2016-01-01

    Cryptococcus neoformans and C. gattii are fungal pathogens that are most commonly found in infections of the central nervous system, which cause life-threatening meningoencephalitis and can grow as a biofilm. Biofilms are structures conferring protection and resistance of microorganism to the antifungal drugs. This study compared the virulence of planktonic and biofilm cells of C. neoformans and C. gattii in Galleria mellonella model, as well as, the quantification of gene transcripts LAC1, URE1, and CAP59 by real time PCR. All three of the genes showed significantly increased expressions in the biofilm conditions for two species of Cryptococcus, when compared to planktonic cells. C. neoformans and C. gattii cells in the biofilm forms were more virulent than the planktonic cells in G. mellonella. This suggests that the biofilm conditions may contribute to the virulence profile. Our results contribute to a better understanding of the agents of cryptococcosis in the host-yeast aspects of the interaction.

  17. In vitro susceptibility of amphotericin-B, voriconazole and caspofungin against Candida guilliermondii biofilms, isolated from dentals units water pipes, under different growth phases.

    Science.gov (United States)

    Mazari, W; Boucherit-Otmani, Z; Boucherit, K

    2015-03-01

    The dental units water pipes are a favorable medium for biofilms formation because of the small diameter of the pipe and the duration of water stagnation, but the question which arises is the nature of the biofilms which are formed inside? This article gives a progress report on the nature of this microbial contamination and precisely the fungal biofilms formation by examining their susceptibility to antifungal agents under different growth phases. Sixteen samples of dental units water pipes were taken from public dental clinic and from stomatology unit at the university hospital of Tlemcen (Algeria). The isolated strains were identified by the conventional mycological methods and were analyzed to determine their minimal concentrations inhibiting their growth (planktonic and sessile forms) using three antifungal agents. Five strains type Candida guilliermondii were identified and analyzed for their resistance to antifungal agents. Antifungal susceptibility testing demonstrated the sensitivity of all planktonic Candida guilliermondii cells against amphotericin-B, voriconzole and caspofungin but the sessile cells of these strains revealed a less susceptibility to antifungal agents and even a resistance when the biofilm made mature. Several types of yeast contaminated the dental units water pipes and especially Candida guilliermondii that was the most founded. This specie was susceptible to antifungal agents under planctonic forms and resistance where the biofilm made mature. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  18. Characterization of Biofilm Formation in [Pasteurella] pneumotropica and [Actinobacillus] muris Isolates of Mouse Origin.

    Directory of Open Access Journals (Sweden)

    Martin Sager

    Full Text Available [Pasteurella] pneumotropica biotypes Jawetz and Heyl and [Actinobacillus] muris are the most prevalent Pasteurellaceae species isolated from laboratory mouse. However, mechanisms contributing to their high prevalence such as the ability to form biofilms have not been studied yet. In the present investigation we analyze if these bacterial species can produce biofilms in vitro and investigate whether proteins, extracellular DNA and polysaccharides are involved in the biofilm formation and structure by inhibition and dispersal assays using proteinase K, DNase I and sodium periodate. Finally, the capacity of the biofilms to confer resistance to antibiotics is examined. We demonstrate that both [P.] pneumotropica biotypes but not [A.] muris are able to form robust biofilms in vitro, a phenotype which is widely spread among the field isolates. The biofilm inhibition and dispersal assays by proteinase and DNase lead to a strong inhibition in biofilm formation when added at the initiation of the biofilm formation and dispersed pre-formed [P.] pneumotropica biofilms, revealing thus that proteins and extracellular DNA are essential in biofilm formation and structure. Sodium periodate inhibited the bacterial growth when added at the beginning of the biofilm formation assay, making difficult the assessment of the role of β-1,6-linked polysaccharides in the biofilm formation, and had a biofilm stimulating effect when added on pre-established mature biofilms of [P.] pneumotropica biotype Heyl and a majority of [P.] pneumotropica biotype Jawetz strains, suggesting that the presence of β-1,6-linked polysaccharides on the bacterial surface might attenuate the biofilm production. Conversely, no effect or a decrease in the biofilm quantity was observed by biofilm dispersal using sodium periodate on further biotype Jawetz isolates, suggesting that polysaccharides might be incorporated in the biofilm structure. We additionally show that [P.] pneumotropica cells

  19. Characterization of Biofilm Formation in [Pasteurella] pneumotropica and [Actinobacillus] muris Isolates of Mouse Origin.

    Science.gov (United States)

    Sager, Martin; Benten, W Peter M; Engelhardt, Eva; Gougoula, Christina; Benga, Laurentiu

    2015-01-01

    [Pasteurella] pneumotropica biotypes Jawetz and Heyl and [Actinobacillus] muris are the most prevalent Pasteurellaceae species isolated from laboratory mouse. However, mechanisms contributing to their high prevalence such as the ability to form biofilms have not been studied yet. In the present investigation we analyze if these bacterial species can produce biofilms in vitro and investigate whether proteins, extracellular DNA and polysaccharides are involved in the biofilm formation and structure by inhibition and dispersal assays using proteinase K, DNase I and sodium periodate. Finally, the capacity of the biofilms to confer resistance to antibiotics is examined. We demonstrate that both [P.] pneumotropica biotypes but not [A.] muris are able to form robust biofilms in vitro, a phenotype which is widely spread among the field isolates. The biofilm inhibition and dispersal assays by proteinase and DNase lead to a strong inhibition in biofilm formation when added at the initiation of the biofilm formation and dispersed pre-formed [P.] pneumotropica biofilms, revealing thus that proteins and extracellular DNA are essential in biofilm formation and structure. Sodium periodate inhibited the bacterial growth when added at the beginning of the biofilm formation assay, making difficult the assessment of the role of β-1,6-linked polysaccharides in the biofilm formation, and had a biofilm stimulating effect when added on pre-established mature biofilms of [P.] pneumotropica biotype Heyl and a majority of [P.] pneumotropica biotype Jawetz strains, suggesting that the presence of β-1,6-linked polysaccharides on the bacterial surface might attenuate the biofilm production. Conversely, no effect or a decrease in the biofilm quantity was observed by biofilm dispersal using sodium periodate on further biotype Jawetz isolates, suggesting that polysaccharides might be incorporated in the biofilm structure. We additionally show that [P.] pneumotropica cells enclosed in biofilms

  20. Rifampicin-containing combinations are superior to combinations of vancomycin, linezolid and daptomycin against Staphylococcus aureus biofilm infection in vivo and in vitro.

    Science.gov (United States)

    Jørgensen, Nis Pedersen; Skovdal, Sandra M; Meyer, Rikke L; Dagnæs-Hansen, Frederik; Fuursted, Kurt; Petersen, Eskild

    2016-06-01

    Susceptibility to antibiotics is dramatically reduced when bacteria form biofilms. In clinical settings this has a profound impact on treatment of implant-associated infections, as these are characterized by biofilm formation. Current routine susceptibility testing of microorganisms from infected implants does not reflect the actual susceptibility, and the optimal antibiotic strategy for treating implant-associated infections is not established. In this study of biofilm formation in implant-associated osteomyelitis, we compared thein vitroandin vivoefficacy of selected antibiotics alone and in combination againstStaphylococcus aureus.We tested vancomycin, linezolid, daptomycin and tigecycline alone and in combination with rifampicin, vancomycin, linezolid and daptomycin againstS. aureusIn vitro, biofilm formation dramatically reduced susceptibility by a factor of 500-2000.In vivo, antibiotic combinations were tested in a murine model of implant-associated osteomyelitis. Mice were infected by inserting implants colonized withS. aureustrough their tibia. After 11 days, the animals were divided into different groups (five animals/group) and given 14 days of antibiotic therapy. All antibiotics resulted in a reduced bacterial load in the infected bone surrounding the implant. Overall, the most effective antibiotic combinations contained rifampicin. Combinations containing two non-rifampicin antibiotics were not more active than single drugs. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  1. Deletion of gtfC of Streptococcus mutans has no influence on the composition of a mixed-species in vitro biofilm model of supragingival plaque.

    Science.gov (United States)

    Van Der Ploeg, Jan R; Guggenheim, Bernhard

    2004-10-01

    Glucosyltransferases from Streptococcus mutans are thought to play an important role in bacterial adherence to the tooth surface. The goal of the present study was to determine the effect of the deletion of the gtfC gene, which encodes a glucosyltransferase that catalyses primarily the formation of insoluble glucan (mutan), on colonization of S. mutans in a mixed-species biofilm model of supragingival plaque. A gtfC deletion mutant of S. mutans UA159 grew poorly in biofilms on a polystyrene surface in Todd-Hewitt medium containing sucrose, but biofilm formation in the semi-defined fluid universal medium (FUM) was not affected. The S. mutans gtfC mutant colonized with the same efficiency as the wild-type strain when grown together with five other species in a mixed-species biofilm on hydroxyapatite in a mixture of FUM and saliva with pulses of sucrose and showed the same ability to demineralize enamel in vitro. Colonization of mutant and wild-type strains was also equal in an association experiment in specific-pathogen-free rats. However, the gtfC mutant gave rise to more dentinal fissure lesions and smooth surface caries than the wild-type strain; this could be caused by a change in diffusion properties as a result of to the lack of mutan.

  2. Staphylococcus aureus biofilms prevent macrophage phagocytosis and attenuate inflammation in vivo||

    Science.gov (United States)

    Thurlow, Lance R.; Hanke, Mark L.; Fritz, Teresa; Angle, Amanda; Aldrich, Amy; Williams, Stetson H.; Engebretsen, Ian L.; Bayles, Kenneth W.; Horswill, Alexander R.; Kielian, Tammy

    2011-01-01

    Biofilms are complex communities of bacteria encased in a matrix composed primarily of polysaccharides, extracellular DNA, and protein. Staphylococcus aureus (S. aureus) can form biofilm infections, which are often debilitating due to their chronicity and recalcitrance to antibiotic therapy. Currently, the immune mechanisms elicited during biofilm growth and their impact on bacterial clearance remains to be defined. We utilized a mouse model of catheter-associated biofilm infection to assess the functional importance of Toll-like receptors 2 and 9 in the host immune response during biofilm formation, since ligands for both receptors are present within the biofilm. Interestingly, neither receptor impacted bacterial density or inflammatory mediator secretion during biofilm growth in vivo, suggesting that S. aureus biofilms circumvent these traditional bacterial recognition pathways. Several potential mechanisms were identified to account for biofilm evasion of innate immunity, including significant reductions in IL-1β, TNF-α, CXCL2, and CCL2 expression during biofilm infection compared to the wound healing response elicited by sterile catheters, limited macrophage invasion into biofilms in vivo, and a skewing of the immune response away from a microbicidal phenotype as evidenced by decreases in iNOS expression concomitant with robust arginase-1 induction. Co-culture studies of macrophages with S. aureus biofilms in vitro revealed that macrophages successful at biofilm invasion displayed limited phagocytosis and gene expression patterns reminiscent of alternatively activated M2 macrophages. Collectively, these findings demonstrate that S. aureus biofilms are capable of attenuating traditional host proinflammatory responses, which may explain why biofilm infections persist in an immunocompetent host. PMID:21525381

  3. Comparative genomics in acid mine drainage biofilm communities reveals metabolic and structural differentiation of co-occurring archaea.

    Science.gov (United States)

    Yelton, Alexis P; Comolli, Luis R; Justice, Nicholas B; Castelle, Cindy; Denef, Vincent J; Thomas, Brian C; Banfield, Jillian F

    2013-07-17

    Metal sulfide mineral dissolution during bioleaching and acid mine drainage (AMD) formation creates an environment that is inhospitable to most life. Despite dominance by a small number of bacteria, AMD microbial biofilm communities contain a notable variety of coexisting and closely related Euryarchaea, most of which have defied cultivation efforts. For this reason, we used metagenomics to analyze variation in gene content that may contribute to niche differentiation among co-occurring AMD archaea. Our analyses targeted members of the Thermoplasmatales and related archaea. These results greatly expand genomic information available for this archaeal order. We reconstructed near-complete genomes for uncultivated, relatively low abundance organisms A-, E-, and Gplasma, members of Thermoplasmatales order, and for a novel organism, Iplasma. Genomic analyses of these organisms, as well as Ferroplasma type I and II, reveal that all are facultative aerobic heterotrophs with the ability to use many of the same carbon substrates, including methanol. Most of the genomes share genes for toxic metal resistance and surface-layer production. Only Aplasma and Eplasma have a full suite of flagellar genes whereas all but the Ferroplasma spp. have genes for pili production. Cryogenic-electron microscopy (cryo-EM) and tomography (cryo-ET) strengthen these metagenomics-based ultrastructural predictions. Notably, only Aplasma, Gplasma and the Ferroplasma spp. have predicted iron oxidation genes and Eplasma and Iplasma lack most genes for cobalamin, valine, (iso)leucine and histidine synthesis. The Thermoplasmatales AMD archaea share a large number of metabolic capabilities. All of the uncultivated organisms studied here (A-, E-, G-, and Iplasma) are metabolically very similar to characterized Ferroplasma spp., differentiating themselves mainly in their genetic capabilities for biosynthesis, motility, and possibly iron oxidation. These results indicate that subtle, but important genomic

  4. Influence of artificial saliva in biofilm formation of Candida albicans in vitro

    Directory of Open Access Journals (Sweden)

    Michelle Peneluppi Silva

    2012-02-01

    Full Text Available Due to the increase in life expectancy, new treatments have emerged which, although palliative, provide individuals with a better quality of life. Artificial saliva is a solution that contains substances that moisten a dry mouth, thus mimicking the role of saliva in lubricating the oral cavity and controlling the existing normal oral microbiota. This study aimed to assess the influence of commercially available artificial saliva on biofilm formation by Candida albicans. Artificial saliva I consists of carboxymethylcellulose, while artificial saliva II is composed of glucose oxidase, lactoferrin, lysozyme and lactoperoxidase. A control group used sterile distilled water. Microorganisms from the oral cavity were transferred to Sabouraud Dextrose Agar and incubated at 37°C for 24 hours. Colonies of Candida albicans were suspended in a sterile solution of NaCl 0.9%, and standardisation of the suspension to 106 cells/mL was achieved. The acrylic discs, immersed in artificial saliva and sterile distilled water, were placed in a 24-well plate containing 2 mL of Sabouraud Dextrose Broth plus 5% sucrose and 0.1 mL aliquot of the Candida albicans suspension. The plates were incubated at 37°C for 5 days, the discs were washed in 2 mL of 0.9% NaCl and placed into a tube containing 10 mL of 0.9% NaCl. After decimal dilutions, aliquots of 0.1 mL were seeded on Sabouraud Dextrose Agar and incubated at 37°C for 48 hours. Counts were reported as CFU/mL (Log10. A statistically significant reduction of 29.89% (1.45 CFU/mL of Candida albicans was observed in saliva I when compared to saliva II (p = 0.002, considering p≤0.05.

  5. Role of extracellular matrix protein CabA in resistance of Vibrio vulnificus biofilms to decontamination strategies.

    Science.gov (United States)

    Park, Jin Hwan; Lee, Byungho; Jo, Youmi; Choi, Sang Ho

    2016-11-07

    Biofilms are recalcitrant and raise safety problems in the food industry. In this study, the role of CabA, an extracellular matrix protein, in the resistance of the biofilms of Vibrio vulnificus, a foodborne pathogen, to decontamination strategies was investigated. Biofilms of the cabA mutant revealed reduced resistance to detachment by vibration and disinfection by sodium hypochlorite compared to the biofilms of the parental wild type in vitro. The reduced resistance of the cabA mutant biofilms was complemented by introducing a recombinant cabA, indicating that the reduced resistance of the cabA mutant biofilms is caused by the inactivation of cabA. The expression of cabA was induced in cells bound to oyster, the primary vehicle of the pathogen. The cabA mutant biofilms on oyster are defective in biomass and resistance to detachment and disinfection. The bacterial cells in the wild-type biofilms are clustered by filaments which are not apparent in the cabA mutant biofilms. The combined results indicated that CabA contributes to the structural integrity of V. vulnificus biofilms possibly by forming filaments in the matrix and thus rendering the biofilms robust, suggesting that CabA could be a target to control V. vulnificus biofilms on oyster. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Biofilm-growing intestinal anaerobic bacteria.

    Science.gov (United States)

    Donelli, Gianfranco; Vuotto, Claudia; Cardines, Rita; Mastrantonio, Paola

    2012-07-01

    Sessile growth of anaerobic bacteria from the human intestinal tract has been poorly investigated, so far. We recently reported data on the close association existing between biliary stent clogging and polymicrobial biofilm development in its lumen. By exploiting the explanted stents as a rich source of anaerobic bacterial strains belonging to the genera Bacteroides, Clostridium, Fusobacterium, Finegoldia, Prevotella, and Veillonella, the present study focused on their ability to adhere, to grow in sessile mode and to form in vitro mono- or dual-species biofilms. Experiments on dual-species biofilm formation were planned on the basis of the anaerobic strains isolated from each clogged biliary stent, by selecting those in which a couple of anaerobic strains belonging to different species contributed to the polymicrobial biofilm development. Then, strains were investigated by field emission scanning electron microscopy and confocal laser scanning microscopy to reveal if they are able to grow as mono- and/or dual-species biofilms. As far as we know, this is the first report on the ability to adhere and form mono/dual-species biofilms exhibited by strains belonging to the species Bacteroides oralis, Clostridium difficile, Clostridium baratii, Clostridium fallax, Clostridium bifermentans, Finegoldia magna, and Fusobacterium necrophorum. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  7. Removal of naturally grown human biofilm with an atmospheric pressure plasma jet: An in-vitro study.

    Science.gov (United States)

    Jablonowski, Lukasz; Fricke, Katja; Matthes, Rutger; Holtfreter, Birte; Schlüter, Rabea; von Woedtke, Thomas; Weltmann, Klaus-Dieter; Kocher, Thomas

    2017-05-01

    The removal of biofilm is a prerequisite for a successful treatment of biofilm-associated diseases. In this study, we compared the feasibility of an atmospheric pressure plasma device with a sonic powered brush to remove naturally grown supragingival biofilm from extracted teeth. Twenty-four periodontally hopeless teeth were extracted. Argon jet plasma with an oxygen admixture of 1 vol% and a sonically driven brush were used to remove biofilm with application times of 60 s, 180 s and 300 s. The treatment efficiency was assessed with light microscopy, scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The highest biofilm removal rate was observed after an application time of 180 s/300 s with the sonic brush (80.4%/86.2%), plasma (75.5%/89.0%). These observations were confirmed by SEM. According to XPS analysis, plasma treatment decreased the amount of carbon and nitrogen, indicative of an extensive removal of proteins. Plasma treatment of naturally grown biofilm resulted in an effective cleaning of the tooth surface and was comparable to mechanical treatment. Treatment time had a significant influence on plaque reduction. These results showed that plasma could be a useful adjuvant treatment modality in cases where biofilm removal or reduction plays a decisive role, such as periodontitis and peri-implantitis. Plasma-treated biofilm on an extracted tooth. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Characterization of Fusarium Keratitis Outbreak Isolates: Contribution of Biofilms to Antimicrobial Resistance and Pathogenesis

    Science.gov (United States)

    Mukherjee, Pranab K.; Chandra, Jyotsna; Yu, Changping; Sun, Yan; Pearlman, Eric; Ghannoum, Mahmoud A.

    2012-01-01

    Purpose. Fusarium is a major cause of microbial keratitis, and its ability to form biofilms was suggested as a contributing factor in recent outbreaks. We investigated the ability of outbreak Fusarium isolates (F. solani species complex [FSSC] and F. oxysporum species complex [FOSC]) to form biofilms in vitro and in vivo, and evaluated their antifungal susceptibilities. Methods. Biofilm formation was assessed using our in vitro contact lens model and in vivo murine model. Biofilm architecture was assessed using confocal laser scanning microscopy (CLSM). Susceptibility against amphotericin B (AmB), voriconazole (VCZ), and natamycin (NAT) was determined using the CLSI-M38-A2 method and XTT metabolic assay. Results. FSSC strains formed more biofilms than FOSC, in a strain- and clade-dependent manner. CLSM analyses revealed that “high biofilm forming” (HBF) strains had denser and thicker biofilms than “low biofilm forming” (LBF) strains of both species (thickness 51 vs. 41 μm for FSSC and 61 vs. 45 μm for FOSC strains, P Fusarium biofilms exhibited species-dependent antifungal susceptibilities (e.g., FSSC biofilms AmB minimal inhibitory concentrations [MIC] ≥16 μg/mL, while NAT or VCZ MICs were 2–8 μg/mL). FSSC-infected mice had severe corneal opacification independent of biofilm thickness, while FOSC infection resulted in moderate corneal opacification. Corneal fungal burden of mice infected with HBF strains was higher than those of the LBF strains. In contrast, the reference ATCC isolate was unable to cause infection. Conclusions. The ability to form biofilms is a key pathogenicity determinant of Fusarium, irrespective of the thickness of these biofilms. Further studies are warranted to explore this association in greater detail. PMID:22669723

  9. An in vitro study of the anti-biofilm properties of proanthocyanidin and chitosan in Pseudomonas syringae pv. papulans

    Science.gov (United States)

    Song, Kai

    Biofilm-forming bacteria are a form of planktonic microorganisms that can become resistant against conventional antibiotics. Because they are difficult to eradicate, biofilm-forming bacteria are extremely problematic for the medical industry areas. Thus, materials that can distort biofilm structure would be helpful for eliminating chronic infection and decreasing bacterial resistance. The primary objective of this study is to evaluate the anti-biofilm effect of two bio-derived substances, proanthocyanidin and chitosan. Proanthocyanidins are secondary plant metabolites that are reported to have antibiotic and antioxidant functions. Chitosan (poly [beta-(1, 4)-amino-2-deoxy-beta-D-glucose]) is a deacetylated derivative of chitin, which is abundant in the exoskeleton of crustaceans and insects. It is reported to be a suitable substitute for conventional fungicides and can enhance the proanthocyanidin content in plants when used as an agrochemical. Chitosan-tripolyphosphate (TPP) nanoparticles, which have good neutral water solubility and are nanoscale in size, can be used as carriers for gene and drug therapy and are thus favorable to be tested as a treatment method against bacterial biofilms. In this study, the anti-biofilm and antibacterial properties of proanthocyanidin, chitosan-TPP nanoparticles and proanthocyanidins-loaded chitosan-TPP nanoparticles were tested using the model plant bacterium, Pseudomonas syringae pv. papulans (Psp), a pathogen isolated from infected apples. At a lower concentration (1 mg/mL and 2.5 mg/mL), both chitosan nanoparticles and proanthocyanidins can postpone the formation of biofilms and eventually disrupted part of the biofilm. While higher concentration (above 5 mg/mL) of chitosan nanoparticles or proanthocyanidins can eliminate most of the biofilm in this study. PAC-loaded chitosan nanoparticles also can also distort biofilms. Both proanthocyanidins and chitosan-TPP nanoparticle showed a mild antibacterial property. PAC

  10. In vitro efficacy of cold atmospheric pressure plasma on S. sanguinis biofilms in comparison of two test models

    Directory of Open Access Journals (Sweden)

    Gorynia, Susanne

    2013-04-01

    Full Text Available [english] Dental plaque critically affects the etiology of caries, periodontitis and periimplantitis. The mechanical removal of plaque can only be performed partially due to limited accessibility. Therefore, plaque still represents one of the major therapeutic challenges. Even though antiseptic mouth rinses reduce the extent of biofilm temporarily, plaque removal remains incomplete and continuous usage can even result in side effects. Here we tested argon plasma produced by kinpen09 as one option to inactivate microorganisms and to eliminate plaque. biofilms cultivated in either the European Biofilm Reactor (EUREBI or in 24 well plates were treated with argon plasma. In both test systems a homogeneous, good analyzable and stable biofilm was produced on the surface of titan plates within 72 h (>6,9 log CFU/ml. Despite the significantly more powerful biofilm production in EUREBI, the difference of 0.4 log CFU/ml between EUREBI and the 24 well plates was practically not relevant. For that reason both test models were equally qualified for the analysis of efficacy of cold atmospheric pressure plasma. We demonstrate a significant reduction of the biofilm compared to the control in both test models. After plasma application of 180 s the biofilm produced in EUREBI or in 24 well plates was decreased by 0.6 log CFU/ml or 0.5 log CFU/ml, respectively. In comparison to recently published studies analyzing the efficacy of kinpen09, produces a hardly removable biofilm. Future investigations using reduced distances between plasma source and biofilm, various compositions of plasma and alternative plasma sources will contribute to further optimization of the efficacy against biofilms.

  11. Bacterial biofilms formed in vitro and in vivo on orthodontic appliances. Effect of antimicrobial agents

    OpenAIRE

    María Cecilia Cortizo; María Elisa Lagares; Mónica Fernández L. de Mele

    2006-01-01

    Los procesos cariogénicos y las infecciones gingivales y en la vecindad de los implantes pueden ocurrir como una consecuencia de la formación de biopelículas. A fin de prevenir estos procesos, se utilizan productos profilácticos tales como fluoruro de sodio (F), clorhexidina (C) y xilitol (X). El objetivo de este trabajo fue estudiar el efecto de las mezclas F + X y C + X sobre las biopelículas orales formadas en dispositivos para Ortodoncia. En los experimentos in vitro se utilizó un consorc...

  12. Effectiveness of antimicrobial photodynamic therapy using a 660 nm laser and methyline blue dye for inactivating Staphylococcus aureus biofilms in compact and cancellous bones: An in vitro study.

    Science.gov (United States)

    Rosa, Luciano Pereira; Silva, Francine Cristina da; Nader, Sumaia Alves; Meira, Giselle Andrade; Viana, Magda Souza

    2015-06-01

    New therapeutic modalities such as antimicrobial photodynamic therapy (APDT) has been investigated in order to be a valid alternative to the treatment of infections caused by different microorganisms. This work evaluated the in vitro effectiveness of Antimicrobial Photodynamic Therapy (APDT) using 660 nm laser combined with methylene blue dye to inactivate Staphylococcus aureus (ATCC 25923) biofilms in compact and cancellous bones specimens. Eighty specimens of compact bone and 80 specimens of cancellous bone were contaminated with a standard suspension of S. aureus and incubated for 14 days at 37°C to induce the formation of biofilms. The specimens were then divided into groups (n = 10) according to the established treatment: PS-L- (control--no treatment), PS+L- (only AM for 5 min in the dark), PS-L+90 (only laser irradiation for 90 s), PS-L+180 (only laser irradiation for 180 s), PS-L+300 (only laser irradiation for 300 s), APDT90 (APDT for 90 s), APDT180 (APDT for 180 s), and APDT300 (APDT for 300 s). The findings were statistically analyzed by ANOVA 5%. All of the experimental treatments showed a significant reduction (log 10 CFU/mL) of S. aureus biofilms in compact and cancellous bones specimens compared with the control group, and the APDT group was the most effective. Compact specimens treated with APDT showed the greatest reduction in biofilms compared with cancellous specimens, regardless of length of treatment. APDT with methylene blue dye and a 660 nm laser proved to be effective in inactivating S. aureus biofilms formed in compact and cancellous bone. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Influence of physico-chemical material characteristics on staphylococcal biofilm formation--a qualitative and quantitative in vitro analysis of five different calcium phosphate bone grafts.

    Science.gov (United States)

    Clauss, M; Furustrand Tafin, U; Betrisey, B; van Garderen, N; Trampuz, A; Ilchmann, T; Bohner, M

    2014-07-18

    Various compositions of synthetic calcium phosphates (CaP) have been proposed and their use has considerably increased over the past decades. Besides differences in physico-chemical properties, resorption and osseointegration, artificial CaP bone graft might differ in their resistance against biofilm formation. We investigated standardised cylinders of 5 different CaP bone grafts (cyclOS, chronOS (both β-TCP (tricalcium phosphate)), dicalcium phosphate (DCP), calcium-deficient hydroxyapatite (CDHA) and α-TCP). Various physico-chemical characterisations e.g., geometrical density, porosity, and specific surface area were investigated. Biofilm formation was carried out in tryptic soy broth (TSB) and human serum (SE) using Staphylococcus aureus (ATCC 29213) and S. epidermidis RP62A (ATCC 35984). The amount of biofilm was analysed by an established protocol using sonication and microcalorimetry. Physico-chemical characterisation showed marked differences concerning macro- and micropore size, specific surface area and porosity accessible to bacteria between the 5 scaffolds. Biofilm formation was found on all scaffolds and was comparable for α-TCP, chronOS, CDHA and DCP at corresponding time points when the scaffolds were incubated with the same germ and/or growth media, but much lower for cyclOS. This is peculiar because cyclOS had an intermediate porosity, mean pore size, specific surface area, and porosity accessible to bacteria. Our results suggest that biofilm formation is not influenced by a single physico-chemical parameter alone but is a multi-step process influenced by several factors in parallel. Transfer from in vitro data to clinical situations is difficult; thus, advocating the use of cyclOS scaffolds over the four other CaP bone grafts in clinical situations with a high risk of infection cannot be clearly supported based on our data.

  14. Effect of Simulated Gastrointestinal Conditions on Biofilm Formation by Salmonella 1,4,[5],12:i:-

    Science.gov (United States)

    Seixas, R.; Gabriel, M.; Machado, J.; Tavares, L.; Bernardo, F.; Oliveira, M.

    2014-01-01

    Salmonella Typhimurium 1,4,[5],12:i:- is a major serovar responsible for human salmonellosis whose biofilm-forming ability, influenced by environmental conditions like those found in the gastrointestinal tract, is one of the main contributing factors to its ability to persist in the host and thus one of the main causes of chronic relapsing infections. Most studies to evaluate biofilm formation are performed in microtiter assays using standard media. However, no reports are available on the ability of this serovar to produce biofilm under in vitro simulated gastrointestinal conditions which better correlate with the environment found in the gastrointestinal tract. To address this, a modified biofilm assay simulating intestinal fluid was conceived to assess the biofilm formation of 133 Salmonella Typhimurium 1,4,[5],12:i:- isolates with and without agitation and at three different time points (24 h, 48 h, and 72 h). The results were then compared to the existing microtiter method using conventional biofilm growth medium (Mueller Hinton Broth). Statistical analysis revealed significant differences in the results obtained between the three protocols used. The simulated human intestinal environment impaired biofilm production demonstrating that conditions like pH, agitation or the presence of enzymes can influence biofilm production. Therefore, results from in vitro simulation of in vivo conditions may contribute to unravelling factors relating to biofilm formation and persistence in the context of the human host. PMID:25093197

  15. In vitro model of production of antibodies; a new approach to reveal the presence of key bacteria in polymicrobial environments.

    Science.gov (United States)

    Wu, Chongcong; Nakka, Sravya; Mansouri, Sepahdar; Bengtsson, Torbjörn; Nayeri, Tayeb; Nayeri, Fariba

    2016-09-09

    There is a rapid emergence of multiple resistant gram-negative bacteria due to overuse of antibiotics in the treatment of infections. Biofilms consist of polymicrobial communities that survive the host's defense system. The key bacteria in biofilms are slow growing and support an attachment and rapid growth of other microorganisms. Current antimicrobial strategies often fail due to poor diagnosis of key pathogens in biofilms. The study aims to develop anti-bacterial human antibodies in vitro from patients who had recently undergone a systemic infection by pathogenic bacteria and to use these antibodies as a tool for detecting bacteria in biofilms. Lymphocytes were separated from whole blood of patients (n = 10) and stimulated with heat-killed bacteria to produce antibodies in vitro. The specificity of antibodies in recognizing the bacteria against which they were directed was evaluated by surface plasmon resonance system (SPR) and electron microscopy. The ulcer secretions from patients with chronic and acute leg ulcers and healthy controls were analyzed by the SPR system and the results were compared with culture studies. The produced antibodies recognized bacteria with high sensitivity (SPR). The antibodies against Enterococcus fecalis bound specifically to the microorganism in a bacterial co-culture that was visualized by electron microscopy. In the present work, a method for producing specific antibodies against bacteria is introduced to recognize bacterial components in body fluids of patients suffering from pathogenic biofilms. This diagnostic technique may be most useful in clinical microbiology and in the choice of antibiotics in the treatment of serious infections.

  16. Susceptibility of Candida albicans, Staphylococcus aureus, and Streptococcus mutans biofilms to photodynamic inactivation: an in vitro study.

    Science.gov (United States)

    Pereira, Cristiane Aparecida; Romeiro, Rogério Lima; Costa, Anna Carolina Borges Pereira; Machado, Ana Karina Silva; Junqueira, Juliana Campos; Jorge, Antonio Olavo Cardoso

    2011-05-01

    The purpose of this study was to evaluate specific effects of photodynamic inactivation (PDI) using methylene blue as photosensitizer and low-power laser irradiation on the viability of single-, dual-, and three-species biofilms formed by C. albicans, S. aureus, and S. mutans. Biofilms were grown in acrylic discs immersed in sterile brain heart infusion broth (BHI) containing 5% sucrose, inoculated with microbial suspension (10(6) cells/ml) and incubated for 5 days. On the fifth day, the effects of the methylene blue (MB) photosensitizer at a concentration of 0.1 mg/ml for 5 min and InGaAlP laser (660 nm) for 98 s, alone and conjugated were evaluated. Next, the discs were placed in tubes with sterile physiological solution [0.9% sodium chloride (NaCl)] and sonicated for to disperse the biofilms. Ten-fold serial dilutions were carried and aliquots seeded in selective agar, which were then incubated for 48 h. Then the numbers CFU/ml (log(10)) were counted and analyzed statistically (ANOVA, Tukey test, p < 0.05). Scanning electron microscopy (SEM) on discs treated with PDI and control biofilms groups was performed. Significant decreases in the viability of all microorganisms were observed for biofilms exposed to PDI mediated by MB dye. Reductions (log(10)) of single-species biofilms were greater (2.32-3.29) than the association of biofilms (1.00-2.44). Scanning electron microscopy micrographs suggested that lethal photosensitization occurred predominantly in the outermost layers of the biofilms. The results showed that PDI mediated by MB dye, might be a useful approach for the control of oral biofilms.

  17. Efeito antibacteriano e antiaderente in vitro do extrato da Punica granatum Linn. sobre microrganismos do biofilme dental

    Directory of Open Access Journals (Sweden)

    Jozinete V. Pereira

    Full Text Available Nesta pesquisa foi avaliada a atividade antimicrobiana e a capacidade de inibição da síntese de glucano in vitro do extrato da casca do fruto da romã (Punica granatum Linn. sobre linhagens bacterianas de Streptococcus mitis, Streptococcus mutans, Streptococcus sanguis, Streptococcus sobrinus e Lactobacillus casei. Os ensaios foram realizados pelas técnicas de ágar-difusão em placas para determinação da Concentração Inibitória Mínima (CIM e técnica dos tubos inclinados para determinação da Concentração Inibitória Mínima de Aderência (CIMA ao vidro, na presença de 5% de sacarose. Os mesmos procedimentos foram realizados com a clorexidina à 0,12%. As CIMs (mg/mL do extrato da P. granatum frente ao S. mitis, S. mutans, S. sanguis, S. sobrinus e L. casei foram 1:32, 1:8, 1:8, 1:512, e 1:128, respectivamente. Para as CIMAs o extrato da P. granatum mostrou resultados melhores que a clorexidina, exceto para o S. mutans com achados semelhantes na diluição de 1:256. Os resultados mostram a potencialidade da P. granatum na inibição do crescimento bacteriano e síntese de glucano representada pela aderência ao vidro, sugerindo o emprego do extrato da romã, como meio alternativo, no controle desses microrganismos na formação do biofilme.

  18. Preliminary observations on influence of dairy products on biofilm removal from silicone rubber voice prostheses in vitro

    NARCIS (Netherlands)

    Busscher, HJ; Free, RH; Van Weissenbruch, R; Albers, FWJ; Van der Mei, HC

    We determined oropharyngeal biofilm removal from silicone rubber voice prostheses in an artificial throat after perfusion with different commercially available dairy products, including buttermilk, Lactobacillus casei Shirota fermented milk (Yakult, Yakult Netherlands BV, Almere, The Netherlands),

  19. Influence of different instrumentation modalities on the surface characteristics and biofilm formation on dental implant neck, in vitro.

    Science.gov (United States)

    Schmidt, Kristina Emily; Auschill, Thorsten Mathias; Heumann, Christian; Frankenberger, Roland; Eick, Sigrun; Sculean, Anton; Arweiler, Nicole Birgit

    2017-04-01

    To evaluate surface characteristics of implants after using different instruments and biofilm formation following instrumentation. Thirty-five commercially available dental implants were embedded into seven plastic models, attached to a phantom head and randomly assigned to seven instrumentation groups: (1) stainless steel (SSC) or (2) titanium curettes (TC); air-polisher using glycine-based (3) perio (PP) or (4) soft (SP) powders or (5) erythritol powder (EP); and an ultrasonic device using (6) stainless steel (PS) or (7) plastic-coated instruments (PI). Half of each implant neck in each group (n = 5) was treated once (30 s), while the other half was left uninstrumented (control). An eighth (8) treatment group used a bur/polisher to smooth two implants (SM). Following instrumentation implants were rinsed (5 ml Ringer's solution), analysed under a scanning electron microscope (SEM) and subjected twice (separately) to bacterial colonization with Streptococcus gordonii (2 h) and a mixed culture (S. gordonii, Actinomyces naeslundii, Fusobacterium nucleatum, Porphyromonas gingivalis and Tannerella forsythia; 24 h). Visual assessment of SEM pictures revealed surface modifications (smoothening to roughening) following instrumentation. These alterations differed between the instrument groups and from the control. Quantitative scoring of the photographs revealed that SSC caused a significantly rougher surface compared to other instruments (P  0.05) were evident between instrumented or control surfaces in either culture. Overall, no significant differences were observed in the surface characteristics (except for SSC) or bacterial colonization based on one-time instrumentation. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. Evaluation of environmental and nutritional factors and sua gene on in vitro biofilm formation of Streptococcus uberis isolates.

    Science.gov (United States)

    Moliva, Melina Vanesa; Cerioli, Florencia; Reinoso, Elina Beatriz

    2017-06-01

    The pathogenesis of Streptococcus uberis is attributed to a combination of extracellular factors and properties such as adherence and biofilm formation. The aim of this work was to evaluate the influence of different factors, additives and bovine milk compounds on S. uberis biofilm formation, as the presence of the sua gene by PCR. Additionally, extracellular DNA and the effect of DNaseI were evaluated in the biofilms yielded. Optimal biofilm development was observed when the pH was adjusted to 7.0 and 37 °C. Additives as glucose and lactose reduced biofilm formation as bovine milk compounds tested. PCR assay showed that not all the isolates yielded sua gene. Extrachromosomal ADN was found in cell-free supernatants, suggesting that DNA released spontaneously to the medium. The results contribute to a better understanding of the factors involved in biofilm production of this important pathogen associated with mastitis in order to promote the design of new therapeutic approaches. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Antimicrobial resistance and in vitro biofilm-forming ability of enterococci from intensive and extensive farming broilers.

    Science.gov (United States)

    Oliveira, M; Santos, V; Fernandes, A; Bernardo, F; Vilela, C L

    2010-05-01

    Enterococci, major broiler intestinal colonizers, play a recognized role in antimicrobial resistance transmission. Several virulence mechanisms, such as biofilm expression, have been identified. Minimum inhibitory concentrations of vancomycin, enrofloxacin, oxytetracycline, streptomycin, and gentamicin and biofilm production of 34 isolates from intensive and extensive farming system broilers were evaluated. All isolates were susceptible to vancomycin. In extensive-reared broilers (n = 18), resistance to enrofloxacin, oxytetracycline, streptomycin, and gentamicin was high (83.33, 55.56, 100, and 83.33%, respectively). Intensive farming broilers (n = 16) showed a lower resistance level for enrofloxacin and streptomycin and a higher resistance level for oxytetracycline and gentamicin. The relation between antimicrobial susceptibility and farming system was not significant for all drugs tested (P > or = 0.05). Enterococci produced biofilm at 24 h (47.0%), 48 h (55.9%), and 72 h (58.8%). Resistance to gentamicin and streptomycin was related to biofilm production at all time points (P or = 0.05). Poultry are colonized by biofilm-producing and antimicrobial-resistant enterococci, independently of the farming system. Results show a relation between resistance to the majority of the drugs tested and biofilm production, which reenforces the importance of these virulence factors in animal and public health.

  2. In vitro studies of the effect of antiseptic-containing mouthwashes on the formation and viability of Streptococcus sanguis biofilms.

    Science.gov (United States)

    Pratten, J; Wills, K; Barnett, P; Wilson, M

    1998-06-01

    The aims of this study were to evaluate the growth of Streptococcus sanguis on hydroxyapatite, bovine enamel and polytetrafluoroethylene substrata in a constant depth film fermentor, and to determine the effects of three antimicrobial-containing mouthwashes on biofilm formation and bacterial viability on hydroxyapatite and enamel. There was little difference in the final cell density (5 x 10(4) cfu mm-2) of the Strep. sanguis biofilm on the three substrata. When hydroxyapatite-grown biofilms were exposed to the mouthwashes for 1 min, the one containing triclosan (T) proved the most effective. The chlorhexidine-containing mouthwash (CX) also achieved significant kills. The T-containing mouthwash was the most effective at killing biofilms grown on enamel. Pre-treatment of hydroxyapatite with CX, cetylpyridium chloride (CPC) or T for 1 min resulted in undetectable biofilm formation after 8 h. After 8 h of growth, only biofilms grown on enamel discs pre-treated with CX showed a reduction in the number of viable organisms. In conclusion, the results of this study have shown that while growth of Strep. sanguis on hydroxyapatite and enamel were similar, the ability of antimicrobial agents to prevent the accumulation of viable bacteria depended on the nature of the substratum.

  3. Transcriptome profiling reveals stage-specific production and requirement of flagella during biofilm development in Bordetella bronchiseptica

    Science.gov (United States)

    In this report we have used microarray analysis to study the transcriptome of B. bronchiseptica over the course of five time points representing distinct stages of biofilm development. The results suggest that B. bronchiseptica undergoes a coordinately regulated gene expression program similar to a ...

  4. Carbohydrate derived fulvic acid (CHD-FA: an in vitro investigation of a novel membrane active antiseptic agent against Candida albicans biofilms

    Directory of Open Access Journals (Sweden)

    Leighann eSherry

    2012-03-01

    Full Text Available Carbohydrate derived fulvic acid (CHD-FA is a heat stable low molecular weight, water soluble, cationic, colloidal material with proposed therapeutic properties. The aim of this study was to evaluate the antifungal activity of CHD-FA against Candida albicans, and to characterise its mode of action. A panel of C. albicans isolates (n=50 derived from a range of clinical specimens were grown planktonically and as biofilms, and the minimum inhibitory concentrations (MICs determined. Scanning electron microscopy was performed to examine ultrastructural changes and different cell membrane assays were used to determine its mode of action. In addition, the role of C. albicans biofilm resistance mechanisms were investigated to determine their effects of CHD-FA activity. CHD-FA was active against planktonic and sessile C. albicans at concentrations 0.125% and 0.25% respectively, and was shown to be fungicidal, acting through disruption of the cell membrane activity. Resistance mechanisms, including matrix, efflux and stress, had a limited role upon CHD-FA activity. Overall, based on the promising in vitro spectrum of activity and minimal biofilm resistance of the natural and cheap antiseptic CHD-FA, further studies are required to determine its applicability for clinical use.

  5. Nanoparticle conversion to biofilms: in vitro demonstration using serum-derived mineralo-organic nanoparticles.

    Science.gov (United States)

    Wong, Tsui-Yin; Peng, Hsin-Hsin; Wu, Cheng-Yeu; Martel, Jan; Ojcius, David M; Hsu, Fu-Yung; Young, John D

    2015-01-01

    Mineralo-organic nanoparticles (NPs) detected in biological fluids have been described as precursors of physiological and pathological calcifications in the body. Our main objective was to examine the early stages of mineral NP formation in body fluids. A nanomaterial approach based on atomic force microscopy, dynamic light scattering, electron microscopy and spectroscopy was used. The mineral particles, which contain the serum proteins albumin and fetuin-A, initially precipitate in the form of round amorphous NPs that gradually grow in size, aggregate and coalesce to form crystalline mineral films similar to the structures observed in calcified human arteries. Our study reveals the early stages of particle formation and provides a platform to analyze the role(s) of mineralo-organic NPs in human tissues.

  6. Biofilms promote survival and virulence of Salmonella enterica sv. Tennessee during prolonged dry storage and after passage through an in vitro digestion system.

    Science.gov (United States)

    Aviles, Bryan; Klotz, Courtney; Eifert, Joseph; Williams, Robert; Ponder, Monica

    2013-04-01

    Salmonella enterica serotypes have been linked to outbreaks associated with low water activity foods. While the biofilm-forming abilities of Salmonella improve its survival during thermal processing and sanitation it is unclear whether biofilms enhance survival to desiccation and gastric stresses. The purpose of this study was to quantify the effect of physiological state (planktonic versus biofilm) and prior exposure to desiccation and storage in dry milk powder on Salmonella survival and gene expression after passage through an in vitro digestion model. Planktonic cells of Salmonella enterica serotype Tennessee were deposited onto membranes while biofilms were formed on glass beads. The cells were subsequently dried at room temperature and stored in dried milk powder (a(w)=0.3) for up to 30 days. Salmonella survival was quantified by serial dilution onto Brilliant Green Agar before desiccation, after desiccation, after 1-day storage and after 30-day storage. At each sampling period both physiological states were tested for survival through a simulated gastrointestinal system. RNA was extracted at the identical time points and Quantitative Real-Time PCR was used to determine relative expression for genes associated with stress response (rpoS, otsB), virulence (hilA, invA, sipC) and a housekeeping gene 16S rRNA. The physiological state and length of storage affected the survival and gene expression of Salmonella within the desiccated milk powder environment and after passage through an in vitro digestion system (pSalmonella genomes detected by qPCR were not significantly different suggesting entry of the planktonic cells of S. Tennessee into a viable but non-culturable state. The increased expression of stress response genes rpoS and otsB correlated with survival, indicating cross-protection to low water activity and acid stress. Increased expression of virulence-associated genes was seen in cells exposed to dry storage for short periods, however the largest amount

  7. Effects of 5-aminolevulinic acid-mediated photodynamic therapy on antibiotic-resistant staphylococcal biofilm: an in vitro study.

    Science.gov (United States)

    Li, Xin; Guo, Hao; Tian, Qingzhong; Zheng, Gang; Hu, Yangchao; Fu, Yu; Tan, Honglue

    2013-10-01

    Treatments of infections are not always successful because of multi-antibiotic-resistant organisms. It is therefore particularly urgent to provide more effective anti-infective strategy against these organisms. 5-Aminolevulinic acid (ALA), with the chemical structure C5H9NO3, is the only photodynamic therapy agent that is a biochemical precursor of a photosensitizer (protoporphyrin IX [PpIX]), which is naturally produced by the body. 5-Aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) has been shown to have a strong effect on the treatment of localized cancerous and precancerous lesions, and further study demonstrated its efficacy for gram-positive and gram-negative bacteria. However, its effect on biofilm formed by antibiotic-resistant strains has not been reported. In this study, we evaluated the effectiveness of ALA-PDT on biofilms formed by methicillin-resistant Staphylococcus aureus (ATCC 43300) and methicillin-resistant S epidermidis (MRSE 287). The strains were cultured with 40 mM of ALA in 24-well microtiter plates containing coverslips at 37°C for 24 h in the dark. PpIX fluorescence in biofilms formed by the two strains was observed by confocal laser scanning microscopy (CLSM). ALA-treated biofilms were irradiated at different doses (0, 100, 200, and 300 J/cm(2)) using a semiconductor laser. Biofilm exposed only to Tryptone Soy Broth or irradiation (300 J/cm(2)) was investigated. Viability determination, CLSM, and scanning electron microscopy were used to investigate the photodynamic inactivation of ALA-PDT. ALA was absorbed and converted to PpIX by both methicillin-resistant S aureus and methicillin-resistant S epidermidis. No cell inactivation was detectable in biofilms of either strain incubated with ALA without exposure to light, incubated with Tryptone Soy Broth only, or irradiated with red light only. However, a significant number of cells within biofilms were inactivated during irradiation with different doses of red light in the presence

  8. In Vitro Activities of Moxifloxacin, 127I-Moxifloxacin and 131I-Moxifloxacin Against Staphylococcus Aureus Biofilms

    Directory of Open Access Journals (Sweden)

    Hasan Demiroğlu

    2015-02-01

    Full Text Available Objective: The aim of the study was to investigate the antimicrobial effect of Moxifloxacin (MXF, radio (Na131I and cold (K127I iodinated MXF on methicillin susceptible Staphylococcus aureus ATCC 35556 (MSSA biofilms. Methods: MXF was labeled with Na131I using the iodogen method. The optimum radiodination conditions for 131I-MXF was determined by thin-layer radio chromatography studies. Thin-layer radio chromatography (TLRC chromatograms were obtained by using Cyclone Plus Storage Phosphor System. The MICs of MXF, 127I-MXF and 131I-MXF were determined using the microdilution broth method according to CLSI criteria. Time kill curves were performed over 24 h using an inoculum of 2×105 (CFU/mL. Biofilms were grown in microtitre plates, dyed with crystal violet and the mean optical density (OD630 was used for quantification. Biofilms were incubated MXF, 127I-MXF and 131I-MXF at various concentration (0.03 to 64 µg/mL. Results: MXF was labeled with 131I iodogen method. 131I-MXF was obtained with high a yield 95±3%. The MIC values for MXF, 127I-MXF and 131I-MXF was 0.06 µg/mL. Bactericidal activity was demonstrated at 0.25 µg/mL 4 hour for MXF, 127I-MXF and 131I-MXF. At MIC levels, MXF, 127I-MXF and 131I-MXF was not showed a marked reduction of metabolic activity in the S. aureus biofilm. The ODs of biofilm after incubation with an increasing antibiotic concentration were significantly lower than the ODs of biofilms without antibiotic p≤005. The radiolabeled MXF was most effective than MXF, 127I-MXF in reducing the number of bacteria in biofilm. After 24 h incubation Log10 CFU/mL values for 32 µg/mL antibiotic concentration: Control, MXF, 127I-MXF and 131I-MXF were 9.5, 4.3, 4.8 and 3.1, respectively. Conclusion: 131I and 127I were used alone there was no penetration of the S. aureus biofilm and no damage. In contrast our results demonstrate that the radiolabeled Moxifloxacin (131I-MXF have potent anti-biofilm activity against S. aureus

  9. Antimicrobial photodynamic inactivation of Staphylococcus aureus biofilms in bone specimens using methylene blue, toluidine blue ortho and malachite green: An in vitro study.

    Science.gov (United States)

    Rosa, Luciano Pereira; da Silva, Francine Cristina; Nader, Sumaia Alves; Meira, Giselle Andrade; Viana, Magda Souza

    2015-05-01

    To evaluate the in vitro effectiveness of APDI with a 660 nm laser combined with methylene blue (MB), toluidine blue ortho (TBO) and malachite green (MG) dyes to inactivate Staphylococcus aureus (ATCC 25923) biofilms in compact and cancellous bone specimens. Eighty specimens of compact and 80 of cancellous bone were contaminated with a standard suspension of the microorganism and incubated for 14 days at 37°C to form biofilms. After this period, the specimens were divided into groups (n=10) according to established treatment: PS-L- (control - no treatment); PSmb+L-, PStbo+L-, PSmg+L- (only MB, TBO or MG for 5 min in the dark); PS-L+ (only laser irradiation for 180 s); and APDImb, APDItbo and APDImg (APDI with MB, TBO or MG for 180 s). The findings were statistically analyzed by ANOVA at 5% significance levels. All experimental treatments showed significant reduction of log CFU/mL S. aureus biofilms when compared with the control group for compact and cancellous bones specimens; the APDI group's treatment was more effective. The APDI carried out for the compact specimens showed better results when compared with cancellous specimens at all times of application. For the group of compact bone, APDImg showed greater reductions in CFU/mL (4.46 log 10). In the group of cancellous bone, the greatest reductions were found in the APDImb group (3.06 log 10). APDI with methylene blue, toluidine blue ortho and malachite green dyes and a 660 nm laser proved to be effective in the inactivation of S. aureus biofilms formed in compact and cancellous bone. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Studying bacterial multispecies biofilms

    DEFF Research Database (Denmark)

    Røder, Henriette Lyng; Sørensen, Søren Johannes; Burmølle, Mette

    2016-01-01

    the approaches used to study these complex communities. This review focuses on the establishment of multispecies biofilms in vitro, interspecies interactions in microhabitats, and how to select communities for evaluation. Studies have used different experimental approaches; here we evaluate the benefits......, but the identity and significance of interspecies bacterial interactions is neglected in these analyses. There is therefore an urgent need for bridging the gap between metagenomic analysis and in vitro models suitable for studies of bacterial interactions.Bacterial interactions and coadaptation are important...... at the microscale of complex communities, including biofilms.Studies of multispecies biofilms and the interactions shaping these are conducted in traditional approaches used for single-species biofilms with some adjustments; but a crucial point for consideration is which strains to combine and where these should...

  11. In vitro activity of alpha-mangostin in killing and eradicating Staphylococcus epidermidis RP62A biofilms.

    Science.gov (United States)

    Sivaranjani, Murugesan; Prakash, Manivannan; Gowrishankar, Shanmugaraj; Rathna, Janarthanam; Pandian, Shunmugiah Karutha; Ravi, Arumugam Veera

    2017-04-01

    Alpha-mangostin (α-MG) has been reported to be an effective antibacterial agent against planktonic cells of many Gram-positive bacteria. However, the antibiofilm potency of α-MG remains unexplored till date. In this study, the antibiofilm and mature biofilm eradication ability of α-MG against Staphylococcus epidermidis RP62A (ATCC 35984) biofilms were evaluated. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of α-MG against S. epidermidis RP62A were found to be 1.25 and 5 μg/mL, respectively. α-MG exhibited a phenomenal concentration dependent rapid bactericidal activity (>4-log reduction within 5 min). In a multi-passage resistance analysis using S. epidermidis, no development of resistance to α-MG as well as antibiotics was observed in its habituation. α-MG at its 1/2 MIC effectively inhibited the initial biofilm formation of S. epidermidis, which was further confirmed through scanning electron microscopic (SEM) analysis that portrayed a lucid reduction in the aggregation and the spread of biofilm. The crystal violet staining and viable cell quantification results confirmed the eradication of preformed immature and mature biofilms of S. epidermidis by α-MG in a concentration dependent manner. Besides, the biofilm eradication ability was also confirmed through SEM and live/dead BacLight staining using confocal laser scanning microscopy (CLSM). Thus, the present study exemplifies that α-MG could plausibly assist to eliminate biofilm infections associated with multidrug-resistance staphylococci.

  12. Combating biofilms

    DEFF Research Database (Denmark)

    Yang, Liang; Liu, Yang; Wu, Hong

    2012-01-01

    Biofilms are complex microbial communities consisting of microcolonies embedded in a matrix of self-produced polymer substances. Biofilm cells show much greater resistance to environmental challenges including antimicrobial agents than their free-living counterparts. The biofilm mode of life...... is believed to significantly contribute to successful microbial survival in hostile environments. Conventional treatment, disinfection and cleaning strategies do not proficiently deal with biofilm-related problems, such as persistent infections and contamination of food production facilities. In this review......, strategies to control biofilms are discussed, including those of inhibition of microbial attachment, interference of biofilm structure development and differentiation, killing of biofilm cells and induction of biofilm dispersion....

  13. Saccharomyces cerevisiae biofilm tolerance towards systemic antifungals depends on growth phase.

    Science.gov (United States)

    Bojsen, Rasmus; Regenberg, Birgitte; Folkesson, Anders

    2014-12-04

    Biofilm-forming Candida species cause infections that can be difficult to eradicate, possibly because of antifungal drug tolerance mechanisms specific to biofilms. In spite of decades of research, the connection between biofilm and drug tolerance is not fully understood. We used Saccharomyces cerevisiae as a model for drug susceptibility of yeast biofilms. Confocal laser scanning microscopy showed that S. cerevisiae and C. glabrata form similarly structured biofilms and that the viable cell numbers were significantly reduced by treatment of mature biofilms with amphotericin B but not voriconazole, flucytosine, or caspofungin. We showed that metabolic activity in yeast biofilm cells decreased with time, as visualized by FUN-1 staining, and mature, 48-hour biofilms contained cells with slow metabolism and limited growth. Time-kill studies showed that in exponentially growing planktonic cells, voriconazole had limited antifungal activity, flucytosine was fungistatic, caspofungin and amphotericin B were fungicidal. In growth-arrested cells, only amphotericin B had antifungal activity. Confocal microscopy and colony count viability assays revealed that the response of growing biofilms to antifungal drugs was similar to the response of exponentially growing planktonic cells. The response in mature biofilm was similar to that of non-growing planktonic cells. These results confirmed the importance of growth phase on drug efficacy. We showed that in vitro susceptibility to antifungal drugs was independent of biofilm or planktonic growth mode. Instead, drug tolerance was a consequence of growth arrest achievable by both planktonic and biofilm populations. Our results suggest that efficient strategies for treatment of yeast biofilm might be developed by targeting of non-dividing cells.

  14. Comparative proteomic analysis of Streptococcus suis biofilms and planktonic cells that identified biofilm infection-related immunogenic proteins.

    Science.gov (United States)

    Wang, Yang; Yi, Li; Wu, Zongfu; Shao, Jing; Liu, Guangjin; Fan, Hongjie; Zhang, Wei; Lu, Chengping

    2012-01-01

    Streptococcus suis (SS) is a zoonotic pathogen that causes severe disease symptoms in pigs and humans. Biofilms of SS bind to extracellular matrix proteins in both endothelial and epithelial cells and cause persistent infections. In this study, the differences in the protein expression profiles of SS grown either as planktonic cells or biofilms were identified using comparative proteomic analysis. The results revealed the existence of 13 proteins of varying amounts, among which six were upregulated and seven were downregulated in the Streptococcus biofilm compared with the planktonic controls. The convalescent serum from mini-pig, challenged with SS, was applied in a Western blot assay to visualize all proteins from the biofilm that were grown in vitro and separated by two-dimensional gel electrophoresis. A total of 10 immunoreactive protein spots corresponding to nine unique proteins were identified by MALDI-TOF/TOF-MS. Of these nine proteins, five (Manganese-dependent superoxide dismutase, UDP-N-acetylglucosamine 1-carboxyvinyltransferase, ornithine carbamoyltransferase, phosphoglycerate kinase, Hypothetical protein SSU05_0403) had no previously reported immunogenic properties in SS to our knowledge. The remaining four immunogenic proteins (glyceraldehyde-3-phosphate dehydrogenase, hemolysin, pyruvate dehydrogenase and DnaK) were identified under both planktonic and biofilm growth conditions. In conclusion, the protein expression pattern of SS, grown as biofilm, was different from the SS grown as planktonic cells. These five immunogenic proteins that were specific to SS biofilm cells may potentially be targeted as vaccine candidates to protect against SS biofilm infections. The four proteins common to both biofilm and planktonic cells can be targeted as vaccine candidates to protect against both biofilm and acute infections.

  15. The effect of propolis honey candy on C. Albicans and clinical isolate biofilms viability (in-vitro)

    Science.gov (United States)

    Soekanto, Sri Angky; Bachtiar, Endang W.; Ramadhan, Amatul Firdaus; Febrina, Riri; Sahlan, Muhamad

    2018-02-01

    The objective of this study was to analyze the effectiveness of Propolis honey candy on the formation of C. Albicans ATCC 10231 and Clinical Isolate biofilms. C. Albicans ATCC 10231 and Clinical Isolate were cultured on 96-wellplates that were previously coated with saliva and serum on each well plate. On each group, a solution of Propolis honey candy, X candy, and honey candy was distributed with a 50% concentration of solution. The well plates were then tested using MTT assay. For the X Candy, both C. Albicans ATCC 10231 and Clinical Isolate biofilms that were coated with saliva and serum showed a significant increase of biofilm formation (0.669±0.320) compared to the control (0.223±0.138). However, there were no significant differences between Propolis honey candy (0.171±0.120) and honey candy (0.217±0.112) in the formation of C. Albicans ATCC 10231 and Clinical Isolate biofilms compared to control. Propolis honey candy has a tendency to decrease the formation of C. Albicans ATCC 10231 and Clinical Isolate biofilms.

  16. In vitro activity of an engineered honey, medical-grade honeys, and antimicrobial wound dressings against biofilm-producing clinical bacterial isolates.

    Science.gov (United States)

    Halstead, F D; Webber, M A; Rauf, M; Burt, R; Dryden, M; Oppenheim, B A

    2016-02-01

    Honey is recognised to be a good topical wound care agent owing to a broad-spectrum of antimicrobial activity combined with healing properties. Surgihoney RO (SH1) is a product based on honey that is engineered to produce enhanced reactive oxygen species (ROS) and has been reported to be highly antimicrobial. The objective was to investigate the ability of the engineered honey and its comparators to prevent biofilm formation in vitro. We tested the ability of three medical-grade honeys SH1, Activon manuka honey (MH) and Medihoney manuka honey (Med), alongside five antimicrobial dressings (AMDs) to prevent the formation of biofilms by 16 isolates. Honeys were serially double diluted from 1:3 down to 1:6144 and the lowest dilution achieving a statistically significant reduction in biomass of at least 50%, compared with untreated controls, was recorded. Although all the honeys were antibacterial and were able to prevent the formation of biofilms, SH1 was the most potent, with efficacy at lower dilutions than the medical honeys for five isolates, and equivalent dilutions for a further six. Additionally, SH1 was superior in antibacterial potency to three commercially available AMDs that contain honey. SH1 is effective at preventing bioflms from forming and is superior to medical honeys and AMDs in in vitro tests. Surgihoney RO was provided free of charge for testing by Matoke Holdings, UK and the hospital pharmacy provided the other honeys and dressings. This paper presents independent research funded by the National Institute for Health Research (NIHR). The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health.

  17. Stress conditions triggering mucoid morphotype variation in Burkholderia species and effect on virulence in Galleria mellonella and biofilm formation in vitro.

    Directory of Open Access Journals (Sweden)

    Inês N Silva

    Full Text Available Burkholderia cepacia complex (Bcc bacteria are opportunistic pathogens causing chronic respiratory infections particularly among cystic fibrosis patients. During these chronic infections, mucoid-to-nonmucoid morphotype variation occurs, with the two morphotypes exhibiting different phenotypic properties. Here we show that in vitro, the mucoid clinical isolate Burkholderia multivorans D2095 gives rise to stable nonmucoid variants in response to prolonged stationary phase, presence of antibiotics, and osmotic and oxidative stresses. Furthermore, in vitro colony morphotype variation within other members of the Burkholderia genus occurred in Bcc and non-Bcc strains, irrespectively of their clinical or environmental origin. Survival to starvation and iron limitation was comparable for the mucoid parental isolate and the respective nonmucoid variant, while susceptibility to antibiotics and to oxidative stress was increased in the nonmucoid variants. Acute infection of Galleria mellonella larvae showed that, in general, the nonmucoid variants were less virulent than the respective parental mucoid isolate, suggesting a role for the exopolysaccharide in virulence. In addition, most of the tested nonmucoid variants produced more biofilm biomass than their respective mucoid parental isolate. As biofilms are often associated with increased persistence of pathogens in the CF lungs and are an indicative of different cell-to-cell interactions, it is possible that the nonmucoid variants are better adapted to persist in this host environment.

  18. A controlled release of antibiotics from calcium phosphate-coated poly(lactic-co-glycolic acid) particles and their in vitro efficacy against Staphylococcus aureus biofilm.

    Science.gov (United States)

    Bastari, Kelsen; Arshath, Mohamed; Ng, Zhi Hui Melissa; Chia, Jia Hua; Yow, Zhi Xian Daniel; Sana, Barindra; Tan, Meng Fong Cherine; Lim, Sierin; Loo, Say Chye Joachim

    2014-03-01

    Ceramic-polymer hybrid particles, intended for osteomyelitis treatment, were fabricated by preparing poly(lactic-co-glycolic acid) particles through an emulsion solvent evaporation technique, followed by calcium phosphate (CaP) coating via a surface adsorption-nucleation method. The presence of CaP coating on the surface of the particles was confirmed by scanning electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray photoelectron spectroscopy. Subsequently, two antibiotics for treating bone infection, nafcillin (hydrophilic) and levofloxacin (amphiphilic), were loaded into these hybrid particles and their in vitro drug release studies were investigated. The CaP coating was shown to reduce burst release, while providing sustained release of the antibiotics for up to 4 weeks. In vitro bacterial study against Staphylococcus aureus demonstrated the capability of these antibiotic-loaded hybrid particles to inhibit biofilm formation as well as deteriorate established biofilm, making this hybrid system a potential candidate for further investigation for osteomyelitis treatment.

  19. Analysis of Candida albicans mutants defective in the Cdk8 module of mediator reveal links between metabolism and biofilm formation.

    Directory of Open Access Journals (Sweden)

    Allia K Lindsay

    2014-10-01

    Full Text Available Candida albicans biofilm formation is a key virulence trait that involves hyphal growth and adhesin expression. Pyocyanin (PYO, a phenazine secreted by Pseudomonas aeruginosa, inhibits both C. albicans biofilm formation and development of wrinkled colonies. Using a genetic screen, we identified two mutants, ssn3Δ/Δ and ssn8Δ/Δ, which continued to wrinkle in the presence of PYO. Ssn8 is a cyclin-like protein and Ssn3 is similar to cyclin-dependent kinases; both proteins are part of the heterotetrameric Cdk8 module that forms a complex with the transcriptional co-regulator, Mediator. Ssn3 kinase activity was also required for PYO sensitivity as a kinase dead mutant maintained a wrinkled colony morphology in the presence of PYO. Furthermore, similar phenotypes were observed in mutants lacking the other two components of the Cdk8 module-Srb8 and Srb9. Through metabolomics analyses and biochemical assays, we showed that a compromised Cdk8 module led to increases in glucose consumption, glycolysis-related transcripts, oxidative metabolism and ATP levels even in the presence of PYO. In the mutant, inhibition of respiration to levels comparable to the PYO-treated wild type inhibited wrinkled colony development. Several lines of evidence suggest that PYO does not act through Cdk8. Lastly, the ssn3 mutant was a hyperbiofilm former, and maintained higher biofilm formation in the presence of PYO than the wild type. Together these data provide novel insights into the role of the Cdk8 module of Mediator in regulation of C. albicans physiology and the links between respiratory activity and both wrinkled colony and biofilm development.

  20. Structural and Functional Analysis of Bacillus subtilis YisP Reveal a Role of its Product in Biofilm Production

    Science.gov (United States)

    Feng, Xinxin; Hu, Yumei; Zheng, Yingying; Zhu, Wei; Li, Kai; Huang, Chun-Hsiang; Ko, Tzu-Ping; Ren, Feifei; Chan, Hsiu-Chien; Nega, Mulugeta; Bogue, Shannon; López, Daniel; Kolter, Roberto; Götz, Friedrich; Guo, Rey-Ting; Oldfield, Eric

    2014-01-01

    Summary YisP is involved in biofilm formation in Bacillus subtilis and has been predicted to produce C30 isoprenoids. We determined the structure of YisP and observe that it adopts the same fold as squalene and dehydrosqualene synthases. However, the first aspartate-rich motif found in essentially all isoprenoid synthases is aspartate-poor in YisP, and cannot catalyze head-to-head condensation reactions. We find that YisP acts as a phosphatase, catalyzing formation of farnesol from farnesyl diphosphate, and is the first phosphatase to adopt the fold seen in the head-to-head prenyl synthases. Farnesol restores biofilm formation in a Δyisp mutant, and modifies lipid membrane structure similarly to the virulence factor, staphyloxanthin. The work clarifies the role of YisP in biofilm formation and suggests an intriguing possibility that many of the YisP-like homologs found in other bacteria may also have interesting products and functions. PMID:25308276

  1. Structural and functional analysis of Bacillus subtilis YisP reveals a role of its product in biofilm production.

    Science.gov (United States)

    Feng, Xinxin; Hu, Yumei; Zheng, Yingying; Zhu, Wei; Li, Kai; Huang, Chun-Hsiang; Ko, Tzu-Ping; Ren, Feifei; Chan, Hsiu-Chien; Nega, Mulugeta; Bogue, Shannon; López, Daniel; Kolter, Roberto; Götz, Friedrich; Guo, Rey-Ting; Oldfield, Eric

    2014-11-20

    YisP is involved in biofilm formation in Bacillus subtilis and has been predicted to produce C30 isoprenoids. We determined the structure of YisP and observed that it adopts the same fold as squalene and dehydrosqualene synthases. However, the first aspartate-rich motif found in essentially all isoprenoid synthases is aspartate poor in YisP and cannot catalyze head-to-head condensation reactions. We find that YisP acts as a phosphatase, catalyzing formation of farnesol from farnesyl diphosphate, and that it is the first phosphatase to adopt the fold seen in the head-to-head prenyl synthases. Farnesol restores biofilm formation in a Δyisp mutant and modifies lipid membrane structure similarly to the virulence factor staphyloxanthin. This work clarifies the role of YisP in biofilm formation and suggests an intriguing possibility that many of the YisP-like homologs found in other bacteria may also have interesting products and functions. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Changes in gene expression of Actinobacillus pleuropneumoniae in response to anaerobic stress reveal induction of central metabolism and biofilm formation.

    Science.gov (United States)

    Li, Lu; Zhu, Jiawen; Yang, Kui; Xu, Zhuofei; Liu, Ziduo; Zhou, Rui

    2014-06-01

    Actinobacillus pleuropneumoniae is an important porcine respiratory pathogen causing great economic losses in the pig industry worldwide. Oxygen deprivation is a stress that A. pleuropneumoniae will encounter during both early infection and the later, persistent stage. To understand modulation of A. pleuropneumoniae gene expression in response to the stress caused by anaerobic conditions, gene expression profiles under anaerobic and aerobic conditions were compared in this study. The microarray results showed that 631 genes (27.7% of the total ORFs) were differentially expressed in anaerobic conditions. Many genes encoding proteins involved in glycolysis, carbon source uptake systems, pyruvate metabolism, fermentation and the electron respiration transport chain were up-regulated. These changes led to an increased amount of pyruvate, lactate, ethanol and acetate in the bacterial cells as confirmed by metabolite detection. Genes encoding proteins involved in cell surface structures, especially biofilm formation, peptidoglycan biosynthesis and lipopolysaccharide biosynthesis were up-regulated as well. Biofilm formation was significantly enhanced under anaerobic conditions. These results indicate that induction of central metabolism is important for basic survival of A. pleuropneumoniae after a shift to an anaerobic environment. Enhanced biofilm formation may contribute to the persistence of this pathogen in the damaged anaerobic host tissue and also in the early colonization stage. These discoveries give new insights into adaptation mechanisms of A. pleuropneumoniae in response to environmental stress.

  3. A new model of in vitro fungal biofilms formed on human nail fragments allows reliable testing of laser and light therapies against onychomycosis.

    Science.gov (United States)

    Vila, Taissa Vieira Machado; Rozental, Sonia; de Sá Guimarães, Claudia Maria Duarte

    2015-04-01

    Onychomycoses represent approximately 50 % of all nail diseases worldwide. In warmer and more humid countries like Brazil, the incidence of onychomycoses caused by non-dermatophyte molds (NDM, including Fusarium spp.) or yeasts (including Candida albicans) has been increasing. Traditional antifungal treatments used for the dermatophyte-borne disease are less effective against onychomycoses caused by NDM. Although some laser and light treatments have demonstrated clinical efficacy against onychomycosis, their US Food and Drug Administration (FDA) approval as "first-line" therapy is pending, partly due to the lack of well-demonstrated fungicidal activity in a reliable in vitro model. Here, we describe a reliable new in vitro model to determine the fungicidal activity of laser and light therapies against onychomycosis caused by Fusarium oxysporum and C. albicans. Biofilms formed in vitro on sterile human nail fragments were treated with 1064 nm neodymium-doped yttrium aluminum garnet laser (Nd:YAG), 420 nm intense pulsed light (IPL) IPL 420, followed by Nd:YAG, or near-infrared light ((NIR) 700-1400 nm). Light and laser antibiofilm effects were evaluated using cell viability assay and scanning electron microscopy (SEM). All treatments were highly effective against C. albicans and F. oxysporum biofilms, resulting in decreases in cell viability of 45-60 % for C. albicans and 92-100 % for F. oxysporum. The model described here yielded fungicidal activities that matched more closely to those observed in the clinic, when compared to published in vitro models for laser and light therapies. Thus, our model might represent an important tool for the initial testing, validation, and "fine-tuning" of laser and light therapies against onychomycosis.

  4. A Phytoanticipin Derivative, Sodium Houttuyfonate, Induces in Vitro Synergistic Effects with Levofloxacin against Biofilm Formation by Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Jing Shao

    2012-09-01

    Full Text Available Antibiotic resistance has become the main deadly factor in infections, as bacteria can protect themselves by hiding in a self-constructed biofilm. Consequently, more attention is being paid to the search for “non-antibiotic drugs” to solve this problem. Phytoanticipins, the natural antibiotics from plants, could be a suitable alternative, but few works on this aspect have been reported. In this study, a preliminary study on the synergy between sodium houttuyfonate (SH and levofloxacin (LFX against the biofilm formation of Pseudomonas aeruginosa was performed. The minimal inhibitory concentrations (MIC of LFX and SH, anti-biofilm formation and synergistic effect on Pseudomonas aeruginosa, and quantification of alginate were determined by the microdilution method, crystal violet (CV assay, checkerboard method, and hydroxybiphenyl colorimetry. The biofilm morphology of Pseudomonas aeruginosa was observed by fluorescence microscope and scanning electric microscope (SEM. The results showed that: (i LFX and SH had an obvious synergistic effect against Pseudomonas aeruginosa with MIC values of 0.25 μg/mL and 128 μg/mL, respectively; (ii ½ × MIC SH combined with 2 × MIC LFX could suppress the biofilm formation of Pseudomonas aeruginosa effectively, with up to 73% inhibition; (iii the concentration of alginate decreased dramatically by a maximum of 92% after treatment with the combination of antibiotics; and (iv more dead cells by fluorescence microscope and more removal of extracellular polymeric structure (EPS by SEM were observed after the combined treatment of LFX and SH. Our experiments demonstrate the promising future of this potent antimicrobial agent against biofilm-associated infections.

  5. Transcriptional Profiling of C. albicans in a Two Species Biofilm with Rothia dentocariosa

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    Priya Uppuluri

    2017-07-01

    Full Text Available Biofilms on silicone rubber voice prostheses are the major cause for frequent failure and replacement of these devices. The presence of both bacterial and yeast strains has been suggested to be crucial for the development of voice prosthetic biofilms. Polymicrobial biofilms that include Candida albicans and Rothia dentocariosa are the leading cause of voice prosthesis failure. An in vitro biofilm comprising these two organisms was developed on silicone rubber, a material used for Groningen button voice prosthesis. We found that this biofilm environment was not conducive for C. albicans growth or differentiation. Global transcriptional analyses of C. albicans biofilm cells grown with R. dentocariosa revealed that genes with functions related to cell cycle progression and hyphal development were repressed >2-fold. The mixed species biofilms were more compact and less robust compared to C. albicans mono-species biofilms, even when developed under conditions of continuous nutrient flow. Under these conditions R. dentocariosa also significantly inhibited C. albicans biofilm dispersal. Preferential adherence of R. dentocariosa to candidal hyphae was mediated by the adhesin Als3.

  6. The efficacy of photodynamic and photothermal therapy on biofilm formation of Streptococcus mutans: An in vitro study.

    Science.gov (United States)

    Beytollahi, Leili; Pourhajibagher, Maryam; Chiniforush, Nasim; Ghorbanzadeh, Roghayeh; Raoofian, Reza; Pourakbari, Babak; Bahador, Abbas

    2017-03-01

    The alternative antibacterial treatments of photodynamic therapy (PDT) and photothermal therapy (PTT) significantly affect microbiota inactivation. The aim of the present research was the assessment of the antimicrobial and anti-biofilm effects of PDT with toluidine blue O (TBO) and PTT with indocyanine green (ICG) on Streptococcus mutans as a cariogenic bacterium. The S. mutans ATCC 35668 strain was treated with final concentrations of 0.1mg/mL TBO and 1mg/mL ICG with energy densities of 17.18 and 15.62J/cm2, respectively. Cell viability was evaluated after culturing and anti-biofilm potential was analyzed using crystal violet assay and scanning electron microscopy. The number of S. mutans colony forming unit (CFU)/mL was significantly lower in the groups submitted to PDT (12.5-100μg/mL TBO) and PTT (62.5-1000μg/mL) compared to the control (untreated group). 0.1mg/mL TBO-PDT and 1mg/mL ICG-PTT showed stronger inhibitory effects on biofilm formation in S. mutans than other concentration levels, with a reduction of 63.87% and 67.3%, respectively. Photo-elimination by high concentrations of TBO-PDT and ICG-PTT exhibited significantly stronger inhibitory effects on biofilm formation and cell viability in S. mutans. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Hibiscus sabdariffa extract inhibits in vitro biofilm formation capacity of Candida albicans isolated from recurrent urinary tract infections.

    Science.gov (United States)

    Alshami, Issam; Alharbi, Ahmed E

    2014-02-01

    To explore the prevention of recurrent candiduria using natural based approaches and to study the antimicrobial effect of Hibiscus sabdariffa (H. sabdariffa) extract and the biofilm forming capacity of Candida albicans strains in the present of the H. sabdariffa extract. In this particular study, six strains of fluconazole resistant Candida albicans isolated from recurrent candiduria were used. The susceptibility of fungal isolates, time-kill curves and biofilm forming capacity in the present of the H. sabdariffa extract were determined. Various levels minimum inhibitory concentration of the extract were observed against all the isolates. Minimum inhibitory concentration values ranged from 0.5 to 2.0 mg/mL. Time-kill experiment demonstrated that the effect was fungistatic. The biofilm inhibition assay results showed that H. sabdariffa extract inhibited biofilm production of all the isolates. The results of the study support the potential effect of H. sabdariffa extract for preventing recurrent candiduria and emphasize the significance of the plant extract approach as a potential antifungal agent.

  8. NOSOCOMIAL INFECTION AND MICROBIAL BIOFILMS IN SURGERY

    Directory of Open Access Journals (Sweden)

    N. I. Gabrielyan

    2012-01-01

    Full Text Available The review presents data on the role of biofilms formation by opportunistic microbes in surgery. It gives in- formation about the microbial structure of biofilms, their architecture and physiology. The attention was payed to significal importance of microbial communities, which form biofilms in surgery. Mechanisms of increased resistance of biofilms bacteria are compared with plankton. The review includes literature data on the process of formation biofilms on intravascular catheters and methods of inhibition and protection. Methods of studing formation and inhibition of biofilms in vitro and in vivo are presented. Different biotechnology methods, based on using antiadhesive, antiseptic, biophysical resources and biomaterials are discussed. 

  9. Metagenome survey of a multispecies and alga-associated biofilm revealed key elements of bacterial-algal interactions in photobioreactors.

    Science.gov (United States)

    Krohn-Molt, Ines; Wemheuer, Bernd; Alawi, Malik; Poehlein, Anja; Güllert, Simon; Schmeisser, Christel; Pommerening-Röser, Andreas; Grundhoff, Adam; Daniel, Rolf; Hanelt, Dieter; Streit, Wolfgang R

    2013-10-01

    Photobioreactors (PBRs) are very attractive for sunlight-driven production of biofuels and capturing of anthropogenic CO2. One major problem associated with PBRs however, is that the bacteria usually associated with microalgae in nonaxenic cultures can lead to biofouling and thereby affect algal productivity. Here, we report on a phylogenetic, metagenome, and functional analysis of a mixed-species bacterial biofilm associated with the microalgae Chlorella vulgaris and Scenedesmus obliquus in a PBR. The biofilm diversity and population dynamics were examined through 16S rRNA phylogeny. Overall, the diversity was rather limited, with approximately 30 bacterial species associated with the algae. The majority of the observed microorganisms were affiliated with Alphaproteobacteria, Betaproteobacteria, and Bacteroidetes. A combined approach of sequencing via GS FLX Titanium from Roche and HiSeq 2000 from Illumina resulted in the overall production of 350 Mbp of sequenced DNA, 165 Mbp of which was assembled in larger contigs with a maximum size of 0.2 Mbp. A KEGG pathway analysis suggested high metabolic diversity with respect to the use of polymers and aromatic and nonaromatic compounds. Genes associated with the biosynthesis of essential B vitamins were highly redundant and functional. Moreover, a relatively high number of predicted and functional lipase and esterase genes indicated that the alga-associated bacteria are possibly a major sink for lipids and fatty acids produced by the microalgae. This is the first metagenome study of microalga- and PBR-associated biofilm bacteria, and it gives new clues for improved biofuel production in PBRs.

  10. Evaluation of Cold Plasma Treatment and Safety in Disinfecting 3-week Root Canal Enterococcus faecalis Biofilm In Vitro.

    Science.gov (United States)

    Li, Yinglong; Sun, Ke; Ye, Guopin; Liang, Yongdong; Pan, Hong; Wang, Guomin; Zhao, Yijiao; Pan, Jie; Zhang, Jue; Fang, Jing

    2015-08-01

    Although endodontic infection is caused by multi-bacteria species, Enterococcus faecalis is usually isolated in chronic apical periodontitis. The aim of this study was to evaluate the effectiveness and mechanical safety of cold plasma therapy in disinfecting 3-week E. faecalis biofilms. Teeth with 3-week E. faecalis biofilm were treated with AC argon/oxygen (Ar/O2) cold plasma for various treatment times and compared with those treated with Ca(OH)2, 2% chlorhexidine gel, and Ca(OH)2/chlorhexidine for a week. Antimicrobial efficacy was assessed by colony-forming unit method. Scanning electron microscopy was used to assess the morphologic changes of E. faecalis biofilm by plasma. Confocal laser scanning microscopy was used to confirm the viability of the biofilm after the plasma treatment. Microhardness and roughness changes of root canal dentin caused by plasma were verified with Vickers Hardness Tester and 3D Profile Measurement Laser Microscope, respectively. There were no detectable live bacteria after 12 minutes of cold plasma treatment. This was further confirmed by scanning electron microscopy and confocal laser scanning microscopy results. Microhardness and roughness of root canal dentin showed no significant difference after plasma treatment. Atmospheric pressure cold plasma is an effective therapy in endodontics for its strong sterilization effect on fully matured biofilm within a few minutes. Meanwhile, it has an accepted mechanical safety for its low temperature and not affecting the microhardness and roughness of root canal dentin significantly. Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  11. Persea americana Glycolic Extract: In Vitro Study of Antimicrobial Activity against Candida albicans Biofilm and Cytotoxicity Evaluation

    Directory of Open Access Journals (Sweden)

    D. Jesus

    2015-01-01

    Full Text Available This study evaluated the antifungal activity of Persea americana extract on Candida albicans biofilm and its cytotoxicity in macrophage culture (RAW 264.7. To determine the minimum inhibitory concentration (MIC, microdilution in broth (CLSI M27-S4 protocol was performed. Thereafter, the concentrations of 12.5, 25, 50, 100, and 200 mg/mL (n=10 with 5 min exposure were analyzed on mature biofilm in microplate wells for 48 h. Saline was used as control (n=10. After treatment, biofilm cells were scraped off and dilutions were plated on Sabouraud dextrose agar. After incubation (37°C/48 h, the values of colony forming units per milliliter (CFU/mL were converted to log10 and analyzed (ANOVA and Tukey test, 5%. The cytotoxicity of the P. americana extract was evaluated on macrophages by MTT assay. The MIC of the extract was 6.25 mg/mL and with 12.5 mg/mL there was elimination of 100% of planktonic cultures. Regarding the biofilms, a significant reduction (P<0.001 of the biofilm at concentrations of 50 (0.580±0.209 log10, 100 (0.998±0.508 log10, and 200 mg/mL (1.093±0.462 log10 was observed. The concentrations of 200 and 100 mg/mL were cytotoxic for macrophages, while the concentrations of 50, 25, and 12.5 mg/mL showed viability higher than 55%.

  12. Persea americana Glycolic Extract: In Vitro Study of Antimicrobial Activity against Candida albicans Biofilm and Cytotoxicity Evaluation.

    Science.gov (United States)

    Jesus, D; Oliveira, J R; Oliveira, F E; Higa, K C; Junqueira, J C; Jorge, A O C; Back-Brito, G N; Oliveira, L D

    2015-01-01

    This study evaluated the antifungal activity of Persea americana extract on Candida albicans biofilm and its cytotoxicity in macrophage culture (RAW 264.7). To determine the minimum inhibitory concentration (MIC), microdilution in broth (CLSI M27-S4 protocol) was performed. Thereafter, the concentrations of 12.5, 25, 50, 100, and 200 mg/mL (n = 10) with 5 min exposure were analyzed on mature biofilm in microplate wells for 48 h. Saline was used as control (n = 10). After treatment, biofilm cells were scraped off and dilutions were plated on Sabouraud dextrose agar. After incubation (37°C/48 h), the values of colony forming units per milliliter (CFU/mL) were converted to log10 and analyzed (ANOVA and Tukey test, 5%). The cytotoxicity of the P. americana extract was evaluated on macrophages by MTT assay. The MIC of the extract was 6.25 mg/mL and with 12.5 mg/mL there was elimination of 100% of planktonic cultures. Regarding the biofilms, a significant reduction (P < 0.001) of the biofilm at concentrations of 50 (0.580 ± 0.209 log10), 100 (0.998 ± 0.508 log10), and 200 mg/mL (1.093 ± 0.462 log10) was observed. The concentrations of 200 and 100 mg/mL were cytotoxic for macrophages, while the concentrations of 50, 25, and 12.5 mg/mL showed viability higher than 55%.

  13. Piper betle and its bioactive metabolite phytol mitigates quorum sensing mediated virulence factors and biofilm of nosocomial pathogen Serratia marcescens in vitro.

    Science.gov (United States)

    Srinivasan, Ramanathan; Devi, Kannan Rama; Kannappan, Arunachalam; Pandian, Shunmugiah Karutha; Ravi, Arumugam Veera

    2016-12-04

    Piper betle, a tropical creeper plant belongs to the family Piperaceae. The leaves of this plant have been well known for their therapeutic, religious and ceremonial value in South and Southeast Asia. It has also been reported to possess several biological activities including antimicrobial, antioxidant, antinociceptive, antidiabetic, insecticidal and gastroprotective activities and used as a common ingredient in indigenous medicines. In Indian system of ayurvedic medicine, P. betle has been well recognized for its antiseptic properties and is commonly applied on wounds and lesions for its healing effects. To evaluate the anti-quorum sensing (anti-QS) and antibiofilm efficacy of P. betle and its bioactive metabolite phytol against Serratia marcescens. The P. betle ethyl acetate extract (PBE) was evaluated for its anti-QS efficacy against S. marcescens by assessing the prodigiosin and lipase production at 400 and 500µgml(-1) concentrations. In addition, the biofilm biomass quantification assay was performed to evaluate the antibiofilm activity of PBE against S. marcescens. Besides, the influence of PBE on bacterial biofilm formation was assessed through microscopic techniques. The biofilm related phenomenons like exopolysaccharides (EPS) production, hydrophobicity and swarming motility were also examined to support the antibiofilm activity of PBE. Transcriptional analysis of QS regulated genes in S. marcescens was also done. Characterization of PBE was done by separation through column chromatography and identification of active metabolites by gas chromatography -mass spectrometry. The major compounds of active fractions such as hexadecanoic acid, eugenol and phytol were assessed for their anti-QS activity against S. marcescens. Further, the in vitro bioassays such as protease, biofilm and HI quantification were also carried out to confirm the anti-QS and antibiofilm potential of phytol in PBE. PBE inhibits QS mediated prodigiosin pigment production in S

  14. Role of gallium and silver from phosphate-based glasses on in vitro dual species oral biofilm models of Porphyromonas gingivalis and Streptococcus gordonii.

    Science.gov (United States)

    Valappil, Sabeel P; Coombes, Marc; Wright, Lucy; Owens, Gareth J; Lynch, Richard J M; Hope, Christopher K; Higham, Susan M

    2012-05-01

    Phosphate-based glasses (PBGs) are excellent controlled delivery agents for antibacterial ions such as silver and gallium. The aim of this study was to assess the potential utility of novel PBGs combining both gallium and silver for use in periodontal therapy. To this end, an in vitro biofilm model with the putative periodontal pathogen, Porphyromonas gingivalis, and an initial colonizer, Streptococcus gordonii, was established. The effect of increasing calcium content in gallium-silver-doped PBG on the susceptibility of P. gingivalis was examined. A decrease in degradation rates (30.34, 25.19, 21.40 μg mm(-2) h(-1)) with increasing PBG calciumcontent (10, 11, 12 mol.% respectively) was observed, correlating well with gallium and silver ion release and antimicrobial activity against planktonic P. gingivalis (approximately 5.4log(10) colony-forming units (CFU) reduction after 24h by the C10 glass compared with controls) and S. gordonii (total growth inhibition after 32h by C10, C11 and C12 glasses compared with controls). The most potent PBG (C10) was evaluated for its ability to inhibit the biofilm growth of P. gingivalis in a newly established constant-depth film fermentor model. The simultaneous release of silver and gallium from the glass reduced P. gingivalis biofilm growth with a maximum effect (1.92log(10) CFU reduction) after 168 h. Given the emergence of antibiotic-resistant bacteria and dearth of new antibiotics in development, the glasses, especially C10, would offer effective alternatives to antibiotics or may complement current therapies through controlled, localized delivery of gallium and silver ions at infected sites in the oral cavity. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  15. The effects of diode laser on Staphylococcus aureus biofilm and Escherichia coli lipopolysaccharide adherent to titanium oxide surface of dental implants. An in vitro study.

    Science.gov (United States)

    Giannelli, Marco; Landini, Giulia; Materassi, Fabrizio; Chellini, Flaminia; Antonelli, Alberto; Tani, Alessia; Zecchi-Orlandini, Sandra; Rossolini, Gian Maria; Bani, Daniele

    2016-11-01

    Effective decontamination of biofilm and bacterial toxins from the surface of dental implants is a yet unresolved issue. This in vitro study aims at providing the experimental basis for possible use of diode laser (λ 808 nm) in the treatment of peri-implantitis. Staphylococcus aureus biofilm was grown for 48 h on titanium discs with porous surface corresponding to the bone-implant interface and then irradiated with a diode laser (λ 808 nm) in noncontact mode with airflow cooling for 1 min using a Ø 600-μm fiber. Setting parameters were 2 W (400 J/cm2) for continuous wave mode; 22 μJ, 20 kHz, 7 μs (88 J/cm2) for pulsed wave mode. Bactericidal effect was evaluated using fluorescence microscopy and counting the residual colony-forming units. Biofilm and titanium surface morphology were analyzed by scanning electron microscopy (SEM). In parallel experiments, the titanium discs were coated with Escherichia coli lipopolysaccharide (LPS), laser-irradiated and seeded with RAW 264.7 macrophages to quantify LPS-driven inflammatory cell activation by measuring the enhanced generation of nitric oxide (NO). Diode laser irradiation in both continuous and pulsed modes induced a statistically significant reduction of viable bacteria and nitrite levels. These results indicate that in addition to its bactericidal effect laser irradiation can also inhibit LPS-induced macrophage activation and thus blunt the inflammatory response. The λ 808-nm diode laser emerges as a valuable tool for decontamination/detoxification of the titanium implant surface and may be used in the treatment of peri-implantitis.

  16. Effectiveness of intracanal dressing protocols on Enterococcus faecalis biofilm in a bovine teeth model: an in vitro study = Eficácia de protocolos de medicação intracanal sobre o biofilme de Enterococcus faecalis em um modelo de dentes bovinos: estudo in vitro

    OpenAIRE

    Borba, Maristela Gutiérrez de; Souza,Matheus Albino; Vier-Pelisser,Fabiana Vieira; Ghisi,Alexandre Correa; Nasário, Jéssica Stéphanie Rodrigues; Oliveira,Silvia Dias de; Figueiredo,José Antonio Poli de

    2015-01-01

    Objetivo: O propósito deste estudo foi avaliar, in vitro, a eficácia de diferentes protocolos de medicação intracanal em canais radiculares infectados com Enterococcus faecalis. Métodos: Oitenta e oito incisivos bovinos foram contaminados com Enterococcus faecalis, permanecendo em cultura por 30 dias para a formação do biofilme. Os dentes foram divididos em dez grupos de acordo com a presença de penetração desinfetante, medicação intracanal utilizada e o local de colocação desta medicação: G1...

  17. Polymicrobial Ventilator-Associated Pneumonia: Fighting In Vitro Candida albicans-Pseudomonas aeruginosa Biofilms with Antifungal-Antibacterial Combination Therapy

    OpenAIRE

    Rodrigues, Maria E.; Lopes, Susana P.; Pereira, Cl?udia R.; Azevedo, Nuno F.; Louren?o, An?lia; Henriques, Mariana; Pereira, Maria O.

    2017-01-01

    The polymicrobial nature of ventilator-associated pneumonia (VAP) is now evident, with mixed bacterial-fungal biofilms colonizing the VAP endotracheal tube (ETT) surface. The microbial interplay within this infection may contribute for enhanced pathogenesis and exert impact towards antimicrobial therapy. Consequently, the high mortality/morbidity rates associated to VAP and the worldwide increase in antibiotic resistance has promoted the search for novel therapeutic strategies to fight VAP po...

  18. High in vitro antimicrobial activity of β-peptoid-peptide hybrid oligomers against planktonic and biofilm cultures of Staphylococcus epidermidis

    DEFF Research Database (Denmark)

    Liu, Yang; Knapp, Kolja Michael; Yang, Liang

    2013-01-01

    antibiotic vancomycin. Susceptibility and time-kill assays were performed to investigate activity against planktonic cells, whilst confocal laser scanning microscopy was used to investigate the dynamics of the activity against cells within biofilms. All tested peptidomimetics were bactericidal against both...... killing of bacterial cells. This class of peptidomimetics may constitute promising antimicrobial alternatives for the prevention and treatment of multidrug-resistant S. epidermidis infections....

  19. Effect of desensitising paste containing 8% arginine and calcium carbonate on biofilm formation of Streptococcus mutans in vitro.

    Science.gov (United States)

    Fu, Dongjie; Pei, Dandan; Huang, Cui; Liu, Yinchen; Du, Xijin; Sun, Hualing

    2013-07-01

    To evaluate the influence of desensitising paste containing 8% arginine and calcium carbonate (Ar-Ca) on biofilm formation on dentine. Dentine discs were cut from extracted third molars and divided into the following three groups: no treatment, pumice treatment and Ar-Ca treatment. Surface topography and roughness were examined using scanning electron microscopy (SEM) and non-contact 3D surface profiler. After sterilisation, samples were incubated with Streptococcus mutans (S. mutans) for 4 h, 24 h and 72 h. Bacterial adhesion and biofilm formation were analysed using SEM, whereas MTT and lactic acid production assays were used to analyse the metabolic activity of S. mutans. After polishing with either pumice or Ar-Ca, the surfaces of the samples became smoother than in the control group. The Ra values of the three experimental groups decreased significantly to 0.43 μm, 0.3 μm and 0.26 μm, respectively. Compared to the control group, fewer bacteria adhered to the dentine surface in the Ar-Ca group, while biofilm thickness decreased significantly for both groups after incubating for 24 h and 72 h. MTT and lactic acid production levers also showed a significant reduction in the Ar-Ca group. Ar-Ca appears to present antibiofilm efficacy and may provide a promising approach to combat bacterial infection in hypersensitive dentinal lesions. As a clinical application of desensitising polishing paste, the paste containing 8% arginine and calcium carbonate could also inhibit the biofilm formation effectively. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

  20. Molecular basis of in-vivo biofilm formation by bacterial pathogens

    OpenAIRE

    Joo, Hwang-Soo; Otto, Michael

    2012-01-01

    Bacterial biofilms are involved in a multitude of serious chronic infections. In recent years, modeling biofilm infection in vitro led to the identification of microbial determinants governing biofilm development. However, we lack information as to whether biofilm formation mechanisms identified in vitro have relevance for biofilm-associated infection. Here, we discuss the molecular basis of biofilm formation using staphylococci and Pseudomonas aeruginosa to illustrate key points, as their bi...

  1. Effectiveness of repeated photodynamic therapy in the elimination of intracanal Enterococcus faecalis biofilm: an in vitro study.

    Science.gov (United States)

    Prażmo, Ewa Joanna; Godlewska, Renata Alicja; Mielczarek, Agnieszka Beata

    2017-04-01

    The study aimed to investigate the effectiveness of photodynamic therapy in the elimination of intracanal Enterococcus faecalis biofilm and to analyse how a repeated light irradiation, replenishment of oxygen and photosensitiser affect the results of the photodynamic disinfecting protocol. After chemomechanical preparation, 46 single-rooted human teeth were infected with a clinical strain of E. faecalis and incubated for a week in microaerobic conditions. The experimental procedures included groups of single application of photodynamic therapy, two cycles of PDT, irrigation with 5.25% NaOCl solution and negative and positive control. The number of residing bacterial colonies in the root canals was determined based on the CFU/ml method. In the group of preparations irrigated with NaOCl, bacterial colonies were not observed. A single PDT eliminated 45% of the initial CFU/ml. Repeated PDT eradicated 95% of the intracanal bacterial biofilm. Photodynamic therapy has a high potential for the elimination of E. faecalis biofilm. There is a safe therapeutic window where photoinduced disinfection can be used as an adjuvant to conventional endodontic treatment, which remains the most effective.

  2. Efficacy of a marine bacterial nuclease against biofilm forming microorganisms isolated from chronic rhinosinusitis.

    Directory of Open Access Journals (Sweden)

    Robert C Shields

    Full Text Available BACKGROUND: The persistent colonization of paranasal sinus mucosa by microbial biofilms is a major factor in the pathogenesis of chronic rhinosinusitis (CRS. Control of microorganisms within biofilms is hampered by the presence of viscous extracellular polymers of host or microbial origin, including nucleic acids. The aim of this study was to investigate the role of extracellular DNA in biofilm formation by bacteria associated with CRS. METHODS/PRINCIPAL FINDINGS: Obstructive mucin was collected from patients during functional endoscopic sinus surgery. Examination of the mucous by transmission electron microscopy revealed an acellular matrix punctuated occasionally with host cells in varying states of degradation. Bacteria were observed in biofilms on mucosal biopsies, and between two and six different species were isolated from each of 20 different patient samples. In total, 16 different bacterial genera were isolated, of which the most commonly identified organisms were coagulase-negative staphylococci, Staphylococcus aureus and α-haemolytic streptococci. Twenty-four fresh clinical isolates were selected for investigation of biofilm formation in vitro using a microplate model system. Biofilms formed by 14 strains, including all 9 extracellular nuclease-producing bacteria, were significantly disrupted by treatment with a novel bacterial deoxyribonuclease, NucB, isolated from a marine strain of Bacillus licheniformis. Extracellular biofilm matrix was observed in untreated samples but not in those treated with NucB and extracellular DNA was purified from in vitro biofilms. CONCLUSION/SIGNIFICANCE: Our data demonstrate that bacteria associated with CRS form robust biofilms which can be reduced by treatment with matrix-degrading enzymes such as NucB. The dispersal of bacterial biofilms with NucB may offer an additional therapeutic target for CRS sufferers.

  3. Bacterial biofilm and associated infections.

    Science.gov (United States)

    Jamal, Muhsin; Ahmad, Wisal; Andleeb, Saadia; Jalil, Fazal; Imran, Muhammad; Nawaz, Muhammad Asif; Hussain, Tahir; Ali, Muhammad; Rafiq, Muhammad; Kamil, Muhammad Atif

    2018-01-01

    Microscopic entities, microorganisms that drastically affect human health need to be thoroughly investigated. A biofilm is an architectural colony of microorganisms, within a matrix of extracellular polymeric substance that they produce. Biofilm contains microbial cells adherent to one-another and to a static surface (living or non-living). Bacterial biofilms are usually pathogenic in nature and can cause nosocomial infections. The National Institutes of Health (NIH) revealed that among all microbial and chronic infections, 65% and 80%, respectively, are associated with biofilm formation. The process of biofilm formation consists of many steps, starting with attachment to a living or non-living surface that will lead to formation of micro-colony, giving rise to three-dimensional structures and ending up, after maturation, with detachment. During formation of biofilm several species of bacteria communicate with one another, employing quorum sensing. In general, bacterial biofilms show resistance against human immune system, as well as against antibiotics. Health related concerns speak loud due to the biofilm potential to cause diseases, utilizing both device-related and non-device-related infections. In summary, the understanding of bacterial biofilm is important to manage and/or to eradicate biofilm-related diseases. The current review is, therefore, an effort to encompass the current concepts in biofilm formation and its implications in human health and disease. Copyright © 2017. Published by Elsevier Taiwan LLC.

  4. Bacterial biofilm and associated infections

    Directory of Open Access Journals (Sweden)

    Muhsin Jamal

    2018-01-01

    Full Text Available Microscopic entities, microorganisms that drastically affect human health need to be thoroughly investigated. A biofilm is an architectural colony of microorganisms, within a matrix of extracellular polymeric substance that they produce. Biofilm contains microbial cells adherent to one-another and to a static surface (living or non-living. Bacterial biofilms are usually pathogenic in nature and can cause nosocomial infections. The National Institutes of Health (NIH revealed that among all microbial and chronic infections, 65% and 80%, respectively, are associated with biofilm formation. The process of biofilm formation consists of many steps, starting with attachment to a living or non-living surface that will lead to formation of micro-colony, giving rise to three-dimensional structures and ending up, after maturation, with detachment. During formation of biofilm several species of bacteria communicate with one another, employing quorum sensing. In general, bacterial biofilms show resistance against human immune system, as well as against antibiotics. Health related concerns speak loud due to the biofilm potential to cause diseases, utilizing both device-related and non-device-related infections. In summary, the understanding of bacterial biofilm is important to manage and/or to eradicate biofilm-related diseases. The current review is, therefore, an effort to encompass the current concepts in biofilm formation and its implications in human health and disease.

  5. Rapid identification of bacterial biofilms and biofilm wound models using a multichannel nanosensor.

    Science.gov (United States)

    Li, Xiaoning; Kong, Hao; Mout, Rubul; Saha, Krishnendu; Moyano, Daniel F; Robinson, Sandra M; Rana, Subinoy; Zhang, Xinrong; Riley, Margaret A; Rotello, Vincent M

    2014-12-23

    Identification of infectious bacteria responsible for biofilm-associated infections is challenging due to the complex and heterogeneous biofilm matrix. To address this issue and minimize the impact of heterogeneity on biofilm identification, we developed a gold nanoparticle (AuNP)-based multichannel sensor to detect and identify biofilms based on their physicochemical properties. Our results showed that the sensor can discriminate six bacterial biofilms including two composed of uropathogenic bacteria. The capability of the sensor was further demonstrated through discrimination of biofilms in a mixed bacteria/mammalian cell in vitro wound model.

  6. Permeabilizing biofilms

    Science.gov (United States)

    Soukos, Nikolaos S [Revere, MA; Lee, Shun [Arlington, VA; Doukas,; Apostolos, G [Belmont, MA

    2008-02-19

    Methods for permeabilizing biofilms using stress waves are described. The methods involve applying one or more stress waves to a biofilm, e.g., on a surface of a device or food item, or on a tissue surface in a patient, and then inducing stress waves to create transient increases in the permeability of the biofilm. The increased permeability facilitates delivery of compounds, such as antimicrobial or therapeutic agents into and through the biofilm.

  7. Antimicrobial resistance and in vitro biofilm-forming ability of Enterococci spp. isolated from urinary tract infection in a tertiary care hospital in Dhaka.

    Science.gov (United States)

    Akhter, J; Ahmed, S; Saleh, A A; Anwar, S

    2014-04-01

    The biofilm mode of life conveys a survival advantage to the microorganism associated with it. Biofilm on an indwelling urinary catheter consists of adherent microorganisms, their extra cellular products, and host components deposited on the catheter and thus biofilm on urinary catheters results in persistent infections that are resistant to antimicrobial therapy. This study was done during the period of January 2010 to December 2010. Fifty nine enterococci isolated from 1203 urine samples were speciated by conventional microbiological methods and examined for their ability to form biofilm by microtitre plate assay and antimicrobial susceptibility testing by disc diffusion method for 10 clinically relevant antibiotics respectively. Biofilm producing Enterococci were more frequently found in catheterized than in non catheterized patient (p biofilm production with antibiotic resistance to amoxicillin, co-trimoxazole, ciprofloxacin, gentamycin, cefotaxime, and cefuroxime. This study demonstrated a high propensity among the isolates of Enterococci to form biofilm and a significant association of biofilms with multiple drug resistance.

  8. Biofilm inhibition by Cymbopogon citratus and Syzygium aromaticum essential oils in the strains of Candida albicans.

    Science.gov (United States)

    Khan, Mohd Sajjad Ahmad; Ahmad, Iqbal

    2012-03-27

    Oils of Cymbopogon citratus and Syzygium aromaticum have been used in traditional medicine to treat fungal infections of skin, mouth, urinary and vaginal tract in Asian countries particularly India and other developing countries. To evaluate essential oils of Cymbopogon citratus and Syzygium aromaticum for their anti-biofilm activity against strong biofilm forming strains of Candida albicans. XTT reduction assay, Time kill assays, light microscopy and scanning electron microscopy (SEM) were employed to determine the effect of test oils on the Candida albicans biofilms. Most of the Candida albicans strains tested displayed formation of moderate to strong biofilms. Preformed Candida biofilms showed ≥1024 times increased resistance to antifungal drugs, 2 times to Syzygium aromaticum, but no increased tolerance for Cymbopogon citratus. Test oils were more active against preformed biofilms compared to amphotericin B and fluconazole. At 0.5× MIC, Cymbopogon citratus followed by Syzygium aromaticum were most inhibitory against biofilm formation. Light and electron microscopic studies revealed the deformity of three dimensional structures of biofilms formed in the presence of sub-MICs of Cymbopogon citratus. The cell membranes appeared to be the target site of compounds in sessile cells as displayed by SEM observations. Our data had demonstrated promising in vitro anti-biofilm activity by Cymbopogon citratus and Syzygium aromaticum and confirm the ethnopharmacological use of these oils in muco-cutaneous Candida infections. Furthermore, it suggests exploitation of these oils as new anti-biofilm products to deal with the problem of drug-resistance and recurrent infection associated with biofilm mode of growth of Candida spp. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  9. Streptococcus gordonii Biofilm Formation: Identification of Genes that Code for Biofilm Phenotypes

    OpenAIRE

    Loo, C. Y.; Corliss, D. A.; Ganeshkumar, N.

    2000-01-01

    Viridans streptococci, which include Streptococcus gordonii, are pioneer oral bacteria that initiate dental plaque formation. Sessile bacteria in a biofilm exhibit a mode of growth that is distinct from that of planktonic bacteria. Biofilm formation of S. gordonii Challis was characterized using an in vitro biofilm formation assay on polystyrene surfaces. The same assay was used as a nonbiased method to screen isogenic mutants generated by Tn916 transposon mutagenesis for defective biofilm fo...

  10. Escherichia coli and Enterococcus faecalis are able to incorporate and enhance a pre-formed Gardnerella vaginalis biofilm.

    Science.gov (United States)

    Castro, Joana; Machado, Daniela; Cerca, Nuno

    2016-04-01

    Gardnerella vaginalis is the most frequent microorganism found in bacterial vaginosis (BV), while Escherichia coli and Enterococcus faecalis are amongst the most frequent pathogens found in urinary tract infections (UTIs). This study aimed to evaluate possible interactions between UTIs pathogens and G. vaginalis using an in vitro dual-species biofilm model. Our results showed that dual-species biofilms reached significantly higher bacterial concentration than monospecies biofilms. Moreover, visualization of dual-populations species in the biofilms, using the epifluorescence microscopy, revealed that all of the urogenital pathogens coexisted with G. vaginalis. In conclusion, our work demonstrates that uropathogens can incorporate into mature BV biofilms. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  11. Relative abundance ofCandida albicansandCandida glabratainin vitroco-culture biofilms impacts biofilm structure and formation.

    Science.gov (United States)

    Olson, Michelle L; Jayaraman, Arul; Kao, Katy C

    2018-02-02

    Candida is a member of the normal human microbiota and often resides on mucosal surfaces such as the oral cavity or the gastrointestinal tract. In addition to their commensality, Candida species can opportunistically become pathogenic if the host microbiota is disrupted or if the host immune system becomes compromised. An important factor for Candida pathogenesis is its ability to form biofilm communities. The two most medically important species - Candida albicans and Candida glabrata - are often co-isolated from infection sites, suggesting the importance of Candida co-culture biofilms. In this work, we report that biofilm formation of the co-culture population depends on the relative ratio of starting cell concentrations of C. albicans (Ca) and C. glabrata (Cg). When using a starting ratio of Ca:Cg of 1:3, a ∼6.5- and ∼2.5-fold increase in biofilm biomass was observed relative to Ca monoculture and a Ca:Cg ratio of 1:1, respectively. Confocal microscopy analysis revealed heterogeneity and complex structures composed of long Ca hyphae and Cg cell clusters in the co-culture biofilms, and qRT-PCR studies showed an increase in the relative expression of the HWP1 and ALS3 adhesion genes in the Ca:Cg 1:3 biofilm compared to Ca monoculture biofilm. Additionally, only the 1:3 Ca:Cg biofilm demonstrated increased resistance to the antifungal drug caspofungin. Overall, the results suggest that interspecific interactions between these two fungal pathogens increase biofilm formation and virulence-related gene expression in a co-culture composition-dependent manner. IMPORTANCE C. albicans and C. glabrata are often co-isolated during infection and the occurrence of co-isolation increases with increasing inflammation, suggesting possible synergistic interactions between the two Candida species in pathogenesis. During the course of an infection, the prevalence of each Candida species may change over time due to differences in metabolism and in resistance of each species to

  12. Effect of Negative Pressure on Proliferation, Virulence Factor Secretion, Biofilm Formation, and Virulence-Regulated Gene Expression of Pseudomonas aeruginosa In Vitro

    Directory of Open Access Journals (Sweden)

    Guo-Qi Wang

    2016-01-01

    Full Text Available Objective. To investigate the effect of negative pressure conditions induced by NPWT on P. aeruginosa. Methods. P. aeruginosa was cultured in a Luria–Bertani medium at negative pressure of −125 mmHg for 24 h in the experimental group and at atmospheric pressure in the control group. The diameters of the colonies of P. aeruginosa were measured after 24 h. ELISA kit, orcinol method, and elastin-Congo red assay were used to quantify the virulence factors. Biofilm formation was observed by staining with Alexa Fluor® 647 conjugate of concanavalin A (Con A. Virulence-regulated genes were determined by quantitative RT-PCR. Results. As compared with the control group, growth of P. aeruginosa was inhibited by negative pressure. The colony size under negative pressure was significantly smaller in the experimental group than that in the controls (p<0.01. Besides, reductions in the total amount of virulence factors were observed in the negative pressure group, including exotoxin A, rhamnolipid, and elastase. RT-PCR results revealed a significant inhibition in the expression level of virulence-regulated genes. Conclusion. Negative pressure could significantly inhibit the growth of P. aeruginosa. It led to a decrease in the virulence factor secretion, biofilm formation, and a reduction in the expression level of virulence-regulated genes.

  13. Structure-based virtual screening and biological evaluation of LuxT inhibitors for targeting quorum sensing through an in vitro biofilm formation

    Science.gov (United States)

    Sasikala, Dakshinamurthy; Jeyakanthan, Jeyaraman; Srinivasan, Pappu

    2017-01-01

    The LuxT regulator protein is a potential drug target for regulating quorum sensing (QS) in Vibrio alginolyticus. There is no substantiation of a 3D structure of LuxT in this particular species, thus the 3D model was constructed using molecular modeling and its stability was verified under solvation effect using molecular dynamics simulation. Further, exploration of the drug candidate against the LuxT binding site through structure-based approaches identified four Bitter compounds, viz., quercetin, cnicin, 5, 5‧ methylenedisalicyclic acid, and flufenamic acid with high docking score and optimum binding energy. Remarkably, these compounds established agreeable interactions with the amino acid residues Phe67, Asp63, Thr59, Gly64, Ile66, Phe99, Arg123, Asn119, and Gly120; which are found to play a vital role in the LuxT mechanistic inhibition. In addition to the scoring and energy parameters, MD simulation and ADME prediction suggested that the proposed compounds may be promising drug candidates against V. alginolyticus. Therefore, the lead candidate quercetin was scrutinized to validate its potential in biofilm inhibition of V. alginolyticus. The results revealed that the compound had better efficacy in biofilm destruction; as a consequence, there was a significant effect on motility and EPS synthesis at an effective concentration of 100 μM, which was further confirmed by microscopic studies. Hence, the present study will provide an insight into the design of high potent drug molecules to combat dreadful pathogenic diseases caused by V. alginolyticus.

  14. Bacterial resistance in biofilm-associated bacteria.

    Science.gov (United States)

    Venkatesan, Nandakumar; Perumal, Govindaraj; Doble, Mukesh

    2015-01-01

    Biofilms are structured groups of different bacterial species that are responsible for most chronic and recurrent infections. Biofilm-related infections reoccur in approximately 65-80% of cases. Bacteria associated with the biofilm are highly resistant to antibiotics. Knowledge of biofilm formation, its propagation and the resistance associated with it is scant and a multidisciplinary approach is followed to understand the science and develop strategies to address this problem. This article discusses the role of various biochemical factors, molecular mechanisms and altered host environment causes associated with bacterial resistance in biofilm. It also reveals the target sites and different multidisciplinary strategies adapted for destroying or preventing the formation of biofilms.

  15. Medical biofilms.

    Science.gov (United States)

    Bryers, James D

    2008-05-01

    For more than two decades, Biotechnology and Bioengineering has documented research focused on natural and engineered microbial biofilms within aquatic and subterranean ecosystems, wastewater and waste-gas treatment systems, marine vessels and structures, and industrial bioprocesses. Compared to suspended culture systems, intentionally engineered biofilms are heterogeneous reaction systems that can increase reactor productivity, system stability, and provide inherent cell:product separation. Unwanted biofilms can create enormous increases in fluid frictional resistances, unacceptable reductions in heat transfer efficiency, product contamination, enhanced material deterioration, and accelerated corrosion. Missing from B&B has been an equivalent research dialogue regarding the basic molecular microbiology, immunology, and biotechnological aspects of medical biofilms. Presented here are the current problems related to medical biofilms; current concepts of biofilm formation, persistence, and interactions with the host immune system; and emerging technologies for controlling medical biofilms. Copyright 2007 Wiley Periodicals, Inc.

  16. Whole transcriptome analysis of Acinetobacter baumannii assessed by RNA-sequencing reveals different mRNA expression profiles in biofilm compared to planktonic cells.

    Directory of Open Access Journals (Sweden)

    Soraya Rumbo-Feal

    Full Text Available Acinetobacterbaumannii has emerged as a dangerous opportunistic pathogen, with many strains able to form biofilms and thus cause persistent infections. The aim of the present study was to use high-throughput sequencing techniques to establish complete transcriptome profiles of planktonic (free-living and sessile (biofilm forms of A. baumannii ATCC 17978 and thereby identify differences in their gene expression patterns. Collections of mRNA from planktonic (both exponential and stationary phase cultures and sessile (biofilm cells were sequenced. Six mRNA libraries were prepared following the mRNA-Seq protocols from Illumina. Reads were obtained in a HiScanSQ platform and mapped against the complete genome to describe the complete mRNA transcriptomes of planktonic and sessile cells. The results showed that the gene expression pattern of A. baumannii biofilm cells was distinct from that of planktonic cells, including 1621 genes over-expressed in biofilms relative to stationary phase cells and 55 genes expressed only in biofilms. These differences suggested important changes in amino acid and fatty acid metabolism, motility, active transport, DNA-methylation, iron acquisition, transcriptional regulation, and quorum sensing, among other processes. Disruption or deletion of five of these genes caused a significant decrease in biofilm formation ability in the corresponding mutant strains. Among the genes over-expressed in biofilm cells were those in an operon involved in quorum sensing. One of them, encoding an acyl carrier protein, was shown to be involved in biofilm formation as demonstrated by the significant decrease in biofilm formation by the corresponding knockout strain. The present work serves as a basis for future studies examining the complex network systems that regulate bacterial biofilm formation and maintenance.

  17. Clinical implications of microbial biofilms in chronic rhinosinusitis and orbital cellulitis.

    Science.gov (United States)

    Nayak, Niranjan; Satpathy, Gita; Prasad, Sujata; Thakar, Alok; Chandra, Mahesh; Nag, T C

    2016-09-21

    Discovery of sessile mode of microbial existence (Biofilm state) focussed much interest, during the recent years, on the study of biofilms in many recurring and chronic infections. However, the exact role of microbial biofilms in chronic rhinosinusitis and orbital cellulitis were not elucidated earlier. The purpose of the present study was to look for the adherent property and biofilm producing ability of the clinical isolates in chronic rhinosinusitis and orbital cellulitis, and to look for the effects of antimicrobial agents on these biofilms by colorimetric assay and ultrastructural analysis. Organisms were isolated and identified from various clinical samples in patients with chronic sinusitis and orbital cellulitis. Antimicrobial sensitivity testing was carried out by the standard protocol. Biofilms were developed; quantified and antimicrobial drug perfusion through the biofilm model was evaluated by the earlier devised procedure. Electronmicroscopic study of the biofilm was performed by the recommended technique. Of the total of 70 clinical samples processed, 48 i.e. 68.5 % grew bacteria and 13 i.e.(18.6 %) fungi. Staphylococcus aureus (20), S epidermidis (16) and Pseudomonas aeruginosa (6) accounted for the majority of the bacterial isolates. Aspergillus flavus (8), however was the commonest amongst the fungi. A total of 40 bacteria and 8 fungi could be tested for biofilm production. Eighteen (45 %) of the 40 bacterial isolates and 4(50 %) out of the 8 A flavus isolates were found to be biofilm producers. In vitro adherence testing revealed that majority i.e. 16 (88.8 %) of the 18 biofilm positive bacteria were adherent to artificial surfaces. Antimicrobial drug perfusion through the biofilm model was poor. Antimicrobial treatment was totally ineffective against strong biofilm producers, whose electron microscopic picture was quite similar to that observed for biofilm producers without any antimicrobial pre-treatment. Filamentous fungi, like bacteria

  18. Symbiotic Relationship between Streptococcus mutans and Candida albicans Synergizes Virulence of Plaque Biofilms In Vivo

    Science.gov (United States)

    Falsetta, Megan L.; Klein, Marlise I.; Colonne, Punsiri M.; Scott-Anne, Kathleen; Gregoire, Stacy; Pai, Chia-Hua; Gonzalez-Begne, Mireya; Watson, Gene; Krysan, Damian J.; Bowen, William H.

    2014-01-01

    Streptococcus mutans is often cited as the main bacterial pathogen in dental caries, particularly in early-childhood caries (ECC). S. mutans may not act alone; Candida albicans cells are frequently detected along with heavy infection by S. mutans in plaque biofilms from ECC-affected children. It remains to be elucidated whether this association is involved in the enhancement of biofilm virulence. We showed that the ability of these organisms together to form biofilms is enhanced in vitro and in vivo. The presence of C. albicans augments the production of exopolysaccharides (EPS), such that cospecies biofilms accrue more biomass and harbor more viable S. mutans cells than single-species biofilms. The resulting 3-dimensional biofilm architecture displays sizeable S. mutans microcolonies surrounded by fungal cells, which are enmeshed in a dense EPS-rich matrix. Using a rodent model, we explored the implications of this cross-kingdom interaction for the pathogenesis of dental caries. Coinfected animals displayed higher levels of infection and microbial carriage within plaque biofilms than animals infected with either species alone. Furthermore, coinfection synergistically enhanced biofilm virulence, leading to aggressive onset of the disease with rampant carious lesions. Our in vitro data also revealed that glucosyltransferase-derived EPS is a key mediator of cospecies biofilm development and that coexistence with C. albicans induces the expression of virulence genes in S. mutans (e.g., gtfB, fabM). We also found that Candida-derived β1,3-glucans contribute to the EPS matrix structure, while fungal mannan and β-glucan provide sites for GtfB binding and activity. Altogether, we demonstrate a novel mutualistic bacterium-fungus relationship that occurs at a clinically relevant site to amplify the severity of a ubiquitous infectious disease. PMID:24566629

  19. In vitro protease cleavage and computer simulations reveal the HIV-1 capsid maturation pathway

    Science.gov (United States)

    Ning, Jiying; Erdemci-Tandogan, Gonca; Yufenyuy, Ernest L.; Wagner, Jef; Himes, Benjamin A.; Zhao, Gongpu; Aiken, Christopher; Zandi, Roya; Zhang, Peijun

    2016-12-01

    HIV-1 virions assemble as immature particles containing Gag polyproteins that are processed by the viral protease into individual components, resulting in the formation of mature infectious particles. There are two competing models for the process of forming the mature HIV-1 core: the disassembly and de novo reassembly model and the non-diffusional displacive model. To study the maturation pathway, we simulate HIV-1 maturation in vitro by digesting immature particles and assembled virus-like particles with recombinant HIV-1 protease and monitor the process with biochemical assays and cryoEM structural analysis in parallel. Processing of Gag in vitro is accurate and efficient and results in both soluble capsid protein and conical or tubular capsid assemblies, seemingly converted from immature Gag particles. Computer simulations further reveal probable assembly pathways of HIV-1 capsid formation. Combining the experimental data and computer simulations, our results suggest a sequential combination of both displacive and disassembly/reassembly processes for HIV-1 maturation.

  20. Biofilm Dispersal

    Science.gov (United States)

    2010-01-01

    Like all sessile organisms, surface-attached communities of bacteria known as biofilms must release and disperse cells into the environment to colonize new sites. For many pathogenic bacteria, biofilm dispersal plays an important role in the transmission of bacteria from environmental reservoirs to human hosts, in horizontal and vertical cross-host transmission, and in the exacerbation and spread of infection within a host. The molecular mechanisms of bacterial biofilm dispersal are only beginning to be elucidated. Biofilm dispersal is a promising area of research that may lead to the development of novel agents that inhibit biofilm formation or promote biofilm cell detachment. Such agents may be useful for the prevention and treatment of biofilms in a variety of industrial and clinical settings. This review describes the current status of research on biofilm dispersal, with an emphasis on studies aimed to characterize dispersal mechanisms, and to identify environmental cues and inter- and intracellular signals that regulate the dispersal process. The clinical implications of biofilm dispersal and the potential therapeutic applications of some of the most recent findings will also be discussed. PMID:20139339

  1. A commensal streptococcus hijacks a Pseudomonas aeruginosa exopolysaccharide to promote biofilm formation.

    Directory of Open Access Journals (Sweden)

    Jessica A Scoffield

    2017-04-01

    Full Text Available Pseudomonas aeruginosa causes devastating chronic pulmonary infections in cystic fibrosis (CF patients. Although the CF airway is inhabited by diverse species of microorganisms interlaced within a biofilm, many studies focus on the sole contribution of P. aeruginosa pathogenesis in CF morbidity. More recently, oral commensal streptococci have been identified as cohabitants of the CF lung, but few studies have explored the role these bacteria play within the CF biofilm. We examined the interaction between P. aeruginosa and oral commensal streptococci within a dual species biofilm. Here we report that the CF P. aeruginosa isolate, FRD1, enhances biofilm formation and colonization of Drosophila melanogaster by the oral commensal Streptococcus parasanguinis. Moreover, production of the P. aeruginosa exopolysaccharide, alginate, is required for the promotion of S. parasanguinis biofilm formation and colonization. However, P. aeruginosa is not promoted in the dual species biofilm. Furthermore, we show that the streptococcal adhesin, BapA1, mediates alginate-dependent enhancement of the S. parasanguinis biofilm in vitro, and BapA1 along with another adhesin, Fap1, are required for the in vivo colonization of S. parasanguinis in the presence of FRD1. Taken together, our study highlights a new association between streptococcal adhesins and P. aeruginosa alginate, and reveals a mechanism by which S. parasanguinis potentially colonizes the CF lung and interferes with the pathogenesis of P. aeruginosa.

  2. A commensal streptococcus hijacks a Pseudomonas aeruginosa exopolysaccharide to promote biofilm formation.

    Science.gov (United States)

    Scoffield, Jessica A; Duan, Dingyu; Zhu, Fan; Wu, Hui

    2017-04-01

    Pseudomonas aeruginosa causes devastating chronic pulmonary infections in cystic fibrosis (CF) patients. Although the CF airway is inhabited by diverse species of microorganisms interlaced within a biofilm, many studies focus on the sole contribution of P. aeruginosa pathogenesis in CF morbidity. More recently, oral commensal streptococci have been identified as cohabitants of the CF lung, but few studies have explored the role these bacteria play within the CF biofilm. We examined the interaction between P. aeruginosa and oral commensal streptococci within a dual species biofilm. Here we report that the CF P. aeruginosa isolate, FRD1, enhances biofilm formation and colonization of Drosophila melanogaster by the oral commensal Streptococcus parasanguinis. Moreover, production of the P. aeruginosa exopolysaccharide, alginate, is required for the promotion of S. parasanguinis biofilm formation and colonization. However, P. aeruginosa is not promoted in the dual species biofilm. Furthermore, we show that the streptococcal adhesin, BapA1, mediates alginate-dependent enhancement of the S. parasanguinis biofilm in vitro, and BapA1 along with another adhesin, Fap1, are required for the in vivo colonization of S. parasanguinis in the presence of FRD1. Taken together, our study highlights a new association between streptococcal adhesins and P. aeruginosa alginate, and reveals a mechanism by which S. parasanguinis potentially colonizes the CF lung and interferes with the pathogenesis of P. aeruginosa.

  3. Mammalian cell growth versus biofilm formation on biomaterial surfaces in an in vitro post-operative contamination model

    NARCIS (Netherlands)

    Subbiahdoss, Guruprakash; Kuijer, Roel; Busscher, Henk J.; van der Mei, Henny C.

    2010-01-01

    Biomaterial-associated infections are the major cause of implant failure and can develop many years after implantation. Success or failure of an implant depends on the balance between host tissue integration and bacterial colonization. Here, we describe a new in vitro model for the postoperative

  4. In Vitro Oral Biofilm Formation on Triclosan-Coated Sutures in the Absence and Presence of Additional Antiplaque Treatment

    NARCIS (Netherlands)

    Venema, Sebastiaan; Abbas, Frank; van de Belt-Gritter, Betsy; van der Mei, Henny C.; Busscher, Henk J.; van Hoogmoed, Chris G.

    Purpose: This study evaluated the in vitro plaque inhibitory effect of triclosan-coated polyglactin 910 sutures in the absence and presence of an additional antiplaque agent commonly used after oral surgery. Materials and Methods: Triclosan-coated sutures were incubated for 4 hours in freshly

  5. Effect of bacterial components of mixed culture supernatants of planktonic and biofilm Pseudomonas aeruginosa with commensal Escherichia coli on the neutrophil response in vitro.

    Science.gov (United States)

    Maslennikova, Irina L; Kuznetsova, Marina V; Nekrasova, Irina V; Shirshev, Sergei V

    2017-11-30

    Pseudomonas aeruginosa (PA) responsible for acute and chronic infections often forms a well-organized bacterial population with different microbial species including commensal strains of Escherichia coli. Bacterial extracellular components of mixed culture can modulate the influence of bacteria on the neutrophil functions. The objective of this study was to compare the effect of pyocyanin, pyoverdine, LPS, exopolysaccharide of single species and mixed culture supernatants of PA strains and E. coli K12 on microbicidal, secretory activity of human neutrophils in vitro. Bacterial components of E. coli K12 in mixed supernatants with 'biofilm' PA strains (PA ATCC, PA BALG) enhanced short-term microbicidal mechanisms and inhibited neutrophil secretion delayed in time. The influence of 'planktonic' PA (PA 9-3) exometabolites in mixed culture is almost mimicked by E. coli K12 effect on functional neutrophil changes. This investigation may help to understand some of the mechanisms of neutrophil response to mixed infections of different PA with other bacteria species. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  6. Influence of exposure time on the release of bacteria from a biofilm on Ti6Al4V discs using sonication: An in vitro model.

    Science.gov (United States)

    Prieto-Borja, Laura; Conde, Ana; Arenas, María A; de Damborenea, Juan J; Esteban, Jaime

    2017-12-01

    Implant sonication is considered a useful method for the diagnosis of implant-related infections. We designed an in vitro study using Ti6Al4V discs and 5 different bacteria to determine the optimal sonication time for recovery of most bacteria tested to enable use of sonication in clinical practice for microbiological diagnosis of implant-related infections. We carried out a specific protocol for the adherence and subsequent biofilm formation on the materials used. The discs were then sonicated and the retrieved bacteria were quantified. From minute 1 to 5, the amount of recovered organisms grew progressively for all bacteria. Between minute 6 and minute 10, the number was irregular for all strains except E. coli, though no pattern was evidenced. E. coli was the only microorganism with a progressive increase in liberation throughout the process. Significant differences were observed in each of the 10minutes analyzed as concerns the release of the 5 strains (PTi6Al4V discs. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Equisetum giganteum influences the ability of Candida albicans in forming biofilms over the denture acrylic resin surface.

    Science.gov (United States)

    da Silva, Rafaela Alves; Bernardo, Laura Pozato; Moreno, Jessica Monique Lopes; Lara, Vanessa Soares; Porto, Vinicius Carvalho

    2017-12-01

    Equisetum giganteum L. (Equisetaceae) is an endemic plant of Central and South America used in traditional medicine. Natural drugs have been frequently used in the treatment of a myriad of diseases, proving to be an alternative to synthetic chemicals, and have been intensively studied in the prevention of sicknesses, including oral diseases. This study evaluated the in vitro antiadherent activity of E. giganteum extract against Candida albicans biofilms. Crystal violet and colony-forming units assays were used to quantify the total biofilm biomass and biofilm living cells on a denture base acrylic resin pretreated with hydroethanolic extract of E. giganteum in different concentrations (50, 25, 16, 8, and 4 mg/mL), after 24 h of biofilm development. Equisetum giganteum affected biofilms by reduction of biomass and living cells per area of acrylic specimens. The results revealed reduction of 15-44% of the biofilm mass and reduction of numbers of colony-forming units (CFUs) present in biofilms (79%) compared to the untreated control (CTRL/PBS). At all concentrations, it demonstrated important antiadherent activity on Candida albicans biofilms, the main microbe in denture stomatitis. The present findings show that E. giganteum antimicrobial effects may qualify the extract as a promising natural alternative for topical treatment or prevention of denture stomatitis. The usage of drugs made of natural products shows advantages in relation to synthetic drugs on the market, such as lower cost, lower toxicity, and in relation to the occurrence of microbial resistance.

  8. Bursting the bubble on bacterial biofilms

    DEFF Research Database (Denmark)

    Crusz, Shanika A; Popat, Roman; Rybtke, Morten Theil

    2012-01-01

    The flow cell biofilm system is an important and widely used tool for the in vitro cultivation and evaluation of bacterial biofilms under hydrodynamic conditions of flow. This paper provides an introduction to the background and use of such systems, accompanied by a detailed guide to the assembly...... methodology amongst researchers would enable findings to be shared and replicated amongst the biofilm research community, with the overall aim of advancing understanding and management of these complex and widespread bacterial communities....

  9. The in Vitro Antibacterial Efficacy of Persian Green Tea Extract as an Intracanal Irrigant on Enterococcus faecalis Biofilm

    Science.gov (United States)

    Ramezanali, Fatemeh; Samimi, Shiva; Kharazifard, Mohammadjavad; Afkhami, Farzaneh

    2016-01-01

    Introduction: The aim of this study was to compare the antibacterial effect of Persian green tea extract (GTE) and 2.5% sodium hypochlorite (NaOCl) against Enterococcus faecalis (E. faecalis) as an intracanal irrigant. Methods and Materials: Thirty freshly extracted teeth were instrumented and sectioned into mesial and distal segments. The specimens were put into wells containing 2 mL of E. faecalis-containing medium. After 3 weeks, the specimens were removed and divided randomly into three groups (n=20). Each group was exposed to 3 mL of different irrigants for 3 min. Groups 1, 2 and 3 were irrigated with GTE, 2.5% NaOCl and normal saline, respectively. Biofilm formed in the middle third of the root canal was carved by sterile scalpel and cultured in Mueller-Hinton medium. Number of colony forming units (CFU) was counted on each plate. In addition, antimicrobial activity of the irrigants was evaluated by the agar disc diffusion test. The diameter of inhibition zone (IZ) around each irrigant was evaluated. The Kruskal-Wallis and Dunn tests were used to analysis the data. Results: While in NaOCl group no bacterial colonies were observed, the mean number of E. faecalis in GTE and control groups were 275±74 CFU/mL (P<0.001) and 119×108±11×108 (P<0.001), respectively. The mean of IZ in NaOCl and GTE groups were 24.35±0.78 and 6.9±0.87 mm, in order of appearance (P<0.001). Zone of inhibition was not observed around the control group (P<0.001). Conclusion: This research highlighted the potential role of plant extracts in antimicrobial root canal irrigation protocol. PMID:27790260

  10. Evaluation of the Effect of Enterococcus faecalis Biofilm on the 2% Chlorhexidine Substantivity: An In Vitro Study.

    Science.gov (United States)

    Böttcher, Daiana Elisabeth; Sehnem, Nicole T; Montagner, Francisco; Fatturi Parolo, Clarissa Cavalcanti; Grecca, Fabiana Soares

    2015-08-01

    The aim of this study was to correlate the bacterial viability and the presence of 2% chlorhexidine (CHX) solution on dentin by means of confocal laser scanning microscopy and high-performance liquid chromatography for 48 hours, 7 days, and 30 days. One hundred twenty-three extracted human teeth were used. Samples were divided into 4 groups according to the solution (CHX or saline) and the presence of Enterococus faecalis biofilm. Samples were kept in contact with 5 mL of the solution for 5 minutes. Each group was divided into 3 subgroups according to the evaluation period (n = 10). Statistical analysis was performed by using the Kruskal-Wallis test, the Mann-Whitney U test (P < .05), and the Spearman rank correlation coefficient (P < .01). There was a negative correlation between the percentage of live cells and the amount of remaining CHX (P = .000). CHX significantly reduced the percentage of viable cells compared with saline after 48 hours (P = .007). Differences were maintained in the 7-day evaluation period (P = .001). After 30 days, the CHX group presented an increase of viable cells, thereby becoming similar to saline (P = .623). Simultaneously, the remaining CHX was significantly reduced in the 30-day specimens (P = .000). The results of this study indicate that 2% CHX solution was detected for 48 hours and 7 days with a low percentage of viable cells. The presence of microorganisms on human dentin did not affect 2% CHX maintenance. Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  11. Evaluation of antibacterial efficacy of Chitosan, Chlorhexidine, Propolis and Sodium hypochlorite on Enterococcus faecalis biofilm : An in vitro study.

    Science.gov (United States)

    Jaiswal, Natasha; Sinha, Dakshita-Joy; Singh, Udai-Pratap; Singh, Kanwardeep; Jandial, Urja-Ahuja; Goel, Shivika

    2017-09-01

    Long term successful root canal treatment requires effective debridement and disinfection of root canal system. Persistent periradicular lesions are usually associated with Enterococccus faecalis. Prompt research for natural alternatives for irrigation is mainly due to the constant increase in antibiotic resistant strains and side effects caused by synthetic drugs. Sodium hypochlorite; the gold standard for irrigation has many disadvantages. Therefore, the present study was aimed to explore newer irrigants probably be as more effective and at the same time would be less irritating to the tissues than NaOCl. Ninety extracted human mandibular premolars were biomechanically prepared, vertically sectioned, placed in tissue culture wells exposing the root canal surface to E. faecalis to form a biofilm. At the end of 3rd week, all groups were irrigated with 3 ml of test solutions and control for 10 minutes. The samples were then scraped with a scalpel, inoculated on tryptone soy agar plates and incubated for 24 hours at 37ºC. The plates were then subjected to digital colony counter and evaluated for E. faecalisgrowth. The growth was statistically analysed by ANOVA & Post Hoc Tukey tests. Chitosan + Chlorhexidine, NaOCl and Chlorhexidine showed no statistically significant difference, whereas all the other inter‑group differences were statistically significant (PChlorhexidine, Chlorhexidine and Propolis were found to be as efficacious as sodium hypochlorite. The use of natural alternatives as root canal irrigation solutions might prove to be advantageous considering several unfavorable properties of NaOCl. Key words:Antibacterial efficacy, Chitosan, Enterococcus faecalis, Root canal irrigation.

  12. Medical Biofilms

    OpenAIRE

    Bryers, James D.

    2008-01-01

    For more than two decades, Biotechnology and Bioengineering has documented research focused on natural and engineered microbial biofilms within aquatic and subterranean ecosystems, wastewater and waste-gas treatment systems, marine vessels and structures, and industrial bioprocesses. Compared to suspended culture systems, intentionally engineered biofilms are heterogeneous reaction systems that can increase reactor productivity, system stability, and provide inherent cell: product separation....

  13. Salmonella biofilms

    NARCIS (Netherlands)

    Castelijn, G.A.A.

    2013-01-01

    Biofilm formation by Salmonellaspp. is a problem in the food industry, since biofilms may act as a persistent source of product contamination. Therefore the aim of this study was to obtain more insight in the processes involved and the factors contributing to Salmonellabiofilm formation. A

  14. Bacterial Biofilms in Jones Tubes.

    Science.gov (United States)

    Ahn, Eric S; Hauck, Matthew J; Kirk Harris, Jonathan; Robertson, Charles E; Dailey, Roger A

    To investigate the presence and microbiology of bacterial biofilms on Jones tubes (JTs) by direct visualization with scanning electron microscopy and polymerase chain reaction (PCR) of representative JTs, and to correlate these findings with inflammation and/or infection related to the JT. In this study, prospective case series were performed. JTs were recovered from consecutive patients presenting to clinic for routine cleaning or recurrent irritation/infection. Four tubes were processed for scanning electron microscopy alone to visualize evidence of biofilms. Two tubes underwent PCR alone for bacterial quantification. One tube was divided in half and sent for scanning electron microscopy and PCR. Symptoms related to the JTs were recorded at the time of recovery. Seven tubes were obtained. Five underwent SEM, and 3 out of 5 showed evidence of biofilms (60%). Two of the 3 biofilms demonstrated cocci and the third revealed rods. Three tubes underwent PCR. The predominant bacteria identified were Pseudomonadales (39%), Pseudomonas (16%), and Staphylococcus (14%). Three of the 7 patients (43%) reported irritation and discharge at presentation. Two symptomatic patients, whose tubes were imaged only, revealed biofilms. The third symptomatic patient's tube underwent PCR only, showing predominantly Staphylococcus (56%) and Haemophilus (36%) species. Two of the 4 asymptomatic patients also showed biofilms. All symptomatic patients improved rapidly after tube exchange and steroid antibiotic drops. Bacterial biofilms were variably present on JTs, and did not always correlate with patients' symptoms. Nevertheless, routine JT cleaning is recommended to treat and possibly prevent inflammation caused by biofilms.

  15. High frequency ultrasound imaging of a single-species biofilm

    NARCIS (Netherlands)

    Shemesh, H.; Goertz, D. E.; van der Sluis, L. W. M.; de Jong, N.; Wu, M. K.; Wesselink, P. R.

    Objective: This study evaluated the feasibility of a high frequency ultrasound scan to examine the 3D morphology of Streptococcus mutans biofilms grown in vitro. Methods: Six 2-day S. mutans biofilms and six 7-day biofilms were grown on tissue culture membranes and on bovine dentine discs. A sterile

  16. The Clostridium difficile Protease Cwp84 Modulates both Biofilm Formation and Cell-Surface Properties.

    Directory of Open Access Journals (Sweden)

    Véronique Pantaléon

    Full Text Available Clostridium difficile is responsible for 15-20% of antibiotic-associated diarrheas, and nearly all cases of pseudomembranous colitis. Among the cell wall proteins involved in the colonization process, Cwp84 is a protease that cleaves the S-layer protein SlpA into two subunits. A cwp84 mutant was previously shown to be affected for in vitro growth but not in its virulence in a hamster model. In this study, the cwp84 mutant elaborated biofilms with increased biomass compared with the parental strain, allowing the mutant to grow more robustly in the biofilm state. Proteomic analyses of the 630Δerm bacteria growing within the biofilm revealed the distribution of abundant proteins either in cell surface, matrix or supernatant fractions. Of note, the toxin TcdA was found in the biofilm matrix. Although the overall proteome differences between the cwp84 mutant and the parental strains were modest, there was still a significant impact on bacterial surface properties such as altered hydrophobicity. In vitro and in vivo competition assays revealed that the mutant was significantly impaired for growth only in the planktonic state, but not in biofilms or in vivo. Taken together, our results suggest that the phenotypes in the cwp84 mutant come from either the accumulation of uncleaved SlpA, or the ability of Cwp84 to cleave as yet undetermined proteins.

  17. Phytotherapeutic prevention of dental biofilm formation.

    Science.gov (United States)

    Rasooli, Iraj; Shayegh, Shojaedin; Taghizadeh, Massoud; Astaneh, Shakiba Darvish Alipoor

    2008-09-01

    The antimicrobial and biofilm formation preventive properties of Mentha piperita and Rosmarinus officinalis essential oils and chlorhexidine were assessed against Streptococcus mutans and Streptococcus pyogenes. 26 and 20 compounds were identified by GC and GC-MS analysis in hydrodistilled oils from M. piperita and R. officinalis, respectively. The minimal bactericidal concentrations (MBC) of the M. piperita and R. officinalis oils and chlorhexidine were (6000, 2000, 8000 ppm) and (1000, 4000, 1000 ppm) for S. mutans and S. pyogenes, respectively. The decimal reduction time (D) of S. mutans exposed to the oils at their MBC levels was 2.8 min while chlorhexidine showed a longer time. The D values of S. pyogenes on exposure to the MBC levels of M. piperita and R. officinalis oils and of chlorhexidine were 2.14, 4.28 and 2.8 min, indicating a higher efficacy of M. piperita oil. Biofilm formation was performed by growing S. mutans culture with and without essential oils in LB medium in polystyrene tubes. In vitro biofilm inhibitory properties were in the order M. piperita > R. officinalis > chlorhexidine. In vivo experiments on the antibiofilm properties revealed that all concentrations of the oils were significantly (p < 0.001) more effective than chlorhexidine. In conclusion, essential oils may be considered as safe agents in the development of novel antibiofilm agents.

  18. Biofilm Development

    DEFF Research Database (Denmark)

    Tolker-Nielsen, Tim

    2015-01-01

    During the past decade we have gained much knowledge about the molecular mechanisms that are involved in initiation and termination of biofilm formation. In many bacteria, these processes appear to occur in response to specific environmental cues and result in, respectively, induction or terminat......During the past decade we have gained much knowledge about the molecular mechanisms that are involved in initiation and termination of biofilm formation. In many bacteria, these processes appear to occur in response to specific environmental cues and result in, respectively, induction...... or termination of biofilm matrix production via the second messenger molecule c-di-GMP. In between initiation and termination of biofilm formation we have defined specific biofilm stages, but the currently available evidence suggests that these transitions are mainly governed by adaptive responses......, and not by specific genetic programs. It appears that biofilm formation can occur through multiple pathways and that the spatial structure of the biofilms is species dependent as well as dependent on environmental conditions. Bacterial subpopulations, e.g., motile and nonmotile subpopulations, can develop...

  19. Fungal Biofilms and Polymicrobial Diseases.

    Science.gov (United States)

    Costa-Orlandi, Caroline B; Sardi, Janaina C O; Pitangui, Nayla S; de Oliveira, Haroldo C; Scorzoni, Liliana; Galeane, Mariana C; Medina-Alarcón, Kaila P; Melo, Wanessa C M A; Marcelino, Mônica Y; Braz, Jaqueline D; Fusco-Almeida, Ana Marisa; Mendes-Giannini, Maria José S

    2017-05-10

    Biofilm formation is an important virulence factor for pathogenic fungi. Both yeasts and filamentous fungi can adhere to biotic and abiotic surfaces, developing into highly organized communities that are resistant to antimicrobials and environmental conditions. In recent years, new genera of fungi have been correlated with biofilm formation. However, Candida biofilms remain the most widely studied from the morphological and molecular perspectives. Biofilms formed by yeast and filamentous fungi present differences, and studies of polymicrobial communities have become increasingly important. A key feature of resistance is the extracellular matrix, which covers and protects biofilm cells from the surrounding environment. Furthermore, to achieve cell-cell communication, microorganisms secrete quorum-sensing molecules that control their biological activities and behaviors and play a role in fungal resistance and pathogenicity. Several in vitro techniques have been developed to study fungal biofilms, from colorimetric methods to omics approaches that aim to identify new therapeutic strategies by developing new compounds to combat these microbial communities as well as new diagnostic tools to identify these complex formations in vivo. In this review, recent advances related to pathogenic fungal biofilms are addressed.

  20. Fungal Biofilms and Polymicrobial Diseases

    Science.gov (United States)

    Costa-Orlandi, Caroline B.; Sardi, Janaina C. O.; Pitangui, Nayla S.; de Oliveira, Haroldo C.; Scorzoni, Liliana; Galeane, Mariana C.; Medina-Alarcón, Kaila P.; Melo, Wanessa C. M. A.; Marcelino, Mônica Y.; Braz, Jaqueline D.; Fusco-Almeida, Ana Marisa; Mendes-Giannini, Maria José S.

    2017-01-01

    Biofilm formation is an important virulence factor for pathogenic fungi. Both yeasts and filamentous fungi can adhere to biotic and abiotic surfaces, developing into highly organized communities that are resistant to antimicrobials and environmental conditions. In recent years, new genera of fungi have been correlated with biofilm formation. However, Candida biofilms remain the most widely studied from the morphological and molecular perspectives. Biofilms formed by yeast and filamentous fungi present differences, and studies of polymicrobial communities have become increasingly important. A key feature of resistance is the extracellular matrix, which covers and protects biofilm cells from the surrounding environment. Furthermore, to achieve cell–cell communication, microorganisms secrete quorum-sensing molecules that control their biological activities and behaviors and play a role in fungal resistance and pathogenicity. Several in vitro techniques have been developed to study fungal biofilms, from colorimetric methods to omics approaches that aim to identify new therapeutic strategies by developing new compounds to combat these microbial communities as well as new diagnostic tools to identify these complex formations in vivo. In this review, recent advances related to pathogenic fungal biofilms are addressed. PMID:29371540

  1. Global expression profile of biofilm resistance to antimicrobial compounds in the plant-pathogenic bacterium Xylella fastidiosa reveals evidence of persister cells.

    Science.gov (United States)

    Muranaka, Lígia S; Takita, Marco A; Olivato, Jacqueline C; Kishi, Luciano T; de Souza, Alessandra A

    2012-09-01

    Investigations of biofilm resistance response rarely focus on plant-pathogenic bacteria. Since Xylella fastidiosa is a multihost plant-pathogenic bacterium that forms biofilm in the xylem, the behavior of its biofilm in response to antimicrobial compounds needs to be better investigated. We analyzed here the transcriptional profile of X. fastidiosa subsp. pauca in response to inhibitory and subinhibitory concentrations of copper and tetracycline. Copper-based products are routinely used to control citrus diseases in the field, while antibiotics are more widely used for bacterial control in mammals. The use of antimicrobial compounds triggers specific responses to each compound, such as biofilm formation and phage activity for copper. Common changes in expression responses comprise the repression of genes associated with metabolic functions and movement and the induction of toxin-antitoxin systems, which have been associated with the formation of persister cells. Our results also show that these cells were found in the population at a ca. 0.05% density under inhibitory conditions for both antimicrobial compounds and that pretreatment with subinhibitory concentration of copper increases this number. No previous report has detected the presence of these cells in X. fastidiosa population, suggesting that this could lead to a multidrug tolerance response in the biofilm under a stressed environment. This is a mechanism that has recently become the focus of studies on resistance of human-pathogenic bacteria to antibiotics and, based on our data, it seems to be more broadly applicable.

  2. Enhanced Removal of Enterococcus faecalis Biofilms in the Root Canal Using Sodium Hypochlorite Plus Photon-Induced Photoacoustic Streaming: An In Vitro Study

    Science.gov (United States)

    Al Shahrani, Mohammed; DiVito, Enrico; Hughes, Christopher V.; Nathanson, Dan

    2014-01-01

    Abstract Objective: The purpose of this study was to determine the effectiveness of laser-activated irrigation by photon-induced photoacoustic streaming (PIPS) using Er:YAG laser energy in decontaminating heavily colonized root canal systems in vitro. Materials and methods: Extracted single-rooted human teeth (n=60) were mechanically and chemically prepared, sterilized, inoculated with Enterococcus faecalis for 3 weeks, and randomly assigned to four groups (n=15): Group I (control, no decontamination), Group II (PIPS+6% NaOCl), Group III (PIPS+saline), and Group IV (6% NaOCl). PIPS settings were all preset to 50 μsec pulse, 20 mJ, 15 Hz, for an average power of 0.3 W. After decontamination, the remaining live microbes from all specimens were collected and recovered via plate counting of the colony-forming units (CFUs). Randomized root canal surfaces were examined with scanning electron microscopy and confocal laser microscopy. Mean variance and Dunnett's t test (post-hoc test) comparisons were used to compare mean scores for the three groups with the control group. Results: The CFU analysis showed the following measurements (mean±SE): Group I (control), 336.8±1.8; Group II (PIPS+NaOCl), 0.27±0.21; Group III (PIPS+saline), 225.0±21; and Group IV (NaOCl), 46.9±20.29. Group II had significantly lower CFUs than any other groups (p<0.05). Both imaging analyses confirmed levels of remaining bacteria on examined root surfaces. Conclusions: The use of the PIPS system along with NaOCl showed the most efficient eradication of the bacterial biofilm. It appears that laser-activated irrigation (LAI) utilizing PIPS may enhance the disinfection of the root canal system. PMID:24717113

  3. Protective Mechanisms of Respiratory Tract Streptococci against Streptococcus pyogenes Biofilm Formation and Epithelial Cell Infection

    Science.gov (United States)

    Fiedler, Tomas; Riani, Catur; Koczan, Dirk; Standar, Kerstin

    2013-01-01

    Streptococcus pyogenes (group A streptococci [GAS]) encounter many streptococcal species of the physiological microbial biome when entering the upper respiratory tract of humans, leading to the question how GAS interact with these bacteria in order to establish themselves at this anatomic site and initiate infection. Here we show that S. oralis and S. salivarius in direct contact assays inhibit growth of GAS in a strain-specific manner and that S. salivarius, most likely via bacteriocin secretion, also exerts this effect in transwell experiments. Utilizing scanning electron microscopy documentation, we identified the tested strains as potent biofilm producers except for GAS M49. In mixed-species biofilms, S. salivarius dominated the GAS strains, while S. oralis acted as initial colonizer, building the bottom layer in mixed biofilms and thereby allowing even GAS M49 to form substantial biofilms on top. With the exception of S. oralis, artificial saliva reduced single-species biofilms and allowed GAS to dominate in mixed biofilms, although the overall two-layer structure was unchanged. When covered by S. oralis and S. salivarius biofilms, epithelial cells were protected from GAS adherence, internalization, and cytotoxic effects. Apparently, these species can have probiotic effects. The use of Affymetrix array technology to assess HEp-2 cell transcription levels revealed modest changes after exposure to S. oralis and S. salivarius biofilms which could explain some of the protective effects against GAS attack. In summary, our study revealed a protection effect of respiratory tract bacteria against an important airway pathogen and allowed a first in vitro insight into local environmental processes after GAS enter the respiratory tract. PMID:23241973

  4. Identification of Genes Controlled by the Essential YycFG Two-Component System Reveals a Role for Biofilm Modulation in Staphylococcus epidermidis

    Directory of Open Access Journals (Sweden)

    Ying Zhang

    2017-04-01

    Full Text Available Biofilms play a crucial role in the pathogenicity of Staphylococcus epidermidis, while little is known about whether the essential YycFG two-component signal transduction system (TCS is involved in biofilm formation. We used antisense RNA (asRNA to silence the yycFG TCS in order to study its regulatory functions in S. epidermidis. Strain 1457 expressing asRNAyycF exhibited a significant delay (~4–5 h in entry to log phase, which was partially complemented by overexpressing ssaA. The expression of asRNAyycF and asRNAyycG resulted in a 68 and 50% decrease in biofilm formation at 6 h, respectively, while they had no significant inhibitory effect on 12 h biofilm formation. The expression of asRNAyycF led to a ~5-fold increase in polysaccharide intercellular adhesion (PIA production, but it did not affect the expression of accumulation-associated protein (Aap or the release of extracellular DNA. Consistently, quantitative real-time PCR showed that silencing yycF resulted in an increased transcription of biofilm-related genes, including icaA, arlR, sarA, sarX, and sbp. An in silico search of the YycF regulon for the conserved YycF recognition pattern and a modified motif in S. epidermidis, along with additional gel shift and DNase I footprinting assays, showed that arlR, sarA, sarX, and icaA are directly regulated by YycF. Our data suggests that YycFG modulates S. epidermidis biofilm formation in an ica-dependent manner.

  5. Identification of Genes Controlled by the Essential YycFG Two-Component System Reveals a Role for Biofilm Modulation in Staphylococcus epidermidis

    Science.gov (United States)

    Xu, Tao; Wu, Yang; Lin, Zhiwei; Bertram, Ralph; Götz, Friedrich; Zhang, Ying; Qu, Di

    2017-01-01

    Biofilms play a crucial role in the pathogenicity of Staphylococcus epidermidis, while little is known about whether the essential YycFG two-component signal transduction system (TCS) is involved in biofilm formation. We used antisense RNA (asRNA) to silence the yycFG TCS in order to study its regulatory functions in S. epidermidis. Strain 1457 expressing asRNAyycF exhibited a significant delay (~4–5 h) in entry to log phase, which was partially complemented by overexpressing ssaA. The expression of asRNAyycF and asRNAyycG resulted in a 68 and 50% decrease in biofilm formation at 6 h, respectively, while they had no significant inhibitory effect on 12 h biofilm formation. The expression of asRNAyycF led to a ~5-fold increase in polysaccharide intercellular adhesion (PIA) production, but it did not affect the expression of accumulation-associated protein (Aap) or the release of extracellular DNA. Consistently, quantitative real-time PCR showed that silencing yycF resulted in an increased transcription of biofilm-related genes, including icaA, arlR, sarA, sarX, and sbp. An in silico search of the YycF regulon for the conserved YycF recognition pattern and a modified motif in S. epidermidis, along with additional gel shift and DNase I footprinting assays, showed that arlR, sarA, sarX, and icaA are directly regulated by YycF. Our data suggests that YycFG modulates S. epidermidis biofilm formation in an ica-dependent manner. PMID:28491057

  6. Molecular Basis for Saccharomyces cerevisiae Biofilm Development

    DEFF Research Database (Denmark)

    Andersen, Kaj Scherz

    of translation of FLO11. In conclusion, I have conducted the first global study of the genetic program for yeast biofilm formation on polystyrene. This work provide several target genes as good basis for further research of biofilm, that I believe can contribute to fields such as cell biology, genetics, system......In this study, I sought to identify genes regulating the global molecular program for development of sessile multicellular communities, also known as biofilm, of the eukaryotic microorganism, Saccharomyces cerevisiae (yeast). Yeast biofilm has a clinical interest, as biofilms can cause chronic......, but only a small subset is previously described as regulators of FLO11. These results reveal that the regulation of biofilm formation and FLO11 is even more complex than what has previously been described. I find that the molecular program for biofilm formation shares many essential components with two...

  7. New insights in Staphylococcus pseudintermedius pathogenicity: antibiotic-resistant biofilm formation by a human wound-associated strain.

    Science.gov (United States)

    Pompilio, Arianna; De Nicola, Serena; Crocetta, Valentina; Guarnieri, Simone; Savini, Vincenzo; Carretto, Edoardo; Di Bonaventura, Giovanni

    2015-05-21

    Staphylococcus pseudintermedius is an opportunistic pathogen recognized as the leading cause of skin, ear, and post-operative bacterial infections in dogs and cats. Zoonotic infections have also recently been reported causing endocarditis, infection of surgical wounds, rhinosinusitis, and catheter-related bacteremia. The aim of the present study is to evaluate, for the first time, the pathogenic potential of S. pseudintermedius isolated from a human infection. To this end, strain DSM 25713, which was recently isolated from a wound of a leukemic patient who underwent a bone marrow transplantation, was investigated for biofilm formation and antibiotic-resistance under conditions relevant for wound infection. The effect of pH (5.5, 7.1, and 8.7) and the presence of serum (diluted at 1:2, 1:10, and 1:100) on biofilm formation was assessed through a crystal violet assay. The presence of serum significantly reduced the ability to form biofilm, regardless of the pH value tested. In vitro activity of eight antibiotics against biofilm formation and mature 48 h-old biofilms was comparatively assessed by crystal violet assay and viable cell count, respectively. Antibiotics at sub-inhibitory concentrations reduced biofilm formation in a dose-dependent manner, although cefoxitin was the most active, causing a significant reduction already at 1/8xMIC. Rifampicin showed the highest activity against preformed biofilms (MBEC90: 2xMIC). None of the antibiotics completely eradicated the preformed biofilms, regardless of tested concentrations. Confocal and electron microscopy analyses of mature biofilm revealed a complex "mushroom-like" architecture consisting of microcolonies embedded in a fibrillar extracellular matrix. For the first time, our results show that human wound-associated S. pseudintermedius is able to form inherently antibiotic-resistant biofilms, suggestive of its pathogenic potential, and consistent with recent reports of zoonotic infections.

  8. In Vitro Efficacy of XP-endo Finisher with 2 Different Protocols on Biofilm Removal from Apical Root Canals.

    Science.gov (United States)

    Bao, Pingping; Shen, Ya; Lin, James; Haapasalo, Markus

    2017-02-01

    The purpose of this study was to evaluate the effectiveness of the XP-endo Finisher (XPF; FKG Dentaire SA, La Chaux-de-Fonds, Switzerland) in biofilm removal in comparison with conventional needle irrigation (CNI) and passive ultrasonic irrigation (PUI) using an infected tooth model with an artificial apical groove. Fifty-four extracted human single-rooted premolars were selected. Each tooth was split longitudinally into 2 halves, with a groove made in the apical segment of the canal wall. After growing mixed bacteria biofilm for 4 weeks, the split halves were reassembled and instrumented using Vortex Blue files (Dentsply Tulsa Dental, Tulsa, OK) to size 40/.06. The instrumented teeth were randomly assigned to 6 groups (n = 8) according to the final irrigation protocol. Three different techniques (CNI, PUI, and XPF) were performed each with either continuous irrigation or 3-step irrigation. Scanning electron microscopic images were taken to evaluate the amount of residual biofilm inside and outside the groove. Robust growth of biofilm was observed in each canal of the controls after 4 weeks. XPF showed the best biofilm removal efficacy inside and outside the groove followed by PUI and CNI (P biofilm from hard-to-reach areas in the root canal system. The 3-step irrigation protocol was more effective than continuous irrigation when XPF was used. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  9. Contribution of cell elongation to the biofilm formation of Pseudomonas aeruginosa during anaerobic respiration.

    Science.gov (United States)

    Yoon, Mi Young; Lee, Kang-Mu; Park, Yongjin; Yoon, Sang Sun

    2011-01-18

    Pseudomonas aeruginosa, a gram-negative bacterium of clinical importance, forms more robust biofilm during anaerobic respiration, a mode of growth presumed to occur in abnormally thickened mucus layer lining the cystic fibrosis (CF) patient airway. However, molecular basis behind this anaerobiosis-triggered robust biofilm formation is not clearly defined yet. Here, we identified a morphological change naturally accompanied by anaerobic respiration in P. aeruginosa and investigated its effect on the biofilm formation in vitro. A standard laboratory strain, PAO1 was highly elongated during anaerobic respiration compared with bacteria grown aerobically. Microscopic analysis demonstrated that cell elongation likely occurred as a consequence of defective cell division. Cell elongation was dependent on the presence of nitrite reductase (NIR) that reduces nitrite (NO(2) (-)) to nitric oxide (NO) and was repressed in PAO1 in the presence of carboxy-PTIO, a NO antagonist, demonstrating that cell elongation involves a process to respond to NO, a spontaneous byproduct of the anaerobic respiration. Importantly, the non-elongated NIR-deficient mutant failed to form biofilm, while a mutant of nitrate reductase (NAR) and wild type PAO1, both of which were highly elongated, formed robust biofilm. Taken together, our data reveal a role of previously undescribed cell biological event in P. aeruginosa biofilm formation and suggest NIR as a key player involved in such process.

  10. Reactive oxygen species mediated bacterial biofilm inhibition via zinc oxide nanoparticles and their statistical determination.

    Directory of Open Access Journals (Sweden)

    Sourabh Dwivedi

    Full Text Available The formation of bacterial biofilm is a major challenge in clinical applications. The main aim of this study is to describe the synthesis, characterization and biocidal potential of zinc oxide nanoparticles (NPs against bacterial strain Pseudomonas aeruginosa. These nanoparticles were synthesized via soft chemical solution process in a very short time and their structural properties have been investigated in detail by using X-ray diffraction and transmission electron microscopy measurements. In this work, the potential of synthesized ZnO-NPs (∼ 10-15 nm has been assessed in-vitro inhibition of bacteria and the formation of their biofilms was observed using the tissue culture plate assays. The crystal violet staining on biofilm formation and its optical density revealed the effect on biofilm inhibition. The NPs at a concentration of 100 µg/mL significantly inhibited the growth of bacteria and biofilm formation. The biofilm inhibition by ZnO-NPs was also confirmed via bio-transmission electron microscopy (Bio-TEM. The Bio-TEM analysis of ZnO-NPs treated bacteria confirmed the deformation and damage of cells. The bacterial growth in presence of NPs concluded the bactericidal ability of NPs in a concentration dependent manner. It has been speculated that the antibacterial activity of NPs as a surface coating material, could be a feasible approach for controlling the pathogens. Additionally, the obtained bacterial solution data is also in agreement with the results from statistical analytical methods.

  11. Biofilm-Forming Methicillin-Resistant Staphylococcus aureus Survive in Kupffer Cells and Exhibit High Virulence in Mice

    Directory of Open Access Journals (Sweden)

    Takuto Oyama

    2016-06-01

    Full Text Available Although Staphylococcus aureus is part of the normal body flora, heavy usage of antibiotics has resulted in the emergence of methicillin-resistant strains (MRSA. MRSA can form biofilms and cause indwelling foreign body infections, bacteremia, soft tissue infections, endocarditis, and osteomyelitis. Using an in vitro assay, we screened 173 clinical blood isolates of MRSA and selected 20 high-biofilm formers (H-BF and low-biofilm formers (L-BF. These were intravenously administered to mice and the general condition of mice, the distribution of bacteria, and biofilm in the liver, lung, spleen, and kidney were investigated. MRSA count was the highest in the liver, especially within Kupffer cells, which were positive for acid polysaccharides that are associated with intracellular biofilm. After 24 h, the general condition of the mice worsened significantly in the H-BF group. In the liver, bacterial deposition and aggregation and the biofilm-forming spot number were all significantly greater for H-BF group than for L-BF. CFU analysis revealed that bacteria in the H-BF group survived for long periods in the liver. These results indicate that the biofilm-forming ability of MRSA is a crucial factor for intracellular persistence, which could lead to chronic infections.

  12. Staphylococcus epidermidis surfactant peptides promote biofilm maturation and dissemination of biofilm-associated infection in mice.

    Science.gov (United States)

    Wang, Rong; Khan, Burhan A; Cheung, Gordon Y C; Bach, Thanh-Huy L; Jameson-Lee, Max; Kong, Kok-Fai; Queck, Shu Y; Otto, Michael

    2011-01-01

    Biofilms are surface-attached agglomerations of microorganisms embedded in an extracellular matrix. Biofilm-associated infections are difficult to eradicate and represent a significant reservoir for disseminating and recurring serious infections. Infections involving biofilms frequently develop on indwelling medical devices in hospitalized patients, and Staphylococcus epidermidis is the leading cause of infection in this setting. However, the molecular determinants of biofilm dissemination are unknown. Here we have demonstrated that specific secreted, surfactant-like S. epidermidis peptides--the β subclass of phenol-soluble modulins (PSMs)--promote S. epidermidis biofilm structuring and detachment in vitro and dissemination from colonized catheters in a mouse model of device-related infection. Our study establishes in vivo significance of biofilm detachment mechanisms for the systemic spread of biofilm-associated infection and identifies the effectors of biofilm maturation and detachment in a premier biofilm-forming pathogen. Furthermore, by demonstrating that antibodies against PSMβ peptides inhibited bacterial spread from indwelling medical devices, we have provided proof of principle that interfering with biofilm detachment mechanisms may prevent dissemination of biofilm-associated infection.

  13. Proteomic analysis of Marinobacter hydrocarbonoclasticus SP17 biofilm formation at the alkane-water interface reveals novel proteins and cellular processes involved in hexadecane assimilation.

    Science.gov (United States)

    Vaysse, Pierre-Joseph; Prat, Laure; Mangenot, Sophie; Cruveiller, Stéphane; Goulas, Philippe; Grimaud, Régis

    2009-12-01

    Many hydrocarbon-degrading bacteria form biofilms at the hydrocarbon-water interface to overcome the weak accessibility of these poorly water-soluble substrates. In order to gain insight into the cellular functions involved, we undertook a proteomic analysis of Marinobacter hydrocarbonoclasticus SP17 biofilm developing at the hexadecane-water interface. Biofilm formation on hexadecane led to a global change in cell physiology involving modulation of the expression of 576 out of 1144 detected proteins when compared with planktonic cells growing on acetate. Biofilm cells overproduced a protein encoded by MARHY0478 that contains a conserved domain belonging to the family of the outer membrane transporters of hydrophobic compounds. Homologs of MARHY0478 were exclusively found in marine bacteria degrading alkanes or possessing alkane degradation genes, and hence presumably constitute a family of alkane transporters specific to marine bacteria. Interestingly, we also found that sessile cells growing on hexadecane overexpressed type VI secretion system components. This secretion system has been identified as a key factor in virulence and in symbiotic interaction with host organisms. This observation is the first experimental evidence of the contribution of a type VI secretion system to environmental adaptation, and raises the intriguing question about the role of this secretion machine in alkane assimilation.

  14. Wound biofilms: lessons learned from oral biofilms

    Science.gov (United States)

    Mancl, Kimberly A.; Kirsner, Robert S.; Ajdic, Dragana

    2013-01-01

    Biofilms play an important role in the development and pathogenesis of many chronic infections. Oral biofilms, more commonly known as dental plaque,are a primary cause of oral diseases including caries, gingivitis and periodontitis. Oral biofilms are commonly studied as model biofilm systems as they are easily accessible, thus biofilm research in oral diseases is advanced with details of biofilm formation and bacterial interactions being well-elucidated. In contrast, wound research has relatively recently directed attentionto the role biofilms have in chronic wounds. This review discusses the biofilms in periodontal disease and chronic wounds with comparisons focusing on biofilm detection, biofilm formation, the immune response to biofilms, bacterial interaction and quorum sensing. Current treatment modalities used by both fields as well as future therapies are also discussed. PMID:23551419

  15. Functional and structural studies of the disulfide isomerase DsbC from the plant pathogen Xylella fastidiosa reveals a redox-dependent oligomeric modulation in vitro.

    Science.gov (United States)

    Santos, Clelton A; Toledo, Marcelo A S; Trivella, Daniela B B; Beloti, Lilian L; Schneider, Dilaine R S; Saraiva, Antonio M; Crucello, Aline; Azzoni, Adriano R; Souza, Alessandra A; Aparicio, Ricardo; Souza, Anete P

    2012-10-01

    Xylella fastidiosa is a Gram-negative bacterium that grows as a biofilm inside the xylem vessels of susceptible plants and causes several economically relevant crop diseases. In the present study, we report the functional and low-resolution structural characterization of the X. fastidiosa disulfide isomerase DsbC (XfDsbC). DsbC is part of the disulfide bond reduction/isomerization pathway in the bacterial periplasm and plays an important role in oxidative protein folding. In the present study, we demonstrate the presence of XfDsbC during different stages of X. fastidiosa biofilm development. XfDsbC was not detected during X. fastidiosa planktonic growth; however, after administering a sublethal copper shock, we observed an overexpression of XfDsbC that also occurred during planktonic growth. These results suggest that X. fastidiosa can use XfDsbC in vivo under oxidative stress conditions similar to those induced by copper. In addition, using dynamic light scattering and small-angle X-ray scattering, we observed that the oligomeric state of XfDsbC in vitro may be dependent on the redox environment. Under reducing conditions, XfDsbC is present as a dimer, whereas a putative tetrameric form was observed under nonreducing conditions. Taken together, our findings demonstrate the overexpression of XfDsbC during biofilm formation and provide the first structural model of a bacterial disulfide isomerase in solution. © 2012 The Authors Journal compilation © 2012 FEBS.

  16. Spatial and temporal variability of biomarkers and microbial diversity reveal metabolic and community flexibility in Streamer Biofilm Communities in the Lower Geyser Basin, Yellowstone National Park.

    Science.gov (United States)

    Schubotz, F; Meyer-Dombard, D R; Bradley, A S; Fredricks, H F; Hinrichs, K-U; Shock, E L; Summons, R E

    2013-11-01

    Detailed analysis of 16S rRNA and intact polar lipids (IPLs) from streamer biofilm communities (SBCs), collected from geochemically similar hot springs in the Lower Geyser Basin, Yellowstone National Park, shows good agreement and affirm that IPLs can be used as reliable markers for the microbial constituents of SBCs. Uncultured Crenarchaea are prominent in SBS, and their IPLs contain both glycosidic and mixed glyco-phospho head groups with tetraether cores, having 0-4 rings. Archaeal IPL contributions increase with increasing temperature and comprise up to one-fourth of the total IPL inventory at >84 °C. At elevated temperatures, bacterial IPLs contain abundant glycosidic glycerol diether lipids. Diether and diacylglycerol (DAG) lipids with aminopentanetetrol and phosphatidylinositol head groups were identified as lipids diagnostic of Aquificales, while DAG glycolipids and glyco-phospholipids containing N-acetylgycosamine as head group were assigned to members of the Thermales. With decreasing temperature and concomitant changes in water chemistry, IPLs typical of phototrophic bacteria, such as mono-, diglycosyl, and sulfoquinovosyl DAG, which are specific for cyanobacteria, increase in abundance, consistent with genomic data from the same samples. Compound-specific stable carbon isotope analysis of IPL breakdown products reveals a large isotopic diversity among SBCs in different hot springs. At two of the hot springs, 'Bison Pool' and Flat Cone, lipids derived from Aquificales are enriched in (13) C relative to biomass and approach values close to dissolved inorganic carbon (DIC) (approximately 0‰), consistent with fractionation during autotrophic carbon fixation via the reversed tricarboxylic acid pathway. At a third site, Octopus Spring, the same Aquificales-diagnostic lipids are 10‰ depleted relative to biomass and resemble stable carbon isotope values of dissolved organic carbon (DOC), indicative of heterotrophy. Other bacterial and archaeal lipids show

  17. Metabolomic Studies of Oral Biofilm, Oral Cancer, and Beyond.

    Science.gov (United States)

    Washio, Jumpei; Takahashi, Nobuhiro

    2016-06-02

    Oral diseases are known to be closely associated with oral biofilm metabolism, while cancer tissue is reported to possess specific metabolism such as the 'Warburg effect'. Metabolomics might be a useful method for clarifying the whole metabolic systems that operate in oral biofilm and oral cancer, however, technical limitations have hampered such research. Fortunately, metabolomics techniques have developed rapidly in the past decade, which has helped to solve these difficulties. In vivo metabolomic analyses of the oral biofilm have produced various findings. Some of these findings agreed with the in vitro results obtained in conventional metabolic studies using representative oral bacteria, while others differed markedly from them. Metabolomic analyses of oral cancer tissue not only revealed differences between metabolomic profiles of cancer and normal tissue, but have also suggested a specific metabolic system operates in oral cancer tissue. Saliva contains a variety of metabolites, some of which might be associated with oral or systemic disease; therefore, metabolomics analysis of saliva could be useful for identifying disease-specific biomarkers. Metabolomic analyses of the oral biofilm, oral cancer, and saliva could contribute to the development of accurate diagnostic, techniques, safe and effective treatments, and preventive strategies for oral and systemic diseases.

  18. In vitro FRAP reveals the ATP-dependent nuclear mobilization of the exon junction complex protein SRm160

    OpenAIRE

    Wagner, Stefan; Chiosea, Simion; Ivshina, Maria; Nickerson, Jeffrey A.

    2004-01-01

    We present a new in vitro system for characterizing the binding and mobility of enhanced green fluorescent protein (EGFP)–labeled nuclear proteins by fluorescence recovery after photobleaching in digitonin-permeabilized cells. This assay reveals that SRm160, a splicing coactivator and component of the exon junction complex (EJC) involved in RNA export, has an adenosine triphosphate (ATP)–dependent mobility. Endogenous SRm160, lacking the EGFP moiety, could also be released from sites at splic...

  19. Plasticity of Candida albicans Biofilms

    Science.gov (United States)

    Daniels, Karla J.

    2016-01-01

    SUMMARY Candida albicans, the most pervasive fungal pathogen that colonizes humans, forms biofilms that are architecturally complex. They consist of a basal yeast cell polylayer and an upper region of hyphae encapsulated in extracellular matrix. However, biofilms formed in vitro vary as a result of the different conditions employed in models, the methods used to assess biofilm formation, strain differences, and, in a most dramatic fashion, the configuration of the mating type locus (MTL). Therefore, integrating data from different studies can lead to problems of interpretation if such variability is not taken into account. Here we review the conditions and factors that cause biofilm variation, with the goal of engendering awareness that more attention must be paid to the strains employed, the methods used to assess biofilm development, every aspect of the model employed, and the configuration of the MTL locus. We end by posing a set of questions that may be asked in comparing the results of different studies and developing protocols for new ones. This review should engender the notion that not all biofilms are created equal. PMID:27250770

  20. SarA is a negative regulator of Staphylococcus epidermidis biofilm formation

    DEFF Research Database (Denmark)

    Martin, Christer; Heinze, C.; Busch, M.

    2012-01-01

    Biofilm formation is essential for Staphylococcus epidermidis pathogenicity in implant-associated infections. Nonetheless, large proportions of invasive S. epidermidis isolates fail to show accumulative biofilm growth in vitro. We here tested the hypothesis that this apparent paradox is related...

  1. A Candida albicans early stage biofilm detachment event in rich medium

    Directory of Open Access Journals (Sweden)

    Nantel Andre

    2009-02-01

    Full Text Available Abstract Background Dispersal from Candida albicans biofilms that colonize catheters is implicated as a primary factor in the link between contaminated catheters and life threatening blood stream infections (BSI. Appropriate in vitro C. albicans biofilm models are needed to probe factors that induce detachment events. Results Using a flow through system to culture C. albicans biofilms we characterized a detachment process which culminates in dissociation of an entire early stage biofilm from a silicone elastomer surface. We analyzed the transcriptome response at time points that bracketed an abrupt transition in which a strong adhesive association with the surface is weakened in the initial stages of the process, and also compared batch and biofilm cultures at relevant time points. K means analysis of the time course array data revealed categories of genes with similar patterns of expression that were associated with adhesion, biofilm formation and glycoprotein biosynthesis. Compared to batch cultures the biofilm showed a pattern of expression of metabolic genes that was similar to the C. albicans response to hypoxia. However, the loss of strong adhesion was not obviously influenced by either the availability of oxygen in the medium or at the silicone elastomer surface. The detachment phenotype of mutant strains in which selected genes were either deleted or overexpressed was characterized. The microarray data indicated that changes associated with the detachment process were complex and, consistent with this assessment, we were unable to demonstrate that transcriptional regulation of any single gene was essential for loss of the strong adhesive association. Conclusion The massive dispersal of the early stage biofilm from a biomaterial surface that we observed is not orchestrated at the level of transcriptional regulation in an obvious manner, or is only regulated at this level by a small subpopulation of cells that mediate adhesion to the

  2. Ascorbic acid augments colony spreading by reducing biofilm formation of methicillin-resistantStaphylococcus aureus.

    Science.gov (United States)

    Ali Mirani, Zulfiqar; Khan, Muhammad Naseem; Siddiqui, Anila; Khan, Fouzia; Aziz, Mubashir; Naz, Shagufta; Ahmed, Ayaz; Khan, Seema Ismat

    2018-02-01

    Staphylococcus aureus is a Gram-positive pathogen, well known for its resistance and versatile lifestyle. Under unfavourable conditions, it adapts biofilm mode of growth. For staphylococcal biofilm formation, production of extracellular polymeric substances (EPS) is a pre-requisite, which is regulated by ica operon-encoded enzymes. This study was designed to know the impact of ascorbic acid on biofilm formation and colony spreading processes of S. aureus and MRSA. The isolates of methicillin-resistant S. aureus (MRSA) used in present study, were recovered from different food samples. Various selective and differential media were used for identification and confirmation of S. aureus . Agar dilution method was used for determination of oxacillin and ascorbic acid resistance level. MRSA isolates were re-confirmed by E-test and by amplification of mecA gene. Tube methods and Congo-Red agar were used to study biofilm formation processes. Gene expression studies were carried on real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The results revealed the presence of mecA gene belonging to SCC mecA type IV along with agr type II in the isolates. In vitro studies showed the sub-inhibitory concentration of oxacillin induced biofilm production. However, addition of sub-inhibitory dose of ascorbic acid was found to inhibit EPS production, biofilm formation and augment colony spreading on soft agar plates. The inhibition of biofilm formation and augmentation of colony spreading observed with ascorbic acid alone or in combination with oxacillin. Moreover, gene expression studies showed that ascorbic acid increases agr expression and decreases icaA gene expression. The present study concluded that ascorbic acid inhibits biofilm formation, promotes colony spreading and increases agr gene expression in MRSA.

  3. APPLICATIONS OF BIOTECHNOLOGY IN DEVELOPMENT OF BIOMATERIALS: NANOTECHNOLOGY AND BIOFILMS

    Energy Technology Data Exchange (ETDEWEB)

    Brigmon, R.; Berry, T.; Narayan, R.

    2010-11-29

    Biotechnology is the application of biological techniques to develop new tools and products for medicine and industry. Due to various properties including chemical stability, biocompatibility, and specific activity, e.g. antimicrobial properties, many new and novel materials are being investigated for use in biosensing, drug delivery, hemodialysis, and other medical applications. Many of these materials are less than 100 nanometers in size. Nanotechnology is the engineering discipline encompassing designing, producing, testing, and using structures and devices less than 100 nanometers. One of the challenges associated with biomaterials is microbial contamination that can lead to infections. In recent work we have examined the functionalization of nanoporous biomaterials and antimicrobial activities of nanocrystalline diamond materials. In vitro testing has revealed little antimicrobial activity against Pseudomonas fluorescens bacteria and associated biofilm formation that enhances recalcitrance to antimicrobial agents including disinfectants and antibiotics. Laser scanning confocal microscopy studies further demonstrated properties and characteristics of the material with regard to biofilm formation.

  4. Estudos com o extrato da Punica granatum Linn. (romã: efeito antimicrobiano in vitro e avaliação clínica de um dentifrício sobre microrganismos do biofilme dental = Studies with the extract of the punica granatum linn. (pomegranate: effect antimicrobial “in vitro” and trial avaliation of a toothpaste upon microrganisms of the oral biofilm

    Directory of Open Access Journals (Sweden)

    Pereira, Jozinete Vieira

    2005-01-01

    Full Text Available A atividade antimicrobiana in vitro, CIM (Concentração Inibitória Mínima, CIMA (Concentração Inibitória Mínima de Aderência e a CBM (Concentração Mínima Bactericida do extrato da romã (Punica granatum Linn. foi avaliada em cinco linhagens bacterianas do biofilme dental: Streptococcus mutans, Streptococcus mitis, Streptococcus sanguis, Streptococcus sobrinus e Lactobacillus casei. Em estudo comparativo com o gluconato de clorexidina, os resultados demonstraram efetiva ação inibitória de ambas as substâncias. O extrato da romã foi efetivo na CIMA das cinco linhagens ensaiadas, representada pela ausência de aderência ao vidro na presença de sacarose. A atividade antimicrobiana in vivo do extrato da romã foi testada em 13 pacientes, através do uso de um dentrifício a base desse extrato e foi observadas redução do número de Streptococcus mutans (53,84% e Índice de Sangramento Gengival. Conclui-se, portanto, que o extrato da Punica granatum Linn. , apresentou potente atividade antimicrobiana in vivo e in vitro sobre as linhagens do biofilme dental. Os resultados sugerem a utilização do extrato da romã em indicações terapêuticas na prática odontológica

  5. Bacterial biofilms. Bacteria Quorum sensing in biofilms

    Directory of Open Access Journals (Sweden)

    E. S. Vorobey

    2012-03-01

    Full Text Available Data on biofilms, their structure and properties, peculiarities of formation and interaction between microorganisms in the film are presented. Information on discovery and study of biofilms, importance of biofilms in the medical and clinical microbiology are offered. The data allow to interpret biofilm as a form of existence of human normal microflora. For the exchange of information within the biofilm between the individual cells of the same or different species bacteria use the signal molecules of the Quorum sensing system. Coordination of bacterial cells activity in the biofilms gives them significant advantages: in the biofilms bacteria are protected from the influence of the host protective factors and the antibacterial drugs.

  6. Urinary catheter indwelling clinical pathogen biofilm formation, exopolysaccharide characterization and their growth influencing parameters.

    Science.gov (United States)

    Murugan, Kasi; Selvanayaki, Krishnasamy; Al-Sohaibani, Saleh

    2016-01-01

    Self-reproducing microbial biofilm community mainly involved in the contamination of indwelling medical devices including catheters play a vital role in nosocomial infections. The catheter-associated urinary tract infection (CA-UTI) causative Staphylococcus aureus, Enterobacter faecalis, and Pseudomonas aeruginosa were selectively isolated, their phenotypic as well as genotypic biofilm formation, production and monomeric sugar composition of EPS as well as sugar, salt, pH and temperature influence on their in vitro biofilm formation were determined. From 50 culture positive urinary catheters S. aureus (24%), P. aeruginosa (18%), E. faecalis (14%) and others (44%) were isolated. The performed assays revealed their varying biofilm forming ability. The isolated S. aureus ica, E. faecalis esp, and P. aeruginosa cup A gene sequencing and phylogenetic analysis showed their close branching and genetic relationship. The analyzed sugar, salt, pH, and temperature showed that the degree of CA-UTI isolates biofilm formation is an environmentally sensitive process. EPS monosaccharide HPLC analysis showed the presence of neutral sugars (ng/μl) as follows: glucose (P. aeruginosa: 44.275; E. faecalis: 4.23), lactose (P. aeruginosa: 7.29), mannitol (P. aeruginosa: 2.53; S. aureus: 2.62; E. faecalis: 2.054) and maltose (E. faecalis: 7.0042) revealing species-specific presence and variation. This study may have potential clinical relevance for the easy diagnosis and management of CA-UTI.

  7. Pseudomonas aeruginosa forms Biofilms in Acute InfectionIndependent of Cell-to-Cell Signaling

    Energy Technology Data Exchange (ETDEWEB)

    Schaber, J. Andy; Triffo, W.J.; Suh, Sang J.; Oliver, Jeffrey W.; Hastert, Mary C.; Griswold, John A.; Auer, Manfred; Hamood, Abdul N.; Rumbaugh, Kendra P.

    2006-09-20

    Biofilms are bacterial communities residing within a polysaccharide matrix that are associated with persistence and antibiotic resistance in chronic infections. We show that the opportunistic pathogen Pseudomonas aeruginosa forms biofilms within 8 hours of infection in thermally-injured mice, demonstrating that biofilms contribute to bacterial colonization in acute infections. P. aeruginosa biofilms were visualized within burned tissue surrounding blood vessels and adipose cells. Although quorum sensing (QS), a bacterial signaling mechanism, coordinates differentiation of biofilms in vitro, wild type and QS-deficient P. aeruginosa formed similar biofilms in vivo. Our findings demonstrate that P. aeruginosa forms biofilms on specific host tissues independent of QS.

  8. Biofilm disruption with rotating microrods enhances antimicrobial efficacy

    Science.gov (United States)

    Mair, Lamar O.; Nacev, Aleksandar; Hilaman, Ryan; Stepanov, Pavel Y.; Chowdhury, Sagar; Jafari, Sahar; Hausfeld, Jeffrey; Karlsson, Amy J.; Shirtliff, Mark E.; Shapiro, Benjamin; Weinberg, Irving N.

    2017-04-01

    Biofilms are a common and persistent cause of numerous illnesses. Compared to planktonic microbes, biofilm residing cells often demonstrate significant resistance to antimicrobial agents. Thus, methods for dislodging cells from the biofilm may increase the antimicrobial susceptibility of such cells, and serve as a mechanical means of increasing antimicrobial efficacy. Using Aspergillus fumigatus as a model microbe, we magnetically rotate microrods in and around biofilm. We show that such rods can improve the efficacy of antimicrobial Amphotericin B treatments in vitro. This work represents a first step in using kinetic magnetic particle therapy for disrupting fungal biofilms.

  9. Quercetin Assists Fluconazole to Inhibit Biofilm Formations of Fluconazole-Resistant Candida Albicans in In Vitro and In Vivo Antifungal Managements of Vulvovaginal Candidiasis

    Directory of Open Access Journals (Sweden)

    Mei Gao

    2016-11-01

    Full Text Available Background: Vulvovaginal candidiasis (VVC is a common gynecological disease. Candida albicans is believed to be mainly implicated in VVC occurrence, the biofilm of which is one of the virulence factors responsible for resistance to traditional antifungal agents especially to fluconazole (FCZ. Quercetin (QCT is a dietary flavonoid and has been demonstrated to be antifungal against C. albicans biofilm. Methods: 17 C. albicans isolates including 15 clinical ones isolated from VVC patients were employed to investigate the effects of QCT and/or FCZ on the inhibition of C. albicans biofilm. Results: We observed that 64 µg/mL QCT and/or 128 µg/mL FCZ could (i be synergistic against 10 FCZ-resistant planktonic and 17 biofilm cells of C. albicans, (ii inhibit fungal adherence, cell surface hydrophobicity (CSH, flocculation, yeast-to-hypha transition, metabolism, thickness and dispersion of biofilms; (iii down-regulate the expressions of ALS1, ALS3, HWP1, SUN41, UME6 and ECE1 and up-regulate the expressions of PDE2, NRG1 and HSP90, and we also found that (iv the fungal burden was reduced in vaginal mucosa and the symptoms were alleviated in a murine VVC model after the treatments of 5 mg/kg QCT and/or 20 mg/kg FCZ. Conclusion: Together with these results, it could be demonstrated that QCT could be a favorable antifungal agent and a promising synergist with FCZ in the clinical management of VVC caused by C. albicans biofilm.

  10. Estudo in vitro da atividade antimicrobiana do hipoclorito de sodio e da clorexidina usados como substancias quimicas auxiliares frente a biofilmes de especie unica

    OpenAIRE

    NeyllaTeixeira Sena

    2004-01-01

    Resumo: O objetivo deste estudo foi investigar a atividade antimicrobiana do hipoclorito de sódio (NaOCl) 2,5% e 5,25% e da clorexidina (CLX) 2.0% tanto na forma gel como líquida utilizados como substância química auxiliar durante o preparo químico-mecânico frente a biofilmes de espécies única. Biofilmes simples dos microrganismos Enterococcus faecalis, Staphylococcus aureus, Prevotella intermedia, Porphyromonas gingivalis, Porphyromonas endodontalis, Fusobacterium nucleatum e Candida albican...

  11. Characterization of the TolB-Pal trans-envelope complex from Xylella fastidiosa reveals a dynamic and coordinated protein expression profile during the biofilm development process.

    Science.gov (United States)

    Santos, Clelton A; Janissen, Richard; Toledo, Marcelo A S; Beloti, Lilian L; Azzoni, Adriano R; Cotta, Monica A; Souza, Anete P

    2015-10-01

    The intriguing roles of the bacterial Tol-Pal trans-envelope protein complex range from maintenance of cell envelope integrity to potential participation in the process of cell division. In this study, we report the characterization of the XfTolB and XfPal proteins of the Tol-Pal complex of Xylella fastidiosa. X. fastidiosa is a major plant pathogen that forms biofilms inside xylem vessels, triggering the development of diseases in important cultivable plants around the word. Based on functional complementation experiments in Escherichia coli tolB and pal mutant strains, we confirmed the role of xftolB and xfpal in outer membrane integrity. In addition, we observed a dynamic and coordinated protein expression profile during the X. fastidiosa biofilm development process. Using small-angle X-ray scattering (SAXS), the low-resolution structure of the isolated XfTolB-XfPal complex in solution was solved for the first time. Finally, the localization of the XfTolB and XfPal polar ends was visualized via immunofluorescence labeling in vivo during bacterial cell growth. Our results highlight the major role of the components of the cell envelope, particularly the TolB-Pal complex, during the different phases of bacterial biofilm development. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Revealing the relationship between microbial community structure in natural biofilms and the pollution level in urban rivers: a case study in the Qinhuai River basin, Yangtze River Delta.

    Science.gov (United States)

    Cai, Wei; Li, Yi; Wang, Peifang; Niu, Lihua; Zhang, Wenlong; Wang, Chao

    River pollution is one of the most challenging environmental issues, but the effect of river pollution levels on the biofilm communities has not been well-studied. Spatial and temporal distribution characteristics of environmental parameters and the biofilm communities were investigated in the Qinhuai River basin, Nanjing, China. Water samples were grouped into three clusters reflecting their varying pollution levels of relatively slight pollution, moderated pollution, and high pollution by hierarchical cluster analysis. In different clusters, the biofilm communities mainly differed in the proportion of Actinobacteria, Firmicutes, and Proteobacteria. As the dominant classes of Proteobacteria, Alpha-, Beta- and Gammaproteobacteria seemed to show an upward trend followed by a small fluctuation in the abundance with the escalation of water pollution level. Results of redundancy analysis demonstrated that temperature, total nitrogen to total phosphorus ratios (TN/TP) and concentrations of ammonia nitrogen (NH3-N) and TN were mainly responsible for the variation in bacterial community structure. The occurrences of Alpha-, Beta- and Gammaproteobacteria were closely associated with higher temperature, higher concentrations of NH3-N and TN and a lower TN/TP ratio. This study may provide a theoretical basis for the water pollution control and ecological restoration in urban rivers under different pollution levels.

  13. Red and Green Fluorescence from Oral Biofilms.

    Directory of Open Access Journals (Sweden)

    Catherine M C Volgenant

    Full Text Available Red and green autofluorescence have been observed from dental plaque after excitation by blue light. It has been suggested that this red fluorescence is related to caries and the cariogenic potential of dental plaque. Recently, it was suggested that red fluorescence may be related to gingivitis. Little is known about green fluorescence from biofilms. Therefore, we assessed the dynamics of red and green fluorescence in real-time during biofilm formation. In addition, the fluorescence patterns of biofilm formed from saliva of eight different donors are described under simulated gingivitis and caries conditions. Biofilm formation was analysed for 12 hours under flow conditions in a microfluidic BioFlux flow system with high performance microscopy using a camera to allow live cell imaging. For fluorescence images dedicated excitation and emission filters were used. Both green and red fluorescence were linearly related with the total biomass of the biofilms. All biofilms displayed to some extent green and red fluorescence, with higher red and green fluorescence intensities from biofilms grown in the presence of serum (gingivitis simulation as compared to the sucrose grown biofilms (cariogenic simulation. Remarkably, cocci with long chain lengths, presumably streptococci, were observed in the biofilms. Green and red fluorescence were not found homogeneously distributed within the biofilms: highly fluorescent spots (both green and red were visible throughout the biomass. An increase in red fluorescence from the in vitro biofilms appeared to be related to the clinical inflammatory response of the respective saliva donors, which was previously assessed during an in vivo period of performing no-oral hygiene. The BioFlux model proved to be a reliable model to assess biofilm fluorescence. With this model, a prediction can be made whether a patient will be prone to the development of gingivitis or caries.

  14. Red and Green Fluorescence from Oral Biofilms.

    Science.gov (United States)

    Volgenant, Catherine M C; Hoogenkamp, Michel A; Krom, Bastiaan P; Janus, Marleen M; Ten Cate, Jacob M; de Soet, Johannes J; Crielaard, Wim; van der Veen, Monique H

    2016-01-01

    Red and green autofluorescence have been observed from dental plaque after excitation by blue light. It has been suggested that this red fluorescence is related to caries and the cariogenic potential of dental plaque. Recently, it was suggested that red fluorescence may be related to gingivitis. Little is known about green fluorescence from biofilms. Therefore, we assessed the dynamics of red and green fluorescence in real-time during biofilm formation. In addition, the fluorescence patterns of biofilm formed from saliva of eight different donors are described under simulated gingivitis and caries conditions. Biofilm formation was analysed for 12 hours under flow conditions in a microfluidic BioFlux flow system with high performance microscopy using a camera to allow live cell imaging. For fluorescence images dedicated excitation and emission filters were used. Both green and red fluorescence were linearly related with the total biomass of the biofilms. All biofilms displayed to some extent green and red fluorescence, with higher red and green fluorescence intensities from biofilms grown in the presence of serum (gingivitis simulation) as compared to the sucrose grown biofilms (cariogenic simulation). Remarkably, cocci with long chain lengths, presumably streptococci, were observed in the biofilms. Green and red fluorescence were not found homogeneously distributed within the biofilms: highly fluorescent spots (both green and red) were visible throughout the biomass. An increase in red fluorescence from the in vitro biofilms appeared to be related to the clinical inflammatory response of the respective saliva donors, which was previously assessed during an in vivo period of performing no-oral hygiene. The BioFlux model proved to be a reliable model to assess biofilm fluorescence. With this model, a prediction can be made whether a patient will be prone to the development of gingivitis or caries.

  15. Investigating the Complex Conductivity Response of Different Biofilm Components

    Science.gov (United States)

    Atekwana, E. A.; Abdel Aal, G. Z.; Sarkisova, S. A.; Patrauchan, M.

    2013-12-01

    Microbial biofilms are structured communities of microorganisms commonly attached to a surface and embedded in a self-produced matrix. The matrix is composed of extracellular polymeric substances (EPS), which commonly include extracellular DNA, proteins, and polysaccharides. In addition, the biofilm structure may contain some other components such as metabolic byproducts and biogenic nanoparticle minerals. Biogeophysical studies have demonstrated the sensitivity of spectral induced polarization (SIP) measurements to the growth and development of biofilm in saturated porous media. However, the mechanisms are not very well understood. The overarching goal of this study is to determine the contribution of the different biofilm components to the spectral induced polarization (SIP) signatures in aqueous and/or porous media. We investigated the SIP response of different biofilm components including bacterial cells, alginate (exopolysaccharide), phenazine (redox-active metabolite) and magnetite (semi-conductive particulate matter). The porous media was glass beads with grain diameter of 1 mm. Each of the biofilm components was suspended in a low salt growth medium with electrolytic conductivity of 513 μS/cm. Using Pseudomonas aeruginosa PAO1 cells in suspension and in porous media, we observed the increase in SIP parameters with increasing cell density with a very well defined relaxation peak at a frequency of ~10 Hz, which was predicted by recently developed quantitative models. However, this characteristic relaxation peak was minimized in the presence of porous media. We also observed that cells suspended in alginate enhance the polarization and show a peak frequency at ~10 Hz. The study of alginate gelation in liquid phase and porous media in vitro revealed that solidified (gelated) alginate (from brown algae) increased the magnitude of imaginary conductivity while real conductivity increased very moderately. In contrast, the study of the SIP response within a porous

  16. Efficacy of NVC-422 against Staphylococcus aureus biofilms in a sheep biofilm model of sinusitis.

    Science.gov (United States)

    Singhal, Deepti; Jekle, Andreas; Debabov, Dmitri; Wang, Lu; Khosrovi, Bez; Anderson, Mark; Foreman, Andrew; Wormald, Peter-John

    2012-01-01

    Bacterial biofilms are a major obstacle in management of recalcitrant chronic rhinosinusitis. NVC-422 is a potent, fast-acting, broad-spectrum, nonantibiotic, antimicrobial with a new mechanism of action effective against biofilm bacteria in in vitro conditions. The aim of this study was to investigate the safety and efficacy of NVC-422 as local antibiofilm treatment in a sheep model of rhinosinusitis. After accessing and occluding frontal sinus ostia in 24 merino sheep via staged endoscopic procedures, S. aureus clinical isolate was instilled in frontal sinuses. Following biofilm formation, ostial obstruction was removed and sinuses irrigated with 0.1% and 0.5% NVC-422 in 5 mM acetate isotonic saline at pH 4.0. Sheep were monitored for adverse effects and euthanized 24 hours after treatment. Frontal sinuses were assessed for infection and changes in mucosa after the treatment. S. aureus biofilms were identified with Baclight-confocal scanning microscopy protocol and the biofilm biomass assayed by applying the COMSTAT2 program to recorded image stacks. After 2 irrigations with 0.1% NVC-422, S. aureus biofilm biomass was reduced when compared to control sinuses (p = 0.0001), though this effect was variable in samples. NVC-422 0.5% solution irrigations reduced biofilm even more significantly and consistently over all samples (p biofilm biomass (p biofilms, with dose-dependent efficacy in this animal model of biofilm-associated sinusitis. Copyright © 2012 American Rhinologic Society-American Academy of Otolaryngic Allergy, LLC.

  17. Biofilm Risks

    DEFF Research Database (Denmark)

    Wirtanen, Gun Linnea; Salo, Satu

    2016-01-01

    in food factories. In the prevention of biofilm formation microbial control in process lines should both limit the number of microbes on surfaces and reduce microbial activity in the process. Thus the hygienic design of process equipment and process lines is important in improving the process hygiene...

  18. Biofilm extracellular DNA enhances mixed species biofilms of Staphylococcus epidermidis and Candida albicans.

    Science.gov (United States)

    Pammi, Mohan; Liang, Rong; Hicks, John; Mistretta, Toni-Ann; Versalovic, James

    2013-11-14

    Polymicrobial infections are responsible for significant mortality and morbidity in adults and children. Staphylococcus epidermidis and Candida albicans are the most frequent combination of organisms isolated from polymicrobial infections. Vascular indwelling catheters are sites for mixed species biofilm formation and pose a significant risk for polymicrobial infections. We hypothesized that enhancement of biofilms in a mixed species environment increases patient mortality and morbidity. Mixed species biofilms of S. epidermidis and C. albicans were evaluated in vitro and in a subcutaneous catheter infection model in vivo. Mixed species biofilms were enhanced compared to single species biofilms of either S. epidermidis or C. albicans. A mixed species environment increased catheter infection and increased dissemination of S. epidermidis in mice. Microarrays were used to explore differential gene expression of S. epidermidis in the mixed species biofilms. In mixed species biofilms, compared to single species S. epidermidis biofilms, 2.7% of S. epidermidis genes were upregulated and 6% were down regulated. Staphylococcal autolysis repressors lrgA and lrgB were down regulated 36-fold and 27-fold respectively. The role of biofilm extracellular DNA was investigated by quantitation and by evaluating the effects of DNAse in a concentration and time dependent manner. S. epidermidis specific eDNA was increased in mixed species biofilms and further confirmed by degradation with DNAse. Mixed-species biofilms are enhanced and associated with increased S. epidermidis-specific eDNA in vitro and greater systemic dissemination of S. epidermidis in vivo. Down regulation of the lrg operon, a repressor of autolysis, associated with increased eDNA suggests a possible role for bacterial autolysis in mixed species biofilms. Enhancement and systemic dissemination of S. epidermidis may explain adverse outcomes after clinical polymicrobial infections of S. epidermidis and C. albicans.

  19. Biofilm extracellular DNA enhances mixed species biofilms of Staphylococcus epidermidis and Candida albicans

    Science.gov (United States)

    2013-01-01

    Background Polymicrobial infections are responsible for significant mortality and morbidity in adults and children. Staphylococcus epidermidis and Candida albicans are the most frequent combination of organisms isolated from polymicrobial infections. Vascular indwelling catheters are sites for mixed species biofilm formation and pose a significant risk for polymicrobial infections. We hypothesized that enhancement of biofilms in a mixed species environment increases patient mortality and morbidity. Results Mixed species biofilms of S. epidermidis and C. albicans were evaluated in vitro and in a subcutaneous catheter infection model in vivo. Mixed species biofilms were enhanced compared to single species biofilms of either S. epidermidis or C. albicans. A mixed species environment increased catheter infection and increased dissemination of S. epidermidis in mice. Microarrays were used to explore differential gene expression of S. epidermidis in the mixed species biofilms. In mixed species biofilms, compared to single species S. epidermidis biofilms, 2.7% of S. epidermidis genes were upregulated and 6% were down regulated. Staphylococcal autolysis repressors lrgA and lrgB were down regulated 36-fold and 27-fold respectively. The role of biofilm extracellular DNA was investigated by quantitation and by evaluating the effects of DNAse in a concentration and time dependent manner. S. epidermidis specific eDNA was increased in mixed species biofilms and further confirmed by degradation with DNAse. Conclusions Mixed-species biofilms are enhanced and associated with increased S. epidermidis-specific eDNA in vitro and greater systemic dissemination of S. epidermidis in vivo. Down regulation of the lrg operon, a repressor of autolysis, associated with increased eDNA suggests a possible role for bacterial autolysis in mixed species biofilms. Enhancement and systemic dissemination of S. epidermidis may explain adverse outcomes after clinical polymicrobial infections of S

  20. Synthesis and activity of biomimetic biofilm disruptors.

    Science.gov (United States)

    Böttcher, Thomas; Kolodkin-Gal, Ilana; Kolter, Roberto; Losick, Richard; Clardy, Jon

    2013-02-27

    Biofilms are often associated with human bacterial infections, and the natural tolerance of biofilms to antibiotics challenges treatment. Compounds with antibiofilm activity could become useful adjuncts to antibiotic therapy. We used norspermidine, a natural trigger for biofilm disassembly in the developmental cycle of Bacillus subtilis , to develop guanidine and biguanide compounds with up to 20-fold increased potency in preventing biofilm formation and breaking down existing biofilms. These compounds also were active against pathogenic Staphylococcus aureus . An integrated approach involving structure-activity relationships, protonation constants, and crystal structure data on a focused synthetic library revealed that precise spacing of positively charged groups and the total charge at physiological pH distinguish potent biofilm inhibitors.

  1. AFM Structural Characterization of Drinking Water Biofilm ...

    Science.gov (United States)

    Due to the complexity of mixed culture drinking water biofilm, direct visual observation under in situ conditions has been challenging. In this study, atomic force microscopy (AFM) revealed the three dimensional morphology and arrangement of drinking water relevant biofilm in air and aqueous solution. Operating parameters were optimized to improve imaging of structural details for a mature biofilm in liquid. By using a soft cantilever (0.03 N/m) and slow scan rate (0.5 Hz), biofilm and individual bacterial cell’s structural topography were resolved and continuously imaged in liquid without loss of spatial resolution or sample damage. The developed methodology will allow future in situ investigations to temporally monitor mixed culture drinking water biofilm structural changes during disinfection treatments. Due to the complexity of mixed culture drinking water biofilm, direct visual observation under in situ conditions has been challenging. In this study, atomic force microscopy (AFM) revealed the three dimensional morphology and arrangement of drinking water relevant biofilm in air and aqueous solution. Operating parameters were optimized to improve imaging of structural details for a mature biofilm in liquid. By using a soft cantilever (0.03 N/m) and slow scan rate (0.5 Hz), biofilm and individual bacterial cell’s structural topography were resolved and continuously imaged in liquid without loss of spatial resolution or sample damage. The developed methodo

  2. Role of multicellular aggregates in biofilm formation

    DEFF Research Database (Denmark)

    Kragh, Kasper N.; Hutchison, Jaime B.; Melaugh, Gavin

    2016-01-01

    In traditional models of in vitro biofilm development, individual bacterial cells seed a surface, multiply, and mature into multicellular, three-dimensional structures. Much research has been devoted to elucidating the mechanisms governing the initial attachment of single cells to surfaces. However......, in natural environments and during infection, bacterial cells tend to clump as multicellular aggregates, and biofilms can also slough off aggregates as a part of the dispersal process. This makes it likely that biofilms are often seeded by aggregates and single cells, yet how these aggregates impact biofilm...... initiation and development is not known. Here we use a combination of experimental and computational approaches to determine the relative fitness of single cells and preformed aggregates during early development of Pseudomonas aeruginosa biofilms. We find that the relative fitness of aggregates depends...

  3. In vitro and in vivo comparisons of staphylococcal biofilm formation on a cross-linked poly(ethylene glycol)-based polymer coating

    NARCIS (Netherlands)

    Fernandez, Isabel C. Saldarriaga; van der Mei, Henny C.; Metzger, Steve; Grainger, David W.; Engelsman, Anton F.; Nejadnik, M. Reza; Busscher, Henk J.

    Poly(ethylene glycol) (PEG) coatings are known to reduce microbial adhesion ill terms of numbers and binding strength However, bacterial adhesion remains of the order of 10(4) cm(-2) It is unknown whether this density of bacteria will eventually grow into a biofilm This study investigates the

  4. An in vitro study on the effect of free amino acids alone or in combination with nisin on biofilms as well as on planktonic bacteria of Streptococcus mutans

    NARCIS (Netherlands)

    Tong, Z.; Zhang, L.; Ling, J.; Jian, Y.; Huang, L.; Deng, D.

    2014-01-01

    Free D-amino acids (D-AAs) are one of the most striking features of the peptidoglycan composition in bacteria and play a key role in regulating and disassembling bacterial biofilms. Previous studies have indicated that the antimicrobial peptide nisin can inhibit the growth of the cariogenic bacteria

  5. In vitro photodynamic inactivation effects of cationic benzylidene cyclopentanone photosensitizers on clinical fluconazole-resistant Candida albicans planktonic cells and biofilms

    Science.gov (United States)

    Zhou, Shaona; Fang, Yanyan; Ye, Zulin; Wang, Ying; Zhao, Yuxia; Gu, Ying

    2016-10-01

    Background: An increasing prevalence of Candida infections has emerged with the wide use of immune-suppressants and antibiotics. Photodynamic inactivation (PDI) as a new approach to treat localized Candida infections is an emerging and promising field nowadays. This study evaluated the efficacy of photodynamic therapy using two new Cationic benzylidene cyclopentanone photosensitizers(P1 and P2) against strains of clinical fluconazole-resistant Candida albicans. Methods: Suspensions and biofilms of Candida species were incubated with P1 and P2 concentrations (0.25 50 μM) for 30 min followed by 532nm laser irradiation. For planktonic suspensions, viability of cells was assayed by CFU counting. For biofilms, the metabolic activity was evaluated by XTT. Results: In PDI of a planktonic culture of clinical fluconazole-resistant Candida albicans, P2 showed the higher efficacy. After incubation with 25 μM of P2 for 30 min and irradiation with 532nm laser (36 J cm-2), the viability of C. albicans planktonic cells decreased by 3.84 log10. For biofilm cells, a higher light dose of 75 mW cm-2 was necessary to achieve 97.71% metabolic activity reduction. Conclusions: The results of this investigation demonstrated that benzylidene cyclopentanone photosensitizer, P2, is an efficient photosensitizer to kill C. albicans. Moreover, single-species biofilms were less susceptible to PDT than their planktonic counterparts.

  6. Efficacy of a surfactant-based wound dressing on biofilm control.

    Science.gov (United States)

    Percival, Steven L; Mayer, Dieter; Salisbury, Anne-Marie

    2017-09-01

    The aim of this study was to evaluate the efficacy of both a nonantimicrobial and antimicrobial (1% silver sulfadiazine-SSD) surfactant-based wound dressing in the control of Pseudomonas aeruginosa, Enterococcus sp, Staphylococcus epidermidis, Staphylococcus aureus, and methicillin-resistant S. aureus (MRSA) biofilms. Anti-biofilm efficacy was evaluated in numerous adapted American Standards for Testing and Materials (ASTM) standard biofilm models and other bespoke biofilm models. The ASTM standard models employed included the Minimum biofilm eradication concentration (MBEC) biofilm model (ASTM E2799) and the Centers for Disease Control (CDC) biofilm reactor model (ASTM 2871). Such bespoke biofilm models included the filter biofilm model and the chamberslide biofilm model. Results showed complete kill of microorganisms within a biofilm using the antimicrobial surfactant-based wound dressing. Interestingly, the nonantimicrobial surfactant-based dressing could disrupt existing biofilms by causing biofilm detachment. Prior to biofilm detachment, we demonstrated, using confocal laser scanning microscopy (CLSM), the dispersive effect of the nonantimicrobial surfactant-based wound dressing on the biofilm within 10 minutes of treatment. Furthermore, the non-antimicrobial surfactant-based wound dressing caused an increase in microbial flocculation/aggregation, important for microbial concentration. In conclusion, this nonantimicrobial surfactant-based wound dressing leads to the effective detachment and dispersion of in vitro biofilms. The use of surfactant-based wound dressings in a clinical setting may help to disrupt existing biofilm from wound tissue and may increase the action of antimicrobial treatment. © 2017 by the Wound Healing Society.

  7. In vitro studies reveal antiurolithic effect of Terminalia arjuna using quantitative morphological information from computerized microscopy

    Directory of Open Access Journals (Sweden)

    A. Mittal

    2015-10-01

    Full Text Available ABSTRACT Purpose: For most cases, urolithiasis is a condition where excessive oxalate is present in the urine. Many reports have documented free radical generation followed by hyperoxaluria as a consequence of which calcium oxalate (CaOx deposition occurs in the kidney tissue. The present study is aimed to exam the antilithiatic potency of the aqueous extract (AE of Terminalia arjuna (T. arjuna. Materials and Methods: The antilithiatic activity of Terminalia arjuna was investigated in vitro nucleation, aggregation and growth of the CaOx crystals as well as the morphology of CaOx crystals using the inbuilt software ‘Image-Pro Plus 7.0’ of Olympus upright microscope (BX53. Antioxidant activity of AE of Terminalia arjuna bark was also determined in vitro. Results: Terminalia arjuna extract exhibited a concentration dependent inhibition of nucleation and aggregation of CaOx crystals. The AE of Terminalia arjuna bark also inhibited the growth of CaOx crystals. At the same time, the AE also modified the morphology of CaOx crystals from hexagonal to spherical shape with increasing concentrations of AE and reduced the dimensions such as area, perimeter, length and width of CaOx crystals in a dose dependent manner. Also, the Terminalia arjuna AE scavenged the DPPH (2, 2-diphenyl-1-picrylhydrazyl radicals with an IC50 at 13.1µg/mL. Conclusions: The study suggests that Terminalia arjuna bark has the potential to scavenge DPPH radicals and inhibit CaOx crystallization in vitro. In the light of these studies, Terminalia arjuna can be regarded as a promising candidate from natural plant sources of antilithiatic and antioxidant activity with high value.

  8. Role of biofilm roughness and hydrodynamic conditions in Legionella pneumophila adhesion to and detachment from simulated drinking water biofilms.

    Science.gov (United States)

    Shen, Yun; Monroy, Guillermo L; Derlon, Nicolas; Janjaroen, Dao; Huang, Conghui; Morgenroth, Eberhard; Boppart, Stephen A; Ashbolt, Nicholas J; Liu, Wen-Tso; Nguyen, Thanh H

    2015-04-07

    Biofilms in drinking water distribution systems (DWDS) could exacerbate the persistence and associated risks of pathogenic Legionella pneumophila (L. pneumophila), thus raising human health concerns. However, mechanisms controlling adhesion and subsequent detachment of L. pneumophila associated with biofilms remain unclear. We determined the connection between L. pneumophila adhesion and subsequent detachment with biofilm physical structure characterization using optical coherence tomography (OCT) imaging technique. Analysis of the OCT images of multispecies biofilms grown under low nutrient condition up to 34 weeks revealed the lack of biofilm deformation even when these biofilms were exposed to flow velocity of 0.7 m/s, typical flow for DWDS. L. pneumophila adhesion on these biofilm under low flow velocity (0.007 m/s) positively correlated with biofilm roughness due to enlarged biofilm surface area and local flow conditions created by roughness asperities. The preadhered L. pneumophila on selected rough and smooth biofilms were found to detach when these biofilms were subjected to higher flow velocity. At the flow velocity of 0.1 and 0.3 m/s, the ratio of detached cell from the smooth biofilm surface was from 1.3 to 1.4 times higher than that from the rough biofilm surface, presumably because of the low shear stress zones near roughness asperities. This study determined that physical structure and local hydrodynamics control L. pneumophila adhesion to and detachment from simulated drinking water biofilm, thus it is the first step toward reducing the risk of L. pneumophila exposure and subsequent infections.

  9. Esp-independent biofilm formation by Enterococcus faecalis.

    Science.gov (United States)

    Kristich, Christopher J; Li, Yung-Hua; Cvitkovitch, Dennis G; Dunny, Gary M

    2004-01-01

    Enterococcus faecalis is a gram-positive opportunistic pathogen known to form biofilms in vitro. In addition, this organism is often isolated from biofilms on the surfaces of various indwelling medical devices. However, the molecular mechanisms regulating biofilm formation in these clinical isolates are largely unknown. Recent work has suggested that a specific cell surface protein (Esp) of E. faecalis is critical for biofilm formation by this organism. However, in the same study, esp-deficient strains of E. faecalis were found to be capable of biofilm formation. To test the hypothesis that Esp is dispensable for biofilm formation by E. faecalis, we used microtiter plate assays and a chemostat-based biofilm fermentor assay to examine biofilm formation by genetically well-defined, non-Esp-expressing strains. Our results demonstrate that in vitro biofilm formation occurs, not only in the absence of esp, but also in the absence of the entire pathogenicity island that harbors the esp coding sequence. Using scanning electron microscopy to evaluate biofilms of E. faecalis OG1RF grown in the fermentor system, biofilm development was observed to progress through multiple stages, including attachment of individual cells to the substratum, microcolony formation, and maturation into complex multilayered structures apparently containing water channels. Microtiter plate biofilm analyses indicated that biofilm formation or maintenance was modulated by environmental conditions. Furthermore, our results demonstrate that expression of a secreted metalloprotease, GelE, enhances biofilm formation by E. faecalis. In summary, E. faecalis forms complex biofilms by a process that is sensitive to environmental conditions and does not require the Esp surface protein.

  10. Microbial pathogenesis and biofilm development

    DEFF Research Database (Denmark)

    Reisner, A.; Høiby, N.; Tolker-Nielsen, Tim

    2004-01-01

    cycles of different microorganisms will eventually lead to improved treatments. Several bacteria have evolved specific strategies for virulent colonization of humans in addition to their otherwise harmless establishment as environmental inhabitants. In many such cases biofilm development seems to play...... permit bacterial growth to occur. In laboratory model systems the growth of the surface-associated bacteria is supported by the nutrient supply in the moving or standing liquid. A benchmark of biofilm formation by several organisms in vitro is the development of three-dimensional structures that have...... been termed 'maturation', which is thought to be mediated by a differentiation process. Maturation into late stages of biofilm development resulting in stable and robust structures may require the formation of a matrix of extracellular polymeric substances (EPS), which are most often assumed to consist...

  11. Comparative evaluation of sutures coated with triclosan and chlorhexidine for oral biofilm inhibition potential and antimicrobial activity against periodontal pathogens: An in vitro study

    Directory of Open Access Journals (Sweden)

    Kunal Sunder Sethi

    2016-01-01

    Full Text Available Background: Surgical site plaque accumulation is one of the challenging problems leading to unfavorable healing. The antibacterial sutures can be used to reduce or inhibit plaque formation. Presently there is no study comparing efficacy of sutures coated with triclosan and chlorhexidine in terms of oral biofilm inhibition and antimicrobial property against periodontal pathogens. Aim: The aim of present study was to evaluate the antibacterial efficacy and oral biofilm inhibition around chlorhexidine and triclosan coated polyglactin sutures in comparison to uncoated sutures. Materials and Method: Equal segments of chlorhexidine and triclosan coated polyglactin sutures (3-0 were incubated at 370°C in saliva collected from 10 chronic periodontitis patients for 7 days. Plain uncoated suture served as control. Biofilm formation was analyzed with Confocal Laser-Scanning Microscopy (CLSM and Scanning Electron Microscopy (SEM. Quantitative assessment was done using Colony Forming Units (CFU/mL.The antibacterial efficacy of the sutures was tested against specific periodontal pathogens (S.mutans, F.nucleatum, A.actinomycetomcomitans, P.intermedia, P.gingivalis using agar diffusion method. CLSM and SEM were not subjected to statistical analysis. ANOVA test was used for colony forming units and agar diffusion test. (P < 0.05 Results: CLSM and SEM showed substantial biofilm inhibition around chlorhexidine-coated sutures followed by triclosan-coated when compared to plain uncoated suture. The antibacterial coated sutures showed statistically significant difference in CFUs/ml and zone of inhibition compared to plain uncoated sutures. Among coated sutures, chlorhexidine-coated sutures showed better results. Conclusion: The antibacterial coated sutures have a promising potential in preventing the colonization of periodontal pathogens around it thereby inhibiting biofilm formation.

  12. An in vitro study on the effect of free amino acids alone or in combination with nisin on biofilms as well as on planktonic bacteria of Streptococcus mutans.

    Directory of Open Access Journals (Sweden)

    Zhongchun Tong

    Full Text Available Free D-amino acids (D-AAs are one of the most striking features of the peptidoglycan composition in bacteria and play a key role in regulating and disassembling bacterial biofilms. Previous studies have indicated that the antimicrobial peptide nisin can inhibit the growth of the cariogenic bacteria Streptococcus mutans. The present study investigated the effect of free amino acids either alone or in combination with nisin on biofilm and on planktonic S. mutans bacteria. The results of the MIC and MBC analyses showed that D-cysteine (Cys, D- or L-aspartic acid (Asp, and D- or L-glutamic acid (Glu significantly improve the antibacterial activity of nisin against S. mutans and that the mixture of D-Cys, D-Asp, and D-Glu (3D-AAs and the mixture of L-Cys, L-Asp, and L-Glu (3L-AAs at a concentration of 40 mM can prevent S. mutans growth. Crystal violet staining showed that the D- or L-enantiomers of Cys, Asp, and Glu at a concentration of 40 mM can inhibit the formation of S. mutans biofilms, and their mixture generated a stronger inhibition than the components alone. Furthermore, the mixture of the three D-AAs or L-AAs may improve the antibacterial activity of nisin against S. mutans biofilms. This study underscores the potential of free amino acids for the enhancement of the antibacterial activity of nisin and the inhibition of the cariogenic bacteria S. mutans and biofilms.

  13. Pseudomonas aeruginosa variants obtained from veterinary clinical samples reveal a role for cyclic di-GMP in biofilm formation and colony morphology.

    Science.gov (United States)

    Brock, Maria T; Fedderly, Galya C; Borlee, Grace I; Russell, Michael M; Filipowska, Liliana K; Hyatt, Doreene R; Ferris, Ryan A; Borlee, Bradley R

    2017-10-16

    Overuse of antibiotics is contributing to an emerging antimicrobial resistance crisis. To better understand how bacteria adapt tolerance and resist antibiotic treatment, Pseudomonas aeruginosa isolates obtained from infection sites sampled from companion animals were collected and evaluated for phenotypic differences. Selected pairs of clonal isolates were obtained from individual infection samples and were assessed for antibiotic susceptibility, cyclic di-GMP levels, biofilm production, motility and genetic-relatedness. A total of 18 samples from equine, feline and canine origin were characterized. A sample from canine otitis media produced a phenotypically heterogeneous pair of P. aeruginosa isolates, 42121A and 42121B, which during growth on culture medium respectively exhibited hyper dye-binding small colony morphology and wild-type phenotypes. Antibiotic susceptibility to gentamicin and ciprofloxacin also differed between this pair of clonal isolates. Sequence analysis of gyrA, a gene known to be involved in ciprofloxacin resistance, indicated that 42121A and 42121B both contained mutations that confer ciprofloxacin resistance, but this did not explain the differences in ciprofloxacin resistance that were observed. Cyclic di-GMP levels also varied between this pair of isolates and were shown to contribute to the observed colony morphology variation and ability to form a biofilm. Our results demonstrate the role of cyclic di-GMP in generating the observed morphological phenotypes that are known to contribute to biofilm-mediated antibiotic tolerance. The generation of phenotypic diversity may go unnoticed during standard diagnostic evaluation, which potentially impacts the therapeutic strategy chosen to treat the corresponding infection and may contribute to the spread of antibiotic resistance.

  14. AtlA Mediates Extracellular DNA Release, Which Contributes to Streptococcus mutans Biofilm Formation in an Experimental Rat Model of Infective Endocarditis.

    Science.gov (United States)

    Jung, Chiau-Jing; Hsu, Ron-Bin; Shun, Chia-Tung; Hsu, Chih-Chieh; Chia, Jean-San

    2017-09-01

    Host factors, such as platelets, have been shown to enhance biofilm formation by oral commensal streptococci, inducing infective endocarditis (IE), but how bacterial components contribute to biofilm formation in vivo is still not clear. We demonstrated previously that an isogenic mutant strain of Streptococcus mutans deficient in autolysin AtlA (ΔatlA) showed a reduced ability to cause vegetation in a rat model of bacterial endocarditis. However, the role of AtlA in bacterial biofilm formation is unclear. In this study, confocal laser scanning microscopy analysis showed that extracellular DNA (eDNA) was embedded in S. mutans GS5 floes during biofilm formation on damaged heart valves, but an ΔatlA strain could not form bacterial aggregates. Semiquantification of eDNA by PCR with bacterial 16S rRNA primers demonstrated that the ΔatlA mutant strain produced dramatically less eDNA than the wild type. Similar results were observed with in vitro biofilm models. The addition of polyanethol sulfonate, a chemical lysis inhibitor, revealed that eDNA release mediated by bacterial cell lysis is required for biofilm initiation and maturation in the wild-type strain. Supplementation of cultures with calcium ions reduced wild-type growth but increased eDNA release and biofilm mass. The effect of calcium ions on biofilm formation was abolished in ΔatlA cultures and by the addition of polyanethol sulfonate. The VicK sensor, but not CiaH, was found to be required for the induction of eDNA release or the stimulation of biofilm formation by calcium ions. These data suggest that calcium ion-regulated AtlA maturation mediates the release of eDNA by S. mutans, which contributes to biofilm formation in infective endocarditis. Copyright © 2017 American Society for Microbiology.

  15. Next generation sequencing analysis reveals that the ribonucleases RNase II, RNase R and PNPase affect bacterial motility and biofilm formation in E. coli.

    Science.gov (United States)

    Pobre, Vânia; Arraiano, Cecília M

    2015-02-14

    The RNA steady-state levels in the cell are a balance between synthesis and degradation rates. Although transcription is important, RNA processing and turnover are also key factors in the regulation of gene expression. In Escherichia coli there are three main exoribonucleases (RNase II, RNase R and PNPase) involved in RNA degradation. Although there are many studies about these exoribonucleases not much is known about their global effect in the transcriptome. In order to study the effects of the exoribonucleases on the transcriptome, we sequenced the total RNA (RNA-Seq) from wild-type cells and from mutants for each of the exoribonucleases (∆rnb, ∆rnr and ∆pnp). We compared each of the mutant transcriptome with the wild-type to determine the global effects of the deletion of each exoribonucleases in exponential phase. We determined that the deletion of RNase II significantly affected 187 transcripts, while deletion of RNase R affects 202 transcripts and deletion of PNPase affected 226 transcripts. Surprisingly, many of the transcripts are actually down-regulated in the exoribonuclease mutants when compared to the wild-type control. The results obtained from the transcriptomic analysis pointed to the fact that these enzymes were changing the expression of genes related with flagellum assembly, motility and biofilm formation. The three exoribonucleases affected some stable RNAs, but PNPase was the main exoribonuclease affecting this class of RNAs. We confirmed by qPCR some fold-change values obtained from the RNA-Seq data, we also observed that all the exoribonuclease mutants were significantly less motile than the wild-type cells. Additionally, RNase II and RNase R mutants were shown to produce more biofilm than the wild-type control while the PNPase mutant did not form biofilms. In this work we demonstrate how deep sequencing can be used to discover new and relevant functions of the exoribonucleases. We were able to obtain valuable information about the

  16. In vitro effectiveness of antimicrobial photodynamic therapy (APDT) using a 660 nm laser and malachite green dye in Staphylococcus aureus biofilms arranged on compact and cancellous bone specimens.

    Science.gov (United States)

    Rosa, Luciano Pereira; da Silva, Francine Cristina; Nader, Sumaia Alves; Meira, Giselle Andrade; Viana, Magda Souza

    2014-11-01

    The aim of this study was to evaluate the in vitro effectiveness of antimicrobial photodynamic therapy (APDT) using a 660 nm visible laser combined with malachite green (MG) dye in the inactivation of Staphylococcus aureus (ATCC 25923) biofilms formed within compact and cancellous bone specimens. Specimens of 80 compact bones and 80 cancellous bones were contaminated with a standard suspension of S. aureus and incubated for 14 days at 37 °C to allow for the formation of biofilms. The specimens were divided into the following groups (n = 10) according to the treatment conditions: PS-L - (control - no treatment), PS+L - (only MG for 5 min), PS-L + 90 (only laser irradiation for 90 s), PS-L + 180 (only laser irradiation for 180 s), PS-L + 300 (only laser irradiation for 300 s), APDT90 (APDT for 90 s), APDT180 (APDT for 180 s), and APDT300 (APDT for 300 s). The findings were statistically analyzed using an ANOVA 5%. All of the experimental groups were significantly different from the control group for both the compact and cancellous bone specimens. The compact bone specimens that received APDT treatment (for either 90, 180, or 300 s) showed reductions in the log10 CFU/ml of S. aureus by a magnitude of 4 log10. Cancellous bone specimens treated with 300 s of APDT showed the highest efficacy, and these specimens had a reduction in S. aureus CFU/ml by a factor of 3 log10. APDT treatment using these proposed parameters in combination with MG was effective at inactivating S. aureus biofilms in compact and cancellous bone specimens.

  17. Nanohybrids of silver particles on clay platelets delaminate Pseudomonas biofilms.

    Science.gov (United States)

    Tseng, Li-Ping; Juan, Chih-Yin; Lin, Shiun-Long; Doran, Michael R; Lin, Jiang-Jen; Hsu, Shan-Hui; Liao, Jiunn-Wang; Shen, Ching-I; Su, Hong-Lin

    2014-05-01

    To evaluate the effectiveness of novel nanohybrids, composed of silver nanoparticles and nanoscale silicate platelets, to clear Pseudomonas aeruginosa biofilms. The nanohybrids were manufactured from an in situ reduction of silver salts in the silicate platelet dispersion, and then applied to biofilms in vitro and in vivo. In reference to the biocidal effects of gentamycin, the nanohybrids mitigated the spreading of the biofilms, and initiated robust cell death and exfoliation from the superficial layers of the biofilms in vitro. In vivo, the nanohybrids exhibited significant therapeutic effects by eliminating established biofilms from infected corneas and promoting the recovery of corneal integrity. All of the evaluations indicate the high potency of the newly developed silver nanoparticle/nanoscale silicate platelet nanohybrids for eliminating biofilms.

  18. Evaluation of combinations of putative anti-biofilm agents and antibiotics to eradicate biofilms of Staphylococcus aureus and Pseudomonas aeruginosa.

    Science.gov (United States)

    Belfield, Katherine; Bayston, Roger; Hajduk, Nadzieja; Levell, Georgia; Birchall, John P; Daniel, Matija

    2017-09-01

    To evaluate potential anti-biofilm agents for their ability to enhance the activity of antibiotics for local treatment of localized biofilm infections. Staphylococcus aureus and Pseudomonas aeruginosa in vitro biofilm models were developed. The putative antibiotic enhancers N-acetylcysteine, acetylsalicylic acid, sodium salicylate, recombinant human deoxyribonuclease I, dispersin B, hydrogen peroxide and Johnson's Baby Shampoo (JBS) were tested for their anti-biofilm activity alone and their ability to enhance the activity of antibiotics for 7 or 14 days, against 5 day old biofilms. The antibiotic enhancers were paired with rifampicin and clindamycin against S. aureus and gentamicin and ciprofloxacin against P. aeruginosa. Isolates from biofilms that were not eradicated were tested for antibiotic resistance. Antibiotic levels 10× MIC and 100× MIC significantly reduced biofilm, but did not consistently eradicate it. Antibiotics at 100× MIC with 10% JBS for 14 days was the only treatment to eradicate both staphylococcal and pseudomonal biofilms. Recombinant human deoxyribonuclease I significantly reduced staphylococcal biofilm. Emergence of resistance of surviving isolates was minimal and was often associated with the small colony variant phenotype. JBS enhanced the activity of antibiotics and several other promising anti-biofilm agents were identified. Antibiotics with 10% JBS eradicated biofilms produced by both organisms. Such combinations might be useful in local treatment of localized biofilm infections.

  19. Biofilm disruption with rotating microrods enhances antimicrobial efficacy

    Energy Technology Data Exchange (ETDEWEB)

    Mair, Lamar O., E-mail: Lamar.Mair@gmail.com [Weinberg Medical Physics, Inc., North Bethesda, MD (United States); Nacev, Aleksandar; Hilaman, Ryan; Stepanov, Pavel Y.; Chowdhury, Sagar; Jafari, Sahar [Weinberg Medical Physics, Inc., North Bethesda, MD (United States); Hausfeld, Jeffrey [School of Medicine and Health Sciences, George Washington University, WA (United States); Karlsson, Amy J. [Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, MD (United States); Shirtliff, Mark E. [School of Dentistry, University of Maryland, Baltimore, MD (United States); Shapiro, Benjamin [Fischell Department of Bioengineering, University of Maryland, College Park, MD (United States); Weinberg, Irving N. [Weinberg Medical Physics, Inc., North Bethesda, MD (United States)

    2017-04-01

    Biofilms are a common and persistent cause of numerous illnesses. Compared to planktonic microbes, biofilm residing cells often demonstrate significant resistance to antimicrobial agents. Thus, methods for dislodging cells from the biofilm may increase the antimicrobial susceptibility of such cells, and serve as a mechanical means of increasing antimicrobial efficacy. Using Aspergillus fumigatus as a model microbe, we magnetically rotate microrods in and around biofilm. We show that such rods can improve the efficacy of antimicrobial Amphotericin B treatments in vitro. This work represents a first step in using kinetic magnetic particle therapy for disrupting fungal biofilms. - Highlights: • Fungal biofilms have been implicated in a variety of medical ailments. • Magnetic microrods, grown via electroplating, were rotated in and around fungal biofilms. • Rotating microrods potentiate the effectiveness of antimicrobial drug. • Antimicrobial efficacy may be enhanced due to increased mixing.

  20. Inhibition of quorum sensing mediated biofilm development and virulence in uropathogens by Hyptis suaveolens.

    Science.gov (United States)

    Salini, Ramesh; Sindhulakshmi, Muthukrishnan; Poongothai, Thirumaran; Pandian, Shunmugiah Karutha

    2015-04-01

    Bacterial urinary tract infections (UTIs) are the most common nosocomial infections, accounting for about 40 % of all hospital-acquired infections. The bacterial spectrum of nosocomial UTIs is broad and the treatment of UTIs is becoming difficult owing to the emergence of drug resistance. Therefore, it is reasonable to investigate novel and alternative therapeutic strategies to treat UTIs. Since UTIs are caused by uropathogens with quorum sensing (QS)-dependent biofilm forming abilities, interruption of QS systems may be a novel approach to combat drug resistance. In the present study, a methanol extract (and hexane extract derived from it) of the medicinal plant Hyptis suaveolens (L.) were shown to have anti-QS activity against the biosensor strain Chromobacterium violaceum (ATCC 12472). Furthermore, the hexane extract of H. suaveolens (HEHS) inhibited biofilm formation by uropathogens such as Escherichia coli, Proteus vulgaris, Proteus mirabilis, Klebsiella pneumoniae and Serratia marcescens. HEHS promotes the loosening of biofilm architecture and strongly inhibits in vitro biofilm formation by uropathogens, which was more apparent from microscopic images. In addition to this, HEHS reduces the production of QS-dependent virulence factors like protease and hemolysin, along with motility. The partial purification and GC-MS analysis of the active fraction revealed the presence of several therapeutically important compounds which may synergistically act on the uropathogens and possibly reduce the QS-dependent phenotypes. These findings suggest HEHS as potential phytotherapeutic agent which can be employed to formulate protective strategies against biofilm linked infections caused by uropathogens.

  1. RNA profiles of porcine embryos during genome activation reveal complex metabolic switch sensitive to in vitro conditions.

    Directory of Open Access Journals (Sweden)

    Olga Østrup

    Full Text Available Fertilization is followed by complex changes in cytoplasmic composition and extensive chromatin reprogramming which results in the abundant activation of totipotent embryonic genome at embryonic genome activation (EGA. While chromatin reprogramming has been widely studied in several species, only a handful of reports characterize changing transcriptome profiles and resulting metabolic changes in cleavage stage embryos. The aims of the current study were to investigate RNA profiles of in vivo developed (ivv and in vitro produced (ivt porcine embryos before (2-cell stage and after (late 4-cell stage EGA and determine major metabolic changes that regulate totipotency. The period before EGA was dominated by transcripts responsible for cell cycle regulation, mitosis, RNA translation and processing (including ribosomal machinery, protein catabolism, and chromatin remodelling. Following EGA an increase in the abundance of transcripts involved in transcription, translation, DNA metabolism, histone and chromatin modification, as well as protein catabolism was detected. The further analysis of members of overlapping GO terms revealed that despite that comparable cellular processes are taking place before and after EGA (RNA splicing, protein catabolism, different metabolic pathways are involved. This strongly suggests that a complex metabolic switch accompanies EGA. In vitro conditions significantly altered RNA profiles before EGA, and the character of these changes indicates that they originate from oocyte and are imposed either before oocyte aspiration or during in vitro maturation. IVT embryos have altered content of apoptotic factors, cell cycle regulation factors and spindle components, and transcription factors, which all may contribute to reduced developmental competence of embryos produced in vitro. Overall, our data are in good accordance with previously published, genome-wide profiling data in other species. Moreover, comparison with mouse and

  2. Antibiotic susceptibility of Aggregatibacter actinomycetemcomitans JP2 in a biofilm

    Directory of Open Access Journals (Sweden)

    Orit Oettinger-Barak

    2013-05-01

    Full Text Available Background: Localized aggressive periodontitis (LAgP is an inflammatory disease associated with specific bacteria, particularly Aggregatibacter actinomycetemcomitans, which can result in early tooth loss. The bacteria grow as a biofilm known as subgingival plaque. Treatment includes mechanical debridement of the biofilm, often associated with empirical antibiotic treatment. Objective: The aims of this study were to test in vitro the sensitivity of A. actinomycetemcomitans JP2 during planktonic and biofilm growth to doxycycline and to the combination of metronidazole and amoxicillin, which are two antibiotic protocols commonly used in clinical practice. Design: Two in vitro biofilm models were used to test the effects of the antibiotics: a static 96-well plate assay was used to investigate the effect of these antibiotics on biofilm formation whilst a flow chamber model was used to examine the effect on established biofilms. Results: Of the antibiotics tested in this model system, doxycycline was most efficacious with a minimal inhibitory concentration (MIC against planktonic cells of 0.21 mg/L and minimal biofilm inhibitory concentration (MBIC of 2.10 mg/L. The most commonly prescribed antibiotic regimen, amoxicillin + metronidazole, was much less effective against both planktonic and biofilm cells with an MIC and MBIC of 12.0 mg/L and 20.2 mg/L, respectively. A single treatment of the clinically achievable concentration of 10 mg/L doxycycline to sparse A. actinomycetemcomitans biofilms in the flow chamber model resulted in significant decreases in biofilm thickness, biovolume, and cell viability. Dense A. actinomycetemcomitans biofilms were significantly more resistant to doxycycline treatment. Low concentrations of antibiotics enhanced biofilm formation. Conclusion: A. actinomycetemcomitans JP2 homotypic biofilms were more susceptible in vitro to doxycycline than amoxicillin + metronidazole.

  3. Biofilm formation by Staphylococcus aureus isolates from a dental clinic in Konya, Turkey.

    Science.gov (United States)

    Torlak, Emrah; Korkut, Emre; Uncu, Ali T; Şener, Yağmur

    The ability of Staphylococcus aureus to form biofilm is considered to be a major virulence factor influencing its survival and persistence in both the environment and the host. Biofilm formation in S. aureus is most frequently associated with production of polysaccharide intercellular adhesion by ica operon-encoded enzymes. The present work aimed at evaluating the in vitro biofilm production and presence of the icaA and icaD genes in S. aureus isolates from a dental clinic in Konya, Turkey. The surfaces of inanimate objects were sampled over a period of six months. S. aureus isolates were subjected to Congo Red Agar (CRA) and crystal violet (CV) staining assays to evaluate their ability of biofilm production, while the presence of the icaA and icaD genes was determined by polymerase chain reaction. S. aureus contamination was detected in 13.2% of the environmental samples. All the 32 isolates were observed to be positive for both the icaA and icaD genes. Phenotypic evaluations revealed that CV staining assay is a more reliable alternative to CRA assay to determine biofilm formation ability. A high percentage of agreement (91%) was observed between the results from CV staining and ica genes' detection assays. Phenotypic and genotypic evaluations should be combined to detect biofilm formation in S. aureus. Our findings indicate that dental clinic environments should be considered as potential reservoir for biofilm-producing S. aureus and thus cross contamination. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  4. Human Plasma Enhances the Expression of Staphylococcal Microbial Surface Components Recognizing Adhesive Matrix Molecules Promoting Biofilm Formation and Increases Antimicrobial Tolerance In Vitro

    Science.gov (United States)

    2014-07-17

    Material Command to JCW. Author details 1Department of Medicine, Infectious Disease Service, Brooke Army Medical Center, 3551 Roger Brooke Drive, JBSA...infections. Science 1999, 284:1318 1322. 3. Hall Stoodley L, Costerton JW, Stoodley P: Bacterial biofilms: from the natural environment to infectious ...infection and invasion in Staphylococcus aureus experimental endocarditis . J Exp Med 2005, 201:1627 1635. 23. Atshan SS, Shamsudin MN, Karunanidhi A, van

  5. Comparative evaluation of sutures coated with triclosan and chlorhexidine for oral biofilm inhibition potential and antimicrobial activity against periodontal pathogens: An in vitro study.

    Science.gov (United States)

    Sethi, Kunal Sunder; Karde, Prerna Ashok; Joshi, Chaitanya Pradeep

    2016-01-01

    Surgical site plaque accumulation is one of the challenging problems leading to unfavorable healing. The antibacterial sutures can be used to reduce or inhibit plaque formation. Presently there is no study comparing efficacy of sutures coated with triclosan and chlorhexidine in terms of oral biofilm inhibition and antimicrobial property against periodontal pathogens. The aim of present study was to evaluate the antibacterial efficacy and oral biofilm inhibition around chlorhexidine and triclosan coated polyglactin sutures in comparison to uncoated sutures. Equal segments of chlorhexidine and triclosan coated polyglactin sutures (3-0) were incubated at 370°C in saliva collected from 10 chronic periodontitis patients for 7 days. Plain uncoated suture served as control. Biofilm formation was analyzed with Confocal Laser-Scanning Microscopy (CLSM) and Scanning Electron Microscopy (SEM). Quantitative assessment was done using Colony Forming Units (CFU/mL).The antibacterial efficacy of the sutures was tested against specific periodontal pathogens (S.mutans, F.nucleatum, A.actinomycetomcomitans, P.intermedia, P.gingivalis) using agar diffusion method. CLSM and SEM were not subjected to statistical analysis. ANOVA test was used for colony forming units and agar diffusion test. (P chlorhexidine-coated sutures followed by triclosan-coated when compared to plain uncoated suture. The antibacterial coated sutures showed statistically significant difference in CFUs/ml and zone of inhibition compared to plain uncoated sutures. Among coated sutures, chlorhexidine-coated sutures showed better results. The antibacterial coated sutures have a promising potential in preventing the colonization of periodontal pathogens around it thereby inhibiting biofilm formation.

  6. High extensibility of stress fibers revealed by in vitro micromanipulation with fluorescence imaging

    Energy Technology Data Exchange (ETDEWEB)

    Matsui, Tsubasa S. [Department of Biomolecular Sciences, Tohoku University (Japan); Sato, Masaaki [Department of Biomedical Engineering, Tohoku University (Japan); Department of Bioengineering and Robotics, Tohoku University (Japan); Deguchi, Shinji, E-mail: deguchi@nitech.ac.jp [Department of Bioengineering and Robotics, Tohoku University (Japan)

    2013-05-10

    Highlights: •We isolate contractile stress fibers from vascular smooth muscle cells. •We measure the extensibility of individual stress fibers. •We present the first direct evidence that individual stress fibers are highly extensible. •We quantitatively determine the local strain along the length of stress fibers. •The high extensibility we found is beyond that explained by a conventional model. -- Abstract: Stress fibers (SFs), subcellular bundles of actin and myosin filaments, are physically connected at their ends to cell adhesions. The intracellular force transmitted via SFs plays an essential role in cell adhesion regulation and downstream signaling. However, biophysical properties intrinsic to individual SFs remain poorly understood partly because SFs are surrounded by other cytoplasmic components that restrict the deformation of the embedded materials. To characterize their inherent properties independent of other structural components, we isolated SFs from vascular smooth muscle cells and mechanically stretched them by in vitro manipulation while visualizing strain with fluorescent quantum dots attached along their length. SFs were elongated along their entire length, with the length being approximately 4-fold of the stress-free length. This surprisingly high extensibility was beyond that explained by the tandem connection of actin filaments and myosin II bipolar filaments present in SFs, thus suggesting the involvement of other structural components in their passive biophysical properties.

  7. Identification of bacterial strains isolated from the Mediterranean Sea exhibiting different abilities of biofilm formation.

    Science.gov (United States)

    Brian-Jaisson, Florence; Ortalo-Magné, Annick; Guentas-Dombrowsky, Linda; Armougom, Fabrice; Blache, Yves; Molmeret, Maëlle

    2014-07-01

    The Mediterranean Sea has rarely been investigated for the characterization of marine bacteria as compared to other marine environments such as the Atlantic or Pacific Ocean. Bacteria recovered from inert surfaces are poorly studied in these environments, when it has been shown that the community structure of attached bacteria can be dissimilar from that of planktonic bacteria present in the water column. The objectives of this study were to identify and characterize marine bacteria isolated from biofilms developed on inert surfaces immersed in the Mediterranean Sea and to evaluate their capacity to form a biofilm in vitro. Here, 13 marine bacterial strains have been isolated from different supports immersed in seawater in the Bay of Toulon (France). Phylogenetic analysis and different biological and physico-chemical properties have been investigated. Among the 13 strains recovered, 8 different genera and 12 different species were identified including 2 isolates of a novel bacterial species that we named Persicivirga mediterranea and whose genus had never been isolated from the Mediterranean Sea. Shewanella sp. and Pseudoalteromonas sp. were the most preponderant genera recovered in our conditions. The phenotypical characterization revealed that one isolate belonging to the Polaribacter genus differed from all the other ones by its hydrophobic properties and poor ability to form biofilms in vitro. Identifying and characterizing species isolated from seawater including from Mediterranean ecosystems could be helpful for example, to understand some aspects of bacterial biodiversity and to further study the mechanisms of biofilm (and biofouling) development in conditions approaching those of the marine environment.

  8. Diffusion of antimicrobials in multispecies biofilms evaluated in a new biofilm model.

    Science.gov (United States)

    van der Waal, S V; de Almeida, J; Krom, B P; de Soet, J J; Crielaard, W

    2017-04-01

    To describe the application of a newly-developed in vitro model in which the diffusion of antimicrobials in oral biofilms can be studied. In a flow chamber consisting of three parallel feeding channels connected with each other by eight perpendicular side channels, multispecies biofilms were grown from saliva of a single donor for 48 h. The dimensions of the side channels were 100 μm × 100 μm × 5130 μm (H × W × L). When one or more side channels were filled with biofilm, the biofilms were stained with fluorescent stains. Then, one side-channel biofilm was selected and treated with phosphate buffered saline, 2% sodium hypochlorite (NaOCl), 17% ethylenediaminetetra-acetic acid (EDTA) or modified salt solution (MSS). Diffusion of the irrigants was observed by acquiring fluorescence images at 10× objective every 15 s for 30 min. It was possible to culture biofilms in the narrow (100 μm) channels. The biofilms varied in phenotype. In this model, no diffusion of NaOCl into the biofilms was seen after its application. Seventeen-percentage EDTA only diffused into the biofilm up to 200 μm in 30 min. MSS did diffuse in the biofilm over a distance of 450 μm within 2 min after a single application. This new model enables the investigation of the diffusion of antimicrobials in biofilms. Other applications to improve our understanding of the characteristics of biofilms are now possible. © 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  9. Widely Used Pesticides with Previously Unknown Endocrine Activity Revealed as in Vitro Antiandrogens

    Science.gov (United States)

    Orton, Frances; Rosivatz, Erika; Scholze, Martin; Kortenkamp, Andreas

    2011-01-01

    Background Evidence suggests that there is widespread decline in male reproductive health and that antiandrogenic pollutants may play a significant role. There is also a clear disparity between pesticide exposure and data on endocrine disruption, with most of the published literature focused on pesticides that are no longer registered for use in developed countries. Objective We used estimated human exposure data to select pesticides to test for antiandrogenic activity, focusing on highest use pesticides. Methods We used European databases to select 134 candidate pesticides based on highest exposure, followed by a filtering step according to known or predicted receptor-mediated antiandrogenic potency, based on a previously published quantitative structure–activity relationship (QSAR) model. In total, 37 pesticides were tested for in vitro androgen receptor (AR) antagonism. Of these, 14 were previously reported to be AR antagonists (“active”), 4 were predicted AR antagonists using the QSAR, 6 were predicted to not be AR antagonists (“inactive”), and 13 had unknown activity, which were “out of domain” and therefore could not be classified with the QSAR (“unknown”). Results All 14 pesticides with previous evidence of AR antagonism were confirmed as antiandrogenic in our assay, and 9 previously untested pesticides were identified as antiandrogenic (dimethomorph, fenhexamid, quinoxyfen, cyprodinil, λ-cyhalothrin, pyrimethanil, fludioxonil, azinphos-methyl, pirimiphos-methyl). In addition, we classified 7 compounds as androgenic. Conclusions Due to estimated antiandrogenic potency, current use, estimated exposure, and lack of previous data, we strongly recommend that dimethomorph, fludioxonil, fenhexamid, imazalil, ortho-phenylphenol, and pirimiphos-methyl be tested for antiandrogenic effects in vivo. The lack of human biomonitoring data for environmentally relevant pesticides presents a barrier to current risk assessment of pesticides on humans. PMID

  10. Anti-inflammatory Effects of Fungal Metabolites in Mouse Intestine as Revealed by In vitro Models

    Science.gov (United States)

    Schreiber, Dominik; Marx, Lisa; Felix, Silke; Clasohm, Jasmin; Weyland, Maximilian; Schäfer, Maximilian; Klotz, Markus; Lilischkis, Rainer; Erkel, Gerhard; Schäfer, Karl-Herbert

    2017-01-01

    Inflammatory bowel diseases (IBD), which include Crohn's disease and ulcerative colitis, are chronic inflammatory disorders that can affect the whole gastrointestinal tract or the colonic mucosal layer. Current therapies aiming to suppress the exaggerated immune response in IBD largely rely on compounds with non-satisfying effects or side-effects. Therefore, new therapeutical options are needed. In the present study, we investigated the anti-inflammatory effects of the fungal metabolites, galiellalactone, and dehydrocurvularin in both an in vitro intestinal inflammation model, as well as in isolated myenteric plexus and enterocyte cells. Administration of a pro-inflammatory cytokine mix through the mesenteric artery of intestinal segments caused an up-regulation of inflammatory marker genes. Treatment of the murine intestinal segments with galiellalactone or dehydrocurvularin by application through the mesenteric artery significantly prevented the expression of pro-inflammatory marker genes on the mRNA and the protein level. Comparable to the results in the perfused intestine model, treatment of primary enteric nervous system (ENS) cells from the murine intestine with the fungal compounds reduced expression of cytokines such as IL-6, TNF-α, IL-1β, and inflammatory enzymes such as COX-2 and iNOS on mRNA and protein levels. Similar anti-inflammatory effects of the fungal metabolites were observed in the human colorectal adenocarcinoma cell line DLD-1 after stimulation with IFN-γ (10 ng/ml), TNF-α (10 ng/ml), and IL-1β (5 ng/ml). Our results show that the mesenterially perfused intestine model provides a reliable tool for the screening of new therapeutics with limited amounts of test compounds. Furthermore, we could characterize the anti-inflammatory effects of two novel active compounds, galiellalactone, and dehydrocurvularin which are interesting candidates for studies with chronic animal models of IBD. PMID:28824460

  11. Anti-inflammatory Effects of Fungal Metabolites in Mouse Intestine as Revealed by In vitro Models

    Directory of Open Access Journals (Sweden)

    Dominik Schreiber

    2017-08-01

    Full Text Available Inflammatory bowel diseases (IBD, which include Crohn's disease and ulcerative colitis, are chronic inflammatory disorders that can affect the whole gastrointestinal tract or the colonic mucosal layer. Current therapies aiming to suppress the exaggerated immune response in IBD largely rely on compounds with non-satisfying effects or side-effects. Therefore, new therapeutical options are needed. In the present study, we investigated the anti-inflammatory effects of the fungal metabolites, galiellalactone, and dehydrocurvularin in both an in vitro intestinal inflammation model, as well as in isolated myenteric plexus and enterocyte cells. Administration of a pro-inflammatory cytokine mix through the mesenteric artery of intestinal segments caused an up-regulation of inflammatory marker genes. Treatment of the murine intestinal segments with galiellalactone or dehydrocurvularin by application through the mesenteric artery significantly prevented the expression of pro-inflammatory marker genes on the mRNA and the protein level. Comparable to the results in the perfused intestine model, treatment of primary enteric nervous system (ENS cells from the murine intestine with the fungal compounds reduced expression of cytokines such as IL-6, TNF-α, IL-1β, and inflammatory enzymes such as COX-2 and iNOS on mRNA and protein levels. Similar anti-inflammatory effects of the fungal metabolites were observed in the human colorectal adenocarcinoma cell line DLD-1 after stimulation with IFN-γ (10 ng/ml, TNF-α (10 ng/ml, and IL-1β (5 ng/ml. Our results show that the mesenterially perfused intestine model provides a reliable tool for the screening of new therapeutics with limited amounts of test compounds. Furthermore, we could characterize the anti-inflammatory effects of two novel active compounds, galiellalactone, and dehydrocurvularin which are interesting candidates for studies with chronic animal models of IBD.

  12. Staphylococcus aureus biofilm formation at the physiologic glucose concentration depends on the S. aureus lineage

    NARCIS (Netherlands)

    Croes, Sander; Deurenberg, Ruud H; Boumans, Marie-Louise L; Beisser, Patrick S; Neef, Cees; Stobberingh, Ellen E

    2009-01-01

    BACKGROUND: Since bacteria embedded in biofilms are far more difficult to eradicate than planktonic infections, it would be useful to know whether certain Staphylococcus aureus lineages are especially involved in strong biofilm formation. For this reason, in vitro biofilm formation of 228 clinical

  13. The pulsed light inactivation of veterinary relevant microbial biofilms ...

    African Journals Online (AJOL)

    tulyasys

    2016-01-27

    Jan 27, 2016 ... The presence of pathogenic organisms namely parasite species and bacteria in biofilms in veterinary settings, is a public health concern in ..... medicine. Vet. Microbiol. 121, 1-17. Coenye, T. and Nelis J.J. 2010. In vitro and in vivo model systems to study microbial biofilm formation. J. Microbiol. Methods 83 ...

  14. Biofilm formation on dental restorative and implant materials.

    Science.gov (United States)

    Busscher, H J; Rinastiti, M; Siswomihardjo, W; van der Mei, H C

    2010-07-01

    Biomaterials for the restoration of oral function are prone to biofilm formation, affecting oral health. Oral bacteria adhere to hydrophobic and hydrophilic surfaces, but due to fluctuating shear, little biofilm accumulates on hydrophobic surfaces in vivo. More biofilm accumulates on rough than on smooth surfaces. Oral biofilms mostly consist of multiple bacterial strains, but Candida species are found on acrylic dentures. Biofilms on gold and amalgam in vivo are thick and fully covering, but barely viable. Biofilms on ceramics are thin and highly viable. Biofilms on composites and glass-ionomer cements cause surface deterioration, which enhances biofilm formation again. Residual monomer release from composites influences biofilm growth in vitro, but effects in vivo are less pronounced, probably due to the large volume of saliva into which compounds are released and its continuous refreshment. Similarly, conflicting results have been reported on effects of fluoride release from glass-ionomer cements. Finally, biomaterial-associated infection of implants and devices elsewhere in the body is compared with oral biofilm formation. Biomaterial modifications to discourage biofilm formation on implants and devices are critically discussed for possible applications in dentistry. It is concluded that, for dental applications, antimicrobial coatings killing bacteria upon contact are more promising than antimicrobial-releasing coatings.

  15. Candida biofilms: is adhesion sexy?

    Science.gov (United States)

    Soll, David R

    2008-08-26

    The development of Candida albicans biofilms requires two types of adhesion molecule - the Als proteins and Hwp1. Mutational analyses have recently revealed that these molecules play complementary roles, and their characteristics suggest that they may have evolved from primitive mating agglutinins.

  16. Sticking together: building a biofilm the Bacillus subtilis way.

    Science.gov (United States)

    Vlamakis, Hera; Chai, Yunrong; Beauregard, Pascale; Losick, Richard; Kolter, Roberto

    2013-03-01

    Biofilms are ubiquitous communities of tightly associated bacteria encased in an extracellular matrix. Bacillus subtilis has long served as a robust model organism to examine the molecular mechanisms of biofilm formation, and a number of studies have revealed that this process is regulated by several integrated pathways. In this Review, we focus on the molecular mechanisms that control B. subtilis biofilm assembly, and then briefly summarize the current state of knowledge regarding biofilm disassembly. We also discuss recent progress that has expanded our understanding of B. subtilis biofilm formation on plant roots, which are a natural habitat for this soil bacterium.

  17. Shaping the growth behaviour of biofilms initiated from bacterial aggregates

    DEFF Research Database (Denmark)

    Melaugh, Gavin; Hutchison, Jaime; Kragh, Kasper Nørskov

    2016-01-01

    Bacterial biofilms are usually assumed to originate from individual cells deposited on a surface. However, many biofilm-forming bacteria tend to aggregate in the planktonic phase so that it is possible that many natural and infectious biofilms originate wholly or partially from pre-formed cell ag...... outcomes are governed by a trade-off between aggregate surface area and height. Our results provide new insight into biofilm formation and development, and reveal new factors that may be at play in the social evolution of biofilm communities....

  18. Fungal Biofilms: Inside Out.

    Science.gov (United States)

    La