Pettit George R
Full Text Available Abstract Background Staphylococcus aureus and S. epidermidis biofilms differ in structure, growth and regulation, and thus the high-throughput method of evaluating biofilm susceptibility that has been published for S. epidermidis cannot be applied to S. aureus without first evaluating the assay's reproducibility and reliability with S. aureus biofilms. Methods Staphylococcus aureus biofilms were treated with eleven approved antibiotics, lysostaphin, or Conflikt®, exposed to the oxidation reduction indicator Alamar blue, and reduction relative to untreated controls was determined visually and spectrophotometrically. The minimum biofilm inhibitory concentration (MBIC was defined as ≤ 50% Alamar blue reduction and a purple/blue well 60 min after the addition of Alamar blue. Because all of the approved antibiotics had MBICs >128 μg/ml (most >2048 μg/ml, lysostaphin and Conflikt®, with relatively low MBICs, were used to correlate Alamar blue reduction with 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl-2H-tetrazolium-5-carboxanilide (XTT reduction and viable counts (CFU/ml for S. aureus ATCC 29213 and three clinical isolates. Alamar blue's stability and lack of toxicity allowed CFU/ml to be determined from the same wells as Alamar blue absorbances. Results Overall, Alamar blue reduction had excellent correlation with XTT reduction and with CFU/ml. For ATCC 29213 and two clinical isolates treated with lysostaphin or Conflikt®, Alamar blue reduction had excellent correlation with XTT reduction (r = 0.93-0.99 and with CFU/ml (r = 0.92-0.98. For one of the clinical isolates, the results were moderately correlated for Conflikt® (r = 0.76, Alamar blue vs. XTT; r = 0.81, Alamar blue vs. CFU/ml and had excellent correlation for lysostaphin (r = 0.95, Alamar blue vs. XTT; r = 0.97, Alamar blue vs. CFU/ml. Conclusion A reliable, reproducible method for evaluating biofilm susceptibility was successfully applied to S. aureus biofilms. The described method
Turner Raymond J
Full Text Available Abstract Background Microbial biofilms exist all over the natural world, a distribution that is paralleled by metal cations and oxyanions. Despite this reality, very few studies have examined how biofilms withstand exposure to these toxic compounds. This article describes a batch culture technique for biofilm and planktonic cell metal susceptibility testing using the MBEC assay. This device is compatible with standard 96-well microtiter plate technology. As part of this method, a two part, metal specific neutralization protocol is summarized. This procedure minimizes residual biological toxicity arising from the carry-over of metals from challenge to recovery media. Neutralization consists of treating cultures with a chemical compound known to react with or to chelate the metal. Treated cultures are plated onto rich agar to allow metal complexes to diffuse into the recovery medium while bacteria remain on top to recover. Two difficulties associated with metal susceptibility testing were the focus of two applications of this technique. First, assays were calibrated to allow comparisons of the susceptibility of different organisms to metals. Second, the effects of exposure time and growth medium composition on the susceptibility of E. coli JM109 biofilms to metals were investigated. Results This high-throughput method generated 96-statistically equivalent biofilms in a single device and thus allowed for comparative and combinatorial experiments of media, microbial strains, exposure times and metals. By adjusting growth conditions, it was possible to examine biofilms of different microorganisms that had similar cell densities. In one example, Pseudomonas aeruginosa ATCC 27853 was up to 80 times more resistant to heavy metalloid oxyanions than Escherichia coli TG1. Further, biofilms were up to 133 times more tolerant to tellurite (TeO32- than corresponding planktonic cultures. Regardless of the growth medium, the tolerance of biofilm and planktonic
Biofilm, Porphyromonas gingivalis, susceptibility testing, amoxicillin, doxycycline, metronidazole......Biofilm, Porphyromonas gingivalis, susceptibility testing, amoxicillin, doxycycline, metronidazole...
Traba, Christian; Liang, Jun F.
Formation of bacterial biofilms at solid-liquid interfaces creates numerous problems in both industrial and biomedical sciences. In this study, the susceptibility of Staphylococcus aureus biofilms to discharge gas generated from plasma was tested. It was found that despite distinct chemical/physical properties, discharge gases from oxygen, nitrogen, and argon demonstrated very potent and almost the same anti-biofilm activity. The bacterial cells in S. aureus biofilms were killed (>99.9%) by d...
Li, Xiaobao; Lu, Nanxi; Brady, Hannah R; Packman, Aaron I
Ureolytic biomineralization induced by urease-producing bacteria, particularly Proteus mirabilis, is responsible for the formation of urinary tract calculi and the encrustation of indwelling urinary catheters. Such microbial biofilms are challenging to eradicate and contribute to the persistence of catheter-associated urinary tract infections, but the mechanisms responsible for this recalcitrance remain obscure. In this study, we characterized the susceptibility of wild-type (ure+) and urease-negative (ure-) P. mirabilis biofilms to killing by ciprofloxacin. Ure+ biofilms produced fine biomineral precipitates that were homogeneously distributed within the biofilm biomass in artificial urine, while ure- biofilms did not produce biomineral deposits under identical growth conditions. Following exposure to ciprofloxacin, ure+ biofilms showed greater survival (less killing) than ure- biofilms, indicating that biomineralization protected biofilm-resident cells against the antimicrobial. To evaluate the mechanism responsible for this recalcitrance, we observed and quantified the transport of Cy5-conjugated ciprofloxacin into the biofilm by video confocal microscopy. These observations revealed that the reduced susceptibility of ure+ biofilms resulted from hindered delivery of ciprofloxacin into biomineralized regions of the biofilm. Further, biomineralization enhanced retention of viable cells on the surface following antimicrobial exposure. These findings together show that ureolytic biomineralization induced by P. mirabilis metabolism strongly regulates antimicrobial susceptibility by reducing internal solute transport and increasing biofilm stability. PMID:26953206
Djordjevic, D.; Wiedmann, M.; McLandsborough, L. A.
Listeria monocytogenes has the ability to form biofilms on food-processing surfaces, potentially leading to food product contamination. The objective of this research was to standardize a polyvinyl chloride (PVC) microtiter plate assay to compare the ability of L. monocytogenes strains to form biofilms. A total of 31 coded L. monocytogenes strains were grown in defined medium (modified Welshimer's broth) at 32°C for 20 and 40 h in PVC microtiter plate wells. Biofilm formation was indirectly a...
Full Text Available Staphylococci are frequently isolated from food processing environments, and it has been speculated whether survival after cleaning and disinfection with benzalkonium chloride-containing disinfectants is due to biofilm formation, matrix composition or benzalkonium chloride efflux mechanisms. Out of 35 food associated staphylococci, eight produced biofilm in a microtiter plate assay and were identified as Staphylococcus capitis (2, S. cohnii, S. epidermidis, S. lentus (2, and S. saprophyticus (2. The eight biofilm producing strains were characterized using whole genome sequencing. Three of these strains contained the ica operon responsible for production of a polysaccharide matrix, and formed a biofilm which was detached upon exposure to the polysaccharide degrading enzyme Dispersin B, but not Proteinase K or trypsin. These strains were more tolerant to the lethal effect of benzalkonium chloride both in suspension and biofilm than the remaining five biofilm producing strains. The five benzalkonium chloride susceptible strains were characterized by lack of the ica operon, and their biofilms were detached by Proteinase K or trypsin, but not Dispersin B, indicating that proteins were major structural components of their biofilm matrix. Several novel cell wall anchored repeat domain proteins with domain structures similar to that of MSCRAMM adhesins were identified in the genomes of these strains, potentially representing novel mechanisms of ica-independent biofilm accumulation. Biofilms from all strains showed similar levels of detachment after exposure to alkaline chlorine, which is used for cleaning in the food industry. Strains with qac genes encoding benzalkonium chloride efflux pumps could grow at higher concentrations of benzalkonium chloride than strains without these genes, but no differences were observed at biocidal concentrations. In conclusion, the biofilm matrix of food associated staphylococci varies with respect to protein or
Fagerlund, Annette; Langsrud, Solveig; Heir, Even; Mikkelsen, Maria I; Møretrø, Trond
Staphylococci are frequently isolated from food processing environments, and it has been speculated whether survival after cleaning and disinfection with benzalkonium chloride (BC)-containing disinfectants is due to biofilm formation, matrix composition, or BC eﬄux mechanisms. Out of 35 food associated staphylococci, eight produced biofilm in a microtiter plate assay and were identified as Staphylococcus capitis (2), S. cohnii, S. epidermidis, S. lentus (2), and S. saprophyticus (2). The eight biofilm producing strains were characterized using whole genome sequencing. Three of these strains contained the ica operon responsible for production of a polysaccharide matrix, and formed a biofilm which was detached upon exposure to the polysaccharide degrading enzyme Dispersin B, but not Proteinase K or trypsin. These strains were more tolerant to the lethal effect of BC both in suspension and biofilm than the remaining five biofilm producing strains. The five BC susceptible strains were characterized by lack of the ica operon, and their biofilms were detached by Proteinase K or trypsin, but not Dispersin B, indicating that proteins were major structural components of their biofilm matrix. Several novel cell wall anchored repeat domain proteins with domain structures similar to that of MSCRAMM adhesins were identified in the genomes of these strains, potentially representing novel mechanisms of ica-independent biofilm accumulation. Biofilms from all strains showed similar levels of detachment after exposure to alkaline chlorine, which is used for cleaning in the food industry. Strains with qac genes encoding BC eﬄux pumps could grow at higher concentrations of BC than strains without these genes, but no differences were observed at biocidal concentrations. In conclusion, the biofilm matrix of food associated staphylococci varies with respect to protein or polysaccharide nature, and this may affect the sensitivity toward a commonly used disinfectant. PMID:27375578
Xu, Zhenbo; Liang, Yanrui; Lin, Shiqi; Chen, Dingqiang; Li, Bing; Li, Lin; Deng, Yang
Staphylococcus aureus (S. Aureus) is a common food-borne pathogenic microorganism. Biofilm formation remains the major obstruction for bacterial elimination. The study aims at providing a basis for determining S. aureus biofilm formation. 257 clinical samples of S. aureus isolates were identified by routine analysis and multiplex PCR detection and found to contain 227 MRSA, 16 MSSA, 11 MRCNS, and 3 MSCNS strains. Two assays for quantification of S. aureus biofilm formation, the crystal violet (CV) assay and the XTT (tetrazolium salt reduction) assay, were optimized, evaluated, and further compared. In CV assay, most isolates formed weak biofilm 74.3 %), while the rest formed moderate biofilm (23.3 %) or strong biofilm (2.3 %). However, most isolates in XTT assay showed weak metabolic activity (77.0 %), while the rest showed moderate metabolic activity (17.9 %) or high metabolic activity (5.1 %). In this study, we found a distinct strain-to-strain dissimilarity in terms of both biomass formation and metabolic activity, and it was concluded from this study that two assays were mutual complementation rather than being comparison. PMID:27324342
Rich, Joseph O; Leathers, Timothy D; Nunnally, Melinda S; Bischoff, Kenneth M
Bacterial contaminants from commercial fuel ethanol production facilities were previously shown to form biofilms as mixed cultures under laboratory conditions. In this study, a rapid assay was developed to simultaneously compare isolates for their ability to form biofilms as pure cultures. A total of 10 strains were isolated from a dry-grind fuel ethanol plant that routinely doses with virginiamycin. These were identified by sequence analysis as six strains of Lactobacillus fermentum, two strains of L. johnsonii, and one strain each of L. mucosae and L. amylovorus. Isolates exhibited a range of susceptibility to virginiamycin in a planktonic assay, with MIC's (minimum inhibitory concentration) of ≤0.5-16 μg/ml. Even though all strains were isolated from a mixed culture biofilm, they varied greatly in their ability to form biofilms as pure cultures. Surprisingly, growth as biofilms did not appear to provide resistance to virginiamycin, even if biofilms were grown for 144 h prior to antibiotic challenge. PMID:20855199
Marcos-Zambrano, Laura Judith; Escribano, Pilar; Bouza, Emilio; Guinea, Jesús
Micafungin is more active against biofilms with high metabolic activity; however, it is unknown whether this observation applies to caspofungin and anidulafungin and whether it is also dependent on the biomass production. We compare the antifungal activity of anidulafungin, caspofungin, and micafungin against preformed Candida albicans biofilms with different degrees of metabolic activity and biomass production from 301 isolates causing fungemia in patients admitted to Gregorio Marañon Hospital (January 2007 to September 2014). Biofilms were classified as having low, moderate, or high metabolic activity according XTT reduction assay or having low, moderate, or high biomass according to crystal violet assay. Echinocandin MICs for planktonic and sessile cells were measured using the EUCAST E.Def 7.2 procedure and XTT reduction assay, respectively. Micafungin showed the highest activity against biofilms classified according to the metabolic activity and biomass production (P < .001). The activity of caspofungin and anidulafungin was not dependent on the metabolic activity of the biofilm or the biomass production. These observations were confirmed by scanning electron microscopy. None of the echinocandins produced major changes in the structure of biofilms with low metabolic activity and biomass production when compared with the untreated biofilms. However, biofilm with high metabolic activity or high biomass production was considerably more susceptible to micafungin; this effect was not shown by caspofungin or anidulafungin. PMID:26543157
Li, Xiaobao; Chopp, David L; Russin, William A; Brannon, Paul T; Parsek, Matthew R; Packman, Aaron I
Microbial biofilms and mineral precipitation commonly co-occur in engineered water systems, such as cooling towers and water purification systems, and both decrease process performance. Microbial biofilms are extremely challenging to control and eradicate. We previously showed that in situ biomineralization and the precipitation and deposition of abiotic particles occur simultaneously in biofilms under oversaturated conditions. Both processes could potentially alter the essential properties of biofilms, including susceptibility to biocides. However, the specific interactions between mineral formation and biofilm processes remain poorly understood. Here we show that the susceptibility of biofilms to chlorination depends specifically on internal transport processes mediated by biomineralization and the accumulation of abiotic mineral deposits. Using injections of the fluorescent tracer Cy5, we show that Pseudomonas aeruginosa biofilms are more permeable to solutes after in situ calcite biomineralization and are less permeable after the deposition of abiotically precipitated calcite particles. We further show that biofilms are more susceptible to chlorine killing after biomineralization and less susceptible after particle deposition. Based on these observations, we found a strong correlation between enhanced solute transport and chlorine killing in biofilms, indicating that biomineralization and particle deposition regulate biofilm susceptibility by altering biocide penetration into the biofilm. The distinct effects of in situ biomineralization and particle deposition on biocide killing highlight the importance of understanding the mechanisms and patterns of biomineralization and scale formation to achieve successful biofilm control. PMID:26944848
Jiang, Lei-Meng; Hoogenkamp, Michel A.; van der Sluis, Lucas W. M.; Wesselink, Paul R.; Crielaard, Wim; Deng, Dong Mei
Introduction: Endodontic infections are caused by polymicrobial biofilms. Therefore, novel root canal disinfectants should be evaluated not only on single-species biofilms but also on dual- or mixed-species biofilms. A simple, high-throughput assay is urgently needed for this. In this study, the app
Aslan, Hacer; Gülmez, Dolunay
Frequency of Candida species causing urinary tract infections is increasing, and this increase is outstanding in nosocomial urinary tract infections especially in intensive care units. The ability of biofilm formation that is contributed to the virulence of the yeast, plays a role in the pathogenesis of biomaterial-related infections and also constitutes a risk for treatment failure. The aims of this study were to compare biofilm forming abilities of Candida strains isolated from urine cultures of patients with and without urinary catheters, and to investigate the change of antifungal susceptibility in the presence of biofilm. A total of 50 Candida strains isolated from urine cultures of 25 patients with urinary catheters (10 C.tropicalis, 6 C.glabrata, 4 C.albicans, 4 C.parapsilosis, 1 C.krusei) and 25 without urinary catheters (8 C.tropicalis, 6 C.albicans, 4 C.krusei, 3 C.parapsilosis, 2 C.kefyr, 1 C.glabrata, 1 C.lusitaniae) were included in the study. Biofilm forming ability was tested by Congo red agar (CRA) and microplate XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction methods. Fluconazole (FLU) and amphotericin B (AMP-B) susceptibilities of the isolates were determined by reference microdilution method recommended by Clinical and Laboratory Standards Institute for planktonic cells and by XTT reduction assay in case of biofilm presence. Biofilm formation was detected in 12 (24%) by CRA and 50 (100%) of the isolates by XTT reduction method. None of the C.albicans (n= 10) and C.tropicalis (n= 18) strains were detected as biofilm positive by CRA, however, these strains were strongly positive by XTT reduction method. No statistically significant correlation was detected between the presence of urinary catheter and biofilm forming ability of the isolate (p> 0.05). This might be caused by the advantage of biofilm forming strains in adhesion to bladder mucosa at the initial stages of infection. For all of the isolates in
Muszanska, L.H.; Nejadnik, M.R.; Chen, Y.; Heuvel, van den E.R.; Busscher, H.J.; Mei, van der H.C.; Norde, W.
Biofilms causing biomaterial-associated infection resist antibiotic treatment and usually necessitate the replacement of infected implants. Here we relate bacterial adhesion forces and the antibiotic susceptibility of biofilms on uncoated and polymer brush-coated silicone rubber. Nine strains of Sta
Poovendran Ponnusamy; Vidhya Natarajan; Murugan Sevanan
Objective:To detect in vitro biofilm formation of uropathogenic Escherichia coli(E. coli)(UPEC) strains isolated from urine specimens and also to determine their antimicrobial susceptibility pattern using 13 commonly used antibiotics.Methods: The present study comprised of166 urine specimens collected from tertiary care hospitals in and around Coimbatore, South India. All the specimens were subjected to gram staining, bacterial culture and theE. coli strains were screened for biofilm formation using Tube Method(TM), Congo Red Agar(CRA) and Tissue Culture Plate method(TCP) respectively. Subsequently, the antimicrobial susceptibility test was performed by Kirby Bauer-disk diffusion method for the biofilm and non-biofilm producingE. colistrains.Results: Of the100 (60.2 %)E. coli strains,72 strains displayed a biofilm positive phenotype under the optimized conditions in the Tube Method and the strains were classified as highly positive(17, 23.6%), moderate positive(19, 26.3 %) and weakly positive(36, 50.0 %), similarly under the optimized conditions on Congo Red agar medium, biofilm positive phenotype strains were classified as highly positive(23, 23 %), moderate positive(37, 37 %)and weakly positive (40, 40%). While inTCP method, the biofilm positive phenotype strains were also classified as highly positive(6, 6 %), moderate positive (80, 80 %)and weakly positive(14, 14 %), it didn’t not correlate well with the tube method for detecting biofilm formation in E. coli. The rates of antibiotic resistance of biofilm producingE. coliwere found to be 100 % for chloramphenicol and amoxyclav (amoxicillin and clavulanic acid),86% for gentamicin and cefotaxime,84% for ceftazidime,83% for cotrimoxazole and piperacillin/tazobactam,75% for tetracycline and70% for amikacin.Conclusions: This study reveals the prevalence and antimicrobial susceptibility pattern of biofilm and non-biofilm producing uropathogenic E. coli strains.
A field method is described for the assay of [35S]sulfate reduction by sulfate-reducing bacteria in biofilms on metal surfaces. The assay is such that the biofilm can be studied without removing it from the substratum. The presence of the metal coupons, however, required preliminary optimisation of conditions for accurate determination of in situ sulfate reduction rates. Modifications to the radiorespirometric assay are described and successful field trials are presented. 18 refs.; 4 figs
Otter, J A; Vickery, K; Walker, J T; deLancey Pulcini, E; Stoodley, P; Goldenberg, S D; Salkeld, J A G; Chewins, J; Yezli, S; Edgeworth, J D
Microbes tend to attach to available surfaces and readily form biofilms, which is problematic in healthcare settings. Biofilms are traditionally associated with wet or damp surfaces such as indwelling medical devices and tubing on medical equipment. However, microbes can survive for extended periods in a desiccated state on dry hospital surfaces, and biofilms have recently been discovered on dry hospital surfaces. Microbes attached to surfaces and in biofilms are less susceptible to biocides, antibiotics and physical stress. Thus, surface attachment and/or biofilm formation may explain how vegetative bacteria can survive on surfaces for weeks to months (or more), interfere with attempts to recover microbes through environmental sampling, and provide a mixed bacterial population for the horizontal transfer of resistance genes. The capacity of existing detergent formulations and disinfectants to disrupt biofilms may have an important and previously unrecognized role in determining their effectiveness in the field, which should be reflected in testing standards. There is a need for further research to elucidate the nature and physiology of microbes on dry hospital surfaces, specifically the prevalence and composition of biofilms. This will inform new approaches to hospital cleaning and disinfection, including novel surfaces that reduce microbial attachment and improve microbial detachment, and methods to augment the activity of biocides against surface-attached microbes such as bacteriophages and antimicrobial peptides. Future strategies to address environmental contamination on hospital surfaces should consider the presence of microbes attached to surfaces, including biofilms. PMID:25447198
Nitis; Smanthong; Ratree; Tavichakorntrakool; Phitsamai; Saisud; Vitoon; Prasongwatana; Pipat; Sribenjalux; Aroonlug; Lulitanond; Orathai; Tunkamnerdthai; Chaisiri; Wongkham; Patcharee; Boonsiri
Objective: To compare bioi lm formation in trimethoprim/sulfamethoxazole(SXT)-susceptible Escherichia coli(E. coli)(SSEC) and SXT-resistant E. coli(SREC) isolated from patients with urinary tract infections, and study the motile ability and physical characteristics of the isolates.Methods: A total of 74 E. coli isolates were tested for antimicrobial susceptibility with the disc diffusion assay. Based on the SXT-susceptibility test, the E. coli isolates were divided into SSEC(N = 30) and SREC(N = 44) groups. All E. coli isolates were examined for motile ability by using a motility test medium, and for checking bioi lm formation a scanning electron microscope was used. Bacterial colony size was measured with a vernier caliper and bacterial cell length was measured under a light microscope. The bacterial growth rate was studied by plotting the cell growth(absorbance) versus the incubation time. Results: The frequencies of non-motility and biofilm formation in the SREC group were signii cantly higher than that in the SSEC group(P < 0.01). The SREC bacterial cell length was shorter than that in the SSEC group [(1.35 ± 0.05) vs.(1.53 ± 0.05) μm, P < 0.05)], whereas the bacterial colony size and mid-log phase of the growth curve were not signii cantly dif erent. Conclusions: The present study indicated that bioi lm formation and phenotypic change of uropathogenic E. coli can be attributed to the mechanism of E. coli SXT resistance.
Full Text Available Minimum inhibitory concentration (MIC, minimum bactericidal concentration (MBC, and minimum biofilm eradication concentration (MBEC and kill kinetics were established for vancomycin, rifampicin, trimethoprim, gentamicin, and ciprofloxacin against the biofilm forming bacteria Staphylococcus epidermidis (ATCC 35984, Staphylococcus aureus (ATCC 29213, Methicillin Resistant Staphylococcus aureus (MRSA (ATCC 43300, Pseudomonas aeruginosa (PAO1, and Escherichia coli (NCTC 8196. MICs and MBCs were determined via broth microdilution in 96-well plates. MBECs were studied using the Calgary Biofilm Device. Values obtained were used to investigate the kill kinetics of conventional antimicrobials against a range of planktonic and biofilm microorganisms over a period of 24 hours. Planktonic kill kinetics were determined at 4xMIC and biofilm kill kinetics at relative MBECs. Susceptibility of microorganisms varied depending on antibiotic selected and phenotypic form of bacteria. Gram-positive planktonic isolates were extremely susceptible to vancomycin (highest MBC: 7.81 mg L−1: methicillin sensitive and resistant S. aureus but no MBEC value was obtained against all biofilm pathogens tested (up to 1000 mg L−1. Both gentamicin and ciprofloxacin displayed the broadest spectrum of activity with MIC and MBCs in the mg L−1 range against all planktonic isolates tested and MBEC values obtained against all but S. epidermidis (ATCC 35984 and MRSA (ATCC 43300.
Rahim, Muhammad Imran; Rohde, Manfred; Rais, Bushra; Seitz, Jan-Marten; Mueller, Peter P
Magnesium alloys have promising mechanical and biological properties as biodegradable medical implant materials for temporary applications during bone healing or as vascular stents. Whereas conventional implants are prone to colonization by treatment resistant microbial biofilms in which bacteria are embedded in a protective matrix, magnesium alloys have been reported to act antibacterial in vitro. To permit a basic assessment of antibacterial properties of implant materials in vivo an economic but robust animal model was established. Subcutaneous magnesium implants were inoculated with bacteria in a mouse model. Contrary to the expectations, bacterial activity was enhanced and prolonged in the presence of magnesium implants. Systemic antibiotic treatments were remarkably ineffective, which is a typical property of bacterial biofilms. Biofilm formation was further supported by electron microscopic analyses that revealed highly dense bacterial populations and evidence for the presence of extracellular matrix material. Bacterial agglomerates could be detected not only on the implant surface but also at a limited distance in the peri-implant tissue. Therefore, precautions may be necessary to minimize risks of metallic magnesium-containing implants in prospective clinical applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1489-1499, 2016. PMID:26860452
Muszanska, Agnieszka K.; Nejadnik, M. Reza; Chen, Yun; van den Heuvel, Edwin R; Busscher, Henk J.; van der Mei, Henny C; Norde, Willem
Biofilms causing biomaterial-associated infection resist antibiotic treatment and usually necessitate the replacement of infected implants. Here we relate bacterial adhesion forces and the antibiotic susceptibility of biofilms on uncoated and polymer brush-coated silicone rubber. Nine strains of Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa adhered more weakly to brush-coated silicone rubber (−0.05 ± 0.03 to −0.51 ± 0.62 nN) than to uncoated silicone rubber (−1...
Herrling, Maria Pia
To contribute to a better understanding of the interaction between engineered nanoparticles (ENP) and real biofilms, a new analytical approach was demonstrated within this dissertation using a magnetic susceptibility balance and magnetic resonance imaging for in-situ and non-invasive measurements. The focus was to examine the role of the water matrix, particle properties and exposure time on the ENP-biofilm-interactions and biosorption in biofilm systems with compact and fluffy structures.
HUANG Zi-kun; LUO Qing; JIANG Bi-xia; LI Wei-ting; XU Xiao-meng; XIONG Guo-liang; LI Jun-ming
Background Drug susceptibility assay is very important in tuberculosis therapy.Pyrazinamide is a first line antituberculosis drug and diagnosis of its resistance in Mycobacterium tuberculosis (M.tuberculosis) is difficult and time consuming by conventional methods.In this study,we aimed to evaluate the performance of the microscopic observation drug susceptibility (MODS) assay in the detection of pyrazinamide resistance in M.tuberculosis relative to the conventional Wayne assay and Lowenstein-Jensen (L J) proportion method.Methods M.tuberculosis clinical isolates (n=132) were tested by the MODS and the Wayne assay:the results were compared with those obtained by the LJ proportion method.Mutations in the gene were identified by direct sequencing of the pncA genes of all isolates in which pyrazinamide resistance was detected by any of the three methods.Results Compared to the LJ results,the sensitivity and specificity of the MODS assay were 97.8％ and 96.5％ respectively; the sensitivity and specificity of the Wayne assay were 87.0％ and 97.7％ respectively.Mutations in the pncA gene were found in 41 of 46 strains that were pyrazinamide resistant (3 tests),in 1 of the 4 strains (LJ only),in 42 of 48 strains (at least 1 test),but no mutations in 1 strain sensitive according to the MODS assay only.The MODS assay,Wayne assay and LJ proportion method provided results in a median time of 6,7 and 26 days respectively.Conclusions MODS assay offers a rapid,simple and reliable method for the detection of pyrazinamide resistance in M.tuberculosis and is an optimal alternative method in resource limited countries.
Garvey, M.; Coughlan, G.; Murphy, N.; Rowan, N.
The presence of pathogenic organisms namely parasite species and bacteria in biofilms in veterinary settings, is a public health concern in relation to human and animal exposure. Veterinary clinics represent a significant risk factor for the transfer of pathogens from housed animals to humans, especially in cases of wound infection and the shedding of faecal matter. This study aims to provide a means of detecting veterinary relevant parasite species in bacterial biofilms, and to provide a means of disinfecting these biofilms. A real time PCR assay was utilized to detect parasite DNA in Bacillus cereus biofilms on stainless steel and PVC surfaces. Results show that both Cryptosporidium and Giardia attach to biofilms in large numbers (100-1000 oo/cysts) in as little as 72 hours. Pulsed light successfully inactivated all test species (Listeria, Salmonella, Bacillus, Escherichia) in planktonic and biofilm form with an increase in inactivation for every increase in UV dose. PMID:26862516
Queiroz, Paula Assis; Godoy, Janine Silva Ribeiro; Mendonça, Patrícia de Souza Bonfim; Pedroso, Raíssa Bocchi; Svidzinski, Terezinha Inez Estivalet; Negri, Melyssa
Yeasts of the genera Candida and Saccharomyces are opportunist pathogens and cause oral lesions, especially in immunocompromised patients. This study assessed yeasts isolated from chronic kidney patients undergoing haemodialysis for their adhesion capacity, biofilm formation and susceptibility to antifungal agents. Ten isolates of Candida spp. and one isolate of Saccharomyces cerevisiae were tested for adhesion to buccal epithelial cells (BECs), adhesion and formation of biofilm in artificial saliva and their susceptibility profile to antifungal agents. Adhesion and biofilm formation were undertaken in polystyrene plates with artificial saliva, whilst susceptibility to antifungal agents was evaluated by broth microdilution. Candida parapsilosis had the highest adhesion index in BECs (154.55 ± 22.13) and Candida rugosa was the species with the highest adhesion capacity (18 398 Abs cm(-2)) in abiotic surface with artificial saliva. Candida albicans provided the greatest biofilm formation (2035 Abs cm(-2) ± 0.09) but was revealed to be susceptible to the five antifungal agents under analysis. However, some non-albicans Candida isolates showed a lower susceptibility for the antifungal agents itraconazole, fluconazole and voriconazole. All of the species were sensitive to amphotericin B and nystatin. The current analysis showed that yeasts isolated from the mouth of chronic kidney patients undergoing haemodialysis varied significantly with regard to their capacity for adherence, biofilm formation and susceptibility to antifungal agents, underscoring the high virulence of non-albicans Candida species. PMID:26297016
Sugnaux, Marc; Fischer, Fabian
A novel method was developed to assay the antimicrobial capacity of nanostructured surfaces for medical implants in a bicathodic microbial fuel cell. Nano-structured gold surfaces with protruding nanopillars and nanorings were investigated. Escherichia coli K12 were used as a model microbe to record electronic effects caused by the interaction with nanosurfaces. The nanostructured gold surfaces enabled power density maxima up to 1910mW/m(2), indicating fair vivacity, while flat surfaces on the nanoscale provided almost no power 0.35mW/m(2). The biofilm presence on antimicrobial nanosurfaces was confirmed by the addition of ampicillin and its bactericidal effect resulted in oscillating and declining potentiometric signals. Current density experiments showed that biofilms on antimicrobial nanostructured electrodes caused low currents, indicating that E.coli biofilm remained functional before destruction. The bicathodic microbial fuel cell sensor is a novel tool for evaluating antimicrobial effects caused by nanosurfaces and antibiotics. PMID:27071334
Younis, Khansa Mohammed; Usup, Gires; Ahmad, Asmat
This study is aimed to isolate, detect biofilm formation ability and antibiotic susceptibility of urinary catheter adherent microorganisms from elderly hospitalized patient at the Universiti Kebangsaan Malaysia Medical Center. Microorganisms were isolated from three samples of urinary catheters (UC) surface; one of the acute vascular rejection patient (UCB) and two from benign prostate hyperplasia patients (UCC and UCD). A total of 100 isolates was isolated with 35 from UCB, 38 (UCC) and 28 (UCD). Ninety six were identified as Gram-negative bacilli, one Gram-positive bacilli and three yeasts. Results of biofilm forming on sterile foley catheter showed that all the isolates can form biofilm at different degrees; strong biofilm forming: 32% from the 35 isolates (UCB), 25% out of 38 isolates (UCC), 26% out of 28 isolates (UCD). As for moderate biofilm forming; 3% from UCB, 10% from UCC and 2% from UCD. Weak biofilm forming in UCC (3%). The antibiotic susceptibility for (UCB) isolates showed highly resistant to ampicillin, novobiocin and penicillin 100 (%), kanamycin (97%), tetracycline (94%), chloramphenicol (91%), streptomycin (77%) and showed low level of resistance to gentamycin (17%), while all the isolates from (UCC-D) showed high resistant towards ampicillin and penicillin, novobiocin (94%), tetracycline (61%), streptomycin (53%), gentamycin (50%) and low level of resistance to kanamycin (48%), chloramphenicol (47%). The findings indicate that these isolates can spread within the community on urinary catheters surface and produce strong biofilm, therefore, monitoring antibiotic susceptibility of bacteria isolated in the aggregation is recommended.
López, Daniel; Vlamakis, Hera; Kolter, Roberto
The ability to form biofilms is a universal attribute of bacteria. Biofilms are multicellular communities held together by a self-produced extracellular matrix. The mechanisms that different bacteria employ to form biofilms vary, frequently depending on environmental conditions and specific strain attributes. In this review, we emphasize four well-studied model systems to give an overview of how several organisms form biofilms: Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and ...
Sarkar, Sohinee; Vagenas, Dimitrios; Schembri, Mark A; Totsika, Makrina
Escherichia coli sequence type 131 (ST131) has emerged as a pandemic lineage of important multidrug resistant pathogens worldwide. Despite many studies examining the epidemiology of ST131, only a few studies to date have investigated the capacity of ST131 strains to form biofilms. Some of these studies have reported contrasting findings, with no specific ST131 biofilm-promoting factors identified. Here, we examined a diverse collection of ST131 isolates for in vitro biofilm formation in different media and assay conditions, including urine from healthy adult women. We found significant differences among strains and assay conditions, which offers an explanation for the contrasting findings reported by previous studies using a single condition. Importantly, we showed that expression of type 1 fimbriae is a critical determinant for biofilm formation by ST131 strains and that inhibition of the FimH adhesin significantly reduces biofilm formation. We also offer direct genetic evidence for the contribution of type 1 fimbriae in biofilm formation by the reference ST131 strain EC958, a representative of the clinically dominant H30-Rx ST131 subgroup. This is the first study of ST131 biofilm formation in biologically relevant conditions and paves the way for the application of FimH inhibitors in treating drug resistant ST131 biofilm infections. PMID:26940589
Cerca, Nuno; Martins, Silvia; Cerca, Filipe; Jefferson, Kimberly K.; Pier, Gerald B.; Oliveira, Rosário; Azeredo, Joana
Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy Objectives: To quantitatively compare the antibiotic susceptibility of biofilms formed by the coagulasenegative staphylococci (CoNS) Staphylococcus epidermidis and Staphylococcus haemolyticus with the susceptibility of planktonic cultures. Methods: SeveralCoNSstrains were grown planktonically or as biofilms to determine the effect of themode of growth on the level of susceptibility to...
Efficacy of ultraviolet C light at sublethal dose in combination with antistaphylococcal antibiotics to disinfect catheter biofilms of methicillin-susceptible and methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis in vitro
Full Text Available Mohamed El-Azizi,1 Nancy Khardori2 1Department of Microbiology, Immunology and Biotechnology, Faculty of Pharmacy and Biotechnology, German University in Cairo, New Cairo City, Egypt; 2Department of Internal Medicine, Division of Infectious Diseases, Eastern Virginia Medical School, Norfolk, VA, USA Background: Biofilm formation inside inserted medical devices leads to their failure and acts as a source of refractory infections. The ultraviolet C (UVC light is a potential therapy that can be used against the biofilm of bacterial pathogens. Objective: We evaluated the efficacy of sublethal dose of UVC light with anti-staphylococcal antibiotics against biofilms made from 30 isolates of methicillin-susceptible Staphylococcus aureus and methicillin-resistant S. aureus and S. epidermidis on vascular catheters. Materials and methods: A novel biofilm device was used to assess the combined approach. The biofilms on the catheters were irradiated with the UVC light at 254 nm and irradiance of 6.4 mW followed by treatment with vancomycin or quinupristin/dalfopristin at twice their minimum bactericidal concentrations or with linezolid at 64 µg/mL for 24 hours. The catheters were cut into segments and sonicated, and the number of the sessile cells was determined colorimetrically using XTT viable cells assay. The effect of UVC radiation followed by treatment with an antistaphylococcal antibiotic on the viability of the bacteria in the biofilm was visualized using LIVE/DEAD BacLight bacterial viability stain and confocal laser scanning microscopy. Results: Exposure of the bacterial biofilms to the UVC light or each of the antibiotics alone was ineffective in killing the bacteria. Treatment of the biofilms with the antibiotics following their exposure to UVC light significantly (P<0.001 reduced the number of viable cells within the biofilms but did not completely eradicate them. Conclusion: To our knowledge, this combinatorial approach has not been
Baillie, George S.; Douglas, L. Julia
Biofilms of Candida albicans were grown in vitro under iron limitation and at a low growth rate to simulate conditions for implant-associated biofilms in vivo. Their properties were compared with those of glucose-limited biofilms grown under analogous conditions. At steady state, the adherent cell populations of iron-limited biofilms were double those of glucose-limited biofilms, although the growth rates were similar (0.038 to 0.043 h−1). Both biofilm types were resistant to amphotericin B, ...
Joanna Kwiecińska-Piróg; Krzysztof Skowron; Katarzyna Zniszczol; Eugenia Gospodarek
Rods of the Proteus genus are commonly isolated from patients, especially from the urinary tracts of the catheterised patients. The infections associated with biomaterials are crucial therapeutic obstacles, due to the bactericidal resistance of the biofilm. The aim of this study was to assess the susceptibility of P. mirabilis planktonic forms to ciprofloxacin and ceftazidime, the ability to form biofilm, and the impact of chosen sub-MIC concentrations of these antibiotics on biofilm at diffe...
Pratten, J.; Barnett, P.; Wilson, M
Using a constant-depth film fermentor, we have grown a six-membered biofilm community with a bacterial composition similar to that found in supragingival dental plaque. Cryosectioning revealed the distribution of bacteria throughout the biofilm. Exposure to 0.2% chlorhexidine for up to 5 min had little effect on biofilm viability.
Full Text Available Background: Coryneform bacteria constitute an important segment of male urogenital microbiota. They have been generally considered as saprophytes, although some species have been associated with prostatitis as well. At the same time, biofilm infections have been suspected as a cause of prostatitis. Objective: To identify a set of coryneform bacteria isolated from semen of either healthy men or prostatitis patients applying different methods to reveal inter-assay variability and to determine their ability of adhesion and biofilm production. Design: Coryneform bacteria were identified by API Coryne 2.0 biochemical identification system and 16S rDNA sequencing using different primer sets. Quantitative assessment of biofilm production was performed using crystal violet binding assay method. Results: The most common species were Corynebacterium seminale, C. minutissimum, and Dermabacter hominis. Altogether 14 species and related genera were found. We observed the best inter-assay agreement when identifying C. seminale. Biofilm was observed in 7 out of 24 strains. The biofilm-producing strains belonged to Arthrobacter cumminsii, Dermabacter hominis, C. minutissimum, and Actinomyces neuii. No differences were found between the strains originating from prostatitis patients and healthy men. Dermabacter hominis strains were more potent biofilm producers than C. seminale strains (p=0.048. Conclusions: We can conclude that a wide variety of coryneform bacteria can be found from the male genital tract, although their exact identification is problematic due to insufficient representation in databases. Nearly one third of the strains are able to form biofilm that may give them an advantage for surviving several host- and treatment-related conditions.
Berlin: Springer, 2015 - (Amils, R.; Gargaud, M.; Cernicharo Quintanilla, J.; James Claves, H.; Irvine, W.; Pinti, D.; Viso, M.), s. 1-3 ISBN 978-3-642-27833-4 Institutional support: RVO:67985939 Keywords : biofilm * microbial mat * astrobiology Subject RIV: EF - Botanics
Full Text Available In this study, 24 standard nontuberculous mycobacteria (NTM species strains including 12 slowly growing mycobacteria strains and 12 rapidly growing mycobacteria strains were subjected to drug susceptibility testing using microplate Alamar Blue assay-based 7H9 broth. The most active antimicrobial agents against the 24 NTM strains were streptomycin, amikacin, the fluoroquinolones, and the tetracyclines. Mycobacterium chelonae, Mycobacterium abscessus, Mycobacterium bolletii, and Mycobacterium simiae are resistant to most antimicrobial agents. The susceptibility results of this study from 24 NTM standard strains can be referenced by clinicians before susceptibility testing for clinical isolates is performed or when conditions do not allow for susceptibility testing. The application of broth-based methods is recommended by the Clinical and Laboratory Standards Institute, and the documentation of the susceptibility patterns of standard strains of mycobacteria can improve the international standardization of susceptibility testing methods.
Turovskiy, Yevgeniy; Cheryian, Thomson; Algburi, Ammar; Wirawan, Ruth E; Takhistov, Paul; Sinko, Patrick J; Chikindas, Michael L
Bacterial vaginosis is a common vaginal infection associated with numerous gynecological and obstetric complications. This condition is characterized by the presence of thick adherent vaginal biofilms, composed mainly of Gardnerella vaginalis. This organism is thought to be the primary aetiological cause of the infection paving the way for various opportunists to colonize the niche. Previously, we reported that the natural antimicrobials subtilosin, ε-poly-L-lysine, and lauramide arginine ethyl ester selectively inhibit the growth of this pathogen. In this study, we used plate counts to evaluate the efficacy of these antimicrobials against established biofilms of G. vaginalis. Additionally, we validated and compared two rapid methods (ATP viability and resazurin assays) for the assessment of cell viability in the antimicrobial-treated G. vaginalis biofilms. Out of the tested antimicrobials, lauramide arginine ethyl ester had the strongest bactericidal effect, followed by subtilosin, with clindamycin and polylysine showing the weakest effect. In comparison to plate counts, ATP viability and resazurin assays considerably underestimated the bactericidal effect of some antimicrobials. Our results indicate that these assays should be validated for every new application. PMID:23024575
Morandi, S; Cremonesi, P; Capra, E; Silvetti, T; Decimo, M; Bianchini, V; Alves, A C; Vargas, A C; Costa, G M; Ribeiro, M G; Brasca, M
Bovine mastitis caused by Prototheca is a serious and complex problem that accounts for high economic losses in the dairy industry. The main objective of this study was to identify and characterize at genetic level different Prototheca strains and provide the most complete data about protothecal antibiotic resistance. The study involves 46 isolates from Italian (13 strains) and Brazilian (33 strains) mastitic milk. These strains were identified by multiplex PCR and single strand conformation polymorphism analysis and characterized by randomly amplified polymorphic DNA (RAPD)-PCR. Moreover, biofilm production and antibiotic susceptibility were evaluated. Forty-two strains resulted as Prototheca zopfii genotype 2, whereas 4 isolates could belong to a potential new Prototheca species. The RAPD-PCR, performed with 3 primers (M13, OPA-4, and OPA-18), showed a notable heterogeneity among isolates and grouped the strains according to the species and geographical origin. Biofilm production was species-dependent and P. zopfii genotype 2 strains were classified as strong biofilm producers. In vitro antibiotic susceptibility tests indicated that Prototheca strains were susceptible to antibacterial drugs belonging to aminoglycosides group; the highest activity against Prototheca strains was observed in the case of colistin sulfate, gentamicin, and netilmicin (100% of susceptible strains). It is interesting to note that all the Italian P. zopfii genotype 2 strains showed lower minimum inhibitory concentration values than the Brazilian ones. Nisin showed more efficacy than lysozyme and potassium sorbate, inhibiting 31% of the strains. Results obtained in this study confirmed that RAPD-PCR is a rapid, inexpensive, and highly discriminating tool for Prototheca strains characterization and could give a good scientific contribution for better understanding the protothecal mastitis in dairy herd. PMID:27236754
Full Text Available Microorganisms adhere to non-living material or living tissue, and form biofilms made up of extracellular polymers/slime. Biofilm-associated microorganisms behave differently from free-floating bacteria with respect to growth rates and ability to resist antimicrobial treatments and therefore pose a public health problem. The objective of this study is to detect the prevalence of biofilm producers among Gram positive and Gram negative bacteria isolated from clinical specimens, and to study their antimicrobial susceptibility pattern. The study was carried out from October 2009 to March 2010, at the Department of Microbiology, Army Medical College/ National University of Sciences and Technology (NUST, Rawalpindi, Pakistan. Clinical specimens were received from various wards of a tertiary care hospital. These were dealt by standard microbiological procedures. Gram positive and Gram negative bacteria isolated were subjected to biofilm detection by congo red agar method (CRA. Antimicrobial susceptibility testing of those isolates, which showed positive results (slime production, was done according to the Kirby-Bauer disc diffusion technique. A total of 150 isolates were tested for the production of biofilm/slime. Among them, 81 isolates showed positive results. From these 81, 51 were Gram positive and 30 were Gram negative. All the 81(54% slime producers showed reduced susceptibility to majority of antibiotics. Bacterial biofilms are an important virulence factor associated with chronic nosocomial infection. Detection of biofilm forming organisms can help in appropriate antibiotic choice.
ABalasubramanian; KChairman; AJARanjit Singh; GAlagumuthu
Objective: Bacterial species colonize indwelling catheters as biofilm induce complications in patients care. Methods: From the biofilm matrix seven species of microbes were isolated. The predominant bacteria seen in catheters were E.coli, (27 percent) P.mirabilis (20 percent) and S.epidermis (18 percent). Results: The biomass of microbes associated with the biofilm was estimated. The mean dry weight of biomass of bacteria associated with a catheter that was used for over a month time was in the range 2.5±0.04g - 3.1 ± 0.6g. Conclusion: But it was found to colonize the microtitre plate to attain a peak growth at 84h. P.mirabilis isolated from the biofilm was able to tolerate the antibiotics tetracycline, Penicillin, Kanamycin and Gentamycin at a dose level of 20μg/ml. The study indicated that the catheter has to be replaced if biofilm formation was noticed.
Cavaliere, Rosalia; Ball, Jessica L; Turnbull, Lynne; Whitchurch, Cynthia B.
Nontypeable Hemophilus influenzae (NTHi) is a Gram-negative bacterial pathogen that causes chronic biofilm infections of the ears and airways. The biofilm matrix provides structural integrity to the biofilm and protects biofilm cells from antibiotic exposure by reducing penetration of antimicrobial compounds into the biofilm. Extracellular DNA (eDNA) has been found to be a major matrix component of biofilms formed by many species of Gram-positive and Gram-negative bacteria, including NTHi. In...
ABSTRACT Mycobacterium avium and Mycobacterium intracellulare are environmental opportunistic pathogens whose source for human infection is water and soil. M. avium and M. intracellulare cause pulmonary infections (tuberculosis) in immunocompetent individuals and bacteremia in immunodeficient individuals (e.g. AIDS). One factor likely influencing the lack of success of antibiotic therapy in patients would be their ability to form biofilms. Growth in biofilms might result in antimicrob...
Laverty, Garry; Gorman, Sean P; Gilmore, Brendan F
In this study, we report the antimicrobial planktonic and biofilm kill kinetics of ultrashort cationic lipopeptides previously demonstrated by our group to have a minimum biofilm eradication concentration (MBEC) in the microgram per mL (μg/mL) range against clinically relevant biofilm-forming micro-organisms. We compare the rate of kill for the most potent of these lipopeptides, dodecanoic (lauric) acid-conjugated C12 -Orn-Orn-Trp-Trp-NH2 against the tetrapeptide amide H-Orn-Orn-Trp-Trp-NH2 motif and the amphibian peptide Maximin-4 via a modification of the MBEC Assay(™) for Physiology & Genetics (P&G). Improved antimicrobial activity is achieved upon N-terminal lipidation of the tetrapeptide amide. Increased antimicrobial potency was demonstrated against both planktonic and biofilm forms of Gram-positive micro-organisms. We hypothesize rapid kill to be achieved by targeting of microbial membranes. Complete kill against established 24-h Gram-positive biofilms occurred within 4 h of exposure to C12 -OOWW-NH2 at MBEC values [methicillin-resistant Staphylococcus epidermidis (ATCC 35984): 15.63 μg/mL] close to the values for the planktonic minimum inhibitory concentration (MIC) [methicillin-resistant Staphylococcus epidermidis (ATCC 35984): 1.95 μg/mL]. Such rapid kill, especially against sessile biofilm forms, is indicative of a reduction in the likelihood of resistant strains developing with the potential for quicker resolution of pathogenic infection. Ultrashort antimicrobial lipopeptides have high potential as antimicrobial therapy. PMID:25264285
Full Text Available Rods of the Proteus genus are commonly isolated from patients, especially from the urinary tracts of the catheterised patients. The infections associated with biomaterials are crucial therapeutic obstacles, due to the bactericidal resistance of the biofilm. The aim of this study was to assess the susceptibility of P. mirabilis planktonic forms to ciprofloxacin and ceftazidime, the ability to form biofilm, and the impact of chosen sub-MIC concentrations of these antibiotics on biofilm at different stages of its formation. The research included 50 P. mirabilis strains isolated from wounds and the urinary tracts from patients of the University Hospital No. 1 in Bydgoszcz. The assessment of susceptibility to ciprofloxacin and ceftazidime was conducted using micromethods. The impact of sub-MIC concentrations of the chosen antibiotics on the biofilm was measured using the TTC method. The resistance to ciprofloxacin was confirmed for 20 strains (40.0% while to ceftazidime for 32 (64.0% of the tested P. mirabilis strains. All of the tested strains formed biofilm: 24.0% weakly, 26.0% moderately, and 50.0% strongly. It was determined that ciprofloxacin and ceftazidime caused eradication of the biofilm. Moreover, the connection between origin of the strains, biofilm maturity level, and resistance to antibiotics was proved.
Baba, Sevinç; Aydın, Mustafa Derya
Streptococcus agalactiae (Group B streptococcus, GBS), a member of normal flora of human gastrointestinal and genitourinary systems, is a leading cause of sepsis, meningitis, and pneumonia particularly in newborn. GBS can also cause severe infections in pregnant women and adults with underlying disease, as well as mild diseases, such as urinary tract infections (UTIs). GBS strains exhibit 10 different serotypes, and the identification of serotype distribution is important epidemiologically. The role of biofilm production is one of the virulence factors that has been discussed in the pathogenesis of GBS infections. Although resistance to penicillin and ampicillin has not been documented in GBS, different rates of resistance has been reported for the alternative antibiotics to penicillin. The aim of this study was to investigate the serotype distribution, the ability of biofilm formation and the antibiotic susceptibilities of S.agalactiae strains isolated from urine cultures. A total of 60 strains were included in the study, 40 of them were isolated from patients (38 female 2 male; mean age: 36.7 years) with urinary tract complaints whose cultures yielded single type of colonies in the number of ≥ 50.000 cfu/ml, whereas 20 of them were isolated from patients (19 female 1 male; mean age: 37.2 years) without urinary tract complaints whose cultures yielded mixed colonies in the number of ≤ 20.000 cfu/ml. Chromogenic media were used for the isolation and the isolates were identified by conventional methods. The isolates were then serotyped by latex agglutination method and their antibiotic susceptibilities were determined by disk diffusion method recommended by CLSI documents. Biofilm formation of the strains were investigated by microplate and Congo red agar (CRA) methods. In our study, the most frequently detected serotypes were V (n= 18; 30%) and II (n= 14; 23.3%), followed by serotype Ia (n= 10; 16.7%), III (n= 9; 15%), Ib (n= 3; 5%), VI (n= 1; 1.7%) and VII (n
Nuryastuti, Titik; Krom, Bastiaan P.; Aman, Abu T.; Busscher, Henk J.; van der Mei, Henny C.
Ica-expression by Staphylococcus epidermidis and slime production depends on environmental conditions such as implant material and presence of antibiotics. Here, we evaluate biofilm formation and ica-expression of S. epidermidis strains on biomaterials involved in total hip-and knee arthroplasty [po
Garland Suzanne M
Full Text Available Abstract Background Coagulase-negative staphylococci are major causes of bloodstream infections in very low birth weight babies cared for in Neonatal Intensive Care Units. The virulence of these bacteria is mainly due to their ability to form biofilms on indwelling medical devices. Biofilm-related infections often fail to respond to antibiotic chemotherapy guided by conventional antibiotic susceptibility tests. Methods Coagulase-negative staphylococcal blood culture isolates were grown in different phases relevant to biofilm formation: planktonic cells at mid-log phase, planktonic cells at stationary phase, adherent monolayers and mature biofilms and their susceptibilities to conventional antibiotics were assessed. The effects of oxacillin, gentamicin, and vancomycin on preformed biofilms, at the highest achievable serum concentrations were examined. Epifluorescence microscopy and confocal laser scanning microscopy in combination with bacterial viability staining and polysaccharide staining were used to confirm the stimulatory effects of antibiotics on biofilms. Results Most coagulase-negative staphylococcal clinical isolates were resistant to penicillin G (100%, gentamicin (83.3% and oxacillin (91.7% and susceptible to vancomycin (100%, ciprofloxacin (100%, and rifampicin (79.2%. Bacteria grown as adherent monolayers showed similar susceptibilities to their planktonic counterparts at mid-log phase. Isolates in a biofilm growth mode were more resistant to antibiotics than both planktonic cultures at mid-log phase and adherent monolayers; however they were equally resistant or less resistant than planktonic cells at stationary phase. Moreover, for some cell-wall active antibiotics, concentrations higher than conventional MICs were required to prevent the establishment of planktonic cultures from biofilms. Finally, the biofilm-growth of two S. capitis isolates could be enhanced by oxacillin at the highest achievable serum concentration. Conclusion
Mycobacterial growth was monitored by bioluminescence assay of mycobacterial ATP. Cultures of Mycobacterium tuberculosis H37Rv and of 25 clinical isolates of the same species were exposed to serial dilutions of ethambutol, isoniazid, rifampin, and streptomycin. A suppression of ATP, indicating growth inhibition, occurred for susceptible but not resistant strains within 5 to 7 days of incubation. Breakpoint concentrations between susceptibility and resistance were determined by comparing these results with those obtained by reference techniques. Full agreement was found in 99% of the assays with the resistance ratio method on Lowenstein-Jensen medium, and 98% of the assays were in full agreement with the radiometric system (BACTEC). A main advantage of the bioluminescence method is its rapidity, with results available as fast as with the radiometric system but at a lower cost and without the need for radioactive culture medium. The method provides kinetic data concerning drug effects within available in vivo drug concentrations and has great potential for both rapid routine susceptibility testing and research applications in studies of drug effects on mycobacteria
Peterson, S W; Martin, I; Demczuk, W; Bharat, A; Hoang, L; Wylie, J; Allen, V; Lefebvre, B; Tyrrell, G; Horsman, G; Haldane, D; Garceau, R; Wong, T; Mulvey, M R
The incidence of antimicrobial-resistant Neisseria gonorrhoeae continues to rise in Canada; however, antimicrobial resistance data are lacking for approximately 70% of gonorrhea infections that are diagnosed directly from clinical specimens by nucleic acid amplification tests (NAATs). We developed a molecular assay for surveillance use to detect mutations in genes associated with decreased susceptibility to cephalosporins that can be applied to both culture isolates and clinical samples. Real-time PCR assays were developed to detect single nucleotide polymorphisms (SNPs) in ponA, mtrR, penA, porB, and one N. gonorrhoeae-specific marker (porA). We tested the real-time PCR assay with 252 gonococcal isolates, 50 nongonococcal isolates, 24 N. gonorrhoeae-negative NAAT specimens, and 34 N. gonorrhoeae-positive NAAT specimens. Twenty-four of the N. gonorrhoeae-positive NAAT specimens had matched culture isolates. Assay results were confirmed by comparison with whole-genome sequencing data. For 252 N. gonorrhoeae strains, the agreement between the DNA sequence and real-time PCR was 100% for porA, ponA, and penA, 99.6% for mtrR, and 95.2% for porB. The presence of ≥2 SNPs correlated with decreased susceptibility to ceftriaxone (sensitivities of >98%) and cefixime (sensitivities of >96%). Of 24 NAAT specimens with matched cultures, the agreement between the DNA sequence and real-time PCR was 100% for porB, 95.8% for ponA and mtrR, and 91.7% for penA. We demonstrated the utility of a real-time PCR assay for sensitive detection of known markers for the decreased susceptibility to cephalosporins in N. gonorrhoeae. Preliminary results with clinical NAAT specimens were also promising, as they correlated well with bacterial culture results. PMID:25878350
Lopes, H.; Machado, Idalina; Lopes, Susana Patrícia; Alves, D.; Pereira, Maria Olívia
P.aeruginosa (PA) is an opportunistic pathogen responsible for high percentage of nosocomial infections. Its virulence and persistence rises when bacteria switch from planktonic to biofilm state and when facing antimicrobial pressures. This study aimed to evaluate the antimicrobial tolerance and regrowth of several biofilmproducing PA after antibiotic treatment, and the occurrence of cross-resistance to other antimicrobials. PAO, ATCC, CGCT and an isolated strain were used to form...
Radiotherapy is one of the mainstays in the treatment for cancer, but its success can be limited due to inherent or acquired resistance. Mechanisms underlying radioresistance in various cancers are poorly understood and available radiosensitizers have shown only modest clinical benefit. There is thus a need to identify new targets and drugs for more effective sensitization of cancer cells to irradiation. Compound and RNA interference high-throughput screening technologies allow comprehensive enterprises to identify new agents and targets for radiosensitization. However, the gold standard assay to investigate radiosensitivity of cancer cells in vitro, the colony formation assay (CFA), is unsuitable for high-throughput screening. We developed a new high-throughput screening method for determining radiation susceptibility. Fast and uniform irradiation of batches up to 30 microplates was achieved using a Perspex container and a clinically employed linear accelerator. The readout was done by automated counting of fluorescently stained nuclei using the Acumen eX3 laser scanning cytometer. Assay performance was compared to that of the CFA and the CellTiter-Blue homogeneous uniform-well cell viability assay. The assay was validated in a whole-genome siRNA library screening setting using PC-3 prostate cancer cells. On 4 different cancer cell lines, the automated cell counting assay produced radiation dose response curves that followed a linear-quadratic equation and that exhibited a better correlation to the results of the CFA than did the cell viability assay. Moreover, the cell counting assay could be used to detect radiosensitization by silencing DNA-PKcs or by adding caffeine. In a high-throughput screening setting, using 4 Gy irradiated and control PC-3 cells, the effects of DNA-PKcs siRNA and non-targeting control siRNA could be clearly discriminated. We developed a simple assay for radiation susceptibility that can be used for high-throughput screening. This will aid
Perrier, Marine; Désiré, Nathalie; Deback, Claire; Agut, Henri; Boutolleau, David; Burrel, Sonia
The emergence of varicella-zoster virus (VZV) resistance to current antivirals as acyclovir (ACV) constitutes a hindrance to antiviral treatment effectiveness of VZV infections, especially in immunocompromised patients. The molecular mechanisms of VZV resistance reported so far rely on the presence of mutations within thymidine kinase (TK, ORF36) and DNA polymerase (ORF28) viral genes. The aim of this work was to develop reliable and complementary diagnostic methods to detect VZV antiviral resistance: (i) a genotypic assay based on TK and DNA polymerase genes sequencing, (ii) a plaque reduction assay to determine antiviral 50% effective concentrations, and (iii) a functional assay to evaluate in vitro phosphorylation activity of recombinant TKs. As a whole, this study included the analysis of 21 VZV clinical isolates and 62 biological samples from patients experiencing VZV infection. Genetic analysis revealed 3 and 9 new amino acid changes that have not been previously described within the highly conserved TK and DNA polymerase, respectively. Then, VZV isolates bearing newly identified mutations considered as natural polymorphisms were characterized as susceptible to ACV using plaque-reduction assay in MeWo cells. In parallel, the impact of TK changes on ACV phosphorylation activity was examined using a nonradioactive in vitro enzymatic assay. PMID:26994966
Highlights: • Biofilm stages in static batch conditions were similar to dynamic conditions. • Expression of csgA gene increased earlier than alg8 gene in biofilm maturation. • AgNPs had higher effect on less mature biofilms. • Removal of extracellular polymeric substance made biofilms susceptible to AgNPs. - Abstract: This study determined the effect of silver nanoparticles (AgNPs) on Pseudomonas putida KT2440 biofilms at different stages of maturity. Three biofilm stages (1–3, representing early to late stages of development) were identified from bacterial adenosine triphosphate (ATP) activity under static (96-well plate) and dynamic conditions (Center for Disease Control and Prevention biofilm reactor). Extracellular polymeric substance (EPS) levels, measured using crystal violet and total carbohydrate assays, and expression of the EPS-associated genes, csgA and alg8, supported the conclusion that biofilms at later stages were older than those at earlier stages. More mature biofilms (stages 2 and 3) showed little to no reduction in ATP activity following exposure to AgNPs. In contrast, the same treatment reduced ATP activity by more than 90% in the less mature stage 1 biofilms. Regardless of maturity, biofilms with EPS stripped off were more susceptible to AgNPs than controls with intact EPS, demonstrating that EPS is critical for biofilm tolerance of AgNPs. The findings from this study show that stage of maturity is an important factor to consider when studying effect of AgNPs on biofilms
Thuptimdang, Pumis, E-mail: email@example.com [International Program in Hazardous Substance and Environmental Management, Graduate School, Chulalongkorn University, Bangkok 10330 (Thailand); Center of Excellence on Hazardous Substance Management, Bangkok 10330 (Thailand); Limpiyakorn, Tawan, E-mail: firstname.lastname@example.org [Center of Excellence on Hazardous Substance Management, Bangkok 10330 (Thailand); Department of Environmental Engineering, Chulalongkorn University, Bangkok 10330 (Thailand); Research Unit Control of Emerging Micropollutants in Environment, Chulalongkorn University, Bangkok 10330 (Thailand); McEvoy, John, E-mail: email@example.com [Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo, ND 58108 (United States); Prüß, Birgit M., E-mail: firstname.lastname@example.org [Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo, ND 58108 (United States); Khan, Eakalak, E-mail: email@example.com [Department of Civil and Environmental Engineering, North Dakota State University, Fargo, ND 58108 (United States)
Highlights: • Biofilm stages in static batch conditions were similar to dynamic conditions. • Expression of csgA gene increased earlier than alg8 gene in biofilm maturation. • AgNPs had higher effect on less mature biofilms. • Removal of extracellular polymeric substance made biofilms susceptible to AgNPs. - Abstract: This study determined the effect of silver nanoparticles (AgNPs) on Pseudomonas putida KT2440 biofilms at different stages of maturity. Three biofilm stages (1–3, representing early to late stages of development) were identified from bacterial adenosine triphosphate (ATP) activity under static (96-well plate) and dynamic conditions (Center for Disease Control and Prevention biofilm reactor). Extracellular polymeric substance (EPS) levels, measured using crystal violet and total carbohydrate assays, and expression of the EPS-associated genes, csgA and alg8, supported the conclusion that biofilms at later stages were older than those at earlier stages. More mature biofilms (stages 2 and 3) showed little to no reduction in ATP activity following exposure to AgNPs. In contrast, the same treatment reduced ATP activity by more than 90% in the less mature stage 1 biofilms. Regardless of maturity, biofilms with EPS stripped off were more susceptible to AgNPs than controls with intact EPS, demonstrating that EPS is critical for biofilm tolerance of AgNPs. The findings from this study show that stage of maturity is an important factor to consider when studying effect of AgNPs on biofilms.
Machado, António; Salgueiro, Débora; Rodrigues, L. R.; Cerca, Nuno; Jefferson, Kimberly K
Bacterial vaginosis (BV) is the most common vaginal disorder of women of reproductive age. It is commonly accepted that the microbial switch from normal to BV state is characterized by a decrease in vaginal colonization by Lactobacillus species together with an increase in the number of Gardnerella vaginalis and others anaerobes. G. vaginalis can be found associated with normal vaginal epithelium but recent findings suggest that it is the biofilm produced by G. vaginalis that leads to establi...
Full Text Available Background: Candida albicans co-aggregates with Streptococcus gordonii to form biofilms and their interactions in mucosal biofilms may lead to pathogenic synergy. Although the functions of glucosyltransferases (Gtf of Mutans streptococci have been well characterized, the biological roles of these enzymes in commensal oral streptococci, such as S. gordonii, in oral biofilm communities are less clear. Objective: The objective of this work was to explore the role of GtfG, the single Gtf enzyme of S. gordonii, in biofilm interactions with C. albicans. Design: Biofilms were grown under salivary flow in flow cells in vitro, or under static conditions in 96 well plates. A panel of isogenic S. gordonii CH1 gtfG mutants and complemented strains were co-inoculated with C. albicans strain SC5314 to form mixed biofilms. Biofilm accretion and binding interactions between the two organisms were tested. Biofilms were quantified using confocal microscopy or the crystal violet assay. Results: The presence of GtfG enhanced dual biofilm accretion, and sucrose supplementation further augmented dual biofilm formation, pointing to a role of newly synthesized glucans. GtfG also promoted binding to C. albicans preformed biofilms. Soluble α-1,6-glucans played a role in these interactions since: 1 a strain producing only soluble glucans (CH107 formed robust dual biofilms under conditions of salivary flow; and 2 the dual biofilm was susceptible to enzymatic breakdown by dextranase which specifically degrades soluble α-1,6-glucans. Conclusion: Our work identified a novel molecular mechanism for C. albicans and S. gordonii biofilm interactions, mediated by GtfG. This protein promotes early biofilm binding of S. gordonii to C. albicans which leads to increased accretion of streptococcal cells in mixed biofilms. We also showed that soluble glucans, with α-1,6-linkages, promoted inter-generic adhesive interactions.
Full Text Available Periodic monitoring of Staphylococcus aureus characteristics in a locality is imperative as their drug-resistant variants cause treatment problem. In this study, antibiograms, prevalence of toxin genes (sea-see, seg-ser, seu, tsst-1, eta, etb, and etd, PFGE types, accessory gene regulator (agr groups, and ability to form biofilm of 92 S. aureus Thailand clinical isolates were investigated. They were classified into 10 drug groups: groups 1–7 (56 isolates were methicillin resistant (MRSA and 8–10 (36 isolates were methicillin sensitive (MSSA. One isolate did not have any toxin gene, 4 isolates carried one toxin gene (seq, and 87 isolates had two or more toxin genes. No isolate had see, etb, or tsst-1; six isolates had eta or etd. Combined seg-sei-sem-sen-seo of the highly prevalent egc locus was 26.1%. The seb, sec, sel, seu, and eta associated significantly with MSSA; sek was more in MRSA. The sek-seq association was 52.17% while combined sed-sej was not found. Twenty-three PFGE types were revealed, no association of toxin genes with PFGE types. All four agr groups were present; agr group 1 was predominant (58.70% but agr group 2 strains carried more toxin genes and were more frequent toxin producers. Biofilm formation was found in 72.83% of the isolates but there was no association with antibiograms. This study provides insight information on molecular and phenotypic markers of Thailand S. aureus clinical isolates which should be useful for future active surveillance that aimed to control a spread of existing antimicrobial resistant bacteria and early recognition of a newly emerged variant.
Angela V. Holguín
Full Text Available Pseudomonas aeruginosa is one of the Multi-Drug-Resistant organisms most frequently isolated worldwide and, because of a shortage of new antibiotics, bacteriophages are considered an alternative for its treatment. Previously, P. aeruginosa phages were isolated and best candidates were chosen based on their ability to form clear plaques and their host range. This work aimed to characterize one of those phages, ΦPan70, preliminarily identified as a good candidate for phage-therapy. We performed infection curves, biofilm removal assays, transmission-electron-microscopy, pulsed-field-gel-electrophoresis, and studied the in vivo ΦPan70 biological activity in the burned mouse model. ΦPan70 was classified as a member of the Myoviridae family and, in both planktonic cells and biofilms, was responsible for a significant reduction in the bacterial population. The burned mouse model showed an animal survival between 80% and 100%, significantly different from the control animals (0%. However, analysis of the ΦPan70 genome revealed that it was 64% identical to F10, a temperate P. aeruginosa phage. Gene annotation indicated ΦPan70 as a new, but possible temperate phage, therefore not ideal for phage-therapy. Based on this, we recommend genome sequence analysis as an early step to select candidate phages for potential application in phage-therapy, before entering into a more intensive characterization.
Full Text Available The lack of predictable in vitro methods to analyze antimicrobial activity could play a role in the development of resistance to antibiotics. Current used methods analyze planktonic cells but for the method to be clinically relevant, biofilm in in vivo like conditions ought to be studied. Hence, our group has developed a qualitative and quantitative method with in vivo like 3D tissue for prediction of antimicrobial activity in reality. Devices (wound dressings were applied on top of Pseudomonas aeruginosa inoculated Muller-Hinton (MH agar or 3D synthetic soft tissues (SST and incubated for 24 hours. The antibacterial activity was then analyzed visually and by viable counts. On MH agar two out of three silver containing devices showed zone of inhibitions (ZOI and on SST, ZOI were detected for all three. Corroborating results were found upon evaluating the bacterial load in SST and shown to be silver concentration dependent. In conclusion, a novel method was developed combining visual rapid screening and quantitative evaluation of the antimicrobial activity in both tissue and devices. It uses tissue allowing biofilm formation thus mimicking reality closely. These conditions are essential in order to predict antimicrobial activity of medical devices in the task to prevent device related infections.
Menzner, Ann-Katrin; Abolpour Mofrad, Sepideh; Friedrich, Oliver; Gilbert, Daniel F
Human pluripotent embryonal carcinoma (NT2) cells are increasingly considered as a suitable model for in vitro toxicity testing, e.g. developmental toxicity and neurotoxicity (DT/DNT) studies, as they undergo neuronal differentiation upon stimulation with retinoic acid (RA) and permit toxicity testing at different stages of maturation. NT2 cells have recently been reported to show specific changes in dielectric resistance profiles during differentiation which can be observed as early as 24h upon RA-stimulation. These observations suggest altered susceptibility to chemicals at an early stage of differentiation. However, chemical susceptibility of early differentiating NT cells has not yet been studied. To address this question, we have established a cell fitness screening assay based on the analysis of intracellular ATP levels and we applied the assay in a large-scale drug screening experiment in NT2 stem cells and early differentiating NT2 cells. Subsequent analysis of ranked fitness phenotypes revealed 19 chemicals with differential toxicity profile in early differentiating NT2 cells. To evaluate whether any of the identified drugs have previously been associated with DT/DNT, we conducted a literature search on the identified molecules and quantified the fraction of chemicals assigned to the FDA (Food and Drug Administration) pregnancy risk categories (PRC) N, A, B, C, D, and X in the hit list and the small molecule library. While the fractions of the categories N and B were decreased (0.81 and 0.35-fold), the classes C, D and X were increased (1.35, 1.47 and 3.27-fold) in the hit list compared to the chemical library. From these data as well as from the literature review, identifying large fractions of chemicals being directly (∼42%) and indirectly associated with DT/DNT (∼32%), we conclude that our method may be beneficial to systematic in vitro-based primary screening for developmental toxicants and neurotoxicants and we propose cell fitness screening in
Manavathu, Elias K.; Vager, Dora L; Vazquez, Jose A
Background Mixed microbial infections of the respiratory tracts with P. aeruginosa and A. fumigatus capable of producing biofilms are commonly found in cystic fibrosis patients. 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. Simultaneous static cocultures of P. aeruginosa and sporelings were used for the d...
Qi, Lihua; Li, Hao; Zhang, Chuanfu; Liang, Beibei; Li, Jie; Wang, Ligui; Du, Xinying; Liu, Xuelin; Qiu, Shaofu; Song, Hongbin
In this study, we aimed to examine the relationships between antibiotic resistance, biofilm formation, and biofilm-specific resistance in clinical isolates of Acinetobacter baumannii. The tested 272 isolates were collected from several hospitals in China during 2010-2013. Biofilm-forming capacities were evaluated using the crystal violet staining method. Antibiotic resistance/susceptibility profiles to 21 antibiotics were assessed using VITEK 2 system, broth microdilution method or the Kirby-Bauer disc diffusion method. The minimum inhibitory concentration (MIC) and minimum biofilm eradication concentration (MBEC) to cefotaxime, imipenem, and ciprofloxacin were evaluated using micro dilution assays. Genetic relatedness of the isolates was also analyzed by pulsed-field gel electrophoresis (PFGE) and plasmid profile. Among all the 272 isolates, 31 were multidrug-resistant (MDR), and 166 were extensively drug-resistant (XDR). PFGE typing revealed 167 pattern types and 103 clusters with a similarity of 80%. MDR and XDR isolates built up the main prevalent genotypes. Most of the non-MDR isolates were distributed in a scattered pattern. Additionally, 249 isolates exhibited biofilm formation, among which 63 were stronger biofilm formers than type strain ATCC19606. Population that exhibited more robust biofilm formation likely contained larger proportion of non-MDR isolates. Isolates with higher level of resistance tended to form weaker biofilms. The MBECs for cefotaxime, imipenem, and ciprofloxacin showed a positive correlation with corresponding MICs, while the enhancement in resistance occurred independent of the quantity of biofilm biomass produced. Results from this study imply that biofilm acts as a mechanism for bacteria to get a better survival, especially in isolates with resistance level not high enough. Moreover, even though biofilms formed by isolates with high level of resistance are always weak, they could still provide similar level of protection for the
Cotter, John J.; O'Gara, James P.; CASEY, Eoin
Biofilm-related research using 96-well microtiter plates involves static incubation of plates indiscriminate of environmental conditions, making oxygen availability an important variable which has not been considered to date. By directly measuring dissolved oxygen concentration over time we report here that dissolved oxygen is rapidly consumed in Staphylococcus epidermidis biofilm cultures grown in 96-well plates irrespective of the oxygen concentration in the gaseous environment in which the...
Full Text Available Bacterial vaginosis (BV, a condition defined by increased vaginal discharge without significant inflammation, is characterized by a change in the bacterial composition of the vagina. Lactobacillus spp., associated with a healthy vaginal microbiome, are outnumbered by BV-associated organisms. These bacteria could form a polymicrobial biofilm which allows them to persist in spite of antibiotic treatment. In this study, we examined the presence of Gardnerella vaginalis and Atopobium vaginae in vaginal biofilms using Peptide Nucleic Acid (PNA probes targeting these bacteria. For this purpose, we developed three new PNA probes for A. vaginae. The most specific A. vaginae probe, AtoITM1, was selected and then used in an assay with two existing probes, Gard162 and BacUni-1, to evaluate multiplex FISH on clinical samples. Using quantitative polymerase chain reaction (qPCR as the gold standard, we demonstrated a sensitivity of 66.7% (95% confidence interval: 54.5% - 77.1% and a specificity of 89.4% (95% confidence interval: 76.1% - 96% of the new AtoITM1 probe. FISH enabled us to show the presence of a polymicrobial biofilm in bacterial vaginosis, in which Atopobium vaginae is part of a Gardnerella vaginalis-dominated biofilm. We showed that the presence of this biofilm is associated with high bacterial loads of A. vaginae and G. vaginalis.
Hardy, Liselotte; Jespers, Vicky; Dahchour, Nassira; Mwambarangwe, Lambert; Musengamana, Viateur; Vaneechoutte, Mario; Crucitti, Tania
Bacterial vaginosis (BV), a condition defined by increased vaginal discharge without significant inflammation, is characterized by a change in the bacterial composition of the vagina. Lactobacillus spp., associated with a healthy vaginal microbiome, are outnumbered by BV-associated organisms. These bacteria could form a polymicrobial biofilm which allows them to persist in spite of antibiotic treatment. In this study, we examined the presence of Gardnerella vaginalis and Atopobium vaginae in vaginal biofilms using Peptide Nucleic Acid (PNA) probes targeting these bacteria. For this purpose, we developed three new PNA probes for A. vaginae. The most specific A. vaginae probe, AtoITM1, was selected and then used in an assay with two existing probes, Gard162 and BacUni-1, to evaluate multiplex FISH on clinical samples. Using quantitative polymerase chain reaction (qPCR) as the gold standard, we demonstrated a sensitivity of 66.7% (95% confidence interval: 54.5% - 77.1%) and a specificity of 89.4% (95% confidence interval: 76.1% - 96%) of the new AtoITM1 probe. FISH enabled us to show the presence of a polymicrobial biofilm in bacterial vaginosis, in which Atopobium vaginae is part of a Gardnerella vaginalis-dominated biofilm. We showed that the presence of this biofilm is associated with high bacterial loads of A. vaginae and G. vaginalis. PMID:26305575
Full Text Available We have developed a high-density microarray platform consisting of nano-biofilms of Candida albicans. A robotic microarrayer was used to print yeast cells of C. albicans encapsulated in a collagen matrix at a volume as low as 50 nL onto surface-modified microscope slides. Upon incubation, the cells grow into fully formed "nano-biofilms". The morphological and architectural complexity of these biofilms were evaluated by scanning electron and confocal scanning laser microscopy. The extent of biofilm formation was determined using a microarray scanner from changes in fluorescence intensities due to FUN 1 metabolic processing. This staining technique was also adapted for antifungal susceptibility testing, which demonstrated that, similar to regular biofilms, cells within the on-chip biofilms displayed elevated levels of resistance against antifungal agents (fluconazole and amphotericin B. Thus, results from structural analyses and antifungal susceptibility testing indicated that despite miniaturization, these biofilms display the typical phenotypic properties associated with the biofilm mode of growth. In its final format, the C. albicans biofilm chip (CaBChip is composed of 768 equivalent and spatially distinct nano-biofilms on a single slide; multiple chips can be printed and processed simultaneously. Compared to current methods for the formation of microbial biofilms, namely the 96-well microtiter plate model, this fungal biofilm chip has advantages in terms of miniaturization and automation, which combine to cut reagent use and analysis time, minimize labor intensive steps, and dramatically reduce assay costs. Such a chip should accelerate the antifungal drug discovery process by enabling rapid, convenient and inexpensive screening of hundreds-to-thousands of compounds simultaneously.
Pulcrano, Giovanna; Panellis, Dimitrios; De Domenico, Giovanni; Rossano, Fabio; Catania, Maria Rosaria
The ability to form biofilm on different surfaces is typical of most Candida species. Microscopic structure and genetic aspects of fungal biofilms have been the object of many studies because of very high resistance to antimycotic agents because of the scarce permeability of the external matrix and to the alterations in cell metabolism. In our study, 31 isolates of Candida parapsilosis, isolated from bloodstream infections, were tested for their ability to produce biofilm and were found to be good producers. The susceptibility to voriconazole, assayed by colorimetrical XTT assay, revealed a very elevated minimum inhibitory concentrations for sessile cells in comparison with planktonic ones. The addition of ambroxol, a mucolytic agent, increased the susceptibility of biofilm forming cells to voriconazole. Expression of the efflux pump genes CDR and MDR was analyzed in biofilms alone or treated with ambroxol, evidencing a role of ambroxol in the expression of genes involved in azole resistance mechanisms of C. parapsilosis biofilms. In conclusion, our data seem to encourage the use of different substances in combination with classical antimycotics, with the aim of finding a solution to the increasing problem of the resistance of biofilms formed on medical devices by nonalbicans Candida species. PMID:22315984
Comparative Efficacies of Tedizolid Phosphate, Linezolid, and Vancomycin in a Murine Model of Subcutaneous Catheter-Related Biofilm Infection Due to Methicillin-Susceptible and -Resistant Staphylococcus aureus.
Bayer, Arnold S; Abdelhady, Wessam; Li, Liang; Gonzales, Rachelle; Xiong, Yan Q
Tedizolid, a novel oxazolidinone, exhibits bacteriostatic activity through inhibition of protein synthesis. The efficacies of tedizolid, linezolid, and vancomycin were compared in a murine catheter-related biofilm infection caused by methicillin-susceptible and -resistant Staphylococcus aureus (MSSA and MRSA, respectively) strains engineered for bioluminescence. We observed significantly improved efficacy in terms of decreased S. aureus densities and bioluminescent signals in the tedizolid-treated group versus the linezolid- and vancomycin-treated groups in the model of infection caused by the MSSA and MRSA strains. PMID:27297485
Cells and tissue of plants carrying the hygromycin phosphotransferase gene (HPT) are tolerant to antibiotic hygromycin and hence become the common selection gene for genetic transformation of crop plants, particularly monocots. The present study aimed at establishing a rapid method for in planta screening of hygromycin tolerant plants in transgenic breeding. At heading stage transgenic and conventional rice plants were sprayed with hygromycin solutions of different concentrations (25 ∼ 100 mg/L) and toxic symptoms were observed in the following days. It was observed that yellow-brown necrotic spots appeared in the leaves and grains of conventional rice varieties after foliar spraying of 50 mg/L hygromycin, and the more spots were occurred with the higher hygromycin solution. There were 21.1 and 19.2 spots per cm2 on flag leaves of indica varieties Jiayou 99 and C10, respectively; while 27.6% and 23.5% grains were yellow-brown in the varieties, respectively. The corresponding data for two japonica varieties Jiayou 5 and R5 were 11.8 and 10.7 for leave spots and 11.2% and 11.6% for yellow-brown grains, respectively. These results indicated that the two indica varieties are more sensitive to hygromycin than the two japonica varieties. In the treatment of hygromycin with above levels, the transgenic rice line KMD1 with the HPT gene showed no toxic symptoms on leaf or panicles. The γ-rays treated M2 population of KMD1 was sprayed with 100 mg/L hygromycin solution at heading stage and 42 plants were screened out with toxic symptoms in leaves and panicles form 120000 plants. Among the 42 hygromycin susceptible plants, 14 plants with leaves available for in vitro feeding assay were subjected to the feeding of rice striped steam borers [Chilo supperssalis (Walker)] and all showed high resistance. The above results showed that spraying of 100 mg/L hygromycin solution at heading stage would lead to visually apparent toxic symptoms in susceptibility rice plants and hence
Kirby, Amy E.; Garner, Kimberly; Levin, Bruce R.
For many bacterial infections, noninherited mechanisms of resistance are responsible for extending the term of treatment and in some cases precluding its success. Among the most important of these noninherited mechanisms of resistance is the ability of bacteria to form biofilms. There is compelling evidence that bacteria within biofilms are more refractory to antibiotics than are planktonic cells. Not so clear, however, is the extent to which this resistance can be attributed to the structure...
Muli, F.; Struthers, J. K.
Gardnerella vaginalis and Lactobacillus acidophilus have been shown to grow to high titers in a simple biofilm system. This system was used in the present investigation to compare the biofilm-eradicating concentrations (BECs) of amoxicillin, clindamycin, erythromycin, and metronidazole to standard tube MIC and minimum bactericidal concentration (MBC) results. With the lactobacillus, the BEC/tube MBC ratio was at least 16:1, while for G. vaginalis the ratio varied from 2:1 to 512:1. The simple...
Wang, Yung-Chih; Kuo, Shu-Chen; Yang, Ya-Sung; Lee, Yi-Tzu; Chiu, Chun-Hsiang; Chuang, Ming-Fen; Lin, Jung-Chung; Chang, Feng-Yee; Chen, Te-Li
Acinetobacter baumannii biofilms are difficult to eradicate. We investigated the effects of meropenem (2 mg/liter), imipenem (2 mg/liter), sulbactam (4 mg/liter), colistin (2 mg/liter), and tigecycline (2 mg/liter), alone or in combination, on biofilm-embedded carbapenem-resistant and carbapenem-susceptible A. baumannii (CRAb and CSAb, respectively) cells, as well as on the architecture of the biofilms. A. baumannii ATCC 15151 (Ab15151) and its OXA-82-overproducing transformant, along with two clinical CSAb and two clinical CRAb isolates of differing clonalities, were used. The minimal bactericidal concentrations for biofilm-embedded cells of the six tested isolates were >50-fold those of their planktonic cells. When used individually, meropenem exhibited a higher killing effect than the other four antimicrobials on biofilm-embedded CSAb cells in the colony biofilm assay. For two clinical CRAb isolates, meropenem plus sulbactam or sulbactam plus tigecycline showed >100-fold the bactericidal effect exhibited by these agents used alone after 48 h of treatment. The effect of antimicrobials on the architecture of Ab15151 biofilm emitting green fluorescence was determined by confocal laser scanning microscopy using COMSTAT software. Significant decreases in the maximum biofilm thickness were observed after exposure to meropenem and imipenem. Meropenem plus sulbactam significantly decreased the biomass and mean thickness and increased the roughness coefficient of biofilms, but sulbactam plus tigecycline only decreased the maximum and mean biofilm thickness compared to any of these agents used alone. Meropenem was active against biofilm-embedded CSAb, whereas meropenem plus sulbactam exhibited synergism against biofilm-embedded CRAb and caused significantly more damage to the biofilm architecture than did any of the agents used alone. PMID:27216052
Petrelli, Dezemona; Repetto, Antonella; D'Ercole, Stefania; Rombini, Silvia; Ripa, Sandro; Prenna, Manuela; Vitali, Luca Agostino
Several characteristics were analysed in 37 Staphylococcus aureus isolates from nosocomial catheter infections: the PFGE profile after SmaI digestion of chromosomal DNA, the ability to form a biofilm on a polystyrene surface, antibiotic susceptibility patterns (penicillin, oxacillin, erythromycin, tetracycline, clindamycin, telithromycin, gentamicin, ciprofloxacin, quinupristin/dalfopristin, rifampicin, vancomycin and linezolid), and the presence of genetic determinants of antibiotic resistance and biofilm formation. All strains but three (92 %) were able to grow on a plastic surface as a biofilm. An almost complete association was found between phenotypes and genotypic traits of antibiotic resistance, whilst PFGE profiling showed the highly polyclonal composition of the set of strains under study. Sixteen isolates (43 %) were meticillin-resistant and were subjected to staphylococcal cassette chromosome mec (SCCmec) and cassette chromosome recombinase (ccr) complex type determination by multiplex PCR. Only a subgroup of six strains belonged to the archaic clone PFGE type and bore the SCCmec/ccrAB type I structure. Among the remaining strains some presented small rearrangements of the SCCmec/ccrAB genetic locus, whilst others could barely be traced back to a known structural type. These observations suggest that, at the local level and at a particular site of infection, S. aureus may show great genetic variability and escape the general rule of expansion of the S. aureus pandemic clones. PMID:18287301
Vellend, H.; Tuttle, S. A.; Barza, M.; Weinstein, L.; Picciolo, G. L.; Chappelle, E. W.
Luciferase assay for adenosine triphosphate (ATP) was optimized for pure bacteria in broth in order to evaluate if changes in bacterial ATP content could be used as a rapid measure of antibiotic effect on microorganisms. Broth cultures of log phase bacteria were incubated at 310 K (37 C) for 2.5 hours at antimicrobial concentrations which resulted in the best discrimination between sensitive and resistant strains. Eighty-seven strains of 11 bacterial species were studied for their susceptibility to 12 commonly used antimicrobial agents: ampicillin, Penicillin G, nafcillin, carbenicillin, cephalothin, tetracycline, erythromycin, clindamycin, gentamicin, nitrofurantoin, colistin, and chloramplenicol. The major advantage of the ATP system over existing methods of rapid microbial susceptibility testing is that the assay can be made specific for bacterial ATP.
Tasse, Jason; Croisier, Delphine; Badel-Berchoux, Stéphanie; Chavanet, Pascal; Bernardi, Thierry; Provot, Christian; Laurent, Frédéric
Biofilms are complex communities of microorganisms embedded in an extracellular matrix and adherent to a surface. The development was described as a four-stage process leading to the formation of a mature biofilm which was resistant to immune system and antibiotic actions. In bone and joint infections (BJIs), the formation of biofilms is a leading cause of treatment failure. Here we study the capacity of 11 antibiotics commonly used in the treatment of BJIs to inhibit the biofilm formation on 29 clinical Staphylococcus aureus isolates by a new test called Antibiofilmogram(®) The minimal inhibitory concentration (MIC) and biofilm MIC (bMIC) were determined in vitro and showed similar values for clindamycin, fusidic acid, linezolid and rifampin. Reversely, daptomycin, fosfomycin, gentamicin and ofloxacin showed a bMIC distribution different from MIC with bMIC above breakpoint. Finally, cloxacillin, teicoplanin and vancomycin revealed an intermediate bMIC distribution with a strain-dependent pattern. A murine in vivo model of catheter-associated S. aureus infection was made and showed a significant reduction, but not total prevention, of catheter colonization with cloxacillin at bMIC, and no or limited reduction with cloxacillin at MIC. Antibiofilmogram(®) could be of great interest after surgical operations on contaminated prostheses and after bacteremia in order to prevent the colonization of the device. PMID:27316688
Full Text Available BACKGROUND: Microorganisms growing in a biofilm are associated with chronic and recurrent human infections and are highly resistant to antimicrobial agents. There are various methods to detect biofilm production like Tissue Culture Plate (TCP, Tube method (TM, Congo Red Agar method (CRA, bioluminescent assay, piezoelectric sensors, and fluorescent microscopic examination. OBJECTIVE: This study was conducted to compare three methods for the detection of biofilms. METHOD: The study was carried out at the Department of Microbiology, Army Medical College, National University of Sciences and Technology, Pakistan, from January 2010 to June 2010. A total of 110 clinical isolates were subjected to biofilm detection methods. Isolates were identified by standard microbiological procedures. Biofilm detection was tested by TCP, TM and CRA. Antibiotic susceptibility test of biofilm producing bacteria was performed by using the Kirby-Bauer disc diffusion technique according to CLSI guidelines. RESULTS: The TCP method was considered to be superior to TM and CRA. From the total of 110 clinical isolates, TCP method detected 22.7% as high, 41% moderate and 36.3% as weak or non-biofilm producers. We have observed higher antibiotic resistance in biofilm producing bacteria than non-biofilm producers. CONCLUSION: We can conclude from our study that the TCP method is a more quantitative and reliable method for the detection of biofilm forming microorganisms as compared to TM and CRA methods, and it can be recommended as a general screening method for detection of biofilm producing bacteria in laboratories.
Dordrecht: Springer, 2007 - (Gubbins, D.; Herrero-Bervera, E.), s. 931-933. (Encyclopedia of Earth sciences series). ISBN 978-1-4020-3992-8 Institutional research plan: CEZ:AV0Z30120515 Keywords : magnetic susceptibility * magnetic field * magnetization curve Subject RIV: DE - Earth Magnetism, Geodesy, Geography
Felkel, Michael; Exner, Robert; Schleucher, Regina; Lay, Helga; Autenrieth, Ingo B; Kempf, Volkhard A J; Frick, Julia-Stefanie
The incidence of tuberculosis (TB) and especially multidrug-resistant TB (MDR) continues to increase alarmingly worldwide, and reliable and fast diagnosis of MDR is essential for the adequate treatment of patients. In contrast to the standard culture methods, nucleid acid amplification tests (NAATs) provide information about presence of Mycobacterium tuberculosis complex (MTBC) DNA and a potential resistance pattern within hours. We analyzed specimens of 110 patients from Nigeria comparing culture-based drug susceptibility testing (DST) to NAAT assays detecting isoniazid (INH), rifampicin (RMP) (GenoType MTBDRplus), and ethambutol (EMB) (GenoType MTBDRsl) resistance. Compared to DST, the GenoType MTBDRplus and MTBDRsl showed a specificity of 100% (86.3-100) and a sensitivity of 86% (42.1-99.6%) for detection of INH and a specificity of 100% (86.3-100) and a sensitivity of 83% (35.9-99.6%) for detection of RMP, and a sensitivity 100% (47.8-100%) for EMB resistance. However, in two strains, the NAAT assays provided false susceptible results as the mutations causing resistance were in genomic regions not covered by the probes of the GenoType MTBDRplus assay. We show that, in combination to DST, application of the GenoType MTBDRplus and GenoType MTBDRsl assays might be a useful additional tool to allow a rapid and safe diagnosis of MDR and extensively drug-resistant (XDR) MTBC. PMID:24294494
Ebrahim Babapour; Azam Haddadi; Reza Mirnejad; Seyed-Abdolhamid Angaji; Nour Amirmozafari
Objective: To check biofilm formation by Acinetobacter baumannii (A. baumannii) clinical isolates and show their susceptibility to different antibiotics and investigate a possible link between establishment of biofilm and multidrug resistance. Methods: This study was performed on clinical samples collected from patients with nosocomial infections in three hospitals of Tehran. Samples were initially screened by culture and biochemical tests for the presence of different species of Acinetobacter. Iden-tifications were further confirmed by PCR assays. Their susceptibilities to 11 antibiotics of different classes were determined by disc diffusion method according to Clinical and Laboratory Standards Institute guidelines. The ability to produce biofilm was investigated using methods:culture on Congo red agar, microtiter plate, and test tube method. Results: From the overall clinical samples, 156 specimens were confirmed to contain A. baumannii. The bacteria were highly resistant to most antibiotics except polymyxin B. Of these isolates, 10.26% were able to produce biofilms as shown on Congo red agar. However, the percentage of bacteria with positive biofilm in test tube, standard microtiter plate, and modified microtiter plate assays were 48.72%, 66.66%, and 73.72%, respec-tively. At least 92%of the biofilm forming isolates were multidrug resistant. Conclusions: Since most of the multidrug resistant strains produce biofilm, it seems necessary to provide continuous monitoring and determination of antibiotic susceptibility of clinical A. baumannii. This would help to select the most appropriate antibiotic for treatment.
Muli, F; Struthers, J K
Gardnerella vaginalis and Lactobacillus acidophilus have been shown to grow to high titers in a simple biofilm system. This system was used in the present investigation to compare the biofilm-eradicating concentrations (BECs) of amoxicillin, clindamycin, erythromycin, and metronidazole to standard tube MIC and minimum bactericidal concentration (MBC) results. With the lactobacillus, the BEC/tube MBC ratio was at least 16:1, while for G. vaginalis the ratio varied from 2:1 to 512:1. The simple continuous-culture system used in the present investigation is ideal for investigating the BEC for bacteria involved in complex ecological situations such as bacterial vaginosis and may be useful for the identification of the most effective and selective antibiotic therapy. PMID:9624489
Al-Fattani, Mohammed A.; Douglas, L. Julia
A filter disk assay was used to investigate the penetration of antifungal agents through biofilms containing single and mixed-species biofilms containing Candida. Fluconazole permeated all single-species Candida biofilms more rapidly than flucytosine. The rates of diffusion of either drug through biofilms of three strains of Candida albicans were similar. However, the rates of drug diffusion through biofilms of C. glabrata or C. krusei were faster than those through biofilms of C. parapsilosi...
Singh, Rachna; Sahore, Simmi; Kaur, Preetinder; Rani, Alka; Ray, Pallab
Bacterial biofilms are implicated in a wide range of implant-based and chronic infections. These infections are often associated with adverse therapeutic outcomes, owing to the decreased antibiotic susceptibility of biofilms compared with their planktonic counterparts. This altered biofilm susceptibility has been attributed to multiple factors, including a reduced antibiotic penetration. Although several studies have addressed the role of penetration barrier in biofilm-associated drug resistance, it remains inconclusive. This study was done to elucidate antibiotic penetration through biofilms formed by Staphylococcus aureus, S. epidermidis, Escherichia coli and Klebsiella pneumoniae, using an agar disk diffusion assay. Penetration capacity of six antimicrobial drugs from different classes (β-lactams, aminoglycosides, tetracyclines, phenicols, fluoroquinolones and glycopeptides) through biofilms formed by standard strains and clinical isolates from catheter-related bloodstream infections (CRBSI) was elucidated by measuring their growth-inhibition zones in lawn cultures on Mueller-Hinton agar, following diffusion of an antibiotic from an overlying disk through their biofilm to the agar medium. Penetration of only select antimicrobials (vancomycin and chloramphenicol) was hindered through biofilms. There was considerable variation in biofilm-permeating capacity depending upon the genus, strain/CRBSI isolate and antibiotic tested. Furthermore, antibiotics failed to kill the biofilm cells independent of penetration, indicating that other factors contributed substantially to biofilm resistance. PMID:27402781
Yin, Liusong; Stern, Lawrence J
HLA-DM (DM) functions as a peptide editor that mediates the exchange of peptides loaded onto MHCII molecules by accelerating peptide dissociation and association kinetics. The relative DM-susceptibility of peptides bound to MHCII molecules correlates with antigen presentation and immunodominance hierarchy, and measurement of DM-susceptibility has been a key effort in this field. Current assays of DM-susceptibility, based on differential peptide dissociation rates measured for individually labeled peptides over a long time base, are difficult and cumbersome. Here, we present a novel method to measure DM-susceptibility based on peptide binding competition assays performed in the presence and absence of DM, reported as a delta-IC(50) (change in 50% inhibition concentration) value. We simulated binding competition reactions of peptides with various intrinsic and DM-catalyzed kinetic parameters and found that under a wide range of conditions the delta-IC(50) value is highly correlated with DM-susceptibility as measured in off-rate assay. We confirmed experimentally that DM-susceptibility measured by delta-IC(50) is comparable to that measured by traditional off-rate assay for peptides with known DM-susceptibility hierarchy. The major advantage of this method is that it allows simple, fast and high throughput measurement of DM-susceptibility for a large set of unlabeled peptides in studies of the mechanism of DM action and for identification of CD4+ T cell epitopes. PMID:24583195
Raimunda Sâmia Nogueira Brilhante
Full Text Available ABSTRACTThe antifungal activity of some statins against different fungal species has been reported. Thus, at the first moment, the in vitro antifungal activity of simvastatin, atorvastatin and pravastatin was tested againstCandida spp. and Cryptococcus spp. Then, in a second approach, considering that the best results were obtained for simvastatin, this drug was evaluated in combination with antifungal drugs against planktonic growth and tested against biofilms ofCandida spp. and Cryptococcus spp. Drug susceptibility testing was performed using the microdilution broth method, as described by the Clinical and Laboratory Standards Institute. The interaction between simvastatin and antifungals against planktonic cells was analyzed by calculating the fractional inhibitory concentration index. Regarding biofilm susceptibility, simvastatin was tested against growing biofilm and mature biofilm of one strain of each tested yeast species. Simvastatin showed inhibitory effect against Candida spp. andCryptococcus spp. with minimum inhibitory concentration values ranging from 15.6 to 1000 mg L-1 and from 62.5 to 1000 mg L-1, respectively. The combination of simvastatin with itraconazole and fluconazole showed synergism against Candidaspp. and Cryptococcus spp., while the combination of simvastatin with amphotericin B was synergistic only againstCryptococcus spp. Concerning the biofilm assays, simvastatin was able to inhibit both growing biofilm and mature biofilm ofCandida spp. and Cryptococcus spp. The present study showed that simvastatin inhibits planktonic cells and biofilms ofCandida and Cryptococcus species.
Bader, Chris; Jesudoss Chelladurai, Jeba; Thompson, Kylie; Hall, Cindy; Carlson, Steve A; Brewer, Matthew T
Tritrichomonas foetus is a sexually transmitted protozoan parasite that causes abortions in cattle and results in severe economic losses. In the United States, there are no safe and effective treatments for this parasite and infected animals are typically culled. In order to expedite drug discovery efforts, we investigated in vitro trophozoite killing assays amenable to high-throughput screening in 96 well plate formats. We evaluated the reduction of resorufin, incorporation of propidium iodide, and a luminescence-based ATP detection assay. Of these methods, reduction of resorufin was found to be the most reliable predictor of trophozoite concentrations. We further validated this method by conducting dose-response experiments suitable for calculation of EC50 values for two established compounds with known activity against trophozoites in vitro, namely, metronidazole and ronidazole. Our results demonstrate that the resorufin method is suitable for high-throughput screening and could be used to enhance efforts targeting new treatments for bovine trichomoniasis. PMID:27198774
R L Kanoshiki
Full Text Available Background : The incidence of Candida tropicalis less susceptible to fluconazole (FLC has been reported in many parts of the world. Objectives : The aim of this study was to examine the changes of putative virulence attributes of Candida tropicalis accompanying the development of resistance to FLC in vitro and in vivo. Materials and Methods : A FLC-resistant strain (FLC-R was obtained after sequential exposure of a clinical isolate FLC-sensitive (FLC-S to increasing concentrations of the antifungal. The course of infection by both strains was analyzed in BALB/c mice. Analyses of gene expression were performed by real-time polymerase chain reaction PCR. The cell surface hydrophobicity, adhesion and biofilm formation were also determined. Results : Development of resistance to FLC could be observed after 15 days of subculture in azole-containing medium. Overexpression of MDR1 and ERG11 genes were observed in FLC-R, and this strain exhibited enhanced virulence in mice, as assessed by the mortality rate. All mice challenged with the FLC-R died and FLC-treatment caused earlier death in mice infected with this strain. All animals challenged with FLC-S survived the experiment, regardless of FLC-treatment. Overall, FLC-R derivatives strains were significantly more hydrophobic than FLC-S strains and showed greater adherence and higher capacity to form biofilm on polystyrene surface. Conclusions : The expression of virulence factors was higher in FLC-R-C. tropicalis and it was enhanced after FLC-exposure. These data alert us to the importance of identifying microorganisms that show resistance to the antifungals to establish an appropriate management of candidiasis therapy.
Fock, Jeppe; Parmvi, Mattias; Strömberg, Mattias;
can be used to accelerate assay kinetics. We present the first study and comparison of the performance of magnetic susceptibility measurements and a newly proposed optomagnetic method. For the comparison we use the C-reactive protein (CRP) induced agglutination of identical samples of 100 nm MNPs...... laser light transmitted through the sample. The two techniques provided highly correlated results upon agglutination when they measure the decrease of the signal from the individual MNPs (turn-off detection strategy), whereas the techniques provided different results, strongly depending on the read......-out frequency, when detecting the signal due to MNP agglomerates (turn-on detection strategy). These observations are considered to be caused by differences in the volumedependence of the magnetic and optical signals from agglomerates. The highest signal from agglomerates was found in the optomagnetic signal at...
Much of the ultraviolet-B radiation (UV-B) research on plants has concentrated on vegetative plant parts, and only a small fraction has dealt with the reproductive system. The present study analyzed pollen grains of 34 taxa germinated and grown under two levels of UV-B radiation (187 and 460 mW/m2) or no UV-B (control group). Visible radiation at 260 micromoles m-2s-1 was present in all treatments. Taxa included those with binucleate and trinucleate pollen types. We detected differences among species. A significant reduction in pollen germination occurred in only five species. Pollen tubes of 50% of the species showed significant reduction in length. Trinucleate pollen types were more likely to exhibit tube length reduction than the binucleate types. Proportionately more monocotyledonous species were sensitive to UV-B treatment than dicotyledonous species, and proportionately more wild species were sensitive than cultivated species and pollen collected from plants growing in the field were somewhat more sensitive than pollen collected from plants grown in the greenhouse. Species in which pollination occurred earlier in the season were more likely to be susceptible to UV-B radiation than those for which anthesis took place later in the season, suggesting a possible adaptation to UV-B radiation. (author)
Full Text Available The most common method for detection of drug resistant (DR TB in resource-limited settings (RLSs is indirect susceptibility testing on Lowenstein-Jensen medium (LJ which is very time consuming with results available only after 2-3 months. Effective therapy of DR TB is therefore markedly delayed and patients can transmit resistant strains. Rapid and accurate tests suitable for RLSs in the diagnosis of DR TB are thus highly needed. In this study we compared two direct techniques--Nitrate Reductase Assay (NRA and Microscopic Observation Drug Susceptibility (MODS for rapid detection of MDR-TB in a high burden RLS. The sensitivity, specificity, and proportion of interpretable results were studied. Smear positive sputum was collected from 245 consecutive re-treatment TB patients attending a TB clinic in Kampala, Uganda. Samples were processed at the national reference laboratory and tested for susceptibility to rifampicin and isoniazid with direct NRA, direct MODS and the indirect LJ proportion method as reference. A total of 229 specimens were confirmed as M. tuberculosis, of these interpretable results were obtained in 217 (95% with either the NRA or MODS. Sensitivity, specificity and kappa agreement for MDR-TB diagnosis was 97%, 98% and 0.93 with the NRA; and 87%, 95% and 0.78 with the MODS, respectively. The median time to results was 10, 7 and 64 days with NRA, MODS and the reference technique, respectively. The cost of laboratory supplies per sample was low, around 5 USD, for the rapid tests. The direct NRA and MODS offered rapid detection of resistance almost eight weeks earlier than with the reference method. In the study settings, the direct NRA was highly sensitive and specific. We consider it to have a strong potential for timely detection of MDR-TB in RLS.
Dang Thi Minh Ha
Full Text Available MODS is a novel liquid culture based technique that has been shown to be effective and rapid for early diagnosis of tuberculosis (TB. We evaluated the MODS assay for diagnosis of TB in children in Viet Nam. 217 consecutive samples including sputum (n = 132, gastric fluid (n = 50, CSF (n = 32 and pleural fluid (n = 3 collected from 96 children with suspected TB, were tested by smear, MODS and MGIT. When test results were aggregated by patient, the sensitivity and specificity of smear, MGIT and MODS against "clinical diagnosis" (confirmed and probable groups as the gold standard were 28.2% and 100%, 42.3% and 100%, 39.7% and 94.4%, respectively. The sensitivity of MGIT and MODS was not significantly different in this analysis (P = 0.5, but MGIT was more sensitive than MODS when analysed on the sample level using a marginal model (P = 0.03. The median time to detection of MODS and MGIT were 8 days and 13 days, respectively, and the time to detection was significantly shorter for MODS in samples where both tests were positive (P<0.001. An analysis of time-dependent sensitivity showed that the detection rates were significantly higher for MODS than for MGIT by day 7 or day 14 (P<0.001 and P = 0.04, respectively. MODS is a rapid and sensitive alternative method for the isolation of M.tuberculosis from children.
Ha, Dang Thi Minh; Lan, Nguyen Thi Ngoc; Wolbers, Marcel; Duong, Tran Ngoc; Quang, Nguyen Dang; Thi Van Thinh, Tran; Thi Hong Ngoc, Le; Thi Ngoc Anh, Nguyen; Van Quyet, Tran; Thi Bich Tuyen, Nguyen; Thi Ha, Vo; Day, Jeremy; Thi Thanh Hang, Hoang; Kiet, Vo Sy; Thi Nho, Nguyen; Hoa, Dai Viet; Dung, Nguyen Huy; Huu Lan, Nguyen; Farrar, Jeremy; Caws, Maxine
MODS is a novel liquid culture based technique that has been shown to be effective and rapid for early diagnosis of tuberculosis (TB). We evaluated the MODS assay for diagnosis of TB in children in Viet Nam. 217 consecutive samples including sputum (n = 132), gastric fluid (n = 50), CSF (n = 32) and pleural fluid (n = 3) collected from 96 children with suspected TB, were tested by smear, MODS and MGIT. When test results were aggregated by patient, the sensitivity and specificity of smear, MGIT and MODS against “clinical diagnosis” (confirmed and probable groups) as the gold standard were 28.2% and 100%, 42.3% and 100%, 39.7% and 94.4%, respectively. The sensitivity of MGIT and MODS was not significantly different in this analysis (P = 0.5), but MGIT was more sensitive than MODS when analysed on the sample level using a marginal model (P = 0.03). The median time to detection of MODS and MGIT were 8 days and 13 days, respectively, and the time to detection was significantly shorter for MODS in samples where both tests were positive (P<0.001). An analysis of time-dependent sensitivity showed that the detection rates were significantly higher for MODS than for MGIT by day 7 or day 14 (P<0.001 and P = 0.04), respectively. MODS is a rapid and sensitive alternative method for the isolation of M.tuberculosis from children. PMID:20020056
Decousser, Jean-Winoc; Methlouthi, Imen; Pina, Patrick; Collignon, Anne; Allouch, Pierre
A real-time PCR assay with locked nucleic acid probes was developed to screen mutations at codons 79 and 83 of the Streptococcus pneumoniae parC gene. Only silent mutations were detected among 236 French invasive fluoroquinolone-susceptible strains. This test could be useful for some high-risk patients or in national surveys.
Decousser, Jean-Winoc; Methlouthi, Imen; Pina, Patrick; Collignon, Anne; Allouch, Pierre
A real-time PCR assay with locked nucleic acid probes was developed to screen mutations at codons 79 and 83 of the Streptococcus pneumoniae parC gene. Only silent mutations were detected among 236 French invasive fluoroquinolone-susceptible strains. This test could be useful for some high-risk patients or in national surveys. PMID:16569894
Full Text Available Abstract Background Apparent emerging artemisinin-resistant Plasmodium falciparum malaria in Southeast Asia requires development of practical tools to monitor for resistant parasites. Although in vitro anti-malarial susceptibility tests are widely used, uncertainties remain regarding interpretation of P. falciparum field isolate values. Methods Performance parameters of the W2 P. falciparum clone (considered artemisinin “sensitive” were evaluated as a reference for the HRP-2 immediate ex vivo assay. Variability in W2 IC50s was assessed, including intra- and inter-assay variability among and between technicians in multiple experiments, over five freeze-thaw cycles, over five months of continuous culture, and before and after transport of drug-coated plates to remote field sites. Nominal drug plate concentrations of artesunate (AS and dihydroartemisinin (DHA were verified by LC-MS analysis. Plasmodium falciparum field isolate IC50s for DHA from subjects in an artemisinin-resistant area in Cambodia were compared with W2 susceptibility. Results Plate drug concentrations and day-to-day technical assay performance among technicians were important sources of variability for W2 IC50s within and between assays. Freeze-thaw cycles, long-term continuous culture, and transport to and from remote sites had less influence. Despite variability in W2 susceptibility, the median IC50s for DHA for Cambodian field isolates were higher (p Conclusion The W2 reference clone improved the interpretability of field isolate susceptibility from the immediate ex vivo HRP-2 assay from areas of artemisinin resistance. Methods to increase the reproducibility of plate coating may improve overall assay interpretability and utility.
Yang, Liang; Liu, Yang; Wu, Hong;
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....
Shinde Ravikumar B
Full Text Available Abstract Background Biofilms formed by Candida albicans are resistant towards most of the available antifungal drugs. Therefore, infections associated with Candida biofilms are considered as a threat to immunocompromised patients. Combinatorial drug therapy may be a good strategy to combat C. albicans biofilms. Methods Combinations of five antifungal drugs- fluconazole (FLC, voriconazole (VOR, caspofungin (CSP, amphotericin B (AmB and nystatin (NYT with cyclosporine A (CSA were tested in vitro against planktonic and biofilm growth of C. albicans. Standard broth micro dilution method was used to study planktonic growth, while biofilms were studied in an in vitro biofilm model. A chequerboard format was used to determine fractional inhibitory concentration indices (FICI of combination effects. Biofilm growth was analyzed using XTT-metabolic assay. Results MICs of various antifungal drugs for planktonic growth of C. albicans were lowered in combination with CSA by 2 to 16 fold. Activity against biofilm development with FIC indices of 0.26, 0.28, 0.31 and 0.25 indicated synergistic interactions between FLC-CSA, VOR-CSA, CSP-CSA and AmB-CSA, respectively. Increase in efficacy of the drugs FLC, VOR and CSP against mature biofilms after addition of 62.5 μg/ml of CSA was evident with FIC indices 0.06, 0.14 and 0.37, respectively. Conclusions The combinations with CSA resulted in increased susceptibility of biofilms to antifungal drugs. Combination of antifungal drugs with CSA would be an effective prophylactic and therapeutic strategy against biofilm associated C. albicans infections.
Full Text Available ABSTRACT BACKGROUND: Microorganisms growing in multilayered cell cluste rs embedded in a matrix of extracellular polysaccharide (slime which facilitat es the adherence of these microorganisms to biomedical surfaces and protect them from host immun e system and antimicrobial therapy. There are various methods to detect biofilm producti on like Tissue Culture Plate (TCP ,Tube method (TM ,Modified Congo Red Agar Method (MCRA, bio luminescent assay ,piezoelectric sensors and fluorescent microscopic examination. OBJECTIVES : This study was conducted to compare three methods f or the detection of biofilms and compare with antibiotic sensitivity pat tern, in uropathogenic Escherichia coli. METHOD: This study was carried out at the Department of Microbiology Dr. B. R. Ambedkar Medical College from Dec 2011 to June 2012. Total n umber of 107 clinical Escherichia coli isolates were randomly selected from all age groups were subjected to biofilm detection methods and their antibiotic resistance pattern w as compared. Isolates were identified by standard phenotypic methods. Biofilm detection was te sted by TCP, TM and MCRA methods . Antibiotic susceptibility test of uropathogenic E co li was performed using Kirby –Bauer disc diffusion method according to CLSI guidelines. RESULTS: From the total of 107 clinical isolate 74 (69.1 % isolates showed biofilm formation by all the TCP, TM, CRP methods. Biofilm forming i solates from catheter associated UTI showed drug resistance to more than 6 drugs. Only 2(13.3% isolates from Asymptomatic UTI showed biofilm by TM & MCRA methods & were sensitive all d rugs. Biofilm forming isolates from symptomatic UTI showed mixed drug resistance pattern. CONCLUSION: We conclude from our study that biofilm formation is more common in catheterized patients. TCP method is more quantitati ve and reliable method for the detection of biofilm forming micro-organisms as compared to TM a nd MCRA methods. So TCP method can be recommended
Castro, J.; Alves, P.; Sousa, Cármen; Cereija, Tatiana Barros Reis; Cerca, Nuno
Bacterial vaginosis (BV) is one of the most common gynaecological disorder affecting women in the reproductive age. Microbiological analysis of BV has shown Gardnerella vaginalis to be the most frequent organism in BV. However, G. vaginalis colonization do not always lead to BV. This raised the question whether there are pathogenic and commensal lineages within this species. In an effort to understand the differences between G. vaginalis strains, we performed in vitro assays to compare virule...
Loo, C. Y.; Corliss, D. A.; Ganeshkumar, N.
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...
Moore, David A. J.; Mendoza, Daniel; Gilman, Robert H.; Evans, Carlton A. W.; Delgado, María-Graciela Hollm; Guerra, Jose; Caviedes, Luz; Vargas, Daniel; Ticona, Eduardo; Ortiz, Jaime; Soto, Giselle; Serpa, Jose
There is an urgent need for new tools to improve our ability to diagnose tuberculosis (TB) and multidrug-resistant TB (MDR-TB) in resource-poor settings. In a retrospective analysis undertaken in a region with a high incidence of TB, we evaluated the performance of the microscopic observation drug susceptibility assay (MODS), a novel assay developed in Perú which uses an inverted light microscope and culture in Middlebrook 7H9 broth to detect mycobacterial growth. MODS detected 94.0% of 1,908...
Quishida, Cristiane Campos Costa; De Oliveira Mima, Ewerton Garcia; Jorge, Janaina Habib; Vergani, Carlos Eduardo; Bagnato, Vanderlei Salvador; Pavarina, Ana Cláudia
This study evaluated the potential of curcumin-mediated antimicrobial photodynamic inactivation (API) on multispecies biofilms of Candida albicans, Candida glabrata, and Streptococcus mutans of different ages. Acrylic samples (n = 480) were made with standardized rough surfaces and incubated with bacteria and yeast for 24 or 48 h. API was performed with curcumin (80, 100, 120 μM) and LED light. Additional acrylic samples were treated with curcumin or LED light only. Positive control samples received neither light nor curcumin. After API, colony counts were quantified (CFU/mL), cell metabolism was determined by means of XTT assay, and the total biofilm biomass was evaluated using Crystal Violet (CV) staining assay and images were obtained by confocal laser scanning microscopy (CLSM). The data were analyzed by nonparametric two-way ANOVA and post hoc Tukey tests (α < 0.05). For 24-h biofilm, API resulted in statistically significant difference (ρ < 0.001) of viability of C. albicans compared with control (P-L-) for all Cur concentrations. For 48-h biofilm, API resulted in statistically significant difference (ρ < 0.001) compared with control only when Cur at 120 μM was used. API promoted statistically significant difference (ρ ≤ 0.001) in the viability of S. mutans and C. glabrata for all Cur concentrations in the two biofilm ages. In addition, API produced a statistically significant difference (ρ < 0.001) of metabolic activity and of total biomass (ρ < 0.001) of multispecies biofilms compared with control for all Cur concentrations. It can be concluded that both 24- and 48-h biofilms were susceptible to API mediated by Cur; however, 24-h biofilm was more sensitive than the 48-h biofilm. PMID:27126412
Redelman, Carly V.; Marrs, Kathleen; Anderson, Gregory G.
In nature, bacteria exist in and adapt to different environments by forming microbial communities called "biofilms." We propose simple, inquiry-based laboratory exercises utilizing a biofilm formation assay, which allows controlled biofilm growth. Students will be able to qualitatively assess biofilm growth via staining. Recently, we developed a…
OBJECTIVES: Decreased susceptibility to fluoroquinolones has become a major problem for the successful therapy of human infections caused by Salmonella enterica, especially the life-threatening typhoid and paratyphoid fevers. METHODS: By using Luminex xTAG beads, we developed a rapid, reliable and cost-effective multiplexed genotyping assay for simultaneously detecting 11 mutations in gyrA, gyrB and parE of S. enterica serovars Typhi and Paratyphi A that result in nalidixic acid resistance (Nal(R)) and\\/or decreased susceptibility to fluoroquinolones. RESULTS: This assay yielded unambiguous single nucleotide polymorphism calls on extracted DNA from 292 isolates of Salmonella Typhi (Nal(R) = 223 and Nal(S) = 69) and 106 isolates of Salmonella Paratyphi A (Nal(R) = 24 and Nal(S) = 82). All of the 247 Nal(R) Salmonella Typhi and Salmonella Paratyphi A isolates were found to harbour at least one of the target mutations, with GyrA Phe-83 as the most common one (143\\/223 for Salmonella Typhi and 18\\/24 for Salmonella Paratyphi A). We also identified three GyrB mutations in eight Nal(S) Salmonella Typhi isolates (six for GyrB Phe-464, one for GyrB Leu-465 and one for GyrB Asp-466), and mutations GyrB Phe-464 and GyrB Asp-466 seem to be related to the decreased ciprofloxacin susceptibility phenotype in Salmonella Typhi. This assay can also be used directly on boiled single colonies. CONCLUSIONS: The assay presented here would be useful for clinical and reference laboratories to rapidly screen quinolone-resistant isolates of Salmonella Typhi and Salmonella Paratyphi A, and decipher the underlying genetic changes for epidemiological purposes.
Zou, Yunyun; Lee, Yoon; Huh, Jinyoung
Objectives This study was designed to evaluate the synergistic antibacterial effect of xylitol and ursolic acid (UA) against oral biofilms in vitro. Materials and Methods S. mutans UA 159 (wild type), S. mutans KCOM 1207, KCOM 1128 and S. sobrinus ATCC 33478 were used. The susceptibility of S. mutans to UA and xylitol was evaluated using a broth microdilution method. Based on the results, combined susceptibility was evaluated using optimal inhibitory combinations (OIC), optimal bactericidal combinations (OBC), and fractional inhibitory concentrations (FIC). The anti-biofilm activity of xylitol and UA on Streptococcus spp. was evaluated by growing cells in 24-well polystyrene microtiter plates for the biofilm assay. Significant mean differences among experimental groups were determined by Fisher's Least Significant Difference (p < 0.05). Results The synergistic interactions between xylitol and UA were observed against all tested strains, showing the FICs < 1. The combined treatment of xylitol and UA inhibited the biofilm formation significantly and also prevented pH decline to critical value of 5.5 effectively. The biofilm disassembly was substantially influenced by different age of biofilm when exposed to the combined treatment of xylitol and UA. Comparing to the single strain, relatively higher concentration of xylitol and UA was needed for inhibiting and disassembling biofilm formed by a mixed culture of S. mutans 159 and S. sobrinus 33478. Conclusions This study demonstrated that xylitol and UA, synergistic inhibitors, can be a potential agent for enhancing the antimicrobial and anti-biofilm efficacy against S. mutans and S. sobrinus in the oral environment. PMID:25383348
Isabel Antonieta Iturrieta-González
Full Text Available Invasive infections caused by Trichosporon spp. have increased considerably in recent years, especially in neutropenic and critically ill patients using catheters and antibiotics. The genus presents limited sensitivity to different antifungal agents, but triazoles are the first choice for treatment. Here, we investigated the biofilm production and antifungal susceptibility to triazoles and amphotericin B of 54 Trichosporon spp. isolates obtained from blood samples (19, urine (20 and superficial mycosis (15. All isolates and 7 reference strains were identified by sequence analysis and phylogenetic inferences of the IGS1 region of the rDNA. Biofilms were grown on 96-well plates and quantitation was performed using crystal violet staining, complemented with Scanning Electron Microscopy (SEM. Susceptibility tests for fluconazole, itraconazole, voriconazole and amphotericin B were processed using the microdilution broth method (CLSI for planktonic cells and XTT reduction assay for biofilm-forming cells. Our results showed that T. asahii was the most frequent species identified (66.7%, followed by T. faecale (11.1%, T. asteroides (9.3%, T. inkin (7.4%, T. dermatis (3.7% and one T. coremiiforme (1.8%. We identified 4 genotypes within T. asahii isolates (G1, G3, G4 and G5 and 2 genotypes within T. faecale (G1 and G3. All species exhibited high adhesion and biofilm formation capabilities, mainly T. inkin, T. asteroides and T. faecale. Microscopy images of high biofilm-producing isolates showed that T. asahii presented mainly hyphae and arthroconidia, whereas T. asteroides exhibited mainly short arthroconidia and few filaments. Voriconazole exhibited the best in vitro activity against all species tested. Biofilm-forming cells of isolates and reference strains were highly resistant to all antifungals tested. We concluded that levels of biofilm formation by Trichosporon spp. were similar or even greater than those described for the Candida genus. Biofilm
Soukos, Nikolaos S.; Lee, Shun; Doukas, Apostolos G.
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.
Fungal keratitis is commonly caused by Fusarium species, while cases of Candida-associated keratitis are less frequent. Recent outbreaks of Fusarium keratitis were associated with contact lens wear and with MoistureLoc contact lens care solution, and biofilm formation on contact lens/lens cases was...
Simões, Lúcia C; Simões, M; Vieira, M. J.
Single- and multispecies biofilms formed by six drinking water-isolated bacterial species were used to assess their susceptibilities to sodium hypochlorite (SHC). In general, multispecies biofilms were more resistant to inactivation and removal than single biofilms. Total biofilm inactivation was achieved only for Acinetobacter calcoaceticus single-species biofilms and for those multispecies biofilms without A. calcoaceticus. Biofilms with all bacteria had the highest resistance t...
Increased tolerance to antimicrobial agents is thought to be an important feature of microbes growing in biofilms. We study the dynamics of antibiotic action within hydrodynamic flow chamber biofilms of Escherichia coli and Pseudomonas aeruginosa using isogenic mutants and fluorescent gene...... expression reporters and we address the question of how biofilm organization affects antibiotic susceptibility. The dynamics of microbial killing is monitored by viable count determination, and confocal laser microscopy. Our work shows that the apparent increased antibiotic tolerance is due to the formation...... of antibiotic tolerant subpopulations within the biofilm. The formation of these subpopulations is highly variable and dependent on the antibiotic used, the biofilm structural organization and the induction of specific tolerance mechanisms....
Osman, Kamelia M; Amer, Aziza M; Badr, Jihan M; Helmy, Nashwa M; Elhelw, Rehab A; Orabi, Ahmed; Bakry, Magdy; Saad, Aalaa S A
Methicillin-resistant Staphylococcus aureus (MRSA) have been found in various farm animal species throughout the world. Yet, methicillin-susceptible S. aureus (MSSA), methicillin-susceptible non-S. aureus (MS-NSA), and methicillin-resistant non-S. aureus (MR-NSA) were not investigated. Therefore, we persued to determine the diversity in their phenotypic virulence assay, phenotypic antimicrobial resistance profile and molecular characterization in one of the food chains in Egypt. Samples were collected during 2013 from beef meat at retail. Twenty seven isolates comprising five species (S. hyicus, S. aureus, S. schleiferi subsp. coagulans, S. intermedius, and S. lentus) were characterized for their antibiotic resistance phenotypic profile and antibiotic resistance genes (mecA, cfr, gyrA, gyrB, and grlA). Out of the 27 Staphylococcus isolates only one isolate was resistant to the 12 antibiotics representing nine classes. Raw beef meat sold across the Great Cairo zone, contains 66.7% of MRS, with highest prevalence was reported in S. aureus (66.7%), while the MRS non-S. aureus strains constituted 66.7% from which S. hyicus (60%), S. intermedius (33.3%), S. schleiferi subsp. coagulans (100%), and S. lentus (100%) were MRS. Seven S. aureus, six S. hyicus, four S. schleiferi subsp. coagulans, three S. intermedius, and one S. lentus isolates although being resistant to oxacillin yet, 11/27 (40.7%) carried the mecA gene. At the same time, the cfr gene was present in 2 of the nine S. aureus isolates, and totally undetectable in S. hyicus, S. schleiferi subsp. coagulans, S. intermedius, and S. lentus. Although, global researches largely focused into MRSA and MR-NSA in animals on pigs, the analysis of our results stipulates, that buffaloes and cattle could be MRSA dispersers and that this theme is not specific to pigs. Detection of MSSA virulence determinants is a must, as although oxacillin resistance may be absent yet, the MSSA may carry the virulence determinants which
Full Text Available Background: The converging epidemics of HIV and tuberculosis (TB pose one of the greatest public health challenges of our time. Rapid diagnosis of TB is essential in view of its infectious nature, high burden of cases, and emergence of drug resistance. Objective: The purpose of this present study was to evaluate the feasibility of implementing the microscopic observation drug susceptibility (MODS assay, a novel assay for the diagnosis of TB and multi-drug-resistant tuberculosis (MDR-TB directly from sputum specimens, in the Indian setting. Materials and Methods: This study involved a cross-sectional, blinded assessment of the MODS assay on 1036 suspected cases of pulmonary TB in HIV-positive and HIV-negative patients against the radiometric method, BD-BACTEC TB 460 system. Results: Overall, the sensitivity, specificity, positive predictive value, and negative predictive value of the MODS assay in detecting MTB among TB suspected patients were 89.1%, 99.1%, 94.2%, 95.8%, respectively. In addition, in the diagnosis of drug-resistant TB, the MODS assay was 84.2% sensitive for those specimens reporting MDR, 87% sensitivity for those specimens reporting INH mono-resistance, and 100% sensitive for specimens reporting RIF mono-resistance. The median time to detection of TB in the MODS assay versus BACTEC was 9 versus 21 days (P < 0.001. Conclusion: Costing 5 to 10 times lesser than the automated culture methods, the MODS assay has the potential clinical utility as a simple and rapid method. It could be effectively used as an alternative method for diagnosing TB and detection of MDR-TB in a timely and affordable way in resource-limited settings.
Disk with High Oxacillin Content Discriminates between Methicillin-Resistant and Borderline Methicillin-Susceptible Staphylococcus aureus Strains in Disk Diffusion Assays Using a Low Salt Concentration
Petersson, Ann Cathrine; Kamme, Carl; Miörner, Håkan
A separation between mecA+ strains of Staphylococcus aureus and strains lacking mecA was achieved by the disk diffusion assay and the agar dilution method, utilizing disks containing 5 μg of oxacillin and inocula of approximately 5 × 105 CFU/spot, respectively, provided that agar with 0 to 0.5% NaCl and incubation at 30°C were employed. The 5-μg oxacillin disks clearly discriminated between borderline methicillin-susceptible and mecA+ strains. The oxacillin MICs were more affected by the inoc...
Preparation of target cells from tissue culture lines which grow adherent to tissue culture vessels is often desirable for tests of cell-mediated cytotoxicity (CMC). In the present study the authors used cells derived from adherent tissue culture lines to compare the merits of suspension vs. adherent target cells in short-term 51Cr-release assays. Cytotoxic activity of murine spleen cells sensitized in vitro against allogeneic spleen cells or syngeneic sarcoma cells was tested with fibroblast or sarcoma target cells. In parallel tests, aliquots of tissue culture lines were detached and used as either suspension or adherent target cells in CMC assays, matching the concentrations of suspension and adherent target cells. In both allogeneic and syngeneic combinations adherent target cells released less 51Cr spontaneously and were more susceptible to CMC than their suspension counterparts. (Auth.)
Sara R Robertson
Full Text Available Surface-adherent biofilm growth is a common trait of bacteria and other microorganisms in nature. Within biofilms, organisms are present in high density and are enmeshed in an organic matrix containing polysaccharides and other molecules. The close proximity of organisms within biofilms facilitates microbial interactions and signaling, including many metabolic processes in which consortia rather than individual organisms participate. Biofilm growth also enables microorganisms to withstand chemical and biological stresses. Here, we review some current literature and document representative beneficial aspects of biofilms using examples from wastewater treatment, microbial fuel cells, biological repair (biocementation of stonework, and biofilm protection against Candida albicans infections. Finally, we address a chemical ecology strategy whereby desired microbial succession and beneficial biofilm formation can be encouraged via manipulation of culture conditions and bacterial signaling.
Montelongo-Jauregui, Daniel; Srinivasan, Anand; Ramasubramanian, Anand K.; Lopez-Ribot, Jose L.
As a member of the normal human oral microbiota, the fungus Candida albicans is often found in association with Streptococcus gordonii, a member of dental plaque forming bacteria. Evidence suggests that S. gordonii serves as a facilitator of C. albicans adherence to dental tissues, which represents a clinically relevant problem, particularly for immunocompromised individuals that could subsequently develop fungal infections. In this study we describe the development of a relatively simple and economical in vitro model that allows for the growth of mixed bacterial/fungal biofilms in 96-well microtiter plates. We have applied this method to test and compare the growth characteristics of single and dual species biofilms in traditional microbiological media versus a synthetic saliva medium (basal medium mucin, BMM) that more closely resembles physiological conditions within the oral cavity. Results indicated a synergistic effect for the formation of biofilms when both microorganisms were seeded together under all conditions tested. The structural and architectural features of the resulting biofilms were further characterized using scanning electron microscopy and confocal scanning laser microscopy. We also performed drug susceptibility assays against single and mixed species biofilms using commonly used antifungals and antibacterial antibiotics, both in monotherapy and in combination therapy, for a direct comparison of resistance against antimicrobial treatment. As expected, mixed species biofilms displayed higher levels of resistance to antimicrobial treatment at every dose tested in both traditional media and BMM synthetic saliva, as compared to single-species biofilms.
Escherichia coli is one of the most commonly used fecal indicator organisms for drinking water and groundwater systems. In order to understand various biogeochemical and biophysical factors affecting its interactions with biofilms, E. coli K12 was chosen as a model organism. A Ta...
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 as...
Bjarnsholt, Thomas; Jensen, Peter Østrup; Moser, Claus Ernst; Høiby, Niels
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......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...... 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 as...
Full Text Available Increased tolerance to antimicrobial agents is thought to be an important feature of microbes growing in biofilms. We address the question of how biofilm organization affects antibiotic susceptibility. We established Escherichia coli biofilms with differential structural organization due to the presence of IncF plasmids expressing altered forms of the transfer pili in two different biofilm model systems. The mature biofilms were subsequently treated with two antibiotics with different molecular targets, the peptide antibiotic colistin and the fluoroquinolone ciprofloxacin. The dynamics of microbial killing were monitored by viable count determination, and confocal laser microscopy. Strains forming structurally organized biofilms show an increased bacterial survival when challenged with colistin, compared to strains forming unstructured biofilms. The increased survival is due to genetically regulated tolerant subpopulation formation and not caused by a general biofilm property. No significant difference in survival was detected when the strains were challenged with ciprofloxacin. Our data show that biofilm formation confers increased colistin tolerance to cells within the biofilm structure, but the protection is conditional being dependent on the structural organization of the biofilm, and the induction of specific tolerance mechanisms.
Ahmet Yilmaz Coban
The aim of this study was to investigate the performance of a new and accurate method for the detection of isoniazid (INH) and rifampicin (RIF) resistance among Mycobacterium tuberculosis isolates using a crystal violet decolourisation assay (CVDA). Fifty-five M. tuberculosis isolates obtained from culture stocks stored at -80ºC were tested. After bacterial inoculation, the samples were incubated at 37ºC for seven days and 100 µL of CV (25 mg/L stock solution) was then added to the control an...
Hardy, Liselotte; Jespers, Vicky; Dahchour, Nassira; Mwambarangwe, Lambert; Musengamana, Viateur; Vaneechoutte, Mario; Crucitti, Tania
Bacterial vaginosis (BV), a condition defined by increased vaginal discharge without significant inflammation, is characterized by a change in the bacterial composition of the vagina. Lactobacillus spp., associated with a healthy vaginal microbiome, are outnumbered by BV-associated organisms. These bacteria could form a polymicrobial biofilm which allows them to persist in spite of antibiotic treatment. In this study, we examined the presence of Gardnerella vaginalis and Atopobium vaginae in ...
Bryers, James D.
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....
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 collectio
, 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...
Hawser, S. P.; Douglas, L J
Biofilms formed by Candida albicans on small discs of catheter material were resistant to the action of five clinically important antifungal agents as determined by [3H]leucine incorporation and tetrazolium reduction assays. Fluconazole showed the greatest activity, and amphotericin B showed the least activity against biofilm cells. These findings were confirmed by scanning electron microscopy of the biofilms.
Donné, Joke; Dewilde, Sylvia
Worldwide, infectious diseases are one of the leading causes of death among children. At least 65% of all infections are caused by the biofilm mode of bacterial growth. Bacteria colonise surfaces and grow as multicellular biofilm communities surrounded by a polymeric matrix as a common survival strategy. These sessile communities endow bacteria with high tolerance to antimicrobial agents and hence cause persistent and chronic bacterial infections, such as dental caries, periodontitis, otitis media, cystic fibrosis and pneumonia. The highly complex nature and the rapid adaptability of the biofilm population impede our understanding of the process of biofilm formation, but an important role for oxygen-binding proteins herein is clear. Much research on this bacterial lifestyle is already performed, from genome/proteome analysis to in vivo antibiotic susceptibility testing, but without significant progress in biofilm treatment or eradication. This review will present the multiple challenges of biofilm research and discuss possibilities to cross these barriers in future experimental studies. PMID:26616519
Ebrahimi, Azizollah; Moatamedi, Azar; Lotfalian, Sharareh; Mirshokraei, Pejhman
Streptococcus agalactiae is a major contagious pathogen causing bovine sub-clinical mastitis. The present investigation was carried out to determine some phenotypic characteristics of the S. agalactiae strains isolated from bovine mastitis cases in dairy cows of Shahrekord in the west-center of Iran. One hundred eighty California mastitis test (CMT) positive milk samples were bacteriologically studied. A total of 31 (17.2%) S. agalactiae isolated. Twenty eight (90.3%) of the isolates were biofilm producers. This finding may indicate the high potential of pathogenicity in isolated strains. Sixteen (51.6%) isolates were α hemolysin producers. Only 19.3%, 22.5% and 29.0% of the isolates were sensitive to streptomycin, flumequine and kanamycin, respectively. None of these three agents is recommended for treatment of mastitis cases. PMID:25568683
Full Text Available Streptococcus agalactiae is a major contagious pathogen causing bovine sub-clinical mastitis. The present investigation was carried out to determine some phenotypic characteristics of the S. agalactiae strains isolated from bovine mastitis cases in dairy cows of Shahrekord in the west-center of Iran. One hundred eighty California mastitis test (CMT positive milk samples were bacteriologically studied. A total of 31 (17.2% S. agalactiae isolated. Twenty eight (90.3% of the isolates were biofilm producers. This finding may indicate the high potential of pathogenicity in isolated strains. Sixteen (51.6% isolates were α hemolysin producers. Only 19.3%, 22.5% and 29.0% of the isolates were sensitive to streptomycin, flumequine and kanamycin, respectively. None of these three agents is recommended for treatment of mastitis cases.
Bharadwaj, Anuja; Stafford, Kirby C
An emulsifiable concentrate (EC) and granular (G) formulation of the entomopathogenic fungus, Metarhizium brunneum strain F52 (formerly Metarhizium anisopliae strain F52) were tested against unfed adults and nymphs of Ixodes scapularis Say in the laboratory. Three exposure methods; dip, surface contact, and direct spray application, and three exposure time intervals (3, 30, and 300 min) were used to evaluate the EC formulation. Application rates ranged from 2.6 x 10(2) to 2.6 x 10(8) conidia/cm2. The surface treatment was used for granular formulation with concentrations ranging from 2.3 x 10(5) to 2.3 x 10(7) conidia/cm2 for same three exposure times. Both the EC and G formulations of this fungus were highly pathogenic against I. scapularis adults and nymphs. Logistic regression analysis found formulation, spore concentration, time of exposure, and observation period were significant or highly significant factors influencing tick mortality. For adult I. scapularis, the spray application with the EC formulation of M. brunneum F52 resulted in a lower LC50 (5.9 x 10(4) conidia/cm2) at 30 min than surface exposure to the EC (LC50 = 1.3 x 10(6) conidia/cm2) or G formulation (LC50 = 8.1 x 10(5) conidia/cm2). At higher concentrations, fungal activity was evident in adult I. scapularis held at 5 degrees C suggesting the fungus may provide control in the cooler fall season. While the observed pathogenicity of a fungus against ticks can be dependent upon the bioassay assessment, we found nymphs and adults of I. scapularis to be highly susceptible to M. brunneum F52, regardless of the exposure method used. PMID:22420275
Ahmet Yilmaz Coban
Full Text Available The aim of this study was to investigate the performance of a new and accurate method for the detection of isoniazid (INH and rifampicin (RIF resistance among Mycobacterium tuberculosis isolates using a crystal violet decolourisation assay (CVDA. Fifty-five M. tuberculosis isolates obtained from culture stocks stored at -80ºC were tested. After bacterial inoculation, the samples were incubated at 37ºC for seven days and 100 µL of CV (25 mg/L stock solution was then added to the control and sample tubes. The tubes were incubated for an additional 24-48 h. CV (blue/purple was decolourised in the presence of bacterial growth; thus, if CV lost its colour in a sample containing a drug, the tested isolate was reported as resistant. The sensitivity, specificity, positive predictive value, negative predictive value and agreement for INH were 92.5%, 96.4%, 96.1%, 93.1% and 94.5%, respectively, and 88.8%, 100%, 100%, 94.8% and 96.3%, respectively, for RIF. The results were obtained within eight-nine days. This study shows that CVDA is an effective method to detect M. tuberculosis resistance to INH and RIF in developing countries. This method is rapid, simple and inexpensive. Nonetheless, further studies are necessary before routine laboratory implementation.
Maria Helena Galdino Figueiredo-Carvalho
Full Text Available This study evaluated the antifungal susceptibility profile and the production of potential virulence attributes in a clinical strain of Candida nivariensis for the first time in Brazil, as identified by sequencing the internal transcribed spacer (ITS1-5.8S-ITS2 region and D1/D2 domains of the 28S of the rDNA. For comparative purposes, tests were also performed with reference strains. All strains presented low planktonic minimal inhibitory concentrations (PMICs to amphotericin B (AMB, caspofungin (CAS, and voriconazole. However, our strain showed elevated planktonic MICs to posaconazole (POS and itraconazole, in addition to fluconazole resistance. Adherence to inert surfaces was conducted onto glass and polystyrene. The biofilm formation and antifungal susceptibility on biofilm-growing cells were evaluated by crystal violet staining and a XTT reduction assay. All fungal strains were able to bind both tested surfaces and form biofilm, with a binding preference to polystyrene (p < 0.001. AMB promoted significant reductions (≈50% in biofilm production by our C. nivariensis strain using both methodologies. This reduction was also observed for CAS and POS, but only in the XTT assay. All strains were excellent protease producers and moderate phytase producers, but lipases were not detected. This study reinforces the pathogenic potential of C. nivariensis and its possible resistance profile to the azolic drugs generally used for candidiasis management.
Perez, A.; Grabenbauer, G.G.; Sauer, R.; Distel, L.V.R. [Dept. of Radiation Oncology, Friedrich Alexander Univ. Erlangen-Nuremberg (Germany); Sprung, C.N. [Div. of Research, Peter MacCallum Cancer Centre, and Dept. of Biochemistry and Molecular Biology, Melbourne Univ., VIC (Australia)
Background and purpose: cell-cycle regulation and checkpoint activation are crucial factors for radiation-induced DNA damage processing. The G2/M phase arrest was assessed in lymphoblastoid cell lines and phytohemagglutinin-stimulated T-lymphocytes of different radiosensitivities to study the relationship of G2/M arrest to radiosensitivity. Material and methods: G2/M arrest was analyzed after in vitro irradiation by 2 and 5 Gy of ionizing radiation up to 6 days using 17 lymphoblastoid cell lines from healthy individuals, ataxia-telangiectasia (AT) patients, Nijmegen breakage syndrome (NBS) patients and cancer patients with clinically increased radiosensitivity. In a second approach, phytohemagglutinin-stimulated T-lymphocytes from 15 healthy individuals, twelve cancer patients, and five cancer patients hypersensitive to ionizing radiation were studied. Image cytometry was performed to analyze G2/M arrest. Results: two of the three AT cell lines showed markedly increased G2/M arrest compared to controls. NBS cells were comparable to controls up to day 3, but then demonstrated a slightly increased G2/M arrest. Two of the six radiosensitive lymphoblast cell lines and the five radiosensitive cancer patients' T-lymphocytes assayed showed a reduction in G2/M arrest, while healthy individuals showed no difference from cancer patients. Conclusion: the interrelation between G2/M arrest and radiosensitivity is not readily apparent since a variety of radiosensitive cells from patients with radiosensitive syndromes and patients identified as radiosensitive following radiation treatment showed inconsistent G2/M arrest dynamics. Secondary effects, like loss of clonogenicity, G1/S phase arrest and failure of G2/M arrest may contribute to variation of the G2/M arrest endpoint and obscure assessment of cellular radiosensitivity using this method. (orig.)
structured aggregation consisting of multiple species of bacteria whose function relies on a complex web of cooperative and/or competitive interactions between community members, indicating that research in “whole-entity” should not be based on the assembled results from “mono pieces”. As one of the best...... by transcriptomic analysis are also presented. Due to the poor reproducibility of most biofilm quantification assays, the first part of my work is to develop a rapid, reproducible and sensitive approach for quantitative screening of biofilm formation by bacteria when cultivated as mono- and multispecies biofilms......, followed by species specific qPCR based on SYBR Green I fluorescence to measure the relative proportion of individual species in mixed-species biofilms. The reported approach was described in Manuscript 1 which can be used as a standard procedure for evaluating interspecies interactions in defined...
Stepanović, Srdjan; Vuković, Dragana; Hola, Veronika; Di Bonaventura, Giovanni; Djukić, Slobodanka; Cirković, Ivana; Ruzicka, Filip
The details of all steps involved in the quantification of biofilm formation in microtiter plates are described. The presented protocol incorporates information on assessment of biofilm production by staphylococci, gained both by direct experience as well as by analysis of methods for assaying biofilm production. The obtained results should simplify quantification of biofilm formation in microtiter plates, and make it more reliable and comparable among different laboratories. PMID:17696944
Mancl, Kimberly A.; Kirsner, Robert S.; Ajdic, Dragana
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 relati...
Diagnostic multiplex polymerase chain reaction assay for the identification of Pseudomonas aeruginosa from the skin biopsy specimens in burn wound infections and detection of antibiotic susceptibility
Objective was to identify Pseudomonas aeruginosa (P. aeruginosa) from the skin biopsy specimens in burn wound infections by multiplex polymerase chain reaction (M-PCR) and detection of antimicrobial susceptibility of isolates from culture. We conducted the cross-sectional study in 140 patients with wound infections who admitted to referral burn center of Motahari, Tehran, Iran, during a 12-month period from 2005-2006. Skin biopsy specimens were aseptically taken from each patient, one for PCR and one for bacterial culture. A M-PCR test based on simultaneous amplification of 2 lipoprotein genes: oprI and oprL, was used to directly detect fluorescent pseudomonades and P. aeruginosa in skin biopsy specimens. The susceptibility of P. aeruginosa isolates to 16 antibiotics was determined using the disc diffusion method. Out of 140 biopsy specimens, M-PCR detected 66 (47.2%) isolates, while culture detected 57 (40.7%) isolates as P. aeruginosa. Positive results for both genes which observed only for P. aeruginosa, while only one gene, oprI, was amplified from other fluorescent pseudomonades (n=12) and all other bacterial tested (n=62) were negative by the amplification test. The most effective antibiotics against isolate of P. aeruginosa were cefepime (79%), azetreonam (76%), ticarcillin-clavulanic acid (68%), tobramycin (62%) and amikacin (61%). Multiplex PCR assay appears promising for the rapid and sensitive detection of P. aeruginosa from the burned skin biopsy specimens. Simultaneous amplification of 2 lipoprotein genes: oprI and oprL could detect P. aeruginosa and oprI gene only for other fluorescent pseudomonades. (author)
LIN Mao-hu; HE Lei; GAO Jie; LIU Yun-xi; SUO Ji-jiang; XING Yu-bin; JIA Ning
Background The catheter related infection caused by Staphylococcus epiderrnidis biofilm is increasing and difficult to treat by antimicrobial chemotherapy.The properties of biofilms that give rise to antibiotic resistance are only partially understood.This study aimed to elucidate the penetration of erythromycin through Staphylococcus epidermidis biofilm.Methods The penetration ratio of erythromycin through Staphylococcus epidermidis biofilms of 1457,1457-msrA,and wild isolate S68 was detected by biofilm penetration model at different time points according to the standard regression curve.The RNNDNA ratio and the cell density within the biofilms were observed by confocal laser microscope and transmission electromicroscope,respectively.Results The penetration ratios of erythromycin through the biofilms of 1457,1457-msrA,and S68 after cultivation for 36 hours were 0.93,0.55 and 0.4,respectively.The erythromycin penetration ratio through 1457 biofilm (0.58 after 8 hours)was higher than that through the other two (0.499 and 0.31 after 24 hours).Lower growth rate of the cells in biofilm was shown,with reduction of RNA/DNA proportion observed by confocal laser microscope through acridine orange stain.Compared with the control group observed by transmission electrmicroscope,the cell density of biofilm air face was lower than that of agar face,with more cell debris.Conclusions Erythromycin could penetrate to the Staphylococcus epidermidis biofilm,but could not kill the cells thoroughly.The lower growth rate of the cells within biofilm could help decreasing the erythromycin susceptibility.
Sharab, Lina Y.
In dental settings, as well as in other natural systems, plaque-forming microorganisms develop biofilms in which the microbes become protected via their own phenotypic changes and their polymeric exudates from disinfection by washes and antibiotics. Photodynamic Therapy (PDT) is variably effective against these microorganisms, depending on such factors as whether the bacteria are Gram positive or Gram negative, plaque age and thickness, and internal biofilm oxygen concentration. This investigation applied a novel combination of PDT and water-jet impingement techniques to Streptococcus mutans (ATCC strain 27351)-formed biofilms on commercially pure titanium (cpTi) starting with three different phases (ages) of the bacteria, to examine whether the detachment shear stress --as a signature for the work required for removal of the biofilms- would be affected by prior PDT treatment independently from microbial viability. Biofilms were grown with sucrose addition to Brain Heart Infusion media, producing visible thick films and nearly invisible thin films (within the same piece) having the same numbers of culturable microorganisms, the thicker films having greater susceptibility to detachment by water--jet impingement. Colony-forming-unit (CFU) counts routinely correlated well with results from a spectrophotometric Alamar Blue (AB) assay. Use of Methylene Blue (MB) as a photosensitizer (PS) for PDT of biofilms did not interfere with the AB assay, but did mask AB reduction spectral changes when employed with planktonic organisms. It was discovered in this work that PD-treated microbial biofilms, independently from starting or PS-influenced microorganism viability, were significantly (p<0.05) and differentially more easily delaminated and ultimately removed from their substrata biomaterials by the hydrodynamic forces of water-jet impingement. Control biofilms of varying thickness, not receiving PDT treatment, required between 144 and 228 dynes/cm2 of shear stress to
Nguyen, Uyen T; Burrows, Lori L
Current sanitation methods in the food industry are not always sufficient for prevention or dispersal of Listeria monocytogenes biofilms. Here, we determined if prevention of adherence or dispersal of existing biofilms could occur if biofilm matrix components were disrupted enzymatically. Addition of DNase during biofilm formation reduced attachment (bromelain and papain were less effective dispersants than proteinase K. In a time course assay, complete dispersal of L. monocytogenes biofilms from both polystyrene and type 304H food-grade stainless steel occurred within 5min at proteinase K concentrations above 25μg/ml. These data confirm that both DNA and proteins are required for L. monocytogenes biofilm development and maintenance, and that these components of the biofilm matrix can be targeted for effective prevention and removal of biofilms. PMID:25043896
E. S. Vorobey
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.
E. S. Vorobey; O. S. Voronkova; A. I. Vinnikov
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 molec...
齐鲁青; 汪晓军; 詹德明
利用氯化三苯基四氮唑(TTC)-脱氢酶还原法定量测定了曝气生物滤池中陶粒负载生物膜的活性,并对影响生物活性测定的因素进行了分析和优化.实验表明,经优化后得出最佳培养条件是:陶粒采用原位法制样,依次加入pH为8.0的Tris-HCl缓冲液,质量分数为0.4％的TTC溶液,0.1 mol/L葡萄糖溶液,在温度40℃下培养4h,最后生成的三苯基甲臢(TriphenylFormazone,TF)用甲苯萃取可以除去有色废水的颜色影响.采用TTC-脱氢酶还原法可以快速、方便的检测曝气生物滤池中陶粒负载生物膜的生物活性,并能很好的进行定量化测定.%The biofilms activity in Biological Aerated Filters (BAF) is quantitatively determined by 2,3,5-triphenyl tetrazolium chloride (TTC) -dehydrogenase assay and some major factors influencing the determination are studied in this paper. The optimum parameters are shown as follows: Tris-HCl buffers and 8. 0 of pH, 0. 4% ( mass fraction) of TTC and 0. 1 mol/L of dextrose solution. The incubation time and temperature are 4 h and 401 , respectively. The formazan reduction product can be efficiently extracted by methylbenzene, which can reduce the disturbance of dye color. This study demonstrates that the TTC-dehydrogenase assay can be successfully applied to the determination of biofilms activity in BAF.
Li, Jinyun; Wang, Nian
Citrus canker caused by the bacterium Xanthomonas citri subsp. citri is an economically important disease of citrus worldwide. Biofilm formation plays an important role in early infection of X. citri subsp. citri on host leaves. In this study, we assessed the hypothesis that small molecules inhibiting biofilm formation reduce X. citri subsp. citri infection and enhance the control of citrus canker disease. D-leucine and 3-indolylacetonitrile (IAN) were found to prevent biofilm formation by X. citri subsp. citri on different abiotic surfaces and host leaves at a concentration lower than the minimum inhibitory concentration (MIC). Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis indicated that IAN repressed expression of chemotaxis/motility-related genes in X. citri subsp. citri. In laboratory experiments, planktonic and biofilm cells of X. citri subsp. citri treated with D-leucine and IAN, either alone or in combination, were more susceptible to copper (CuSO4) than those untreated. In greenhouse assays, D-leucine and IAN applied alone or combined with copper reduced both the number of canker lesions and bacterial populations of X. citri subsp. citri on citrus host leaves. This study provides the basis for the use of foliar-applied biofilm inhibitors for the control of citrus canker alone or combined with copper-based bactericides. PMID:23901828
Kanchanapoom Termkiat; Rao Suma; El-Azizi Mohamed; Khardori Nancy
Abstract Shed cells or disrupted parts of the biofilm may enter the circulation causing serious and very hard to treat biofilm-associated infections. The activity of antimicrobial agents against the shed cells/disrupted biofilms is largely unknown. Methods We studied the in vitro susceptibility of intact and disrupted biofilms of thirty clinical isolates of methicillin-resistant and methicillin–susceptible Staphylococcus aureus (MRSA and MSSA) and Staphylococcus epidermidis to vancomycin, qui...
Wang, TianMing; Shi, GaoXiang; Shao, Jing; Wu, DaQiang; Yan, YuanYuan; Zhang, MengXiang; Cui, YanYan; Wang, ChangZhong
The aim of this study was to investigate the antifungal activity of baicalin and its potential mechanism of action against Candida albicans biofilms. The standard techniques including microdilution method and checkerboard assay were employed to evaluate the susceptibilities of baicalin alone and in combination with fluconazole against planktonic and biofilm cells of C. albicans. Transmission electron microscope (TEM), scanning electron microscope (SEM), fluorescent microscope and flow cytometry were used to assess the apoptotic incidences induced by baicalin in biofilm cells. The expressions of four genes (RAS1, CAP1, PDE2 and TPK1) related to Ras-cAMP-PKA pathway were also analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The results showed that minimum inhibitory concentration (MIC) and sessile minimum inhibitory concentration (SMIC50) of baicalin were 500 and 2000 μg/mL with fractional inhibitory concentration indexs (FICIs) ranging from 0.28 to 0.75. A series of events related to apoptosis were observed in baicalin-treated C. albicans biofilms, including extensive chromatin condensation along the nuclear envelope, ROS accumulation, MMP reduction, PS externalization, nuclear fragmentation, chromatin condensation, metacaspase activation and Cyt C release. Additionally, the expressions of RAS1 and TPK1 were up-regulated by 3.2 and 2.9 folds respectively, while those of CAP1 and PDE2 were down-regulated by 3.3 and 6.6 folds respectively after exposure to baicalin in biofilm cells. In conclusion, baicalin can suppress the development of C. albicans biofilms most likely due to inducing cell death via apoptosis. PMID:26169236
Høiby, Niels; Johansen, Helle Krogh; Ciofu, Oana;
. Bacteria in biofilms communicate by means of quorum sensing which activates genes for virulence factors. Biofilms can be prevented by antibiotic prophylaxis or early therapy or by quorum sensing inhibitors which make them susceptible to antibiotics and phagocytosis. Udgivelsesdato: 2007-Nov-26...
Souza, Caio Marcelo Cury; Pereira Junior, Silvio Alves; Moraes, Thaís da Silva; Damasceno, Jaqueline Lopes; Amorim Mendes, Suzana; Dias, Herbert Júnior; Stefani, Ricardo; Tavares, Denise Crispim; Martins, Carlos Henrique Gomes; Crotti, Antônio Eduardo Miller; Mendes-Giannini, Maria José Soares; Pires, Regina Helena
Dental prosthesis supports Candida species growth and may predispose the oral cavity to lesions. C. tropicalis has emerged as a colonizer of prosthesis and has shown resistance to clinically used antifungal agents, which has increased the search for new antifungals. This work describes the effectiveness of fifteen essential oils (EOs) against C. tropicalis The EOs were obtained by hydrodistillation and were chemically characterized by gas chromatography-mass spectrometry. The antifungal activities of the EOs were evaluated by the microdilution method and showed that Pelargonium graveolens (Geraniaceae) (PG-EO) was the most effective oil. Geraniol and linalool were the major constituents of PG-EO. The 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide (XTT) assay showed that all the clinical C. tropicalis strains formed viable biofilms. Scanning electron microscopy examination of the biofilms revealed a complex architecture with basal layer of yeast cells and an upper layer of filamentous cells. Treatments with PG-EO, linalool, and geraniol significantly reduced the number of viable biofilm cells and inhibited biofilm formation after exposure for 48 h. PG-EO, geraniol, and linalool were not toxic to normal human lung fibroblasts (GM07492A) at the concentrations they were active against C. tropicalis Together, our results indicated that C. tropicalis is susceptible to treatment with PG-EO, geraniol, and linalool, which could become options to prevent or treat this infection. PMID:26868902
Jensen, E T; Kharazmi, A; Garred, P;
In chronic infections, such as the bronchopulmonary Pseudomonas aeruginosa infection in cystic fibrosis (CF) patients, bacteria persist despite an intact host immune defense and frequent antibiotic treatment. An important reason for the persistence of the bacteria is their capacity for the biofilm...... mode of growth. In this study we investigated the role of biofilms in activation of complement, a major contributor to the inflammatory process. Complement activation by P. aeruginosa was examined in a complement consumption assay, production of C3 and factor B conversion products assessed by crossed...... immuno-electrophoresis, C5a generation tested by a PMN chemotactic assay, and terminal complement complex formation measured by ELISA. Two of the four assays showed that P. aeruginosa grown in biofilm activated complement less than planktonic bacteria, and all assays showed that activation by intact...
Fong, Jiunn N. C.; Yildiz, Fitnat H.
Proteinaceous components of the biofilm matrix include secreted extracellular proteins, cell surface adhesins and protein subunits of cell appendages such as flagella and pili. Biofilm matrix proteins play diverse roles in biofilm formation and dissolution. They are involved in attaching cells to surfaces, stabilizing the biofilm matrix via interactions with exopolysaccharide and nucleic acid components, developing three-dimensional biofilm architectures, and dissolving biofilm matrix via enz...
Cruz, H.; Henriques, Mariana; Teughels, Wim; Celis, Jean-Pierre; L. A. Rocha
Dental implant failures occur mainly due to biofilm infections and mastication micro-movements or load stresses that may induce wear degradation. Dental implants microgaps are highly susceptible areas for biofilm formation and fluoride retention. Although fluoride is an antimicrobial agent widely used in caries prevention, its influence on biofilms under sliding wear remains unknown. The main aim of this work was to assess the effect of mixed biofilms and fluoride on the tribocorrosion behavi...
Kroukamp, Otini; Dumitrache, Romeo G.; Wolfaardt, Gideon M
Biofilm formation renders sessile microbial populations growing in continuous-flow systems less susceptible to variation in dilution rate than planktonic cells, where dilution rates exceeding an organism's maximum growth rate (μmax) results in planktonic cell washout. In biofilm-dominated systems, the biofilm's overall μmax may therefore be more relevant than the organism's μmax, where the biofilm μmax is considered as a net process dependent on the adsorption rate, growth rate, and removal r...
Maheshwari, Meenu; Ahmad, Iqbal; Althubiani, Abdullah Safar
This study aimed to investigate the occurrence of biofilm-forming extended-spectrum β-lactamase (ESBL)-producing enteric bacteria in hospital wastewater and to evaluate their antibiotic resistance behaviour and transferability of the plasmid-encoded blaCTX-M gene in biofilm. ESBL production was confirmed using the combined disc test and Etest. Amplification of blaCTX-M was performed by PCR. Antibiotic susceptibility was evaluated using the disc diffusion assay and broth dilution method. Transfer of blaCTX-M in planktonic and biofilm state was performed by broth mating and filter mating experiments, respectively. Among 110 enteric bacteria, 24 (21.8%) isolates belonging to Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae were found to produce ESBL and formed varying levels of biofilm in vitro. Presence of blaCTX-M was detected in 18 (75%) ESBL-producing isolates. A many fold increase in resistance to antibiotics was observed in biofilm. Among ESBL-producers, seven isolates could transfer the blaCTX-M gene by conjugation, with transfer frequencies ranging from 2.22×10(-4) to 7.14×10(-2) transconjugants/recipient cell in the planktonic state and from 3.04×10(-3) to 9.15×10(-1) in biofilm. The transfer frequency of blaCTX-M was significantly higher in biofilm compared with the planktonic state, and co-transfer of ciprofloxacin resistance was also detected in five isolates. This study demonstrates that biofilm-forming ESBL-producing enteric bacteria with a greater transfer frequency of resistance genes will lead to frequent dissemination of β-lactam and fluoroquinolone resistance genes in environmental settings. The emergence and spread of such multidrug resistance is a serious threat to animal and public health. PMID:27530857
A cost-saving algorithm for rapid diagnosis of Staphylococcus aureus and susceptibility to oxacillin directly from positive blood culture bottles by combined testing with BinaxNOW® S. aureus and Xpert MRSA/SA Assay.
Yossepowitch, Orit; Dan, Michael; Kutchinsky, Anuta; Gottesman, Tamar; Schwartz-Harari, Orna
We studied an algorithm combining 2 rapid methods to detect Staphylococcus aureus and its susceptibility to oxacillin directly from positive blood cultures; our goal was to reduce the cost of the procedure, while maintaining accuracy and a short turnaround time. A total of 581 blood cultures containing gram-positive cocci in clusters were tested by BinaxNOW® Staphylococcus aureus Test. Positive samples were further assessed by the Xpert MRSA/SA BC Assay. Phenotypic methods have identified coagulase-negative staphylococci in 505 samples and S. aureus in 76 samples, of which 51 were oxacillin sensitive and 25 were oxacillin resistant. Sensitivity and specificity of the BinaxNOW® Test were 92% and 99%, respectively, compared to the phenotypic method. The Xpert MRSA/SA BC Assay showed complete concordance with phenotypic identification and antimicrobial susceptibility results. The combined rapid assays produced results within 2 hours and reduced the cost by 75% compared with the Xpert MRSA/SA BC Assay if used alone on all blood bottles. PMID:24503507
Alhede, Maria; Alhede, Morten
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....
Stahlhut, Steen G; Schroll, Casper; Harmsen, Morten;
compared with the E. coli parent strain using a biofilm microtiter plate assay. Nine clones with significantly enhanced biofilm formation were identified, subjected to random Tn5 transposon mutagenesis, screened for biofilm deficiency and the biofilm-promoting genes identified. Five of the clones contained...... the type 3 fimbriae gene cluster, a well-known K. pneumoniae virulence factor and biofilm promoter. Thus, the effectiveness of our approach was confirmed. Furthermore, genes encoding cell surface proteins and proteins involved in metabolism, none of them previously associated with biofilm formation in...
Kerkar, S.; Raiker, L.; Tiwari, A.; Mayilraj, S.; Dastager, S.
viz. Nerul and Curca to find a possible reason for the rapid proliferation of these solar biofilms. Out of the 125 bacteria isolated from these biofilms, 16 produced indole-3-acetic acid (IAA). Rapid in-situ assay with Salkowski reagent and HPLC...
Stewart, Philip S
This article examines a likely basis of the tenacity of biofilm infections that has received relatively little attention: the resistance of biofilms to mechanical clearance. One way that a biofilm infection persists is by withstanding the flow of fluid or other mechanical forces that work to wash or sweep microorganisms out of the body. The fundamental criterion for mechanical persistence is that the biofilm failure strength exceeds the external applied stress. Mechanical failure of the biofilm and release of planktonic microbial cells is also important in vivo because it can result in dissemination of infection. The fundamental criterion for detachment and dissemination is that the applied stress exceeds the biofilm failure strength. The apparent contradiction for a biofilm to both persist and disseminate is resolved by recognizing that biofilm material properties are inherently heterogeneous. There are also mechanical aspects to the ways that infectious biofilms evade leukocyte phagocytosis. The possibility of alternative therapies for treating biofilm infections that work by reducing biofilm cohesion could (1) allow prevailing hydrodynamic shear to remove biofilm, (2) increase the efficacy of designed interventions for removing biofilms, (3) enable phagocytic engulfment of softened biofilm aggregates, and (4) improve phagocyte mobility and access to biofilm. PMID:24376149
Noguchi, Nobuo; Noiri, Yuichiro; Narimatsu, Masahiro; Ebisu, Shigeyuki
Bacterial biofilms have been found to develop on root surfaces outside the apical foramen and be associated with refractory periapical periodontitis. However, it is unknown which bacterial species form extraradicular biofilms. The present study aimed to investigate the identity and localization of bacteria in human extraradicular biofilms. Twenty extraradicular biofilms, used to identify bacteria using a PCR-based 16S rRNA gene assay, and seven root-tips, used to observe immunohistochemical l...
Xie, Zhihong; Thompson, Angela; Sobue, Takanori; Kashleva, Helena; Xu, Hongbin; Vasilakos, John; Dongari-Bagtzoglou, Anna
Neutrophils are found within Candida albicans biofilms in vivo and could play a crucial role in clearing the pathogen from biofilms forming on catheters and mucosal surfaces. Our goal was to compare the antimicrobial activity of neutrophils against developing and mature C. albicans biofilms and identify biofilm-specific properties mediating resistance to immune cells. Antibiofilm activity was measured with the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)2H-tetrazolium-5-carboxanilide assay and a ...
Issam Alshami; Ahmed E Alharbi
Objective: 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.Methods: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. Results: 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. Conclusions: 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.
Huang, Lijia; van Loveren, Cor; Ling, Junqi; Wei, Xi; Crielaard, Wim; Deng, Dong Mei
Porphyromonas gingivalis is present as a biofilm at the sites of periodontal infections. The detachment of gingival epithelial cells induced by P. gingivalis biofilms was examined using planktonic cultures as a comparison. Exponentially grown planktonic cultures or 40-h biofilms were co-incubated with epithelial cells in a 24-well plate for 4 h. Epithelial cell detachment was assessed using imaging. The activity of arginine-gingipain (Rgp) and gene expression profiles of P. gingivalis cultures were examined using a gingipain assay and quantitative PCR, respectively. P. gingivalis biofilms induced significantly higher cell detachment and displayed higher Rgp activity compared to the planktonic cultures. The genes involved in gingipain post-translational modification, but not rgp genes, were significantly up-regulated in P. gingivalis biofilms. The results underline the importance of including biofilms in the study of bacterial and host cell interactions. PMID:26963862
Full Text Available The work reviewed here was published between 2008 and 2010 and describes research that involved aerobic and anoxic biofilm treatment of water pollutants. Biofilm denitrification systems are covered when appropriate. References catalogued here are divided on the basis of fundamental research area or reactor types. Fundamental research into biofilms is presented in two sections, Biofilm Measurement and Characterization and Growth and Modeling. The reactor types covered are: trickling filters, rotating biological contactors, fluidized bed bioreactors, submerged bed biofilm reactors, biological granular activated carbon, membrane bioreactors, and immobilized cell reactors. Innovative reactors, not easily classified, are then presented, followed by a section on biofilms on sand, soil and sediment.
Harmsen, Morten; Lappann, Martin; Knøchel, S;
(eDNA) may be the only central component of the biofilm matrix and that it is necessary for both initial attachment and early biofilm formation for 41 L. monocytogenes strains that were tested. DNase I treatment resulted in dispersal of biofilms, not only in microtiter tray assays but also in flow......Listeria monocytogenes is a food-borne pathogen that is capable of living in harsh environments. It is believed to do this by forming biofilms, which are surface-associated multicellular structures encased in a self-produced matrix. In this paper we show that in L. monocytogenes extracellular DNA...... cell biofilm assays. However, it was also demonstrated that in a culture without eDNA, neither Listeria genomic DNA nor salmon sperm DNA by itself could restore the capacity to adhere. A search for additional necessary components revealed that peptidoglycan (PG), specifically N-acetylglucosamine (NAG...
Meyer, M. T.; Subramanian, S.; Kim, Y. W.; Ben-Yoav, H.; Gnerlich, M.; Gerasopoulos, K.; Bentley, W. E.; Ghodssi, R.
Bacterial biofilms present a societal challenge, as they occur in the majority of infections but are highly resistant to both immune mechanisms and traditional antibiotics. In the pursuit of better understanding biofilm biology for developing new treatments, there is a need for streamlined, controlled platforms for biofilm growth and evaluation. We leverage advantages of microfluidics to develop a system in which biofilms are formed and sectioned, allowing parallel assays on multiple sections of one biofilm. A microfluidic testbed with multiple depth profiles was developed to accommodate biofilm growth and sectioning by hydraulically actuated valves. In realization of the platform, a novel fabrication technique was developed for creating multi-depth microfluidic molds using sequentially patterned photoresist separated and passivated by conformal coatings using atomic layer deposition. Biofilm thickness variation within three separately tested devices was less than 13% of the average thickness in each device, while variation between devices was 23% of the average thickness. In a demonstration of parallel experiments performed on one biofilm within one device, integrated valves were used to trisect the uniform biofilms with one section maintained as a control, and two sections exposed to different concentrations of sodium dodecyl sulfate. The technology presented here for multi-depth microchannel fabrication can be used to create a host of microfluidic devices with diverse architectures. While this work focuses on one application of such a device in biofilm sectioning for parallel experimentation, the tailored architectures enabled by the fabrication technology can be used to create devices that provide new biological information.
Soto, Esteban; Halliday-Simmonds, Iona; Francis, Stewart; Kearney, Michael T; Hansen, John D
Francisella noatunensis subsp. orientalis (Fno) is an emergent fish pathogen in both marine and fresh water environments. The bacterium is suspected to persist in the environment even without the presence of a suitable fish host. In the present study, the influence of different abiotic factors such as salinity and temperature were used to study the biofilm formation of different isolates of Fno including intracellular growth loci C (iglC) and pathogenicity determinant protein A (pdpA) knockout strains. Finally, we compared the susceptibility of planktonic and biofilm to three disinfectants used in the aquaculture and ornamental fish industry, namely Virkon(®), bleach and hydrogen peroxide. The data indicates that Fno is capable of producing biofilms within 24 h where both salinity as well as temperature plays a role in the growth and biofilm formation of Fno. Mutations in the iglC or pdpA, both known virulence factors, do not appear to affect the capacity of Fno to produce biofilms, and the minimum inhibitory concentration, and minimum biocidal concentration for the three disinfectants were lower than the minimum biofilm eradication concentration values. This information needs to be taken into account if trying to eradicate the pathogen from aquaculture facilities or aquariums. PMID:26507830
Soto, Esteban; Halliday-Wimmonds, Iona; Kearney, Michael T; Hansen, John D.
Francisella noatunensis subsp. orientalis (Fno) is an emergent fish pathogen in both marine and fresh water environments. The bacterium is suspected to persist in the environment even without the presence of a suitable fish host. In the present study, the influence of different abiotic factors such as salinity and temperature were used to study the biofilm formation of different isolates of Fno including intracellular growth loci C (iglC)and pathogenicity determinant protein A (pdpA) knockout strains. Finally, we compared the susceptibility of planktonic and biofilm to three disinfectants used in the aquaculture and ornamental fish industry, namely Virkon®, bleach and hydrogen peroxide. The data indicates that Fno is capable of producing biofilms within 24 h where both salinity as well as temperature plays a role in the growth and biofilm formation of Fno. Mutations in theiglC or pdpA, both known virulence factors, do not appear to affect the capacity of Fno to produce biofilms, and the minimum inhibitory concentration, and minimum biocidal concentration for the three disinfectants were lower than the minimum biofilm eradication concentration values. This information needs to be taken into account if trying to eradicate the pathogen from aquaculture facilities or aquariums.
Escherichia coli O157:H7 strains isolated from “High Event Period” beef contamination have strong biofilm forming ability and low sanitizer susceptibility which are associated with high pO157 plasmid copy number
In the meat industry, a “High Event Period” (HEP) is defined as a time period when beef processing establishments experience an increased occurrence of product contamination by E. coli O157:H7. Our previous studies suggested that bacterial biofilm formation and sanitizer resistance might contribute...
Manu Chaudhary; Anurag Payasi
Growth of bacterial cells within a biofilm complicate the treatment of infections. Therefore, in the present study biofilm eradication efficacy of (ceftriaxone and sulbactam plus EDTA; CSE1034) was compared with ceftriaxone alone, ceftriaxone plus EDTA and ceftriaxone plus sulbactam against biofilms of ESBL producing Escherichia coli, Klebsiella pneumoniae and Salmonella typhi. Susceptibility testing of each drug was performed on planktonic and biofilm cells in non ESBL producing and ESBL pro...
Harris, Patrick N A; Oltvolgyi, Csongor; Islam, Aminul; Hussain-Yusuf, Hazizul; Loewenthal, Mark R; Vincent, Gemma; Stenos, John; Graves, Stephen
Scrub typhus is caused by the obligate intracellular bacterium Orientia tsutsugamushi and is endemic to many countries in the Asia-Pacific region, including tropical Australia. We describe a recent large outbreak amongst military personnel in north Queensland. A total of 45 clinical cases were identified (36% of all potentially exposed individuals). This occurred despite existing military protocols stipulating the provision of doxycycline prophylaxis. Doxycycline resistance in O. tsutsugamushi has been described in South-East Asia, but not Australia. In one case, O. tsutsugamushi was cultured from eschar tissue and blood. Using quantitative real-time PCR to determine susceptibility to doxycycline for the outbreak strain, a minimum inhibitory concentration (MIC) of ≤0.04 μg/mL was found, indicating susceptibility to this agent. It seems most probable that failure to adhere to adequate prophylaxis over the duration of the military exercise accounted for the large number of cases encountered rather than doxycycline resistance. PMID:27005452
Dipesh Das; Yung-Tse Hung; Charles Moretti; Hasibul Hasan; Harvey Gullicks
The work reviewed here was published between 2008 and 2010 and describes research that involved aerobic and anoxic biofilm treatment of water pollutants. Biofilm denitrification systems are covered when appropriate. References catalogued here are divided on the basis of fundamental research area or reactor types. Fundamental research into biofilms is presented in two sections, Biofilm Measurement and Characterization and Growth and Modeling. The reactor types covered are: trickling filters, r...
Winston, M.; Rupp, C.J.; Vinogradov, A.; Towler, B.W.; Adams, H; Stoodley, P
The paper describes an experimental study concerning the mechanical properties of bacterial biofilms formed from the early dental plaque colonizer Streptoccocus mutans and pond water biofilms. Experiments reported in this paper demonstrate that both types of biofilms exhibit mechanical behavior similar to that of rheological fluids. The time-dependent properties of both biofilms have been modeled using the principles of viscoelasticity theory. The Burger model has been found to accurately re...
Stewart, Philip S.
This article examines a likely basis of the tenacity of biofilm infections that has received relatively little attention: the resistance of biofilms to mechanical clearance. One way that a biofilm infection persists is by withstanding the flow of fluid or other mechanical forces that work to wash or sweep microorganisms out of the body. The fundamental criterion for mechanical persistence is that the biofilm failure strength exceeds the external applied stress. Mechanical failure of the biofi...
Chandki, Rita; Banthia, Priyank; Banthia, Ruchi
Microbial biofilms are mainly implicated in etiopathogenesis of caries and periodontal disease. Owing to its properties, these pose great challenges. Continuous and regular disruption of these biofilms is imperative for prevention and management of oral diseases. This essay provides a detailed insight into properties, mechanisms of etiopathogenesis, detection and removal of these microbial biofilms.
use of conventional antimicrobial compounds in many cases cannot eradicate biofilms, there is an urgent need to develop alternative measures to combat biofilm infections. The present review is focussed on the important opportunistic pathogen and biofilm model organism Pseudomonas aeruginosa. Initially...
Chiang, Wen-Chi; Nilsson, Martin; Jensen, Peter Østrup;
Within recent years, it has been established that extracellular DNA is a key constituent of the matrix of microbial biofilms. In addition, it has recently been demonstrated that DNA binds positively charged antimicrobials such as aminoglycosides and antimicrobial peptides. In the present study, we...... provide evidence that extracellular DNA shields against aminoglycosides in Pseudomonas aeruginosa biofilms. We show that exogenously supplemented DNA integrates into P. aeruginosa biofilms and increases their tolerance toward aminoglycosides. We provide evidence that biofilms formed by a DNA release......-deficient P. aeruginosa quorum-sensing mutant are more susceptible to aminoglycoside treatment than wild-type biofilms but become rescued from the detrimental action of aminoglycosides upon supplementation with exogenous DNA. Furthermore, we demonstrate that exposure to lysed polymorphonuclear leukocytes...
Full Text Available In this study we present a scheme for quantitative determination of biofilm viability offering significant improvement over existing methods with metabolic assays. Existing metabolic assays for quantifying viable bacteria in biofilms usually utilize calibration curves derived from planktonic bacteria, which can introduce large errors due to significant differences in the metabolic and/or growth rates of biofilm bacteria in the assay media compared to their planktonic counterparts. In the presented method we derive the specific growth rate of Streptococcus mutans bacteria biofilm from a series of metabolic assays using the pH indicator phenol red, and show that this information could be used to more accurately quantify the relative number of viable bacteria in a biofilm. We found that the specific growth rate of S. mutans in biofilm mode of growth was 0.70 h−1, compared to 1.09 h−1 in planktonic growth. This method should be applicable to other bacteria types, as well as other metabolic assays, and, for example, to quantify the effect of antibacterial treatments or the performance of bactericidal implant surfaces.
Sasha J Rose
Full Text Available Mycobacterium avium subsp. hominissuis is an opportunistic pathogen that is associated with biofilm-related infections of the respiratory tract and is difficult to treat. In recent years, extracellular DNA (eDNA has been found to be a major component of bacterial biofilms, including many pathogens involved in biofilm-associated infections. To date, eDNA has not been described as a component of mycobacterial biofilms. In this study, we identified and characterized eDNA in a high biofilm-producing strain of Mycobacterium avium subsp. hominissuis (MAH. In addition, we surveyed for presence of eDNA in various MAH strains and other nontuberculous mycobacteria. Biofilms of MAH A5 (high biofilm-producing strain and MAH 104 (reference strain were established at 22°C and 37°C on abiotic surfaces. Acellular biofilm matrix and supernatant from MAH A5 7 day-old biofilms both possess abundant eDNA, however very little eDNA was found in MAH 104 biofilms. A survey of MAH clinical isolates and other clinically relevant nontuberculous mycobacterial species revealed many species and strains that also produce eDNA. RAPD analysis demonstrated that eDNA resembles genomic DNA. Treatment with DNase I reduced the biomass of MAH A5 biofilms when added upon biofilm formation or to an already established biofilm both on abiotic surfaces and on top of human pharyngeal epithelial cells. Furthermore, co-treatment of an established biofilm with DNase 1 and either moxifloxacin or clarithromycin significantly increased the susceptibility of the bacteria within the biofilm to these clinically used antimicrobials. Collectively, our results describe an additional matrix component of mycobacterial biofilms and a potential new target to help treat biofilm-associated nontuberculous mycobacterial infections.
Shailes, Josh; Ejaz, Rooshanie Nadia; Charalambous, Nayia Karen; Holm, Anne Marie Rubæk; Ganes, Mikkel Coff; Maphosa, Tafadzwa Lucky
Biofilm infections are currently of serious societal concern and implicated in different medical procedures such as surgery with implants and catheters, as well as being implicated in various respiratory diseases. Since a biofilm formation has proven to be less susceptible to traditional antibiotic treatment, finding a viable solution to this problem is being heavily researched. For this paper, research was performed by reviewing scientific literature concerning this topic area with a par...
Jacquelin, L F; Le Magrex, E; Brisset, L; Carquin, J; Berthet, A; Choisy, C
Disrupting bacterial biofilms is necessary for a wide application domains such as reusable medical devices, or systems of pipes for water or fluids in cosmetics, food and chemicals industry. Bacterial cells embedded in a biofilm are less susceptible to disinfectants than suspended cells. This property is referable to the structure of the biofilm itself. The gangue of exopolymers and the thickness of a 5-day-old biofilm of Escherichia coli (more than 200 layers of bacteria), contribute to this decrease of susceptibility. The present work deals with the release of an Escherichia coli biofilm by the sequential action of enzymes and a phenolic disinfectant on the one hand, and by the sequential or simultaneous action of surfactants and the previous disinfectant on the other hand. The decrease of bacteria count per mm2 and the Scanning Electron Microscope observations exhibited a synergic action in every case. Nevertheless, Escherichia coli biofilms quickly reconstructed even after exposition to the previous treatment. PMID:7824307
He, Miao; Du, Minquan; Fan, Mingwen; Bian, Zhuan
Most manifestations of candidiasis are associated with biofilm formation occurring on the surfaces of host tissues and medical devices. Candida albicans is the most frequently isolated causative pathogen of candidiasis, and the biofilms display significantly increased levels of resistance to the conventional antifungal agents. Eugenol, the major phenolic component of clove essential oil, possesses potent antifungal activity. The aim of this study was to investigate the effects of eugenol on preformed biofilms, adherent cells, subsequent biofilm formation and cell morphogenesis of C. albicans. Eugenol displayed in vitro activity against C. albicans cells within biofilms, when MIC(50) for sessile cells was 500 mg/L. C. albicans adherent cell populations (after 0, 1, 2 and 4 h of adherence) were treated with various concentrations of eugenol (0, 20, 200 and 2,000 mg/L). The extent of subsequent biofilm formation were then assessed with the tetrazolium salt reduction assay. Effect of eugenol on morphogenesis of C. albicans cells was observed by scanning electron microscopy (SEM). The results indicated that the effect of eugenol on adherent cells and subsequent biofilm formation was dependent on the initial adherence time and the concentration of this compound, and that eugenol can inhibit filamentous growth of C. albicans cells. In addition, using human erythrocytes, eugenol showed low hemolytic activity. These results indicated that eugenol displayed potent activity against C. albicans biofilms in vitro with low cytotoxicity and therefore has potential therapeutic implication for biofilm-associated candidal infections. PMID:17356790
... page helpful? Also known as: Sensitivity Testing; Drug Resistance Testing; Culture and Sensitivity; C & S; Antimicrobial Susceptibility Formal name: Bacterial and Fungal Susceptibility Testing Related tests: Urine Culture ; ...
Nuryastuti, Titik; van der Mei, Henny C.; Henk J Busscher; Iravati, Susi; Aman, Abu T.; Krom, Bastiaan P.
Staphylococcus epidermidis is notorious for its biofilm formation on medical devices, and novel approaches to prevent and kill S. epidermidis biofilms are desired. In this study, the effect of cinnamon oil on planktonic and biofilm cultures of clinical S. epidermidis isolates was evaluated. Initially, susceptibility to cinnamon oil in planktonic cultures was compared to the commonly used antimicrobial agents chlorhexidine, triclosan, and gentamicin. The MIC of cinnamon oil, defined as the low...
Pereira, Maria Olívia; Machado, Idalina; Lopes, Susana Patrícia; H. Costa
In actual situations bacteria can live nicely in hostile environments in part due to its ability to live in biofilms and to regulate gene expression as an adaptive response to a variety of stresses. This altered profile, compared to planktonic counterparts, as well as the interactions amongst the several strains existing within the biofilm seems to be responsible for the gradual loss of susceptibility to antimicrobials. The presence of biofilms on the surfaces of various types of medical surf...
Florjanič, Maja; Kristl, Julijana
Systems for storage and distribution of purified water at ambient temperature are highly susceptible to microbial contamination. The water flow, microbial content and chemical quality of the purified water in an industrial water system have been simulated in a biofilm annular reactor (BAR) to study the impact of different hydrodynamic conditions on biofilm development. Our results reveal the potential of stagnant purified water at total organic compounds (TOC) below 50 ppb to develop biofilm ...
Full Text Available Abstract Objective The main objective of this study was to assess the influence of gas mixtures on in vitro Plasmodium falciparum growth and 50% inhibitory concentration (IC50 for chloroquine. Methods The study was performed between February 2004 and December 2005. 136 Plasmodium falciparum isolates were used to evaluate gas mixtures effect on IC50 for chloroquine by isotopic microtest. The oxygen effect on asexual blood cycle of 3D7 and W2 clones was determined by thin blood smears examination and tritiated hypoxanthine uptake. Results From 5% O2 to 21% O2 conditions, no parasiticide effect of O2 concentration was observed in vitro on the clones 3D7 and W2. A parasitostatic effect was observed during the exposure of mature trophozoïtes and schizonts at 21% O2 with an increase in the length of schizogony. The chloroquine IC50 at 10% O2 were significantly higher than those at 21% O2, means of 173.5 nM and 121.5 nM respectively (p in vitro resistant to chloroquine (IC50 > 100 nM at 10% O2, 17 were sensitive to chloroquine (IC50 2. Conclusion Based on these results, laboratories should use the same gas mixture to realize isotopic microtest. Further studies on comparison of isotopic and non-isotopic assays are needed to establish a standardized in vitro assay protocol to survey malaria drug resistance.
Head, Nathan E.; Yu, Hongwei
Chronic lung infections with Pseudomonas aeruginosa biofilms are associated with refractory and fatal pneumonia in cystic fibrosis (CF). In this study, a group of genomically diverse P. aeruginosa isolates were compared with the reference strain PAO1 to assess the roles of motility, twitching, growth rate, and overproduction of a capsular polysaccharide (alginate) in biofilm formation. In an in vitro biofilm assay system, P. aeruginosa displayed strain-specific biofilm formation that was not ...
Shelud'ko, A V; Filip'echeva, Yu A; Shumiliva, E M; Khlebtsov, B N; Burov, A M; Petrova, L P; Katsy, E I
Azospirillum brasilense Sp245 with mixed flagellation are able to form biofilms on various surfaces. A nonflagellated mutant of this strain with inactivated chromosomal copy of the flhB gene (flhB1) was shown to exhibit specific traits at the later stages of biofilm formation on a hydrophilic (glass) surface. Mature biofilms of the flhB1::Omegon-Km mutant Sp245.1063 were considerably thinner than those of the parent strain Sp245. The biofilms of the mutant were more susceptible to the forces of hydrodynamic shear. A. brasilense Sp245 cells in biofilms were not found to possess lateral flagella. Cells with polar flagella were, however, revealed by atomic force microscopy of mature native biofilms of strain Sp245. Preservation of a polar flagellum (probably nonmotile) on the cells of A. brasilense Sp245 may enhance the biofilm stability. PMID:26263623
Jackson, Lindsay M D; Kroukamp, Otini; Wolfaardt, Gideon M
Biofilms typically exist as complex communities comprising multiple species with the ability to adapt to a variety of harsh conditions. In clinical settings, antibiotic treatments based on planktonic susceptibility tests are often ineffective against biofilm infections. Using a CO2 evolution measurement system we delineated the real-time metabolic response in continuous flow biofilms to streptomycin doses much greater than their planktonic susceptibilities. Stable biofilms from a multispecies culture (containing mainly Pseudomonas aeruginosa and Stenotrophomonas maltophilia), Gram-negative environmental isolates, and biofilms formed by pure culture P. aeruginosa strains PAO1 and PAO1 ΔMexXY (minimum planktonic inhibitory concentrations between 1.5 and 3.5 mg/l), were exposed in separate experiments to 4000 mg/l streptomycin for 4 h after which growth medium resumed. In complex medium, early steady state multispecies biofilms were susceptible to streptomycin exposure, inferred by a cessation of CO2 production. However, multispecies biofilms survived high dose exposures when there was extra carbon in the antibiotic medium, or when they were grown in defined citrate medium. The environmental isolates and PAO1 biofilms showed similar metabolic profiles in response to streptomycin; ceasing CO2 production after initial exposure, with CO2 levels dropping toward baseline levels prior to recovery back to steady state levels, while subsequent antibiotic exposure elicited increased CO2 output. Monitoring biofilm metabolic response in real-time allowed exploration of conditions resulting in vulnerability after antibiotic exposure compared to the resistance displayed following subsequent exposures. PMID:26441887
Full Text Available A total of 14 Escherichia coli isolates were assessed for their ability to produce biofilm in-vitro by slime production on Congo red agar medium (CRA and microtitre plate assay. Out of 14 isolates tested, 12 were slime producing on CRA as indicated by black colonies. The isolates of E.coli varied in their ability to produce biofilm on the surface of microtitre plate ranging from 0.101 to 0.543 ODm. Out of 14 isolates tested, 10 were positive for biofilm production employing criterion of blank corrected ODs9s > 0.1. Two of slime negative isolated were also negative for biofilm production where as the two slime positive isolates were found to be negative for biofilm production. [Veterinary World 2010; 3(8.000: 364-366
Zheng, Sha; Chang, Wenqiang; Li, Chen; Lou, Hongxiang
Copper surfaces possess efficient antimicrobial effect. Here, we reported that copper surfaces could inactivate Candida albicans biofilms within 40 min. The intracellular reactive oxygen species in C. albicans biofilms were immediately stimulated during the contact of copper surfaces, which might be an important factor for killing the mature biofilms. Copper release assay demonstrated that the copper ions automatically released from the surface of 1 mm thick copper coupons with over 99.9% purity are not the key determinant for the copper-mediated killing action. The susceptibility test to copper surfaces by using C. albicans mutant strains, which were involved in efflux pumps, adhesins, biofilms formation or osmotic stress response showed that als1/als1 and als3/als3 displayed higher resistance to the copper surface contact than other mutants did. The intracellular concentration of copper ions was lower in als1/als1 and als3/als3 than that in wild-type strain. Transcriptional analysis revealed that the expression of copper transporter-related gene, CRP1, was significantly increased in als1/als1, als3/als3, suggesting a potential role of ALS1 and ALS3 in absorbing ions by regulating the expression of CRP1 This study provides a potential application in treating pathogenic fungi by using copper surfaces and uncovers the roles of ALS1 and ALS3 in absorbing copper ions for C. albicans. PMID:27189057
Full Text Available Introduction: Medical devices are made from a variety of materials such as polypropylene, polycarbonate, poly styrene, glass and etc. by attaching to this surfaces, Acinetobacter baumannii can form biofilms and then cause several device associated infections. Biofilms are communities of bacteria attached to the surfaces. In this study, biofilm formation ability in clinical isolates of Acinetobacter baumannii was assessed by two methods on different surfaces. Materials and methods: Biofilm formation by 75 clinical isolates of A. baumannii was evaluated on polycarbonate surface (microtiter plate and polypropylene surface (falcon by crystal violet and 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl-2H-tetrazolium-5-carboxanilide salt (XTT tetrazolium sodium salt assay methods. Falcon or tube method was carried out under static and agitation conditions. Results: Results showed the most isolates can form biofilm but higher numbers of isolates form biofilm on polypropylene surface under agitation. XTT method confirmed strong biofilm formation ability of 10 isolates. Discussion and conclusion: Each of the two assays showed an excellent applicability for the quantification of biofilms. The Crystal violet assay is cheap, easy and is usually used for the quantification of biofilms formed by microorganisms but XTT is more reliable and repeatable. Most of A. baumannii isolates have potential to form biofilm on the medical devices which may result in device-associated infections.
Cheng, X; Mei, Y; Ji, X; Xue, Q; Chen, D
The human leukocyte antigen HLA-B27 is directly involved in the disease pathogenesis of ankylosing spondylitis (AS). HLA-B27 has a high degree of genetic polymorphism, with 105 currently known subtypes; the presence of aspartic acid at residue 116 (Asp116) has been found to play an essential role in AS susceptibility. Here, we systematically investigated the molecular mechanism of the susceptibility difference between the AS-associated subtypes HLA-B*27:02/04/05 and AS-unassociated subtypes HLA-B*27:06/09 to AS at sequence, structure, energetic and dynamic levels. In total seven variable residues were identified among the five studied HLA-B27 subtypes, in which Asp116 can be largely stabilized by a spatially vicinal, positively charged His114 through a salt bridge, while five other variable residues seem to have only a marginal effect on AS susceptibility. We also employed a quantitative structure-activity relationship approach to model the statistical correlation between peptide structure and affinity to HLA-B*27:05, a genetic ancestor of all other HLA-B27 subtypes and associated strongly with AS. The built regression predictor was verified rigorously through both internal cross-validation and external blind validation, and was then employed to identify potential HLA-B*27:05 binders from >20,000 cartilage-derived self-peptides. Subsequently, the binding potency of the top five antigenic peptides to HLA-B*27:05 was assayed in vitro using a FACS-based MHC stabilization experiment. Consequently, two (QRVGSDEFK and LRGAGTNEK) out of the five peptides were determined to have high affinity (BL50 = 5.5 and 15.8 nM, respectively) and, as expected, both of them possess positively charged Lys at the C-terminus. PMID:27228481
Masi, Elisa; Ciszak, Marzena; Santopolo, Luisa; Frascella, Arcangela; Giovannetti, Luciana; Marchi, Emmanuela; Viti, Carlo; Mancuso, Stefano
In nature, biofilms are the most common form of bacterial growth. In biofilms, bacteria display coordinated behaviour to perform specific functions. Here, we investigated electrical signalling as a possible driver in biofilm sociobiology. Using a multi-electrode array system that enables high spatio-temporal resolution, we studied the electrical activity in two biofilm-forming strains and one non-biofilm-forming strain. The action potential rates monitored during biofilm-forming bacterial gro...
Maria José Alves
Full Text Available Microorganisms can colonize a wide variety of medical devices, putting patients in risk for local and systemic infectious complications, including local-site infections, catheter-related bloodstream infections, and endocarditis. These microorganisms are able to grow adhered to almost every surface, forming architecturally complex communities termed biofilms. The use of natural products has been extremely successful in the discovery of new medicine, and mushrooms could be a source of natural antimicrobials. The present study reports the capacity of wild mushroom extracts to inhibit in vitro biofilm formation by multi-resistant bacteria. Four Gram-negative bacteria biofilm producers (Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, and Acinetobacter baumannii isolated from urine were used to verify the activity of Russula delica, Fistulina hepatica, Mycena rosea, Leucopaxilus giganteus, and Lepista nuda extracts. The results obtained showed that all tested mushroom extracts presented some extent of inhibition of biofilm production. Pseudomonas aeruginosa was the microorganism with the highest capacity of biofilm production, being also the most susceptible to the extracts inhibition capacity (equal or higher than 50%. Among the five tested extracts against E. coli, Leucopaxillus giganteus (47.8% and Mycenas rosea (44.8% presented the highest inhibition of biofilm formation. The extracts exhibiting the highest inhibitory effect upon P. mirabilis biofilm formation were Sarcodon imbricatus (45.4% and Russula delica (53.1%. Acinetobacter baumannii was the microorganism with the lowest susceptibility to mushroom extracts inhibitory effect on biofilm production (highest inhibition—almost 29%, by Russula delica extract. This is a pioneer study since, as far as we know, there are no reports on the inhibition of biofilm production by the studied mushroom extracts and in particular against multi-resistant clinical isolates; nevertheless, other
Bacteria in natural, industrial and clinical settings predominantly live in biofilms, i.e., sessile structured microbial communities encased in self-produced extracellular matrix material. One of the most important characteristics of microbial biofilms is that the resident bacteria display a...... remarkable increased tolerance toward antimicrobial attack. Biofilms formed by opportunistic pathogenic bacteria are involved in devastating persistent medical device-associated infections, and chronic infections in individuals who are immune-compromised or otherwise impaired in the host defense. Because the...... use of conventional antimicrobial compounds in many cases cannot eradicate biofilms, there is an urgent need to develop alternative measures to combat biofilm infections. The present review is focussed on the important opportunistic pathogen and biofilm model organism Pseudomonas aeruginosa. Initially...
WANG Dong; WANG Ying; LIU You-ning
Background It is recognized that Haemophilus influenzae isolated from patients with otitis media forms biofilms both in vitro and in vivo, suggesting that biofilm formation in vivo might play an important role in the pathogenesis and chronicity of otitis media, but the effect of antibiotics on biofilm has not been well studied. We investigated the impact of ciprofloxacin and azithromycin on bacterial biofilms formed by Haemophilus influenzae in vitro in this study.Methods Eleven strains of Haemophilus influenzae were isolated from sputum specimens collected from patients with acute exacerbation of chronic obstructive pulmonary diseases. Formation of bacterial biofilm was examined by crystal violet assay and a scanning electron microscope. Alterations of biofilms were measured under varying concentrations of azithromycin and ciprofloxacin.Results Striking differences were observed among strains with regard to the ability to form biofilm. Typical membrane-like structure formed by bacterial cells and extracellular matrix was detected. Initial biofilm synthesis was inhibited by azithromycin and ciprofloxacin at concentrations higher than two-fold minimal inhibitory concentration.Disruption of mature biofilms could be achieved at relatively higher concentration, and ciprofloxacin displayed more powerful activity.Conclusions Haemophilus influenzae is capable of forming biofilm in vitro. Sufficient dosage might control early formation of biofilms. Ciprofloxacin exerts better effects on breakdown of biofilm than azithromycin at conventional concentration in clinics.
Harper, David R; Helena M. R. T. Parracho; James Walker; Richard Sharp; Gavin Hughes; Maria Werthén; Susan Lehman; Sandra Morales
Biofilms are an extremely common adaptation, allowing bacteria to colonize hostile environments. They present unique problems for antibiotics and biocides, both due to the nature of the extracellular matrix and to the presence within the biofilm of metabolically inactive persister cells. Such chemicals can be highly effective against planktonic bacterial cells, while being essentially ineffective against biofilms. By contrast, bacteriophages seem to have a greater ability to target this commo...
Scavone, Paola; Iribarnegaray, Victoria; Caetano, Ana Laura; Schlapp, Geraldine; Härtel, Steffen; Zunino, Pablo
Proteus mirabilis is one of the most common etiological agents of complicated urinary tract infections, especially those associated with catheterization. This is related to the ability of P. mirabilis to form biofilms on different surfaces. This pathogen encodes 17 putative fimbrial operons, the highest number found in any sequenced bacterial species so far. The present study analyzed the role of four P. mirabilis fimbriae (MR/P, UCA, ATF and PMF) in biofilm formation using isogenic mutants. Experimental approaches included migration over catheter, swimming and swarming motility, the semiquantitative assay based on adhesion and crystal violet staining, and biofilm development by immunofluorescence and confocal microscopy. Different assays were performed using LB or artificial urine. Results indicated that the different fimbriae contribute to the formation of a stable and functional biofilm. Fimbriae revealed particular associated roles. First, all the mutants showed a significantly reduced ability to migrate across urinary catheter sections but neither swimming nor swarming motility were affected. However, some mutants formed smaller biofilms compared with the wild type (MRP and ATF) while others formed significantly larger biofilms (UCA and PMF) showing different bioarchitecture features. It can be concluded that P. mirabilis fimbriae have distinguishable roles in the generation of biofilms, particularly in association with catheters. PMID:27091004
Elborn Stuart J
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
Stiefel, Philipp; Mauerhofer, Stefan; Schneider, Jana; Maniura-Weber, Katharina; Rosenberg, Urs; Ren, Qun
Efficient removal of biofilms from medical devices is a big challenge in health care to avoid hospital-acquired infections, especially from delicate devices like flexible endoscopes, which cannot be reprocessed using harsh chemicals or high temperatures. Therefore, milder solutions such as enzymatic cleaners have to be used, which need to be carefully developed to ensure efficacious performance. In vitro biofilm in a 96-well-plate system was used to select and optimize the formulation of novel enzymatic cleaners. Removal of the biofilm was quantified by crystal violet staining, while the disinfecting properties were evaluated by a BacTiter-Glo assay. The biofilm removal efficacy of the selected cleaner was further tested by using European standard (EN) for endoscope cleaning EN ISO 15883, and removal of artificial blood soil was investigated by treating TOSI (Test Object Surgical Instrument) cleaning indicators. Using the process described here, a novel enzymatic endoscope cleaner was developed, which removed 95% of Staphylococcus aureus and 90% of Pseudomonas aeruginosa biofilms in the 96-well plate system. With a >99% reduction of CFU and a >90% reduction of extracellular polymeric substances, this cleaner enabled subsequent complete disinfection and fulfilled acceptance criteria of EN ISO 15883. Furthermore, it efficiently removed blood soil and significantly outperformed comparable commercial products. The cleaning performance was stable even after storage of the cleaner for 6 months. It was demonstrated that incorporation of appropriate enzymes into the cleaner enhanced performance significantly. PMID:27044552
Pemmaraju, Suma C; Pruthi, Parul A; Prasad, R; Pruthi, Vikas
In the present investigation, the role of carbon sources (glucose, lactate, sucrose, and arabinose) on Candida albicans biofilm development and virulence factors was studied on polystyrene microtiter plates. Besides this, structural changes in cell wall component β-glucan in presence of different carbon sources have also been highlighted. Biofilm formation was analyzed by XTT (2,3-bis[2-Methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) reduction assay. Glucose-grown cells exhibited the highest metabolic activity during adhesion among all carbon sources tested (p albicans biofilm development and modulate virulence factors and structural organization of cell wall component β-glucan. PMID:26899861
Watters, C; Fleming, D; Bishop, D; Rumbaugh, K P
From birth to death the human host immune system interacts with bacterial cells. Biofilms are communities of microbes embedded in matrices composed of extracellular polymeric substance (EPS), and have been implicated in both the healthy microbiome and disease states. The immune system recognizes many different bacterial patterns, molecules, and antigens, but these components can be camouflaged in the biofilm mode of growth. Instead, immune cells come into contact with components of the EPS matrix, a diverse, hydrated mixture of extracellular DNA (bacterial and host), proteins, polysaccharides, and lipids. As bacterial cells transition from planktonic to biofilm-associated they produce small molecules, which can increase inflammation, induce cell death, and even cause necrosis. To survive, invading bacteria must overcome the epithelial barrier, host microbiome, complement, and a variety of leukocytes. If bacteria can evade these initial cell populations they have an increased chance at surviving and causing ongoing disease in the host. Planktonic cells are readily cleared, but biofilms reduce the effectiveness of both polymorphonuclear neutrophils and macrophages. In addition, in the presence of these cells, biofilm formation is actively enhanced, and components of host immune cells are assimilated into the EPS matrix. While pathogenic biofilms contribute to states of chronic inflammation, probiotic Lactobacillus biofilms cause a negligible immune response and, in states of inflammation, exhibit robust antiinflammatory properties. These probiotic biofilms colonize and protect the gut and vagina, and have been implicated in improved healing of damaged skin. Overall, biofilms stimulate a unique immune response that we are only beginning to understand. PMID:27571696
Nazik, Hasan; Penner, John C; Ferreira, Jose A; Haagensen, Janus A J; Cohen, Kevin; Spormann, Alfred M; Martinez, Marife; Chen, Vicky; Hsu, Joe L; Clemons, Karl V; Stevens, David A
Iron acquisition is crucial for the growth of Aspergillus fumigatus. A. fumigatus biofilm formation occurs in vitro and in vivo and is associated with physiological changes. In this study, we assessed the effects of Fe chelators on biofilm formation and development. Deferiprone (DFP), deferasirox (DFS), and deferoxamine (DFM) were tested for MIC against a reference isolate via a broth macrodilution method. The metabolic effects (assessed by XTT [2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt]) on biofilm formation by conidia were studied upon exposure to DFP, DFM, DFP plus FeCl3, or FeCl3 alone. A preformed biofilm was exposed to DFP with or without FeCl3. The DFP and DFS MIC50 against planktonic A. fumigatus was 1,250 μM, and XTT gave the same result. DFM showed no planktonic inhibition at concentrations of ≤2,500 μM. By XTT testing, DFM concentrations of biofilms forming in A. fumigatus or preformed biofilms (P biofilm formation (P Biofilm formation with 625 μM DFP plus any concentration of FeCl3 was lower than that in the controls (P biofilms, DFP in the range of ≥625 to 1,250 μM was inhibitory compared to the controls (P biofilm formation (P biofilm increased with 2,500 μM FeCl3 only (P biofilms of A. fumigatus clinical isolates to DFP were noted. In conclusion, iron stimulates biofilm formation and preformed biofilms. Chelators can inhibit or enhance biofilms. Chelation may be a potential therapy for A. fumigatus, but we show here that chelators must be chosen carefully. Individual isolate susceptibility assessments may be needed. PMID:26239975
Vital-Lopez, Francisco G; Reifman, Jaques; Wallqvist, Anders
A hallmark of Pseudomonas aeruginosa is its ability to establish biofilm-based infections that are difficult to eradicate. Biofilms are less susceptible to host inflammatory and immune responses and have higher antibiotic tolerance than free-living planktonic cells. Developing treatments against biofilms requires an understanding of bacterial biofilm-specific physiological traits. Research efforts have started to elucidate the intricate mechanisms underlying biofilm development. However, many aspects of these mechanisms are still poorly understood. Here, we addressed questions regarding biofilm metabolism using a genome-scale kinetic model of the P. aeruginosa metabolic network and gene expression profiles. Specifically, we computed metabolite concentration differences between known mutants with altered biofilm formation and the wild-type strain to predict drug targets against P. aeruginosa biofilms. We also simulated the altered metabolism driven by gene expression changes between biofilm and stationary growth-phase planktonic cultures. Our analysis suggests that the synthesis of important biofilm-related molecules, such as the quorum-sensing molecule Pseudomonas quinolone signal and the exopolysaccharide Psl, is regulated not only through the expression of genes in their own synthesis pathway, but also through the biofilm-specific expression of genes in pathways competing for precursors to these molecules. Finally, we investigated why mutants defective in anthranilate degradation have an impaired ability to form biofilms. Alternative to a previous hypothesis that this biofilm reduction is caused by a decrease in energy production, we proposed that the dysregulation of the synthesis of secondary metabolites derived from anthranilate and chorismate is what impaired the biofilms of these mutants. Notably, these insights generated through our kinetic model-based approach are not accessible from previous constraint-based model analyses of P. aeruginosa biofilm
Lebeer, Sarah; Verhoeven, Tine L. A.; Perea Vélez, Mónica; Vanderleyden, Jos; De Keersmaecker, Sigrid C. J.
Lactobacillus rhamnosus GG (ATCC 53103) is one of the clinically best-studied probiotic organisms. Moreover, L. rhamnosus GG displays very good in vitro adherence to epithelial cells and mucus. Here, we report that L. rhamnosus GG is able to form biofilms on abiotic surfaces, in contrast to other strains of the Lactobacillus casei group tested under the same conditions. Microtiter plate biofilm assays indicated that in vitro biofilm formation by L. rhamnosus GG is strongly modulated by cultur...
Thomas K. Wood
Biofilms transform independent cells into specialized cell communities. Here are presented some insights into biofilm formation ascertained with the best-characterized strain, Escherichia coli. Investigations of biofilm formation and inhibition with this strain using whole-transcriptome profiling coupled to phenotypic assays, in vivo DNA binding studies, and isogenic mutants have led to discoveries related to the role of stress, to the role of intra- and interspecies cell signaling, to the im...
Full Text Available Aim: 127 S.aureus and 65 CoNS strains were isolated from patients noses%u2019. To produce a biofilm ability was investigated using three different methods. Slime-positive and negative staphylococcies%u2019 resistance were evaluated against different antibiotics. Material and Method: Swap samples puted 7% blood agar. Staphylococcus aureus and coagulase-negative staphylococci (CoNS isolates biofilm produced ability were investigated using Congo Red Agar (CRA, microplates (MP and Standard Tube (ST methods. In addition to that, presence of antibiotic resistance of the staphylococcal isolates are determined agar disc diffusion method. Results: The rate of biofilm producing Staphylococcus spp strains was found to be 72.4%, 67.7%, and 62.9%, respectively with CRA, MP, and ST tests. There was no significant relationship among the tests (p>0.05. In addition, antibiotic resistance of Staphylococcus spp. against various antibiotics was also determined by the agar disk diffusion method. Resistance rates of biofilm positive (BP Staphylococcus spp for penicilin G, ampicilin, amocycilin/clavulanic acid, tetracyclin, eritromycin, gentamycin, and enrofloxacin 71.7%, 69.7%, 6.2%, 20.7%, 21.4%, 1.4%, and 0.7%, respectively. Resistance rates of biofilm negative (BN spp for 42.6%, 23.4%, 4.3%, 14.9%, 19.1%, 0.0%, 0.0% respectively. All Staphylococcus isolates were found to be susceptible to vancomycin and teicaplonin. Although BP strains antibiotic resistance rates were observed higher than BN strains. But resistance rates were not found statistically significant (p>0.05. Discussion: CRA is the reliablity and specifity method to determine Staphylococcus spp. biofilm produce ability.
Gómez, Natacha C; Ramiro, Juan M P; Quecan, Beatriz X V; de Melo Franco, Bernadette D G
Use of probiotic biofilms can be an alternative approach for reducing the formation of pathogenic biofilms in food industries. The aims of this study were (i) to evaluate the probiotic properties of bacteriocinogenic (Lactococcus lactis VB69, L. lactis VB94, Lactobacillus sakei MBSa1, and Lactobacillus curvatus MBSa3) and non-bacteriocinogenic (L. lactis 368, Lactobacillus helveticus 354, Lactobacillus casei 40, and Weissela viridescens 113) lactic acid bacteria (LAB) isolated from Brazilian's foods and (ii) to develop protective biofilms with these strains and test them for exclusion of Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella Typhimurium. LAB were tested for survival in acid and bile salt conditions, surface properties, biosurfactant production, β-galactosidase and gelatinase activity, antibiotic resistance and presence of virulence genes. Most strains survived exposure to pH 2 and 4% bile salts. The highest percentages of auto-aggregation were obtained after 24 h of incubation. Sixty-seven percentage auto-aggregation value was observed in W. viridescens 113 and Lactobacillus curvatus MBSa3 exhibited the highest co-aggregation (69% with Listeria monocytogenes and 74.6% with E. coli O157:H7), while the lowest co-aggregation was exhibited by W. viridescens 113 (53.4% with Listeria monocytogenes and 38% with E. coli O157:H7). Tests for hemolytic activity, bacterial cell adherence with xylene, and drop collapse confirmed the biosurfactant-producing ability of most strains. Only one strain (L. lactis 368) produced β-galactosidase. All strains were negative for virulence genes cob, ccf, cylLL, cylLs, cyllM, cylB, cylA and efaAfs and gelatinase production. The antibiotic susceptibility tests indicated that the MIC for ciprofloxacin, clindamycin, gentamicin, kanamycin, and streptomycin did not exceed the epidemiological cut-off suggested by the European Food Safety Authority. Some strains were resistant to one or more antibiotics and resistance
Biofilms may interfere with membrane performance in at least three ways: (i) increase of the transmembrane pressure drop, (ii) increase of feed channel (feed-concentrate) pressure drop, and (iii) increase of transmembrane passage. Given the relevance of biofouling, it is surprising how few data exist about the hydraulic resistance of biofilms that may affect the transmembrane pressure drop and membrane passage. In this study, biofilms were generated in a lab scale cross flow microfiltration system at two fluxes (20 and 100Lm-2h-1) and constant cross flow (0.1ms-1). As a nutrient source, acetate was added (1.0mgL-1 acetate C) besides a control without nutrient supply. A microfiltration (MF) membrane was chosen because the MF membrane resistance is very low compared to the expected biofilm resistance and, thus, biofilm resistance can be determined accurately. Transmembrane pressure drop was monitored. As biofilm parameters, thickness, total cell number, TOC, and extracellular polymeric substances (EPS) were determined, it was demonstrated that no internal membrane fouling occurred and that the fouling layer actually consisted of a grown biofilm and was not a filter cake of accumulated bacterial cells. At 20Lm-2h-1 flux with a nutrient dosage of 1mgL-1 acetate C, the resistance after 4 days reached a value of 6×1012m-1. At 100Lm-2h-1 flux under the same conditions, the resistance was 5×1013m-1. No correlation of biofilm resistance to biofilm thickness was found; Biofilms with similar thickness could have different resistance depending on the applied flux. The cell number in biofilms was between 4×107 and 5×108 cellscm-2. At this number, bacterial cells make up less than a half percent of the overall biofilm volume and therefore did not hamper the water flow through the biofilm significantly. A flux of 100Lm-2h-1 with nutrient supply caused higher cell numbers, more biomass, and higher biofilm resistance than a flux of 20Lm-2h-1. However, the biofilm thickness
Lappann, M.; Haagensen, Janus Anders Juul; Claus, H.;
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......, 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...... PilX 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....
Almaris N Alonso
Full Text Available Listeria monocytogenes is a Gram-positive, food-borne pathogen of humans and animals. L. monocytogenes is considered to be a potential public health risk by the U.S. Food and Drug Administration (FDA, as this bacterium can easily contaminate ready-to-eat (RTE foods and cause an invasive, life-threatening disease (listeriosis. Bacteria can adhere and grow on multiple surfaces and persist within biofilms in food processing plants, providing resistance to sanitizers and other antimicrobial agents. While whole genome sequencing has led to the identification of biofilm synthesis gene clusters in many bacterial species, bioinformatics has not identified the biofilm synthesis genes within the L. monocytogenes genome. To identify genes necessary for L. monocytogenes biofilm formation, we performed a transposon mutagenesis library screen using a recently constructed Himar1 mariner transposon. Approximately 10,000 transposon mutants within L. monocytogenes strain 10403S were screened for biofilm formation in 96-well polyvinyl chloride (PVC microtiter plates with 70 Himar1 insertion mutants identified that produced significantly less biofilms. DNA sequencing of the transposon insertion sites within the isolated mutants revealed transposon insertions within 38 distinct genetic loci. The identification of mutants bearing insertions within several flagellar motility genes previously known to be required for the initial stages of biofilm formation validated the ability of the mutagenesis screen to identify L. monocytogenes biofilm-defective mutants. Two newly identified genetic loci, dltABCD and phoPR, were selected for deletion analysis and both ΔdltABCD and ΔphoPR bacterial strains displayed biofilm formation defects in the PVC microtiter plate assay, confirming these loci contribute to biofilm formation by L. monocytogenes.
Alkasir, Rashad; Wang, Jianfang; Gao, Jian; Ali, Tariq; Zhang, Limei; Szenci, Ottó; Bajcsy, Árpád Csaba; Han, Bo
Trueperella (T.) pyogenes is an opportunistic pathogen that causes suppurative diseases in domestic animals. In this work, the properties, pathogenesis and phenotypic diversity of T. pyogenes isolates from bovine mastitis were studied. Both pyolysin (plo) and collagen-binding protein (cbp) virulence factor genes were detected by PCR in all T. pyogenes isolates (n = 50). Using the tissue culture plate method, 90% of T. pyogenes isolates were able to form biofilms. The minimum inhibitory concentrations (MICs) of 13 antimicrobials against T. pyogenes isolates were determined. High susceptibility was observed to rifampin (96%), ampicillin (94%), ciprofloxacin (94%), and penicillin (92%), while low susceptibility was found to trimethoprim-sulphamethoxazole (10%) and bacitracin (2%). The intracellular assay revealed that T. pyogenes isolates had different cytopathogenic effects on cells. The high percentage (28.6%) of T. pyogenes isolates suggests that this bacterium is an important contributor to mastitis. Moreover, the high occurrence of multidrug resistance, biofilm production, intracellular survival, and the temporal dynamics of T. pyogenes interactions are key factors for a better understanding of how immunity acts on infections with these bacteria and how they evade immune surveillance, thus highlighting the need for the prudent use of antimicrobial agents in veterinary medicine. PMID:26919137
Leiman, Sara A; May, Janine M; Lebar, Matthew D; Kahne, Daniel; Kolter, Roberto; Losick, Richard
The soil bacterium Bacillus subtilis forms biofilms on surfaces and at air-liquid interfaces. It was previously reported that these biofilms disassemble late in their life cycle and that conditioned medium from late-stage biofilms inhibits biofilm formation. Such medium contained a mixture of D-leucine, D-methionine, D-tryptophan, and D-tyrosine and was reported to inhibit biofilm formation via the incorporation of these D-amino acids into the cell wall. Here, we show that L-amino acids were able to specifically reverse the inhibitory effects of their cognate D-amino acids. We also show that D-amino acids inhibited growth and the expression of biofilm matrix genes at concentrations that inhibit biofilm formation. Finally, we report that the strain routinely used to study biofilm formation has a mutation in the gene (dtd) encoding D-tyrosyl-tRNA deacylase, an enzyme that prevents the misincorporation of D-amino acids into protein in B. subtilis. When we repaired the dtd gene, B. subtilis became resistant to the biofilm-inhibitory effects of D-amino acids without losing the ability to incorporate at least one noncanonical D-amino acid, D-tryptophan, into the peptidoglycan peptide side chain. We conclude that the susceptibility of B. subtilis to the biofilm-inhibitory effects of D-amino acids is largely, if not entirely, due to their toxic effects on protein synthesis. PMID:24097941
Full Text Available Background: Reports on the association of nosocomial bacterial infections with indwelling medical devices such as intravenous catheters (IVC has increased in recent years. The potential to form biofilm on these devices seems to be the main reason for establishment of such infections. The aim of this study was to measure the potential of biofilm formation by bacterialisolates from IVCs.Methods: Seventy-one IVCs were collected from hospitalized patients in ICU, NICU, hematology and oncology wards at Taleghani Hospital from Jan 2010 to Jan 2011. The bacterial isolates were identified using the standard biochemical tests and the potential to form biofilms was determined by the microtiter plate assay method (MTP and colony morphology using Congo red agar plates (CRA.Results: Overall, 54 (71% IVCs were colonized and 76 bacteria were isolated among which, 64 (84.2% were coagulase negative staphylococci (CoNS, 3 (3.9% S. aureus, 3 (3.9% Enterococcus spp., 2 (2.6% E. coli and 4 (5.3% were miscellaneous isolates not further identified. Among the CoNS, biofilm formation was observed in 68.7% and 82.8% of bacteriausing MTP and CRA methods, respectively. S. aureus and E. coli isolates also were biofilm producers but Enterococcus and other unknown isolates were biofilm negative.Conclusions: Our results confirm that the prevalent biofilm forming bacteria on IVCs were CoNS and that was the reason for high rates of nosocomial infections.
Many types of bacteria form colonies that grow into physically robust and strongly adhesive aggregates known as biofilms. A distinguishing characteristic of bacterial biofilms is an extracellular polymeric substance (EPS) matrix that encases the cells and provides physical integrity to the colony. The EPS matrix consists of a large amount of polysaccharide, as well as protein filaments, DNA and degraded cellular materials. The genetic pathways that control the transformation of a colony into a biofilm have been widely studied, and yield a spatiotemporal heterogeneity in EPS production. Spatial gradients in metabolites parallel this heterogeneity in EPS, but nutrient concentration as an underlying physiological initiator of EPS production has not been explored. Here, we study the role of nutrient depletion in EPS production in Bacillus subtilis biofilms. By monitoring simultaneously biofilm size and matrix production, we find that EPS production increases at a critical colony thickness that depends on the initial amount of carbon sources in the medium. Through studies of individual cells in liquid culture we find that EPS production can be triggered at the single-cell level by reducing nutrient concentration. To connect the single-cell assays with conditions in the biofilm, we calculate carbon concentration with a model for the reaction and diffusion of nutrients in the biofilm. This model predicts the relationship between the initial concentration of carbon and the thickness of the colony at the point of internal nutrient deprivation. (paper)
da Silva, Anderson Ramos; de Andrade Neto, João Batista; da Silva, Cecília Rocha; Campos, Rosana de Sousa; Costa Silva, Rose Anny; Freitas, Daniel Domingues; do Nascimento, Francisca Bruna Stefany Aires; de Andrade, Larissa Nara Dantas; Sampaio, Letícia Serpa; Grangeiro, Thalles Barbosa; Magalhães, Hemerson Iury Ferreira; Cavalcanti, Bruno Coêlho; de Moraes, Manoel Odorico; Nobre Júnior, Hélio Vitoriano
The incidence of fungal infections and, in particular, the incidence of fungal antibiotic resistance, which is associated with biofilm formation, have significantly increased, contributing to morbidity and mortality. Thus, new therapeutic strategies need to be developed. In this context, natural products have emerged as a major source of possible antifungal agents. Berberine is a protoberberine-type isoquinoline alkaloid isolated from the roots, rhizomes, and stem bark of natural herbs, such as Berberis aquifolium, Berberis vulgaris, Berberis aristata, and Hydrastis canadensis, and of Phellodendron amurense Berberine has been proven to have broad antibacterial and antifungal activity. In the present study, the potential antifungal effect of berberine against fluconazole-resistant Candida and Cryptococcus neoformans strains, as well as against the biofilm form of Candida spp., was assessed. The antifungal effect of berberine was determined by a broth microdilution method (the M27-A3 method of the Clinical and Laboratory Standards Institute) and flow cytometry techniques, in which the probable mechanism of action of the compound was also assessed. For biofilm assessment, a colorimetric 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to determine the susceptibility of sessile cells. The isolates used in the study belonged to the Laboratory of Bioprospection and Experiments in Yeast (LABEL) of the Federal University of Ceará. After 24 and 72 h, fluconazole-resistant Candida and Cryptococcus neoformans strains showed berberine MICs equal to 8 μg/ml and 16 μg/ml, respectively. Cytometric analysis showed that treatment with berberine caused alterations to the integrity of the plasma and mitochondrial membranes and DNA damage, which led to cell death, probably by apoptosis. Assessment of biofilm-forming isolates after treatment showed statistically significant reductions in biofilm cell activity (P < 0.001). PMID:27021328
Cierech, Mariusz; Kolenda, Adam; Grudniak, Anna M; Wojnarowicz, Jacek; Woźniak, Bartosz; Gołaś, Marlena; Swoboda-Kopeć, Ewa; Łojkowski, Witold; Mierzwińska-Nastalska, Elżbieta
The objective of this study was to obtain a material composite with antifungal properties for dentures to be used as an alternative protocol in denture stomatitis treatment and prevention. Denture stomatitis is still a clinical problem in patients particularly vulnerable to this disease. Composites of PMMA and doped ZnO-NPs (weight concentrations, 2.5%, 5%, 7.5%) and PMMA with sprayed solvothermal and hydrothermal ZnO-NPs were tested. The following investigations of newly formed biomaterials were undertaken: influence on Candida albicans solution, biofilm staining, XTT analysis and a quantitative analysis of adhered C. albicans. These studies evidenced the antifungal activity of both nanocomposites PMMA-ZnO-NPs and the efficacy of sputtering of zinc oxide nanoparticles on the PMMA. The study of the biofilm deposition on the surface showed that antifungal properties increase with increasing concentration of ZnO-NPs. The XTT assay in conjunction with testing the turbidity of solutions may indicate the mechanism by which ZnO-NPs exert their effect on the increased induction of antioxidative stress in microorganism cells. The denture base made of the aforesaid materials may play a preventive role in patients susceptible to fungal infections. Based on the results obtained a modified treatment of stomatitis Type II (Newton's classification) complicated by fungal infection was proposed. PMID:27346417
Dlugaszewska, Jolanta; Leszczynska, Malgorzata; Lenkowski, Marcin; Tatarska, Agnieszka; Pastusiak, Tomasz; Szyfter, Witold
Chronic rhinosinusitis (CRS) is a very common disorder that remains poorly understood from a pathogenic standpoint. Recent research on the pathogenesis of CRS has been focused on the potential role of biofilms in this chronic infection. The aim of this study was to assess the sinuses' microflora and biofilm formation on the sino-nasal mucosa in patients with CRS. Paranasal sinus mucosa specimens were harvested at the time of functional endoscopic sinus surgery (FESS). Classical microbiology techniques for the isolation and identification of sinus mucosa microbial flora were used. Scanning electron microscopy (SEM) was used to detect biofilm on the surface of mucosa. A microtiter plate assay for in vitro biofilm formation was employed, divided into three aliquots. One part was assessed for bacterial presence, utilizing an API manual system and the Vitek(®) 2 Compact system. The two remaining aliquots were tested by in vitro conventional microbiological assay with the use of the Infinite M200 (Tecan) microtiter plate reader, and also by scanning electron microscopy (SEM). A microbiological examination of mucosal specimens had taken during FESS operation revealed the presence of various types of bacteria in 29 out of 30 tested samples. Out of 62 different strains isolated from patients with CRS, 23 strains of coagulase-negative Staphylococcus epidermidis and 6 strains of Escherichia coli were the most frequently isolated microorganisms, accounting for 37.1 and 9.7 %, respectively. Among the 62 isolated strains, 58 were used to assess biofilm formation. From the total of 58 isolates, 8.6 % were strong biofilm producers, 20.7 % were moderate, and 70.7 % of isolates were considered to be non- or weak biofilm producers. SEM of the 30 nasal concha mucosal samples taken from patients with CRS revealed biofilm in 23 specimens. A marked destruction of the epithelium was observed, with variation in degrees of severity, from disarrayed cilia to complete absence of cilia
Silva, Ana; Sousa, Ana Margarida; Alves, Diana; Lourenço, Anália; Pereira, Maria Olívia
The emergence of Klebsiella pneumoniae multidrug-resistant strains paves the way to the re-introduction of colistin as a salvage therapy. However, recent planktonic studies have reported several cases of heteroresistance to this antimicrobial agent. The aim of this present work was to gain better understanding about the response of K. pneumoniae biofilms to colistin antibiotherapy and inspect the occurrence of heteroresistance in biofilm-derived cells. Biofilm formation and its susceptibility to colistin were evaluated through the determination of biofilm-cells viability. The profiling of planktonic and biofilm cell populations was conducted to assess the occurrence of heteroresistance. Colony morphology was further characterized in order to inspect the potential role of colistin in K. pneumoniae phenotypic differentiation. Results show that K. pneumoniae was susceptible to colistin in its planktonic form, but biofilms presented enhanced resistance. Population analysis profiles pointed out that K. pneumoniae manifest heteroresistance to colistin only when grown in biofilm arrangements, and it was possible to identify a resistant sub-population presenting a small colony morphology (diameter around 5 mm). To the best of our knowledge, this is the first report linking heteroresistance to biofilm formation and a morphological distinctive sub-population. Moreover, this is the first evidence that biofilm formation can trigger the emergence of heteroresistance in an apparently susceptible strain. PMID:27140200
Nowak, Jessika; Cruz, Cristina D; Palmer, Jon; Fletcher, Graham C; Flint, Steve
Listeria monocytogenes 15G01, a strain belonging to the persistent pulsotype 5132, was isolated from a seafood processing plant in New Zealand. Simple monoculture assays using crystal violet staining showed good biofilm formation for this strain and it was therefore chosen to be further investigated in regard to its biofilm forming ability. To evaluate its behaviour in different conditions commonly encountered in food processing environments, biofilm assays and growth studies were performed using common laboratory media under a range of temperatures (20 °C, 30 °C and 37 °C). Furthermore, the effects of incubation time and different environmental conditions including static, dynamic and anaerobic incubation on biofilm formation were investigated. Changes in the environmental conditions resulted in different biofilm phenotypes of L. monocytogenes 15G01. We demonstrated that increasing temperature and incubation time led to a higher biofilm mass and that dynamic incubation has little effect on biofilm formation at 37 °C but encourages biofilm formation at 30 °C. Biofilm production at 20 °C was minimal regardless of the medium used. We furthermore observed that anaerobic environment led to reduced biofilm mass at 30 °C for all tested media but not at 37 °C. Biofilm formation could not be narrowed down to one factor but was rather dependent on multiple factors with temperature and medium having the biggest effects. PMID:26524221
Controlling Biofilm Formation by Inhibiting the Quorum-Sensing Activity of Pseudomonas aeruginosa using the Ethanolic Extracts of Piper nigrum (Piperaceae Fruit, Punica granatum (Lythraceae Pericarp, and Pisum sativum (Fabaceae Seed
Full Text Available Bacterial biofilm formation can cause serious problems in clinical and industrial settings, which drives the development or screening of biofilm inhibitors. Pseudomonas aeruginosa is a well-known pathogen that exhibit biofilm formation through quorum-sensing, which is a bacterial cell-to-cell communication that regulates the production of many virulence factors. The inhibition of biofilm formation is a viable option for bacterial eradication. The antibacterial effect of Piper nigrum is related to the presence of phenolic and flavonoid components. Punica granatum has been reported to possess a wide range of biological actions, with tannins and alkaloids stated to be the reason of its antibacterial property. Pisum sativum, on the other hand, contains various constituents, but the tannins and phenolic compounds stated as responsible for its antibacterial property. The minimum inhibitory concentration using the susceptibility testing of P. nigrum, P. granatum, P. sativum ethanolic extracts were 6.67×10-4 g/mL, 2.1978×10-5 g/mL, and 6.25×10-4 g/mL, respectively. On the swarming assay, P. granatum and P. sativum inhibits swarming motility at concentrations of 2.1978×10-2 up to 2.1978×10-4 g/mL, and 6.25×10-2 to 6.25×10-3 g/mL, respectively. The P. nigrum extract did not inhibit the motility.
Migration radionuclides in an underground environment are one of the major concerns in the safety assessment of a geological repository. Biofilms can have an impact on the transport of radionuclides in several ways: (1) by acting as a barrier to radionuclide sorption onto geological surfaces, or (2) by providing a sorption site for radionuclides, or (3) by trapping many things, including radionuclides. Little is known about bacterial effects on the biofilm formation deep underground. In this study, we isolated bacterial strains from deep groundwater and evaluated the biofilm formation abilities of these strains by crystal violet assay. Bacterial strains were isolated from ground-water collected at -140 m in the 07-V140-M01 borehole at the Horonobe Underground Research Center, Japan. The crystal violet assay showed that 98% of the isolated strains had biofilm formation abilities under tested conditions. This result suggested that biofilm formation must not be neglected in the study of migration radionuclides in nuclear waste repositories. The isolated strains produced differential amounts of biofilm, although they were identified as the same Pseudomonas species, suggesting that biofilm formation abilities varied at different strain levels. These results support the conclusion that the assessment of biofilm impact on the transport of radionuclides in a geological repository must consider the variation in biofilm formation as a function of strain level. (author)
Valladares Linares, R.
Operation of membrane systems for water treatment can be seriously hampered by biofouling. A better characterization of biofilms in membrane systems and their impact on membrane performance may help to develop effective biofouling control strategies. The objective of this study was to determine the occurrence, extent and timescale of biofilm compaction and relaxation (decompaction), caused by permeate flux variations. The impact of permeate flux changes on biofilm thickness, structure and stiffness was investigated in situ and non-destructively with optical coherence tomography using membrane fouling monitors operated at a constant crossflow velocity of 0.1 m s−1 with permeate production. The permeate flux was varied sequentially from 20 to 60 and back to 20 L m−2 h−1. The study showed that the average biofilm thickness on the membrane decreased after elevating the permeate flux from 20 to 60 L m−2 h−1 while the biofilm thickness increased again after restoring the original flux of 20 L m−2 h−1, indicating the occurrence of biofilm compaction and relaxation. Within a few seconds after the flux change, the biofilm thickness was changed and stabilized, biofilm compaction occurred faster than the relaxation after restoring the original permeate flux. The initial biofilm parameters were not fully reinstated: the biofilm thickness was reduced by 21%, biofilm stiffness had increased and the hydraulic biofilm resistance was elevated by 16%. Biofilm thickness was related to the hydraulic biofilm resistance. Membrane performance losses are related to the biofilm thickness, density and morphology, which are influenced by (variations in) hydraulic conditions. A (temporarily) permeate flux increase caused biofilm compaction, together with membrane performance losses. The impact of biofilms on membrane performance can be influenced (increased and reduced) by operational parameters. The article shows that a (temporary) pressure increase leads to more
Røder, Henriette Lyng; Sørensen, Søren Johannes; Burmølle, Mette
The high prevalence and significance of multispecies biofilms have now been demonstrated in various bacterial habitats with medical, industrial, and ecological relevance. It is highly evident that several species of bacteria coexist and interact in biofilms, which highlights the need for evaluating...... 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 and drawbacks of varying the degree of complexity. This review aims to facilitate multispecies biofilm research in order to expand the current limited knowledge on interspecies interactions. Recent technological advances have enabled total diversity analysis of highly complex and diverse microbial...
Burmølle, Mette; Ren, Dawei; Bjarnsholt, Thomas;
The recent focus on complex bacterial communities has led to the recognition of interactions across species boundaries. This is particularly pronounced in multispecies biofilms, where synergistic interactions impact the bacterial distribution and overall biomass produced. Importantly, in a number...... of settings, the interactions in a multispecies biofilm affect its overall function, physiology, or surroundings, by resulting in enhanced resistance, virulence, or degradation of pollutants, which is of significant importance to human health and activities. The underlying mechanisms causing these...
Watamoto, T; Samaranayake, L P; Egusa, H; Yatani, H; Seneviratne, C J
Biofilm formation is a major virulence attribute of Candida albicans and is directly associated with therapeutic failure. One method by which Candida acquires antifungal resistance is the expression of drug-resistance genes. This study aimed to evaluate the transcriptional regulation of several genes associated with antifungal resistance of C. albicans under planktonic, recently adhered and biofilm growth modes and in C. albicans biofilms in response to antifungal agents. Initially, the antifungal susceptibility of C. albicans cultures in different growth modes was evaluated by standard antifungal susceptibility testing. Next, to assess CDR1, CDR2, MDR1, ERG11, FKS1 and PIL1 expression, RNA was harvested from cells in each growth mode, and from biofilms after drug treatment, and subjected to quantitative real-time RT-PCR (qRT-PCR). Biofilm C. albicans was more resistant to antifungals than recently adhered cells and stationary-phase planktonic cultures. Transcriptional expression of CDR1, CDR2, MDR1, ERG11 and FKS1 was lower in recently adhered C. albicans than in the stationary-phase planktonic cultures. In contrast, PIL1 levels were significantly increased in recently adhered and biofilm modes of growth. The expression of MDR1 in biofilms greatly increased on challenge with amphotericin B but not with the other drugs tested (PERG11 was significantly upregulated by ketoconazole (PCandida biofilms, and lay a foundation for future large-scale genome-wide expression analysis. PMID:21474609
Cho, Kyu Hong; Caparon, Michael G
The ability of Streptococcus pyogenes to form biofilm-like bacterial communities during infection of soft tissue has suggested that the capacity to produce biofilm may be important for pathogenesis. To examine this relationship, a panel of mutants was evaluated for their ability to form biofilm on abiotic surfaces in several assays. Several established virulence factors were crucial for biofilm formation, including the M protein, required for initial cell-surface interactions, and the hyaluronic acid capsule, required for subsequent maturation into a three-dimensional structure. Mutants lacking the transcription regulators Mga and CovR (CsrR) also failed to form biofilm. Comparison of transcriptional profiles revealed differential regulation of approximately 25% of the genome upon adaptation to biofilm. During infection of zebrafish, several virulence factors (notably cysteine protease and streptokinase) were regulated in a biofilm-like manner. However, the overall profile of virulence factor expression indicated that tissue communities have a pattern of gene expression different from biofilm. Taken together, these data show that while biofilm and tissue communities have many characteristics in common, that biofilm reproduces only a subset of the myriad cues used by tissue communities for regulation of virulence. PMID:16135223
Zezhang T Wen
Full Text Available Streptococcus mutans, a key etiological agent of human dental caries, lives almost exclusively on the tooth surface in plaque biofilms and is known for its ability to survive and respond to various environmental insults, including low pH, and antimicrobial agents from other microbes and oral care products. In this study, a penicillin-binding protein (PBP1a-deficient mutant, strain JB467, was generated by allelic replacement mutagenesis and analyzed for the effects of such a deficiency on S. mutans' stress tolerance response and biofilm formation. Our results so far have shown that PBP1a-deficiency in S. mutans affects growth of the deficient mutant, especially at acidic and alkaline pHs. As compared to the wild-type, UA159, the PBP1a-deficient mutant, JB467, had a reduced growth rate at pH 6.2 and did not grow at all at pH 8.2. Unlike the wild-type, the inclusion of paraquat in growth medium, especially at 2 mM or above, significantly reduced the growth rate of the mutant. Acid killing assays showed that the mutant was 15-fold more sensitive to pH 2.8 than the wild-type after 30 minutes. In a hydrogen peroxide killing assay, the mutant was 16-fold more susceptible to hydrogen peroxide (0.2%, w/v after 90 minutes than the wild-type. Relative to the wild-type, the mutant also had an aberrant autolysis rate, indicative of compromises in cell envelope integrity. As analyzed using on 96-well plate model and spectrophotometry, biofilm formation by the mutant was decreased significantly, as compared to the wild-type. Consistently, Field Emission-SEM analysis also showed that the PBP1a-deficient mutant had limited capacity to form biofilms. TEM analysis showed that PBP1a mutant existed primarily in long rod-like cells and cells with multiple septa, as compared to the coccal wild-type. The results presented here highlight the importance of pbp1a in cell morphology, stress tolerance, and biofilm formation in S. mutans.
Vandecandelaere, Ilse; Van Acker, Heleen; Coenye, Tom
We describe a 96-well microtiter plate-based system as an in vitro model for biofilm formation and quantification. Although in vitro assays are artificial systems and thus significantly differ from in vivo conditions, they represent an important tool to evaluate biofilm formation and the effect of compounds on biofilms. Stainings to evaluate the amount of biomass (crystal violet staining) and the number of metabolically active cells (resazurin assay) are discussed and specific attention is paid to the use of this model to quantify persisters in sessile populations. PMID:26468099
Kim, Soo-Kyoung; Lee, Joon-Hee
In recent decades, many researchers have written numerous articles about microbial biofilms. Biofilm is a complex community of microorganisms and an example of bacterial group behavior. Biofilm is usually considered a sessile mode of life derived from the attached growth of microbes to surfaces, and most biofilms are embedded in self-produced extracellular matrix composed of extracellular polymeric substances (EPSs), such as polysaccharides, extracellular DNAs (eDNA), and proteins. Dispersal, a mode of biofilm detachment indicates active mechanisms that cause individual cells to separate from the biofilm and return to planktonic life. Since biofilm cells are cemented and surrounded by EPSs, dispersal is not simple to do and many researchers are now paying more attention to this active detachment process. Unlike other modes of biofilm detachment such as erosion or sloughing, which are generally considered passive processes, dispersal occurs as a result of complex spatial differentiation and molecular events in biofilm cells in response to various environmental cues, and there are many biological reasons that force bacterial cells to disperse from the biofilms. In this review, we mainly focus on the spatial differentiation of biofilm that is a prerequisite for dispersal, as well as environmental cues and molecular events related to the biofilm dispersal. More specifically, we discuss the dispersal-related phenomena and mechanisms observed in Pseudomonas aeruginosa, an important opportunistic human pathogen and representative model organism for biofilm study. PMID:26832663
Abdulamir, Ahmed Sahib; Jassim, Sabah A. A.; Hafidh, Rand R; Bakar, Fatimah Abu
Background This study assessed novel approach of using highly lytic phages against methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) biofilms with and without biofilm extracellular matrix- disrupting chemical. Method The resultant phage-based control was assessed in relation to the type of biofilm extracellular matrix namely, polysaccharide intercellular adhesion (PIA) or proteinacious fibronectin-binding protein A (FnBPA). The biofilm...
Full Text Available Legionnaires’ disease is a potentially fatal pneumonia caused by Legionella pneumophila, an aquatic bacterium often found within the biofilm niche. In man-made water systems microbial biofilms increase the resistance of legionella to disinfection, posing a significant threat to public health. Disinfection methods currently used in water systems have been shown to be ineffective against legionella over the long-term, allowing recolonization by the biofilm-protected microorganisms. In this study, the anti-biofilm activity of previously fabricated polyamino-phenolic ligands and polyamidoamine dendrimers was investigated against legionella mono-species and multi-species biofilms formed by L. pneumophila in association with other bacteria that can be found in tap water (Aeromonas hydrophila, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae. Bacterial ability to form biofilms was verified using a crystal violet colorimetric assay and testing cell viability by real-time quantitative PCR and Plate Count assay. The concentration of the chemicals tested as anti-biofilm agents was chosen based on cytotoxicity assays: the highest non-cytotoxic chemical concentration was used for biofilm inhibition assays, with dendrimer concentration ten-fold higher than polyamino-phenolic ligands. While Macrophen and Double Macrophen were the most active substances among polyamino-phenolic ligands, dendrimers were overall two-fold more effective than all other compounds with a reduction up to 85% and 73% of legionella and multi-species biofilms, respectively. Chemical interaction with matrix molecules is hypothesized, based on SEM images and considering the low or absent anti-microbial activity on planktonic bacteria showed by flow cytometry. These data suggest that the studied compounds, especially dendrimers, could be considered as novel molecules in the design of research projects aimed at the development of efficacious anti-biofilm disinfection
Andreozzi, Elisa; Barbieri, Federica; Ottaviani, Maria F; Giorgi, Luca; Bruscolini, Francesca; Manti, Anita; Battistelli, Michela; Sabatini, Luigia; Pianetti, Anna
Legionnaires' disease is a potentially fatal pneumonia caused by Legionella pneumophila, an aquatic bacterium often found within the biofilm niche. In man-made water systems microbial biofilms increase the resistance of legionella to disinfection, posing a significant threat to public health. Disinfection methods currently used in water systems have been shown to be ineffective against legionella over the long-term, allowing recolonization by the biofilm-protected microorganisms. In this study, the anti-biofilm activity of previously fabricated polyamino-phenolic ligands and polyamidoamine dendrimers was investigated against legionella mono-species and multi-species biofilms formed by L. pneumophila in association with other bacteria that can be found in tap water (Aeromonas hydrophila, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae). Bacterial ability to form biofilms was verified using a crystal violet colorimetric assay and testing cell viability by real-time quantitative PCR and Plate Count assay. The concentration of the chemicals tested as anti-biofilm agents was chosen based on cytotoxicity assays: the highest non-cytotoxic chemical concentration was used for biofilm inhibition assays, with dendrimer concentration 10-fold higher than polyamino-phenolic ligands. While Macrophen and Double Macrophen were the most active substances among polyamino-phenolic ligands, dendrimers were overall twofold more effective than all other compounds with a reduction up to 85 and 73% of legionella and multi-species biofilms, respectively. Chemical interaction with matrix molecules is hypothesized, based on SEM images and considering the low or absent anti-microbial activity on planktonic bacteria showed by flow cytometry. These data suggest that the studied compounds, especially dendrimers, could be considered as novel molecules in the design of research projects aimed at the development of efficacious anti-biofilm disinfection treatments of water systems in
Andreozzi, Elisa; Barbieri, Federica; Ottaviani, Maria F.; Giorgi, Luca; Bruscolini, Francesca; Manti, Anita; Battistelli, Michela; Sabatini, Luigia; Pianetti, Anna
Legionnaires’ disease is a potentially fatal pneumonia caused by Legionella pneumophila, an aquatic bacterium often found within the biofilm niche. In man-made water systems microbial biofilms increase the resistance of legionella to disinfection, posing a significant threat to public health. Disinfection methods currently used in water systems have been shown to be ineffective against legionella over the long-term, allowing recolonization by the biofilm-protected microorganisms. In this study, the anti-biofilm activity of previously fabricated polyamino-phenolic ligands and polyamidoamine dendrimers was investigated against legionella mono-species and multi-species biofilms formed by L. pneumophila in association with other bacteria that can be found in tap water (Aeromonas hydrophila, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae). Bacterial ability to form biofilms was verified using a crystal violet colorimetric assay and testing cell viability by real-time quantitative PCR and Plate Count assay. The concentration of the chemicals tested as anti-biofilm agents was chosen based on cytotoxicity assays: the highest non-cytotoxic chemical concentration was used for biofilm inhibition assays, with dendrimer concentration 10-fold higher than polyamino-phenolic ligands. While Macrophen and Double Macrophen were the most active substances among polyamino-phenolic ligands, dendrimers were overall twofold more effective than all other compounds with a reduction up to 85 and 73% of legionella and multi-species biofilms, respectively. Chemical interaction with matrix molecules is hypothesized, based on SEM images and considering the low or absent anti-microbial activity on planktonic bacteria showed by flow cytometry. These data suggest that the studied compounds, especially dendrimers, could be considered as novel molecules in the design of research projects aimed at the development of efficacious anti-biofilm disinfection treatments of water systems
Simões, Lúcia C; Lemos, Madalena; Pereira, Ana M; Abreu, Ana C; Saavedra, Maria J; Simões, Manuel
This study investigated the physiology and behaviour following treatment with ortho-phthalaldehyde (OPA), of Pseudomonas fluorescens in both the planktonic and sessile states. Steady-state biofilms and planktonic cells were collected from a bioreactor and their extracellular polymeric substances (EPS) were extracted using a method that did not destroy the cells. Cell structure and physiology after EPS extraction were compared in terms of respiratory activity, morphology, cell protein and polysaccharide content, and expression of the outer membrane proteins (OMP). Significant differences were found between the physiological parameters analysed. Planktonic cells were more metabolically active, and contained greater amounts of proteins and polysaccharides than biofilm cells. Moreover, biofilm formation promoted the expression of distinct OMP. Additional experiments were performed with cells after EPS extraction in order to compare the susceptibility of planktonic and biofilm cells to OPA. Cells were completely inactivated after exposure to the biocide (minimum bactericidal concentration, MBC = 0.55 ± 0.20 mM for planktonic cells; MBC = 1.7 ± 0.30 mM for biofilm cells). After treatment, the potential of inactivated cells to recover from antimicrobial exposure was evaluated over time. Planktonic cells remained inactive over 48 h while cells from biofilms recovered 24 h after exposure to OPA, and the number of viable and culturable cells increased over time. The MBC of the recovered biofilm cells after a second exposure to OPA was 0.58 ± 0.40 mM, a concentration similar to the MBC of planktonic cells. This study demonstrates that persister cells may survive in biocide-treated biofilms, even in the absence of EPS. PMID:21547756
Lutz, Carla; Thomas, Torsten; Steinberg, Peter; Kjelleberg, Staffan; Egan, Suhelen
Interspecific competition between bacteria shapes community dynamics, causing evolutionary changes that affect life history traits. Here, we studied the role of interspecific competition on the generation of trait diversity using a two-species model system of marine, surface-associated bacteria. Bacterial biofilms of Phaeobacter inhibens were established alone or in competition with Pseudoalteromonas tunicata and phenotypic traits of dispersal cells were assessed during biofilm development. P. inhibens dispersal isolates from competition biofilms displayed less phenotypic variation, were superior competitors, were less susceptible to predation, and reached higher planktonic biomass than isolates from noncompetition biofilms. Moreover, the maintenance of competitive ability exhibited by individual dispersal isolates from competition biofilms did not result in an obvious reduction (measured as a negative trait correlation) in other traits, but was rather positively correlated with planktonic growth. However, where negative correlations between traits were found, they were exhibited by individuals derived from noncompetitive biofilms, whose populations also had a higher degree of trait variation than those from biofilms experiencing competition. Our observations indicate that interspecific competition during biofilm formation is important for maintaining both competitive ability and affects variation in ecologically relevant traits. Given that most bacteria in biofilms exist in diverse, multispecies communities, an understanding of how bacteria respond to biotic factors such as interspecific competition is critical for understanding the dynamics of bacterial populations in both ecological and evolutionary time. PMID:26914307
Full Text Available In the past, interest in biofilms was limited to research related to water distribution systems, waste water treatment and dental plaques. Biofilm has become a more popular research topic in many other areas in recent years including food safety. Biofilm formation can compromise the sanitation of food surfaces and environmental surfaces by spreading detached organisms to other areas of processing plants. Unfortunately, these detached organisms are not similar to normal microorganisms suspended in an aquatic environment but are more resistant to several stresses or microbial inactivation including some food preservation methods. Microstructures of biofilms as revealed by different types of microscopic techniques showed that biofilms are highly complex and consist of many symbiotic organisms, some of which are human pathogens. This article reviewed the process of biofilm formation, the significance of biofilms on food or food contact surfaces, their ability to protect foodborne pathogens from environmental stresses and recent methods for the study of biofilms on food contact surfaces.
Prevention of biofilm development and its removal has crucial meaning in membrane reactor. Biofilm causes pore blocking on membranes, which causes a drop in efficiency of mixed liquor filtration and consequently deteriorates the efficiency of whole membrane bioreactor. This thesis deals with factors that affect biofilm development in membrane bioreactors. Structure and growth of biofilm are presented from its initial attachment of individual particles, their parameters of adhesion, hydrodynam...
Ganguly, Shantanu; Mitchell, Aaron P.
Biofilms are microbial communities that form on surfaces and are embedded in an extracellular matrix. C. albicans forms pathogenic mucosal biofilms that are evoked by changes in host immunity or mucosal ecology. Mucosal surfaces are inhabited by many microbial species; hence these biofilms are polymicrobial. Several recent studies have applied paradigms of biofilm analysis to study mucosal C. albicans infections. These studies reveal that the Bcr1 transcription factor is a master regulator of...
The objective of this thesis was to explore the possibilities of using microalgal biofilms for the treatment of municipal wastewater, with a focus on the post-treatment of municipal wastewater effluent. The potential of microalgal biofilms for wastewater treatment was first investigated using a scenario analysis. Then biofilms were grown on wastewater treatment plant effluent in horizontal flow cells under different nutrient loads to determine the maximum uptake capacity of the biofilms for N...
Mitchell, A. C.; Phillips, A.; Hiebert, R.; Gerlach, R.; Kaszuba, J.; Cunningham, A.
In order to develop subsurface CO2 storage as a viable engineered mechanism to reduce concentrations of atmospheric CO2, any potential ¡°leakage¡± of injected supercritical CO2 (scCO2) from the ground to the atmosphere must be reduced. Here, we investigate the utility of biofilms, which are microorganism assemblages firmly attached to a surface, as a means of reducing scCO2 leakage. Firstly, experiments were performed to test whether biofilms were more resilient than planctonic cells to scCO2. Bacillus mojavensis biofilms were grown on a sand support matrix in scCO2 extractor cartridges at 30°C. B. mojavensis was also grown under suspended planctonic conditions in the same media overnight and aliquots were decanted into scCO2 extractor cartridges. Biofilm and suspended B. mojavensis samples were processed on a Supercritical Fluid Extractor with pressurization to 2000 psi at 35°C, and a 20 minute flow of scCO2. Suspended growth samples revealed a 3 log reduction in cell viability while biofilm only showed a 1 log reduction, demonstrating that B. mojavensis biofilms are more resilient than planctonic cells to scCO2. Protective extra cellular polymeric substances which make up the biofilm matrix likely provide a protective barrier against scCO2. Secondly, the ability of biofilms to grow under high pressure and reduce the permeability of porous geological matrices was investigated using a unique high pressure (8.9MPa), moderate temperature (¡Ý 32°C) flow reactor containing 40 millidarcy Berea sandstone cores. The flow reactor was inoculated with the biofilm forming organism Shewanella fridgidimarina. Electron microscopy of the rock core revealed substantial biofilm accumulation in rock pores which resulted in core permeability. Permeability did not increase in response to starvation and scCO2 challenges. Viable population assays of organisms in the effluent indicated survival of the microorganisms following scCO2 challenges of <71h and starvation for <363h
Smets, Barth F.; D'Alvise, Paul; Yankelovich, T.;
Adherent growth of Pseudomonas putida KT2440 with and without the TOL plasmid (pWWO) at the solid-liquid and air-liquid interface was examined. We compared biofilm formation on glass in flow cells, and assayed pellicle (air-liquid interface biofilm) formation in stagnant liquid cultures by confocal...... laser scanning microscopy. The TOL-carrying strains formed pellicles and thick biofilms, whereas the same strains without the plasmid displayed little adherent growth. Microscopy using fluorescent nucleic acid- specific stains (cytox orange, propidium iodide) revealed differences in production...... combined with specific cytostains; release of cytoplasmic material was assayed by a β-glucosidase assay. Enhanced cell lysis due to plasmid carriage was ruled out as the mechanism for eDNA release. We report, for the first time, that carriage of a conjugative plasmid leads to increased biofilm formation...
Full Text Available Objective(sMethicillin-resistant Staphylococcus aureus (MRSA is an important cause of nosocomial and community infections. Biofilm formation, mediated by a polysaccharide intercellular adhesin (PIA and encoded by the ica operon, is considered to be an important virulence factor in both S. epidermidis and S. aureus. However, the clinical impact of the ica locus and PIA production is less well described in S. aureus. We studied biofilm formation in clinical isolates of MRSA in relation to the presence of the ica operon. Materials and MethodsForty five MRSA were studied for biofilm formation by colony morphology on Congo red agar (CRA and the microtitre plate assay (MtP. Presence of the ica genes was detected by PCR and specific primers. ResultsThe results showed that 53.3% of the isolates had the potential to form biofilm by colony morphology of which, 75% carried the ica operon. Weak biofilm production was observed in the MtP assay by 57.8%, of which 53.8% harbored the ica operon. However, about 70% of biofilm non-producers also carried the ica operon. ConclusionOverall, there was no agreement between the icaAB gene carriage and biofilm phenotype by either of the two phenotypic methods. However, 91% of biofilm formers on CRA also produced biofilm in the MtP assay.
Sturtevant, Rachael A; Sharma, Prannda; Pavlovsky, Leonid; Stewart, Elizabeth J; Solomon, Michael J; Younger, John G
Given the increasing evidence of safe application of elevated temperature in other clinical contexts, we consider the potential for supplemental hyperthermia to augment the effects of vancomycin against staphylococci, a major source of postoperative and posttraumatic sepsis. Laboratory reference strains and libraries of clinical blood isolates of Staphylococcus epidermidis and methicillin-resistant Staphylococcus aureus, both as planktonic cells and as established biofilms, were assessed for thermosensitivity and increased susceptibility to vancomycin in the setting of thermal treatment. In addition to viability measures, patterns of stress gene expression were assessed with quantitative polymerase chain reaction, and structural changes were measured using quantitative transmission electron microscopy. Laboratory strains of both species had reduced growth and biofilm viability at 45°C, a temperature commonly used in other domains such as adjuvant treatments of malignancy. Blood isolates of S. epidermidis were consistent in this regard as well, but significant between-isolate variability in thermosensitivity was seen in blood isolates of S. aureus. Expression profiling and ultrastructural measurements confirmed that elevated temperature was a substantial stressor with or without vancomycin treatment. Our findings suggest that temperature elevations shown to be tolerated in humans in other settings hold the potential to be used as an adjuvant to antibiotic therapy against staphylococcal biofilms. PMID:25784524
Muzny, Christina A; Schwebke, Jane R
Biofilms are microbial communities of surface-attached cells embedded in a self-produced extracellular matrix. They are of major medical significance because they decrease susceptibility to antimicrobial agents and enhance the spread of antimicrobial resistance. Biofilm-associated bacterial and fungal microorganisms have increasingly been recognized to play a role in multiple infectious diseases, particularly in their persistence and recurrence. More recently, biofilms have also been implicated in vaginal infections, notably bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC), particularly in the setting of treatment failure and recurrence. The purpose of this review is to discuss the impact of biofilms on the management and treatment of BV and recurrent VVC and highlight the need for additional research and development of novel therapeutics targeting pathogenic vaginal biofilms. PMID:25935553
Jorge, Paula; Lourenço, Anália; Pereira, Maria Olívia
Biofilm research is growing more diverse and dependent on high-throughput technologies, and the large-scale production of results aggravates data substantiation. In particular, experimental protocols are often adapted to meet the needs of a particular laboratory, and no statistical validation of the modified method is provided. This paper discusses the impact of intralaboratory adaptation and non-rigorous documentation of experimental protocols on biofilm data interchange and validation. The case study is a non-standard, but widely used, workflow for Pseudomonas aeruginosa biofilm development considering three analysis assays: the crystal violet (CV) assay for biomass quantification, the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) assay for respiratory activity assessment, and the colony forming units (CFU) assay for determination of cell viability. The ruggedness of the protocol was assessed by introducing small changes in the biofilm growth conditions, which simulate minor protocol adaptations and non- rigorous protocol documentation. Results show that even minor variations in the biofilm growth conditions may affect the results considerably, and that the biofilm analysis assays lack repeatability. Intralaboratory validation of non-standard protocols is found critical to ensure data quality and enable the comparison of results within and among laboratories. PMID:26651585
Srijan Aggarwal; Philip S. Stewart; Hozalski, Raymond M.
Bacterial biofilms are highly resistant to common antibacterial treatments, and several physiological explanations have been offered to explain the recalcitrant nature of bacterial biofilms. Herein, a biophysical aspect of biofilm recalcitrance is being reported on. While engineering structures are often overdesigned with a factor of safety (FOS) usually under 10, experimental measurements of biofilm cohesive strength suggest that the FOS is on the order of thousands. In other words, bacteria...
Wu, C-C; Lin, C-T; Wu, C-Y; Peng, W-S; Lee, M-J; Tsai, Y-C
Dental caries arises from an imbalance of metabolic activities in dental biofilms developed primarily by Streptococcus mutans. This study was conducted to isolate potential oral probiotics with antagonistic activities against S. mutans biofilm formation from Lactobacillus salivarius, frequently found in human saliva. We analysed 64 L. salivarius strains and found that two, K35 and K43, significantly inhibited S. mutans biofilm formation with inhibitory activities more pronounced than those of Lactobacillus rhamnosus GG (LGG), a prototypical probiotic that shows anti-caries activity. Scanning electron microscopy showed that co-culture of S. mutans with K35 or K43 resulted in significantly reduced amounts of attached bacteria and network-like structures, typically comprising exopolysaccharides. Spot assay for S. mutans indicated that K35 and K43 strains possessed a stronger bactericidal activity against S. mutans than LGG. Moreover, quantitative real-time polymerase chain reaction showed that the expression of genes encoding glucosyltransferases, gtfB, gtfC, and gtfD was reduced when S. mutans were co-cultured with K35 or K43. However, LGG activated the expression of gtfB and gtfC, but did not influence the expression of gtfD in the co-culture. A transwell-based biofilm assay indicated that these lactobacilli inhibited S. mutans biofilm formation in a contact-independent manner. In conclusion, we identified two L. salivarius strains with inhibitory activities on the growth and expression of S. mutans virulence genes to reduce its biofilm formation. This is not a general characteristic of the species, so presents a potential strategy for in vivo alteration of plaque biofilm and caries. PMID:24961744