The role of biofilms in persistent infections and factors involved in ica-independent biofilm development and gene regulation in Staphylococcus aureus.

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Figueiredo, Agnes Marie Sá; Ferreira, Fabienne Antunes; Beltrame, Cristiana Ossaille; Côrtes, Marina Farrel

2017-09-01

Staphylococcus aureus biofilms represent a unique micro-environment that directly contribute to the bacterial fitness within hospital settings. The accumulation of this structure on implanted medical devices has frequently caused the development of persistent and chronic S. aureus-associated infections, which represent an important social and economic burden worldwide. ica-independent biofilms are composed of an assortment of bacterial products and modulated by a multifaceted and overlapping regulatory network; therefore, biofilm composition can vary among S. aureus strains. In the microniches formed by biofilms-produced by a number of bacterial species and composed by different structural components-drug refractory cell subpopulations with distinct physiological characteristics can emerge and result in therapeutic failures in patients with recalcitrant bacterial infections. In this review, we highlight the importance of biofilms in the development of persistence and chronicity in some S. aureus diseases, the main molecules associated with ica-independent biofilm development and the regulatory mechanisms that modulate ica-independent biofilm production, accumulation, and dispersion.

Extracellular DNA facilitates the formation of functional amyloids in Staphylococcus aureus biofilms.

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Schwartz, Kelly; Ganesan, Mahesh; Payne, David E; Solomon, Michael J; Boles, Blaise R

2016-01-01

Persistent staphylococcal infections often involve surface-associated communities called biofilms. Staphylococcus aureus biofilm development is mediated by the co-ordinated production of the biofilm matrix, which can be composed of polysaccharides, extracellular DNA (eDNA) and proteins including amyloid fibers. The nature of the interactions between matrix components, and how these interactions contribute to the formation of matrix, remain unclear. Here we show that the presence of eDNA in S. aureus biofilms promotes the formation of amyloid fibers. Conditions or mutants that do not generate eDNA result in lack of amyloids during biofilm growth despite the amyloidogeneic subunits, phenol soluble modulin peptides, being produced. In vitro studies revealed that the presence of DNA promotes amyloid formation by PSM peptides. Thus, this work exposes a previously unacknowledged interaction between biofilm matrix components that furthers our understanding of functional amyloid formation and S. aureus biofilm biology. © 2015 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

Colonization and biofilm formation by Staphylococcus aureus on endothelial cell layers under flow

DEFF Research Database (Denmark)

Grønnemose, Rasmus Birkholm; Antoinette Asferg, Cecilie; Kolmos, Hans Jørn

Staphylococcus aureus is a major human pathogen and known for causing vascular infections such as sepsis and infective endocarditis. It has previously been proposed that S. aureus succeed in colonization of the endothelial wall by specific surface attachment likely followed by biofilm formation....... Furthermore, S. aureus is known to invade human cells, which has been proposed to promote persistence through immune and antibiotic evasion. In the current study, we sought to investigate endothelial colonization, invasion, and biofilm formation by S. aureus using a newly developed in vitro flow chamber model....... We show that under physiological shear rates, S. aureus utilizes cellular invasion to enable the following surface colonization and biofilm formation. These observations might help explain the success of S. aureus as a bloodstream pathogen and guide further studies in S. aureus pathogenesis...

Streptokinase Treatment Reverses Biofilm-Associated Antibiotic Resistance in Staphylococcus aureus

DEFF Research Database (Denmark)

Jørgensen, Nis Pedersen; Zobek, Natalia; Dreier, Cindy

2016-01-01

by combining antibiotic treatment with a fibrinolytic drug. We quantified S. aureus USA300 biofilms grown on peg-lids in brain heart infusion (BHI) broth with 0%–50% human plasma. Young (2 h) and mature (24 h) biofilms were then treated with streptokinase to determine if this lead to dispersal. Then......, the minimal biofilm eradication concentration (MBEC) of 24 h old biofilms was measured for vancomycin and daptomycin alone or in combination with 10 µg/mL rifampicin in the presence or absence of streptokinase in the antibiotic treatment step. Finally, biofilms were visualized by confocal laser scanning...... or daptomycin, which are commonly used antibiotics for treatment of S. aureus infections. Fibronolytic drugs have been used to treat thromboembolic events for decades, and our findings suggest that their use against biofilm infections has the potential to improve the efficacy of antibiotics in treatment of S...

Inhibitory effects of flavonoids on biofilm formation by Staphylococcus aureus that overexpresses efflux protein genes.

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Lopes, Laênia Angélica Andrade; Dos Santos Rodrigues, Jéssica Bezerra; Magnani, Marciane; de Souza, Evandro Leite; de Siqueira-Júnior, José P

2017-06-01

This study evaluated the efficacy of glycone (myricitrin, hesperidin and phloridzin) and aglycone flavonoids (myricetin, hesperetin and phloretin) in inhibiting biofilm formation by Staphylococcus aureus RN4220 and S. aureus SA1199B that overexpress the msrA and norA efflux protein genes, respectively. The minimum inhibitory concentration (MIC) and minimum biofilm inhibitory concentration (MBIC 50 - defined as the lowest concentration that resulted in ≥50% inhibition of biofilm formation) of flavonoids were determined using microdilution in broth procedures. The flavonoids showed MIC >1024 μg/mL against S. aureus RN4220 and S. aureus SA1199B; however, these compounds at lower concentrations (1-256 μg/mL) showed inhibitory effects on biofilm formation by these strains. Aglycone flavonoids showed lower MBIC 50 values than their respective glycone forms. The lowest MBIC 50 values (1 and 4 μg/mL) were observed against S. aureus RN4220. Myricetin, hesperetin and phloretin exhibited biofilm formation inhibition >70% for S. aureus RN4220, and lower biofilm formation inhibition against S. aureus SA1199B. These results indicate that sub-MICs of the tested flavonoids inhibit biofilm formation by S. aureus strains that overexpress efflux protein genes. These effects are more strongly established by aglycone flavonoids. Copyright © 2017 Elsevier Ltd. All rights reserved.

Combined Staining Techniques for Demonstration of Staphylococcus aureus Biofilm in Routine Histopathology

DEFF Research Database (Denmark)

Jensen, Louise Kruse; Henriksen, Nicole Lind; Bjarnsholt, Thomas

2018-01-01

Aim: Visualization of Staphylococcus aureus biofilm using histochemical staining and combined histochemistry (HC) and immunohistochemistry (IHC). Methods: The ability of S. aureus S54F9 to form biofilm was tested in vitro. Hereafter, infected bone tissue was collected from two different porcine m...

Bioactive Compounds Produced by Hypoxylon fragiforme against Staphylococcus aureus Biofilms

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Kamila Tomoko Yuyama

2017-12-01

Full Text Available Treating infections organized in biofilms is a challenge due to the resistance of the pathogens against antibiotics and host immune cells. Many fungi grow in a wet environment, favorable for the growth of bacterial biofilms, and we speculated that fungi possess some strategies to control these bacterial biofilms. A fungus identified as Hypoxylon fragiforme, was collected in the Harz Mountains, Germany, and its mycelial culture was fermented in different culture media for 67 days to test its biological potential against bacterial biofilms. Sclerin, sclerin diacid and its 3-methyl monoester (methyl 1-(5-hydroxy-6-carboxylic-2,3,4-trimethylphenyl propionate are here described for the first time from this fungus. Sclerin and its diacid interfered with the biofilm formation of the pathogen Staphylococcus aureus, inhibiting 86% and 80% of the biofilm at 256 μg mL−1, respectively, but not killing the bacterium. Interestingly, the monomethylester of sclerin diacid was inactive. Although these compounds did not possess any activity against a pre-formed biofilm, they prevented its formation at subtoxic concentrations. Furthermore, sclerin and its diacid displayed a high specificity against Staphylococcus aureus, indicating a good strategy against pathogenic biofilms when combined with antibiotics.

Enhancement of photodynamic inactivation of Staphylococcus aureus biofilms by disruptive strategies.

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Gándara, Lautaro; Mamone, Leandro; Bohm, Gabriela Cervini; Buzzola, Fernanda; Casas, Adriana

2017-11-01

Photodynamic inactivation (PDI) has been used to inactivate microorganisms through the use of photosensitizers and visible light. On the one hand, near-infrared treatment (NIRT) has also bactericidal and dispersal effects on biofilms. In addition, dispersal biological tools such as enzymes have also been employed in antibiotic combination treatments. The aim of this work was to use alternative approaches to increase the PDI efficacy, employing combination therapies aimed at the partial disruption of the biofilms, thus potentially increasing photosensitizer or oxygen penetration and interaction with bacteria. To that end, we applied toluidine blue (TB)-PDI treatment to Staphylococcus aureus biofilms previously treated with NIRT or enzymes and investigated the outcome of the combined therapies. TB employed at 0.5 mM induced per se 2-log drop in S. aureus RN6390 biofilm viability. Each NIRT (980-nm laser) and PDI (635-nm laser) treatment induced a further reduction of 1-log of viable counts. The combination of successive 980- and 635-nm laser treatments on TB-treated biofilms induced additive effects, leading to a 4.5-log viable count decrease. Proteinase K treatment applied to S. aureus of the Newman strain induced an additive effect on PDI mortality, leading to an overall 4-log decrease in S. aureus viability. Confocal scanning laser microscopy after biofilm staining with a fluorescent viability test and scanning electron microscopy observations were correlated with colony counts. The NIRT dose employed (227 J/cm 2 ) led to an increase from 21 to 47 °C in the buffer temperature of the biofilm system, and this NIRT dose also induced 100% keratinocyte death. Further work is needed to establish conditions under which biofilm dispersal occurs at lower NIRT doses.

Metabolic activity, urease production, antibiotic resistance and virulence in dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus

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Vandecandelaere, Ilse; Van Nieuwerburgh, Filip; Deforce, Dieter

2017-01-01

In this paper, the metabolic activity in single and dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus isolates was investigated. Our results demonstrated that there was less metabolic activity in dual species biofilms compared to S. aureus biofilms. However, this was not observed if S. aureus and S. epidermidis were obtained from the same sample. The largest effect on metabolic activity was observed in biofilms of S. aureus Mu50 and S. epidermidis ET-024. A transcriptomic analysis of these dual species biofilms showed that urease genes and genes encoding proteins involved in metabolism were downregulated in comparison to monospecies biofilms. These results were subsequently confirmed by phenotypic assays. As metabolic activity is related to acid production, the pH in dual species biofilms was slightly higher compared to S. aureus Mu50 biofilms. Our results showed that S. epidermidis ET-024 in dual species biofilms inhibits metabolic activity of S. aureus Mu50, leading to less acid production. As a consequence, less urease activity is required to compensate for low pH. Importantly, this effect was biofilm-specific. Also S. aureus Mu50 genes encoding virulence-associated proteins (Spa, SplF and Dps) were upregulated in dual species biofilms compared to monospecies biofilms and using Caenorhabditis elegans infection assays, we demonstrated that more nematodes survived when co-infected with S. epidermidis ET-024 and S. aureus mutants lacking functional spa, splF or dps genes, compared to nematodes infected with S. epidermidis ET-024 and wild- type S. aureus. Finally, S. epidermidis ET-024 genes encoding resistance to oxacillin, erythromycin and tobramycin were upregulated in dual species biofilms and increased resistance was subsequently confirmed. Our data indicate that both species in dual species biofilms of S. epidermidis and S. aureus influence each other’s behavior, but additional studies are required necessary to elucidate the exact

Development of a Standard Test to Assess the Resistance of Staphylococcus aureus Biofilm Cells to Disinfectants

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Luppens, S.B.I.; Reij, M.W.; Heijden, van der R.W.; Rombouts, F.M.; Abee, T.

2002-01-01

A standardized disinfectant test for Staphylococcus aureus cells in biofilms was developed. Two disinfectants, the membrane-active compound benzalkonium chloride (BAC) and the oxidizing agent sodium hypochlorite, were used to evaluate the biofilm test. S. aureus formed biofilms on glass, stainless

Bactericidal and Anti-biofilm Effects of Polyhexamethylene Biguanide in Models of Intracellular and Biofilm of Staphylococcus aureus Isolated from Bovine Mastitis

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Nor F. Kamaruzzaman

2017-08-01

Full Text Available Staphylococcus aureus infection is a common cause of mastitis, reducing milk yield, affecting animal welfare and causing huge economic losses within the dairy industry. In addition to the problem of acquired drug resistance, bacterial invasion into udder cells and the formation of surface biofilms are believed to reduce antibiotic efficacy, leading to treatment failure. Here, we investigated the antimicrobial activities of enrofloxacin, an antibiotic that is commonly used in mastitis therapy and polyhexamethylene biguanide (PHMB, an antimicrobial polymer. The antimicrobial activities were tested against intracellular S. aureus in infected Mac-T cells (host cells. Also, fluorescein-tagged PHMB was used to study PHMB uptake and localization with S. aureus within the infected Mac-T cells. Anti-biofilm activities were tested by treating S. aureus biofilms and measuring effects on biofilm mass in vitro. Enrofloxacin and PHMB at 15 mg/L killed between 42 to 92 and 99.9% of intracellular S. aureus, respectively. PHMB-FITC entered and colocalized with the intracellular S. aureus, suggesting direct interaction of the drug with the bacteria inside the host cells. Enrofloxacin and PHMB at 15 mg/L reduced between 10 to 27% and 28 to 37% of biofilms’ mass, respectively. The half-maximal inhibitory concentrations (IC50 obtained from a cytotoxicity assay were 345 ± 91 and 21 ± 2 mg/L for enrofloxacin and PHMB, respectively; therefore, both compounds were tolerated by the host cells at high concentrations. These findings suggest that both antimicrobials are effective against intracellular S. aureus and can disrupt biofilm structures, with PHMB being more potent against intracellular S. aureus, highlighting the potential application of PHMB in mastitis therapy.

Combinations of maggot excretions/secretions and antibiotics are effective against Staphylococcus aureus biofilms and the bacteria derived therefrom

DEFF Research Database (Denmark)

van der Plas, Mariena J A; Dambrot, Cheryl; Dogterom-Ballering, Heleen C M

2010-01-01

OBJECTIVES: Maggots of the blowfly Lucilia sericata are used for the treatment of chronic wounds. Previously we reported that maggot excretions/secretions (ES) break down Staphylococcus aureus biofilms but do not kill the bacteria. As many antibiotics are not effective against biofilms we assessed...... the effect of combinations of ES and antibiotics on S. aureus biofilms and on the survival of the bacteria released from the biofilms. METHODS: Effects of ES, antibiotics (vancomycin, daptomycin or clindamycin) and combinations thereof on S. aureus ATCC 29 213 biofilms and bacterial viability were determined...... using microtitre plates and in vitro killing assays. RESULTS: Vancomycin and daptomycin dose-dependently enhanced biofilm formation, whereas clindamycin reduced S. aureus biofilm size. Adding ES to antibiotic incubations caused a complete biofilm breakdown. After a lag time the bacteria derived from...

Alpha-Toxin Promotes Mucosal Biofilm Formation by Staphylococcus aureus

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Michele J Anderson

2012-05-01

Full Text Available Staphylococcus aureus causes numerous diseases in humans ranging from the mild skin infections to serious, life-threatening, superantigen-mediated Toxic Shock Syndrome (TSS. S. aureus may also be asymptomatically carried in the anterior nares, vagina or on the skin, which serve as reservoirs for infection. Pulsed-field gel electrophoresis clonal type USA200 is the most widely disseminated colonizer and a major cause of TSS. Our prior studies indicated that α-toxin was a major epithelial proinflammatory exotoxin produced by TSS S. aureus USA200 isolates. It also facilitated the penetration of TSS Toxin-1 (TSST-1 across vaginal mucosa. However, the majority of menstrual TSS isolates produce low α-toxin due to a nonsense point mutation at codon 113, designated hly, suggesting mucosal adaptation. The aim of this study was to characterize the differences between TSS USA200 strains [high (hla+ and low (hly+ α-toxin producers] in their abilities to infect and disrupt vaginal mucosal tissue. A mucosal model was developed using ex vivo porcine vaginal mucosa, LIVE/DEAD® staining and confocal microscropy to characterize biofilm formation and tissue viability of TSS USA 200 isolates CDC587 and MN8, which contain the α-toxin pseudogene (hly, MNPE (hla+ and MNPE isogenic hla knockout (hlaKO. All TSS strains grew to similar bacterial densities (1-5 x 108 CFU on the mucosa and were proinflammatory over 3 days. However, MNPE formed biofilms with significant reductions in the mucosal viability whereas neither CDC587, MN8 (hly+, or MNPE hlaKO, formed biofilms and were less cytotoxic. The addition of exogenous, purified α-toxin to MNPE hlaKO restored the biofilm phenotype. Our studies suggest α-toxin affects S. aureus phenotypic growth on vaginal mucosa, by promoting tissue disruption and biofilm formation; and α–toxin mutants (hly are not benign colonizers, but rather form a different type of infection, which we have termed high density pathogenic

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

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Ashish Kumar Singh

2017-01-01

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

Commensal Protection of Staphylococcus aureus against Antimicrobials by Candida albicans Biofilm Matrix

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Kong, Eric F.; Tsui, Christina; Kucharíková, Sona; Andes, David

2016-01-01

ABSTRACT Biofilm-associated polymicrobial infections, particularly those involving fungi and bacteria, are responsible for significant morbidity and mortality and tend to be challenging to treat. Candida albicans and Staphylococcus aureus specifically are considered leading opportunistic fungal and bacterial pathogens, respectively, mainly due to their ability to form biofilms on catheters and indwelling medical devices. However, the impact of mixed-species biofilm growth on therapy remains largely understudied. In this study, we investigated the influence of C. albicans secreted cell wall polysaccharides on the response of S. aureus to antibacterial agents in biofilm. Results demonstrated significantly enhanced tolerance for S. aureus to drugs in the presence of C. albicans or its secreted cell wall polysaccharide material. Fluorescence confocal time-lapse microscopy revealed impairment of drug diffusion through the mixed biofilm matrix. Using C. albicans mutant strains with modulated cell wall polysaccharide expression, exogenous supplementation, and enzymatic degradation, the C. albicans-secreted β-1,3-glucan cell wall component was identified as the key matrix constituent providing the bacteria with enhanced drug tolerance. Further, antibody labeling demonstrated rapid coating of the bacteria by the C. albicans matrix material. Importantly, via its effect on the fungal biofilm matrix, the antifungal caspofungin sensitized the bacteria to the drugs. Understanding such symbiotic interactions with clinical relevance between microbial species in biofilms will greatly aid in overcoming the limitations of current therapies and in defining potential new targets for treating polymicrobial infections. PMID:27729510

Calcium-chelating alizarin and other anthraquinones inhibit biofilm formation and the hemolytic activity of Staphylococcus aureus.

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Lee, Jin-Hyung; Kim, Yong-Guy; Yong Ryu, Shi; Lee, Jintae

2016-01-14

Staphylococcal biofilms are problematic and play a critical role in the persistence of chronic infections because of their abilities to tolerate antimicrobial agents. Thus, the inhibitions of biofilm formation and/or toxin production are viewed as alternative means of controlling Staphylococcus aureus infections. Here, the antibiofilm activities of 560 purified phytochemicals were examined. Alizarin at 10 μg/ml was found to efficiently inhibit biofilm formation by three S. aureus strains and a Staphylococcus epidermidis strain. In addition, two other anthraquinones purpurin and quinalizarin were found to have antibiofilm activity. Binding of Ca(2+) by alizarin decreased S. aureus biofilm formation and a calcium-specific chelating agent suppressed the effect of calcium. These three anthraquinones also markedly inhibited the hemolytic activity of S. aureus, and in-line with their antibiofilm activities, increased cell aggregation. A chemical structure-activity relationship study revealed that two hydroxyl units at the C-1 and C-2 positions of anthraquinone play important roles in antibiofilm and anti-hemolytic activities. Transcriptional analyses showed that alizarin repressed the α-hemolysin hla gene, biofilm-related genes (psmα, rbf, and spa), and modulated the expressions of cid/lrg genes (the holin/antiholin system). These findings suggest anthraquinones, especially alizarin, are potentially useful for controlling biofilm formation and the virulence of S. aureus.

Effects of Low-Dose Amoxicillin on Staphylococcus aureus USA300 Biofilms.

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Mlynek, Kevin D; Callahan, Mary T; Shimkevitch, Anton V; Farmer, Jackson T; Endres, Jennifer L; Marchand, Mélodie; Bayles, Kenneth W; Horswill, Alexander R; Kaplan, Jeffrey B

2016-05-01

Previous studies showed that sub-MIC levels of β-lactam antibiotics stimulate biofilm formation in most methicillin-resistant Staphylococcus aureus (MRSA) strains. Here, we investigated this process by measuring the effects of sub-MIC amoxicillin on biofilm formation by the epidemic community-associated MRSA strain USA300. We found that sub-MIC amoxicillin increased the ability of USA300 cells to attach to surfaces and form biofilms under both static and flow conditions. We also found that USA300 biofilms cultured in sub-MIC amoxicillin were thicker, contained more pillar and channel structures, and were less porous than biofilms cultured without antibiotic. Biofilm formation in sub-MIC amoxicillin correlated with the production of extracellular DNA (eDNA). However, eDNA released by amoxicillin-induced cell lysis alone was evidently not sufficient to stimulate biofilm. Sub-MIC levels of two other cell wall-active agents with different mechanisms of action-d-cycloserine and fosfomycin-also stimulated eDNA-dependent biofilm, suggesting that biofilm formation may be a mechanistic adaptation to cell wall stress. Screening a USA300 mariner transposon library for mutants deficient in biofilm formation in sub-MIC amoxicillin identified numerous known mediators of S. aureus β-lactam resistance and biofilm formation, as well as novel genes not previously associated with these phenotypes. Our results link cell wall stress and biofilm formation in MRSA and suggest that eDNA-dependent biofilm formation by strain USA300 in low-dose amoxicillin is an inducible phenotype that can be used to identify novel genes impacting MRSA β-lactam resistance and biofilm formation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Characterization of the effect of serum and chelating agents on Staphylococcus aureus biofilm formation; chelating agents augment biofilm formation through clumping factor B

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Abraham, Nabil Mathew

Staphylococcus aureus is the causative agent of a diverse array of acute and chronic infections, and some these infections, including infective endocarditis, joint infections, and medical device-associated bloodstream infections, depend upon its capacity to form tenacious biofilms on surfaces. Inserted medical devices such as intravenous catheters, pacemakers, and artificial heart valves save lives, but unfortunately, they can also serve as a substrate on which S. aureus can form a biofilm, attributing S. aureus as a leading cause of medical device-related infections. The major aim of this work was take compounds to which S. aureus would be exposed during infection and to investigate their effects on its capacity to form a biofilm. More specifically, the project investigated the effects of serum, and thereafter of catheter lock solutions on biofilm formation by S. aureus. Pre-coating polystyrene with serum is frequently used as a method to augment biofilm formation. The effect of pre-coating with serum is due to the deposition of extracellular matrix components onto the polystyrene, which are then recognized by MSCRAMMs. We therefore hypothesized that the major component of blood, serum, would induce biofilm formation. Surprisingly, serum actually inhibited biofilm formation. The inhibitory activity was due to a small molecular weight, heat-stable, non-proteinaceous component/s of serum. Serum-mediated inhibition of biofilm formation may represent a previously uncharacterized aspect of host innate immunity that targets the expression of a key bacterial virulence factor: the ability to establish a resistant biofilm. Metal ion chelators like sodium citrate are frequently chosen to lock intravenous catheters because they are regarded as potent inhibitors of bacterial biofilm formation and viability. We found that, while chelating compounds abolished biofilm formation in most strains of S. aureus, they actually augmented the phenotype in a subset of strains. We

5-aminolevulinic acid-mediated photodynamic therapy and its strain-dependent combined effect with antibiotics on Staphylococcus aureus biofilm.

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Qing-Zhao Zhang

Full Text Available Staphylococcus aureus (S. aureus is hard to be eradicated, not only due to the emergence of antibiotic resistant strains but also because of its ability to form biofilm. Antibiotics are the major approach to treating biofilm infections, but their effects are unsatisfactory. One of the potential alternative treatments for controlling biofilm infections is photodynamic therapy (PDT, which requires the administration of photosensitizer, followed by light activation. 5-aminolevulinic acid (ALA, a natural photosensitizer prodrug, presents favorable characteristics, such as easy penetration and rapid clearance. These advantages enable ALA-based PDT (ALA-PDT to be well-tolerated by patients and it can be repeatedly applied without cumulative toxicity or serious side effects. ALA-PDT has been proven to be an effective treatment for multidrug resistant pathogens; however, the study of its effect on S. aureus biofilm is limited. Here, we established our PDT system based on the utilization of ALA and a light-emitting diode, and we tested the effect of ALA-PDT on S. aureus biofilm as well as the combined effect of ALA-PDT and antibiotics on S. aureus biofilm. Our results showed that ALA-PDT has a strong antibacterial effect on S. aureus biofilm, which was confirmed by the confocal laser scanning microscope. We also found that lethal photosensitization occurred predominantly in the upper layer of the biofilm, while the residual live bacteria were located in the lower layer of the biofilm. In addition, the improved bactericidal effect was observed in the combined treatment group but in a strain-dependent manner. Our results suggest that ALA-PDT is a potential alternative approach for future clinical use to treat S. aureus biofilm-associated infections, and some patients may benefit from the combined treatment of ALA-PDT and antibiotics, but drug sensitivity testing should be performed in advance.

Anti-biofilm formation of a novel stainless steel against Staphylococcus aureus.

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Nan, Li; Yang, Ke; Ren, Guogang

2015-06-01

Staphylococcus aureus (S. aureus) is a bacterium frequently found proliferating on metal surfaces such as stainless steels used in healthcare and food processing facilities. Past research has shown that a novel Cu-bearing 304 type stainless steel (304CuSS) exhibits excellent antibacterial ability (i.e. against S. aureus) in a short time period (24h.). This work was dedicated to investigate the 304CuSS's inhibition ability towards the S. aureus biofilm formation for an extended period of 7days after incubation. It was found that the antibacterial rate of the 304CuSS against sessile bacterial cells reached over 99.9% in comparison with the 304SS. The thickness and sizes of the biofilms on the 304SS surfaces increased markedly with period of contact, and thus expected higher risk of bio-contamination, indicated by the changes of surface free energy between biofilm and the steel surfaces. The results demonstrated that the 304CuSS exhibited strong inhibition on the growth and adherence of the biofilms. The surface free energy of the 304CuSS after contact with sessile bacterial cells was much lower than that of the 304SS towards the same culture times. The continuously dissolved Cu(2+) ions well demonstrated the dissolution ability of Cu-rich precipitates after exposure to S. aureus solution, from 3.1ppm (2days) to 4.5ppm (7days). For this to occur, a hypothesis mechanism might be established for 304CuSS in which the Cu(2+) ions were released from Cu-rich phases that bond with extracellular polymeric substances (EPS) of the microorganisms. And these inhibited the activities of cell protein/enzymes and effectively prevented planktonic bacterial cells attaching to the 304CuSS metal surface. Copyright © 2015. Published by Elsevier B.V.

Anti-biofilm formation of a novel stainless steel against Staphylococcus aureus

International Nuclear Information System (INIS)

Nan, Li; Yang, Ke; Ren, Guogang

2015-01-01

Staphylococcus aureus (S. aureus) is a bacterium frequently found proliferating on metal surfaces such as stainless steels used in healthcare and food processing facilities. Past research has shown that a novel Cu-bearing 304 type stainless steel (304CuSS) exhibits excellent antibacterial ability (i.e. against S. aureus) in a short time period (24 h.). This work was dedicated to investigate the 304CuSS's inhibition ability towards the S. aureus biofilm formation for an extended period of 7 days after incubation. It was found that the antibacterial rate of the 304CuSS against sessile bacterial cells reached over 99.9% in comparison with the 304SS. The thickness and sizes of the biofilms on the 304SS surfaces increased markedly with period of contact, and thus expected higher risk of bio-contamination, indicated by the changes of surface free energy between biofilm and the steel surfaces. The results demonstrated that the 304CuSS exhibited strong inhibition on the growth and adherence of the biofilms. The surface free energy of the 304CuSS after contact with sessile bacterial cells was much lower than that of the 304SS towards the same culture times. The continuously dissolved Cu 2+ ions well demonstrated the dissolution ability of Cu-rich precipitates after exposure to S. aureus solution, from 3.1 ppm (2 days) to 4.5 ppm (7 days). For this to occur, a hypothesis mechanism might be established for 304CuSS in which the Cu 2+ ions were released from Cu-rich phases that bond with extracellular polymeric substances (EPS) of the microorganisms. And these inhibited the activities of cell protein/enzymes and effectively prevented planktonic bacterial cells attaching to the 304CuSS metal surface

Anti-biofilm formation of a novel stainless steel against Staphylococcus aureus

Energy Technology Data Exchange (ETDEWEB)

Nan, Li; Yang, Ke [Institute of Metal Research, Chinese Academy of Sciences, Shenyang 110016 (China); Ren, Guogang, E-mail: g.g.ren@herts.ac.uk [University of Hertfordshire, Hatfield AL10 9AB (United Kingdom)

2015-06-01

Staphylococcus aureus (S. aureus) is a bacterium frequently found proliferating on metal surfaces such as stainless steels used in healthcare and food processing facilities. Past research has shown that a novel Cu-bearing 304 type stainless steel (304CuSS) exhibits excellent antibacterial ability (i.e. against S. aureus) in a short time period (24 h.). This work was dedicated to investigate the 304CuSS's inhibition ability towards the S. aureus biofilm formation for an extended period of 7 days after incubation. It was found that the antibacterial rate of the 304CuSS against sessile bacterial cells reached over 99.9% in comparison with the 304SS. The thickness and sizes of the biofilms on the 304SS surfaces increased markedly with period of contact, and thus expected higher risk of bio-contamination, indicated by the changes of surface free energy between biofilm and the steel surfaces. The results demonstrated that the 304CuSS exhibited strong inhibition on the growth and adherence of the biofilms. The surface free energy of the 304CuSS after contact with sessile bacterial cells was much lower than that of the 304SS towards the same culture times. The continuously dissolved Cu{sup 2+} ions well demonstrated the dissolution ability of Cu-rich precipitates after exposure to S. aureus solution, from 3.1 ppm (2 days) to 4.5 ppm (7 days). For this to occur, a hypothesis mechanism might be established for 304CuSS in which the Cu{sup 2+} ions were released from Cu-rich phases that bond with extracellular polymeric substances (EPS) of the microorganisms. And these inhibited the activities of cell protein/enzymes and effectively prevented planktonic bacterial cells attaching to the 304CuSS metal surface.

Novel application for the prevention and treatment of Staphylococcus aureus biofilm formation

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Traba, Christian

Formation of bacterial biofilms at solid-liquid interfaces creates numerous problems in both industrial and biomedical sciences. In this dissertation, the application of plasma from two very different facets was studied. In part one, the susceptibility of pre-formed Staphylococcus aureus biofilms on biomaterials to different plasmas was investigated. It was found that the distinct chemical/physical properties of plasmas generated from oxygen, nitrogen, and argon all demonstrated very potent but very different anti-biofilm mechanisms of action. An in depth analysis of these results show: 1) different reactive species produced in each plasma demonstrate specific activity, and 2) the commonly associated etching effect could be manipulated and even controlled, depending on experimental conditions and the discharge gas. These studies provide insights into the anti-biofilm mechanisms of plasma as well as the effects of different reactive species on biofilm inactivation. Under experimental parameters, bacterial cells in Staphylococcus aureus biofilms were killed (>99.9%) by plasmas within minutes of exposure and no bacteria nor biofilm re-growth from discharge gas treated biofilms was observed throughout the life-span of the re-growth experiment. The decontamination ability of plasmas for the treatment of biofilm related infections on biomedical materials was confirmed and novel applications involving the use of low power argon and oxygen for the treatment of biofilm contaminated biomaterials and indwelling devices is proposed. The second facet of this dissertation explores the interaction between biofilm forming Staphylococcus aureus bacteria on different antibacterial/anti-biofilm surfaces. The antibiotic-free anti-fouling surfaces constructed in this study were generated from the plasma-assisted graft polymerization technique. These sophisticated surfaces were stable, biocompatible and capable of preventing biofilm formation on biomaterials and medical devices. Under

Effects of Subinhibitory Concentrations of Ceftaroline on Methicillin-Resistant Staphylococcus aureus (MRSA Biofilms.

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María Lázaro-Díez

Full Text Available Ceftaroline (CPT is a novel cephalosporin with in vitro activity against Staphylococcus aureus. Ceftaroline exhibits a level of binding affinity for PBPs in S. aureus including PBP2a of methicillin-resistant S. aureus (MRSA. The aims of this study were to investigate the morphological, physiological and molecular responses of MRSA clinical strains and MRSA biofilms to sub-MICs (1/4 and 1/16 MIC of ceftaroline by using transmission, scanning and confocal microscopy. We have also used quantitative Real-Time PCR to study the effect of sub-MICs of ceftaroline on the expression of the staphylococcal icaA, agrA, sarA and sasF genes in MRSA biofilms. In one set of experiments, ceftaroline was able to inhibit biofilm formation in all strains tested at MIC, however, a strain dependent behavior in presence of sub-MICs of ceftaroline was shown. In a second set of experiments, destruction of preformed biofilms by addition of ceftaroline was evaluated. Ceftaroline was able to inhibit biofilm formation at MIC in all strains tested but not at the sub-MICs. Destruction of preformed biofilms was strain dependent because the biofilm formed by a matrix-producing strain was resistant to a challenge with ceftaroline at MIC, whereas in other strains the biofilm was sensitive. At sub-MICs, the impact of ceftaroline on expression of virulence genes was strain-dependent at 1/4 MIC and no correlation between ceftaroline-enhanced biofilm formation and gene regulation was established at 1/16 MIC. Our findings suggest that sub-MICs of ceftaroline enhance bacterial attachment and biofilm formation by some, but not all, MRSA strains and, therefore, stress the importance of maintaining effective bactericidal concentrations of ceftaroline to fight biofilm-MRSA related infections.

Antimicrobial Resistance Profile of Planktonic and Biofilm Cells of Staphylococcus aureus and Coagulase-Negative Staphylococci

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Adilson de Oliveira

2016-09-01

Full Text Available The objective of the present study was to determine the antimicrobial resistance profile of planktonic and biofilm cells of Staphylococcus aureus and coagulase-negative staphylococci (CoNS. Two hundred Staphylococcus spp. strains were studied, including 50 S. aureus and 150 CoNS strains (50 S. epidermidis, 20 S. haemolyticus, 20 S. warneri, 20 S. hominis, 20 S. lugdunensis, and 20 S. saprophyticus. Biofilm formation was investigated by adherence to polystyrene plates. Positive strains were submitted to the broth microdilution method to determine the minimum inhibitory concentration (MIC for planktonic and biofilm cells and the minimal bactericidal concentration for biofilm cells (MBCB. Forty-nine Staphylococcus spp. strains (14 S. aureus, 13 S. epidermidis, 13 S. saprophyticus, 3 S. haemolyticus, 1 S. hominis, 3 S. warneri, and 2 S. lugdunensis were biofilm producers. These isolates were evaluated regarding their resistance profile. Determination of planktonic cell MIC identified three (21.4% S. aureus strains that were resistant to oxacillin and six (42.8% that were resistant to erythromycin. Among the CoNS, 31 (88.6% strains were resistant to oxacillin, 14 (40% to erythromycin, 18 (51.4% to gentamicin, and 8 (22.8% to sulfamethoxazole/trimethoprim. None of the planktonic isolates were resistant to vancomycin or linezolid. MICs were 2-, 4-, 8-, and up to 16-fold higher for biofilm cells than for planktonic cells. This observation was more common for vancomycin and erythromycin. The MBCB ranged from 8 to >256 µg/mL for oxacillin, 128 to >128 µg/mL for vancomycin, 256 to >256 µg/mL for erythromycin and gentamicin, >64 µg/mL for linezolid, and 32/608 to >32/608 µg/mL for sulfamethoxazole/trimethoprim. The results showed considerably higher MICs for S. aureus and CoNS biofilm cells compared to planktonic cells. Analysis of MBCM confirmed that even high concentrations of vancomycin were unable to eliminate the biofilms of S. aureus and Co

High Glucose Concentration Promotes Vancomycin-Enhanced Biofilm Formation of Vancomycin-Non-Susceptible Staphylococcus aureus in Diabetic Mice.

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Chi-Yu Hsu

Full Text Available We previously demonstrated that vancomycin treatment increased acquisition of eDNA and enhanced biofilm formation of drug-resistant Staphylococcus aureus through a cidA-mediated autolysis mechanism. Recently we found that such enhancement became more significant under a higher glucose concentration in vitro. We propose that besides improper antibiotic treatment, increased glucose concentration environment in diabetic animals may further enhance biofilm formation of drug-resistant S. aureus. To address this question, the diabetic mouse model infected by vancomycin-resistant S. aureus (VRSA was used under vancomycin treatment. The capacity to form biofilms was evaluated through a catheter-associated biofilm assay. A 10- and 1000-fold increase in biofilm-bound bacterial colony forming units was observed in samples from diabetic mice without and with vancomycin treatment, respectively, compared to healthy mice. By contrast, in the absence of glucose vancomycin reduced propensity to form biofilms in vitro through the increased production of proteases and DNases from VRSA. Our study highlights the potentially important role of increased glucose concentration in enhancing biofilm formation in vancomycin-treated diabetic mice infected by drug-resistant S. aureus.

Iron enhances the peptidyl deformylase activity and biofilm formation in Staphylococcus aureus.

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Swarupa, Vimjam; Chaudhury, Abhijit; Sarma, Potukuchi Venkata Gurunadha Krishna

2018-01-01

Staphylococcus aureus plays a major role in persistent infections and many of these species form structured biofilms on different surfaces which is accompanied by changes in gene expression profiles. Further, iron supplementation plays a critical role in the regulation of several protein(s)/enzyme function, which all aid in the development of active bacterial biofilms. It is well known that for each protein, deformylation is the most crucial step in biosynthesis and is catalyzed by peptidyl deformylase (PDF). Thus, the aim of the current study is to understand the role of iron in biofilm formation and deformylase activity of PDF. Hence, the PDF gene of S. aureus ATCC12600 was PCR amplified using specific primers and sequenced, followed by cloning and expression in Escherichia coli DH5α. The deformylase activity of the purified recombinant PDF was measured in culture supplemented with/without iron where the purified rPDF showed K m of 1.3 mM and V max of 0.035 mM/mg/min, which was close to the native PDF ( K m  = 1.4 mM, V max  = 0.030 mM/mg/min). Interestingly, the K m decreased and PDF activity increased when the culture was supplemented with iron, corroborating with qPCR results showing 100- to 150-fold more expression compared to control in S. aureus and its drug-resistant strains. Further biofilm-forming units (BU) showed an incredible increase (0.42 ± 0.005 to 6.3 ± 0.05 BU), i.e., almost 15-fold elevation in anaerobic conditions, indicating the significance of iron in S. aureus biofilms.

Searching for new strategies against biofilm infections: Colistin-AMP combinations against Pseudomonas aeruginosa and Staphylococcus aureus single- and double-species biofilms.

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Paula Jorge

Full Text Available Antimicrobial research is being pressured to look for more effective therapeutics for the ever-growing antibiotic-resistant infections, and antimicrobial peptides (AMP and antimicrobial combinations are promising solutions. This work evaluates colistin-AMP combinations against two major pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, encompassing non- and resistant strains. Colistin (CST combined with the AMP temporin A (TEMP-A, citropin 1.1 (CIT-1.1 and tachyplesin I linear analogue (TP-I-L was tested against planktonic, single- and double-species biofilm cultures. Overall synergy for planktonic P. aeruginosa and synergy/additiveness for planktonic S. aureus were observed. Biofilm growth prevention was achieved with synergy and additiveness. Pre-established 24 h-old biofilms were harder to eradicate, especially for S. aureus and double-species biofilms; still, some synergy and addictiveness was observed for higher concentrations, including for the biofilms of resistant strains. Different treatment times and growth media did not greatly influence AMP activity. CST revealed low toxicity compared with the other AMP but its combinations were toxic for high concentrations. Overall, combinations reduced effective AMP concentrations, mainly in prevention scenarios. Improvement of effectiveness and toxicity of therapeutic strategies will be further investigated.

Phosphorylation of Staphylococcus aureus Protein-Tyrosine Kinase Affects the Function of Glucokinase and Biofilm Formation.

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Vasu, Dudipeta; Kumar, Pasupuleti Santhosh; Prasad, Uppu Venkateswara; Swarupa, Vimjam; Yeswanth, Sthanikam; Srikanth, Lokanathan; Sunitha, Manne Mudhu; Choudhary, Abhijith; Sarma, Potukuchi Venkata Gurunadha Krishna

2017-03-01

When Staphylococcus aureus is grown in the presence of high concentration of external glucose, this sugar is phosphorylated by glucokinase (glkA) to form glucose-6-phosphate. This product subsequently enters into anabolic phase, which favors biofilm formation. The presence of ROK (repressor protein, open reading frame, sugar kinase) motif, phosphate-1 and -2 sites, and tyrosine kinase sites in glkA of S. aureus indicates that phosphorylation must regulate the glkA activity. The aim of the present study was to identify the effect of phosphorylation on the function of S. aureus glkA and biofilm formation. Pure glkA and protein-tyrosine kinase (BYK) of S. aureus ATCC 12600 were obtained by fractionating the cytosolic fractions of glkA1 and BYK-1 expressing recombinant clones through nickel metal chelate column. The pure glkA was used as a substrate for BYK and the phosphorylation of glkA was confirmed by treating with reagent A and resolving in SDS-PAGE, as well as staining with reagent A. The kinetic parameters of glkA and phosphorylated glkA were determined spectrophotometrically, and in silico tools were used for validation. S. aureus was grown in brain heart infusion broth, which was supplemented with glucose, and then biofilm units were calculated. Fourfold elevated glkA activity was observed upon the phosphorylation by BYK. Protein-protein docking analysis revealed that glkA structure docked close to the adenosine triphosphate-binding site of BYK structure corroborating the kinetic results. Further, S. aureus grown in the presence of elevated glucose concentration exhibited an increase in the rate of biofilm formation. The elevated function of glkA is an essential requirement for increased biofilm units in S. aureus, a key pathogenic factor that helps its survival and spread the infection.

Evaluation of combinations of putative anti-biofilm agents and antibiotics to eradicate biofilms of Staphylococcus aureus and Pseudomonas aeruginosa.

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Belfield, Katherine; Bayston, Roger; Hajduk, Nadzieja; Levell, Georgia; Birchall, John P; Daniel, Matija

2017-09-01

To evaluate potential anti-biofilm agents for their ability to enhance the activity of antibiotics for local treatment of localized biofilm infections. Staphylococcus aureus and Pseudomonas aeruginosa in vitro biofilm models were developed. The putative antibiotic enhancers N-acetylcysteine, acetylsalicylic acid, sodium salicylate, recombinant human deoxyribonuclease I, dispersin B, hydrogen peroxide and Johnson's Baby Shampoo (JBS) were tested for their anti-biofilm activity alone and their ability to enhance the activity of antibiotics for 7 or 14 days, against 5 day old biofilms. The antibiotic enhancers were paired with rifampicin and clindamycin against S. aureus and gentamicin and ciprofloxacin against P. aeruginosa. Isolates from biofilms that were not eradicated were tested for antibiotic resistance. Antibiotic levels 10× MIC and 100× MIC significantly reduced biofilm, but did not consistently eradicate it. Antibiotics at 100× MIC with 10% JBS for 14 days was the only treatment to eradicate both staphylococcal and pseudomonal biofilms. Recombinant human deoxyribonuclease I significantly reduced staphylococcal biofilm. Emergence of resistance of surviving isolates was minimal and was often associated with the small colony variant phenotype. JBS enhanced the activity of antibiotics and several other promising anti-biofilm agents were identified. Antibiotics with 10% JBS eradicated biofilms produced by both organisms. Such combinations might be useful in local treatment of localized biofilm infections. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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

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Watters CM

2016-04-01

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

Streptococcus pneumoniae eradicates preformed Staphylococcus aureus biofilms through a mechanism requiring physical contact

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Faidad Khan

2016-09-01

Full Text Available Staphylococcus aureus (Sau strains are a main cause of disease, including nosocomial infections which have been linked to the production of biofilms and the propagation of antibiotic resistance strains such as methicillin-resistant Staphylococcus aureus (MRSA. A previous study found that Streptococcus pneumoniae (Spn strains kill planktonic cultures of Sau strains. In this work, we have further evaluated in detail the eradication of Sau biofilms and investigated ultrastructural interactions of the biofilmicidal effect. Spn strain D39, which produces the competence stimulating peptide 1 (CSP1, reduced Sau biofilms within 8 h of inoculation, while TIGR4, producing CSP2, eradicated Sau biofilms and planktonic cells within 4 h. Differences were not attributed to pherotypes as other Spn strains producing different pheromones eradicated Sau within 4 h. Experiments using Transwell devices, which physically separated both species growing in the same well, demonstrated that direct contact between Spn and Sau was required to efficiently eradicate Sau biofilms and biofilm-released planktonic cells. Physical contact-mediated killing of Sau was not related to production of hydrogen peroxide as an isogenic TIGR4spxB mutant eradicated Sau bacteria within 4 h. Confocal micrographs confirmed eradication of Sau biofilms by TIGR4 and allowed us to visualize ultrastructural point of contacts between Sau and Spn. A time-course study further demonstrated spatial colocalization of Spn chains and Sau tetrads as early as 30 min post-inoculation (Pearson’s coefficient >0.72. Finally, precolonized biofilms produced by Sau strain Newman, or MRSA strain USA300, were eradicated by mid-log phase cultures of washed TIGR4 bacteria within 2 h post-inoculation. In conclusion, Spn strains rapidly eradicate pre-colonized Sau aureus biofilms, including those formed by MRSA strains, by a mechanism(s requiring bacterium-bacterium contact, but independent from the production of

Antimicrobial activity of zinc and titanium dioxide nanoparticles against biofilm-producing methicillin-resistant Staphylococcus aureus

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Jesline, A.; John, Neetu P.; Narayanan, P. M.; Vani, C.; Murugan, Sevanan

2015-02-01

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major nosocomial pathogens responsible for a wide spectrum of infections and the emergence of bacterial resistance to antibiotics has lead to treatment drawbacks towards large number of drugs. Formation of biofilms is the main contributing factor to antibiotic resistance. The development of reliable processes for the synthesis of zinc oxide nanoparticles is an important aspect of nanotechnology today. Zinc oxide and titanium dioxide nanoparticles comprise well-known inhibitory and bactericidal effects. Emergence of antimicrobial resistance by pathogenic bacteria is a major health problem in recent years. This study was designed to determine the efficacy of zinc and titanium dioxide nanoparticles against biofilm producing methicillin-resistant S. aureus. Biofilm production was detected by tissue culture plate method. Out of 30 MRSA isolates, 22 isolates showed strong biofilm production and 2 showed weak and moderate biofilm formation. Two strong and weak biofilm-producing methicillin-resistant S. aureus isolates were subjected to antimicrobial activity using commercially available zinc and titanium dioxide nanoparticles. Thus, the nanoparticles showed considerably good activity against the isolates, and it can be concluded that they may act as promising, antibacterial agents in the coming years.

Antimicrobial activity of essential oils against Staphylococcus aureus biofilms.

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Vázquez-Sánchez, Daniel; Cabo, Marta L; Rodríguez-Herrera, Juan J

2015-12-01

The present study was aimed to evaluate the potential of essential oils to remove the foodborne pathogen Staphylococcus aureus from food-processing facilities. The effectiveness of 19 essential oils against planktonic cells of S. aureus was firstly assessed by minimal inhibitory concentration. Planktonic cells showed a wide variability in resistance to essential oils, with thyme oil as the most effective, followed by lemongrass oil and then vetiver oil. The eight essential oils most effective against planktonic cells were subsequently tested against 48-h-old biofilms formed on stainless steel. All essential oils reduced significantly (p oils were the most effective, but high concentrations were needed to achieve logarithmic reductions over 4 log CFU/cm(2) after 30 min exposure. Alternatively, the use of sub-lethal doses of thyme oil allowed to slow down biofilm formation and to enhance the efficiency of thyme oil and benzalkonium chloride against biofilms. However, some cellular adaptation to thyme oil was detected. Therefore, essential oil-based treatments should be based on the rotation and combination of different essential oils or with other biocides to prevent the emergence of antimicrobial-resistant strains. © The Author(s) 2014.

Detection of Alpha-Toxin and Other Virulence Factors in Biofilms of Staphylococcus aureus on Polystyrene and a Human Epidermal Model.

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den Reijer, P M; Haisma, E M; Lemmens-den Toom, N A; Willemse, J; Koning, R I; Koning, R A; Demmers, J A A; Dekkers, D H W; Rijkers, E; El Ghalbzouri, A; Nibbering, P H; van Wamel, W

2016-01-01

The ability of Staphylococcus aureus to successfully colonize (a)biotic surfaces may be explained by biofilm formation and the actions of virulence factors. The aim of the present study was to establish the presence of 52 proteins, including virulence factors such as alpha-toxin, during biofilm formation of five different (methicillin resistant) S. aureus strains on Leiden human epidermal models (LEMs) and polystyrene surfaces (PS) using a competitive Luminex-based assay. All five S. aureus strains formed biofilms on PS, whereas only three out of five strains formed biofilms on LEMs. Out of the 52 tested proteins, six functionally diverse proteins (ClfB, glucosaminidase, IsdA, IsaA, SACOL0688 and nuclease) were detected in biofilms of all strains on both PS and LEMs. At the same time, four toxins (alpha-toxin, gamma-hemolysin B and leukocidins D and E), two immune modulators (formyl peptide receptor-like inhibitory protein and Staphylococcal superantigen-like protein 1), and two other proteins (lipase and LytM) were detectable in biofilms by all five S. aureus strains on LEMs, but not on PS. In contrast, fibronectin-binding protein B (FnbpB) was detectable in biofilms by all S. aureus biofilms on PS, but not on LEMs. These data were largely confirmed by the results from proteomic and transcriptomic analyses and in case of alpha-toxin additionally by GFP-reporter technology. Functionally diverse virulence factors of (methicillin-resistant) S. aureus are present during biofilm formation on LEMs and PS. These results could aid in identifying novel targets for future treatment strategies against biofilm-associated infections.

Effect of peracetic acid on biofilms formed by Staphylococcus aureus and Listeria monocytogenes isolated from dairy plants.

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Lee, S H I; Cappato, L P; Corassin, C H; Cruz, A G; Oliveira, C A F

2016-03-01

This research investigated the removal of adherent cells of 4 strains of Staphylococcus aureus and 1 Listeria monocytogenes strain (previously isolated from dairy plants) from polystyrene microtiter plates using peracetic acid (PAA, 0.5%) for 15, 30, 60, and 120 s, and the inactivation of biofilms formed by those strains on stainless steel coupons using the same treatment times. In the microtiter plates, PAA removed all S. aureus at 15 s compared with control (no PAA treatment). However, L. monocytogenes biofilm was not affected by any PAA treatment. On the stainless steel surface, epifluorescence microscopy using LIVE/DEAD staining (BacLight, Molecular Probes/Thermo Fisher Scientific, Eugene, OR) showed that all strains were damaged within 15 s, with almost 100% of cells inactivated after 30 s. Results of this trial indicate that, although PAA was able to inactivate both S. aureus and L. monocytogenes monospecies biofilms on stainless steel, it was only able to remove adherent cells of S. aureus from polystyrene microplates. The correct use of PAA is critical for eliminating biofilms formed by S. aureus strains found in dairy plants, although further studies are necessary to determine the optimal PAA treatment for removing biofilms of L. monocytogenes. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

In vitro biofilm formation by methicillin susceptible and resistant Staphylococcus aureus strains isolated from cystic fibrosis patients

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Antonietta Lambiase

2008-12-01

Full Text Available Staphylococcus aureus is one of the most common pathogens isolated from respiratory tracts of Cystic Fibrosis patients (CF. The infection by this pathogen starts in early infancy, often preceding chronic infections by Pseudomonas aeruginosa. The infection and colonization by methicillin-resistant Staphylococcus aureus (MRSA are, by then, events very frequent among CF patients and this bacterial isolation leads to complications in therapeutic management because of the limited treatment options. Strains of Staphylococcus aureus are able to produce biofilms on natural or synthetic surfaces. Biofilms are sophisticated communities of matrix-encased bacteria and infections by biofilm-producing bacteria are particularly problematic because sessile bacteria can often withstand host immune responses and are generally much more tolerant to antibiotics. The first aim of this work is to evaluate the ability of MRSA strains isolated from respiratory secretions of CF patients to develop biofilms in comparison with methicillin-sensitive Staphylococcus aureus (MSSA strains obtained from respiratory secretions of CF patients.Therefore, our second aim is to evaluate the environmental influence on this ability. To evaluate the development of biofilm on solid matrix and the possible environmental influence,we applied the method described by Christensen et al. We found that a significantly higher number of MRSA strains were biofilm positive compared with MSSA strains (p<0.05.The presence of glucose did not influence the ability to form biofilm in our MRSA strains (p=0.165. MSSA strains are not strong biofilm-producers, but, when grown in TSB added with 0.25% glucose, the number of biofilm-forming strains increases, as expected. These data suggest a possible association between methicillin-resistance and biofilm formation.

Impaired respiration elicits SrrAB-dependent programmed cell lysis and biofilm formation in Staphylococcus aureus

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Mashruwala, Ameya A; van de Guchte, Adriana; Boyd, Jeffrey M

2017-01-01

Biofilms are communities of microorganisms attached to a surface or each other. Biofilm-associated cells are the etiologic agents of recurrent Staphylococcus aureus infections. Infected human tissues are hypoxic or anoxic. S. aureus increases biofilm formation in response to hypoxia, but how this occurs is unknown. In the current study we report that oxygen influences biofilm formation in its capacity as a terminal electron acceptor for cellular respiration. Genetic, physiological, or chemical inhibition of respiratory processes elicited increased biofilm formation. Impaired respiration led to increased cell lysis via divergent regulation of two processes: increased expression of the AtlA murein hydrolase and decreased expression of wall-teichoic acids. The AltA-dependent release of cytosolic DNA contributed to increased biofilm formation. Further, cell lysis and biofilm formation were governed by the SrrAB two-component regulatory system. Data presented support a model wherein SrrAB-dependent biofilm formation occurs in response to the accumulation of reduced menaquinone. DOI: http://dx.doi.org/10.7554/eLife.23845.001 PMID:28221135

The Activity of Cotinus coggygria Scop. Leaves on Staphylococcus aureus Strains in Planktonic and Biofilm Growth Forms

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Katarína Rendeková

2015-12-01

Full Text Available The purpose of this study was to detect the effectiveness of Cotinus coggygria Scop. leaves methanol extract against planktonic and biofilm growth forms of Staphylococcus aureus. The antimicrobial activity was determined by the broth microdilution test. Minimal inhibitory concentrations and minimal bactericidal concentrations were detected against two collection and ten clinical S. aureus strains. Anti-biofilm activity of the tested extract was detected using 24 h bacterial biofilm on the surface of microtiter plate wells. The biofilm inhibitory activity was evaluated visually after 24 h interaction of extract with biofilm, and the eradicating activity by a regrowth method. The tested extract showed bactericidal activity against all S. aureus strains (methicillin susceptible or methicillin resistant in concentrations ranging from 0.313 to 0.625 mg·mL−1. Biofilm inhibitory concentrations were 10-times higher and biofilm eradicating concentrations 100-times higher (8 and 32 mg·mL−1, respectively. The phytochemical analysis of C. coggygria leaves 60% methanol extract performed by LC-DAD-MS/MS revealed quercetin rhamnoside, methyl gallate, and methyl trigallate as main constituents. Results of our study indicate that C. coggygria, rich in tannins and flavonoids, seems to be a prospective topical antibacterial agent with anti-biofilm activity.

Efficacy of silk fibroin–nano silver against Staphylococcus aureus biofilms in a rabbit model of sinusitis

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Jia M

2017-04-01

Full Text Available Minghui Jia,1,2 Zhongchun Chen,2 Yongwei Guo,2 Xin Chen,3 Xia Zhao2 1Department of Otolaryngology-Head and Neck Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 2Department of Otolaryngology-Head and Neck Surgery, Huashan Hospital, Fudan University, 3State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Laboratory of Advanced Materials, Fudan University, Shanghai, China Background: Staphylococcus aureus biofilms contribute significantly to the recalcitrant nature of chronic rhinosinusitis. In previous studies, it has been shown that silk fibroin–nano silver solution can eliminate S. aureus biofilms in vitro, which suggests a potential role of this novel agent in the treatment of biofilm-associated diseases, such as sinusitis.Objective: The aim of this study was to investigate the efficacy of silk fibroin–nano silver solution as a topical anti-biofilm agent in a rabbit model of sinusitis.Methods: Biofilm-associated sinusitis models were established in 24 New Zealand White rabbits by gelatin sponge placement and S. aureus inoculation through a hole drilled into the anterolateral wall of the right maxillary sinus. After 4 weeks, indwelling catheters were placed into the maxillary sinus. Different concentrations of silk fibroin–nano silver solution or normal saline were irrigated slowly into the maxillary sinus via the indwelling catheters. After 7 days of irrigation, the rabbits were sacrificed. The sinus mucosa was harvested and examined for biofilm biomass as well as morphological integrity of the epithelium by scanning electron microscopy.Results: Silk fibroin–nano silver solution was found to be most effective in reducing the biomass of the S. aureus biofilms at a concentration of 384 mg/L, followed by the concentration of 153.6 mg/L, when compared with saline. After treatment with 384 mg/L silk fibroin–nano silver solution, the biofilms were completely

Coral-Associated Bacteria as a Promising Antibiofilm Agent against Methicillin-Resistant and -Susceptible Staphylococcus aureus Biofilms

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Shanmugaraj Gowrishankar

2012-01-01

Full Text Available The current study deals with the evaluation of two coral-associated bacterial (CAB extracts to inhibit the biofilm synthesis in vitro as well as the virulence production like hemolysin and exopolysaccharide (EPS, and also to assess their ability to modify the adhesion properties, that is cell surface hydrophobicity (CSH of methicillin-resistant (MRSA and -susceptible Staphylococcus aureus (MSSA. Out of nine CAB screened, the ethyl acetate extract of CAB-E2 (Bacillus firmus and CAB-E4 (Vibrio parahemolyticus have shown excellent antibiofilm activity against S. aureus. CAB-E2 reduced the production of EPS (57–79% and hemolysin (43–70%, which ultimately resulted in the significant inhibition of biofilms (80–87% formed by both MRSA and MSSA. Similarly, CAB-E4 was also found to decrease the production of EPS (43–57%, hemolysin (43–57% and biofilms (80–85% of test pathogens. CLSM analysis also proved the antibiofilm efficacy of CAB extracts. Furthermore, the CAB extracts strongly decreased the CSH of S. aureus. Additionally, FT-IR analysis of S. aureus treated with CAB extracts evidenced the reduction in cellular components compared to their respective controls. Thus, the present study reports for the first time, B. firmus—a coral-associated bacterium, as a promising source of antibiofilm agent against the recalcitrant biofilms formed by multidrug resistant S. aureus.

9 CFR 113.104 - Leptospira Grippotyphosa Bacterin.

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2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Leptospira Grippotyphosa Bacterin. 113... REQUIREMENTS Inactivated Bacterial Products § 113.104 Leptospira Grippotyphosa Bacterin. Leptospira Grippotyphosa Bacterin shall be produced from a culture of Leptospira grippotyphosa which has been inactivated...

Detection of Intracellular Adhesion (ica and Biofilm Formation Genes in Staphylococcus aureus Isolates from Clinical Samples

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Khadije Rezaie Keikhaie

2017-02-01

Full Text Available Introduction: Nosocomial infections that result in the formation of biofilms on the surfaces of biomedical implants are a leading cause of sepsis and are often associated with colonization of the implants by Staphylococcus epidermidis. Biofilm formation is thought to require two sequential steps: adhesion of cells to a solid substrate followed by cell-cell adhesion, creating multiple layers of cells. Intercellular adhesion requires the polysaccharide intercellular adhesion (PIA, which is composed of linear β-1, 6-linked glucosaminylglycans and can be synthesized in vitro from UDP-N-acetylglucosamine by products of the intercellular adhesion (ica locus. We have investigated a variety of Staphylococcus aureus strains and find that all strains tested contain the ica locus and that several can form biofilms in vitro. Material and Method: A total of 31 clinical S. aureus isolates were collected from Zabol, Iran. In vitro biofilm formation ability was determined by microliter tissue culture plates. All clinical isolates were examined for determination the ica locus by using PCR method. Result: The results of this study showed that 40 strains of Staphylococcus aureus, 12 strains carrying the gene Cocos icaA (30% and 8 strains carrying the gene icaD (20% and the number of five strains (12.5% containing both genes ica A and has been ica D. Conclusions:  S. aureus clinical isolates have different ability to form biofilm. This may be caused by the differences in the expression of biofilm related genes, genetic make-up and physiological conditions.

Transcriptional and functional analysis of the effects of magnolol: inhibition of autolysis and biofilms in Staphylococcus aureus.

Science.gov (United States)

Wang, Dacheng; Jin, Qi; Xiang, Hua; Wang, Wei; Guo, Na; Zhang, Kaiyu; Tang, Xudong; Meng, Rizeng; Feng, Haihua; Liu, Lihui; Wang, Xiaohong; Liang, Junchao; Shen, Fengge; Xing, Mingxun; Deng, Xuming; Yu, Lu

2011-01-01

The targeting of Staphylococcus aureus biofilm structures are now gaining interest as an alternative strategy for developing new types of antimicrobial agents. Magnolol (MOL) shows inhibitory activity against S. aureus biofilms and Triton X-100-induced autolysis in vitro, although there are no data regarding the molecular mechanisms of MOL action in bacteria. The molecular basis of the markedly reduced autolytic phenotype and biofilm inhibition triggered by MOL were explored using transcriptomic analysis, and the transcription of important genes were verified by real-time RT-PCR. The inhibition of autolysis by MOL was evaluated using quantitative bacteriolytic assays and zymographic analysis, and antibiofilm activity assays and confocal laser scanning microscopy were used to elucidate the inhibition of biofilm formation caused by MOL in 20 clinical isolates or standard strains. The reduction in cidA, atl, sle1, and lytN transcript levels following MOL treatment was consistent with the induced expression of their autolytic repressors lrgA, lrgB, arlR, and sarA. MOL generally inhibited or reversed the expression of most of the genes involved in biofilm production. The growth of S. aureus strain ATCC 25923 in the presence of MOL dose-dependently led to decreases in Triton X-100-induced autolysis, extracellular murein hydrolase activity, and the amount of extracellular DNA (eDNA). MOL may impede biofilm formation by reducing the expression of cidA, a murein hydrolase regulator, to inhibit autolysis and eDNA release, or MOL may directly repress biofilm formation. MOL shows in vitro antimicrobial activity against clinical and standard S. aureus strains grown in planktonic and biofilm cultures, suggesting that the structure of MOL may potentially be used as a basis for the development of drugs targeting biofilms.

Transcriptional and functional analysis of the effects of magnolol: inhibition of autolysis and biofilms in Staphylococcus aureus.

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Dacheng Wang

Full Text Available BACKGROUND: The targeting of Staphylococcus aureus biofilm structures are now gaining interest as an alternative strategy for developing new types of antimicrobial agents. Magnolol (MOL shows inhibitory activity against S. aureus biofilms and Triton X-100-induced autolysis in vitro, although there are no data regarding the molecular mechanisms of MOL action in bacteria. METHODOLOGY/PRINCIPAL FINDINGS: The molecular basis of the markedly reduced autolytic phenotype and biofilm inhibition triggered by MOL were explored using transcriptomic analysis, and the transcription of important genes were verified by real-time RT-PCR. The inhibition of autolysis by MOL was evaluated using quantitative bacteriolytic assays and zymographic analysis, and antibiofilm activity assays and confocal laser scanning microscopy were used to elucidate the inhibition of biofilm formation caused by MOL in 20 clinical isolates or standard strains. The reduction in cidA, atl, sle1, and lytN transcript levels following MOL treatment was consistent with the induced expression of their autolytic repressors lrgA, lrgB, arlR, and sarA. MOL generally inhibited or reversed the expression of most of the genes involved in biofilm production. The growth of S. aureus strain ATCC 25923 in the presence of MOL dose-dependently led to decreases in Triton X-100-induced autolysis, extracellular murein hydrolase activity, and the amount of extracellular DNA (eDNA. MOL may impede biofilm formation by reducing the expression of cidA, a murein hydrolase regulator, to inhibit autolysis and eDNA release, or MOL may directly repress biofilm formation. CONCLUSIONS/SIGNIFICANCE: MOL shows in vitro antimicrobial activity against clinical and standard S. aureus strains grown in planktonic and biofilm cultures, suggesting that the structure of MOL may potentially be used as a basis for the development of drugs targeting biofilms.

Bacteriophage Therapy for Staphylococcus aureus Biofilm-Infected Wounds: A New Approach to Chronic Wound Care

Science.gov (United States)

2013-02-01

lidocaine and 1:100,000 epineph- rine at the planned wound sites. Six full-thickness dermal wounds, 6 mm in diameter, were created on the ventral ear...action were ineffective against S. aureus biofilm, as was seen with P. aeruginosa biofilm.22 Given the durability of biofilm in the face of a harsh

9 CFR 113.122 - Salmonella Choleraesuis Bacterin.

Science.gov (United States)

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Salmonella Choleraesuis Bacterin. 113... REQUIREMENTS Inactivated Bacterial Products § 113.122 Salmonella Choleraesuis Bacterin. Salmonella Choleraesuis Bacterin shall be prepared from a culture of Salmonella choleraesuis which has been inactivated and is...

9 CFR 113.120 - Salmonella Typhimurium Bacterin.

Science.gov (United States)

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Salmonella Typhimurium Bacterin. 113... REQUIREMENTS Inactivated Bacterial Products § 113.120 Salmonella Typhimurium Bacterin. Salmonella Typhimurium Bacterin shall be prepared from a culture of Salmonella typhimurium which has been inactivated and is...

Biofilms of a Bacillus subtilis hospital isolate protect Staphylococcus aureus from biocide action.

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Arnaud Bridier

Full Text Available The development of a biofilm constitutes a survival strategy by providing bacteria a protective environment safe from stresses such as microbicide action and can thus lead to important health-care problems. In this study, biofilm resistance of a Bacillus subtilis strain (called hereafter ND(medical recently isolated from endoscope washer-disinfectors to peracetic acid was investigated and its ability to protect the pathogen Staphylococcus aureus in mixed biofilms was evaluated. Biocide action within Bacillus subtilis biofilms was visualised in real time using a non-invasive 4D confocal imaging method. The resistance of single species and mixed biofilms to peracetic acid was quantified using standard plate counting methods and their architecture was explored using confocal imaging and electronic microscopy. The results showed that the ND(medical strain demonstrates the ability to make very large amount of biofilm together with hyper-resistance to the concentration of PAA used in many formulations (3500 ppm. Evidences strongly suggest that the enhanced resistance of the ND(medical strain was related to the specific three-dimensional structure of the biofilm and the large amount of the extracellular matrix produced which can hinder the penetration of peracetic acid. When grown in mixed biofilm with Staphylococcus aureus, the ND(medical strain demonstrated the ability to protect the pathogen from PAA action, thus enabling its persistence in the environment. This work points out the ability of bacteria to adapt to an extremely hostile environment, and the necessity of considering multi-organism ecosystems instead of single species model to decipher the mechanisms of biofilm resistance to antimicrobials agents.

9 CFR 113.101 - Leptospira Pomona Bacterin.

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2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Leptospira Pomona Bacterin. 113.101... Inactivated Bacterial Products § 113.101 Leptospira Pomona Bacterin. Leptospira Pomona Bacterin shall be produced from a culture of Leptospira pomona which has been inactivated and is nontoxic. Each serial of...

9 CFR 113.103 - Leptospira Canicola Bacterin.

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2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Leptospira Canicola Bacterin. 113.103... Inactivated Bacterial Products § 113.103 Leptospira Canicola Bacterin. Leptospira Canicola Bacterin shall be produced from a culture of Leptospira canicola which has been inactivated and is nontoxic. Each serial of...

9 CFR 113.105 - Leptospira Hardjo Bacterin.

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2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Leptospira Hardjo Bacterin. 113.105... Inactivated Bacterial Products § 113.105 Leptospira Hardjo Bacterin. Leptospira Hardjo Bacterin shall be produced from a culture of Leptospira hardjo which has been inactivated and is nontoxic. Each serial of...

9 CFR 113.123 - Salmonella Dublin Bacterin.

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2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Salmonella Dublin Bacterin. 113.123... Inactivated Bacterial Products § 113.123 Salmonella Dublin Bacterin. Salmonella Dublin Bacterin shall be prepared from a culture of Salmonella dublin which has been inactivated and is nontoxic. Each serial of...

Pathology and biofilm formation in a porcine model of staphylococcal osteomyelitis

DEFF Research Database (Denmark)

Johansen, L K; Koch, J; Frees, D

2012-01-01

A porcine model was used to examine the potential of human and porcine Staphylococcus aureus isolates to induce haematogenously spread osteomyelitis. Pigs were inoculated in the right femoral artery with one of the following S. aureus strains: S54F9 (from a porcine lung abscess; n = 3 animals), N...... dependent on the strain of bacteria inoculated and on the formation of a biofilm....... with colonies of S. aureus as demonstrated immunohistochemically. By peptide nucleic acid fluorescence in situ hybridization bacterial aggregates were demonstrated to be embedded in an opaque matrix, indicating that the bacteria had formed a biofilm. Development of experimental osteomyelitis was therefore...

Investigation of the antibiotic resistance and biofilm-forming ability of Staphylococcus aureus from subclinical bovine mastitis cases.

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Aslantaş, Özkan; Demir, Cemil

2016-11-01

A total of 112 Staphylococcus aureus isolates obtained from subclinical bovine mastitis cases were examined for antibiotic susceptibility and biofilm-forming ability as well as genes responsible for antibiotic resistance, biofilm-forming ability, and adhesin. Antimicrobial susceptibility of the isolates were determined by disk diffusion method. Biofilm forming ability of the isolates were investigated by Congo red agar method, standard tube method, and microplate method. The genes responsible for antibiotic resistance, biofilm-forming ability, and adhesion were examined by PCR. Five isolates (4.5%) were identified as methicillin-resistant Staph. aureus by antibiotic susceptibility testing and confirmed by mecA detection. The resistance rates to penicillin, ampicillin, tetracycline, erythromycin, trimethoprim-sulfamethoxazole, enrofloxacin, and amoxicillin-clavulanic acid were 45.5, 39.3, 33, 26.8, 5.4, 0.9, and 0.9%, respectively. All isolates were susceptible against vancomycin and gentamicin. The blaZ (100%), tetK (67.6%), and ermA (70%) genes were the most common antibiotic-resistance genes. Using Congo red agar, microplate, and standard tube methods, 70.5, 67, and 62.5% of the isolates were found to be biofilm producers, respectively. The percentage rate of icaA, icaD, and bap genes in Staph. aureus isolates were 86.6, 86.6, and 13.4%, respectively. The adhesion molecules fnbA, can, and clfA were detected in 87 (77.7%), 98 (87.5%), and 75 (70%) isolates, respectively. The results indicated that Staph. aureus from sublinical bovine mastitis cases were mainly resistant to β-lactams and, to a lesser extent, to tetracycline and erythromycin. Also, biofilm- and adhesion-related genes, which are increasingly accepted as an important virulence factor in the pathogenesis of Staph. aureus infections, were detected at a high rate. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Comparison of Biofilm Formation between Methicillin-Resistant and Methicillin-Susceptible Isolates of Staphylococcus aureus.

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Ghasemian, Abdolmajid; Najar Peerayeh, Shahin; Bakhshi, Bita; Mirzaee, Mohsen

2016-07-01

The aim of this study was to compare the biofilm formation and the prevalence of biofilm-associated genes between the isolates of methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) Staphylococcus aureus. In total, 209 S. aureus isolates were collected. The antibiotic susceptibility test was conducted using nine antibiotics according to the guidelines of Clinical and Laboratory Standards Institute. Phenotypic biofilm formation was performed with microtiter plate assay. The polymerase chain reaction was employed to detect icaA, icaD, icaB, icaC, clfA, clfB, fnbA, fnbB, fib, cna, eno, ebps, bbp, mecA, and SCCmec types as well as agr group genes with specific primers. Sixty-four (30.62%) isolates were resistant to methicillin, and 54 (83%) MRSA harbored SCCmec III. Furthermore, 122 (58.3%) isolates belonged to agr group I. Twenty-six (36.1%) MRSA and 42 (28.9%) MSSA isolates were strong biofilm producers (no significant difference). The prevalence of icaA, icaD, icaB, and icaC genes in MSSA isolates was 71, 41, 76, and 72%, respectively. The frequency of clfA, clfB, fnbA, fnbB, fib, cna, eno, ebps, and bbp in MSSA was 100, 100, 56, 46, 74, 54, 78, 11, and 1%, respectively. However, in MRSA isolates, the frequency was 97, 97, 64, 51, 76, 56, 79, and 12% with no track of bbp, respectively. Statistical difference between MSSA and MRSA regarding biofilm formation and the frequency of all biofilm-encoding genes was not significant. The majority of the S. aureus isolates harbored clfA, clfB, eno, fib, icaA, and icaD genes.

Short communication: Effects of lactose and milk on the expression of biofilm-associated genes in Staphylococcus aureus strains isolated from a dairy cow with mastitis.

Science.gov (United States)

Xue, Ting; Chen, Xiaolin; Shang, Fei

2014-10-01

Staphylococcus aureus is the main etiological organism responsible for bovine mastitis. The ability of S. aureus to form biofilms plays an important role in the pathogenesis of mastitis. Biofilm formation in S. aureus is associated with the production of polysaccharide intercellular adhesin (PIA) protein and several other proteins. Several environmental factors, including glucose, osmolarity, oleic acid, temperature, and anaerobiosis, have been reported to affect biofilm formation in S. aureus. This study investigated the influence of lactose and milk on the biofilm formation capacity of 2 clinical bovine isolates of S. aureus. We found that lactose increased biofilm formation predominantly by inducing PIA production, whereas milk increased biofilm formation through PIA as well as by increasing the production of other biofilm-associated proteins, which might be mediated by the transcriptional regulators intercellular adhesion regulator (icaR) and repressor of biofilm (rbf). Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

AzaSite® inhibits Staphylococcus aureus and coagulase-negative Staphylococcus biofilm formation in vitro.

Science.gov (United States)

Wu, Eric C; Kowalski, Regis P; Romanowski, Eric G; Mah, Francis S; Gordon, Y Jerold; Shanks, Robert M Q

2010-12-01

The aim of this study was to analyze the effect of azithromycin (AZM) 1% ophthalmic solution in DuraSite® (AzaSite®) on biofilm formation by Staphylococcus aureus and coagulase-negative staphylococci in vitro. Susceptible and resistant clinical strains (n = 8) of S. aureus and coagulase-negative staphylococci were challenged with serial dilutions of AzaSite® and its components: AZM, benzalkonium chloride (BAK), and the DuraSite drug delivery vehicle. After 20 h of incubation, bacterial growth was quantified using a spectrophotometer (A = 600 nm). Plates were stained with crystal violet and biofilm formation was quantified spectrophotometrically at A = 590 nm. AzaSite® and AZM inhibited bacterial growth (P reduction in biofilm formation (P reduction in biofilm formation at concentrations from 1.25 to 10 mg/mL in most strains. DuraSite® inhibited biofilm formation at concentrations between 10% and 2.5% in all studied strains (P < 0.05), without affecting bacterial growth. BAK inhibited bacterial growth and biofilm formation in all strains between concentrations of 0.042 and 0.375 mg/mL (P < 0.05). AzaSite®, AZM, or BAK prevented biofilm formation by inhibiting growth of AZM-susceptible strains. AzaSite®, AZM, and DuraSite® also reduced biofilm formation at subinhibitory concentrations for growth. Our data indicate that AZM has a moderate inhibitory effect on biofilm formation, whereas DuraSite® appears to play a greater role in the inhibition of staphylococcal biofilm formation by AzaSite®.

Effect of biosurfactants on Pseudomonas aeruginosa and Staphylococcus aureus biofilms in a BioFlux channel.

Science.gov (United States)

Diaz De Rienzo, M A; Stevenson, P S; Marchant, R; Banat, I M

2016-07-01

Recent studies have indicated that biosurfactants play a role both in maintaining channels between multicellular structures in biofilms and in dispersal of cells from biofilms. A combination of caprylic acid (0.01 % v/v) together with rhamnolipids (0.04 % v/v) was applied to biofilms of Pseudomonas aeruginosa ATCC 15442, Staphylococcus aureus ATCC 9144 and a mixed culture under BioFlux flowthrough conditions and caused disruption of the biofilms. The biofilms were also treated with a combination of rhamnolipids (0.04 % v/v) and sophorolipids (0.01 %). Control treatments with PBS 1× had no apparent effect on biofilm disruption. The Gram-positive bacterium (S. aureus ATCC 9144) was more sensitive than P. aeruginosa ATCC 15442 in terms of disruption and viability as shown by Live/Dead staining. Disruption of biofilms of P. aeruginosa ATCC 15442 was minimal. Oxygen consumption by biofilms, after different treatments with biosurfactants, confirms that sophorolipid on its own is unable to kill/inhibit cells of P. aeruginosa ATCC 15442, and even when used in combination with rhamnolipids, under static conditions, no decrease in the cell viability was observed. Cells in biofilms exposed to mono-rhamnolipids (0.04 % v/v) showed behaviour typical of exposure to bacteriostatic compounds, but when exposed to di-rhamnolipids (0.04 % v/v), they displayed a pattern characteristic of bactericidal compounds.

Nisin and lysostaphin activity against preformed biofilm of Staphylococcus aureus involved in bovine mastitis.

Science.gov (United States)

Ceotto-Vigoder, H; Marques, S L S; Santos, I N S; Alves, M D B; Barrias, E S; Potter, A; Alviano, D S; Bastos, M C F

2016-07-01

The biofilm produced by Staphylococcus aureus isolates involved in clinical or subclinical bovine mastitis and the activity of nisin and lysostaphin against the preformed biofilm produced by these strains were investigated. Eighteen strains were tested and all produced biofilm. Eight strains with distinct biofilm composition were selected for the antimicrobial activity assays. The minimal inhibitory concentration of each bacteriocin was determined against the planktonic cells and ranged from 15·6 to 500 μg ml(-1) for nisin, and from 3·9 to 50 μg ml(-1) , for lysostaphin. Lysostaphin treatment (0·4 μg ml(-1) ) for 4 h caused a strong Staph. aureus 4181 biofilm detachment and death of the majority of the sessile cells, while nisin treatment (100 μg ml(-1) ) for the same time caused only a great reduction in cell viability. Additionally, combination of both bacteriocins for 4 h resulted in significant death of the sessile cells but no biofilm detachment. The treatment with lysostaphin alone or in combination with nisin was effective in killing most biofilm sessile cells. The action of lysostaphin, either alone or in combination with nisin, against established staphylococcal biofilm may represent an alternative to bovine mastitis control. However, the duration of the treatment should be considered for its application so that the best effectiveness can be achieved. © 2016 The Society for Applied Microbiology.

Biofilm-forming ability profiling of Staphylococcus aureus and Staphylococcus epidermidis mastitis isolates

DEFF Research Database (Denmark)

Oliveira, M; Bexiga, R; Nunes, S F

2006-01-01

of FISH to artificially contaminated milk samples allowed the direct observation of biofilm production by 37.5% isolates, showing total agreement with the CRA results. This method better mimics the in vivo conditions, especially in terms of the presence of calcium and iron, which in high concentrations...... hybridisation (FISH) protocol that would allow the direct observation of biofilm formation in milk samples. The analysis of phenotypic expression in Congo Red Agar (CRA) and by FISH, showed that 37.5% of the S. aureus isolates produced biofilm, while by optical density measurement only 18.75% isolates revealed...

Contribution of the Staphylococcus aureus Atl AM and GL murein hydrolase activities in cell division, autolysis, and biofilm formation.

Directory of Open Access Journals (Sweden)

Jeffrey L Bose

Full Text Available The most prominent murein hydrolase of Staphylococcus aureus, AtlA, is a bifunctional enzyme that undergoes proteolytic cleavage to yield two catalytically active proteins, an amidase (AM and a glucosaminidase (GL. Although the bifunctional nature of AtlA has long been recognized, most studies have focused on the combined functions of this protein in cell wall metabolism and biofilm development. In this study, we generated mutant derivatives of the clinical S. aureus isolate, UAMS-1, in which one or both of the AM and GL domains of AtlA have been deleted. Examination of these strains revealed that each mutant exhibited growth rates comparable to the parental strain, but showed clumping phenotypes and lysis profiles that were distinct from the parental strain and each other, suggesting distinct roles in cell wall metabolism. Given the known function of autolysis in the release of genomic DNA for use as a biofilm matrix molecule, we also tested the mutants in biofilm assays and found both AM and GL necessary for biofilm development. Furthermore, the use of enzymatically inactive point mutations revealed that both AM and GL must be catalytically active for S. aureus to form a biofilm. The results of this study provide insight into the relative contributions of AM and GL in S. aureus and demonstrate the contribution of Atl-mediated lysis in biofilm development.

Contribution of the Staphylococcus aureus Atl AM and GL murein hydrolase activities in cell division, autolysis, and biofilm formation.

Science.gov (United States)

Bose, Jeffrey L; Lehman, McKenzie K; Fey, Paul D; Bayles, Kenneth W

2012-01-01

The most prominent murein hydrolase of Staphylococcus aureus, AtlA, is a bifunctional enzyme that undergoes proteolytic cleavage to yield two catalytically active proteins, an amidase (AM) and a glucosaminidase (GL). Although the bifunctional nature of AtlA has long been recognized, most studies have focused on the combined functions of this protein in cell wall metabolism and biofilm development. In this study, we generated mutant derivatives of the clinical S. aureus isolate, UAMS-1, in which one or both of the AM and GL domains of AtlA have been deleted. Examination of these strains revealed that each mutant exhibited growth rates comparable to the parental strain, but showed clumping phenotypes and lysis profiles that were distinct from the parental strain and each other, suggesting distinct roles in cell wall metabolism. Given the known function of autolysis in the release of genomic DNA for use as a biofilm matrix molecule, we also tested the mutants in biofilm assays and found both AM and GL necessary for biofilm development. Furthermore, the use of enzymatically inactive point mutations revealed that both AM and GL must be catalytically active for S. aureus to form a biofilm. The results of this study provide insight into the relative contributions of AM and GL in S. aureus and demonstrate the contribution of Atl-mediated lysis in biofilm development.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA isolates of swine origin form robust biofilms.

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Tracy L Nicholson

Full Text Available Methicillin-resistant Staphylococcus aureus (MRSA colonization of livestock animals is common and prevalence rates for pigs have been reported to be as high as 49%. Mechanisms contributing to the persistent carriage and high prevalence rates of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA strains in swine herds and production facilities have not been investigated. One explanation for the high prevalence of MRSA in swine herds is the ability of these organisms to exist as biofilms. In this report, the ability of swine LA-MRSA strains, including ST398, ST9, and ST5, to form biofilms was quantified and compared to several swine and human isolates. The contribution of known biofilm matrix components, polysaccharides, proteins and extracellular DNA (eDNA, was tested in all strains as well. All MRSA swine isolates formed robust biofilms similar to human clinical isolates. The addition of Dispersin B had no inhibitory effect on swine MRSA isolates when added at the initiation of biofilm growth or after pre-established mature biofilms formed. In contrast, the addition of proteinase K inhibited biofilm formation in all strains when added at the initiation of biofilm growth and was able to disperse pre-established mature biofilms. Of the LA-MRSA strains tested, we found ST398 strains to be the most sensitive to both inhibition of biofilm formation and dispersal of pre-formed biofilms by DNaseI. Collectively, these findings provide a critical first step in designing strategies to control or eliminate MRSA in swine herds.

Biofilm production and beta-lactamic resistance in Brazilian Staphylococcus aureus isolates from bovine mastitis

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Viviane Figueira Marques

Full Text Available Abstract Staphylococcus spp. play an important role in the etiology of bovine mastitis. Staphylococcus aureus is considered the most relevant species due to the production of virulence factors such as slime, which is required for biofilm formation. This study aimed to evaluate biofilm production and its possible relation to beta-lactamic resistance in 20 S. aureus isolates from bovine mastitic milk. The isolates were characterized by pheno-genotypic and MALDI TOF-MS assays and tested for genes such as icaA, icaD, bap, agr RNAIII, agr I, agr II, agr III, and agr IV, which are related to slime production and its regulation. Biofilm production in microplates was evaluated considering the intervals determined along the bacterial growth curve. In addition, to determine the most suitable time interval for biofilm analysis, scanning electron microscopy was performed. Furthermore, genes such as mecA and blaZ that are related to beta-lactamic resistance and oxacillin susceptibility were tested. All the studied isolates were biofilm producers and mostly presented icaA and icaD. The Agr type II genes were significantly prevalent. According to the SEM, gradual changes in the bacterial arrangement were observed during biofilm formation along the growth curve phases, and the peak was reached at the stationary phase. In this study, the penicillin resistance was related to the production of beta-lactamase, and the high minimal bactericidal concentration for cefoxitin was possibly associated with biofilm protection. Therefore, further studies are warranted to better understand biofilm formation, possibly contributing to our knowledge about bacterial resistance in vivo.

Biofilm formation by Staphylococcus aureus from food contact surfaces in a meat-based broth and sensitivity to sanitizers

Directory of Open Access Journals (Sweden)

Evandro Leite de Souza

2014-01-01

Full Text Available This study assessed the capacity of adhesion, the detachment kinetic and the biofilm formation by Staphylococcus aureus isolated from food services on stainless steel and polypropylene surfaces (2 x 2 cm when cultivated in a meat-based broth at 28 and 7 ºC. It was also to study the efficacy of the sanitizers sodium hypochlorite (250 mg/L and peracetic acid (30 mg/L in inactivating the bacterial cells in the preformed biofilm. S. aureus strains adhered in high numbers regardless the assayed surface kind and incubation temperature over 72 h. Cells detachment of surfaces revealed high persistence over the incubation period. Number of cells needed for biofilm formation was noted at all experimental systems already after 3 days. Peracetic acid and sodium hypochlorite were not efficient in completely removing the cells of S. aureus adhered on polypropylene and stainless steel surfaces. From these results, the assayed strains revealed high capacity to adhere and form biofilm on polypropylene and stainless steel surfaces under different growth conditions. Moreover, the cells in biofilm matrix were resistant for total removal when submitted to the exposure to sanitizers.

Extended biofilm susceptibility assay for Staphylococcus aureus bovine mastitis isolates: evidence for association between genetic makeup and biofilm susceptibility.

Science.gov (United States)

Melchior, M B; van Osch, M H J; Lam, T J G M; Vernooij, J C M; Gaastra, W; Fink-Gremmels, J

2011-12-01

Staphylococcus aureus is one of the most prevalent causes of bovine mastitis. The antimicrobial treatment of this disease is currently based on antimicrobial susceptibility tests according to Clinical and Laboratory Standards Institute standards. However, various authors have shown a discrepancy between the results of this standard susceptibility test and the actual cure rate of the applied antimicrobial treatment. Increasing evidence suggests that in vivo biofilm formation by Staph. aureus, which is not assessed in the antimicrobial susceptibility tests, is associated with this problem, resulting in disappointing cure rates, especially for infections of longer duration. Previous data obtained with a limited number of strains showed that the extended biofilm antimicrobial susceptibility (EBS) assay reveals differences between strains, which cannot be derived from a standard susceptibility test or from a 24-h biofilm susceptibility test. The objective of this study was to test a collection of Staph. aureus bovine mastitis strains in the EBS assay and to model the effect of antimicrobial exposure, duration of antimicrobial exposure, and genotype profile of the strains on antimicrobial susceptibility. With the results from a previous study with the same collection of strains, the effect of genotype represented by accessory gene regulator gene (agr-type), the presence of insertional sequence 257 (IS257), intercellular adhesion (ica), and the β-lactamase (blaZ) gene were entered as explanatory factors in a logistic regression model. The agr locus of Staph. aureus controls the expression of most of the virulence factors, represses the transcription of several cell wall-associated proteins, and activates several exoproteins during the post-exponential phase. The IS257 gene has been related to biofilm formation in vitro and was found earlier in 50% of the agr-type 2 strains. The ica gene cluster encodes for the production of an extracellular polysaccharide adhesin, termed

A novel in vitro model for hematogenous spreading of S. aureus device biofilms demonstrating clumping dispersal as an advantageous dissemination mechanism

DEFF Research Database (Denmark)

Grønnemose, Rasmus Birkholm; Lindhardt Sæderup (Madsen), Kirstine; Kolmos, Hans Jørn

2017-01-01

Staphylococcus aureus is able to disseminate from vascular device biofilms to the blood and organs, resulting in life-threatening infections such as endocarditis. The mechanisms behind spreading are largely unknown, especially how the bacterium escapes immune effectors and antibiotics in the proc......Staphylococcus aureus is able to disseminate from vascular device biofilms to the blood and organs, resulting in life-threatening infections such as endocarditis. The mechanisms behind spreading are largely unknown, especially how the bacterium escapes immune effectors and antibiotics...... the ability to adhere to and initiate colonization of endothelial cell layers under flow. In vivo experiments showed that the released biofilm material reached the heart similarly as ordinary broth-grown bacteria, but also that clumps to some extend were trapped in the lungs. The clumping dispersal of S....... aureus from in vivo-like vascular biofilms and their specific properties demonstrated here help explain the pathophysiology associated with S. aureus bloodstream infections....

Morphological bactericidal fast-acting effects of peracetic acid, a high-level disinfectant, against Staphylococcus aureus and Pseudomonas aeruginosa biofilms in tubing.

Science.gov (United States)

Chino, T; Nukui, Y; Morishita, Y; Moriya, K

2017-01-01

The bactericidal effect of disinfectants against biofilms is essential to reduce potential endoscopy-related infections caused by contamination. Here, we investigated the bactericidal effect of a high-level disinfectant, peracetic acid (PAA), against Staphylococcus aureus and Pseudomonas aeruginosa biofilm models in vitro. S. aureus and P. aeruginosa biofilms were cultured at 35 °C for 7 days with catheter tubes. The following high-level disinfectants (HLDs) were tested: 0.3% PAA, 0.55% ortho-phthalaldehyde (OPA), and 2.0% alkaline-buffered glutaraldehyde (GA). Biofilms were exposed to these agents for 1-60 min and observed after 5 min and 30 min by transmission and scanning electron microscopy. A Student's t test was performed to compare the exposure time required for bactericidal effectiveness of the disinfectants. PAA and GA were active within 1 min and 5 min, respectively, against S. aureus and P. aeruginosa biofilms. OPA took longer than 10 min and 30 min to act against S. aureus and P. aeruginosa biofilms, respectively ( p  < 0.01). Treatment with PAA elicited changes in cell shape after 5 min and structural damage after 30 min. Amongst the HLDs investigated, PAA elicited the most rapid bactericidal effects against both biofilms. Additionally, treatment with PAA induced morphological alterations in the in vitro biofilm models, suggesting that PAA exerts fast-acting bactericidal effects against biofilms associated with endoscopy-related infections. These findings indicate that the exposure time for bactericidal effectiveness of HLDs for endoscope reprocessing in healthcare settings should be reconsidered.

Quantitative Expression Analysis of SpA, FnbA and Rsp Genes in Staphylococcus aureus: Actively Associated in the Formation of Biofilms.

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Yeswanth, Sthanikam; Chaudhury, Abhijit; Sarma, Potukuchi Venkata Gurunadha Krishna

2017-12-01

In Staphylococcus aureus, adherence and secretory proteins play chief role in the formation of biofilms. This mode of growth exhibits resistance to a variety of antibiotics and spreads its infections. In the present study, secretary and adherence proteins, Protein-A, Fibronectin-binding protein-A (FnbA) and Rsp (a transcription regulator encoding proteolytic property) expression levels were evaluated at different stages of growth in S. aureus ATCC12600 a drug-sensitive strain and multidrug-resistant strains of S. aureus. Initially, the SpA, FnbA and Rsp genes of S. aureus ATCC12600 were cloned, sequenced, expressed and characterized. The proteolytic property of recombinant Rsp was conspicuously shown when this pathogen was grown in aerobic conditions correlating with reduced biofilm units. In anaerobic mode of growth, S. aureus exhibited a higher expression of SpA and FnbA in early and mid adherence phases and finally stabilized at 48 h of incubation. This expression was more pronounced in methicillin-resistant strains (LMV1-8 and D1-4) of S. aureus. In all these stages, Rsp gene expression was at the lowest level and these results concur with the increased biofilm units. The results of the present study explain proteins chiefly contribute in the formation of biofilms.

Norlichexanthone Reduces Virulence Gene Expression and Biofilm Formation in Staphylococcus aureus

DEFF Research Database (Denmark)

Baldry, Mara; Nielsen, Anita; Bojer, Martin S.

2016-01-01

Staphylococcus aureus is a serious human pathogen and antibiotic resistant, community-associated strains, such as the methicillin resistant S. aureus (MRSA) strain USA300, continue to spread. To avoid resistance, anti-virulence therapy has been proposed where toxicity is targeted rather than...... viability. Previously we have shown that norlichexanthone, a small non-reduced tricyclic polyketide produced by fungi and lichens, reduces expression of hla encoding α-hemolysin as well as the regulatory RNAIII of the agr quorum sensing system in S. aureus 8325-4. The aim of the present study was to further...... SaeRS system. Our data show that norlichexanthone treatment reduces expression of key virulence factors in CA-MRSA strain USA300 via AgrA binding and represses biofilm formation....

Staphylococcus aureus biofilm formation on different gentamicin-loaded polymethylmethacrylate bone cements

NARCIS (Netherlands)

van de Belt, H; Neut, D; Schenk, W; van Horn, [No Value; van der Mei, HC; Busscher, HJ

In this in vitro study, the formation of a Staphylococcus aureus biofilm on six gentamicin-loaded bone cements (CMW1, CMW3, CMW Endurance, CMW2000, Palacos. and Palamed) was determined in a modified Robbins device over a 3 days time span and related with previously (Van de Belt et al., Biomaterials

Morphological bactericidal fast-acting effects of peracetic acid, a high-level disinfectant, against Staphylococcus aureus and Pseudomonas aeruginosa biofilms in tubing

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T. Chino

2017-12-01

Full Text Available Abstract Background The bactericidal effect of disinfectants against biofilms is essential to reduce potential endoscopy-related infections caused by contamination. Here, we investigated the bactericidal effect of a high-level disinfectant, peracetic acid (PAA, against Staphylococcus aureus and Pseudomonas aeruginosa biofilm models in vitro. Methods S. aureus and P. aeruginosa biofilms were cultured at 35 °C for 7 days with catheter tubes. The following high-level disinfectants (HLDs were tested: 0.3% PAA, 0.55% ortho-phthalaldehyde (OPA, and 2.0% alkaline-buffered glutaraldehyde (GA. Biofilms were exposed to these agents for 1–60 min and observed after 5 min and 30 min by transmission and scanning electron microscopy. A Student’s t test was performed to compare the exposure time required for bactericidal effectiveness of the disinfectants. Results PAA and GA were active within 1 min and 5 min, respectively, against S. aureus and P. aeruginosa biofilms. OPA took longer than 10 min and 30 min to act against S. aureus and P. aeruginosa biofilms, respectively (p < 0.01. Treatment with PAA elicited changes in cell shape after 5 min and structural damage after 30 min. Conclusions Amongst the HLDs investigated, PAA elicited the most rapid bactericidal effects against both biofilms. Additionally, treatment with PAA induced morphological alterations in the in vitro biofilm models, suggesting that PAA exerts fast-acting bactericidal effects against biofilms associated with endoscopy-related infections. These findings indicate that the exposure time for bactericidal effectiveness of HLDs for endoscope reprocessing in healthcare settings should be reconsidered.

SaeRS Is Responsive to Cellular Respiratory Status and Regulates Fermentative Biofilm Formation in Staphylococcus aureus.

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Mashruwala, Ameya A; Gries, Casey M; Scherr, Tyler D; Kielian, Tammy; Boyd, Jeffrey M

2017-08-01

Biofilms are multicellular communities of microorganisms living as a quorum rather than as individual cells. The bacterial human pathogen Staphylococcus aureus uses oxygen as a terminal electron acceptor during respiration. Infected human tissues are hypoxic or anoxic. We recently reported that impaired respiration elicits a p rogrammed c ell l ysis (PCL) phenomenon in S. aureus leading to the release of cellular polymers that are utilized to form biofilms. PCL is dependent upon the AtlA murein hydrolase and is regulated, in part, by the SrrAB two-component regulatory system (TCRS). In the current study, we report that the SaeRS TCRS also governs fermentative biofilm formation by positively influencing AtlA activity. The SaeRS-modulated factor fibronectin-binding protein A (FnBPA) also contributed to the fermentative biofilm formation phenotype. SaeRS-dependent biofilm formation occurred in response to changes in cellular respiratory status. Genetic evidence presented suggests that a high cellular titer of phosphorylated SaeR is required for biofilm formation. Epistasis analyses found that SaeRS and SrrAB influence biofilm formation independently of one another. Analyses using a mouse model of orthopedic implant-associated biofilm formation found that both SaeRS and SrrAB govern host colonization. Of these two TCRSs, SrrAB was the dominant system driving biofilm formation in vivo We propose a model wherein impaired cellular respiration stimulates SaeRS via an as yet undefined signal molecule(s), resulting in increasing expression of AtlA and FnBPA and biofilm formation. Copyright © 2017 American Society for Microbiology.

Reducing Staphylococcus aureus biofilm formation on stainless steel 316L using functionalized self-assembled monolayers.

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Kruszewski, Kristen M; Nistico, Laura; Longwell, Mark J; Hynes, Matthew J; Maurer, Joshua A; Hall-Stoodley, Luanne; Gawalt, Ellen S

2013-05-01

Stainless steel 316L (SS316L) is a common material used in orthopedic implants. Bacterial colonization of the surface and subsequent biofilm development can lead to refractory infection of the implant. Since the greatest risk of infection occurs perioperatively, strategies that reduce bacterial adhesion during this time are important. As a strategy to limit bacterial adhesion and biofilm formation on SS316L, self-assembled monolayers (SAMs) were used to modify the SS316L surface. SAMs with long alkyl chains terminated with hydrophobic (-CH3) or hydrophilic (oligoethylene glycol) tail groups were used to form coatings and in an orthogonal approach, SAMs were used to immobilize gentamicin or vancomycin on SS316L for the first time to form an "active" antimicrobial coating to inhibit early biofilm development. Modified SS316L surfaces were characterized using surface infrared spectroscopy, contact angles, MALDI-TOF mass spectrometry and atomic force microscopy. The ability of SAM-modified SS316L to retard biofilm development by Staphylococcus aureus was functionally tested using confocal scanning laser microscopy with COMSTAT image analysis, scanning electron microscopy and colony forming unit analysis. Neither hydrophobic nor hydrophilic SAMs reduced biofilm development. However, gentamicin-linked and vancomycin-linked SAMs significantly reduced S. aureus biofilm formation for up to 24 and 48 h, respectively. Copyright © 2013 Elsevier B.V. All rights reserved.

Reducing Staphylococcus aureus biofilm formation on stainless steel 316L using functionalized self-assembled monolayers

International Nuclear Information System (INIS)

Kruszewski, Kristen M.; Nistico, Laura; Longwell, Mark J.; Hynes, Matthew J.; Maurer, Joshua A.; Hall-Stoodley, Luanne; Gawalt, Ellen S.

2013-01-01

Stainless steel 316L (SS316L) is a common material used in orthopedic implants. Bacterial colonization of the surface and subsequent biofilm development can lead to refractory infection of the implant. Since the greatest risk of infection occurs perioperatively, strategies that reduce bacterial adhesion during this time are important. As a strategy to limit bacterial adhesion and biofilm formation on SS316L, self-assembled monolayers (SAMs) were used to modify the SS316L surface. SAMs with long alkyl chains terminated with hydrophobic (− CH 3 ) or hydrophilic (oligoethylene glycol) tail groups were used to form coatings and in an orthogonal approach, SAMs were used to immobilize gentamicin or vancomycin on SS316L for the first time to form an “active” antimicrobial coating to inhibit early biofilm development. Modified SS316L surfaces were characterized using surface infrared spectroscopy, contact angles, MALDI-TOF mass spectrometry and atomic force microscopy. The ability of SAM-modified SS316L to retard biofilm development by Staphylococcus aureus was functionally tested using confocal scanning laser microscopy with COMSTAT image analysis, scanning electron microscopy and colony forming unit analysis. Neither hydrophobic nor hydrophilic SAMs reduced biofilm development. However, gentamicin-linked and vancomycin-linked SAMs significantly reduced S. aureus biofilm formation for up to 24 and 48 h, respectively. - Highlights: ► SS316L was modified with glycol terminated SAMs in order to reduce biofilm growth. ► Antibiotics gentamicin and vancomycin were immobilized on SS316L via SAMs. ► Only the antibiotic modifications reduced biofilm development on SS316L

Reducing Staphylococcus aureus biofilm formation on stainless steel 316L using functionalized self-assembled monolayers

Energy Technology Data Exchange (ETDEWEB)

Kruszewski, Kristen M., E-mail: kruszewskik@duq.edu [Duquesne University, Department of Chemistry and Biochemistry, 600 Forbes Avenue, Pittsburgh, PA 15282 (United States); Nistico, Laura, E-mail: lnistico@wpahs.org [Allegheny General Hospital, Center for Genomic Sciences, Allegheny-Singer Research Institute, 320 East North Avenue, 11th floor, South Tower, Pittsburgh, PA 15212 (United States); Longwell, Mark J., E-mail: mlongwel@wpahs.org [Allegheny General Hospital, Center for Genomic Sciences, Allegheny-Singer Research Institute, 320 East North Avenue, 11th floor, South Tower, Pittsburgh, PA 15212 (United States); Hynes, Matthew J., E-mail: mjhynes@go.wustl.edu [Washington University in St. Louis, Department of Chemistry, One Brookings Drive, St. Louis, MO 63130 (United States); Maurer, Joshua A., E-mail: maurer@wustl.edu [Washington University in St. Louis, Department of Chemistry, One Brookings Drive, St. Louis, MO 63130 (United States); Hall-Stoodley, Luanne, E-mail: L.Hall-Stoodley@soton.ac.uk [Southampton Wellcome Trust Clinical Research Facility/NIHR Respiratory BRU, University of Southampton Faculty of Medicine, Southampton General Hospital, Tremona Road, Southampton, Hampshire SO16 6YD (United Kingdom); Gawalt, Ellen S., E-mail: gawalte@duq.edu [Duquesne University, Department of Chemistry and Biochemistry, McGowan Institute for Regenerative Medicine, 600 Forbes Avenue, Pittsburgh, PA 15282 (United States)

2013-05-01

Stainless steel 316L (SS316L) is a common material used in orthopedic implants. Bacterial colonization of the surface and subsequent biofilm development can lead to refractory infection of the implant. Since the greatest risk of infection occurs perioperatively, strategies that reduce bacterial adhesion during this time are important. As a strategy to limit bacterial adhesion and biofilm formation on SS316L, self-assembled monolayers (SAMs) were used to modify the SS316L surface. SAMs with long alkyl chains terminated with hydrophobic (− CH{sub 3}) or hydrophilic (oligoethylene glycol) tail groups were used to form coatings and in an orthogonal approach, SAMs were used to immobilize gentamicin or vancomycin on SS316L for the first time to form an “active” antimicrobial coating to inhibit early biofilm development. Modified SS316L surfaces were characterized using surface infrared spectroscopy, contact angles, MALDI-TOF mass spectrometry and atomic force microscopy. The ability of SAM-modified SS316L to retard biofilm development by Staphylococcus aureus was functionally tested using confocal scanning laser microscopy with COMSTAT image analysis, scanning electron microscopy and colony forming unit analysis. Neither hydrophobic nor hydrophilic SAMs reduced biofilm development. However, gentamicin-linked and vancomycin-linked SAMs significantly reduced S. aureus biofilm formation for up to 24 and 48 h, respectively. - Highlights: ► SS316L was modified with glycol terminated SAMs in order to reduce biofilm growth. ► Antibiotics gentamicin and vancomycin were immobilized on SS316L via SAMs. ► Only the antibiotic modifications reduced biofilm development on SS316L.

Methicillin-Resistant Staphylococcus aureus Biofilms and Their Influence on Bacterial Adhesion and Cohesion

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Khulood Hamid Dakheel

2016-01-01

Full Text Available Twenty-five methicillin-resistant Staphylococcus aureus (MRSA isolates were characterized by staphylococcal protein A gene typing and the ability to form biofilms. The presence of exopolysaccharides, proteins, and extracellular DNA and RNA in biofilms was assessed by a dispersal assay. In addition, cell adhesion to surfaces and cell cohesion were evaluated using the packed-bead method and mechanical disruption, respectively. The predominant genotype was spa type t127 (22 out of 25 isolates; the majority of isolates were categorized as moderate biofilm producers. Twelve isolates displayed PIA-independent biofilm formation, while the remaining 13 isolates were PIA-dependent. Both groups showed strong dispersal in response to RNase and DNase digestion followed by proteinase K treatment. PIA-dependent biofilms showed variable dispersal after sodium metaperiodate treatment, whereas PIA-independent biofilms showed enhanced biofilm formation. There was no correlation between the extent of biofilm formation or biofilm components and the adhesion or cohesion abilities of the bacteria, but the efficiency of adherence to glass beads increased after biofilm depletion. In conclusion, nucleic acids and proteins formed the main components of the MRSA clone t127 biofilm matrix, and there seems to be an association between adhesion and cohesion in the biofilms tested.

Reduced adhesion of Staphylococcus aureus to ZnO/PVC nanocomposites

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Geilich BM

2013-03-01

Full Text Available Benjamin M Geilich,1 Thomas J Webster21Program in Bioengineering, 2Department of Chemical Engineering, College of Engineering, Northeastern University, Boston, MA, USAAbstract: In hospitals and clinics worldwide, medical device surfaces have become a rapidly growing source of nosocomial infections. In particular, patients requiring mechanical ventilation (and, thus, intubation with an endotracheal tube for extended lengths of time are faced with a high probability of contracting ventilator-associated pneumonia. Once inserted into the body, the endotracheal tube provides a surface to which bacteria can adhere and form a biofilm (a robust, sticky matrix that provides protection against the host immune system and antibiotic treatment. Adding to the severity of this problem is the spread of bacterial genetic tolerance to antibiotics, in part demonstrated by the recent and significant increase in the prevalence of methicillin-resistant Staphylococcus aureus. To combat these trends, different techniques in biomaterial design must be explored. Recent research has shown that nanomaterials (materials with at least one dimension less than 100 nm may have the potential to prevent or disrupt bacterial processes that lead to infections. In this study, polyvinyl chloride (PVC taken from a conventional endotracheal tube was embedded with varying concentrations of zinc oxide (ZnO nanoparticles. S. aureus biofilms were then grown on these nanocomposite surfaces during a 24-hour culture. Following this, biofilms were removed from the surfaces and the number of colony forming units present was assessed. Bacterial proliferation on the samples embedded with the highest concentration of ZnO nanoparticles was 87% less when compared to the control, indicating that this technique is effective at reducing biofilm formation on PVC surfaces without the use of antibiotics.Keywords: nanomaterials, endotracheal tube, biofilm, zinc oxide, nanoparticles, Staphylococcus aureus

Antimicrobial potential of Eucalyptus globulus against biofilms of Staphylococcus aureus isolated from bovine mastitis

OpenAIRE

Gomes, F. I.; Martins, Natália; Ferreira, Isabel C. F. R.; Henriques, Mariana

2016-01-01

Staphylococcus aureus are among the most common species isolated from bovine mastitis. The pathogenesis of this bacterium is facilitated by a number of virulence factors, including the ability to adhere to abiotic surfaces and/or host tissues often leading to biofilms' formation. From the clinical perspective, the most important feature of Staphytococcus species' biofilms is their high tolerance to the conventional antimicrobial therapy. So, the increasing number of bovine m...

Formação de biofilme em aço inoxidável por Aeromonas hydrophila e Staphylococcus aureus usando leite e diferentes condições de cultivo Biofilm formation by Aeromonas hydrophila and Staphylococcus aureus on stainless steel using milk and different conditions of cultivation

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Cleube Andrade Boari

2009-12-01

Full Text Available O objetivo desta pesquisa consistiu em avaliar a formação de biofilme em aço inoxidável por Aeromonas hydrophila e Staphylococcus aureus usando leite e diferentes condições de cultivo. As variáveis em estudo consistem no cultivo monoespécie e combinado, dos referidos microrganismos e nas temperaturas de 4, 7 e 18 °C. Recipientes contendo 1000 mL de leite, densidade populacional de 10(5 UFC.mL-1 de cada microrganismo e 10 cupons de aço inoxidável (10 × 20 mm foram lacrados e armazenados, sob agitação de 60 rpm, por um período de 10 dias. As análises ocorreram a cada 48 horas. Células sésseis de A.hydrophila e S. aureus foram enumeradas através do plaqueamento seletivo em ágar m-Aeromonas selective e Baird-Parker, respectivamente. Estudos sobre o tempo de geração, enumeração de células planctônicas e observação dos cupons através da microscopia eletrônica de varredura foram conduzidos. S. aureus, em monocultivo, formou biofilme a 18 °C e a 7 °C. Para 4 °C, foi observado um processo de adesão. A presença de A. hydrophila reduziu o desempenho de S. aureus. Nesta condição de cultivo multiespécie houve formação de biofilme a 18 °C. A. hydrophila, tanto em monocultivo quanto em presença de S. aureus, formou biofilme em todas as condições pesquisadas.The aim of this research was to verify the capability of biofilm formation on stainless steel by Aeromonas hydrophila and Staphylococcus aureus using milk and different conditions of cultivation. The variables consisted in mono and multi-species cultivation of these microorganisms and in the temperatures of 4, 7 and 18 °C. Containers containing 1000 mL of milk, population density of 10(5 CFU.mL-1 of each microorganism, and ten suspended chips of stainless steel AISI 304 (10 × 20 mm were used to seal up and storage, under 60 rpm of agitation for 10 days. The analyses were conducted every 48 hours. Sessile cells of A. hydrophila and S. aureus and were enumerated

Staphylococcus aureus Quorum Regulator SarA Targeted Compound, 2-[(Methylaminomethyl]phenol Inhibits Biofilm and Down-Regulates Virulence Genes

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P. Balamurugan

2017-07-01

Full Text Available Staphylococcus aureus is a widely acknowledged Gram-positive pathogen for forming biofilm and virulence gene expressions by quorum sensing (QS, a cell to cell communication process. The quorum regulator SarA of S. aureus up-regulates the expression of many virulence factors including biofilm formation to mediate pathogenesis and evasion of the host immune system in the late phases of growth. Thus, inhibiting the production or blocking SarA protein might influence the down-regulation of biofilm and virulence factors. In this context, here we have synthesized 2-[(Methylaminomethyl]phenol, which was specifically targeted toward the quorum regulator SarA through in silico approach in our previous study. The molecule has been evaluated in vitro to validate its antibiofilm activity against clinical S. aureus strains. In addition, antivirulence properties of the inhibitor were confirmed with the observation of a significant reduction in the expression of representative virulence genes like fnbA, hla and hld that are governed under S. aureus QS. Interestingly, the SarA targeted inhibitor showed negligible antimicrobial activity and markedly reduced the minimum inhibitory concentration of conventional antibiotics when used in combination making it a more attractive lead for further clinical tests.

9 CFR 113.102 - Leptospira Icterohaemorrhagiae Bacterin.

Science.gov (United States)

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Leptospira Icterohaemorrhagiae... REQUIREMENTS Inactivated Bacterial Products § 113.102 Leptospira Icterohaemorrhagiae Bacterin. Leptospira Icterohaemorrhagiae Bacterin shall be produced from a culture of Leptospira icterohaemorrhagiae which has been...

Anti-biofilm activity of biogenic selenium nanoparticles and selenium dioxide against clinical isolates of Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus mirabilis.

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Shakibaie, Mojtaba; Forootanfar, Hamid; Golkari, Yaser; Mohammadi-Khorsand, Tayebe; Shakibaie, Mohammad Reza

2015-01-01

The aim of the present study was to investigate the anti-biofilm activity of biologically synthesized selenium nanoparticles (Se NPs) against the biofilm produced by clinically isolated bacterial strains compared to that of selenium dioxide. Thirty strains of Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus mirabilis were isolated from various specimens of the patients hospitalized in different hospitals (Kerman, Iran). Quantification of the biofilm using microtiter plate assay method introduced 30% of S. aureus, 13% of P. aeruginosa and 17% of P. mirabilis isolates as severely adherent strains. Transmission electron micrograph (TEM) of the purified Se NPs (produced by Bacillus sp. MSh-1) showed individual and spherical nano-structure in the size range of 80-220nm. Obtained results of the biofilm formation revealed that selenium nanoparticles inhibited the biofilm of S. aureus, P. aeruginosa, and P. mirabilis by 42%, 34.3%, and 53.4%, respectively, compared to that of the non-treated samples. Effect of temperature and pH on the biofilm formation in the presence of Se NPs and SeO2 was also evaluated. Copyright © 2014 Elsevier GmbH. All rights reserved.

Staphylococcus aureus sarA regulates inflammation and colonization during central nervous system biofilm formation.

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Jessica N Snowden

Full Text Available Infection is a frequent and serious complication following the treatment of hydrocephalus with CSF shunts, with limited therapeutic options because of biofilm formation along the catheter surface. Here we evaluated the possibility that the sarA regulatory locus engenders S. aureus more resistant to immune recognition in the central nervous system (CNS based on its reported ability to regulate biofilm formation. We utilized our established model of CNS catheter-associated infection, similar to CSF shunt infections seen in humans, to compare the kinetics of bacterial titers, cytokine production and inflammatory cell influx elicited by wild type S. aureus versus an isogenic sarA mutant. The sarA mutant was more rapidly cleared from infected catheters compared to its isogenic wild type strain. Consistent with this finding, several pro-inflammatory cytokines and chemokines, including IL-17, CXCL1, and IL-1β were significantly increased in the brain following infection with the sarA mutant versus wild type S. aureus, in agreement with the fact that the sarA mutant displayed impaired biofilm growth and favored a planktonic state. Neutrophil influx into the infected hemisphere was also increased in the animals infected with the sarA mutant compared to wild type bacteria. These changes were not attributable to extracellular protease activity, which is increased in the context of SarA mutation, since similar responses were observed between sarA and a sarA/protease mutant. Overall, these results demonstrate that sarA plays an important role in attenuating the inflammatory response during staphylococcal biofilm infection in the CNS via a mechanism that remains to be determined.

Anti-biofilm, anti-hemolysis, and anti-virulence activities of black pepper, cananga, myrrh oils, and nerolidol against Staphylococcus aureus.

Science.gov (United States)

Lee, Kayeon; Lee, Jin-Hyung; Kim, Soon-Il; Cho, Moo Hwan; Lee, Jintae

2014-11-01

The long-term usage of antibiotics has resulted in the evolution of multidrug-resistant bacteria. Unlike antibiotics, anti-virulence approaches target bacterial virulence without affecting cell viability, which may be less prone to develop drug resistance. Staphylococcus aureus is a major human pathogen that produces diverse virulence factors, such as α-toxin, which is hemolytic. Also, biofilm formation of S. aureus is one of the mechanisms of its drug resistance. In this study, anti-biofilm screening of 83 essential oils showed that black pepper, cananga, and myrrh oils and their common constituent cis-nerolidol at 0.01 % markedly inhibited S. aureus biofilm formation. Furthermore, the three essential oils and cis-nerolidol at below 0.005 % almost abolished the hemolytic activity of S. aureus. Transcriptional analyses showed that black pepper oil down-regulated the expressions of the α-toxin gene (hla), the nuclease genes, and the regulatory genes. In addition, black pepper, cananga, and myrrh oils and cis-nerolidol attenuated S. aureus virulence in the nematode Caenorhabditis elegans. This study is one of the most extensive on anti-virulence screening using diverse essential oils and provides comprehensive data on the subject. This finding implies other beneficial effects of essential oils and suggests that black pepper, cananga, and myrrh oils have potential use as anti-virulence strategies against persistent S. aureus infections.

The anti-biofilm activity of lemongrass (Cymbopogon flexuosus) and grapefruit (Citrus paradisi) essential oils against five strains of Staphylococcus aureus.

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Adukwu, E C; Allen, S C H; Phillips, C A

2012-11-01

To determine the sensitivity of five strains of Staphylococcus aureus to five essential oils (EOs) and to investigate the anti-biofilm activity of lemongrass and grapefruit EOs. Antimicrobial susceptibility screening was carried out using the disk diffusion method. All of the strains tested were susceptible to lemongrass, grapefruit, bergamot and lime EOs with zones of inhibition varying from 2·85 to 8·60 cm although they were resistant to lemon EO. Lemongrass EO inhibited biofilm formation at 0·125% (v/v) as measured by colorimetric assay and at 0·25% (v/v) no metabolic activity was observed as determined by 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction. Grapefruit EO did not show any anti-biofilm activity. Following exposure to lemongrass EO extensive disruption to Staph. aureus biofilms was shown under scanning electron microscopy. In comparison to the other EOs tested, lemongrass exhibited the most effective antimicrobial and anti-biofilm activity. The effect of lemongrass EO highlights its potential against antibiotic resistant Staph. aureus in the healthcare environment. © 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

Controlled Release of Plectasin NZ2114 from a Hybrid Silicone-Hydrogel Material for Inhibition of Staphylococcus aureus Biofilm

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Klein, Kasper; Grønnemose, Rasmus Birkholm; Alm, Martin

2017-01-01

this system with plectasin derivate NZ2114 in an attempt to design an S. aureus biofilm-resistant catheter. The material demonstrated promising antibiofilm properties, including properties against methicillin-resistant S. aureus, thus suggesting a novel application of this antimicrobial peptide....

Mammary Gland Pathology Subsequent to Acute Infection with Strong versus Weak Biofilm Forming Staphylococcus aureus Bovine Mastitis Isolates: A Pilot Study Using Non-Invasive Mouse Mastitis Model.

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Jully Gogoi-Tiwari

Full Text Available Biofilm formation by Staphylococcus aureus is an important virulence attribute because of its potential to induce persistent antibiotic resistance, retard phagocytosis and either attenuate or promote inflammation, depending upon the disease syndrome, in vivo. This study was undertaken to evaluate the potential significance of strength of biofilm formation by clinical bovine mastitis-associated S. aureus in mammary tissue damage by using a mouse mastitis model.Two S. aureus strains of the same capsular phenotype with different biofilm forming strengths were used to non-invasively infect mammary glands of lactating mice. Biofilm forming potential of these strains were determined by tissue culture plate method, ica typing and virulence gene profile per detection by PCR. Delivery of the infectious dose of S. aureus was directly through the teat lactiferous duct without invasive scraping of the teat surface. Both bacteriological and histological methods were used for analysis of mammary gland pathology of mice post-infection.Histopathological analysis of the infected mammary glands revealed that mice inoculated with the strong biofilm forming S. aureus strain produced marked acute mastitic lesions, showing profuse infiltration predominantly with neutrophils, with evidence of necrosis in the affected mammary glands. In contrast, the damage was significantly less severe in mammary glands of mice infected with the weak biofilm-forming S. aureus strain. Although both IL-1β and TNF-α inflammatory biomarkers were produced in infected mice, level of TNF-α produced was significantly higher (p<0.05 in mice inoculated with strong biofilm forming S. aureus than the weak biofilm forming strain.This finding suggests an important role of TNF-α in mammary gland pathology post-infection with strong biofilm-forming S. aureus in the acute mouse mastitis model, and offers an opportunity for the development of novel strategies for reduction of mammary tissue damage

Activity of essential oil-based microemulsions against Staphylococcus aureus biofilms developed on stainless steel surface in different culture media and growth conditions.

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Campana, Raffaella; Casettari, Luca; Fagioli, Laura; Cespi, Marco; Bonacucina, Giulia; Baffone, Wally

2017-01-16

Food safety is a fundamental concern for both consumers and the food industry, especially as the numbers of reported cases of food-associated infections continue to increase. Industrial surfaces can provide a suitable substrate for the development and persistence of bacterial organized in biofilms that represent a potential source of food contamination. The negative consumer perception of chemical disinfectants has shifted the attention to natural substances, such as plant extracts. The aim of this study was to investigate the possibility of using the essential oils (EOs) in the fight against S. aureus biofilms. First, the Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), Minimum Biofilm Inhibitory Concentration (MBIC), Minimum Biofilm Eradication Concentration (MBEC) of eleven EOs against S. aureus were determined. Cinnamomum cassia and Salvia officinalis EOs showed the greatest antibacterial properties with 1.25% MIC and MBC, 1.25% MBIC and 2.5% MBEC respectively. Gas Chromatography/Mass Spectrometry analysis revealed cinnamaldehyde (82.66%) and methoxy cinnamaldehyde (10.12%) as the most abundant substances of C. cassia, while cis-thujone (23.90%), camphor (19.22%) and 1.8-cineole (10.62%) of S. officinalis. Three different microemulsions, formulated with C. cassia, S. officinalis or both, were finally tested against S. aureus biofilms in different culture media and growth conditions, causing a >3 logarithmic reductions in S. aureus 24h-old biofilms and desiccated biofilms, and up to 68% of biofilm removal after 90min of exposure. The obtained data suggest the potential use of EOs, alone or in combination, for the formulation of sanitizers as alternative or in support in the disinfection of contaminated surfaces. Copyright © 2016 Elsevier B.V. All rights reserved.

Changes in the Expression of Biofilm-Associated Surface Proteins in Staphylococcus aureus Food-Environmental Isolates Subjected to Sublethal Concentrations of Disinfectants

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Lenka Cincarova

2016-01-01

Full Text Available Sublethal concentrations (sub-MICs of certain disinfectants are no longer effective in removing biofilms from abiotic surfaces and can even promote the formation of biofilms. Bacterial cells can probably adapt to these low concentrations of disinfectants and defend themselves by way of biofilm formation. In this paper, we report on three Staphylococcus aureus biofilm formers (strong B+++, moderate B++, and weak B+ that were cultivated with sub-MICs of commonly used disinfectants, ethanol or chloramine T, and quantified using Syto9 green fluorogenic nucleic acid stain. We demonstrate that 1.25–2.5% ethanol and 2500 μg/mL chloramine T significantly enhanced S. aureus biofilm formation. To visualize differences in biofilm compactness between S. aureus biofilms in control medium, 1.25% ethanol, or 2500 μg/mL chloramine T, scanning electron microscopy was used. To describe changes in abundance of surface-exposed proteins in ethanol- or chloramine T-treated biofilms, surface proteins were prepared using a novel trypsin shaving approach and quantified after dimethyl labeling by LC-LTQ/Orbitrap MS. Our data show that some proteins with adhesive functions and others with cell maintenance functions and virulence factor EsxA were significantly upregulated by both treatments. In contrast, immunoglobulin-binding protein A was significantly downregulated for both disinfectants. Significant differences were observed in the effect of the two disinfectants on the expression of surface proteins including some adhesins, foldase protein PrsA, and two virulence factors.

Biofilm Removal and Antimicrobial Activities of Agar Hydrogel Containing Colloid Nano-Silver against Staphylococcus aureus and Salmonella typhimurium

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Leyla Sadat Bouryabaf

2017-10-01

Full Text Available Background:    Antibacterial and biofilm removal effects of agar hydrogel incorporating silver nanoparticles (SNP at various concentrations were studied against Staphylococcus aureus and Salmonella typhimurium in vitro.Methods:      The minimum inhibitory concentrations (MIC of SNP was determined by agar dilution method. Then, hydrogels were prepared by mixing of 0.5% w/v agar and SNP (1/2 MIC, MIC, and 2 MIC and their inhibitory efficacies against planktonic and biofilm forms of bacteria were measured using agar spot and microtiter test, respectively.Results:    The MIC value was 125 µg/ mL for both bacteria. All SNP hydrogels represented antibacterial activity against Staphylococcus aureus and S. typhimurium on agar culture, which was significant compared to control group (silver sulfadiazine cream. The developed biofilm of S. aureus and S. typhimurium were strongly (85% reduction and modernly affected (60% reduction by SNP hydrogels during 15 min contact time, respectively. A dose-dependent biofilm reduction was not demonstrated when different SNP concentrations were tested. Moreover, the results from this study confirmed the moderate sanitizing ability of SNP loaded hydrogel against planktonic forms of both bacteria, which SNP (2MIC hydrogel decreased only 2.3 log10 CFU/ mL in a primary population of S. typhimurium during 15 min exposure time.Conclusion:     We recommended SNP incorporated agar hydrogel as an effective biofilm removal sanitizer.

Chemical composition of essential oil in Mosla chinensis Maxim cv. Jiangxiangru and its inhibitory effect on Staphylococcus aureus biofilm formation

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Peng Liang

2018-03-01

Full Text Available The essential oil of Mosla chinensis Maxim cv. Jiangxiangru is known for its antibacterial ability. This study aimed to investigate the chemical composition of Jiangxiangru essential oil and its inhibitory effect on Staphylococcus aureus biofilm formation. Gas chromatography/mass spectrometry (GC–MS was used to determine the chemical composition of Jiangxiangru essential oil. Subsequently, the eight major chemical components were quantitatively analyzed using GC– MS, and their minimum inhibitory concentration (MIC values against S. aureus were tested. Biofilm formation was detected by crystal violet semi-quantitative method and silver staining. Of the 59 peaks detected, 29 were identified by GC–MS. Of these peaks, thymol, carvacrol, p-cymene, γ-terpinene, thymol acetate, α-caryophyllene, 3-carene, and carvacryl acetate were present at a relatively higher concentration. The results of the quantitative test showed that thymol, carvacrol, p-cymene, and γ-terpinene were the major components of the essential oil. Among the eight reference substances, only thymol, carvacrol, and thymol acetate had lower MICs compared with the essential oil. Essential oil, carvacrol, carvacryl acetate, α-caryophyllene, and 3-carene showed the better inhibition of S. aureus biofilm formation. When one fourth of the MIC concentrations were used for these substances (0.0625 mg/mL for essential oil, 0.0305 mg/mL for carvacrol, 1.458 mg/mL for carvacryl acetate, 0.1268 mg/mL for α-caryophyllene, and 2.5975 mg/mL for 3-carene, the inhibition rates were over 80%. However, thymol, γ-terpinene, thymol acetate, and p-cymene showed a relatively poor inhibition of S. aureus biofilm formation. When 1× MIC concentrations of these substances were used, the inhibition rates were less than 50%. In conclusion, Jiangxiangru essential oil and its major components, carvacrol, carvacryl acetate, α-caryophyllene, and 3-carene, strongly inhibited biofilm formation in S. aureus.

Evaluation of biofilm formation using milk in a flow cell model and microarray characterization of Staphylococcus aureus strains from bovine mastitis.

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Snel, G G M; Malvisi, M; Pilla, R; Piccinini, R

2014-12-05

It was hypothesized that biofilm could play an important role in the establishment of chronic Staphylococcus aureus bovine mastitis. The in vitro evaluation of biofilm formation can be performed either in closed/static or in flow-based systems. Efforts have been made to characterize the biofilm-forming ability of S. aureus mastitis isolates, however most authors used static systems and matrices other than UHT milk. It is not clear whether such results could be extrapolated to the mammary gland environment. Therefore, the present study aimed to investigate the biofilm-forming ability of S. aureus strains from subclinical bovine mastitis using the static method and a flow-based one. One hundred and twelve strains were tested by the classic tissue culture plate assay (TCP) and 30 out of them were also tested by a dynamic semi-quantitative assay using commercial UHT milk as culture medium (Milk Flow Culture, MFC) or Tryptic Soy Broth as control medium (TS Flow Culture, TSFC). Only 6 (20%) strains formed biofilm in milk under flow conditions, while 36.6% were considered biofilm-producers in TCP, and 93.3% produced biofilm in TSFC. No agreement was found between TCP, MFC and TSFC results. The association between strain genetic profile, determined by microarray, and biofilm-forming ability in milk was evaluated. Biofilm formation in MFC was significantly associated with the presence of those genes commonly found in bovine-associated strains, assigned to clonal complexes typically detected in mastitis. Based on our results, biofilm-forming potential of bovine strains should be critically analysed and tested applying conditions similar to mammary environment. Copyright © 2014 Elsevier B.V. All rights reserved.

Rapid in Vitro Quantification of S. aureus Biofilms on Vascular Graft Surfaces

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Monika Herten

2017-12-01

Full Text Available Objectives: Increasing resistance of microorganisms and particularly tolerance of bacterial biofilms against antibiotics require the need for alternative antimicrobial substances. S. aureus is the most frequent pathogen causing vascular graft infections. In order to evaluate the antimicrobial efficacy, quantification of the bacterial biofilms is necessary. Aim of the present study was the validation of an in vitro model for quantification of bacterial biofilm on vascular graft surfaces using three different assays.Methods: Standardized discs of vascular graft material (Dacron or PTFE or polystyrene (PS as control surface with 0.25 cm2 surface area were inoculated with 10−3 diluted overnight culture of three biofilm-producing S. aureus isolates (BEB-029, BEB-295, SH1000 in 96-well PS culture plates. After incubation for 4 and 18 h, the biofilm was determined by three different methods: (a mitochondrial ATP concentration as measure of bacterial viability (ATP, (b crystal violet staining (Cry, and (c vital cell count by calculation of colony-forming units (CFU. The experiments were performed three times. Quadruplicates were used for each isolate, time point, and method. In parallel, bacterial biofilms were documented via scanning electron microscopy.Results: All three methods could quantify biofilms on the PS control. Time needed was 0:40, 13:10, and 14:30 h for ATP, Cry, and CFU, respectively. The Cry assay could not be used for vascular graft surfaces due to high unspecific background staining. However, ATP assay and CFU count showed comparable results on vascular graft material and control. The correlations between ATP and CFU assay differed according to the surface and incubation time and were significant only after 4 h on Dacron (BEB-029, p = 0.013 and on PS (BEB-029, p < 0.001. Between ATP and Cry assay on PS, a significant correlation could be detected after 4 h (BEB-295, p = 0.027 and after 18 h (all three strains, p < 0.026. The

Effect of montanide adjuvanted staphylococcus aureus bacterin-toxiod on prevalence and incidence of mastitis in cows

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Yousif, A.; Muhmmad, G.; Masood, M.Z.; Rahman, S.U.; Siddique, M.

2009-01-01

The current study was conducted to investigate the prevalence and incidence of mastitis associated with Montanide adjuvanted Staphylococcus aureus bacterin toxiod in dairy cows in Faisalabad (Punjab, Pakistan). A total of 60 mastitis free dairy cows, in first and second month of lactation, were selected on the basis of results determined by California mastitis test and Surf field mastitis test. The animals were randomly divided into three groups (C1, C2, and C3) each containing twenty cows. The vaccine was administered twice with four weeks interval at the rate of 5 ml intramuscularly in the neck region in all the animals of group C1 and C2. Levamisole HCl (Nilverm at the rate, ICI, Pakistan) at the rate 2.5 mg per kg weight was given orally to animals of group C2 after first and second injections of vaccine, while the animals in group C3 were kept as control (non-vaccinated and non-medicated with levamisole HCl). Prevalence and incidence rates were determined at 1st, 30th , 60th , 120th and 180th days post-vaccination. The results showed that maximum numbers of quarters were found positive in group C3 with maximum cumulative prevalence (27.5%) while minimum (11.25%) was recorded in group C2 followed by vaccinated group (13.75 %). There was no significant variation in cumulative incidence between the vaccinated and vaccinated plus levamisole HCl group. (author)

Chitosan-adjuvanted Mycoplasma gallisepticum bacterin via intraocular administration enhances Mycoplasma gallisepticum protection in commercial layers.

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Limsatanun, A; Sasipreeyajan, J; Pakpinyo, S

2018-06-01

Mycoplasma gallisepticum (MG) causes respiratory signs and economic losses in the poultry industry. MG vaccination is one of the effective prevention and control measures that have been used around the world. Our previous study demonstrated that chitosan-adjuvanted MG bacterin could effectively reduce pathological lesions induced by MG and that chitosan could be used as an adjuvant in MG bacterin. The present study determining the efficacy of MG bacterins against the Thai MG strain was based on vaccine programs. Seven groups (25 layers/group) were received MG bacterins containing 0.5% chitosan or a commercial bacterin via intramuscular (IM) or intraocular (IO) route at 6 and 10 wk of age. Sham-negative and sham-positive controls were groups 1 and 2, respectively. Group 3: IM route of chitosan bacterin followed by IM route of chitosan bacterin; group 4: commercial bacterin via IM route followed by chitosan bacterin via IO route; group 5: commercial bacterin via IM route followed by commercial bacterin via IM route; group 6: chitosan bacterin via IM followed by chitosan bacterin via IO route; and group 7: chitosan bacterin via IO route followed by chitosan bacterin via IO route were determined. At 16 wk of age, all groups, excluding group 1, were challenged intratracheally with 0.1 mL containing Thai MG strain 107 colony-forming unit. At 17, 18, and 20 wk of age, 5 birds in each group were bled for serological testing and swabbed at the choanal cleft for the quantitative real-time PCR assay, the euthanized and necropsied. The results showed that birds vaccinated with a commercial intramuscular bacterin followed by an intraocularly chitosan adjuvant bacterin showed the best protection against the MG challenge. The study indicated that chitosan could be the effective mucosal adjuvant and increased the effectiveness of MG bacterin.

Development of a biofilm inhibitor molecule against multidrug resistant Staphylococcus aureus associated with gestational urinary tract infections

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Balamurugan eP

2015-08-01

Full Text Available Urinary Tract Infection (UTI is a globally widespread human infection caused by an infestation of uropathogens. Eventhough, Escherichia coli is often quoted as being the chief among them, Staphylococcus aureus involvement in UTI especially in gestational UTI is often understated. Staphylococcal accessory regulator A (SarA is a quorum regulator of S. aureus that controls the expression of various virulence and biofilm phenotypes. Since SarA had been a focussed target for antibiofilm agent development, the study aims to develop a potential drug molecule targeting the SarA of S. aureus to combat biofilm associated infections in which it is involved. In our previous studies, we have reported the antibiofilm activity of SarA based biofilm inhibitor, (SarABI with a 50% minimum biofilm inhibitory concentration (MBIC50 value of 200 µg/mL against S. aureus associated with vascular graft infections and also the antibiofilm activity of the root ethanolic extracts of Melia dubia against uropathogenic E. coli. In the present study, in silico design of a hybrid molecule composed of a molecule screened from M. dubia root ethanolic extracts and a modified SarA based inhibitor (SarABIM was undertaken. SarABIM is a modified form of SarABI where the fluorine groups are absent in SarABIM. Chemical synthesis of the hybrid molecule, 4-(Benzylaminocyclohexyl 2-hydroxycinnamate (henceforth referred to as UTI Quorum-Quencher, UTIQQ was then performed, followed by in vitro and in vivo validation. The MBIC¬50 and MBIC90 of UTIQQ were found to be 15 µg/mL and 65 µg/mL respectively. Confocal laser scanning microscopy (CLSM images witnessed biofilm reduction and bacterial killing in either UTIQQ or in combined use of antibiotic gentamicin and UTIQQ. Similar results were observed with in vivo studies of experimental UTI in rat model. So, we propose that the drug UTIQQ would be a promising candidate when used alone or, in combination with an antibiotic for staphylococcal

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

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

2018-03-01

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

Melittin and its potential in the destruction and inhibition of the biofilm formation by Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa isolated from bovine milk.

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Picoli, Tony; Peter, Cristina Mendes; Zani, João Luíz; Waller, Stefanie Bressan; Lopes, Matheus Gomes; Boesche, Kamilla Neutzling; Vargas, Gilberto D Ávila; Hübner, Silvia de Oliveira; Fischer, Geferson

2017-11-01

Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa stand out in veterinary and human medicine for their role in opportunistic infections and their pathogenic mechanisms, including the biofilms formation. It was investigated the antibacterial activity of melittin and antibiofilm of such bacteria. Twelve strains of these microorganisms isolated from bovine milk were used, as well as the strains S. aureus ATCC 12600, E. coli ATCC 8739 and Pseudomonas aeruginosa ATCC 15442. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentration (MBC) were determined by broth microdilution technique. The biofilms were formed in 96-well plates and melittin on these colonies was added at different concentrations and times. Bacteria previously exposed to melittin were evaluated for inhibition of biofilm production. The MIC and MBC were respectively in μg/mL: S. aureus (6-7 and 32-64), E. coli (40-42.5 and 64-128) and P. aeruginosa (65-70 and 64-128). S. aureus biofilms were more sensitive to the action of melittin, since upon exposure to a concentration 10 times lower than the MIC for 4 h, was completely destroyed. In Gram negative bacteria, the pre-formed biofilm was destroyed only when exposed for 4 h under the MIC. With respect to inhibition of biofilm production, S. aureus was the most sensitive again because produced only 37.2% of the biofilm formed by the control (without previous exposure to melittin), when exposed to the MIC, and at a concentration hundred times smaller than MIC, this microorganism produced 75.2% of the biofilm. E. coli was the most resistant bacteria and produced 56.3% of the biofilm, even if previously exposed to melittin MIC. Melittin presents desirable effects in combating microorganisms studied both at your disposal, biofilm destruction and inhibition of the formation, and maybe used in future studies of new strategies to combat infections caused by these pathogens. Copyright © 2017 Elsevier Ltd. All

Low Fluid Shear Culture of Staphylococcus Aureus Represses hfq Expression and Induces an Attachment-Independent Biofilm Phenotype

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Ott, C. Mark; Castro, S. L.; Nickerson, C. A.; Nelman-Gonzalez, M.

2011-01-01

Background: The opportunistic pathogen, Staphylococcus aureus, experiences fluctuations in fluid shear during infection and colonization of a human host. Colonization frequently occurs at mucus membrane sites such as in the gastrointestinal tract where the bacterium may experience low levels of fluid shear. The response of S. aureus to low fluid shear remains unclear. Methods: S. aureus was cultured to stationary phase using Rotating-Wall Vessel (RWV) bioreactors which produce a physiologically relevant low fluid shear environment. The bacterial aggregates that developed in the RWV were evaluated by electron microscopy as well as for antibiotic resistance and other virulence-associated stressors. Genetic expression profiles for the low-shear cultured S. aureus were determined by microarray analysis and quantitative real-time PCR. Results: Planktonic S. aureus cultures in the low-shear environment formed aggregates completely encased in high amounts of extracellular polymeric substances. In addition, these aggregates demonstrated increased antibiotic resistance indicating attachment-independent biofilm formation. Carotenoid production in the low-shear cultured S. aureus was significantly decreased, and these cultures displayed an increased susceptibility to oxidative stress and killing by whole blood. The hfq gene, associated with low-shear growth in Gram negative organisms, was also found to be down-regulated in S. aureus. Conclusions: Collectively, this data suggests that S. aureus decreases virulence characteristics in favor of a biofilm-dwelling colonization phenotype in response to a low fluid shear environment. Furthermore, the identification of an Hfq response to low-shear culture in S. aureus, in addition to the previously reported responses in Gram negative organisms, strongly suggests an evolutionarily conserved response to mechanical stimuli among structurally diverse prokaryotes.

Biofilm formation by Staphylococcus aureus and Salmonella spp. under mono and dual-species conditions and their sensitivity to cetrimonium bromide, peracetic acid and sodium hypochlorite

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Maricarmen Iñiguez-Moreno

Full Text Available Abstract The aim of this study was evaluated the biofilm formation by Staphylococcus aureus 4E and Salmonella spp. under mono and dual-species biofilms, onto stainless steel 316 (SS and polypropylene B (PP, and their sensitivity to cetrimonium bromide, peracetic acid and sodium hypochlorite. The biofilms were developed by immersion of the surfaces in TSB by 10 d at 37 °C. The results showed that in monospecies biofilms the type of surface not affected the cellular density (p > 0.05. However, in dual-species biofilms on PP the adhesion of Salmonella spp. was favored, 7.61 ± 0.13 Log10 CFU/cm2, compared with monospecies biofilms onto the same surface, 5.91 ± 0.44 Log10 CFU/cm2 (p < 0.05. The mono and dual-species biofilms were subjected to disinfection treatments; and the most effective disinfectant was peracetic acid (3500 ppm, reducing by more than 5 Log10 CFU/cm2, while the least effective was cetrimonium bromide. In addition, S. aureus 4E and Salmonella spp. were more resistant to the disinfectants in mono than in dual-species biofilms (p < 0.05. Therefore, the interspecies interactions between S. aureus 4E and Salmonella spp. had a negative effect on the antimicrobial resistance of each microorganism, compared with the monospecies biofilms.

NaOCl effect on biofilm produced by Staphylococcus aureus isolated from the milking environment and mastitis infected cows

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Poliana de Castro Melo

2014-02-01

Full Text Available Biofilms constitute a physical barrier, protecting the encased bacteria from detergents and sanitizers. The objective of this work was to analyze the effectiveness of sodium hypochlorite (NaOCl against strains of Staphylococcus aureus isolated from raw milk of cows with subclinical mastitis and Staphylococcus aureus isolated from the milking environment (blowers and milk conducting tubes. The results revealed that, in the presence of NaOCl (150ppm, the number of adhered cells of the twelve S. aureus strains was significantly reduced. When the same strains were evaluated in biofilm condition, different results were obtained. It was found that, after a contact period of five minutes with NaOCl (150ppm, four strains (two strains from milk , one from the blowers and one from a conductive rubber were still able to grow. Although with the increasing contact time between the bacteria and the NaOCl (150ppm, no growth was detected for any of the strains. Concerning the efficiency of NaOCl on total biofilm biomass formation by each S. aureus strain, a decrease was observed when these strains were in contact with 150 ppm NaOCl for a total period of 10 minutes. This study highlights the importance of a correct sanitation protocol of all the milk processing units which can indeed significantly reduce the presence of microorganisms, leading to a decrease of cow´s mastitis and milk contamination.

XF-70 and XF-73, novel antibacterial agents active against slow-growing and non-dividing cultures of Staphylococcus aureus including biofilms.

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Ooi, Nicola; Miller, Keith; Randall, Christopher; Rhys-Williams, William; Love, William; Chopra, Ian

2010-01-01

Slow-growing and non-dividing bacteria exhibit tolerance to many antibiotics. However, membrane-active agents may act against bacteria in all growth phases. We sought to examine whether the novel porphyrin antibacterial agents XF-70 and XF-73, which have rapid membrane-perturbing activity against Staphylococcus aureus, retained antistaphylococcal activity against growth-attenuated cells. The killing kinetics of XF-70, XF-73 and various comparator agents against exponential phase cultures of S. aureus SH1000 were compared with effects on cells held at 4 degrees C, non-growing cultures expressing the stringent response induced by mupirocin and bacteria in the stationary phase. Biofilms of S. aureus SH1000 were generated with the Calgary device to examine the activities of XF-70 and XF-73 under a further system exhibiting diminished bacterial growth. Cold culture, stringent response and stationary phase cultures remained susceptible to XF-70 and XF-73, which caused > or =5 log reductions in viability over 2 h. During this period the most active comparator agents (chlorhexidine and cetyltrimethylammonium bromide) only promoted a 3 log drop in viability. XF-70 and XF-73 were also highly active against biofilms, with both agents exhibiting low biofilm MICs (1 mg/L) and minimum biofilm eradication concentrations (2 mg/L). XF-70 and XF-73 remained highly active against various forms of slow-growing or non-dividing S. aureus. The results support the hypothesis that membrane-active agents may be particularly effective in eradicating slow- or non-growing bacteria and suggest that XF-70 and XF-73 could be utilized to treat staphylococcal infections where the organisms are only dividing slowly, such as biofilm-associated infections of prosthetic devices.

Topical colloidal silver as an anti-biofilm agent in a Staphylococcus aureus chronic rhinosinusitis sheep model.

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Rajiv, Sukanya; Drilling, Amanda; Bassiouni, Ahmed; James, Craig; Vreugde, Sarah; Wormald, Peter-John

2015-04-01

Treatment of recalcitrant chronic rhinosinusitis (CRS) is a challenge with increasing antibiotic resistance, leading to re-emergence of topical therapies. The aim of this study was to assess safety and efficacy of topical colloidal silver solution for the treatment of Staphylococcus aureus biofilms in a sheep model. In the safety study, normal saline (control) and 30-ppm colloidal silver solution (test) was used to flush the frontal sinuses for 14 days in 8 sheep (4 sheep each). In the efficacy study, following frontal sinus infection with Staphylococcus aureus, sheep were treated with either control saline or topical silver solution of varying concentrations (30 ppm/20 ppm/10 ppm/5 ppm) for 5 days, with 4 sheep in each group. Blood silver level, full blood counts, and biochemical parameters were analyzed in both safety and efficacy studies. Sinus tissue was harvested for histological examination and ciliary structure analysis in safety and for biofilm biomass quantification by fluorescence in situ hybridization (FISH) technique and COMSTAT 2 software in the efficacy study. Results were analyzed using appropriate statistical tests. Sheep treated with silver showed a significant decrease in biofilm biomass (0.004, 0.004, 0.004, and 0.007, in the 4 silver-treated groups, respectively) compared to saline control (0.175), p silver levels were higher in the treated groups compared to controls (p colloidal silver solution has effective antibiofilm activity in Staphylococcus aureus CRS in a sheep model and appears safe. © 2015 ARS-AAOA, LLC.

Inhibitory effects of food additives derived from polyphenols on staphylococcal enterotoxin A production and biofilm formation by Staphylococcus aureus.

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Shimamura, Yuko; Hirai, Chikako; Sugiyama, Yuka; Shibata, Masaharu; Ozaki, Junya; Murata, Masatsune; Ohashi, Norio; Masuda, Shuichi

2017-12-01

In this study, we examined the inhibitory effects of 14 food additives derived from polyphenol samples on staphylococcal enterotoxin A (SEA) production and biofilm formation by Staphylococcus aureus. Tannic acid AL (TA), Purephenon 50 W (PP) and Polyphenon 70A (POP) at 0.25 mg/mL and Gravinol®-N (GN), Blackcurrant polyphenol AC10 (BP), and Resveratrol-P5 (RT) at 1.0 mg/mL significantly decreased SEA production by S. aureus C-29 (p Food additives derived from polyphenols have viability to be used as a means to inhibit the enterotoxin production and control the biofilm formation of foodborne pathogens.

Biofilm formation by Staphylococcus aureus and Salmonella spp. under mono and dual-species conditions and their sensitivity to cetrimonium bromide, peracetic acid and sodium hypochlorite.

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Iñiguez-Moreno, Maricarmen; Gutiérrez-Lomelí, Melesio; Guerrero-Medina, Pedro Javier; Avila-Novoa, María Guadalupe

The aim of this study was evaluated the biofilm formation by Staphylococcus aureus 4E and Salmonella spp. under mono and dual-species biofilms, onto stainless steel 316 (SS) and polypropylene B (PP), and their sensitivity to cetrimonium bromide, peracetic acid and sodium hypochlorite. The biofilms were developed by immersion of the surfaces in TSB by 10 d at 37°C. The results showed that in monospecies biofilms the type of surface not affected the cellular density (p>0.05). However, in dual-species biofilms on PP the adhesion of Salmonella spp. was favored, 7.61±0.13Log 10 CFU/cm 2 , compared with monospecies biofilms onto the same surface, 5.91±0.44Log 10 CFU/cm 2 (pdisinfection treatments; and the most effective disinfectant was peracetic acid (3500ppm), reducing by more than 5Log 10 CFU/cm 2 , while the least effective was cetrimonium bromide. In addition, S. aureus 4E and Salmonella spp. were more resistant to the disinfectants in mono than in dual-species biofilms (p<0.05). Therefore, the interspecies interactions between S. aureus 4E and Salmonella spp. had a negative effect on the antimicrobial resistance of each microorganism, compared with the monospecies biofilms. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Mammary Gland Pathology Subsequent to Acute Infection with Strong versus Weak Biofilm Forming Staphylococcus aureus Bovine Mastitis Isolates: A Pilot Study Using Non-Invasive Mouse Mastitis Model.

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Gogoi-Tiwari, Jully; Williams, Vincent; Waryah, Charlene Babra; Costantino, Paul; Al-Salami, Hani; Mathavan, Sangeetha; Wells, Kelsi; Tiwari, Harish Kumar; Hegde, Nagendra; Isloor, Shrikrishna; Al-Sallami, Hesham; Mukkur, Trilochan

2017-01-01

Biofilm formation by Staphylococcus aureus is an important virulence attribute because of its potential to induce persistent antibiotic resistance, retard phagocytosis and either attenuate or promote inflammation, depending upon the disease syndrome, in vivo. This study was undertaken to evaluate the potential significance of strength of biofilm formation by clinical bovine mastitis-associated S. aureus in mammary tissue damage by using a mouse mastitis model. Two S. aureus strains of the same capsular phenotype with different biofilm forming strengths were used to non-invasively infect mammary glands of lactating mice. Biofilm forming potential of these strains were determined by tissue culture plate method, ica typing and virulence gene profile per detection by PCR. Delivery of the infectious dose of S. aureus was directly through the teat lactiferous duct without invasive scraping of the teat surface. Both bacteriological and histological methods were used for analysis of mammary gland pathology of mice post-infection. Histopathological analysis of the infected mammary glands revealed that mice inoculated with the strong biofilm forming S. aureus strain produced marked acute mastitic lesions, showing profuse infiltration predominantly with neutrophils, with evidence of necrosis in the affected mammary glands. In contrast, the damage was significantly less severe in mammary glands of mice infected with the weak biofilm-forming S. aureus strain. Although both IL-1β and TNF-α inflammatory biomarkers were produced in infected mice, level of TNF-α produced was significantly higher (pmastitis model, and offers an opportunity for the development of novel strategies for reduction of mammary tissue damage, with or without use of antimicrobials and/or anti-inflammatory compounds for the treatment of bovine mastitis.

Investigation of biofilm formation on contact eye lenses caused by methicillin resistant Staphylococcus aureus.

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Khalil, M A; Sonbol, F I

2014-01-01

The objective was to investigate the biofilm-forming capacity of methicillin resistant Staphylococcus aureus (MRSA) isolated from eye lenses of infected patients. A total of 32 MRSA isolated from contact lenses of patients with ocular infections were screened for their biofilm-forming capacity using tube method (TM), Congo red agar (CRA), and microtiter plate (MtP) methods. The effect of some stress factor on the biofilm formation was studied. The biofilm-forming related genes, icaA, icaD and 10 microbial surface components that recognize adhesive matrix molecule (MSCRAMM), of the selected MRSA were also detected using polymerase chain reaction. Of 32 MRSA isolates, 34.37%, 59.37%, and 81.25% showed positive results using CRA, TM or MtP, respectively. Biofilm production was found to be reduced in the presence of ethanol or ethylenediaminetetraacetic acid and at extreme pH values. On the other hand, glucose or heparin leads to a concentration dependent increase of biofilm production by the isolates. The selected biofilm producing MRSA isolate was found to harbor the icaA, icaD and up to nine of 10 tested MSCRAMM genes, whereas the selected non biofilm producing MRSA isolate did not carry any of the tested genes. The MtP method was found to be the most effective phenotypic screening method for detection of biofilm formation by MRSA. Furthermore, the molecular approach should be taken into consideration for the rapid and correct diagnosis of virulent bacteria associated with contact eye lenses.

Effect of a solution containing citrate/Methylene Blue/parabens on Staphylococcus aureus bacteria and biofilm, and comparison with various heparin solutions.

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Sauer, Karin; Steczko, Janusz; Ash, Stephen R

2009-05-01

Some antibiotic solutions increase bacterial resistance and may cause toxic side effects. Heparin, frequently used as an anticoagulant in catheter lock solutions, may cause bleeding and stimulate biofilm formation. The aim of this study was to investigate the effect of a new antibacterial/antithrombotic solution, citrate/Methylene Blue/parabens (C/MB/P), versus various heparin solutions on the viability and the structure of preformed mature biofilms of Staphylococcus aureus bacteria. The degree of eradication of both planktonic and sessile microorganisms was evaluated. The changes in the structure of biofilms after exposure to C/MB/P and several concentrations of heparin were analysed by means of confocal laser scanning microscopy. COMSTAT image analysis was utilized to compare biofilm biomass, average and maximum height, surface coverage and roughness coefficient. Viability studies were performed on both biofilms and supernatant solutions. C/MB/P, in contrast to heparin solutions, significantly reduced biofilm biomass and thickness and reduced viability by 5 log when compared with saline treatment. No viable planktonic bacteria were detected and the few remaining biofilm cells appeared to be lysed. In contrast, most heparin solutions only reduced viability up to 1.0 log and failed to eradicate planktonic bacteria. C/MB/P has a rapid bactericidal effect on the preformed, mature biofilm of S. aureus. The structural changes of biofilms treated with C/MB/P, together with the observed log reduction of viable biofilm cells, confirmed the high potential of this solution to eliminate sessile bacteria. Furthermore, the tested solution entirely eliminated planktonic bacteria detached from the biofilm.

Formation of Staphylococcus aureus Biofilm in the Presence of Sublethal Concentrations of Disinfectants Studied via a Transcriptomic Analysis Using Transcriptome Sequencing (RNA-seq)

Science.gov (United States)

Oppelt, J.; Cincarova, L.

2017-01-01

ABSTRACT Staphylococcus aureus is a common biofilm-forming pathogen. Low doses of disinfectants have previously been reported to promote biofilm formation and to increase virulence. The aim of this study was to use transcriptome sequencing (RNA-seq) analysis to investigate global transcriptional changes in S. aureus in response to sublethal concentrations of the commonly used food industry disinfectants ethanol (EtOH) and chloramine T (ChT) and their combination (EtOH_ChT) in order to better understand the effects of these agents on biofilm formation. Treatment with EtOH and EtOH_ChT resulted in more significantly altered expression profiles than treatment with ChT. Our results revealed that EtOH and EtOH_ChT treatments enhanced the expression of genes responsible for regulation of gene expression (sigB), cell surface factors (clfAB), adhesins (sdrDE), and capsular polysaccharides (cap8EFGL), resulting in more intact biofilm. In addition, in this study we were able to identify the pathways involved in the adaptation of S. aureus to the stress of ChT treatment. Further, EtOH suppressed the effect of ChT on gene expression when these agents were used together at sublethal concentrations. These data show that in the presence of sublethal concentrations of tested disinfectants, S. aureus cells trigger protective mechanisms and try to cope with them. IMPORTANCE So far, the effect of disinfectants is not satisfactorily explained. The presented data will allow a better understanding of the mode of disinfectant action with regard to biofilm formation and the ability of bacteria to survive the treatment. Such an understanding could contribute to the effort to eliminate possible sources of bacteria, making disinfectant application as efficient as possible. Biofilm formation plays significant role in the spread and pathogenesis of bacterial species. PMID:29030437

Systematic Exploration of Natural and Synthetic Flavonoids for the Inhibition of Staphylococcus aureus Biofilms

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Manner, Suvi; Skogman, Malena; Goeres, Darla; Vuorela, Pia; Fallarero, Adyary

2013-01-01

When single-cell (or suspended) bacteria switch into the biofilm lifestyle, they become less susceptible to antimicrobials, imposing the need for anti-biofilms research. Flavonoids are among the most extensively studied natural compounds with an unprecedented amount of bioactivity claims. Most studies focus on the antibacterial effects against suspended cells; fewer reports have researched their anti-biofilm properties. Here, a high throughput phenotypic platform was utilized to screen for the inhibitory activity of 500 flavonoids, including natural and synthetic derivatives, against Staphylococcus aureus biofilms. Since discrepancies among results from earlier antibacterial studies on flavonoids had been noted, the current study aimed to minimize sources of variations. After the first screen, flavonoids were classified as inactive (443), moderately active (47) or highly active (10). Further, exclusion criteria combining bioactivity and selectivity identified two synthetic flavans as the most promising. The body of data reported here serves three main purposes. First, it offers an improved methodological workflow for anti-biofilm screens of chemical libraries taking into account the (many times ignored) connections between anti-biofilm and antibacterial properties. This is particularly relevant for the study of flavonoids and other natural products. Second, it provides a large and freely available anti-biofilm bioactivity dataset that expands the knowledge on flavonoids and paves the way for future structure-activity relationship studies and structural optimizations. Finally, it identifies two new flavans that can successfully act on biofilms, as well as on suspended bacteria and represent more feasible antibacterial candidates. PMID:24071942

msaABCR operon positively regulates biofilm development by repressing proteases and autolysis in Staphylococcus aureus.

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Sahukhal, Gyan S; Batte, Justin L; Elasri, Mohamed O

2015-02-01

Staphylococcus aureus is an important human pathogen that causes nosocomial and community-acquired infections. One of the most important aspects of staphylococcal infections is biofilm development within the host, which renders the bacterium resistant to the host's immune response and antimicrobial agents. Biofilm development is very complex and involves several regulators that ensure cell survival on surfaces within the extracellular polymeric matrix. Previously, we identified the msaABCR operon as an additional positive regulator of biofilm formation. In this study, we define the regulatory pathway by which msaABCR controls biofilm formation. We demonstrate that the msaABCR operon is a negative regulator of proteases. The control of protease production mediates the processing of the major autolysin, Atl, and thus regulates the rate of autolysis. In the absence of the msaABCR operon, Atl is processed by proteases at a high rate, leading to increased cell death and a defect in biofilm maturation. We conclude that the msaABCR operon plays a key role in maintaining the balance between autolysis and growth within the staphylococcal biofilm. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Bacteriophage Lysin CF-301, a Potent Antistaphylococcal Biofilm Agent.

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Schuch, Raymond; Khan, Babar K; Raz, Assaf; Rotolo, Jimmy A; Wittekind, Michael

2017-07-01

Biofilms pose a unique therapeutic challenge because of the antibiotic tolerance of constituent bacteria. Treatments for biofilm-based infections represent a major unmet medical need, requiring novel agents to eradicate mature biofilms. Our objective was to evaluate bacteriophage lysin CF-301 as a new agent to target Staphylococcus aureus biofilms. We used minimum biofilm-eradicating concentration (MBEC) assays on 95 S. aureus strains to obtain a 90% MBEC (MBEC 90 ) value of ≤0.25 μg/ml for CF-301. Mature biofilms of coagulase-negative staphylococci, Streptococcus pyogenes , and Streptococcus agalactiae were also sensitive to disruption, with MBEC 90 values ranging from 0.25 to 8 μg/ml. The potency of CF-301 was demonstrated against S. aureus biofilms formed on polystyrene, glass, surgical mesh, and catheters. In catheters, CF-301 removed all biofilm within 1 h and killed all released bacteria by 6 h. Mixed-species biofilms, formed by S. aureus and Staphylococcus epidermidis on several surfaces, were removed by CF-301, as were S. aureus biofilms either enriched for small-colony variants (SCVs) or grown in human synovial fluid. The antibacterial activity of CF-301 was further demonstrated against S. aureus persister cells in exponential-phase and stationary-phase populations. Finally, the antibiofilm activity of CF-301 was greatly improved in combinations with the cell wall hydrolase lysostaphin when tested against a range of S. aureus strains. In all, the data show that CF-301 is highly effective at disrupting biofilms and killing biofilm bacteria, and, as such, it may be an efficient new agent for treating staphylococcal infections with a biofilm component. Copyright © 2017 American Society for Microbiology.

Bacteriophage Lysin CF-301, a Potent Antistaphylococcal Biofilm Agent

KAUST Repository

Schuch, Raymond; Khan, Babar Khalid; Raz, Assaf; Rotolo, Jimmy A.; Wittekind, Michael

2017-01-01

Biofilms pose a unique therapeutic challenge because of the antibiotic tolerance of constituent bacteria. Treatments for biofilm-based infections represent a major unmet medical need, requiring novel agents to eradicate mature biofilms. Our objective was to evaluate bacteriophage lysin CF-301 as a new agent to target Staphylococcus aureus biofilms. We used minimum biofilm-eradicating concentration (MBEC) assays on 95 S. aureus strains to obtain a 90% MBEC (MBEC90) value of <= 0.25 mu g/ml for CF-301. Mature biofilms of coagulase-negative staphylococci, Streptococcus pyogenes, and Streptococcus agalactiae were also sensitive to disruption, with MBEC90 values ranging from 0.25 to 8 mu g/ml. The potency of CF-301 was demonstrated against S. aureus biofilms formed on polystyrene, glass, surgical mesh, and catheters. In catheters, CF-301 removed all biofilm within 1 h and killed all released bacteria by 6 h. Mixed-species biofilms, formed by S. aureus and Staphylococcus epidermidis on several surfaces, were removed by CF-301, as were S. aureus biofilms either enriched for small-colony variants (SCVs) or grown in human synovial fluid. The antibacterial activity of CF-301 was further demonstrated against S. aureus persister cells in exponential-phase and stationary-phase populations. Finally, the antibiofilm activity of CF-301 was greatly improved in combinations with the cell wall hydrolase lysostaphin when tested against a range of S. aureus strains. In all, the data show that CF-301 is highly effective at disrupting biofilms and killing biofilm bacteria, and, as such, it may be an efficient new agent for treating staphylococcal infections with a biofilm component.

Bacteriophage Lysin CF-301, a Potent Antistaphylococcal Biofilm Agent

KAUST Repository

Schuch, Raymond

2017-05-02

Biofilms pose a unique therapeutic challenge because of the antibiotic tolerance of constituent bacteria. Treatments for biofilm-based infections represent a major unmet medical need, requiring novel agents to eradicate mature biofilms. Our objective was to evaluate bacteriophage lysin CF-301 as a new agent to target Staphylococcus aureus biofilms. We used minimum biofilm-eradicating concentration (MBEC) assays on 95 S. aureus strains to obtain a 90% MBEC (MBEC90) value of <= 0.25 mu g/ml for CF-301. Mature biofilms of coagulase-negative staphylococci, Streptococcus pyogenes, and Streptococcus agalactiae were also sensitive to disruption, with MBEC90 values ranging from 0.25 to 8 mu g/ml. The potency of CF-301 was demonstrated against S. aureus biofilms formed on polystyrene, glass, surgical mesh, and catheters. In catheters, CF-301 removed all biofilm within 1 h and killed all released bacteria by 6 h. Mixed-species biofilms, formed by S. aureus and Staphylococcus epidermidis on several surfaces, were removed by CF-301, as were S. aureus biofilms either enriched for small-colony variants (SCVs) or grown in human synovial fluid. The antibacterial activity of CF-301 was further demonstrated against S. aureus persister cells in exponential-phase and stationary-phase populations. Finally, the antibiofilm activity of CF-301 was greatly improved in combinations with the cell wall hydrolase lysostaphin when tested against a range of S. aureus strains. In all, the data show that CF-301 is highly effective at disrupting biofilms and killing biofilm bacteria, and, as such, it may be an efficient new agent for treating staphylococcal infections with a biofilm component.

Biofilms.

Science.gov (United States)

López, Daniel; Vlamakis, Hera; Kolter, Roberto

2010-07-01

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 Staphylococcus aureus. Using these bacteria as examples, we discuss the key features of biofilms as well as mechanisms by which extracellular signals trigger biofilm formation.

Combined use of Bacteriophage K and a novel Bacteriophage to reduce Staphylococcus aureus biofilm formation

DEFF Research Database (Denmark)

Alves, D.R.; Gaudion, A.; Bean, J.E.

2014-01-01

Biofilms are major causes of impairment of wound healing and patient morbidity. One of the most common and aggressive wound pathogens is Staphylococcus aureus, displaying a large repertoire of virulence factors and commonly reduced susceptibility to antibiotics, such as the spread of methicillin-...

Photodynamic inactivation of Staphylococcus aureus and Escherichia coli biofilms by malachite green and phenothiazine dyes: an in vitro study.

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Vilela, Simone Furgeri Godinho; Junqueira, Juliana Campos; Barbosa, Junia Oliveira; Majewski, Marta; Munin, Egberto; Jorge, Antonio Olavo Cardoso

2012-06-01

The organization of biofilms in the oral cavity gives them added resistance to antimicrobial agents. The action of phenothiazinic photosensitizers on oral biofilms has already been reported. However, the action of the malachite green photosensitizer upon biofilm-organized microorganisms has not been described. The objective of the present work was to compare the action of malachite green with the phenothiazinic photosensitizers (methylene blue and toluidine blue) on Staphylococcus aureus and Escherichia coli biofilms. The biofilms were grown on sample pieces of acrylic resin and subjected to photodynamic therapy using a 660-nm diode laser and photosensitizer concentrations ranging from 37.5 to 3000 μM. After photodynamic therapy, cells from the biofilms were dispersed in a homogenizer and cultured in Brain Heart Infusion broth for quantification of colony-forming units per experimental protocol. For each tested microorganism, two control groups were maintained: one exposed to the laser radiation without the photosensitizer (L+PS-) and other treated with the photosensitizer without exposure to the red laser light (L-PS+). The results were subjected to descriptive statistical analysis. The best results for S. aureus and E. coli biofilms were obtained with photosensitizer concentrations of approximately 300 μM methylene blue, with microbial reductions of 0.8-1.0 log(10); 150 μM toluidine blue, with microbial reductions of 0.9-1.0 log(10); and 3000 μM malachite green, with microbial reductions of 1.6-4.0 log(10). Greater microbial reduction was achieved with the malachite green photosensitizer when used at higher concentrations than those employed for the phenothiazinic dyes. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

Science.gov (United States)

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

2013-06-01

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

Staphylococcus aureus Clinical Isolates: Antibiotic Susceptibility, Molecular Characteristics, and Ability to Form Biofilm

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N. Indrawattana

2013-01-01

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.

Biofilm-Forming Methicillin-Resistant Staphylococcus aureus Survive in Kupffer Cells and Exhibit High Virulence in Mice

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Takuto Oyama

2016-06-01

Full Text Available Although Staphylococcus aureus is part of the normal body flora, heavy usage of antibiotics has resulted in the emergence of methicillin-resistant strains (MRSA. MRSA can form biofilms and cause indwelling foreign body infections, bacteremia, soft tissue infections, endocarditis, and osteomyelitis. Using an in vitro assay, we screened 173 clinical blood isolates of MRSA and selected 20 high-biofilm formers (H-BF and low-biofilm formers (L-BF. These were intravenously administered to mice and the general condition of mice, the distribution of bacteria, and biofilm in the liver, lung, spleen, and kidney were investigated. MRSA count was the highest in the liver, especially within Kupffer cells, which were positive for acid polysaccharides that are associated with intracellular biofilm. After 24 h, the general condition of the mice worsened significantly in the H-BF group. In the liver, bacterial deposition and aggregation and the biofilm-forming spot number were all significantly greater for H-BF group than for L-BF. CFU analysis revealed that bacteria in the H-BF group survived for long periods in the liver. These results indicate that the biofilm-forming ability of MRSA is a crucial factor for intracellular persistence, which could lead to chronic infections.

Antimicrobial blue light inactivation of Methicillin-resistant Staphylococcus aureus

Science.gov (United States)

Wang, Yucheng; Dai, Tianhong; Gu, Ying

2016-10-01

Background: With the increasing emergence of multidrug-resistant (MDR) bacterial strains, there is a pressing need for the development of alternative treatment for infections. Antimicrobial blue light (aBL) has provided a simple and effective approach. Methods: We first investigated the effectiveness of aBL (415 nm) inactivation of USA300 LAClux (a communityacquired Methicillin-resistant Staphylococcus aureus strain) both in the planktonic and biofilm forms. The survival of the bacteria in suspensions was determined by serial dilution and that of the biofilm-embedded bacteria was determined by bioluminescence quantification. Using a mouse model of thermal burn infected with USA300 LAClux, we further assessed the effectiveness of aBL for treating localized infections. Bioluminescence imaging was performed to monitor in real time bacterial viability in vivo. Results: In vitro study showed that, for the planktonic counterpart of the bacteria or the 24-h-old biofilms, an irradiance of 55 mW/cm2 for 60 min resulted in a 4.61 log10 or 2.56 log10 inactivation, respectively. In vivo study using infected mouse burns demonstrated that a 2.56-log10 inactivation was achieved after 100-mW/cm2 irradiation for 62 min. Conclusions: aBL is a potential alternative approach for treating Methicillin-resistant Staphylococcus aureus infections.

Adhesion and biofilm formation by Staphylococcus aureus from food processing plants as affected by growth medium, surface type and incubation temperature

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Heloísa Maria Ângelo Jerônimo

2012-12-01

Full Text Available This study assessed the effect of different growth media [BHI broth, BHI broth plus glucose (10 g/100 mL and BHI broth plus NaCl (5 g/100 mL] and incubation temperatures (28 or 37 ºC on the adherence, detachment and biofilm formation on polypropylene and stainless steel surfaces (2 x 2 cm coupons for a prolonged period (24-72 h by some strains of Staphylococcus aureus (S3, S28 and S54 from food processing plants. The efficacy of the sanitizers sodium hypochlorite (250 mg/mL and peracetic acid (30 mg/mL in reducing the number of viable bacterial cells in a preformed biofilm was also evaluated. S. aureus strains adhered in highest numbers in BHI broth, regardless of the type of surface or incubation temperature. Cell detachment from surfaces revealed high persistence over the incubation period. The number of cells needed for biofilm formation was noted in all experimental systems after 3 days. Peracetic acid and sodium hypochlorite were not efficient in completely removing the cells of S. aureus adhered onto polypropylene and stainless steel surfaces. From these results, the assayed strains revealed high capacities to adhere and form biofilms on polypropylene and stainless steel surfaces under the different growth conditions, and the cells in biofilm matrixes were resistant to total removal when exposed to the sanitizers sodium hypochlorite and peracetic acid.Este estudo teve como objetivo avaliar o efeito de diferentes meios de crescimento [caldo BHI, caldo BHI adicionado de glucose (10 g/100 mL e caldo BHI adicionado de NaCl (5 g/100 mL] e temperaturas de incubação (28 e 37 ºC sobre a adesão, separação e formação de biofilme sobre superfícies (2 x 2 cm de polipropileno e aço inoxidável durante longo tempo de incubação (24-72 h por parte de cepas de Staphylococcus aureus (S3, S58 e S54 isoladas de plantas de processamento de alimentos. Também foi avaliada a eficácia dos sanitizantes hipoclorito de sódio (250 mg/mL e ácido perac

Antimicrobial susceptibility, virulence factors and biofilm formation among Staphylococcus aureus isolates from hospital infections in Kerman, Iran

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Mohammad Reza Shakibaie

2014-12-01

Full Text Available Objective: The aims of present study were to determine the antimicrobial susceptibility, virulence factors and biofilm formation among MRSA hospital isolates of Staphylococcus aureus. Methods: Thirty non-repetitive strains of S. aureus isolated from three hospitals in Kerman, Iran. Antimicrobial sus­ceptibility was determined by disk diffusion breakpoints method according to CLSI guideline. The minimum inhibitory concentration (MIC of vancomycin and methicillin were measured by the broth microdilution and E-test procedures. Virulence factors (protease, DNase, lecithinase, capsule and hemolysis associated with the above isolates was studied. Biofilm was quantified by microtiter technique. Results: In total, 14 (46.7% S. aureus were isolated from lower respiratory tract, six (20.0% from urinary tract and re­maining 10 (33.3% were recovered from wounds, blood and orthopedic patients. All of the isolates were susceptible to tigecycline, eight (26.7% were found to be resistant to methicillin (MRSA and 4 (13.3% showed reduced susceptibility to vancomycin. No any vancomycin resistant isolate was detected (p≤0.05. MIC results showed that four of the isolates (13.3% exhibited MIC 4 μg/mL to vancomycin while, five (16.6% demonstrated MIC 32 μg/mL to methicillin. The iso­lates were also resistant to amoxicillin/clavulanic acid, tetracycline and tobramycin. It was found that, six (75 % of MRSA strains produced lecithinase, seven (96.7% demonstrated protease and DNase activities as compared to MSSA isolates. Biofilm analysis revealed that twenty (66.7% isolates formed strong, seven (23.3% formed moderate and three (10.0% had weak biofilm. Conclusion: From the results, it can be concluded that, treatment options available for these infections are limited; therefore, monitoring, and management of infections due to MRSA with reduced susceptibility to vancomycin, must be done in order to control spread of these strains in the hospital environment. J

Influence of biofilm-forming lactic acid bacteria against methicillin-resistant Staphylococcus aureus (MRSA S547

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Laavanya M. Kumar

2017-12-01

Full Text Available Objective: To investigate the antibacterial effect of selected lactic acid bacteria (LAB biofilms on the planktonic and biofilm population of methicillin-resistant Staphylococcus aureus (MRSA (S547. Methods: In this study, biofilm-forming LAB were isolated from tairu and kefir. Isolate Y1 and isolate KF were selected based on their prominent inhibition against test pathogens (using spot-on-agar method and agar-well-diffusion assay and efficient biofilm production (using tissue culture plate method. They were then identified as Lactobacillus casei (L. casei Y1 and Lactobacillus plantarum (L. plantarum KF, respectively using 16S rDNA gene sequencing. The influence of incubation time, temperature and aeration on the biofilm production of L. casei Y1 and L. plantarum KF was also investigated using tissue culture plate method. The inhibitory activity of both the selected LAB biofilms was evaluated against MRSA (Institute for Medical Research code: S547 using L. plantarum ATCC 8014 as the reference strain. Results: L. casei Y1 showed the highest reduction of MRSA biofilms, by 3.53 log at 48 h while L. plantarum KF records the highest reduction of 2.64 log at 36 h. In inhibiting planktonic population of MRSA (S547, both L. casei Y1 and L. plantarum KF biofilms recorded their maximum reduction of 4.13 log and 3.41 log at 24 h, respectively. Despite their inhibitory effects being time-dependent, both LAB biofilms exhibited good potential in controlling the biofilm and planktonic population of MRSA (S547. Conclusions: The results from this study could highlight the importance of analysing biofilms of LAB to enhance their antibacterial efficacy. Preferably, these protective biofilms of LAB could also be a better alternative to control the formation of biofilms by pathogens such as MRSA. Keywords: MRSA, Biofilms, Lactic acid bacteria, Antibacterial

Formation of biofilms by Staphylococcus aureus on stainless steel and glass surfaces and its resistance to some selected chemical sanitizers Formação de biofilme por Staphylococcus aureus na superfície de aço inoxidável e vidro e sua resistência a alguns sanificantes químicos

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Simone Cristina Marques

2007-09-01

Full Text Available The objectives of this work were to verify the capability of Staphylococcus aureus of forming bio-film on stainless steel and glass surfaces; to evaluate the efficiency of sodium dichloroisocyanurate, hydrogen peroxide and peracetic acid in inactivating Staphylococcus aureus cells adhered onto these surfaces; and to visualize biofilm development by scanning electron microscopy before and after sanitizer treatment. The surfaces studied consisted of 10x20mm chips immersed in Petri dishes containing BHI broth inoculated with S. aureus ATCC 25923. Biofilm formation was observed after 15-day incubation, when the cells were removed using the swab technique, followed by Baird Parker agar plating. Also, the efficiency of the chemical sanitizers on the chip surfaces was tested and the non-removed cells were counted on the Baird-Parker agar. After biofilm formation and use of sanitizers, the chips were respectively observed by scanning electronic microscopy following a pre-existing protocol. The obtained results showed biofilm formation on both surfaces, with bacterial count in the order of 10(7 CFU/cm² on and 10(8 CFU/cm² on stainless steel and glass surfaces, respectively. Peracetic acid was the most efficient in removing adhered cells, presenting 5.26 and 4.5 decimal reduction for adhered cells on stainless steel and glass surfaces, respectively.Os objetivos deste trabalho foram verificar a capacidade de Staphylococcus aureus formar biofilme nas superfícies de aço inoxidável e vidro, avaliar a eficiência do dicloroisocianurato de sódio, peróxido de hidrogênio e ácido peracético na inativação de células de S. aureus aderidas e visualização por microscopia eletrônica de varredura, o desenvolvimento antes e depois do tratamento das superfícies com os sanificantes. As superfícies foram cupons 10x200mm imersos em placas de Petri contendo caldo BHI inoculado com cultura de Staphylococcus aureus ATCC 25923. A formação de biofilme foi

Cross-protection between experimental anti-leptospirosis bacterins

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Cristina Corsi Dib

2014-09-01

Full Text Available We investigated the existence of cross-protection between two anti-leptospirosis monovalent experimental bacterins produced with two strains of Leptospira serogroup Pomona: Fromm strain of serovar Kennewicky, isolated from pigs in the United States, and strain GR6 of serovar Pomona isolated from pigs in Brazil. Both were added of aluminum hydroxide as an adjuvant. Experimental bacterins were tested with the hamster potency test in order to assess protection provided against the disease and against the establishment of kidney infection. Controls were polyvalent commercial vaccine produced with Leptospira strains isolated outside Brazil, which included a representative of Pomona serovar, or Sorensen solution added of aluminum hydroxide adjuvant. The challenge was performed with cross-strains of serogroup Pomona tested in accordance with international standards established for the potency test. After 21 days of the challenge, survivors were killed to evaluate the condition of Leptospira renal carrier. Experimental bacterins protected hamsters against homologous and heterologous strains, demonstrating the existence of cross-protection. The commercial vaccine protected the hamsters challenged with both strains, but there was a high proportion of animals diagnosed as renal carriers when the challenge was performed with strain GR6, isolated from pigs in Brazil.

Cross-protection between experimental anti-leptospirosis bacterins

Science.gov (United States)

Dib, Cristina Corsi; Gonçales, Amane Paldês; de Morais, Zenaide Maria; de Souza, Gisele Oliveira; Miraglia, Fabiana; Abreu, Patricia Antonia Estima; Vasconcellos, Silvio Arruda

2014-01-01

We investigated the existence of cross-protection between two anti-leptospirosis monovalent experimental bacterins produced with two strains of Leptospira serogroup Pomona: Fromm strain of serovar Kennewicky, isolated from pigs in the United States, and strain GR6 of serovar Pomona isolated from pigs in Brazil. Both were added of aluminum hydroxide as an adjuvant. Experimental bacterins were tested with the hamster potency test in order to assess protection provided against the disease and against the establishment of kidney infection. Controls were polyvalent commercial vaccine produced with Leptospira strains isolated outside Brazil, which included a representative of Pomona serovar, or Sorensen solution added of aluminum hydroxide adjuvant. The challenge was performed with cross-strains of serogroup Pomona tested in accordance with international standards established for the potency test. After 21 days of the challenge, survivors were killed to evaluate the condition of Leptospira renal carrier. Experimental bacterins protected hamsters against homologous and heterologous strains, demonstrating the existence of cross-protection. The commercial vaccine protected the hamsters challenged with both strains, but there was a high proportion of animals diagnosed as renal carriers when the challenge was performed with strain GR6, isolated from pigs in Brazil. PMID:25477946

Stimulated phase-shift acoustic nanodroplets enhance vancomycin efficacy against methicillin-resistant Staphylococcus aureus biofilms

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Guo H

2017-06-01

Full Text Available Hao Guo,1 Ziming Wang,1 Quanyin Du,1 Pan Li,2 Zhigang Wang,2 Aimin Wang1 1Department of Orthopedics, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, China; 2Chongqing Key Laboratory of Ultrasound Molecular Imaging, Institute of Ultrasound Imaging, Second Affiliated Hospital of Chongqing Medical University, Chongqing, China Purpose: Bacterial biofilms on the surface of prostheses are becoming a rising concern in managing prosthetic joint infections. The inherent resistant features of biofilms render traditional antimicrobial therapy unproductive and revision surgery outcomes uncertain. This situation has prompted the exploration of novel antimicrobial strategies. The synergy of ultrasound microbubbles and vancomycin has been proposed as an efficient alternative for biofilm eradication. The purpose of this study was to evaluate the anti-biofilm effect of stimulated phase-shift acoustic nanodroplets (NDs combined with vancomycin.Materials and methods: We fabricated lipid phase-shift NDs with a core of liquid perfluoropentane. A new phase change mode for NDs incorporating an initial unfocused low-intensity pulsed ultrasound for 5 minutes and a subsequent incubation at 37°C into a 24-hour duration was developed. Methicillin-resistant Staphylococcus aureus (MRSA biofilms were incubated with vancomycin and NDs under the hybrid stimulation. Biofilm morphology following treatment was determined using confocal laser scanning microscopy and scanning electron microscopy. Resazurin assay was used to quantify bactericidal efficacy against MRSA biofilm bacteria.Results: NDs treated sequentially with ultrasound and heating at 37°C achieved gradual and substantial ND vaporization and cavitation in a successive process. NDs after stimulation were capable of generating stronger destruction on biofilm structure which was best characterized by residual circular arc margins and more dead bacteria. Furthermore, NDs

In vitro production of biofilm in a flow cell system in a strain of Pseudomonas aeruginosa and Staphylococcus aureus and determination of efficiency of ciprofloxacin against them

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Soham Gupta

2011-01-01

Full Text Available Background: Microorganisms develop biofilm on various medical devices. The process is particularly relevant in public health since biofilm associated organisms are much more resistant to antibiotics and have a potential to cause infections in patients with indwelling medical devices. Materials and Methods: To determine the efficiency of an antibiotic against the biofilm it is inappropriate to use traditional technique of determining Minimum Inhibitory Concentration (MIC on the free floating laboratory phenotype. Thus we have induced formation of biofilm in two strains (Pseudomonas aeruginosa and Staphylococcus aureus, which showed heavy growth of biofilm in screening by Tube method in a flow cell system and determined their antibiotic susceptibility against ciprofloxacin by agar dilution method in the range (0.25 mg/ml to 8 mg/ml. The MIC value of ciprofloxacin for the biofilm produced organism was compared with its free form and a standard strain as control on the same plates. Observations: Both the biofilm produced strains showed a higher resistance (MIC > 8 mg/ml than its free form, which were 2 μg/ml for Pseudomonas aeruginosa and 4 mg/ml for Staphylococcus aureus. Thus biofilm can pose a threat in the patient treatment.

The ability of S.aureus to form biofilm on the Ti-6Al-7Nb scaffolds produced by Selective Laser Melting and subjected to the different types of surface modifications.

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Szymczyk, Patrycja; Junka, Adam; Ziółkowski, Grzegorz; Smutnicka, Danuta; Bartoszewicz, Marzenna; Chlebus, Edward

2013-01-01

The Gram-positive coccus, Staphylococcus aureus, is the leading etiologic agent of limb and life-threatening biofilm-related infections in the patients following the orthopaedic implantations. The aim of the present paper is to estimate the ability of S. aureus to form biofilm on titanium alloy (Ti-6Al-7Nb) scaffolds produced by Selective Laser Melting (SLM) and subjected to the different types of surface modifications, including ultrasonic cleaning and chemical polishing. The results obtained indicate significantly the decreased ability of S.aureus to form biofilm on the surface of scaffolds subjected to the chemical polishing in comparison to the scaffolds cleaned ultrasonically. The data provided can be useful for future applications of the SLM technology in production of Ti-6Al-7Nb medical implants.

The in vitro effect of xylitol on chronic rhinosinusitis biofilms.

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

2016-12-01

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

Pseudomonas aeruginosa Alters Staphylococcus aureus Sensitivity to Vancomycin in a Biofilm Model of Cystic Fibrosis Infection

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Giulia Orazi

2017-07-01

Full Text Available The airways of cystic fibrosis (CF patients have thick mucus, which fosters chronic, polymicrobial infections. Pseudomonas aeruginosa and Staphylococcus aureus are two of the most prevalent respiratory pathogens in CF patients. In this study, we tested whether P. aeruginosa influences the susceptibility of S. aureus to frontline antibiotics used to treat CF lung infections. Using our in vitro coculture model, we observed that addition of P. aeruginosa supernatants to S. aureus biofilms grown either on epithelial cells or on plastic significantly decreased the susceptibility of S. aureus to vancomycin. Mutant analyses showed that 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO, a component of the P. aeruginosa Pseudomonas quinolone signal (PQS system, protects S. aureus from the antimicrobial activity of vancomycin. Similarly, the siderophores pyoverdine and pyochelin also contribute to the ability of P. aeruginosa to protect S. aureus from vancomycin, as did growth under anoxia. Under our experimental conditions, HQNO, P. aeruginosa supernatant, and growth under anoxia decreased S. aureus growth, likely explaining why this cell wall-targeting antibiotic is less effective. P. aeruginosa supernatant did not confer additional protection to slow-growing S. aureus small colony variants. Importantly, P. aeruginosa supernatant protects S. aureus from other inhibitors of cell wall synthesis as well as protein synthesis-targeting antibiotics in an HQNO- and siderophore-dependent manner. We propose a model whereby P. aeruginosa causes S. aureus to shift to fermentative growth when these organisms are grown in coculture, leading to reduction in S. aureus growth and decreased susceptibility to antibiotics targeting cell wall and protein synthesis.

Biofilm Formation and Its Relationship with the Molecular Characteristics of Food-Related Methicillin-Resistant Staphylococcus aureus (MRSA).

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Vergara, Alberto; Normanno, Giovanni; Di Ciccio, Pierluigi; Pedonese, Francesca; Nuvoloni, Roberta; Parisi, Antonio; Santagada, Gianfranco; Colagiorgi, Angelo; Zanardi, Emanuela; Ghidini, Sergio; Ianieri, Adriana

2017-10-01

The capability to produce biofilm is an important persistence and dissemination mechanism of some foodborne bacteria. This paper investigates the relationship between some molecular characteristics (SCCmec, ST, spa-type, agr-type, cna, sarA, icaA, icaD, clfA, fnbA, fnbB, hla, hlb) of 22 food-related methicillin-resistant Staphylococcus aureus (MRSA) strains and their ability to form biofilm on stainless steel and polystyrene. Five (22.7%, 5/22) strains were able to synthesize biofilm on polystyrene, and one of these (4.5%, 1/22) strains was also able to synthesize biofilm on stainless steel. The largest amount of biofilm was formed on polystyrene by 2 MRSA strains isolated from cows' milk, thus raising concern about the dairy industry. The majority of MRSA biofilm producers carried SCCmec type IVa, suggesting that the presence of SCCmecIVa and/or agr type III could be related to the ability to form biofilm. In conclusion, in order to achieve an acceptable level of food safety, Good Hygiene Practices should be strictly implemented along the food chain to reduce the risk of colonization and dissemination of MRSA biofilm-producing strains in the food industry. In this study, some assayed isolates of food-related MRSA demonstrated the capacity to form biofilm. Biofilm formation differed according to surface characteristics and MRSA strains. A relationship was observed between some molecular characteristics and the ability to form biofilms. Few studies have investigated the ability of MRSA to form biofilms, and the majority of these studies have investigated clinical aspects. This work was performed to investigate whether or not there is a difference between MRSA food isolates and MRSA clinical isolates in their ability to form biofilm. These initial findings could provide information that will contribute to a better understanding of these aspects. © 2017 Institute of Food Technologists®.

Antibiofilm Effect of Octenidine Hydrochloride on Staphylococcus aureus, MRSA and VRSA

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Mary Anne Roshni Amalaradjou

2014-05-01

Full Text Available Millions of indwelling devices are implanted in patients every year, and staphylococci (S. aureus, MRSA and vancomycin-resistant S. aureus (VRSA are responsible for a majority of infections associated with these devices, thereby leading to treatment failures. Once established, staphylococcal biofilms become resistant to antimicrobial treatment and host response, thereby serving as the etiological agent for recurrent infections. This study investigated the efficacy of octenidine hydrochloride (OH for inhibiting biofilm synthesis and inactivating fully-formed staphylococcal biofilm on different matrices in the presence and absence of serum protein. Polystyrene plates and stainless steel coupons inoculated with S. aureus, MRSA or VRSA were treated with OH (zero, 0.5, one, 2 mM at 37 °C for the prevention of biofilm formation. Additionally, the antibiofilm effect of OH (zero, 2.5, five, 10 mM on fully-formed staphylococcal biofilms on polystyrene plates, stainless steel coupons and urinary catheters was investigated. OH was effective in rapidly inactivating planktonic and biofilm cells of S. aureus, MRSA and VRSA on polystyrene plates, stainless steel coupons and urinary catheters in the presence and absence of serum proteins. The use of two and 10 mM OH completely inactivated S. aureus planktonic cells and biofilm (>6.0 log reduction on all matrices tested immediately upon exposure. Further, confocal imaging revealed the presence of dead cells and loss in biofilm architecture in the OH-treated samples when compared to intact live biofilm in the control. Results suggest that OH could be applied as an effective antimicrobial to control biofilms of S. aureus, MRSA and VRSA on appropriate hospital surfaces and indwelling devices.

Biofilm formation and virulence factor analysis of Staphylococcus aureus isolates collected from ovine mastitis.

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Azara, E; Longheu, C; Sanna, G; Tola, S

2017-08-01

To perform a phenotypic and genotypic characterization of 258 Staphylococcus aureus isolates from clinical ovine mastitis and used for the preparation of inactivated autogenous vaccines. The potential for biofilm production was determined by phenotypic test of Congo Red Agar (CRA) and by PCR for the detection of icaA/D genes. Isolates were also screened by PCR for the presence of enterotoxins (sea, seb, sec, sed and see), toxic shock syndrome toxin (tsst), leukotoxins (lukD-E, lukM and lukPV83), haemolysins (hly-β and hly-γ), autolysin (atlA) genes and encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs: clfA, clfB, fnbA, fnbB, bbp, cna, eno, fib, epbs, sdrC, sdrD and SdrE). None of the 258 isolates showed biofilm-forming ability on CRA and harboured icaA/D genes. The most frequent pyrogenic toxin superantigen genes amplified were sec plus tsst-1, which were found strictly in combination with 71·3% of the Staph. aureus isolates tested. None of the isolates harboured the genes encoding sea and see. Of the 258 isolates tested, 159 (61·6%) possessed all lukD-E/lukM/lukPV83 genes, 123 (47·7%) harboured both hly-β/hly-γ genes, whereas almost all (97·3%) were PCR positive for atlA gene. With respect to adhesion determinants, 179 (69·4%) isolates presented simultaneously four genes (fnbA, fib, clfA and clfB) for fibronectin- and fibrinogen-binding proteins. In this search, several putative virulence determinants have been identified in ovine Staph. aureus isolates collected in Sardinia. Some of the putative virulence determinants could be considered as components of a vaccine because of their role in ovine mastitis pathogenesis. © 2017 The Society for Applied Microbiology.

Cloning, Expression and Characterization of NAD Kinase from Staphylococcus aureus Involved in the Formation of NADP (H: A Key Molecule in the Maintaining of Redox Status and Biofilm Formation

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U Venkateswara Prasad

2017-01-01

Full Text Available Background:Staphylococcus aureus has the ability to form biofilms on any niches, a key pathogenic factor of this organism and this phenomenon is directly related to the concentration of NADPH. The formation of NADP is catalyzed by NAD kinase (NADK and this gene of S. aureus ATCC 12600 was cloned, sequenced, expressed and characterized. Materials and Methods: The NADK gene was polymerase chain reaction amplified from the chromosomal DNA of S. aureus ATCC 12600 and cloned in pQE 30 vector, sequenced and expressed in Escherichia coli DH5α. The pure protein was obtained by passing through nickel metal chelate agarose column. The enzyme kinetics of the enzyme and biofilm assay of the S. aureus was carried out in both aerobic and anaerobic conditions. The kinetics was further confirmed by the ability of the substrates to dock to the NADK structure. Results: The recombinant NADK exhibited single band with a molecular weight of 31kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gene sequence (GenBank: JN645814 revealed presence of only one kind of NADK in all S. aureus strains. The enzyme exhibited very high affinity for NAD compared to adenosine triphosphate concurring with the docking results. A root-mean-square deviation value 14.039Š observed when NADK structure was superimposed with its human counterpart suggesting very low homology. In anaerobic conditions, higher biofilm units were found with decreased NADK activity. Conclusion: The results of this study suggest increased NADPH concentration in S. aureus plays a vital role in the biofilm formation and survival of this pathogen in any environmental conditions.

Plectranthus amboinicus essential oil and carvacrol bioactive against planktonic and biofilm of oxacillin- and vancomycin-resistant Staphylococcus aureus.

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Vasconcelos, Sara Edwirgens Costa Benício; Melo, Hider Machado; Cavalcante, Theodora Thays Arruda; Júnior, Francisco Eduardo Aragão Catunda; de Carvalho, Mário Geraldo; Menezes, Francisca Gleire Rodrigues; de Sousa, Oscarina Viana; Costa, Renata Albuquerque

2017-09-16

The emergence of multidrug-resistant bacteria is a worldwide concern and in order to find an alternative to this problem, the occurrence of antimicrobial compounds in Plectranthus amboinicus essential oil was investigated. Thus, this study aims to determine susceptibility of Staphylococcus aureus isolated from food to antibiotics, P. amboinicus essential oil (PAEO) and carvacrol. Leaves and stem of P. amboinicus were used for extraction of essential oil (PAEO) by hydrodistillation technique and EO chemical analysis was performed by gas chromatography coupled to a mass spectrometer. S. aureus strains (n = 35) isolated from food and S. aureus ATCC 6538 were used to evaluate the antimicrobial and antibiofilm activity of PAEO and carvacrol. All strains (n = 35) were submitted to antimicrobial susceptibility profile by disk diffusion method. Determination of MIC and MBC was performed by microdilution technique and antibiofilm activity was determined by microtiter-plate technique with crystal violet assay and counting viable cells in Colony Forming Units (CFU). Carvacrol (88.17%) was the major component in the PAEO. Antibiotic resistance was detected in 28 S. aureus strains (80%) and 12 strains (34.3%) were oxacillin and vancomycin-resistant (OVRSA). From the 28 resistant strains, 7 (25%) showed resistance plasmid of 12,000 bp. All strains (n = 35) were sensitive to PAEO and carvacrol, with inhibition zones ranging from 16 to 38 mm and 23 to 42 mm, respectively. The lowest MIC (0.25 mg mL -1 ) and MBC (0.5 mg mL -1 ) values were observed when carvacrol was used against OVRSA. When a 0.5 mg mL -1 concentration of PAEO and carvacrol was used, no viable cells were found on S. aureus biofilm. The antibacterial effect of carvacrol and PAEO proves to be a possible alternative against planktonic forms and staphylococcal biofilm.

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

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

2015-12-15

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

Femtosecond laser surface texturing of titanium as a method to reduce the adhesion of Staphylococcus aureus and biofilm formation

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Cunha, Alexandre [Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais 1, 1049-001 Lisboa (Portugal); Bordeaux University, Institute of Chemistry & Biology of Membranes & Nanoobjects (CBMN UMR 5248, CNRS), European Institute of Chemistry and Biology, 2 Rue Robert Escarpit, 33607 Pessac (France); Elie, Anne-Marie [Bordeaux University, CBMN UMR 5248, CNRS, Bordeaux Science Agro, 1 Rue du G. de Gaulle, 33170 Gradignan (France); Plawinski, Laurent [Bordeaux University, Institute of Chemistry & Biology of Membranes & Nanoobjects (CBMN UMR 5248, CNRS), European Institute of Chemistry and Biology, 2 Rue Robert Escarpit, 33607 Pessac (France); Serro, Ana Paula [Instituto Superior Técnico, Universidade de Lisboa, CQE-Centro de Química Estrutural, Av. Rovisco Pais 1, 1049-001 Lisbon (Portugal); Botelho do Rego, Ana Maria [Instituto Superior Técnico, Universidade de Lisboa, CQFM-Centro de Química-Física Molecular and Institute of Nanoscience and Nanotechnology - IN, Av. Rovisco Pais 1, 1049-001 Lisbon (Portugal); Almeida, Amélia [Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais 1, 1049-001 Lisboa (Portugal); Urdaci, Maria C. [Bordeaux University, CBMN UMR 5248, CNRS, Bordeaux Science Agro, 1 Rue du G. de Gaulle, 33170 Gradignan (France); Durrieu, Marie-Christine [Bordeaux University, Institute of Chemistry & Biology of Membranes & Nanoobjects (CBMN UMR 5248, CNRS), European Institute of Chemistry and Biology, 2 Rue Robert Escarpit, 33607 Pessac (France); Vilar, Rui, E-mail: rui.vilar@tecnico.ulisboa.pt [Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais 1, 1049-001 Lisboa (Portugal)

2016-01-01

Graphical abstract: - Highlights: • The short-term adhesion of Staphylococcus aureus onto femtosecond laser textured surfaces of titanium was investigated. • The laser textured surfaces consist of laser-induced periodic surface structures (LIPSS) and nanopillars. • The laser treatment enhances the hydrophilicity and the surface free energy of the material. • The laser treatment reduces significantly the adhesion of S. aureus and biofilm formation. • Femtosecond laser surface texturing of titanium is a simple and promising method for endowing dental and orthopedic implants with antibacterial properties. - Abstract: The aim of the present work was to investigate the possibility of using femtosecond laser surface texturing as a method to reduce the colonization of Grade 2 Titanium alloy surfaces by Staphylococcus aureus and the subsequent formation of biofilm. The laser treatments were carried out with a Yb:KYW chirped-pulse-regenerative amplification laser system with a central wavelength of 1030 nm and a pulse duration of 500 fs. Two types of surface textures, consisting of laser-induced periodic surface structures (LIPSS) and nanopillars, were produced. The topography, chemical composition and phase constitution of these surfaces were investigated by atomic force microscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, micro-Raman spectroscopy, and X-ray diffraction. Surface wettability was assessed by the sessile drop method using water and diiodomethane as testing liquids. The response of S. aureus put into contact with the laser treated surfaces in controlled conditions was investigated by epifluorescence microscopy and scanning electron microscopy 48 h after cell seeding. The results achieved show that the laser treatment reduces significantly the bacterial adhesion to the surface as well as biofilm formation as compared to a reference polished surfaces and suggest that femtosecond laser texturing is a simple and promising method

Boeravinone B, A Novel Dual Inhibitor of NorA Bacterial Efflux Pump of Staphylococcus aureus and Human P-Glycoprotein, Reduces the Biofilm Formation and Intracellular Invasion of Bacteria.

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Singh, Samsher; Kalia, Nitin P; Joshi, Prashant; Kumar, Ajay; Sharma, Parduman R; Kumar, Ashok; Bharate, Sandip B; Khan, Inshad A

2017-01-01

This study elucidated the role of boeravinone B, a NorA multidrug efflux pump inhibitor, in biofilm inhibition. The effects of boeravinone B plus ciprofloxacin, a NorA substrate, were evaluated in NorA-overexpressing, wild-type, and knocked-out Staphylococcus aureus (SA-1199B, SA-1199, and SA-K1758, respectively). The mechanism of action was confirmed using the ethidium bromide accumulation and efflux assay. The role of boeravinone B as a human P -glycoprotein ( P -gp) inhibitor was examined in the LS-180 (colon cancer) cell line. Moreover, its role in the inhibition of biofilm formation and intracellular invasion of S. aureus in macrophages was studied. Boeravinone B reduced the minimum inhibitory concentration (MIC) of ciprofloxacin against S. aureus and its methicillin-resistant strains; the effect was stronger in SA-1199B. Furthermore, time-kill kinetics revealed that boeravinone B plus ciprofloxacin, at subinhibitory concentration (0.25 × MIC), is as equipotent as that at the MIC level. This combination also had a reduced mutation prevention concentration. Boeravinone B reduced the efflux of ethidium bromide and increased the accumulation, thus strengthening the role as a NorA inhibitor. Biofilm formation was reduced by four-eightfold of the minimal biofilm inhibitory concentration of ciprofloxacin, effectively preventing bacterial entry into macrophages. Boeravinone B effectively inhibited P -gp with half maximal inhibitory concentration (IC 50 ) of 64.85 μM. The study concluded that boeravinone B not only inhibits the NorA-mediated efflux of fluoroquinolones but also considerably inhibits the biofilm formation of S. aureus. Its P -gp inhibition activity demonstrates its potential as a bioavailability and bioefficacy enhancer.

Novel Treatment of Staphylococcus aureus Device-Related Infections Using Fibrinolytic Agents.

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Hogan, S; O'Gara, J P; O'Neill, E

2018-02-01

Staphylococcal infections involving biofilms represent a significant challenge in the treatment of patients with device-related infections. Staphylococcus aureus biofilms have been shown to be SaeRS regulated and dependent on the coagulase-catalyzed conversion of fibrinogen into fibrin on surfaces coated with human plasma. Here we investigated the treatment of staphylococcal biofilm device-related infections by digesting the fibrin biofilm matrix with and without existing antimicrobials. The fibrinolytic agents plasmin, streptokinase, and nattokinase, and TrypLE, a recombinant trypsin-like protease, were used to digest and treat S. aureus biofilms grown in vitro using in vivo -like static biofilm assays with and without antimicrobials. Cytotoxicity, the potential to induce a cytokine response in whole human blood, and the risk of induction of tolerance to fibrinolytic agents were investigated. A rat model of intravascular catheter infection was established to investigate the efficacy of selected fibrinolytic agents in vivo Under biomimetic conditions, the fibrinolytic agents effectively dispersed established S. aureus biofilms and, in combination with common antistaphylococcal antimicrobials, effectively killed bacterial cells being released from the biofilm. These fibrinolytic agents were not cytotoxic and did not affect the host immune response. The rat model of infection successfully demonstrated the activity of the selected fibrinolytic agents alone and in combination with antimicrobials on established biofilms in vivo TrypLE and nattokinase most successfully removed adherent cells from plasma-coated surfaces and significantly improved the efficacy of existing antimicrobials against S. aureus biofilms in vitro and in vivo These biofilm dispersal agents represent a viable future treatment option for S. aureus device-related infections. Copyright © 2018 American Society for Microbiology.

Soluble factors from biofilm of Candida albicans and Staphylococcus aureus promote cell death and inflammatory response.

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de Carvalho Dias, Kassia; Barbugli, Paula Aboud; de Patto, Fernanda; Lordello, Virginia Barreto; de Aquino Penteado, Letícia; Medeiros, Alexandra Ivo; Vergani, Carlos Eduardo

2017-06-30

The objective of this study was to better understand the effects of soluble factors from biofilm of single- and mixed-species Candida albicans (C. albicans) and methicillin-sensitive Staphylococcus aureus (MSSA) cultures after 36 h in culture on keratinocytes (NOK-si and HaCaT) and macrophages (J774A.1). Soluble factors from biofilms of C. albicans and MSSA were collected and incubated with keratinocytes and macrophages, which were subsequently evaluated by cell viability assays (MTT). Lactate dehydrogenase (LDH) enzyme release was measured to assess cell membrane damage to keratinocytes. Cells were analysed by brightfield microscopy after 2 and 24 h of exposure to the soluble factors from biofilm. Cell death was detected by labelling apoptotic cells with annexin V and necrotic cells with propidium iodide (PI) and was visualized via fluorescence microscopy. Soluble factors from biofilm were incubated with J774A.1 cells for 24 h; the subsequent production of NO and the cytokines IL-6 and TNF-α was measured by ELISA. The cell viability assays showed that the soluble factors of single-species C. albicans cultures were as toxic as the soluble factors from biofilm of mixed cultures, whereas the soluble factors of MSSA cultures were less toxic than those of C. albicans or mixed cultures. The soluble factors from biofilm of mixed cultures were the most toxic to the NOK-si and HaCaT cells, as confirmed by analyses of PI labelling and cell morphology. Soluble factors from biofilm of single-species MSSA and mixed-species cultures induced the production of IL-6, NO and TNF-α by J744A.1 macrophages. The production of IL-6 and NO induced by the soluble factors from biofilm of mixed cultures was lower than that induced by the soluble factors from biofilm of single-species MSSA cultures, whereas the soluble factors from biofilm of C. albicans cultures induced only low levels of NO. Soluble factors from 36-h-old biofilm of C. albicans and MSSA cultures promoted cell death and

Identification and characterization of an operon, msaABCR, that controls virulence and biofilm development in Staphylococcus aureus.

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Sahukhal, Gyan S; Elasri, Mohamed O

2014-06-11

Community-acquired, methicillin-resistant Staphylococcus aureus strains often cause localized infections in immunocompromised hosts, but some strains show enhanced virulence leading to severe infections even among healthy individuals with no predisposing risk factors. The genetic basis for this enhanced virulence has yet to be determined. S. aureus possesses a wide variety of virulence factors, the expression of which is carefully coordinated by a variety of regulators. Several virulence regulators have been well characterized, but others have yet to be thoroughly investigated. Previously, we identified the msa gene as a regulator of several virulence genes, biofilm development, and antibiotic resistance. We also found evidence of the involvement of upstream genes in msa function. To investigate the mechanism of regulation of the msa gene (renamed msaC), we examined the upstream genes whose expression was affected by its deletion. We showed that msaC is part of a newly defined four-gene operon (msaABCR), in which msaC is a non-protein-coding RNA that is essential for the function of the operon. Furthermore, we found that an antisense RNA (msaR) is complementary to the 5' end of the msaB gene and is expressed in a growth phase-dependent manner suggesting that it is involved in regulation of the operon. These findings allow us to define a new operon that regulates fundamental phenotypes in S. aureus such as biofilm development and virulence. Characterization of the msaABCR operon will allow us to investigate the mechanism of function of this operon and the role of the individual genes in regulation and interaction with its targets. This study identifies a new element in the complex regulatory circuits in S. aureus, and our findings may be therapeutically relevant.

Fluconazole-Pyridoxine Bis-Triazolium Compounds with Potent Activity against Pathogenic Bacteria and Fungi Including Their Biofilm-Embedded Forms

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Marsel R. Garipov

2017-01-01

Full Text Available Two novel quaternary ammonium salts, bis-triazolium derivatives of fluconazole and pyridoxine, were synthesized by reaction of fluconazole with pyridoxine-based synthetic intermediates. The leading compound demonstrated pronounced antimycotic and antibacterial in vitro activity, comparable to or exceeding that of the reference antifungal (fluconazole, terbinafine and antibacterial/antiseptic (miramistin, benzalkonium chloride agents. In contrast to many antimicrobials, the leading compound was also active against biofilm-embedded staphylococci and Escherichia coli. While no biofilm structure destruction occurred, all compounds were able to diffuse into the matrix and reduce the number of colony-forming units by three orders of magnitude at 16 × MBC. The leading compound was significantly less toxic than miramistin and benzalkonium chloride and more toxic than the reference antifungal drugs. The obtained results make the described chemotype a promising starting point for the development of new broad-spectrum antimicrobial therapies with powerful effect on fungal and bacterial pathogens including their biofilm-embedded forms.

Effect of essential oils of Syzygium aromaticum and Cinnamomum zeylanicum and their major components on biofilm production in Staphylococcus aureus strains isolated from milk of cows with mastitis.

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Budri, P E; Silva, N C C; Bonsaglia, E C R; Fernandes Júnior, A; Araújo Júnior, J P; Doyama, J T; Gonçalves, J L; Santos, M V; Fitzgerald-Hughes, D; Rall, V L M

2015-09-01

Bovine mastitis is an inflammation of the mammary glands of cows and causes significant economic losses in dairy cattle. Staphylococcus aureus is one of the microorganisms most commonly isolated. Novel agents are required in agricultural industries to prevent the development of mastitis. The production of biofilm by Staph. aureus facilitates the adhesion of bacteria to solid surfaces and contributes to the transmission and maintenance of these bacteria. The effect of the essential oils of Syzygium aromaticum (clove; EOSA) and Cinnamomum zeylanicum (cinnamon; EOCZ) and their major components, eugenol and cinnamaldehyde, on Staph. aureus biofilm formation on different surfaces was investigated. The results showed a significant inhibition of biofilm production by EOSA on polystyrene and stainless steel surfaces (69.4 and 63.6%, respectively). However, its major component, eugenol, was less effective on polystyrene and stainless steel (52.8 and 19.6%, respectively). Both EOCZ and its major component, cinnamaldehyde, significantly reduced biofilm formation on polystyrene (74.7 and 69.6%, respectively) and on stainless steel surfaces (45.3 and 44.9%, respectively). These findings suggest that EOSA, EOCZ, and cinnamaldehyde may be considered for applications such as sanitization in the food industry. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

The Effect of Tsukamurella inchonensis Bacterin on the Immune Response Against Influenza and Newcastle Disease Vaccines in Broiler Chickens

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Forough Talazadeh

2016-11-01

Full Text Available Background: In poultry production, improving immunity is very important to prevent infectious diseases. One solution to improve the immunity of animals and to decrease their susceptibility to infectious disease is administration of immunostimulants. Surveys have indicated that some bacteria can work as immunomodulators such as Mycobacterium vaccae and can promote Th1-mediated mechanisms, and switch off pre-existing Th2 preponderance (1. Objectives: The aim of this study was to examine the effect of Tsukamurella inchonensis bacterin on the immune response against Influenza and Newcastle disease vaccine in broiler chickens . Materials and Methods: A total of 170 day-old broiler chicks were purchased and divided randomly into 5 equal groups. Chickens of group A received 106 bacterin subcutaneously on two days before vaccination against Newcastle disease and avian influenza. Chickens of group B received 106 bacterin subcutaneously on six days after the first injection of bacterin. Chickens of group C received 106bacterin subcutaneously on six days after the second injection of bacterin. Chickens of group D, vaccinated against Newcastle disease and avian influenza but did not receive bacterin. Chickens of group E, did not vaccinate against Newcastle disease and avian influenza and did not receive bacterin. All groups except group E, were vaccinated with live Newcastle vaccine and AI-ND killed vaccine (subtype H9N2. Blood samples were collected and antibody titer against Newcastle disease vaccine and avian influenza vaccine was determined by HI test. Results: The results of present study showed that receiving of Tsukamurella inchonensis bacterin for 3 times, significantly increased the specific antibody response to avian influenza subtype H9N2 vaccine. Also about Newcastle vaccine, significantly increased the specific antibody response to Newcastle vaccine at 21 and 28 days after vaccination. Conclusions: Receiving of Tsukamurella inchonensis bacterin

A new dry-surface biofilm model: An essential tool for efficacy testing of hospital surface decontamination procedures.

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Almatroudi, Ahmad; Hu, Honghua; Deva, Anand; Gosbell, Iain B; Jacombs, Anita; Jensen, Slade O; Whiteley, Greg; Glasbey, Trevor; Vickery, Karen

2015-10-01

The environment has been shown to be a source of pathogens causing infections in hospitalised patients. Incorporation of pathogens into biofilms, contaminating dry hospital surfaces, prolongs their survival and renders them tolerant to normal hospital cleaning and disinfection procedures. Currently there is no standard method for testing efficacy of detergents and disinfectants against biofilm formed on dry surfaces. The aim of this study was to develop a reproducible method of producing Staphylococcus aureus biofilm with properties similar to those of biofilm obtained from dry hospital clinical surfaces, for use in efficacy testing of decontamination products. The properties (composition, architecture) of model biofilm and biofilm obtained from clinical dry surfaces within an intensive care unit were compared. The CDC Biofilm Reactor was adapted to create a dry surface biofilm model. S. aureus ATCC 25923 was grown on polycarbonate coupons. Alternating cycles of dehydration and hydration in tryptone soy broth (TSB) were performed over 12 days. Number of biofilm bacteria attached to individual coupons was determined by plate culture and the coefficient of variation (CV%) calculated. The DNA, glycoconjugates and protein content of the biofilm were determined by analysing biofilm stained with SYTO 60, Alexa-488-labelled Aleuria aurantia lectin and SyproOrange respectively using Image J and Imaris software. Biofilm architecture was analysed using live/dead staining and confocal microscopy (CM) and scanning electron microscopy (SEM). Model biofilm was compared to naturally formed biofilm containing S. aureus on dry clinical surfaces. The CDC Biofilm reactor reproducibly formed a multi-layered, biofilm containing about 10(7) CFU/coupon embedded in thick extracellular polymeric substances. Within run CV was 9.5% and the between run CV was 10.1%. Protein was the principal component of both the in vitro model biofilm and the biofilms found on clinical surfaces. Continued

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

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

2015-05-01

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

Dynamics of biofilm formation and the interaction between Candida albicans and methicillin-susceptible (MSSA) and -resistant Staphylococcus aureus (MRSA).

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Zago, Chaiene Evelin; Silva, Sónia; Sanitá, Paula Volpato; Barbugli, Paula Aboud; Dias, Carla Maria Improta; Lordello, Virgínia Barreto; Vergani, Carlos Eduardo

2015-01-01

Polymicrobial biofilms are an understudied and a clinically relevant problem. This study evaluates the interaction between C. albicans, and methicillin- susceptible (MSSA) and resistant (MRSA) S. aureus growing in single- and dual-species biofilms. Single and dual species adhesion (90 min) and biofilms (12, 24, and 48 h) were evaluated by complementary methods: counting colony-forming units (CFU mL-1), XTT-reduction, and crystal violet staining (CV). The secretion of hydrolytic enzymes by the 48 h biofilms was also evaluated using fluorimetric kits. Scanning electron microscopy (SEM) was used to assess biofilm structure. The results from quantification assays were compared using two-way ANOVAs with Tukey post-hoc tests, while data from enzymatic activities were analyzed by one-way Welch-ANOVA followed by Games-Howell post hoc test (α = 0.05). C. albicans, MSSA and MRSA were able to adhere and to form biofilm in both single or mixed cultures. In general, all microorganisms in both growth conditions showed a gradual increase in the number of cells and metabolic activity over time, reaching peak values between 12 h and 48 h (ρ<0.05). C. albicans single- and dual-biofilms had significantly higher total biomass values (ρ<0.05) than single biofilms of bacteria. Except for single MRSA biofilms, all microorganisms in both growth conditions secreted proteinase and phospholipase-C. SEM images revealed extensive adherence of bacteria to hyphal elements of C. albicans. C. albicans, MSSA, and MRSA can co-exist in biofilms without antagonism and in an apparent synergistic effect, with bacteria cells preferentially associated to C. albicans hyphal forms.

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

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Masaya Ueno

2016-01-01

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

Decontamination Efficacy of Ultraviolet Radiation against Biofilms of Common Nosocomial Bacteria.

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Tingpej, Pholawat; Tiengtip, Rattana; Kondo, Sumalee

2015-06-01

Ultraviolet radiation (UV) is commonly used to destroy microorganisms in the health-care environment. However, the efficacy of UV radiation against bacteria growing within biofilms has never been studied. To measure the sterilization effectiveness of UV radiation against common healthcare associated pathogens growing within biofilms. Staphylococcus aureus, Methicillin-resistant S. aureus (MRSA), Streptococcus epidermidis, Escherichia coli, ESBL-producing E. coli, Pseudomonas aeruginosa and Acinetobacter baumannii were cultivated in the Calgary Biofilm Device. Their biofilms were placed 50 cm from the UV lamp within the Biosafety Cabinet. Viability test, crystal violet assay and a scanning electron microscope were used to evaluate the germicidal efficacy. Within 5 minutes, UV radiation could kill S. aureus, MRSA, S. epidermidis, A. baumannii and ESBL-producing E. coli completely while it required 20 minutes and 30 minutes respectively to kill E. coli and P. aeruginosa. However, the amounts of biomass and the ultrastructure between UV-exposed biofilms and controls were not significantly different. UV radiation is effective in inactivating nosocomial pathogens grown within biofilms, but not removing biofilms and EPS. The biofilm of P. aeruginosa was the most durable.

Comparative proteomic analysis of extracellular proteins expressed by various clonal types of Staphylococcus aureus and during planktonic growth and biofilm development.

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Atshan, Salman S; Shamsudin, Mariana N; Sekawi, Zamberi; Thian Lung, Leslie T; Barantalab, Fatemeh; Liew, Yun K; Alreshidi, Mateg Ali; Abduljaleel, Salwa A; Hamat, Rukman A

2015-01-01

Staphylococcus aureus is well known for its biofilm formation with rapid emergence of new clones circulating worldwide. The main objectives of the study were (1) to identify possible differences in protein expression among various and closely related clonal types of S. aureus, (2) to establish the differences in protein expression in terms of size of protein spots and its intensities between bacteria which are grown statically (biofilm formation) with that of under aeration and agitation, and (3) to compare the differences in protein expression as a function of time (in hours). In this study, we selected six clinical isolates comprising two similar (MRSA-527 and MRSA-524) and four different (MRSA-139, MSSA-12E, MSSA-22d, and MSSA-10E) types identified by spa typing, MLST and SCCmec typing. We performed 2D gel migration comparison. Also, two MRSA isolates (527 and 139) were selected to determine quantitative changes in the level of extracellular proteins at different biofilm growth time points of 12, 24, and 48 h. The study was done using a strategy that combines 2-DGE and LC-MS/MS analysis for absolute quantification and identification of the extracellular proteins. The 2DGE revealed that the proteomic profiles for the isolates belonging to the similar spa, MLST, and SCCmec types were still quite different. Among the extracellular proteins secreted at different time points of biofilm formation, significant changes in protein expression were observed at 48 h incubation as compared to the exponential growth at 12 h incubation. The main conclusion of the work is that the authors do observe differences among isolates, and growth conditions do influence the protein content at different time points of biofilm formation.

Combating biofilms

DEFF Research Database (Denmark)

Yang, Liang; Liu, Yang; Wu, Hong

2012-01-01

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

In vitro pharmacokinetics of antimicrobial cationic peptides alone and in combination with antibiotics against methicillin resistant Staphylococcus aureus biofilms.

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Dosler, Sibel; Mataraci, Emel

2013-11-01

Antibiotic therapy for methicillin-resistant Staphylococcus aureus (MRSA) infections is becoming more difficult in hospitals and communities because of strong biofilm-forming properties and multidrug resistance. Biofilm-associated MRSA is not affected by therapeutically achievable concentrations of antibiotics. Therefore, we investigated the in vitro pharmacokinetic activities of antimicrobial cationic peptides (AMPs; indolicidin, cecropin [1-7]-melittin A [2-9] amide [CAMA], and nisin), either alone or in combination with antibiotics (daptomycin, linezolid, teicoplanin, ciprofloxacin, and azithromycin), against standard and 2 clinically obtained MRSA biofilms. The minimum inhibitory concentrations (MIC) and minimum biofilm-eradication concentrations (MBEC) were determined by microbroth dilution technique. The time-kill curve (TKC) method was used to determine the bactericidal activities of the AMPs alone and in combination with the antibiotics against standard and clinically obtained MRSA biofilms. The MIC values of the AMPs and antibiotics ranged between 2 to 16 and 0.25 to 512 mg/L, and their MBEC values were 640 and 512 to 5120 mg/L, respectively. The TKC studies demonstrated that synergistic interactions occurred most frequently when using nisin+daptomycin/ciprofloxacin, indolicidin+teicoplanin, and CAMA+ciprofloxacin combinations. No antagonism was observed with any combination. AMPs appear to be good candidates for the treatment of MRSA biofilms, as they act as both enhancers of anti-biofilm activities and help to prevent or delay the emergence of resistance when used either alone or in combination with antibiotics. Copyright © 2013 Elsevier Inc. All rights reserved.

Effects of Fermented Sumach on the Formation of Slime Layer of Staphylococcus aureus

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Sahra Kırmusaoğlu

2012-03-01

Full Text Available Objective: Staphylococcus aureus (S. aureus is one of the most commonly isolated bacterial pathogens in hospitals, and the most frequent cause of nosocomial infections. Nosocomial staphylococcal foreign-body infections related to biofilm formation are a serious threat, demanding new therapeutic and preventive strategies. Implantation of intravenous catheters and surgical implantation of prosthetic implants carry a risk of infection. In order to prevent all these effects of biofilms, a study was designed to observe the possible antibacterial effect of sumach (Rhus coriaria on the biofilm formation of S. aureus. Material and Methods: The influence of varying concentrations of sumach on the formation of biofilms by 13 strains of Staphylococcus aureus was tested by a microelisa assay. Results: The significant differences between varying concentrations of sumach (0.1, 0.2, 0.5 and 1.0 µl/ml were observed in four methicillin resistant Staphylococcus aureus (MRSA and nine methicillin sensitive Staphylococcus aureus (MSSA (p<0.05. In bacteria, a dose-related decrease in the formation of slime, which is a major virulence factor of staphylococcal infections, was observed. Conclusion: In our study, using 0.1, 0.2, 0.5 and 1.0 µl/ml of sumach, thirteen strains lost, 17%, 22%, 28% and 48% respectively of their capacity to produce biofilms. Sumach, which is a herbal product, can decrease the formation of biofilm, which is a major virulence factor in staphylococcal infections.

Effects of Miramistin and Phosprenil on Microbial Biofilms.

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Danilova, T A; Danilina, G A; Adzhieva, A A; Minko, A G; Nikolaeva, T N; Zhukhovitskii, V G; Pronin, A V

2017-08-01

Effects of Miramistin and Phosprenil on biofilms of S. pyogenes, S. aureus, E. coli, L. acidophilus, and L. plantarum were studied. Significant differences in the effects of these substances on mature biofilms of microorganisms and the process of their formation were observed. Miramistin had significant inhibiting effects on the forming of biofilms and on the formed biofilms of all studied microorganisms. Treatment with Miramistin inhibited biofilm formation by 2-3 times compared to the control. This effect was found already after using of Miramistin in the low doses (3.12 μg/ml). Inhibition of the growth of a formed biofilm was observed only after treatment with Miramistin in the high doses (25-50 μg/ml). Phosprenil in the high doses (15-30 mg/ml) inhibited the forming of biofilms, especially the biofilms of S. pyogenes and L. plantarum (by 3-4.5 times). Treatment of formed biofilms with the agent in doses of 6.0 and 0.6 mg/ml was associated with pronounced stimulation of its growth in S. pyogenes, S. aureus, and L. acidophilus.

9 CFR 113.106 - Clostridium Chauvoei Bacterin.

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2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Clostridium Chauvoei Bacterin. 113.106 Section 113.106 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF... Animal and Plant Health Inspection Service, shall be used for challenge 14 to 15 days following the last...

9 CFR 113.107 - Clostridium Haemolyticum Bacterin.

Science.gov (United States)

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Clostridium Haemolyticum Bacterin. 113.107 Section 113.107 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT... challenge 14 to 15 days following the last injection of the product. Each of eight vaccinates and each of...

Antibiofilm and antibacterial effects of specific chitosan molecules on Staphylococcus aureus isolates associated with bovine mastitis

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Asli, Abdelhamid; Brouillette, Eric; Ster, Céline; Ghinet, Mariana Gabriela; Brzezinski, Ryszard; Lacasse, Pierre; Jacques, Mario

2017-01-01

Staphylococcus aureus is one of the major pathogens causing bovine intramammary infections (IMIs) and mastitis. Mastitis is the primary cause for the use of antibiotics in dairy farms but therapeutic failure is often observed. One of the reasons for the lack of effectiveness of antibiotic therapy despite the observed susceptibility of bacterial isolates in vitro are bacterial biofilms. In this study, we used chitosan of well-defined molecular weight (0.4–0.6, 1.3, 2.6 and 4.0 kDa) and investigated their antibiofilm and antibacterial activities in in vitro and in vivo models related to S. aureus IMIs. A chitosan of at least 6 units of glucosamine was necessary for maximum antibacterial activity. The 2.6 and 4.0 kDa forms were able to prevent biofilm production by the biofilm hyperproducer strain S. aureus 2117 and a bovine MRSA (methicillin-resistant S. aureus). The intramammary administration of the 2.6 kDa chitosan showed no adverse effects in mice or in cows, as opposed to the slight inflammatory effect observed in mammary glands with the 4.0 kDa derivative. The 2.6 kDa chitosan killed bacteria embedded in pre-established biofilms in a dose-dependent manner with a >3 log10 reduction in CFU at 4 mg/ml. Also, the 2.6 kDa chitosan could prevent the persistence of the internalized MRSA into the mammary epithelial cell line MAC-T. An in vitro checkerboard assay showed that the 2.6 kDa chitosan produced a synergy with the macrolide class of antibiotics (e.g., tilmicosin) and reduced the MIC of both molecules by 2–8 times. Finally, the intramammary administration of the 2.6 kDa chitosan alone (P<0.01) or in combination with tilmicosin (P<0.0001) reduced the colonization of mammary glands in a murine IMI model. Our results suggest that the use of chitosan alone or in combination with a low dose of a macrolide could help reduce antibiotic use in dairy farms. PMID:28486482

Antibiofilm and antibacterial effects of specific chitosan molecules on Staphylococcus aureus isolates associated with bovine mastitis.

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Abdelhamid Asli

Full Text Available Staphylococcus aureus is one of the major pathogens causing bovine intramammary infections (IMIs and mastitis. Mastitis is the primary cause for the use of antibiotics in dairy farms but therapeutic failure is often observed. One of the reasons for the lack of effectiveness of antibiotic therapy despite the observed susceptibility of bacterial isolates in vitro are bacterial biofilms. In this study, we used chitosan of well-defined molecular weight (0.4-0.6, 1.3, 2.6 and 4.0 kDa and investigated their antibiofilm and antibacterial activities in in vitro and in vivo models related to S. aureus IMIs. A chitosan of at least 6 units of glucosamine was necessary for maximum antibacterial activity. The 2.6 and 4.0 kDa forms were able to prevent biofilm production by the biofilm hyperproducer strain S. aureus 2117 and a bovine MRSA (methicillin-resistant S. aureus. The intramammary administration of the 2.6 kDa chitosan showed no adverse effects in mice or in cows, as opposed to the slight inflammatory effect observed in mammary glands with the 4.0 kDa derivative. The 2.6 kDa chitosan killed bacteria embedded in pre-established biofilms in a dose-dependent manner with a >3 log10 reduction in CFU at 4 mg/ml. Also, the 2.6 kDa chitosan could prevent the persistence of the internalized MRSA into the mammary epithelial cell line MAC-T. An in vitro checkerboard assay showed that the 2.6 kDa chitosan produced a synergy with the macrolide class of antibiotics (e.g., tilmicosin and reduced the MIC of both molecules by 2-8 times. Finally, the intramammary administration of the 2.6 kDa chitosan alone (P<0.01 or in combination with tilmicosin (P<0.0001 reduced the colonization of mammary glands in a murine IMI model. Our results suggest that the use of chitosan alone or in combination with a low dose of a macrolide could help reduce antibiotic use in dairy farms.

Conjugation of Inulin Improves Anti-Biofilm Activity of Chitosan.

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Zhang, Guiqiang; Liu, Jing; Li, Ruilian; Jiao, Siming; Feng, Cui; Wang, Zhuo A; Du, Yuguang

2018-05-04

Bacteria biofilm helps bacteria prevent phagocytosis during infection and increase resistance to antibiotics. Staphylococcus aureus is a Gram-positive pathogenic bacterium and is tightly associated with biofilm-related infections, which have led to great threat to human health. Chitosan, the only cationic polysaccharide in nature, has been demonstrated to have antimicrobial and anti-biofilm activities, which, however, require a relative high dosage of chitosan. Moreover, poor water solubility further restricts its applications on anti-infection therapy. Inulins are a group of polysaccharides produced by many types of plants, and are widely used in processed foods. Compared to chitosan, inulin is very soluble in water and possesses a mild antibacterial activity against certain pathogenic bacteria. In order to develop an effective strategy to treat biofilm-related infections, we introduce a method by covalent conjugation of inulin to chitosan. The physicochemical characterization of the inulin⁻chitosan conjugate was assayed, and the anti-biofilm activity was evaluated against S. aureus biofilm. The results indicated that, as compared to chitosan, this novel polysaccharide⁻polysaccharide conjugate significantly enhanced activities against S. aureus either in a biofilm or planktonic state. Of note, the conjugate also showed a broad spectrum anti-biofilm activity on different bacteria strains and low cellular toxicity to mammalian cells. These results suggested that chitosan conjugation of inulin was a viable strategy for treatment against biofilm-related infections. This finding may further spread the application of natural polysaccharides on treatments of infectious disease.

The ATP-Dependent Protease ClpP Inhibits Biofilm Formation by Regulating Agr and Cell Wall Hydrolase Sle1 in Staphylococcus aureus

Science.gov (United States)

Liu, Qian; Wang, Xing; Qin, Juanxiu; Cheng, Sen; Yeo, Won-Sik; He, Lei; Ma, Xiaowei; Liu, Xiaoyun; Li, Min; Bae, Taeok

2017-01-01

Biofilm causes hospital-associated infections on indwelling medical devices. In Staphylococcus aureus, Biofilm formation is controlled by intricately coordinated network of regulating systems, of which the ATP-dependent protease ClpP shows an inhibitory effect. Here, we demonstrate that the inhibitory effect of ClpP on biofilm formation is through Agr and the cell wall hydrolase Sle1. Biofilm formed by clpP mutant consists of proteins and extracellular DNA (eDNA). The increase of the protein was, at least in part, due to the reduced protease activity of the mutant, which was caused by the decreased activity of agr. On the other hand, the increase of eDNA was due to increased cell lysis caused by the higher level of Sle1. Indeed, as compared with wild type, the clpP mutant excreted an increased level of eDNA, and showed higher sensitivity to Triton-induced autolysis. The deletion of sle1 in the clpP mutant decreased the biofilm formation, the level of eDNA, and the Triton-induced autolysis to wild-type levels. Despite the increased biofilm formation capability, however, the clpP mutant showed significantly reduced virulence in a murine model of subcutaneous foreign body infection, indicating that the increased biofilm formation capability cannot compensate for the intrinsic functions of ClpP during infection. PMID:28555174

The ATP-Dependent Protease ClpP Inhibits Biofilm Formation by Regulating Agr and Cell Wall Hydrolase Sle1 in Staphylococcus aureus

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Qian Liu

2017-05-01

Full Text Available Biofilm causes hospital-associated infections on indwelling medical devices. In Staphylococcus aureus, Biofilm formation is controlled by intricately coordinated network of regulating systems, of which the ATP-dependent protease ClpP shows an inhibitory effect. Here, we demonstrate that the inhibitory effect of ClpP on biofilm formation is through Agr and the cell wall hydrolase Sle1. Biofilm formed by clpP mutant consists of proteins and extracellular DNA (eDNA. The increase of the protein was, at least in part, due to the reduced protease activity of the mutant, which was caused by the decreased activity of agr. On the other hand, the increase of eDNA was due to increased cell lysis caused by the higher level of Sle1. Indeed, as compared with wild type, the clpP mutant excreted an increased level of eDNA, and showed higher sensitivity to Triton-induced autolysis. The deletion of sle1 in the clpP mutant decreased the biofilm formation, the level of eDNA, and the Triton-induced autolysis to wild-type levels. Despite the increased biofilm formation capability, however, the clpP mutant showed significantly reduced virulence in a murine model of subcutaneous foreign body infection, indicating that the increased biofilm formation capability cannot compensate for the intrinsic functions of ClpP during infection.

Bacteriophage-Derived Peptidase CHAPK Eliminates and Prevents Staphylococcal Biofilms

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Mark Fenton

2013-01-01

Full Text Available New antibacterial agents are urgently needed for the elimination of biofilm-forming bacteria that are highly resistant to traditional antimicrobial agents. Proliferation of such bacteria can lead to significant economic losses in the agri-food sector. This study demonstrates the potential of the bacteriophage-derived peptidase, CHAPK, as a biocidal agent for the rapid disruption of biofilm-forming staphylococci, commonly associated with bovine mastitis. Purified CHAPK applied to biofilms of Staphylococcus aureus DPC5246 completely eliminated the staphylococcal biofilms within 4 h. In addition, CHAPK was able to prevent biofilm formation by this strain. The CHAPK lysin also reduced S. aureus in a skin decolonization model. Our data demonstrates the potential of CHAPK as a biocidal agent for prevention and treatment of biofilm-associated staphylococcal infections or as a decontaminating agent in the food and healthcare sectors.

Cross neutralizing antibodies in hamsters vaccinated with leptospiral bacterins produced with three serovars of serogroup Sejroe

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Rosana Tabata

2002-09-01

Full Text Available Three leptospiral bacterins, produced with different serovars of Serogroup Sejroe, namely the hardjo (bacterin A, wolffi (bacterin B and guaricura (bacterin C, were evaluated in male hamsters (Mesocricetus auratus by comparing the agglutinating and neutralizing antibodies titers using microscopic agglutination (MAT and in vitro growth inhibition (GIT tests. The immunization schedule was based on two 1.0 mL doses of non-diluted formalininactivated whole culture bacterin given through subcutaneous route with 10-day interval. The challenge was performed ten days after the second vaccine dose, when the animals were inoculated with 0.2 mL of non-inactivated cultures of each serovar through intraperitoneal route. On the 21st post-challenge day (PCD, all animals were bled and their sera were joined in pools (n=8 and tested by MAT and GIT. All vaccinated and control animals presented no clinical signs of leptospirosis after the challenge, but the serovar guaricura was isolated from the kidneys of control animals on the 21st PCD. The MAT results showed cross agglutinins between serovars hardjo and wolffi, and between wolffi and guaricura. The GIT results revealed the presence of cross neutralizing antibodies between serovars wolffi or guaricura against hardjo, wolffi and guaricura. It was found that the tested strain of serovar hardjo did not produce detectable levels of neutralizing antibodies, indicating its poor immunogenicity.

Maggot excretions inhibit biofilm formation on biomaterials.

Science.gov (United States)

Cazander, Gwendolyn; van de Veerdonk, Mariëlle C; Vandenbroucke-Grauls, Christina M J E; Schreurs, Marco W J; Jukema, Gerrolt N

2010-10-01

Biofilm-associated infections in trauma surgery are difficult to treat with conventional therapies. Therefore, it is important to develop new treatment modalities. Maggots in captured bags, which are permeable for larval excretions/secretions, aid in healing severe, infected wounds, suspect for biofilm formation. Therefore we presumed maggot excretions/secretions would reduce biofilm formation. We studied biofilm formation of Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella oxytoca, Enterococcus faecalis, and Enterobacter cloacae on polyethylene, titanium, and stainless steel. We compared the quantities of biofilm formation between the bacterial species on the various biomaterials and the quantity of biofilm formation after various incubation times. Maggot excretions/secretions were added to existing biofilms to examine their effect. Comb-like models of the biomaterials, made to fit in a 96-well microtiter plate, were incubated with bacterial suspension. The formed biofilms were stained in crystal violet, which was eluted in ethanol. The optical density (at 595 nm) of the eluate was determined to quantify biofilm formation. Maggot excretions/secretions were pipetted in different concentrations to (nonstained) 7-day-old biofilms, incubated 24 hours, and finally measured. The strongest biofilms were formed by S. aureus and S. epidermidis on polyethylene and the weakest on titanium. The highest quantity of biofilm formation was reached within 7 days for both bacteria. The presence of excretions/secretions reduced biofilm formation on all biomaterials. A maximum of 92% of biofilm reduction was measured. Our observations suggest maggot excretions/secretions decrease biofilm formation and could provide a new treatment for biofilm formation on infected biomaterials.

Inactivation of the Autolysis-Related Genes lrgB and yycI in Staphylococcus aureus Increases Cell Lysis-Dependent eDNA Release and Enhances Biofilm Development In Vitro and In Vivo.

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Cristiana Ossaille Beltrame

Full Text Available Staphylococcus aureus ica-independent biofilms are multifactorial in nature, and various bacterial proteins have been associated with biofilm development, including fibronectin-binding proteins A and B, protein A, surface protein SasG, proteases, and some autolysins. The role of extracellular DNA (eDNA has also been demonstrated in some S. aureus biofilms. Here, we constructed a Tn551 library, and the screening identified two genes that affected biofilm formation, lrgB and yycI. The repressive effect of both genes on the development of biofilm was also confirmed in knockout strains constructed by allelic recombination. In contrast, the superexpression of either lrgB or yycI by a cadmium-inducible promoter led to a decrease in biofilm accumulation. Indeed, a significant increase in the cell-lysis dependent eDNA release was detected when lrgB or yycI were inactivated, explaining the enhanced biofilm formed by these mutants. In fact, lrgB and yycI genes belong to distinct operons that repress bacterial autolysis through very different mechanisms. LrgB is associated with the synthesis of phage holin/anti-holin analogues, while YycI participates in the activation/repression of the two-component system YycGF (WalKR. Our in vivo data suggest that autolysins activation lead to increased bacterial virulence in the foreign body animal model since a higher number of attached cells was recovered from the implanted catheters inoculated with lrgB or yycI knockout mutants.

Clonal profile, virulence and resistance of Staphylococcus aureus isolated from sheep milk.

Science.gov (United States)

Martins, Katheryne Benini; Faccioli-Martins, Patricia Yoshida; Riboli, Danilo Flávio Moraes; Pereira, Valéria Cataneli; Fernandes, Simone; Oliveira, Aline A; Dantas, Ariane; Zafalon, Luiz Francisco; da Cunha, Maria de Lourdes Ribeiro de Souza

2015-06-01

The objective of this study was to characterize the clonal profile, virulence factors and antimicrobial resistance, particularly oxacillin resistance, of Staphylococcus aureus isolated from sheep milk. Milk samples were collected from all teats for the California Mastitis Test (CMT), somatic cell count, identification of S. aureus, investigation in these strains of genes encoding toxins (sea, seb, sec, sed, tst), biofilm (icaA, icaC, icaD, bap), leukocidin (luk-PV) oxacillin resistance by mecA gene detection and susceptibility testing (12 antibiotics). Messenger RNA expression was evaluated by RT-PCR in isolates carrying toxin and biofilm genes. Biofilm formation was also evaluated phenotypically by adherence to polystyrene plates. The clonal profile of S. aureus was investigated by pulsed-field gel electrophoresis. A total of 473 milk samples were collected from 242 animals on three farms and 20 S. aureus strains were isolated and none carried the mecA gene. The two sec gene-positive isolates and the isolates carrying the tst and luk-PV genes were positive by RT-PCR. Staphylococcus aureus isolated from the three flocks studied showed high susceptibility to the drugs tested and none was biofilm producer, indicating that biofilm formation was not a virulence factor causing infection by these strains. The typing of 17 S. aureus isolates revealed the presence of a common clone on the three farms studied, and the presence and expression of the sec and tst genes in one strain of this clone suggest the possible acquisition of virulence genes by this clone, a fact that is important for animal health and food hygiene.

Clonal profile, virulence and resistance of Staphylococcus aureus isolated from sheep milk

Science.gov (United States)

Martins, Katheryne Benini; Faccioli-Martins, Patricia Yoshida; Riboli, Danilo Flávio Moraes; Pereira, Valéria Cataneli; Fernandes, Simone; Oliveira, Aline A.; Dantas, Ariane; Zafalon, Luiz Francisco; da Cunha, Maria de Lourdes Ribeiro de Souza

2015-01-01

The objective of this study was to characterize the clonal profile, virulence factors and antimicrobial resistance, particularly oxacillin resistance, of Staphylococcus aureus isolated from sheep milk. Milk samples were collected from all teats for the California Mastitis Test (CMT), somatic cell count, identification of S. aureus, investigation in these strains of genes encoding toxins (sea, seb, sec, sed, tst), biofilm (icaA, icaC, icaD, bap), leukocidin (luk-PV) oxacillin resistance by mecA gene detection and susceptibility testing (12 antibiotics). Messenger RNA expression was evaluated by RT-PCR in isolates carrying toxin and biofilm genes. Biofilm formation was also evaluated phenotypically by adherence to polystyrene plates. The clonal profile of S. aureus was investigated by pulsed-field gel electrophoresis. A total of 473 milk samples were collected from 242 animals on three farms and 20 S. aureus strains were isolated and none carried the mecA gene. The two sec gene-positive isolates and the isolates carrying the tst and luk-PV genes were positive by RT-PCR. Staphylococcus aureus isolated from the three flocks studied showed high susceptibility to the drugs tested and none was biofilm producer, indicating that biofilm formation was not a virulence factor causing infection by these strains. The typing of 17 S. aureus isolates revealed the presence of a common clone on the three farms studied, and the presence and expression of the sec and tst genes in one strain of this clone suggest the possible acquisition of virulence genes by this clone, a fact that is important for animal health and food hygiene. PMID:26273271

Biofilm Risks

DEFF Research Database (Denmark)

Wirtanen, Gun Linnea; Salo, Satu

2016-01-01

This chapter on biofilm risks deals with biofilm formation of pathogenic microbes, sampling and detection methods, biofilm removal, and prevention of biofilm formation. Several common pathogens produce sticky and/or slimy structures in which the cells are embedded, that is, biofilms, on various...... surfaces in food processing. Biofilms of common foodborne pathogens are reviewed. The issue of persistent and nonpersistent microbial contamination in food processing is also discussed. It has been shown that biofilms can be difficult to remove and can thus cause severe disinfection and cleaning problems...... in food factories. In the prevention of biofilm formation microbial control in process lines should both limit the number of microbes on surfaces and reduce microbial activity in the process. Thus the hygienic design of process equipment and process lines is important in improving the process hygiene...

68Ga-labeled phage-display selected peptides as tracers for positron emission tomography imaging of Staphylococcus aureus biofilm-associated infections: Selection, radiolabelling and preliminary biological evaluation

International Nuclear Information System (INIS)

Nielsen, Karin M.; Kyneb, Majbritt H.; Alstrup, Aage K.O.; Jensen, Jakob J.; Bender, Dirk; Schønheyder, Henrik C.; Afzelius, Pia; Nielsen, Ole L.; Jensen, Svend B.

2016-01-01

Introduction: Staphylococcus aureus is a major cause of skin and deep-sited infections, often associated with the formation of biofilms. Early diagnosis and initiated therapy is essential to prevent disease progression and to reduce complications that can be serious. Imaging techniques are helpful combining anatomical with functional data in order to describe and characterize site, extent and activity of the disease. The purpose of the study was to identify and 68 Ga-label peptides with affinity for S. aureus biofilm and evaluate their potential as bacteria-specific positron emission tomography (PET) imaging agents. Methods: Phage-displayed dodecapeptides were selected using an in vitro grown S. aureus biofilm as target. One cyclic (A8) and two linear (A9, A11) dodecapeptides were custom synthesized with 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid (DOTA) conjugated via a lysine linker (K), and for A11 also a glycine–serine–glycine spacer (GSG). The 68 Ga-labeling of A8-K-DOTA, A9-K-DOTA, and A11-GSGK-DOTA were optimized and in vitro bacterial binding was evaluated for 68 Ga-A9-K-DOTA and 68 Ga-A11-GSGK-DOTA. Stability of 68 Ga-A9-K-DOTA was studied in vitro in human serum, while the in vivo plasma stability was analyzed in mice and pigs. Additionally, the whole-body distribution kinetics of 68 Ga-A9-K-DOTA was measured in vivo by PET imaging of pigs and ex vivo in excised mice tissues. Results: The 68 Ga-A9-K-DOTA and 68 Ga-A11-GSGK-DOTA remained stable in product formulation, whereas 68 Ga-A8-K-DOTA was unstable. The S. aureus binding of 68 Ga-A11-GSGK-DOTA and 68 Ga-A9-K-DOTA was observed in vitro, though blocking of the binding was not possible by excess of cold peptide. The 68 Ga-A9-K-DOTA was degraded slowly in vitro, while the combined in vivo evaluation in pigs and mice showed a rapid blood clearance and renal excretion of the 68 Ga-A9-K-DOTA. Conclusion: The preliminary in vitro and in vivo studies of the phage-display S. aureus

ERM booster vaccination of Rainbow trout using diluted bacterin

DEFF Research Database (Denmark)

Schmidt, Jacob Günther; Henriksen, Niels H.; Buchmann, Kurt

2016-01-01

under laboratory conditions extend the protection period. The present field study investigated the applicability of the method under practical farming conditions (freshwater earth ponds supplied by stream water). Primary immersion vaccination of trout (3–4 g) for 30 s in Y. ruckeri bacterin (diluted 1......Enteric Red Mouth Disease ERM caused by Yersinia ruckeri infection is associated with morbidity and mortality in salmonid farming but immersion vaccination of fry may confer some protection for a number of months. Revaccination of rainbow trout, even by use of diluted ERM immersion vaccine, can......:10) in April 2015 was followed 3 months later (July 2015) by 1 h bathing of rainbow trout in bacterin (diluted 1:650 or 1:1700) in order to evaluate if this time saving vaccination methodology can improve immunity and protection. Trout were subjected in farms to natural Y. ruckeri exposure in June and July...

Increased resistance of contact lens related bacterial biofilms to antimicrobial activity of soft contact lens care solutions

Science.gov (United States)

Szczotka-Flynn, Loretta B.; Imamura, Yoshifumi; Chandra, Jyotsna; Yu, Changping; Mukherjee, Pranab K.; Pearlman, Eric; Ghannoum, Mahmoud A.

2014-01-01

PURPOSE To determine if clinical and reference strains of Pseudomonas aeruginosa, Serratia marcescens, and Staphylococcus aureus form biofilms on silicone hydrogel contact lenses, and ascertain antimicrobial activities of contact lens care solutions. METHODS Clinical and American Type Culture Collection (ATCC) reference strains of Pseudomonas aeruginosa, Serratia marcescens, and Staphylococcus aureus were incubated with lotrafilcon A lenses under conditions that facilitate biofilm formation. Biofilms were quantified by quantitative culturing (colony forming units, CFUs), and gross morphology and architecture were evaluated using scanning electron microscopy (SEM) and confocal microscopy. Susceptibilities of the planktonic and biofilm growth phases of the bacteria to five common multipurpose contact lens care solutions and one hydrogen peroxide care solution were assessed. RESULTS P. aeruginosa, S. marcescens, and S. aureus reference and clinical strains formed biofilms on lotrafilcon A silicone hydrogel contact lenses, as dense networks of cells arranged in multiple layers with visible extracellular matrix. The biofilms were resistant to commonly used biguanide preserved multipurpose care solutions. P. aeruginosa and S. aureus biofilms were susceptible to a hydrogen peroxide and a polyquaternium preserved care solution, whereas S. marcescens biofilm was resistant to a polyquaternium preserved care solution but susceptible to hydrogen peroxide disinfection. In contrast, the planktonic forms were always susceptible. CONCLUSIONS P. aeruginosa, S. marcescens, and S. aureus form biofilms on lotrafilcon A contact lenses, which in contrast to planktonic cells, are resistant to the antimicrobial activity of several soft contact lens care products. PMID:19654521

Clonal profile, virulence and resistance of Staphylococcus aureus isolated from sheep milk

Directory of Open Access Journals (Sweden)

Katheryne Benini Martins

2015-06-01

Full Text Available The objective of this study was to characterize the clonal profile, virulence factors and antimicrobial resistance, particularly oxacillin resistance, of Staphylococcus aureus isolated from sheep milk. Milk samples were collected from all teats for the California Mastitis Test (CMT, somatic cell count, identification of S. aureus, investigation in these strains of genes encoding toxins (sea, seb, sec, sed, tst, biofilm (icaA, icaC, icaD, bap, leukocidin (luk-PV oxacillin resistance by mecA gene detection and susceptibility testing (12 antibiotics. Messenger RNA expression was evaluated by RT-PCR in isolates carrying toxin and biofilm genes. Biofilm formation was also evaluated phenotypically by adherence to polystyrene plates. The clonal profile of S. aureus was investigated by pulsed-field gel electrophoresis. A total of 473 milk samples were collected from 242 animals on three farms and 20 S. aureus strains were isolated and none carried the mecA gene. The two sec gene-positive isolates and the isolates carrying the tst and luk-PV genes were positive by RT-PCR. Staphylococcus aureus isolated from the three flocks studied showed high susceptibility to the drugs tested and none was biofilm producer, indicating that biofilm formation was not a virulence factor causing infection by these strains. The typing of 17 S. aureus isolates revealed the presence of a common clone on the three farms studied, and the presence and expression of the sec and tst genes in one strain of this clone suggest the possible acquisition of virulence genes by this clone, a fact that is important for animal health and food hygiene.

Commercial bacterins did not induce detectable levels of antibodies in mice against Mycoplasma hyopneumoniae antigens strongly recognized by swine immune system

Directory of Open Access Journals (Sweden)

Andressa Fisch

2016-01-01

Full Text Available Enzootic Pneumonia (EP caused by Mycoplasma hyopneumoniae results in major economic losses to the swine industry. Hence, the identification of factors that provide protection against EP could help to develop effective vaccines. One such factor that provides partial protection are bacterins. Therefore, the aim of this study was to verify the induction of antibodies against fifteen M. hyopneumoniae antigens, strongly recognized by the swine immune system during natural infection, in mice vaccinated with six commercial bacterins. Each group of mice was inoculated with one bacterin, and seroconversion was assessed by indirect ELISA using recombinant antigens and M. hyopneumoniae 7448 whole cell extract. Sera from one inoculated group recognized antigen MHP_0067, and sera from four inoculated groups recognized antigens MHP_0513 and MHP_0580. None of the bacterins was able to induce seroconversion against the twelve remaining antigens. This absence of a serological response could be attributed to the lack of antigen expression in M. hyopneumoniae strains used in bacterin production. Additionally the partial protection provided by these vaccines could be due to low expression or misfolding of antigens during vaccine preparation. Therefore, the supplementation of bacterins with these recombinant antigens could be a potential alternative in the development of more effective vaccines.

A C-Type Lectin from Bothrops jararacussu Venom Disrupts Staphylococcal Biofilms

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Klein, Raphael Contelli; Fabres-Klein, Mary Hellen; de Oliveira, Leandro Licursi; Feio, Renato Neves; Malouin, François; Ribon, Andréa de Oliveira Barros

2015-01-01

Bovine mastitis is a major threat to animal health and the dairy industry. Staphylococcus aureus is a contagious pathogen that is usually associated with persistent intramammary infections, and biofilm formation is a relevant aspect of the outcome of these infections. Several biological activities have been described for snake venoms, which led us to screen secretions of Bothrops jararacussu for antibiofilm activity against S. aureus NRS155. Crude venom was fractionated by size-exclusion chromatography, and the fractions were tested against S. aureus. Biofilm growth, but not bacterial growth, was affected by several fractions. Two fractions (15 and 16) showed the best activities and were also assayed against S. epidermidis NRS101. Fraction 15 was identified by TripleTOF mass spectrometry as a galactose-binding C-type lectin with a molecular weight of 15 kDa. The lectin was purified from the crude venom by D-galactose affinity chromatography, and only one peak was observed. This pure lectin was able to inhibit 75% and 80% of S. aureus and S. epidermidis biofilms, respectively, without affecting bacterial cell viability. The lectin also exhibited a dose-dependent inhibitory effect on both bacterial biofilms. The antibiofilm activity was confirmed using scanning electron microscopy. A pre-formed S. epidermidis biofilm was significantly disrupted by the C-type lectin in a time-dependent manner. Additionally, the lectin demonstrated the ability to inhibit biofilm formation by several mastitis pathogens, including different field strains of S. aureus, S. hyicus, S. chromogenes, Streptococcus agalactiae, and Escherichia coli. These findings reveal a new activity for C-type lectins. Studies are underway to evaluate the biological activity of these lectins in a mouse mastitis model. PMID:25811661

A C-type lectin from Bothrops jararacussu venom disrupts Staphylococcal biofilms.

Directory of Open Access Journals (Sweden)

Raphael Contelli Klein

Full Text Available Bovine mastitis is a major threat to animal health and the dairy industry. Staphylococcus aureus is a contagious pathogen that is usually associated with persistent intramammary infections, and biofilm formation is a relevant aspect of the outcome of these infections. Several biological activities have been described for snake venoms, which led us to screen secretions of Bothrops jararacussu for antibiofilm activity against S. aureus NRS155. Crude venom was fractionated by size-exclusion chromatography, and the fractions were tested against S. aureus. Biofilm growth, but not bacterial growth, was affected by several fractions. Two fractions (15 and 16 showed the best activities and were also assayed against S. epidermidis NRS101. Fraction 15 was identified by TripleTOF mass spectrometry as a galactose-binding C-type lectin with a molecular weight of 15 kDa. The lectin was purified from the crude venom by D-galactose affinity chromatography, and only one peak was observed. This pure lectin was able to inhibit 75% and 80% of S. aureus and S. epidermidis biofilms, respectively, without affecting bacterial cell viability. The lectin also exhibited a dose-dependent inhibitory effect on both bacterial biofilms. The antibiofilm activity was confirmed using scanning electron microscopy. A pre-formed S. epidermidis biofilm was significantly disrupted by the C-type lectin in a time-dependent manner. Additionally, the lectin demonstrated the ability to inhibit biofilm formation by several mastitis pathogens, including different field strains of S. aureus, S. hyicus, S. chromogenes, Streptococcus agalactiae, and Escherichia coli. These findings reveal a new activity for C-type lectins. Studies are underway to evaluate the biological activity of these lectins in a mouse mastitis model.

(68)Ga-labeled phage-display selected peptides as tracers for positron emission tomography imaging of Staphylococcus aureus biofilm-associated infections: Selection, radiolabelling and preliminary biological evaluation

DEFF Research Database (Denmark)

Nielsen, Karin M; Kyneb, Majbritt H; Alstrup, Aage K O

2016-01-01

, while the in vivo plasma stability was analyzed in mice and pigs. Additionally, the whole-body distribution kinetics of (68)Ga-A9-K-DOTA was measured in vivo by PET imaging of pigs and ex vivo in excised mice tissues. RESULTS: The (68)Ga-A9-K-DOTA and (68)Ga-A11-GSGK-DOTA remained stable in product......INTRODUCTION: Staphylococcus aureus is a major cause of skin and deep-sited infections, often associated with the formation of biofilms. Early diagnosis and initiated therapy is essential to prevent disease progression and to reduce complications that can be serious. Imaging techniques are helpful...... combining anatomical with functional data in order to describe and characterize site, extent and activity of the disease. The purpose of the study was to identify and (68)Ga-label peptides with affinity for S. aureus biofilm and evaluate their potential as bacteria-specific positron emission tomography (PET...

Femtosecond laser surface texturing of titanium as a method to reduce the adhesion of Staphylococcus aureus and biofilm formation

Science.gov (United States)

Cunha, Alexandre; Elie, Anne-Marie; Plawinski, Laurent; Serro, Ana Paula; Botelho do Rego, Ana Maria; Almeida, Amélia; Urdaci, Maria C.; Durrieu, Marie-Christine; Vilar, Rui

2016-01-01

The aim of the present work was to investigate the possibility of using femtosecond laser surface texturing as a method to reduce the colonization of Grade 2 Titanium alloy surfaces by Staphylococcus aureus and the subsequent formation of biofilm. The laser treatments were carried out with a Yb:KYW chirped-pulse-regenerative amplification laser system with a central wavelength of 1030 nm and a pulse duration of 500 fs. Two types of surface textures, consisting of laser-induced periodic surface structures (LIPSS) and nanopillars, were produced. The topography, chemical composition and phase constitution of these surfaces were investigated by atomic force microscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, micro-Raman spectroscopy, and X-ray diffraction. Surface wettability was assessed by the sessile drop method using water and diiodomethane as testing liquids. The response of S. aureus put into contact with the laser treated surfaces in controlled conditions was investigated by epifluorescence microscopy and scanning electron microscopy 48 h after cell seeding. The results achieved show that the laser treatment reduces significantly the bacterial adhesion to the surface as well as biofilm formation as compared to a reference polished surfaces and suggest that femtosecond laser texturing is a simple and promising method for endowing dental and orthopedic titanium implants with antibacterial properties, reducing the risk of implant-associated infections without requiring immobilized antibacterial substances, nanoparticles or coatings.

Effect of essential oils of Syzygium aromaticum and Cinnamomum zeylanicum and their major components on biofilm production in Staphylococcus aureus strains isolated from milk of cows with mastitis.

OpenAIRE

Budri, Paulo E; Silva, Nathalia CC; Bonsaglia, Erika CR; Fernandes Júnior, Ary; Araújo Júnior, Joao P; Doyama, Julio T; Gonçalves, Juliano L; Santos, M V; Fitzgerald-Hughes, Deirdre; Rall, Vera LM

2015-01-01

Bovine mastitis is an inflammation of the mammary glands of cows and causes significant economic losses in dairy cattle. Staphylococcus aureus is one of the microorganisms most commonly isolated. Novel agents are required in agricultural industries to prevent the development of mastitis. The production of biofilm by Staph. aureus facilitates the adhesion of bacteria to solid surfaces and contributes to the transmission and maintenance of these bacteria. The effect of the essential oils of Syz...

Antimicrobial activity of Lactobacillus salivarius and Lactobacillus fermentum against Staphylococcus aureus.

Science.gov (United States)

Kang, Mi-Sun; Lim, Hae-Soon; Oh, Jong-Suk; Lim, You-Jin; Wuertz-Kozak, Karin; Harro, Janette M; Shirtliff, Mark E; Achermann, Yvonne

2017-03-01

The increasing prevalence of methicillin-resistant Staphylococcus aureus has become a major public health threat. While lactobacilli were recently found useful in combating various pathogens, limited data exist on their therapeutic potential for S. aureus infections. The aim of this study was to determine whether Lactobacillus salivarius was able to produce bactericidal activities against S. aureus and to determine whether the inhibition was due to a generalized reduction in pH or due to secreted Lactobacillus product(s). We found an 8.6-log10 reduction of planktonic and a 6.3-log10 reduction of biofilm S. aureus. In contrast, the previously described anti-staphylococcal effects of L. fermentum only caused a 4.0-log10 reduction in planktonic S. aureus cells, with no effect on biofilm S. aureus cells. Killing of S. aureus was partially pH dependent, but independent of nutrient depletion. Cell-free supernatant that was pH neutralized and heat inactivated or proteinase K treated had significantly reduced killing of L. salivarius than with pH-neutralized supernatant alone. Proteomic analysis of the L. salivarius secretome identified a total of five secreted proteins including a LysM-containing peptidoglycan binding protein and a protein peptidase M23B. These proteins may represent potential novel anti-staphylococcal agents that could be effective against S. aureus biofilms. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Antibiotic tolerance and the alternative lifestyles of Staphylococcus aureus.

Science.gov (United States)

Bui, Long M G; Conlon, Brian P; Kidd, Stephen P

2017-02-28

Staphylococcus aureus has an incredible ability to survive, either by adapting to environmental conditions or defending against exogenous stress. Although there are certainly important genetic traits, in part this ability is provided by the breadth of modes of growth S. aureus can adopt. It has been proposed that while within their host, S. aureus survives host-generated and therapeutic antimicrobial stress via alternative lifestyles: a persister sub-population, through biofilm growth on host tissue or by growing as small colony variants (SCVs). Key to an understanding of chronic and relapsing S. aureus infections is determining the molecular basis for its switch to these quasi-dormant lifestyles. In a multicellular biofilm, the metabolically quiescent bacterial community additionally produces a highly protective extracellular polymeric substance (EPS). Furthermore, there are bacteria within a biofilm community that have an altered physiology potentially equivalent to persister cells. Recent studies have directly linked the cellular ATP production by persister cells as their key feature and the basis for their tolerance of a range of antibiotics. In clinical settings, SCVs of S. aureus have been observed for many years; when cultured, these cells form non-pigmented colonies and are approximately ten times smaller than their counterparts. Various genotypic factors have been identified in attempts to characterize S. aureus SCVs and different environmental stresses have been implicated as important inducers. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Single- and Multispecies Biofilms by Escherichia coli, Staphylococcus aureus, and Salmonella spp. Isolated from Raw Fish and a Fish Processing Unit

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Jesieli Braz Frozi

2017-09-01

Full Text Available ABSTRACT: The consumption of fish by the Brazilian population is increasing. However, fish and seafood are highly perishable and can be contaminated with several microorganisms. In addition, the possibility of biofilm formation is a greater cause for concern. In this study, biofilm formation was evaluated in single- and multispecies cultures at 25°C for incubation periods of 0, 4, 8, 24, and 48h in stainless steel coupons (size, 1.0×1.0cm immersed in tryptic soy broth. The characteristics of the formed biofilms after sanitizing by immersing the coupons in 200ppm sodium hypochlorite solution for 10min were also evaluated under the same experimental conditions but with some modifications. Biofilm structure was evaluated using scanning electron microscopy. Analysis of single-species biofilms indicated that all bacterial strains formed biofilms at different intervals without any statistically significant difference. However, comparison of the growth of single- and multispecies cultures indicated a significantly higher biofilm formation by the pure cultures. In multispecies biofilms, compared with the other microorganisms, growth of Salmonella spp. was significantly lower for all tested incubation periods; whereas, of Staphylococcus aureus was significantly higher than that of E. coli until 8h of incubation; the differences in growth were not significantly different after this incubation period. Sanitizing with sodium hypochlorite was effective because no cell growth was observed in the coupons that were treated with 200ppm sodium hypochlorite for 10min. This study demonstrated the ability of isolated microorganisms to form biofilms, reinforcing the need for food handling establishments to adopt good manufacturing practices, developing adequate protocols for cleaning and disinfecting surfaces and equipment used in food production, maintaining and replacing equipment when necessary.

Bacterial biofilm formation in different surfaces of food industries

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Karine Angélica Dalla Costa

2017-06-01

Full Text Available The term biofilm describes the sessile microbial life form, characterized by microorganism adhesion to any surface and with the production of extracellular polymeric substances. In food industries, the formation of biofilms results in serious problems, since it can be a contamination source of the food product, compromising the final product quality and consumer health. The aim of this study was to verify the adhesion of biofilms (sessile cells of pathogenic and/or deteriorating bacteria against surfaces of the food industry. The bacterial species tested were Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213, Listeria monocytogenes ATCC 19117 and Salmonella Typhimurium ATCC 14028. It was used stainless steel and polypropylene coupons as contact surfaces. The results demonstrated that P. aeruginosa and S. Typhimurium showed higher biofilm formation capacity. Statistically, there was no difference in count of P. aeruginosa and S. Typhimurium (p > 0.05 cells. The same occurred between L. monocytogenes and S. aureus. However, the counts of P. aeruginosa and S. Typhimurium cells were statistically higher than S. aureus and L. monocytogenes (p < 0.05. By means of scanning electron microscopy it was also found increased adhesion of P. aeruginosa. The results revealed that P. aeruginosa was the bacterial species with higher biofilm formation capacity among the others.

Host Physiologic Changes Induced by Influenza A Virus Lead to Staphylococcus aureus Biofilm Dispersion and Transition from Asymptomatic Colonization to Invasive Disease

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Ryan M. Reddinger

2016-08-01

Full Text Available Staphylococcus aureus is a ubiquitous opportunistic human pathogen and a major health concern worldwide, causing a wide variety of diseases from mild skin infections to systemic disease. S. aureus is a major source of severe secondary bacterial pneumonia after influenza A virus infection, which causes widespread morbidity and mortality. While the phenomenon of secondary bacterial pneumonia is well established, the mechanisms behind the transition from asymptomatic colonization to invasive staphylococcal disease following viral infection remains unknown. In this report, we have shown that S. aureus biofilms, grown on an upper respiratory epithelial substratum, disperse in response to host physiologic changes related to viral infection, such as febrile range temperatures, exogenous ATP, norepinephrine, and increased glucose. Mice that were colonized with S. aureus and subsequently exposed to these physiologic stimuli or influenza A virus coinfection developed pronounced pneumonia. This study provides novel insight into the transition from colonization to invasive disease, providing a better understanding of the events involved in the pathogenesis of secondary staphylococcal pneumonia.

Does Extracellular DNA Production Vary in Staphylococcal Biofilms Isolated From Infected Implants versus Controls?

Science.gov (United States)

Zatorska, Beata; Groger, Marion; Moser, Doris; Diab-Elschahawi, Magda; Lusignani, Luigi Segagni; Presterl, Elisabeth

2017-08-01

Prosthetic implant infections caused by Staphylococcus aureus and epidermidis are major challenges for early diagnosis and treatment owing to biofilm formation on the implant surface. Extracellular DNA (eDNA) is actively excreted from bacterial cells in biofilms, contributing to biofilm stability, and may offer promise in the detection or treatment of such infections. (1) Does DNA structure change during biofilm formation? (2) Are there time-dependent differences in eDNA production during biofilm formation? (3) Is there differential eDNA production between clinical and control Staphylococcal isolates? (4) Is eDNA production correlated to biofilm thickness? We investigated eDNA presence during biofilm formation in 60 clinical and 30 control isolates of S aureus and S epidermidis. The clinical isolates were isolated from patients with infections of orthopaedic prostheses and implants: 30 from infected hip prostheses and 30 from infected knee prostheses. The control isolates were taken from healthy volunteers who had not been exposed to antibiotics and a hospital environment during the previous 3 and 12 months, respectively. Control S epidermidis was isolated from the skin of the antecubital fossa, and control S aureus was isolated from the nares. For the biofilm experiments the following methods were used to detect eDNA: (1) fluorescent staining with 4',6-diamidino-2-phenylindole (DAPI), (2) eDNA extraction using a commercial kit, and (3) confocal laser scanning microscopy for 24-hour biofilm observation using propidium iodide and concanavalin-A staining; TOTO ® -1 and SYTO ® 60 staining were used for observation and quantification of eDNA after 6 and 24 hours of biofilm formation. Additionally antibiotic resistance was described. eDNA production as observed by confocal laser scanning microscopy was greater in clinical isolates than controls (clinical isolates mean ± SD: 1.84% ± 1.31%; control mean ± SD: 1.17% ± 1.37%; p biofilm formation. After 24 hours, the

Lipopeptide biosurfactants from Paenibacillus polymyxa inhibit single and mixed species biofilms.

Science.gov (United States)

Quinn, Gerry A; Maloy, Aaron P; McClean, Stephen; Carney, Brian; Slater, John W

2012-01-01

Although biofilms are recognised as important in microbial colonisation, solutions to their inhibition are predominantly based on planktonic assays. These solutions have limited efficacy against biofilms. Here, a series of biofilm-orientated tests were used to identify anti-biofilm compounds from marine micro-flora. This led to the isolation of a complex of anti-biofilm compounds from an extract of Paenibacillus polymyxa (PPE). A combination of rpHPLC and mass spectrometry identified the principle components of PPE as fusaricidin B (LI-FO4b) and polymyxin D1, with minor contributions from surfactins. This complex (PPE) reduced the biofilm biomass of Bacillus subtilis, Micrococcus luteus, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus bovis. In contrast, ampicillin was only effective against S. aureus. PPE also inhibited a self-assembling marine biofilm (SAMB) in co-incubation assays by 99.3% ± 1.9 and disrupted established SAMB by 72.4% ± 4.4, while ampicillin showed no significant reduction. The effectiveness of this complex of lipopeptides against single and multispecies biofilms suggests a future role in biofilm prevention strategies.

Effect of Carvacrol on Salmonella Saintpaul Biofilms on Stainless ...

African Journals Online (AJOL)

2025 ... carvacrol on S. saintpaul biofilms on stainless steel surface was evaluated on ... cultures S. saintpaul at 35 ºC were diluted 1:100 .... characteristics of biofilm formation that occur in .... aureus and Salmonella enterica serovar Typhmurium.

Biofilm architecture in a novel pressurized biofilm reactor.

Science.gov (United States)

Jiang, Wei; Xia, Siqing; Duan, Liang; Hermanowicz, Slawomir W

2015-01-01

A novel pure-oxygen pressurized biofilm reactor was operated at different organic loading, mechanical shear and hydrodynamic conditions to understand the relationships between biofilm architecture and its operation. The ultimate goal was to improve the performance of the biofilm reactor. The biofilm was labeled with seven stains and observed with confocal laser scanning microscopy. Unusual biofilm architecture of a ribbon embedded between two surfaces with very few points of attachment was observed. As organic loading increased, the biofilm morphology changed from a moderately rough layer into a locally smoother biomass with significant bulging protuberances, although the chemical oxygen demand (COD) removal efficiency remained unchanged at about 75%. At higher organic loadings, biofilms contained a larger fraction of active cells distributed uniformly within a proteinaceous matrix with decreasing polysaccharide content. Higher hydrodynamic shear in combination with high organic loading resulted in the collapse of biofilm structure and a substantial decrease in reactor performance (a COD removal of 16%). Moreover, the important role of proteins for the spatial distribution of active cells was demonstrated quantitatively.

Antibacterial effect of the laser-generated Se nanocoatings on Staphylococcus aureus and Pseudomonas aeruginosa biofilms

Science.gov (United States)

Ionin, A. A.; Ivanova, A. K.; Khmel'nitskii, R. A.; Klevkov, Yu V.; Kudryashov, S. I.; Levchenko, A. O.; Nastulyavichus, A. A.; Rudenko, A. A.; Saraeva, I. N.; Smirnov, N. A.; Zayarny, D. A.; Gonchukov, S. A.; Tolordava, E. R.

2018-01-01

The antibacterial properties of selenium nanoparticles (Se NPs) were successfully demonstrated in vitro for Staphylococcus aureus and Pseudomonas aeruginosa biofilms. The possible mechanisms of antibacterial impact included the emergence of reactive oxygen species, induced by free radicals on the NP surface and accompanied by subsequent oxidative stress, as well as mechanical decomposition of the mitochondrial membrane. Se nanocoatings were deposited on bare and silver-coated silica glass substrates via inkjet printing with concentrated nanoinks, prepared by infrared laser-ablative processing of a solid Se target in a 50%-isopropyl solution. The resulted porous nanofilms with high-percentage surface coverage, consisting of spherical Se NPs and Se nanorods, were characterized by means of standard microscopy techniques (optical, scanning electron, transmission), UV-vis-IR and EDX spectroscopy.

Propolis-Sahara honeys preparation exhibits antibacterial and anti-biofilm activity against bacterial biofims formed on urinary catheters

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Saad Aissat

2016-11-01

Full Text Available Objective: To evaluate the antibacterial effect of Sahara honeys (SHs against bacterial biofilms formed on urinary catheters in combination with propolis-Sahara honeys (P-SHs. Methods: Three clinical isolates were subjected to biofilm detection methods. The antibacterial and anti-biofilm activity for SHs and P-SHs were determined using agar well diffusion and the percentage of biofilm inhibition (PBI methods. Results: The PBI for Gram-positive bacteria [Staphylococcus aureus (S. aureus] was in the range of 0%–20%, while PBI for Gram-negative bacteria [Pseudomonas aeruginosa and Escherichia coli (E. coli] were in range of 17%–57% and 16%–65%, respectively. The highest PBI (65% was produced by SH2 only on E. coli. In agar well diffusion assay, zones of inhibition ranged from 11–20 mm (S. aureus, 9–19 mm (Pseudomonas aeruginosa and 11–19 mm (E. coli. The highest inhibition (20 mm was produced by SH1 only on S. aureus. In addition, the treatment of SHs and P-SHs catheters with a polymicrobial biofilms reduced biofilm formation after 48 h exposure period. Conclussions: SHs and P-SHs applied as a natural agent can be used as a prophylactic agent to prevent the formation of in vitro biofilm.

Antibacterial and anti-biofilm activity of ginger (Zingiber officinale (Roscoe ethanolic extract

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Nikolić Miloš

2014-01-01

Full Text Available The antibacterial and anti-biofilm activity of ethanolic extract from the rhizome of Zingiber officinale were evaluated. In vitro antibacterial activity was investigated by microdilution method. Minimum inhibitory concentration (MIC and minimum bactericidal concentration (MBC have been determined. The values were in the range from 0.0024 to > 20 mg/ml. The most sensitive bacteria were Gram-positive bacteria: Staphylococcus aureus and Staphylococcus aureus ATCC 25923. Anti-biofilm activity was tested by crystal violet assay. Pseudomonas aeruginosa ATCC 27853, Proteus mirabilis and Escherichia coli ATCC 25922 were used as the test organisms. Ethanolic extract showed the best result on Proteus mirabilis biofilm where biofilm inhibitory concentration (BIC50 was 19 mg/ml.

Bovine mastitis: prevalence and antimicrobial susceptibility profile and detection of genes associated with biofilm formation in Staphylococcus aureus

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Valeska Paula Casanova

2016-06-01

Full Text Available Brazil currently ranks as one of the world leaders in food production and exportation. This scenario encourages the development of animal and plant health programs to ensure the production of safe food, helping the country to become an international provider of food for excellence. However, some health problems in dairy production, such as mastitis, have garnered increasing concern. This study aimed to estimate the prevalence of bovine mastitis in select properties located in the western Santa Catarina region, to assess the susceptibility profile to antimicrobial agents used for treatment and to check for the presence of genes (icaA and icaD associated with biofilm formation in Staphylococcus aureus. In 148 milk samples collected, 72.97% had bacterial growth (n = 108. Among the isolated microorganisms, 21.62% (n = 32 were classified as Staphylococcus aureus, 18.91% (n = 28 as Staphylococcus sp. coagulase negative, 7.43% (n = 11 as Corynebacterium sp., 6.76% (n = 10 as Staphylococcus sp. coagulase positive, 5.41% (n = 8 as Nocardia sp. and 12.83% (n = 19 classified in different bacterial genera. Among the isolates submitted for antimicrobial susceptibility testing, it was observed that 8.95% (n = 6/67 had resistance to amoxicillin, 8.04% (n = 7/87 to ampicillin, 5.88% (n = 5/85 to cephalothin, 3.40% (n = 3/88 to ceftiofur and enrofloxacin, 20.45% (n = 18/88 to streptomycin, 17.04% (n = 15/88 to gentamicin and lincomycin, 31.81% (n = 28/88 to neomycin, 14.94% (n = 13/87 to penicillin and 25% (n = 22/88 to tetracycline. Staphylococcus sp. coagulase negative isolates showed higher multidrug resistance when compared to those of S. aureus and Staphylococcus sp. coagulase positive. Thirty-one strains of S. aureus isolates were genotypically tested by polymerase chain reaction (PCR, yielding a positive result for the icaA gene in 83.87% of the samples, 80.64% positive for icaD and 74.19% of these showed both genes. The results reinforce the importance

Effects of Fermented Sumach on the Formation of Slime Layer of Staphylococcus aureus

OpenAIRE

Kırmusaoğlu, Sahra; Yurdugül, Seyhun; Koçoğlu, Esra

2012-01-01

Objective: Staphylococcus aureus (S. aureus) is one of the most commonly isolated bacterial pathogens in hospitals, and the most frequent cause of nosocomial infections. Nosocomial staphylococcal foreign-body infections related to biofilm formation are a serious threat, demanding new therapeutic and preventive strategies. Implantation of intravenous catheters and surgical implantation of prosthetic implants carry a risk of infection. In order to prevent all these effects of biofilms, a study ...

Direct analysis of bacterial viability in endotracheal tube biofilm from a pig model of methicillin-resistant Staphylococcus aureus pneumonia following antimicrobial therapy.

Science.gov (United States)

Fernández-Barat, Laia; Li Bassi, Gianluigi; Ferrer, Miquel; Bosch, Anna; Calvo, Maria; Vila, Jordi; Gabarrús, Albert; Martínez-Olondris, Pilar; Rigol, Montse; Esperatti, Mariano; Luque, Néstor; Torres, Antoni

2012-07-01

Confocal laser scanning microscopy (CLSM) helps to observe the biofilms formed in the endotracheal tube (ETT) of ventilated subjects and to determine its structure and bacterial viability using specific dyes. We compared the effect of three different treatments (placebo, linezolid, and vancomycin) on the bacterial biofilm viability captured by CLSM. Eight pigs with pneumonia induced by methicillin-resistant Staphylococcus aureus (MRSA) were ventilated up to 96 h and treated with linezolid, vancomycin, or placebo (controls). ETT images were microscopically examined after staining with the live/dead(®) BacLight(™) Kit (Invitrogen, Barcelona, Spain) with a confocal laser scanning microscope. We analyzed 127 images obtained by CLSM. The median ratio of live/dead bacteria was 0.51, 0.74, and 1 for the linezolid, vancomycin, and control groups, respectively (P = 0.002 for the three groups); this ratio was significantly lower for the linezolid group, compared with the control group (P = 0.001). Images showed bacterial biofilm attached and non-attached to the ETT surface but growing within secretions accumulated inside ETT. Systemic treatment with linezolid is associated with a higher proportion of dead bacteria in the ETT biofilm of animals with MRSA pneumonia. Biofilm clusters not necessarily attach to the ETT surface. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Staphylococcus aureus β-Toxin Mutants Are Defective in Biofilm Ligase and Sphingomyelinase Activity, and Causation of Infective Endocarditis and Sepsis.

Science.gov (United States)

Herrera, Alfa; Vu, Bao G; Stach, Christopher S; Merriman, Joseph A; Horswill, Alexander R; Salgado-Pabón, Wilmara; Schlievert, Patrick M

2016-05-03

β-Toxin is an important virulence factor of Staphylococcus aureus, contributing to colonization and development of disease [Salgado-Pabon, W., et al. (2014) J. Infect. Dis. 210, 784-792; Huseby, M. J., et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 14407-14412; Katayama, Y., et al. (2013) J. Bacteriol. 195, 1194-1203]. This cytotoxin has two distinct mechanisms of action: sphingomyelinase activity and DNA biofilm ligase activity. However, the distinct mechanism that is most important for its role in infective endocarditis is unknown. We characterized the active site of β-toxin DNA biofilm ligase activity by examining deficiencies in site-directed mutants through in vitro DNA precipitation and biofilm formation assays. Possible conformational changes in mutant structure compared to that of wild-type toxin were assessed preliminarily by trypsin digestion analysis, retention of sphingomyelinase activity, and predicted structures based on the native toxin structure. We addressed the contribution of each mechanism of action to producing infective endocarditis and sepsis in vivo in a rabbit model. The H289N β-toxin mutant, lacking sphingomyelinase activity, exhibited lower sepsis lethality and infective endocarditis vegetation formation compared to those of the wild-type toxin. β-Toxin mutants with disrupted biofilm ligase activity did not exhibit decreased sepsis lethality but were deficient in infective endocarditis vegetation formation compared to the wild-type protein. Our study begins to characterize the DNA biofilm ligase active site of β-toxin and suggests β-toxin functions importantly in infective endocarditis through both of its mechanisms of action.

Investigation of biofilm forming ability in Staphylococci causing bovine mastitis using phenotypic and genotypic assays.

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Darwish, Samah F; Asfour, Hanaa A E

2013-01-01

A total of 40 S. aureus and 68 coagulase negative Staphylococcus (CNS) isolates from bovine subclinical mastitis were investigated for their ability to form biofilm as one of the most important virulence factors.Using Congo Red Agar (CRA) method, 32.5%, 35%, and 32.5% of S. aureus strains were strong, intermediate, and negative biofilm producers, while in CNS the percentages were 29.5%, 42.6%, and 27.9%, respectively. By microtiter plate (MTP) method, 52.5%, 27.5%, and 20% of S. aureus isolates were strong, moderate, and weak biofilm producers, while in CNS the percentages were 44%, 30.9%, and 19.2%, respectively. Indian ink staining was used to detect the EPS layer of biofilm producers. All isolates were screened for presence of biofilm related genes, eno, icaA, icaD, and bap. In S. aureus isolates, the positive rates of eno, icaA, icaD, and bap genes were 75%, 15%, 62.5%, and 2.5% while in CNS were 92.6%, 5.9%, 47.1%, and 4.4%, respectively. The eno gene had the highest rate while the bap gene had the lowest rate. Presence of icaA and icaD genes was not always correlated with biofilm production. This study demonstrated high prevalence of Staphylococcus biofilm producers among bovine mastitis in Egypt. Therefore, attention must be paid toward implementation of new ways for effective treatment of such infections.

Investigation of Biofilm Forming Ability in Staphylococci Causing Bovine Mastitis Using Phenotypic and Genotypic Assays

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Samah F. Darwish

2013-01-01

Full Text Available A total of 40 S. aureus and 68 coagulase negative Staphylococcus (CNS isolates from bovine subclinical mastitis were investigated for their ability to form biofilm as one of the most important virulence factors.Using Congo Red Agar (CRA method, 32.5%, 35%, and 32.5% of S. aureus strains were strong, intermediate, and negative biofilm producers, while in CNS the percentages were 29.5%, 42.6%, and 27.9%, respectively. By microtiter plate (MTP method, 52.5%, 27.5%, and 20% of S. aureus isolates were strong, moderate, and weak biofilm producers, while in CNS the percentages were 44%, 30.9%, and 19.2%, respectively. Indian ink staining was used to detect the EPS layer of biofilm producers. All isolates were screened for presence of biofilm related genes, eno, icaA, icaD, and bap. In S. aureus isolates, the positive rates of eno, icaA, icaD, and bap genes were 75%, 15%, 62.5%, and 2.5% while in CNS were 92.6%, 5.9%, 47.1%, and 4.4%, respectively. The eno gene had the highest rate while the bap gene had the lowest rate. Presence of icaA and icaD genes was not always correlated with biofilm production. This study demonstrated high prevalence of Staphylococcus biofilm producers among bovine mastitis in Egypt. Therefore, attention must be paid toward implementation of new ways for effective treatment of such infections.

Natural antimicrobial peptide complexes in the fighting of antibiotic resistant biofilms: Calliphora vicina medicinal maggots.

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Natalia Gordya

Full Text Available Biofilms, sedimented microbial communities embedded in a biopolymer matrix cause vast majority of human bacterial infections and many severe complications such as chronic inflammatory diseases and cancer. Biofilms' resistance to the host immunity and antibiotics makes this kind of infection particularly intractable. Antimicrobial peptides (AMPs are a ubiquitous facet of innate immunity in animals. However, AMPs activity was studied mainly on planktonic bacteria and little is known about their effects on biofilms. We studied structure and anti-biofilm activity of AMP complex produced by the maggots of blowfly Calliphora vicina living in environments extremely contaminated by biofilm-forming germs. The complex exhibits strong cell killing and matrix destroying activity against human pathogenic antibiotic resistant Escherichia coli, Staphylococcus aureus and Acinetobacter baumannii biofilms as well as non-toxicity to human immune cells. The complex was found to contain AMPs from defensin, cecropin, diptericin and proline-rich peptide families simultaneously expressed in response to bacterial infection and encoded by hundreds mRNA isoforms. All the families combine cell killing and matrix destruction mechanisms, but the ratio of these effects and antibacterial activity spectrum are specific to each family. These molecules dramatically extend the list of known anti-biofilm AMPs. However, pharmacological development of the complex as a whole can provide significant advantages compared with a conventional one-component approach. In particular, a similar level of activity against biofilm and planktonic bacteria (MBEC/MIC ratio provides the complex advantage over conventional antibiotics. Available methods of the complex in situ and in vitro biosynthesis make this idea practicable.

Natural antimicrobial peptide complexes in the fighting of antibiotic resistant biofilms: Calliphora vicina medicinal maggots.

Science.gov (United States)

Gordya, Natalia; Yakovlev, Andrey; Kruglikova, Anastasia; Tulin, Dmitry; Potolitsina, Evdokia; Suborova, Tatyana; Bordo, Domenico; Rosano, Camillo; Chernysh, Sergey

2017-01-01

Biofilms, sedimented microbial communities embedded in a biopolymer matrix cause vast majority of human bacterial infections and many severe complications such as chronic inflammatory diseases and cancer. Biofilms' resistance to the host immunity and antibiotics makes this kind of infection particularly intractable. Antimicrobial peptides (AMPs) are a ubiquitous facet of innate immunity in animals. However, AMPs activity was studied mainly on planktonic bacteria and little is known about their effects on biofilms. We studied structure and anti-biofilm activity of AMP complex produced by the maggots of blowfly Calliphora vicina living in environments extremely contaminated by biofilm-forming germs. The complex exhibits strong cell killing and matrix destroying activity against human pathogenic antibiotic resistant Escherichia coli, Staphylococcus aureus and Acinetobacter baumannii biofilms as well as non-toxicity to human immune cells. The complex was found to contain AMPs from defensin, cecropin, diptericin and proline-rich peptide families simultaneously expressed in response to bacterial infection and encoded by hundreds mRNA isoforms. All the families combine cell killing and matrix destruction mechanisms, but the ratio of these effects and antibacterial activity spectrum are specific to each family. These molecules dramatically extend the list of known anti-biofilm AMPs. However, pharmacological development of the complex as a whole can provide significant advantages compared with a conventional one-component approach. In particular, a similar level of activity against biofilm and planktonic bacteria (MBEC/MIC ratio) provides the complex advantage over conventional antibiotics. Available methods of the complex in situ and in vitro biosynthesis make this idea practicable.

Growing and analyzing biofilms in flow chambers

DEFF Research Database (Denmark)

Tolker-Nielsen, Tim; Sternberg, Claus

2011-01-01

This unit describes the setup of flow chamber systems for the study of microbial biofilms, and methods for the analysis of structural biofilm formation. Use of flow chambers allows direct microscopic investigation of biofilm formation. The biofilms in flow chambers develop under hydrodynamic......, and disassembly and cleaning of the system. In addition, embedding and fluorescent in situ hybridization of flow chamber-grown biofilms are addressed....

Evaluation of biofilm formation by bacterial strains isolated from milking equipment and milk samples from cows with mastitis

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Laura Gonçalves da Silva Chagas

2017-08-01

Full Text Available The presence of biofilm-forming bacteria from the mammary gland of dairy cows adhered to equipment in the milking environment represents one of the major causes of bacterial resistance during mastitis treatment. The aim of this study was to identify strains of Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli in milk samples from cows with mastitis, as well as in the expansion tank and milking set liners. We aimed to quantify the extracellular proteins and polysaccharides in the biofilm produced by each strain. A total of 294 samples were collected from a dairy farm in the municipality of Uberlândia, Minas Gerais. To identify the S. aureus, S. epidermidis and E. coli isolates responsible for biofilm production, we tested the phenotype using the Congo red agar (CRA and microplate adhesion tests. Protein quantification was performed with a Bicinchoninic Acid Protein Assay Kit (BCA kit, and polysaccharides were quantified by the phenol sulfuric acid method. We identified eight strains of S. aureus, one strain of S. epidermidis and 11 strains of E. coli responsible for biofilm production, all of which showed a higher concentration of polysaccharides than proteins in the matrix. Escherichia coli was considered the most prevalent bacterium among the samples, and S. aureus was determined to be the largest biofilm producer. The results of the CRA and microplate adhesion tests were similar in regard to identification of the biofilm-producing strains according to their phenotype and matrix composition. The classification of S. aureus strains as major biofilm producers is of great concern for producers, as such bacteria are considered one of the predominant contagious etiological agents that cause bovine mastitis. In addition, our observation that E. coli and S. epidermidis can produce biofilms highlights the need to reassess prophylactic measures to avoid the adhesion of biofilm-producing bacteria.

DETECTION OF BIOFILM PRODUCTION IN BLOOD CULTURE ISOLATES OF STAPHYLOCOCCI

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Gupta Puja, Gupta Pratima, Mittal Garima, Agarwal RK, Goyal Rohit

2015-01-01

Full Text Available Background: Biofilm producing bacteria which are inherently resistant to antibiotics and disinfectants are widely associated with implant associated infections. Staphylococcus is the most commonly associated pathogens with bloodstream infection. Aims: The current study was conducted to detect biofilm production in Staphylococci isolated from blood culture specimens. Materials and Methods: 70 clinically significant staphylococcal isolates from blood culture were screened for biofilm production by Tissue culture plate (TCP method, Tube method (TM and Congo red agar (CRA method and their antibiotic susceptibility profile was studied. Results: 59 out of 70 staphylococcal isolates were positive by TCP, out of these 21.4% staphylococci were high biofilm producers, 62.8% staphylococci were moderate biofilm producers and 15.8% were non-biofilm producers. Maximum resistance was observed in biofilm producers to cotrimoxazole (74.5% and erythromycin (62.7% and none were resistant to vancomycin and linezolid. Out of total 59 biofilm producers, 20.3 % (12 were methicillin resistant and all these were S. aureus isolates. 19% (1 out of total 11 biofilm non-producers were methicillin resistant. Conclusion: Biofilm production was seen to be a major virulence factor in most of the staphylococcal isolates obtained from patients with signs and symptoms of septicaemia. S. aureus was found to be the major pathogen and timely detection of biofilm producing phenotype should be carried out using a simple and reproducible method, TCP which is both qualitative and quantitative.

Conjugation of Inulin Improves Anti-Biofilm Activity of Chitosan

OpenAIRE

Guiqiang Zhang; Jing Liu; Ruilian Li; Siming Jiao; Cui Feng; Zhuo A. Wang; Yuguang Du

2018-01-01

Bacteria biofilm helps bacteria prevent phagocytosis during infection and increase resistance to antibiotics. Staphylococcus aureus is a Gram-positive pathogenic bacterium and is tightly associated with biofilm-related infections, which have led to great threat to human health. Chitosan, the only cationic polysaccharide in nature, has been demonstrated to have antimicrobial and anti-biofilm activities, which, however, require a relative high dosage of chitosan. Moreover, poor water solubility...

Anti-Biofilm Efficacy of Nitric Oxide-Releasing Silica Nanoparticles

OpenAIRE

Hetrick, Evan M.; Shin, Jae Ho; Paul, Heather S.; Schoenfisch, Mark H.

2009-01-01

The ability of nitric oxide (NO)-releasing silica nanoparticles to kill biofilm-based microbial cells is reported. Biofilms of Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Candida albicans were formed in vitro and exposed to NO-releasing silica nanoparticles. Replicative viability experiments revealed that ≥ 99% of cells from each type of biofilm were killed via NO release, with the greatest efficacy (≥ 99.999% killing) against gram-negative...

Fremmedlegemeinfektioner--nyt om biofilm og quorum sensing

DEFF Research Database (Denmark)

Høiby, Niels; Johansen, Helle Krogh; Ciofu, Oana

2007-01-01

Biofilms are structured consortia of bacteria embedded in self-produced polymer matrix. Biofilms are resistant to antibiotics, disinfectives and phagocytosis. The persistence of foreign body infections is due to biofilms. Chronic P. aeruginosa lung infection in cystic fibrosis patients is a biofilm....... 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....

Biofilm Formation by Bacteria Isolated from Intravenous Catheters

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Sina Hedayati

2015-10-01

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.

Anti-infective properties of Lactobacillus fermentum against Staphylococcus aureus and Pseudomonas aeruginosa.

Science.gov (United States)

Varma, Parvathi; Nisha, N; Dinesh, Kavitha R; Kumar, Anil V; Biswas, Raja

2011-01-01

Surgical wounds and implant-associated Staphylococcus aureus and Pseudomonas aeruginosa infections are often difficult to treat because of limited susceptibility of several of these strains to conventional antibiotics. As a result, there is a constant need for new alternative drugs. The aim of this study was to investigate the antimicrobial properties of Lactobacillus fermentum, a probiotic bacterium, which we have isolated from colonic biopsies. The inhibition of S. aureus and P. aeruginosa growth was evaluated by coincubating with L. fermentum strains. Growth inhibition was tested for several of their clinical isolates using agar well diffusion assays. For biofilm assay S. aureus and P. aeruginosa were grown on the glass slides and in 96-well plates in presence of 2.5 μg/ml culture filtrate of L. fermentum. Biofilms were photographed using confocal microscope or stained with 0.1% crystal violet. Reduction in the cytotoxicity of S. aureus and P. aeruginosa was observed in presence of 2.5 μg/ml L. fermentum-spent media. Using in vitroexperiments, we showed that L. fermentum-secreted compound(s) inhibits the growth, cytotoxicity and biofilm formation of several S. aureus and P. aeruginosa strains. Compound(s) present in the culture supernatant of L. fermentum may have promising applications in treating hospital-acquired infections. Copyright © 2011 S. Karger AG, Basel.

New quantitative image analysis of staphylococcal biofilms on the surfaces of nontranslucent metallic biomaterials.

Science.gov (United States)

Adachi, Kouichi; Tsurumoto, Toshiyuki; Yonekura, Akihiko; Nishimura, Seisuke; Kajiyama, Shiro; Hirakata, Yoichi; Shindo, Hiroyuki

2007-03-01

Implant-related infection after orthopedic surgery is difficult to cure. One of the causes of infection is the bacterial biofilm that forms around biomaterials used during surgery. Therefore, it is necessary to investigate bacterial biofilms extensively to resolve the problems of these postoperative infections. However, no established culture method or quantification system exists for bacterial biofilms grown on the surface of the metallic biomaterials used in orthopedics, which are nonradiolucent. The purpose of this study was to develop a quantitative method to evaluate the difference in resistance of stainless steel versus titanium to staphylococcal biofilms and the efficacy of antibiotics against biofilms. The bacterial strains used in this study were three Staphylococcus aureus stains: strain Seattle 1945 and two clinical strains cultured from postoperative infections. Staphylococcal biofilms were formed on stainless steel washers (SUS304) and titanium washers (pure titanium). They were stained with crystal violet and were examined with a digital microscope to calculate the bacterial coverage rate (BCR) by NIH imaging. The BCR of S. aureus biofilms formed on stainless steel and titanium washers increased over time. At 24, 48, and 72 h after cultivation, the amount of biofilm on the surface of the stainless steel washers was significantly greater or tended to be greater than that on the titanium. Cefazolin was applied to the obtained biofilms of two clinically isolated S. aureus strains. Cefazolin did not eradicate the biofilms but significantly reduced the biofilm of one strain. The newly developed quantitative method (static microtube culture and measurement system) was useful for assessing the amount of bacterial biofilms on the surface of nontranslucent biomaterial. We found that titanium may be more resistant to bacterial infection than stainless steel. To control implant-related severe infections, the biomaterials should be assessed from the viewpoint of

Experimental model of biofilm implant-related osteomyelitis to test combination biomaterials using biofilms as initial inocula.

Science.gov (United States)

Williams, Dustin L; Haymond, Bryan S; Woodbury, Kassie L; Beck, J Peter; Moore, David E; Epperson, R Tyler; Bloebaum, Roy D

2012-07-01

Currently, the majority of animal models that are used to study biofilm-related infections use planktonic bacterial cells as initial inocula to produce positive signals of infection in biomaterials studies. However, the use of planktonic cells has potentially led to inconsistent results in infection outcomes. In this study, well-established biofilms of methicillin-resistant Staphylococcus aureus were grown and used as initial inocula in an animal model of a Type IIIB open fracture. The goal of the work was to establish, for the first time, a repeatable model of biofilm implant-related osteomyelitis, wherein biofilms were used as initial inocula to test combination biomaterials. Results showed that 100% of animals that were treated with biofilms developed osteomyelitis, whereas 0% of animals not treated with biofilm developed infection. The development of this experimental model may lead to an important shift in biofilm and biomaterials research by showing that when biofilms are used as initial inocula, they may provide additional insights into how biofilm-related infections in the clinic develop and how they can be treated with combination biomaterials to eradicate and/or prevent biofilm formation. Copyright © 2012 Wiley Periodicals, Inc.

Fremmedlegemeinfektioner--nyt om biofilm og quorum sensing

DEFF Research Database (Denmark)

Høiby, Niels; Johansen, Helle Krogh; Ciofu, Oana

2007-01-01

Biofilms are structured consortia of bacteria embedded in self-produced polymer matrix. Biofilms are resistant to antibiotics, disinfectives and phagocytosis. The persistence of foreign body infections is due to biofilms. Chronic P. aeruginosa lung infection in cystic fibrosis patients is a biofilm....... 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...

Effect of biocides on biofilms of some multidrug resistant clinical ...

African Journals Online (AJOL)

The ability of Escherichia coli and Klebsiella aerogenes to form biofilms was most affected. There was little inhibition of biofilm formation by the biocides on Staphylococcus aureus. This study has shown a relationship between biocide and multidrug resistance. Keywords: Biocides, Multi drug resistance, sodium hypochlorite, ...

Chemoinformatics-assisted development of new anti-biofilm compounds

DEFF Research Database (Denmark)

Dürig, Anna; Kouskoumvekaki, Irene; Vejborg, Rebecca Munk

2010-01-01

Bacterial biofilms are associated with a large number of infections. Biofilm-dwelling bacteria are particularly resistant to antibiotics, making it hard to eradicate biofilm-associated infections. Here, we use a novel cross-disciplinary approach combining microbiology and chemoinformatics...... to identify new and efficient anti-biofilm drugs. We found that ellagic acid (present in green tea) significantly inhibited biofilm formation of Streptococcus dysgalactiae. Based on ellagic acid, we performed in silico screening of the Chinese Natural Product Database to predict a 2nd-generation list...... of compounds with similar characteristics. One of these, esculetin, proved to be more efficient in preventing biofilm formation by Staphylococcus aureus. From esculetin a 3rd-generation list of compounds was predicted. One of them, fisetin, was even better to abolish biofilm formation than the two parent...

N-halamine-based rechargeable antimicrobial and biofilm-controlling polyurethane

Science.gov (United States)

Sun, Xinbo; Cao, Zhengbing; Porteous, Nuala; Sun, Yuyu

2012-01-01

An N-halamine precursor, 5, 5-dimethyl hydantoin (DMH), was covalently linked to the surface of polyurethane (PU) with 1,6-hexamethylene diisocyanate (HDI) as a coupling agent. The reaction pathways were investigated using propyl isocyanate (PI) as a model compound, and the results suggested that the imide and amide groups of DMH had very similar reactivity toward the isocyanate groups on PU surfaces activated with HDI. After bleach treatment, the covalently bound DMH moieties were transformed into N-halamines. The new N-halmaine-based PU provided potent antimicrobial effects against Staphylococcus aureus (S. aureus, Gram-positive), Escherichia coli (E. coli, Gram-negative), methicillin-resistant staphylococcus aureus (MRSA, drug resistant Gram-positive bacteria), vancomycin-resistant enterococcus (VRE, drug resistant Gram-positive bacteria), and Candida albicans (C. ablicans, fungi), and successfully prevented bacterial and fungal biofilm formation. The antimicrobial and biofilm-controlling effects were stable for longer than 6 months under normal storage in open air. Furthermore, if the functions were lost due to prolonged use, they could be recharged by another chlorination treatment. The recharging could be repeated as needed to achieve long-term protection against microbial contamination and biofilm-formation. PMID:22244984

[Formation of microbial biofilms in causative agents of acute and chronic pyelonephritis].

Science.gov (United States)

Lagun, L V; Atanasova, Iu V; Tapal'skiĭ, D V

2013-01-01

Study the intensity of formation of microbial biofilms by Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus strains isolated during various forms of pyelonephritis. 150 clinical isolates of microorganisms isolated from urine ofpatientswith acute and chronic pyelonephritiswere included into the study. Determination of intensity of film-formation was carried out by staining of the formed biofilms by crystal violet with consequent extraction of the dye and measurement of its concentration in washout solution. Among causative agents ofpyelonephritis P. aeruginosa isolates had the maximum film-forming ability. The intensity of biofilm formation of these isolates was 2-3 time higher than staphylococcus and enterobacteria strains. Strains isolated from patients with chronic pyelonephritis by ability to form biofilms significantly surpassed strains isolated from acute pyelonephritis patients. A higher ability to form microbial biofilms for microorganisms--causative agents of pyelonephritis progressing against the background ofurolithiasis was noted. The ability to form biofilms is determined by both causative agent species and character of the infectious process in which this microorganism participates. Intensive formation of biofilms by E. coli, P. aeruginosa, K. pneumoniae, S. aureus clinical isolates may be an important factor of chronization of urinary tract infections.

Biofilm inhibition activity of compounds isolated from two Eunicea species collected at the Caribbean Sea

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Yenny Martínez Díaz

Full Text Available Abstract Biofilm has a primary role in the pathogenesis of diseases and in the attachment of multicellular organisms to a fouled surface. Because of that, the control of bacterial biofilms has been identified as an important target. In the present study, five lipid compounds isolated from soft coral Eunicea sp. and three terpenoids together with a mixture of sterols from Eunicea fusca collected at the Colombian Caribbean Sea showed different effectiveness against biofilm formation by three marine bacteria associated with immersed fouled surfaces, Ochrobactrum pseudogringnonense,Alteromona macleodii and Vibrio harveyi, and against two known biofilm forming bacteria, Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 25923. The pure compounds were characterized by NMR, HRESI-MS, HRGC-MS and optical rotation. The most effective compounds were batyl alcohol (1 and fuscoside E peracetate (6, acting against four strains without affecting their microbial growth. Compound 1 showed biofilm inhibition greater than 30% against A. macleodii, and up to 60% against O. pseudogringnonense,V. harveyi and S. aureus. Compound 6 inhibited O. pseudogringnonense and V. harveyi between 25 and 50%, and P. aeruginosa or S. aureus up to 60% at 0.5 mg/ml. The results suggest that these compounds exhibit specific biofilm inhibition with lower antimicrobial effect against the bacterial species assayed.

Spreading of genes encoding enterotoxins, haemolysins, adhesin and biofilm among methicillin resistant Staphylococcus aureus strains with staphylococcal cassette chromosome mec type IIIA isolated from burn patients.

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Motallebi, Mitra; Jabalameli, Fereshteh; Asadollahi, Kheirollah; Taherikalani, Morovat; Emaneini, Mohammad

2016-08-01

The emergence of antibiotic-resistant Staphylococcus aureus in particular methicillin-resistant S. aureus (MRSA) is an important concern in burn medical centers either in Iran or worldwide. A total of 128 S. aureus isolates were collected from wound infection of burn patients during June 2013 to June 2014. Multiplex-polymerase chain reaction (MPCR) assay was performed for the characterization of the staphylococcal cassette chromosome mec (SCCmec). Genes encoding virulence factors and biofilm were targeted by PCR. Of 128 S. aureus isolates, 77 (60.1%) isolates were MRSA. Fifty four (70.1%) isolates were identified as SCCmec type IIIA. The most frequently detected toxin genes among MRSA isolates with SCCmec type IIIA were sea (64.1%) and hla (51.8%). The rate of coexistence of sea with hla and sea with hla and hlb was 37% and12.9%, respectively. The sec, eta, tst, pvl, hla and hlb genes were not detected in any of the MRSA isolates. The most prevalent genes encoding biofilm was eno, found in 61.1% of isolates, followed by fib and icaA found in 48.1% and 38.8% of the isolates, respectively. The rate of coexistence of fib + eno + icaA + icaD and fib + eno was 20.3% and 9.2%, respectively. The ebps gene was not detected in any of the isolates. In conclusion, our study indicated that the sea, hla, fib and icaA were most frequent genes encoding virulence factors among MRSA with SCCmec type IIIA isolated from burn wound infection. Moreover, the results of this study shows that the rate of coexistence of genes encoding different virulence factor were high. Copyright © 2016 Elsevier Ltd. All rights reserved.

Host Proteins Determine MRSA Biofilm Structure and Integrity

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Dreier, Cindy; Nielsen, Astrid; Jørgensen, Nis Pedersen

Human extracellular matrix (hECM) proteins aids the initial attachment and initiation of an infection, by specific binding to bacterial cell surface proteins. However, the importance of hECM proteins in structure, integrity and antibiotic resilience of a biofilm is unknown. This study aims...... to determine how specific hECM proteins affect S. aureus USA300 JE2 biofilms. Biofilms were grown in the presence of synovial fluid from rheumatoid arteritis patients to mimic in vivo conditions, where bacteria incorporate hECM proteins into the biofilm matrix. Difference in biofilm structure, with and without...... addition of hECM to growth media, was visualized by confocal laser scanning microscopy. Two enzymatic degradation experiments were used to study biofilm matrix composition and importance of hECM proteins: enzymatic removal of specific hECM proteins from growth media, before biofilm formation, and enzymatic...

Growing and Analyzing Biofilms in Flow Chambers

DEFF Research Database (Denmark)

Tolker-Nielsen, Tim; Sternberg, Claus

2011-01-01

This unit describes the setup of flow chamber systems for the study of microbial biofilms, and methods for the analysis of structural biofilm formation. Use of flow chambers allows direct microscopic investigation of biofilm formation. The biofilms in flow chambers develop under hydrodynamic......, and disassembly and cleaning of the system. In addition, embedding and fluorescent in situ hybridization of flow chamber–grown biofilms are addressed. Curr. Protoc. Microbiol. 21:1B.2.1-1B.2.17. © 2011 by John Wiley & Sons, Inc....

Association of biofilm production with colonization among clinical isolates of Acinetobacter baumannii.

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Ryu, Seong Yeol; Baek, Won-Ki; Kim, Hyun Ah

2017-03-01

The pathogen Acinetobacter baumannii is increasingly causing healthcare-associated infections worldwide, particularly in intensive care units. Biofilm formation, a factor contributing to the virulence of A. baumannii , is associated with long-term persistence in hospital environments. The present study investigates the clinical impact of biofilm production on colonization and acquisition after patient admission. Forty-nine A. baumannii isolates were obtained between August and November 2013 from Keimyung University Dongsan Medical Center, Daegu, Korea. All isolates were obtained from sputum samples of new patients infected or colonized by A. baumannii . The microtiter plate assay was used to determine biofilm formation. Twenty-four A. baumannii isolates (48%) demonstrated enhanced biofilm formation capacity than that of the standard A. baumannii strain (ATCC 19606). All isolates were resistant to carbapenem, 38 isolates (77%) were collected from patients in an intensive care unit, and 47 isolates (95%) were from patients who had been exposed to antibiotics in the previous month. The median duration of colonization was longer for biofilm-producing isolates than that of the biofilm non-biofilm producing isolates (18 days vs. 12 days, p < 0.05). Simultaneous colonization with other bacteria was more common for biofilm-producing isolates than that for the non-biofilm producing isolates. The most prevalent co-colonizing bacteria was Staphylococcus aureus . Biofilm-producing isolates seem to colonize the respiratory tract for longer durations than the non-biofilm producing isolates. During colonization, biofilm producers promote co-colonization by other bacteria, particularly S. aureus . Additional research is required to determine possible links between biofilm formation and nosocomial infection.

Investigate Nasal Colonize Staphylococcus Species Biofilm Produced

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Cemil Demir

2014-03-01

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.

Disinfection efficiencies of sage and spearmint essential oils against planktonic and biofilm Staphylococcus aureus cells in comparison with sodium hypochlorite.

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Vetas, Dimitrios; Dimitropoulou, Eleni; Mitropoulou, Gregoria; Kourkoutas, Yiannis; Giaouris, Efstathios

2017-09-18

Staphylococcus aureus causes human infections and foodborne intoxications. This study explored the potential antibacterial actions of sage and spearmint essential oils (EOs) against both its planktonic and biofilm cells, in comparison with sodium hypochlorite (NaOCl), a commonly applied chemical sanitizer. Initially, the minimum inhibitory and bactericidal concentrations (MICs, MBCs) of each plant mixture were determined against planktonic cultures, following growth at 30°C for 24h. Stationary phase planktonic bacteria were then individually exposed for 6min to either each EO (applied at 1-2×MBC; 2.5-5%), or NaOCl (250-450ppm). These were also left to form biofilms on 96-well polystyrene microplates, at 30°C for 96h, with medium renewal at 48h, in the presence of 10 different concentrations of each EO, expanding from sub- to super-inhibitory for planktonic growth, and the minimum biofilm inhibitory concentrations (MBICs; >90% inhibition) of each plant mixture were calculated. Formed biofilms were finally exposed for 6min to either each EO (applied at 2-6×MBC; 5-15%), or NaOCl (7500-25,000ppm; applied either alone or in combination with each EO at 5%). Results showed that both EOs presented MIC and MBC equal to 1.25 and 2.5%, respectively. As expected, their application at their MIC and above significantly inhibited biofilm formation, while spearmint EO was still able to cause this at ½ of its MIC, with MBICs equal to 1.25 and 0.63% for sage and spearmint EOs, respectively. Alarmingly, the application of both EOs at 1/8 to 1/16 of their MIC further increased biofilm formation. Regarding biofilm disinfection experiments, the individual application of each EO against the pre-established sessile communities resulted in log decrease ranges of 0.8-3logCFU/cm 2 , while in the case of NaOCl application (either alone or combined with each EO), the observed reductions never exceeded 1.7logCFU/cm 2 . These last results highlight the great antimicrobial recalcitrance of

Identification of the amino acids essential for LytSR-mediated signal transduction in Staphylococcus aureus and their roles in biofilm-specific gene expression

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Lehman, McKenzie K.; Bose, Jeffrey L.; Sharma-Kuinkel, Batu K.; Moormeier, Derek E.; Endres, Jennifer L.; Sadykov, Marat R.; Biswas, Indranil; Bayles, Kenneth W.

2015-01-01

Summary Recent studies have demonstrated that expression of the Staphylococcus aureus lrgAB operon is specifically expressed within tower structures during biofilm development. To gain a better understanding of the mechanisms underlying this spatial control of lrgAB expression, we carried out a detailed analysis of the LytSR two-component system. Specifically, a conserved aspartic acid (Asp53) of the LytR response regulator was shown to be the target of phosphorylation, which resulted in enhanced binding to the lrgAB promoter and activation of transcription. In addition, we identified His390 of the LytS histidine kinase as the site of autophosphorylation and Asn394 as a critical amino acid involved in phosphatase activity. Interestingly, LytS-independent activation of LytR was observed during planktonic growth, with acetyl phosphate acting as a phosphodonor to LytR. In contrast, mutations disrupting the function of LytS prevented tower-specific lrgAB expression, providing insight into the physiologic environment within these structures. In addition, over activation of LytR led to increased lrgAB promoter activity during planktonic and biofilm growth and a change in biofilm morphology. Overall, the results of this study are the first to define the LytSR signal transduction pathway, as well as determine the metabolic context within biofilm tower structures that triggers these signaling events. PMID:25491472

Anti-Biofilm and Immunomodulatory Activities of Peptides That Inhibit Biofilms Formed by Pathogens Isolated from Cystic Fibrosis Patients

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César de la Fuente-Núñez

2014-10-01

Full Text Available Cystic fibrosis (CF patients often acquire chronic respiratory tract infections due to Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc species. In the CF lung, these bacteria grow as multicellular aggregates termed biofilms. Biofilms demonstrate increased (adaptive resistance to conventional antibiotics, and there are currently no available biofilm-specific therapies. Using plastic adherent, hydroxyapatite and flow cell biofilm models coupled with confocal and scanning electron microscopy, it was demonstrated that an anti-biofilm peptide 1018 prevented biofilm formation, eradicated mature biofilms and killed biofilms formed by a wide range of P. aeruginosa and B. cenocepacia clinical isolates. New peptide derivatives were designed that, compared to their parent peptide 1018, showed similar or decreased anti-biofilm activity against P. aeruginosa biofilms, but increased activity against biofilms formed by the Gram-positive bacterium methicillin resistant Staphylococcus aureus. In addition, some of these new peptide derivatives retained the immunomodulatory activity of 1018 since they induced the production of the chemokine monocyte chemotactic protein-1 (MCP-1 and suppressed lipopolysaccharide-mediated tumor necrosis factor-α (TNF-α production by human peripheral blood mononuclear cells (PBMC and were non-toxic towards these cells. Peptide 1018 and its derivatives provide promising leads for the treatment of chronic biofilm infections and hyperinflammatory lung disease in CF patients.

Integrating fluid dynamic and biologic effects on staphylococci bacteria biofilms

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Sherman, Erica; Endres, Jennifer; Bayles, Kenneth; Wei, Timothy

2017-11-01

Staphylococcus aureus bacteria are able to form biofilms and distinctive tower structures that facilitate their ability to tolerate treatment and to spread within the human body. The formation of towers, which break off, get carried downstream and serve to initiate biofilms in other parts of the body are of particular interest here. In previous work on biofilm growth and evolution in steady, laminar microchannel flows, it has been established that tower formation occurs around a very limited range of applied shear stresses centered on 0.6 dynes/cm2. Quantifying cell density characteristics as a function of time during biofilm formation reveals indicators of tower development hours before towers actually form and become visible. The next step in this research is to explore biological factors that might explain why this specific shear is so important. Additional studies with mutants, e.g. ica-A, that have been tied to tower formation have been conducted. The shear dependence of these mutants and their correlation to the behavior of wild type S. aureus is examined.

Elucidating the crucial role of poly N-acetylglucosamine from Staphylococcus aureus in cellular adhesion and pathogenesis.

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Lin, Mei Hui; Shu, Jwu Ching; Lin, Li Ping; Chong, Kowit Yu; Cheng, Ya Wen; Du, Jia Fu; Liu, Shih-Tung

2015-01-01

Staphylococcus aureus is an important pathogen that forms biofilms on the surfaces of medical implants. Biofilm formation by S. aureus is associated with the production of poly N-acetylglucosamine (PNAG), also referred to as polysaccharide intercellular adhesin (PIA), which mediates bacterial adhesion, leading to the accumulation of bacteria on solid surfaces. This study shows that the ability of S. aureus SA113 to adhere to nasal epithelial cells is reduced after the deletion of the ica operon, which contains genes encoding PIA/PNAG synthesis. However, this ability is restored after a plasmid carrying the entire ica operon is transformed into the mutant strain, S. aureus SA113Δica, showing that the synthesis of PIA/PNAG is important for adhesion to epithelial cells. Additionally, S. carnosus TM300, which does not produce PIA/PNAG, forms a biofilm and adheres to epithelial cells after the bacteria are transformed with a PIA/PNAG-expressing plasmid, pTXicaADBC. The adhesion of S. carnosus TM300 to epithelial cells is also demonstrated by adding purified exopolysaccharide (EPS), which contains PIA/PNAG, to the bacteria. In addition, using a mouse model, we find that the abscess lesions and bacterial burden in lung tissues is higher in mice infected with S. aureus SA113 than in those infected with the mutant strain, S. aureus SA113Δica. The results indicate that PIA/PNAG promotes the adhesion of S. aureus to human nasal epithelial cells and lung infections in a mouse model. This study elucidates a mechanism that is important to the pathogenesis of S. aureus infections.

Observed Antagonistic Effect of Linezolid on Daptomycin or Vancomycin Activity against Biofilm-Forming Methicillin-Resistant Staphylococcus aureus in an In Vitro Pharmacodynamic Model

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Luther, Megan K.

2015-01-01

Pharmacodynamic activity in antibiotic combinations of daptomycin, vancomycin, and linezolid was investigated in a 48-h in vitro pharmacodynamic model. Using human-simulated free drug concentrations, activity against clinical biofilm-forming methicillin-resistant Staphylococcus aureus isolates was evaluated. Linezolid antagonized vancomycin activity at 24 and 48 h. Linezolid antagonized daptomycin at 24 and 48 h depending on dose and strain. Adding daptomycin increased vancomycin activity at 48 h (P < 0.03). These results may be strain dependent and require further clinical investigation. PMID:26369963

Diverse modulation of spa transcription by cell wall active antibiotics in Staphylococcus aureus

DEFF Research Database (Denmark)

Nielsen, Lene Nørby; Roggenbuck, Michael; Haaber, Jakob Krause

2012-01-01

ABSTRACT: BACKGROUND: The aim of this study was to investigate the effect of various classes of clinically relevant antibiotics at sub-lethal concentrations on virulence gene expression and biofilm formation in Staphylococcus aureus. FINDINGS: LacZ promoter fusions of genes related to staphylococ......ABSTRACT: BACKGROUND: The aim of this study was to investigate the effect of various classes of clinically relevant antibiotics at sub-lethal concentrations on virulence gene expression and biofilm formation in Staphylococcus aureus. FINDINGS: LacZ promoter fusions of genes related...... to staphylococcal virulence were used to monitor the effects of antibiotics on gene expression in a disc diffusion assay. The selected genes were hla and spa encoding alpha-hemolysin and Protein A, respectively and RNAIII, the effector molecule of the agr quorum sensing system. The results were confirmed...... by quantitative real-time PCR. Additionally, we monitored the effect of subinhibitory concentrations of antibiotics on the ability of S. aureus to form biofilm in a microtiter plate assay. The results show that sub-lethal antibiotic concentrations diversely modulate expression of RNAIII, hla and spa. Consistently...

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

DEFF Research Database (Denmark)

Meyer, Rikke Louise

In vitro biofilm models are the basis for most studies of biofilm biology because they enable high-throughput analyses without the expenditure of animals. But how do we ensure that what we learn from in vitro studies is relevant in vivo? Biofilms grown in standard laboratory media do not interact...... with host factors and are thus profoundly different from in vivo biofilms. We therefore need in vitro models that are as in vivo-like as possible. We investigated how the addition of divalent cations and human plasma to brain heart infusion broth affected biofilm formation by Staphylococcus aureus...

Tolerance of Clostridium perfringens biofilms to disinfectants commonly used in the food industry.

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Charlebois, Audrey; Jacques, Mario; Boulianne, Martine; Archambault, Marie

2017-04-01

Clostridium perfringens is an opportunistic pathogen that can cause food poisoning in humans and various enterotoxemia in animal species. Recently, it was shown to form mono-species biofilms, a structured community of bacterial cells enclosed in a self-produced extracellular matrix. Biofilms have been associated with tolerance to antibiotics, disinfectants, and physical and environmental stresses. Very little is known about the tolerance of C. perfringens biofilm toward disinfectants. In the present study, susceptibilities of C. perfringens biofilms to five types of commonly used disinfectants on farms and in food processing environments were analysed. In this paper, we show that C. perfringens mono-species biofilms can protect the bacterial cells from the action of potassium monopersulfate, quaternary ammonium chloride, hydrogen peroxide and glutaraldehyde solutions. However, sodium hypochlorite solution was shown to be effective on C. perfringens biofilms. Our investigation of dual-species biofilms of C. perfringens with the addition of Staphylococcus aureus or Escherichia coli demonstrated that overall, the mono-species biofilm of C. perfringens was more tolerant to all disinfectants than the dual-species biofilms. For the anaerobic grown biofilms, the mono-species biofilm of C. perfringens was more tolerant to sodium hypochlorite and quaternary ammonium chloride than the dual-species biofilms of C. perfringens with S. aureus or E. coli. This study demonstrates that C. perfringens biofilm is an effective protection mechanism to disinfectants commonly used on farms and in food processing environments. Copyright © 2016 Elsevier Ltd. All rights reserved.

The clinical impact of bacterial biofilms

DEFF Research Database (Denmark)

Høiby, Niels; Ciofu, Oana; Johansen, Helle Krogh

2011-01-01

Bacteria survive in nature by forming biofilms on surfaces and probably most, if not all, bacteria (and fungi) are capable of forming biofilms. A biofilm is a structured consortium of bacteria embedded in a self-produced polymer matrix consisting of polysaccharide, protein and extracellular DNA....... Bacterial biofilms are resistant to antibiotics, disinfectant chemicals and to phagocytosis and other components of the innate and adaptive inflammatory defense system of the body. It is known, for example, that persistence of staphylococcal infections related to foreign bodies is due to biofilm formation....... Likewise, chronic Pseudomonas aeruginosa lung infections in cystic fibrosis patients are caused by biofilm growing mucoid strains. Gradients of nutrients and oxygen exist from the top to the bottom of biofilms and the bacterial cells located in nutrient poor areas have decreased metabolic activity...

Global 3D imaging of Yersinia ruckeri bacterin uptake in rainbow trout fry

DEFF Research Database (Denmark)

Otani, Maki; Villumsen, Kasper Rømer; Koppang, Erling Olaf

2015-01-01

Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM) in rainbow trout, and the first commercially available fish vaccine was an immersion vaccine against ERM consisting of Y. ruckeri bacterin. The ERM immersion vaccine has been successfully used in aquaculture farming of salm...

The alternative sigma factor sigma B of Staphylococcus aureus modulates virulence in experimental central venous catheter-related infections.

Science.gov (United States)

Lorenz, Udo; Hüttinger, Christian; Schäfer, Tina; Ziebuhr, Wilma; Thiede, Arnulf; Hacker, Jörg; Engelmann, Susanne; Hecker, Michael; Ohlsen, Knut

2008-03-01

The impact of the alternative sigma factor sigma B (SigB) on pathogenesis of Staphylococcus aureus is not conclusively clarified. In this study, a central venous catheter (CVC) related model of multiorgan infection was used to investigate the role of SigB for the pathogenesis of S. aureus infections and biofilm formation in vivo. Analysis of two SigB-positive wild-type strains and their isogenic mutants revealed uniformly that the wild-type was significantly more virulent than the SigB-deficient mutant. The observed difference in virulence was apparently not linked to the capability of the strains to form biofilms in vivo since wild-type and mutant strains were able to produce biofilm layers inside of the catheter. The data strongly indicate that the alternative sigma factor SigB plays a role in CVC-associated infections caused by S. aureus.

Several Virulence Factors of Multidrug-Resistant Staphylococcus aureus Isolates From Hospitalized Patients in Tehran

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Abdolmajid Ghasemian

2015-05-01

Full Text Available Background: Biofilm formation plays an important role in resistance of Staphylococcus aureus isolates; especially multidrug-resistant isolates are a threat to healthcare settings. Objectives: The aims of this study were to detect biofilm formation and presence of several related genes among multidrug-resistant (MDR isolates of Staphylococcus aureus. Patients and Methods: A total Of 209 S. aureus strains were isolated from patients and identified by conventional diagnostic tests. The multidrug-resistant MRSA isolates were detected by antibiotic susceptibility test. The phenotypic biofilm formation was detected by micro-titre tissue plate assay. The polymerase chain reaction (PCR was performed to detect the mecA, Staphylococcal Cassette Chromosome mec (SCCmec types, accessory gene regulatory (agr genes, the icaADBC and several genes encoding staphylococcal surface proteins including clfAB, fnbAB, fib, eno, can, ebps and bbp genes with specific primers. Results: Sixty-four (30.6% isolates were methicillin-resistant, among which thirty-six (56.2% were MDR. These isolates were resistant to amoxicillin, tetracycline, ciprofloxacin, gentamicin, erythromycin and trimethoprim-sulfamethoxazole (except to 6 isolates. All the isolates were susceptible to vancomycin and linezolid. All the MDR-MRSA harbored SCCmec type III. All the MDR- MRSA isolates were strong biofilm producers in the phenotypic test. The majority of MDR- MRSA was belonged to agrI (67%, n = 24, followed by agr II (17%, n = 6, agrIV (11%, n = 4 and agrIII (5.5%, n = 2. The frequency of icaADBC genes were 75% (n = 27, 61% (n = 22, 72% (n = 26 and 72% (n = 26, respectively. Furthermore, the prevalence of clfA, clfB, fnbA, fnbB, fib, can, eno, ebps and bbp genes was 100%, 100%, 67%, 56%, 80%, 63%, 78%, 7% and 0%, respectively. Furthermore, approximately all the MRSA was strong biofilm producers. Conclusions: Multidrug-resistant isolates produced biofilm strongly and the majority harbored most

Elucidating the crucial role of poly N-acetylglucosamine from Staphylococcus aureus in cellular adhesion and pathogenesis.

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Mei Hui Lin

Full Text Available Staphylococcus aureus is an important pathogen that forms biofilms on the surfaces of medical implants. Biofilm formation by S. aureus is associated with the production of poly N-acetylglucosamine (PNAG, also referred to as polysaccharide intercellular adhesin (PIA, which mediates bacterial adhesion, leading to the accumulation of bacteria on solid surfaces. This study shows that the ability of S. aureus SA113 to adhere to nasal epithelial cells is reduced after the deletion of the ica operon, which contains genes encoding PIA/PNAG synthesis. However, this ability is restored after a plasmid carrying the entire ica operon is transformed into the mutant strain, S. aureus SA113Δica, showing that the synthesis of PIA/PNAG is important for adhesion to epithelial cells. Additionally, S. carnosus TM300, which does not produce PIA/PNAG, forms a biofilm and adheres to epithelial cells after the bacteria are transformed with a PIA/PNAG-expressing plasmid, pTXicaADBC. The adhesion of S. carnosus TM300 to epithelial cells is also demonstrated by adding purified exopolysaccharide (EPS, which contains PIA/PNAG, to the bacteria. In addition, using a mouse model, we find that the abscess lesions and bacterial burden in lung tissues is higher in mice infected with S. aureus SA113 than in those infected with the mutant strain, S. aureus SA113Δica. The results indicate that PIA/PNAG promotes the adhesion of S. aureus to human nasal epithelial cells and lung infections in a mouse model. This study elucidates a mechanism that is important to the pathogenesis of S. aureus infections.

Preparation, characterization and efficacy of lysostaphin-chitosan gel against Staphylococcus aureus.

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Nithya, Sai; Nimal, T R; Baranwal, Gaurav; Suresh, Maneesha K; C P, Anju; Anil Kumar, V; Gopi Mohan, C; Jayakumar, R; Biswas, Raja

2018-04-15

Lysostaphin (LST) is a bacteriocin that cleaves within the pentaglycine cross bridge of Staphylococcus aureus peptidoglycan. Previous studies have reported the high efficiency of LST even against multi drug resistant S. aureus including methicillin resistant S. aureus (MRSA). In this study, we have developed a new chitosan based hydrogel formulation of LST to exploit its anti-staphylococcal activity. The atomic interactions of LST with chitosan were studied by molecular docking studies. The rheology and the antibacterial properties of the developed LSTC gel were evaluated. The developed LST containing chitosan hydrogel (LSTC gel) was flexible, flows smoothly and remains stable at physiological temperature. The in vitro studies by agar well diffusion and ex vivo studies in porcine skin model exhibited a reduction in S. aureus survival by ∼3 Log 10 CFU/mL in the presence of LSTC gel. The cytocompatibility of the gel was tested in vitro using macrophage RAW 264.7 cell line and in vivo in Drosophila melanogaster. A gradual disruption of S. aureus biofilms with the increase of LST concentrations in the LSTC gel was observed which was confirmed by SEM analysis. We conclude that LSTC gel could be highly effectual and advantageous over antibiotics in treating staphylococcal-topical and biofilm infections. Copyright © 2018 Elsevier B.V. All rights reserved.

Antistaphylococcal and biofilm inhibitory activities of gallic, caffeic, and chlorogenic acids.

Science.gov (United States)

Luís, Ângelo; Silva, Filomena; Sousa, Sónia; Duarte, Ana Paula; Domingues, Fernanda

2014-01-01

Staphylococcus aureus is a Gram-positive pathogen which is able to form biofilms, exhibiting a more pronounced resistance to antibiotics and disinfectants. The hurdles posed in eradicating biofilms have driven the search for new compounds able to fight these structures. Phenolic compounds constitute one of the most numerous and ubiquitous group of plant secondary metabolites with many biological activities. The aim of the present work was to study the potential antimicrobial and antibiofilm properties of gallic, caffeic, and chlorogenic acids against S. aureus as well to elucidate its mechanism of action. It was concluded that the phenolic acids studied in this work have antistaphylococcal properties. For instance, gallic acid is able to influence the adhesion properties of S. aureus. The phenolic acids tested were also able to inhibit the production of α-hemolysin by this microorganism, with the exception of chlorogenic acid. Regarding its mechanism of action, caffeic acid interferes with the stability of the cell membrane and with the metabolic activity of the cells of S. aureus.

Antibiotic resistance of bacterial biofilms

DEFF Research Database (Denmark)

Hoiby, N.; Bjarnsholt, T.; Givskov, M.

2010-01-01

A biofilm is a structured consortium of bacteria embedded in a self-produced polymer matrix consisting of polysaccharide, protein and DNA. Bacterial biofilms cause chronic infections because they show increased tolerance to antibiotics and disinfectant chemicals as well as resisting phagocytosis...... and other components of the body's defence system. The persistence of, for example, staphylococcal infections related to foreign bodies is due to biofilm formation. Likewise, chronic Pseudomonas aeruginosa lung infection in cystic fibrosis patients is caused by biofilm-growing mucoid strains....... Characteristically, gradients of nutrients and oxygen exist from the top to the bottom of biofilms and these gradients are associated with decreased bacterial metabolic activity and increased doubling times of the bacterial cells; it is these more or less dormant cells that are responsible for some of the tolerance...

Ultrastructural changes in biofilm forms of staphylococci cultivated in a mixed culture with lactobacilli

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G. Lavryk

2017-02-01

Full Text Available The capacity of opportunistic bacteria for biofilm formation plays an important role in the development of chronic inflammatory processes, which are difficult to treat. To improve antimicrobial therapy methods, the influence of lactobacilli on the ultrastructure of biofilm-forming clinical strains of staphylococci when co-cultured was investigated. 5 biofilm-forming clinical strains of S. aureus from the skin of acne vulgaris patients (n = 24 were isolated. Using transmission electron microscopy (TEM the morphological changes of S. aureus cells in the mixed culture with standard strains of Lactobacillus plantarum 8P-A3 and clinical strains of L. fermentum (n = 4 were studied. It was found that in 48 hours after the inoculation on the medium of samples of mixed cultures of L. plantarum 8P-A3 and S. aureus growth of staphylococci was not revealed. Only in some cases of mixed cultures of L. fermentum and biofilm-forming staphylococci was growth of S. aureus obtained. In electron diffraction patterns of control samples of 24-hour staphylococcal monocultures and 48-hour lactobacilli monocultures, natural development of the population at the cellular level was observed. Destructive changes under the influence of lactobacilli (probiotic and clinical strains were detected in all ultrathin sections of the cells of biofilm-forming and planktonic staphylococci. Significant destructive changes in the cell wall of the staphylococci were observed: thickening, obtaining of irregular form, detachment of the cytoplasmic membrane, the complete destruction of the peptidoglycan layer and the emergence of "shadow cells". On all electron diffraction patterns fibrillar-threadlike structures of DNA could not be observed, but in some cases mesosome-like formations were poorly contrasted. It was established that the surface S-layer of lactobacilli was expressed on a significantly larger scale in the mixed culture with staphylococci. In mixed culture of clinical strains

[Investigation of biofilm formation properties of staphylococcus isolates].

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Öcal, Duygu Nilüfer; Dolapçı, İştar; Karahan, Zeynep Ceren; Tekeli, Alper

2017-01-01

Biofilm production is an important virulence factor which allows staphylococci to adhere to medical devices. The principal component of biofilm is a "polysaccharide intercellular adhesin (PIA)" which is composed of a beta-1,6-N-acetylglucosamine polymer synthesized by an enzyme (N-acetylglucosamine transferase) encoded by the ica operon found on the bacterial chromosome. This operon is composed of four genes (A, B, C, and D), and a transposable element IS256. In this study, we aimed to determine the biofilm production characteristics of invasive/non-invasive staphylococcus isolates and different staphylococcus species. Biofilm production of 166 staphylococci was phenotypically investigated on Congo Red Agar (CRA); the presence of icaA, icaD and IS256 genes were investigated by polymerase chain reaction (PCR). 74 of the isolates (44.6%) were identified as methicillin resistant Staphylococcus aureus (MRSA), 25 (15.1%) as methicillin sensitive S.aureus (MSSA), 25 (37.3%) as Staphylococcus hominis, 20 (12%) as S.epidermidis, ten (15%) as Staphylococcus haemolyticus, nine (13.4%) as Staphylococcus capitis, two (3%) Staphylococcus saprophyticus and one (1.5%) as Staphylococcus warnerii. Of the MRSA strains, 52 were isolated from blood and 22 from nose; all MSSA strains were isolated from nose cultures. Coagulase-negative staphylococci (CoNS) strains were composed of invasive and non-invasive strains isolated from nose, catheter tip and blood cultures from patients with catheter. Production with CRA method was found to be statistically significant in invasive isolates (paureus isolates produced biofilm on CRA (paureus when compared with CoNS. Carriage of three genes and biofilm formation capacity of invasive isolates can cause refractory infections and the importance of carriage and hospital infections of these bacteria, it is important to prevent the spread of these isolates. A combination of phenotypic and genotypic tests is recommended for the investigation of biofilm

Dual Roles of FmtA in Staphylococcus aureus Cell Wall Biosynthesis and Autolysis

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Qamar, Aneela

2012-01-01

The fmtA gene is a member of the Staphylococcus aureus core cell wall stimulon. The FmtA protein interacts with β-lactams through formation of covalent species. Here, we show that FmtA has weak d-Ala-d-Ala-carboxypeptidase activity and is capable of covalently incorporating C14-Gly into cell walls. The fluorescence microscopy study showed that the protein is localized to the cell division septum. Furthermore, we show that wall teichoic acids interact specifically with FmtA and mediate recruitment of FmtA to the S. aureus cell wall. Subjection of S. aureus to FmtA concentrations of 0.1 μM or less induces autolysis and biofilm production. This effect requires the presence of wall teichoic acids. At FmtA concentrations greater than 0.2 μM, autolysis and biofilm formation in S. aureus are repressed and growth is enhanced. Our findings indicate dual roles of FmtA in S. aureus growth, whereby at low concentrations, FmtA may modulate the activity of the major autolysin (AtlA) of S. aureus and, at high concentrations, may participate in synthesis of cell wall peptidoglycan. These two roles of FmtA may reflect dual functions of FmtA in the absence and presence of cell wall stress, respectively. PMID:22564846

Biofilm formation by clinical isolates and the implications in chronic infections

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Sanchez Carlos J

2013-01-01

Full Text Available Abstract Background Biofilm formation is a major virulence factor contributing to the chronicity of infections. To date few studies have evaluated biofilm formation in infecting isolates of patients including both Gram-positive and Gram-negative multidrug-resistant (MDR species in the context of numerous types of infectious syndromes. Herein, we investigated the biofilm forming capacity in a large collection of single patient infecting isolates and compared the relationship between biofilm formation to various strain characteristics. Methods The biofilm-forming capacity of 205 randomly sampled clinical isolates from patients, collected from various anatomical sites, admitted for treatment at Brooke Army Medical Center (BAMC from 2004–2011, including methicillin-resistant/methicillin susceptible Staphylococcus aureus (MRSA/MSSA (n=23, Acinetobacter baumannii (n=53, Pseudomonas aeruginosa (n=36, Klebsiella pneumoniae (n=54, and Escherichia coli (n=39, were evaluated for biofilm formation using the high-throughput microtiter plate assay and scanning electron microscopy (SEM. Relationships between biofilm formation to clonal type, site of isolate collection, and MDR phenotype were evaluated. Furthermore, in patients with relapsing infections, serial strains were assessed for their ability to form biofilms in vitro. Results Of the 205 clinical isolates tested, 126 strains (61.4% were observed to form biofilms in vitro at levels greater than or equal to the Staphylococcus epidermidis, positive biofilm producing strain, with P. aeruginosa and S. aureus having the greatest number of biofilm producing strains. Biofilm formation was significantly associated with specific clonal types, the site of isolate collection, and strains positive for biofilm formation were more frequently observed to be MDR. In patients with relapsing infections, the majority of serial isolates recovered from these individuals were observed to be strong biofilm producers in vitro

Pseudomonas aeruginosa Biofilms

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Alhede, Maria; Bjarnsholt, Thomas; Givskov, Michael

2014-01-01

biofilms, which protect the aggregated, biopolymer-embedded bacteria from the detrimental actions of antibiotic treatments and host immunity. A key component in the protection against innate immunity is rhamnolipid, which is a quorum sensing (QS)-regulated virulence factor. QS is a cell-to-cell signaling...... mechanism used to coordinate expression of virulence and protection of aggregated biofilm cells. Rhamnolipids are known for their ability to cause hemolysis and have been shown to cause lysis of several cellular components of the human immune system, for example, macrophages and polymorphonuclear leukocytes...

Antibiofilm Effect of Octenidine Hydrochloride on Staphylococcus aureus, MRSA and VRSA

OpenAIRE

Amalaradjou, Mary Anne Roshni; Venkitanarayanan, Kumar

2014-01-01

Millions of indwelling devices are implanted in patients every year, and staphylococci (S. aureus, MRSA and vancomycin-resistant S. aureus (VRSA)) are responsible for a majority of infections associated with these devices, thereby leading to treatment failures. Once established, staphylococcal biofilms become resistant to antimicrobial treatment and host response, thereby serving as the etiological agent for recurrent infections. This study investigated the efficacy of octenidine hydrochlorid...

Characterisation of Lactobacillus plantarum single and multi-strain biofilms

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Fernández Ramírez, Mónica D.

2016-01-01

Biofilms consist of microorganisms attached to a surface and embedded in a protective matrix of extracellular polymeric substances. Within a biofilm, micro-organisms are protected from harsh environmental conditions including those resulting from cleaning and disinfecting agents leading to food

Prevalence and Characterization of Staphylococcus aureus Cultured From Raw Milk Taken From Dairy Cows With Mastitis in Beijing, China

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Wei Wang

2018-06-01

Full Text Available The colonization of dairy herds and subsequent contamination of raw milk by Staphylococcus aureus (S. aureus, especially those expressing a multi-drug resistance (MDR, biofilm and toxins producing ability, remains an important issue for both the dairy producer and public health. In this study, we investigated the prevalence, antimicrobial resistance, virulence, and genetic diversity of S. aureus in raw milk taken from 2 dairy farms in Beijing, China. Ninety (46.2%, 90/195 samples were positive for S. aureus. Resistant to penicillin (PEN (31.3%, ciprofloxacin (18.8% and enrofloxacin (15.6% were the most often observed. Isolates cultured from farm B showed significantly higher resistance to penicillin (73.9%, ciprofloxacin (34.8%, enrofloxacin (34.8%, tilmicosin (17.4%, and erythromycin (17.4% than those from farm A (p < 0.05. Totally, 94.8% S. aureus harbored at least one virulence gene and the pvl (93.8%, sec (65.6%, and sea (60.4% genes were the most frequently detected. The pvl and sec genes were more often detected in isolates from farm A (97.3% and 84.9% respectively than those from farm B (p < 0.05. Of all 77 staphylococcus enterotoxin (SE-positive isolates, more than 90% could produce enterotoxins and 70.1% could produce two types. Biofilm related genes (icaA/D, clf/B, can, and fnbA were detected in all96 isolates. All 96 isolates could produce biofilm with 8.3, 70.8, and 18.8% of the isolates demonstrating weak, moderate and strong biofilm formation, respectively. A total of 5 STs, 7 spa types (1 novel spa type t17182, 3agr types (no agrII, and 14 SmaI-pulso-types were found in this study. PFGE cluster II-CC1-ST1-t127-agr III was the most prevalent clone (56.3%. Isolates of agr III (PFGE Cluster I/II-CC1-ST1-t127/2279 had higher detection of virulence genes than those of agr I and agr IV. TheMSSA-ST398-t1456-agr I clone expressed the greatest MDRbut with no virulence genes and weakly biofilm formation. Our finding indicated a relatively

Efeito dos óleos essenciais de Syzygium aromaticum e Cinnamomum zeylanicume e seus compostos majoritários na produçao de biofilme em cepas de Staphylococcus aureus, isolados de leite de vacas com mastite subclínica

OpenAIRE

Budri, Paulo Eduardo [UNESP

2014-01-01

Bovine Mastitis is an inflammation of the mammary gland of cows causing economic losses in dairy cattle and Staphylococcus aureus is one of the microorganisms most commonly isolated in this kind of disease. The production of biofilms by S. aureus is an important virulence factor and spreed of this microorganism, involving the adhesion of bacteria on solid surfaces. In this work we tested the essential oils of Syzygium aromaticum (OESA) and Cinnamomum zeylanicum (OECZ) and their major compound...

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

DEFF Research Database (Denmark)

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

2015-01-01

Objective: Bacterial biofilms remain difficult to treat. The biofilm mode of growth enables bacteria to survive antibiotic treatment and the inflammatory reaction. Low-frequency ultrasound has recently been shown to improve healing in a variety of settings. It is hypothesised that ultrasound...... disrupts the biofilm leaving bacteria more vulnerable to antiseptic or antibiotic treatment. The objective of this study is to develop a realistic model to elucidate the effect of ultrasound on biofilms. Method: A novel in vitro wound biofilm model was developed. Biofilms of Staphylococcus aureus were...

Antibiofilm activity of Vetiveria zizanioides root extract against methicillin-resistant Staphylococcus aureus.

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Kannappan, Arunachalam; Gowrishankar, Shanmugaraj; Srinivasan, Ramanathan; Pandian, Shunmugiah Karutha; Ravi, Arumugam Veera

2017-09-01

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading human pathogen responsible for causing chronic clinical manifestation worldwide. In addition to antibiotic resistance genes viz. mecA and vanA, biofilm formation plays a prominent role in the pathogenicity of S. aureus by enhancing its resistance to existing antibiotics. Considering the role of folk medicinal plants in the betterment of human health from the waves of multidrug resistant bacterial infections, the present study was intended to explore the effect of Vetiveria zizanioides root on the biofilm formation of MRSA and its clinical counterparts. V. zizanioides root extract (VREX) showed a concentration-dependent reduction in biofilm formation without hampering the cellular viability of the tested strains. Micrographs of scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) portrayed the devastating impact of VREX on biofilm formation. In addition to antibiofilm activity, VREX suppresses the production of biofilm related phenotypes such as exopolysaccharide, slime and α-hemolysin toxin. Furthermore, variation in FT-IR spectra evidenced the difference in cellular factors of untreated and VREX treated samples. Result of mature biofilm disruption assay and down regulation of genes like fnbA, fnbB, clfA suggested that VREX targets these adhesin genes responsible for initial adherence. GC-MS analysis revealed the presence of sesquiterpenes as a major constituent in VREX. Thus, the data of present study strengthen the ethnobotanical value of V. zizanioides and concludes that VREX contain bioactive molecules that have beneficial effect over the biofilm formation of MRSA and its clinical isolates. Copyright © 2017 Elsevier Ltd. All rights reserved.

Norlichexanthone Reduces Virulence Gene Expression and Biofilm Formation in Staphylococcus aureus

DEFF Research Database (Denmark)

Baldry, Mara; Nielsen, Anita; Bojer, Martin S.

2016-01-01

characterise the mode of action of norlichexanthone and its effect on biofilm formation. We find that norlichexanthone reduces expression of both hla and RNAIII also in strain USA300. Structurally, norlichexanthone resembles ω-hydroxyemodin that recently was shown to bind the agr two component response......-hydroxyemodin however, norlichexanthone reduces staphylococcal biofilm formation. Transcriptomic analysis revealed that genes regulated by the SaeRS two-component system are repressed by norlichexanthone when compared to untreated cells, an effect that was mitigated in strain Newman carrying a partially constitutive...... SaeRS system. Our data show that norlichexanthone treatment reduces expression of key virulence factors in CA-MRSA strain USA300 via AgrA binding and represses biofilm formation....

Pseudomonas aeruginosa biofilms in cystic fibrosis

DEFF Research Database (Denmark)

Høiby, Niels; Ciofu, Oana; Bjarnsholt, Thomas

2010-01-01

The persistence of chronic Pseudomonas aeruginosa lung infections in cystic fibrosis (CF) patients is due to biofilm-growing mucoid (alginate-producing) strains. A biofilm is a structured consortium of bacteria, embedded in a self-produced polymer matrix consisting of polysaccharide, protein...... and DNA. In CF lungs, the polysaccharide alginate is the major part of the P. aeruginosa biofilm matrix. Bacterial biofilms cause chronic infections because they show increased tolerance to antibiotics and resist phagocytosis, as well as other components of the innate and the adaptive immune system....... As a consequence, a pronounced antibody response develops, leading to immune complex-mediated chronic inflammation, dominated by polymorphonuclear leukocytes. The chronic inflammation is the major cause of the lung tissue damage in CF. Biofilm growth in CF lungs is associated with an increased frequency...

EXTRATO DE CHÁ VERDE (Camellia sinensis PROMOVE AÇÃO ANTIMICROBIANA SOBRE Staphylococcus aureus

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Leandro Wagner Figueira

2017-01-01

Full Text Available É de grande importância ampliar as pesquisas científicas com plantas medicinais sobre micro-organismos. Foi verificado o efeito antimicrobiano do extrato de C. sinensis , sobre cultura planctônica e biofilme de S. aureus. Neste estudo foi verificada ação do extrato entre 50 a 0,09 mg/mL sobre cultura planctônica, para determinação da CIM e CBM. A ação do extrato sobre biofilme foi analisada pela exposição de 5 min ao produto vegetal a 200mg/mL. Foi utilizado o método de MTT para determinar a redução da viabilidade do biofilme. Nos grupos controles foram utilizadas clorexidina (+ e solução salina (-, sendo n = 12/grupo. A redução do biofilme foi analisada por ANOVA e Tukey Test (p ≤ 0,05. Em cultura planctônica as concentrações avaliadas não foram efetivas, contudo, utilizando-se a concentração de 200 mg/mL sobre o biofilme, foi verificada redução significativa. Com isso, concluiu-se que o extrato de chá verde demonstrou ação sobre biofilme de S. aureus provendo redução significativa em sua viabilidade.

Population structure and antimicrobial profile of Staphylococcus aureus strains associated with bovine mastitis in China.

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Zhang, Lili; Li, Yuchen; Bao, Hongduo; Wei, Ruicheng; Zhou, Yan; Zhang, Hui; Wang, Ran

2016-08-01

Staphylococcus aureus is a significant bacterial pathogen associated with bovine mastitis. The aim of the present study was to investigate and characterize of S. aureus strains isolated from the milk of cows suffering from mastitis in the mid-east of China. Among the 200 milk samples analyzed, 58 were positive for S. aureus, of these isolates, 11 isolates were methicillin-resistant Staphylococcus aureus (MRSA). All of the 58 S. aureus strains were classified in agr group I, while seven different sequence type (ST) patterns were identified and among them the most common was ST630 followed by ST188. All of the S. aureus isolates belonging to ST630 were resistant to more than four antimicrobials, and 22.2% of isolates belonging to ST188 were resistant to eight antimicrobials. Interestingly, while strong biofilm producers demonstrated higher resistance to multiple antimicrobials, they exhibited lower intracellular survival rates. The results of this study illustrated the distribution, antimicrobial susceptibility profiles, genotype, and the ability of biofilm production and mammary epithelial cells invasion of these S. aureus isolates. This study can provide the basis for the development of a disease prevention program in dairy farms to reduce the potential risk in both animal and human health. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

DEFF Research Database (Denmark)

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

2016-01-01

and microcalorimetry methods. Patients and methods - Biofilms of Staphylococcus aureus, S. epidermidis, Escherichia coli, and Propionibacterium acnes were formed on porous glass beads and exposed for 24 h to antibiotic concentrations from 1 to 1,024 times the minimal inhibitory concentration (MIC) of vancomycin......, daptomycin, rifampin, flucloxacillin, or ciprofloxacin. The beads were then sonicated to dislodge biofilm, followed by culture and measurement of growth-related heat flow by microcalorimetry of the resulting sonication fluid. Results - Vancomycin did not inhibit the heat flow of staphylococci and P. acnes...... at concentrations ≤1,024 μg/mL, whereas flucloxacillin at >128 μg/mL inhibited S. aureus. Daptomycin inhibited heat flow of S. aureus, S. epidermidis, and P. acnes at lower concentrations (32-128 times MIC, p

High potential of adhesion to biotic and abiotic surfaces by opportunistic Staphylococcus aureus strains isolated from orthodontic appliances.

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Merghni, Abderrahmen; Ben Nejma, Mouna; Dallel, Ines; Tobji, Samir; Ben Amor, Adel; Janel, Sébastien; Lafont, Frank; Aouni, Mahjoub; Mastouri, Maha

2016-02-01

Orthodontic and other oral appliances act as reservoir of opportunistic pathogens that can easily become resistant to antibiotics and cause systemic infections. The aim of this study was to investigate the ability of Staphylococcus aureus strains isolated from healthy patients with orthodontic appliances, to adhere to biotic (HeLa cells) and abiotic surfaces (polystyrene and dental alloy). Adhesive ability to polystyrene was tested by crystal violet staining and quantitative biofilm production on dental alloy surfaces was evaluated by MTT reduction assay. In addition, the presence of icaA and icaD genes was achieved by polymerase chain reaction (PCR). Qualitative biofilm production revealed that 70.6% of strains were slime producers. The metabolic activity of S. aureus biofilms on dental alloy surfaces was high and did not differ between tested strains. Moreover, all the isolates were adhesive to HeLa cells and 94% of them harbor icaA and icaD genes. Considerable adhesion and internalization capacity to the epithelial HeLa cells and strong biofilm production abilities together, with a high genotypic expression of icaA/icaD genes are an important equipment of S. aureus to colonize orthodontic appliances and eventually to disseminate towards other body areas. Copyright © 2015 Elsevier Ltd. All rights reserved.

Efficacy of Poly-Lactic-Co-Glycolic Acid Micro- and Nanoparticles of Ciprofloxacin Against Bacterial Biofilms.

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Thomas, Nicky; Thorn, Chelsea; Richter, Katharina; Thierry, Benjamin; Prestidge, Clive

2016-10-01

Bacterial biofilms are associated with a number of recurring infectious diseases and are a major cause for antibiotic resistance. Despite the broad use of polymeric microparticles and nanoparticles in biomedical research, it is not clear which particle size is more effective against biofilms. The purpose of this study was to evaluate the efficacy of sustained release poly-lactic-co-glycolic acid (PLGA) micro- and nanoparticles containing ciprofloxacin against biofilms of Staphylococcus aureus and Pseudomonas aeruginosa. The PLGA particles were prepared by the double emulsion solvent evaporation method. The resulting microparticles (12 μm) and nanoparticles (300 nm) contained drug loads of 7.3% and 4.5% (wt/wt) ciprofloxacin, respectively. Drug release was complete within 1 week following comparable release profiles for both particle sizes. Micro- and nanoparticles demonstrated a similar in vitro antibiofilm performance against mature P aeruginosa and S aureus with marked differences between the 2 strains. The sustained release of ciprofloxacin from micro- and nanoparticles over 6 days was equally effective as the continuous treatment with ciprofloxacin solution over the same period resulting in the eradication of culturable S aureus suggesting that reformulation of ciprofloxacin as sustained release PLGA micro- and nanoparticles might be valuable formulation approaches for the treatment of biofilms. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Molecular characteristics of bap-positive Staphylococcus aureus strains from dairy cow mastitis.

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Snel, Gustavo G M; Monecke, Stefan; Ehricht, Ralf; Piccinini, Renata

2015-08-01

The biofilm-associated protein (Bap) of Staphylococcus aureus is a high molecular weight cell-wall-anchored protein involved in biofilm formation, first described in bovine mastitis strains from Spain. So far, studies regarding Bap were mainly based on the Spanish strain V329 and its mutants, but no information on the genetic variability of bap-positive Staph. aureus strains is yet available in the literature. The present study investigated the molecular characteristics of 8 bap-positive Staph. aureus strains from subclinical bovine mastitis, isolated in 5 herds; somatic cell counts (SCC) of milk samples were also registered. Strains were characterised using MLST, SPA typing and microarray and the results were compared with V329. All isolates from this study and V329 were assigned to ST126, t605, but some molecular differences were observed. Only herd A and B strains harboured the genes for β-lactams resistance; the leukocidin D/E gene, a type I site-specific deoxyribonuclease subunit, 3rd locus gene and serin-protease A and B were carried by all strains, but not by V329, while serin-protease E was absent in V329 and in another isolate. Four isolates and V329 harboured the fibronectin-binding protein B gene. SCC showed the highest value in the milk sample affected by the only strain carrying all the virulence factors considered. Potential large variability of virulence was evidenced among V329 and all bap-positive Staph. aureus strains considered: the carriage of fnb could enhance the accumulation of biofilm, but the lack of lukD/E and splA, B or E might decrease the invasiveness of strain.

Antioxidant and antiproliferative potential of biosurfactants isolated from Lactobacillus casei and their anti-biofilm effect in oral Staphylococcus aureus strains.

Science.gov (United States)

Merghni, Abderrahmen; Dallel, Ines; Noumi, Emira; Kadmi, Yassine; Hentati, Hajer; Tobji, Samir; Ben Amor, Adel; Mastouri, Maha

2017-03-01

Biosurfactants also called bioemulsifiers are amphipathic compounds produced by many microorganisms that allow them to exhibit a wide range of biological activities. The aim of this study was to determine the antioxidant and antiproliferative potential of biosurfactants isolated from Lactobacillus casei and to assess their anti-adhesive and anti-biofilm abilities against oral opportunistic Staphylococcus aureus strains. The antioxidant activity of biosurfactant was evaluated using the in vitro scavenging ability on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. The antiproliferative activity was determined on epithelial cell line (HEp-2) by the Methylthiazole tetrazolium (MTT) reduction assay. The anti-adhesive and antibiofilm activity against S. aureus strains were achieved using crystal violet staining. Our results revealed that the DPPH scavenging activity of biosurfactants at 5.0 mg/mL concentration is between 74.6 and 77.3%. Furthermore, biosurfactants showed antiproliferative potency against studied epithelial cells as judged by IC50 and its value ranged from 109.1 ± 0.84 mg/mL to 129.7 ± 0.52 mg/mL. The results of the growth inhibition indicate that biosurfactant BS-LBl was more effective against oral S. aureus strains 9P and 29P with an IC50 of 1.92 ± 0.26 mg/mL and 2.16 ± 0.12 mg/mL respectively. Moreover, both biosurfactants displayed important antibiofilm activity with eradication percentages ranging from 80.22 ± 1.33% to 86.21 ± 2.94% for the BS-LBl, and from 53.38 ± 1.77% to 64.42 ± 2.09% for the BS-LZ9. Our findings demonstrate that biosurfactants from L. casei strains exhibited considerable antioxidant and antiproliferative potencies and were able to inhibit oral S. aureus strains with important antibiofilm efficacy. They could have a promising role in the prevention of oral diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Formation and retention of staphylococcal biofilms on DLC and its hybrids compared to metals used as biomaterials.

Science.gov (United States)

Myllymaa, Katja; Levon, Jaakko; Tiainen, Veli-Matti; Myllymaa, Sami; Soininen, Antti; Korhonen, Hannu; Kaivosoja, Emilia; Lappalainen, Reijo; Konttinen, Yrjö Tapio

2013-01-01

Staphylococcus epidermidis and Staphylococcus aureus cause most of the implant-related infections. Antibiotic treatment often fails and cure requires surgical intervention. It was hypothesized that biomaterial coatings resistant to biofilms offer a preventive option. Physical vapour deposited diamond-like carbon (DLC) and its polytetrafluoroethylene (DLC-PTFE-h) and polydimethylsiloxane (DLC-PDMS-h) hybrids were compared to titanium (Ti), tantalum (Ta) and chromium (Cr) thin films on silicon wafers for their resistance against formation and/or retention of biofilms produced by S. epidermidis and S. aureus in vitro. Sample surfaces were characterized for surface topography, contact angle and zeta-potential, because such properties might affect the biofilm. Biofilm was stained using calcofluor white and analysed in fluorescence microscopy using morphometry. Sixteen hour incubation was selected in pilot tests; at this checkpoint Ti, Ta, Cr and DLC-PDMS-h were almost fully covered by biofilm, but DLC and DLC-PTFE-h were only partially biofilm coated by S. epidermidis (88±26%, pDLC and its PTFE hybrid offer a potential biofilm hostile surface coating for implants and medical devices. This ability to resist biofilm formation and attachment could not be explained by only one factor, but it seems to be related to a combination of various properties, with electrokinetic streaming potential and protein coating being particularly important for its outcome. Copyright © 2012 Elsevier B.V. All rights reserved.

[Biofilms and their significance in medical microbiology].

Science.gov (United States)

Cernohorská, L; Votava, M

2002-11-01

Microorganisms are able to adhere to various surfaces and to form there a three-dimensional structure known as biofilm. In biofilms, microbial cells show characteristics and behaviours different from those of plankton cells. Intercellular signalizations of the quorum-sensing type regulate interaction between members of the biofilm. Bacteria embedded in the biofilm can escape and form well known planktonic forms, that are obviously only a part of the bacterial life cycle. Bacteria adhere also to medically important surfaces such as catheters, either urinary or intravenous ones, artificial heart valves, orthopedic implants and so on and contribute to device-related infections like cystitis, catheter-related sepsis, endocarditis etc. Once a biofilm has been established on a surface, the bacteria harboured inside are less exposed to the host's immune response and less susceptible to antibiotics. As an important cause of nosocomial infections the biofilm must remain in the centre of the microbiologist's attention.

Biofilm infections between Scylla and Charybdis: interplay of host antimicrobial peptides and antibiotics

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Chernysh S

2018-04-01

Full Text Available Sergey Chernysh,* Natalia Gordya,* Dmitry Tulin, Andrey Yakovlev Laboratory of Insect Biopharmacology and Immunology, Faculty of Biology, St. Petersburg State University, St. Petersburg, Russia *These authors contributed equally to this work Purpose: The aim of this study is to improve the anti-biofilm activity of antibiotics. We hypothesized that the antimicrobial peptide (AMP complex of the host’s immune system can be used for this purpose and examined the assumption on model biofilms. Methods: FLIP7, the AMP complex of the blowfly Calliphora vicina containing a combination of defensins, cecropins, diptericins and proline-rich peptides was isolated from the hemolymph of bacteria-challenged maggots. The complex interaction with antibiotics of various classes was studied in biofilm and planktonic cultures of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii by the checkerboard method using trimethyl tetrazolium chloride cell viability and crystal violet biofilm eradication assays supplemented with microscopic analysis. Results: We found that FLIP7 demonstrated: high synergy (fractional inhibitory concentration index <0.25 with meropenem, amikacin, kanamycin, ampicillin, vancomycin and cefotaxime; synergy with clindamycin, erythromycin and chloramphenicol; additive interaction with oxacillin, tetracycline, ciprofloxacin and gentamicin; and no interaction with polymyxin B. The interaction in planktonic cell models was significantly weaker than in biofilms of the same strains. The analysis of the dose–effect curves pointed to persister cells as a likely target of FLIP7 synergistic effect. The biofilm eradication assay showed that the effect also caused total destruction of S. aureus and E. coli biofilm materials. The effect allowed reducing the effective anti-biofilm concentration of the antibiotic to a level well below the one clinically achievable (2–3 orders of magnitude in

On the determining role of network structure titania in silicone against bacterial colonization: Mechanism and disruption of biofilm

International Nuclear Information System (INIS)

Depan, D.; Misra, R.D.K.

2014-01-01

Silicone-based biomedical devices are prone to microbial adhesion, which is the primary cause of concern in the functioning of the artificial device. Silicone exhibiting long-term and effective antibacterial ability is highly desirable to prevent implant related infections. In this regard, nanophase titania was incorporated in silicone as an integral part of the silicone network structure through cross-link mechanism, with the objective to reduce bacterial adhesion to a minimum. The bacterial adhesion was studied using crystal violet assay, while the mechanism of inhibition of biofilm formation was studied via electron microscopy. The incorporation of nanophase titania in silicone dramatically reduced the viability of Staphylococcus aureus (S. aureus) and the capability to adhere on the surface of hybrid silicone by ∼ 93% in relation to stand alone silicone. The conclusion of dramatic reduction in the viability of S. aureus is corroborated by different experimental approaches including biofilm inhibition assay, zone of inhibition, and through a novel experiment that involved incubation of biofilm with titania nanoparticles. It is proposed that the mechanism of disruption of bacterial film in the presence of titania involves puncturing of the bacterial cell membrane. - Highlights: • Network structure titania in silicone imparts antimicrobial activity. • Ability to microbial adhesion is significantly reduced. • Antimicrobial mechanism involves rupture of biofilm

Anti-MRSA activities of Enterocins DD28 and DD93 and evidences on their role in the inhibition of biofilm formation

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Ahmed Khassaf eAL ATYA

2016-05-01

Full Text Available Methicillin-resistant Staphylococcus aureus (MRSA, along with other antibiotic resistant bacteria, has become a worrisome superbug worldwide. This work was aimed at studying the efficacies of two class IIb bacteriocins, enterocins DD28 and DD93, against MRSA-S1 grown in planktonic culture and embedded in biofilms. These bacteriocins were purified, from the cultures supernatants of Enterococcus faecalis 28 and 93, using a simplified purification procedure consisting in a cation exchange chromatography and a reversed-phase high-performance liquid chromatography. The anti-Staphylococcal activity of these bacteriocins was shown in-vitro by the assessment of the minimal inhibitory concentration (MIC. Afterwhich, a checkerboard and time-kill kinetics permitted unveiled a synergistic effect of these bacteriocins in combination with erythromycin and kanamycin against the clinical MRSA-S1. These bacteriocins alone or in combination with erythromycin and kanamycin were able to impede the formation of MRSA-S1 biofilms on stainless steel and glace devices as supported by the microbial cell counts, epifluorescence and Scanning Electron Microscope analyses.

Cathodic voltage-controlled electrical stimulation of titanium for prevention of methicillin-resistant Staphylococcus aureus and Acinetobacter baumannii biofilm infections.

Science.gov (United States)

Canty, Mary; Luke-Marshall, Nicole; Campagnari, Anthony; Ehrensberger, Mark

2017-01-15

Antibiotic resistance of bacterial biofilms limits available treatment methods for implant-associated orthopaedic infections. This study evaluated the effects of applying cathodic voltage-controlled electrical stimulations (CVCES) of -1.5V and -1.8V (vs. Ag/AgCl) to coupons of commercially pure titanium (cpTi) incubated in cultures of methicillin-resistant Staphylococcus aureus (MRSA) and Acinetobacter baumannii (A. baumannii) as a method of preventing bacterial attachment. Stimulations were applied for 2, 4, and 8h and coupon-associated and planktonic colony-forming units (CFU) were enumerated following stimulation. Compared to open circuit potential (OCP) controls, CVCES for 4h at -1.8V significantly reduced coupon-associated MRSA CFU by 99.9% (1.30×10 4 vs. 4.45×10 7 , p=0.047) and A. baumannii coupon-associated CFU by 99.9% (1.64×10 4 vs. 5.93×10 7 , p=0.001) and reduced planktonic CFU below detectable levels for both strains. CVCES at -1.8V for 8h also reduced coupon-associated and planktonic CFU below detectable levels for each strain. CVCES at -1.5V for 4 and 8h, and -1.8V for 2h did not result in clinically relevant reductions. For 4 and 8h stimulations, the current density was significantly higher for -1.8V than -1.5V, an effect directly related to the rate of water and oxygen reduction on the cpTi surface. This significantly increased the pH, a suspected influence in decreased CFU viability. The voltage-dependent electrochemical properties of cpTi likely contribute to the observed antimicrobial effects of CVCES. This study revealed that CVCES of titanium could prevent coupon-associated and planktonic CFU of Gram-positive MRSA and Gram-negative A. baumannii from reaching detectable levels in a magnitude-dependent and time-dependent manner. Periprosthetic joint infection is a devastating outcome of total joint arthroplasty and has led to increased patient morbidity and rising healthcare costs. Current treatments are limited by the growing prevalence of

Chamaecyparis obtusa Essential Oil Inhibits Methicillin-Resistant Staphylococcus aureus Biofilm Formation and Expression of Virulence Factors.

Science.gov (United States)

Kim, Eun-Sook; Kang, Sun-Young; Kim, Young-Hoi; Lee, Young-Eun; Choi, Na-Young; You, Yong-Ouk; Kim, Kang-Ju

2015-07-01

The emergence of antibiotic-resistant bacteria has caused difficulty in treating infectious diseases. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most commonly recognized antibiotic-resistant bacteria. Novel antibiotics are urgently required to treat these bacteria. Raw materials derived from natural sources can be used for the development of novel antibiotics, such as Chamaecyparis obtusa (C. obtusa), which has been traditionally used in treating asthmatic disease. In this study, the antibacterial activity of the essential oil (EO) extracted from C. obtusa leaves against MRSA was investigated. MRSA growth and acid production from glucose metabolism were inhibited at concentrations greater than 0.1 mg/mL C. obtusa EO. MRSA biofilm formation was observed using scanning electron microscopy and safranin staining. C. obtusa EO inhibited MRSA biofilm formation at concentrations greater than 0.1 mg/mL. Using real-time polymerase chain reaction, mRNA expression of virulence factor genes, sea, agrA, and sarA, was observed. agrA expression was inhibited with C. obtusa EO concentrations greater than 0.2 mg/mL, whereas inhibition of sea and sarA expression was also observed at a concentration of 0.3 mg/mL. C. obtusa EO was analyzed by gas chromatography (GC) and GC coupled for mass spectrometry, which identified 59 constituents, accounting to 98.99% of the total EO. These findings suggest that C. obtusa EO has antibacterial effects against MRSA, which might be associated with the major components of C. obtusa EO, such as sabinene (19.06%), α-terpinyl acetate (16.99%), bornyl acetate (10.48%), limonene (8.54%), elemol (7.47%), myrcene (5.86%), γ-terpinene (4.04%), and hibaene (3.01%).

Effect of repeated tracheostomy tube reprocessing on biofilm formation.

Science.gov (United States)

Rodney, Jennifer; Ojano-Dirain, Carolyn P; Antonelli, Patrick J; Silva, Rodrigo C

2016-04-01

To determine the effect of repeated reprocessing of pediatric tracheostomy tubes (TTs) on biofilm formation. In vitro microbiological study. Pediatric, uncuffed, polyvinyl chloride (PVC) TTs from two different manufacturers (Tracoe Mini and Shiley) were reprocessed mechanically with household detergent and soaked in sodium hypochlorite (bleach). Two TTs of each brand were reprocessed 0 (control), 10, or 20 times. Twenty 2-mm coupons were then obtained from each TT, immersed in human mucus, and cultured with either Staphylococcus aureus or Pseudomonas aeruginosa. Biofilm formation was evaluated with bacterial counts. Bacterial counts of S. aureus for both brands were significantly higher on the TTs that were reprocessed 20 times compared to those that were not reprocessed (Tracoe: P = .040, Shiley: P  attachment. Further investigation is needed to determine the optimal technique and limits of reprocessing TTs in clinical practice. NA. Laryngoscope published by Wiley on behalf of the American Laryngological, Rhinological and Otological Society, Inc, “The Triological Society” and American Laryngological Association (the “Owner”).

Evaluation of the biofilm forming ability and its associated genes in Staphylococcus species isolates from bovine mastitis in Argentinean dairy farms.

Science.gov (United States)

Felipe, Verónica; Morgante, Carolina A; Somale, Paola S; Varroni, Florencia; Zingaretti, María L; Bachetti, Romina A; Correa, Silvia G; Porporatto, Carina

2017-03-01

Staphylococcus aureus and coagulase-negative staphylococci (CNS) are important causes of intramammary infection in dairy cattle, and their ability to produce biofilm is considered an important virulence property in the pathogenesis of mastitis. However, the published date on mechanisms and factors involved in infection persistence in the mammary gland remains unclear. The aim of this study was to investigate whether the main Staphylococcus species involved in bovine intramammary infections possess specific characteristics that promote colonization of the udder. We evaluated the biofilm-forming ability and distribution of adhesion- and biofilm-associated genes of Staphylococcus spp. isolated from bovine mastitis infected animals in Argentinean dairy farms. For this purpose, the phenotypic biofilm formation ability of 209 Staphylococcus spp. from bovine mastitis was investigated. All isolates produced biofilm in vitro, being 35,0% and 45,0% of the 127 S. aureus or 51,0% and 29,0% of the 82 CNS strong and moderate biofilm producers respectively. All S. aureus samples were PCR-positive for icaA, icaD, clfA, clfB and fnbpA genes, 76.3% were positive for fnbpB gene and 11.0% were positive for bap gene. In CNS isolates, the positive rates for icaA and icaD were 73.2%, while for clfA, clfB, fnbpA fnbpB and bap genes the percentage were lower. The results demonstrate that in Staphylococcus spp. biofilm formation, the polysaccharide and the adhesion- and biofilm-associated genes are of overall importance on bovine mastitis in Argentina. Therefore, future works should focus on these pathogenic specific factors for the development of more effective therapies of control, being essential to consider the ability of isolates to produce biofilm. Copyright © 2017 Elsevier Ltd. All rights reserved.

New approaches to combat Porphyromonas gingivalis biofilms

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Gerits, Evelien; Verstraeten, Natalie; Michiels, Jan

2017-01-01

ABSTRACT In nature, bacteria predominantly reside in structured, surface-attached communities embedded in a self-produced, extracellular matrix. These so-called biofilms play an important role in the development and pathogenesis of many infections, as they are difficult to eradicate due to their resistance to antimicrobials and host defense mechanisms. This review focusses on the biofilm-forming periodontal bacterium Porphyromonas gingivalis. Current knowledge on the virulence mechanisms underlying P. gingivalis biofilm formation is presented. In addition, oral infectious diseases in which P. gingivalis plays a key role are described, and an overview of conventional and new therapies for combating P. gingivalis biofilms is given. More insight into this intriguing pathogen might direct the development of better strategies to combat oral infections. PMID:28473880

Evaluation of various metallic coatings on steel to mitigate biofilm formation.

Science.gov (United States)

Kanematsu, Hideyuki; Ikigai, Hajime; Yoshitake, Michiko

2009-02-01

In marine environments and water systems, it is easy for many structures to form biofilms on their surfaces and to be deteriorated due to the corrosion caused by biofilm formation by bacteria. The authors have investigated the antibacterial effects of metallic elements in practical steels so far to solve food-related problems, using Escherichia coli and Staphylococcus aureus. However, from the viewpoint of material deterioration caused by bacteria and their antifouling measures, we should consider the biofilm behavior as aggregate rather than individual bacterium. Therefore, we picked up Pseudomonas aeruginosa and Pseudoalteromonas carageenovara in this study, since they easily form biofilms in estuarine and marine environments. We investigated what kind of metallic elements could inhibit the biofilm formation at first and then discussed how the thin films of those inhibitory elements on steels could affect biofilm formation. The information would lead to the establishment of effective antifouling measures against corrosion in estuarine and marine environments.

Evaluation of Various Metallic Coatings on Steel to Mitigate Biofilm Formation

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Hajime Ikigai

2009-02-01

Full Text Available In marine environments and water systems, it is easy for many structures to form biofilms on their surfaces and to be deteriorated due to the corrosion caused by biofilm formation by bacteria. The authors have investigated the antibacterial effects of metallic elements in practical steels so far to solve food-related problems, using Escherichia coli and Staphylococcus aureus. However, from the viewpoint of material deterioration caused by bacteria and their antifouling measures, we should consider the biofilm behavior as aggregate rather than individual bacterium. Therefore, we picked up Pseudomonas aeruginosa and Pseudoalteromonas carageenovara in this study, since they easily form biofilms in estuarine and marine environments. We investigated what kind of metallic elements could inhibit the biofilm formation at first and then discussed how the thin films of those inhibitory elements on steels could affect biofilm formation. The information would lead to the establishment of effective antifouling measures against corrosion in estuarine and marine environments.

Management of dental unit waterline biofilms in the 21st century.

LENUS (Irish Health Repository)

O'Donnell, Mary J

2011-10-01

Dental chair units (DCUs) use water to cool and irrigate DCU-supplied instruments and tooth surfaces, and provide rinsewater during dental treatment. A complex network of interconnected plastic dental unit waterlines (DUWLs) supply water to these instruments. DUWLs are universally prone to microbial biofilm contamination seeded predominantly from microorganisms in supply water. Consequently, DUWL output water invariably becomes contaminated by high densities of microorganisms, principally Gram-negative environmental bacteria including Pseudomonas aeruginosa and Legionella species, but sometimes contain human-derived pathogens such as Staphylococcus aureus. Patients and staff are exposed to microorganisms from DUWL output water and to contaminated aerosols generated by DCU instruments. A wide variety of approaches, many unsuccessful, have been proposed to control DUWL biofilm. More recently, advances in biofilm science, chemical DUWL biofilm treatment agents, DCU design, supply water treatment and development of automated DUWL biofilm control systems have provided effective long-term solutions to DUWL biofilm control.

Phenotypic Variation Is Almost Entirely Independent of the Host-Pathogen Relationship in Clinical Isolates of S. aureus.

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Adrian D Land

Full Text Available A key feature of Staphylococcus aureus biology is its ability to switch from an apparently benign colonizer of ~30% of the population to a cutaneous pathogen, to a deadly invasive pathogen. Little is known about the mechanisms driving this transition or the propensity of different S. aureus strains to engender different types of host-pathogen interactions. At the same time, significant weight has been given to the role of specific in vitro phenotypes in S. aureus virulence. Biofilm formation, hemolysis and pigment formation have all been associated with virulence in mice.To determine if there is a correlation between in vitro phenotype and the three types of host-pathogen relationships commonly exhibited by S. aureus in the context of its natural human host, we assayed 300 clinical isolates for phenotypes implicated in virulence including hemolysis, sensitivity to autolysis, and biofilm formation. For comparative purposes, we also assayed phenotype in 9 domesticated S. aureus strains routinely used for analysis of virulence determinants in laboratory settings.Strikingly, the clinical strains exhibited significant phenotypic uniformity in each of the assays evaluated in this study. One exception was a small, but significant, correlation between an increased propensity for biofilm formation and isolation from skin and soft tissue infections (SSTIs. In contrast, we observed a high degree of phenotypic variation between common laboratory strains that exhibit virulence in mouse models. These data suggest the existence of significant evolutionary pressure on the S. aureus genome and highlight a role for host factors as a strong determinant of the host-pathogen relationship. In addition, the high degree of variation between laboratory strains emphasizes the need for caution when applying data obtained in one lab strain to the analysis of another.

Hygrocin C from marine-derived Streptomyces sp. SCSGAA 0027 inhibits biofilm formation in Bacillus amyloliquefaciens SCSGAB0082 isolated from South China Sea gorgonian.

Science.gov (United States)

Wang, Jie; Nong, Xu-Hua; Amin, Muhammad; Qi, Shu-Hua

2018-02-01

Several ansamycins have been reported to inhibit bacterial biofilm formation and accelerate the eradication of developed biofilms, but little is known about the effect of hygrocin C, an ansamycin, on bacterial biofilm formation. Here, hygrocin C was isolated from the marine-derived Streptomyces sp. SCSGAA 0027 and reported for the first time to be capable of inhibiting the biofilm formation of Staphylococcus aureus and Bacillus amyloliquefaciens SCSGAB0082 with the production of anti-microbial lipopeptides from South China Sea gorgonian Subergorgia suberosa at concentrations of less than minimum inhibitory concentrations. Moreover, hygrocin C also promoted the eradication of developed biofilms, affected the biofilm architecture, and lowered the extracellular polymeric matrix formation, cell motility, and surface hydrophobicity in B. amyloliquefaciens, which was in accordance with the inhibition of biofilm formation. Furthermore, transcriptome analysis revealed that hygrocin C altered the transcripts of several genes associated with bacterial chemotaxis and flagellar, two-component system and the synthesis of arginine and histidine, which are important for bacterial biofilm formation. In conclusion, hygrocin C could be used as a potential biofilm inhibitor against S. aureus and B. amyloliquefaciens. But further genetic investigations are needed to provide more details for elucidation of the molecular mechanisms responsible for the effects of hygrocin C on B. amyloliquefaciens biofilm formation.

Pattern differentiation in co-culture biofilms formed by Staphylococcus aureus and Pseudomonas aeruginosa

DEFF Research Database (Denmark)

Yang, Liang; Liu, Yang; Markussen, Trine

2011-01-01

Biofilm infections may not simply be the result of colonization by one bacterium, but rather the consequence of pathogenic contributions from several bacteria. Interspecies interactions of different organisms in mixed-species biofilms remain largely unexplained, but knowledge of these is very imp...

Role of bacterial efflux pumps in biofilm formation.

Science.gov (United States)

Alav, Ilyas; Sutton, J Mark; Rahman, Khondaker Miraz

2018-02-28

Efflux pumps are widely implicated in antibiotic resistance because they can extrude the majority of clinically relevant antibiotics from within cells to the extracellular environment. However, there is increasing evidence from many studies to suggest that the pumps also play a role in biofilm formation. These studies have involved investigating the effects of efflux pump gene mutagenesis and efflux pump inhibitors on biofilm formation, and measuring the levels of efflux pump gene expression in biofilms. In particular, several key pathogenic species associated with increasing multidrug resistance, such as Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, have been investigated, whilst other studies have focused on Salmonella enterica serovar Typhimurium as a model organism and problematic pathogen. Studies have shown that efflux pumps, including AcrAB-TolC of E. coli, MexAB-OprM of P. aeruginosa, AdeFGH of A. baumannii and AcrD of S. enterica, play important roles in biofilm formation. The substrates for such pumps, and whether changes in their efflux activity affect biofilm formation directly or indirectly, remain to be determined. By understanding the roles that efflux pumps play in biofilm formation, novel therapeutic strategies can be developed to inhibit their function, to help disrupt biofilms and improve the treatment of infections. This review will discuss and evaluate the evidence for the roles of efflux pumps in biofilm formation and the potential approaches to overcome the increasing problem of biofilm-based infections.

Copper-Containing Anti-Biofilm Nanofiber Scaffolds as a Wound Dressing Material.

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Jayesh J Ahire

Full Text Available Copper particles were incorporated into nanofibers during the electrospinning of poly-D,L-lactide (PDLLA and poly(ethylene oxide (PEO. The ability of the nanofibers to prevent Pseudomonas aeruginosa PA01 and Staphylococcus aureus (strain Xen 30 to form biofilms was tested. Nanofibers containing copper particles (Cu-F were thinner (326 ± 149 nm in diameter, compared to nanofibers without copper (CF; 445 ± 93 nm in diameter. The crystalline structure of the copper particles in Cu-F was confirmed by X-ray diffraction (XRD. Copper crystals were encapsulated, but also attached to the surface of Cu-F, as shown scanning transmission electron microscopy (STEM and transmission electron microscopy (TEM, respectively. The copper particles had no effect on the thermal degradation and thermal behaviour of Cu-F, as shown by thermogravimetric analysis (TGA and differential scanning calorimeter (DSC. After 48 h in the presence of Cu-F, biofilm formation by P. aeruginosa PA01 and S. aureus Xen 30 was reduced by 41% and 50%, respectively. Reduction in biofilm formation was ascribed to copper released from the nanofibers. Copper-containing nanofibers may be incorporated into wound dressings.

Silver nanoparticles for the inhibition of Staphylococcus aureus

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Miguel Ángel Ortiz-Gila

2015-01-01

Full Text Available Existe un gran ecosistema microbiano en la cavidad oral donde Staphylococcus aureus ( S. aureus se puede encontrar, causando patologías orales tales como quelitis angular, las paperas y la mucositis estafilocócica. Estas enfermedades producidas por S. aureus en la cavidad oral son consecuencia de los factores de virulencia, toxinas y multiresistencia a los antibióticos, lo que contribuye a la infección. La colonización en la cavidad oral por S. aureus en pacientes sanos es de 24% a 36%. Sin embargo, la incidencia aumenta a 48% en pacientes con prótesis debido a la formación de biofilms en la superficie de las dentaduras postizas. Actualmente, no existe ningún tratamiento para infecciones orales sin el uso de antibióticos. Investigaciones recientes indican que las nanopartículas de plata (AgNPs son un material o estrategia para eliminar S. aureus debido a su efecto antibacteriano. Sin embargo, el mecanismo del efecto inhibidor de los iones de Ag sobre S. aureus es sólo parcialmente conocida y muy poco se ha informado. Por lo tanto, el propósito de la presente revisión sistemática es determinar las estrategias y retos de la utilización de biomateriales antimicrobianos con AgNPs frente a las infecciones orales de S. aureus.

Effect of Carvacrol on Salmonella Saintpaul Biofilms on Stainless ...

African Journals Online (AJOL)

HP

Nostro A, Sudano Roccaro A, Bisignano G, Marino A,. Cannatelli MA, Pizzimenti FC, Cioni PL, Procopio F,. Blanco AR. Effects of oregano, carvacrol and thymol on Staphylococcus aureus and Staphylococcus epidermidis biofilms. J Med Microbiol 2007; 56: 519-. 523. 7. Pérez-Conesa D, Cao J, Chen L, McLandsborough L,.

Staphylococcal biofilm exopolysaccharide protects against Caenorhabditis elegans immune defenses.

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Jakob Begun

2007-04-01

Full Text Available Staphylococcus epidermidis and Staphylococcus aureus are leading causes of hospital-acquired infections that have become increasingly difficult to treat due to the prevalence of antibiotic resistance in these organisms. The ability of staphylococci to produce biofilm is an important virulence mechanism that allows bacteria both to adhere to living and artificial surfaces and to resist host immune factors and antibiotics. Here, we show that the icaADBC locus, which synthesizes the biofilm-associated polysaccharide intercellular adhesin (PIA in staphylococci, is required for the formation of a lethal S. epidermidis infection in the intestine of the model nematode Caenorhabditis elegans. Susceptibility to S. epidermidis infection is influenced by mutation of the C. elegans PMK-1 p38 mitogen-activated protein (MAP kinase or DAF-2 insulin-signaling pathways. Loss of PIA production abrogates nematocidal activity and leads to reduced bacterial accumulation in the C. elegans intestine, while overexpression of the icaADBC locus in S. aureus augments virulence towards nematodes. PIA-producing S. epidermidis has a significant survival advantage over ica-deficient S. epidermidis within the intestinal tract of wild-type C. elegans, but not in immunocompromised nematodes harboring a loss-of-function mutation in the p38 MAP kinase pathway gene sek-1. Moreover, sek-1 and pmk-1 mutants are equally sensitive to wild-type and icaADBC-deficient S. epidermidis. These results suggest that biofilm exopolysaccharide enhances virulence by playing an immunoprotective role during colonization of the C. elegans intestine. These studies demonstrate that C. elegans can serve as a simple animal model for studying host-pathogen interactions involving staphylococcal biofilm exopolysaccharide and suggest that the protective activity of biofilm matrix represents an ancient conserved function for resisting predation.

Bacillus cereus growth and biofilm formation: the impact of substratum, iron sources, and transcriptional regulator Sigma 54

NARCIS (Netherlands)

Hayrapetyan, Hasmik

2017-01-01

Biofilms are surface-associated communities of microbial cells embedded in a matrix of extracellular polymers. It is generally accepted that the biofilm growth mode represents the most common lifestyle of microorganisms. Next to beneficial biofilms used in biotechnology applications, undesired

Brief ultrasonication improves detection of biofilm-formative bacteria around a metal implant.

Science.gov (United States)

Kobayashi, Naomi; Bauer, Thomas W; Tuohy, Marion J; Fujishiro, Takaaki; Procop, Gary W

2007-04-01

Biofilms are complex microenvironments produced by microorganisms on surfaces. Ultrasonication disrupts biofilms and may make the microorganism or its DNA available for detection. We determined whether ultrasonication could affect our ability to detect bacteria adherent to a metal substrate. A biofilm-formative Staphylococcus aureus strain was used for an in vitro implant infection model (biofilm-formative condition). We used quantitative culture and real time-polymerase chain reaction to determine the influence of different durations of ultrasound on bacterial adherence and viability. Sonication for 1 minute increased the yield of bacteria. Sonication longer than 5 minutes led to fewer bacterial colonies by conventional culture but not by polymerase chain reaction. This suggests short periods of sonication help release bacteria from the metal substrate by disrupting the biofilm, but longer periods of sonication lyse bacteria prohibiting their detection in microbiologic cultures. A relatively short duration of sonication may be desirable for maximizing detection of biofilm-formative bacteria around implants by culture or polymerase chain reaction.

Exposure of Bacterial Biofilms to Electrical Current Leads to Cell Death Mediated in Part by Reactive Oxygen Species.

Science.gov (United States)

Brinkman, Cassandra L; Schmidt-Malan, Suzannah M; Karau, Melissa J; Greenwood-Quaintance, Kerryl; Hassett, Daniel J; Mandrekar, Jayawant N; Patel, Robin

2016-01-01

Bacterial biofilms may form on indwelling medical devices such as prosthetic joints, heart valves and catheters, causing challenging-to-treat infections. We have previously described the 'electricidal effect', in which bacterial biofilms are decreased following exposure to direct electrical current. Herein, we sought to determine if the decreased bacterial quantities are due to detachment of biofilms or cell death and to investigate the role that reactive oxygen species (ROS) play in the observed effect. Using confocal and electron microscopy and flow cytometry, we found that direct current (DC) leads to cell death and changes in the architecture of biofilms formed by Gram-positive and Gram-negative bacteria. Reactive oxygen species (ROS) appear to play a role in DC-associated cell death, as there was an increase in ROS-production by Staphylococcus aureus and Staphylococcus epidermidis biofilms following exposure to DC. An increase in the production of ROS response enzymes catalase and superoxide dismutase (SOD) was observed for S. aureus, S. epidermidis and Pseudomonas aeruginosa biofilms following exposure to DC. Additionally, biofilms were protected from cell death when supplemented with antioxidants and oxidant scavengers, including catalase, mannitol and Tempol. Knocking out SOD (sodAB) in P. aeruginosa led to an enhanced DC effect. Microarray analysis of P. aeruginosa PAO1 showed transcriptional changes in genes related to the stress response and cell death. In conclusion, the electricidal effect results in death of bacteria in biofilms, mediated, at least in part, by production of ROS.

Commensal coagulase-negative Staphylococcus from the udder of healthy cows inhibits biofilm formation of mastitis-related pathogens.

Science.gov (United States)

Isaac, Paula; Bohl, Luciana Paola; Breser, María Laura; Orellano, María Soledad; Conesa, Agustín; Ferrero, Marcela Alejandra; Porporatto, Carina

2017-08-01

Bovine mastitis, considered the most important cause of economic losses in the dairy industry, is a major concern in veterinary medicine. Staphylococcus aureus and coagulase-negative staphylococci (CNS) are the main pathogens associated with intramammary infections, and bacterial biofilms are suspected to be responsible for the persistence of this disease. CNS from the udder are not necessarily associated with intramammary infections. In fact, some commensal CNS have been shown to have biological activities. This issue led us to screen exoproducts from commensal Staphylococcus chromogenes for anti-biofilm activity against different mastitis pathogens. The cell-free supernatant from S. chromogenes LN1 (LN1-CFS) was confirmed to display a non-biocidal inhibition of pathogenic biofilms. The supernatant was subjected to various treatments to estimate the nature of the biofilm-inhibiting compounds. The results showed that the bioactive compound >5KDa in mass is sensitive to thermal treatment and proteinase K digestion, suggesting its protein properties. LN1-CFS was able to significantly inhibit S. aureus and CNS biofilm formation in a dose-independent manner and without affecting the viability of bovine cells. These findings reveal a new activity of the udder microflora of healthy animals. Studies are underway to purify and identify the anti-biofilm biocompound and to evaluate its biological activity in vivo. Copyright © 2017 Elsevier B.V. All rights reserved.

Photodynamic inactivation of Staphylococcus aureus and Escherichia coli using a new bacteriochlorin as photosensitizer

Science.gov (United States)

Barboza, Diego D.; Martins, Laura C. A.; Corrêa, Thaila Quatrini; Geralde, Mariana Carreira; Pratavieira, Sebastião.; de Oliveira, Kleber Thiago; Uliana, Marciana P.; de Souza, Clovis W.

2018-02-01

In this study, we used bacteriochlorin as a photosensitizer, characterized by their low toxicity in the absence of light, presenting absorption around 780 nm, with the objective of evaluating their photodynamic inactivation potential on Staphylococcus aureus and Escherichia coli. Bacteriochlorins were synthesized from the extraction of bacteriochlorophylls from non-sulfurous purple bacteria and were then converted to bacteriochlorins. S. aureus and E. coli microorganisms were used in the planktonic and biofilm forms. For the formation of biofilms on glass coverslips, suspensions of the microorganisms at the concentration of 106 CFU/mL were inoculated into each well of a microplate. There was an exchange of culture medium (Tryptic Soy Broth - TSB) every 24 hours for 7 days, pre-washing the coverslips with a phosphate-buffered saline (PBS), to ensure that only adhered microorganisms were grown and then incubated at (36 +/- 1)°C between the middle exchanges. After 7 days of induction, the biofilm was mature, like those normally found in nature, and then it was applied different treatments (light doses associated with FS concentrations). At the end of the treatment, the coverslips underwent an ultrasonic disintegration, and the quantitative evaluation of viable cells was performed by plate counting using the plate method in Tryptic Soy Agar (TSA), incubating at (36 +/- 1)°C for 24 hours. The results showed that the PDI for E. coli was not successful even when it was more susceptible to the planktonic form, whereas for S. aureus the results showed a reduction in cell viability 6 logs for the planktonic forms, but lower to 1 log in biofilms. Therefore, novel studies using bacteriochlorins and surfactants will be performed to verify the potential of this alternative treatment method.

Biofilm synthesis and its relationship with genetic characteristics in clinical methicillin-resistant staphylococci

Directory of Open Access Journals (Sweden)

Nikolaos Giormezis

2015-09-01

Full Text Available Staphylococcus aureus can cause a broad range of infections, including skin infections, pneumonia and bacteraemia. Coagulase-negative staphylococci (CNS, mainly S. epidermidis, have also emerged as important pathogens, especially in immunocompromised patients or those with prosthetic devices, such as intravascular catheters or biomaterials. Of great importance in the initiation of these infections is the ability of staphylococci to adhere to various surfaces, such as host tissues and prosthetic devices and to form biofilm. The staphylococcal adhesins are encoded by a number of genes such as fnbA (S. aureus fibronectin binding protein A, sasG (S. aureus surface protein G, aap (S. epidermidis accumulation associated protein, bhp (Bap homologue protein and fbe (fibrinogen binding protein epidermidis. In this study, 106 methicillin-resistant S. aureus (MRSA, 145 methicillin-resistant S. epidermidis (MRSE and 70 non-epidermidis methicillin-resistant CNS (MR-CNS; 58 S. haemolyticus, 10 S. hominis and two S. lugdunensis were compared in terms of biofilm formation, antimicrobial resistance, clonal distribution and adhesin genes carriage. Isolates were classified into pulsotypes by PFGE and assigned to sequence types by MLST. In total, 121/321 isolates (37.7% produced biofilm and 219 (68.2% carried ica operon. The majority was multidrug resistant (94.7% and carried one or more adhesin genes. MRSE and all other MR-CNS prevailed in biofilm formation (P < 0.001 and antimicrobial resistance (P < 0.05 as compared to MRSA. MRSE also prevailed in ica carriage compared to the other methicillin-resistant staphylococci (P ≤ 0.007 Among MRSE, isolates from bacteraemias prevailed in biofilm formation (P = 0.031, whereas, strains from prosthetic device-associated infections carried more frequently aap (P = 0.003. Even though PFGE showed genetic diversity among MRSE, MLST revealed three major clones (ST2, ST5, ST16. MRSA isolates were less diverse, with five PFGE

Bovine origin Staphylococcus aureus: A new zoonotic agent?

Science.gov (United States)

Rao, Relangi Tulasi; Jayakumar, Kannan; Kumar, Pavitra

2017-10-01

The study aimed to assess the nature of animal origin Staphylococcus aureus strains. The study has zoonotic importance and aimed to compare virulence between two different hosts, i.e., bovine and ovine origin. Conventional polymerase chain reaction-based methods used for the characterization of S. aureus strains and chick embryo model employed for the assessment of virulence capacity of strains. All statistical tests carried on R program, version 3.0.4. After initial screening and molecular characterization of the prevalence of S. aureus found to be 42.62% in bovine origin samples and 28.35% among ovine origin samples. Meanwhile, the methicillin-resistant S. aureus prevalence is found to be meager in both the hosts. Among the samples, only 6.8% isolates tested positive for methicillin resistance. The biofilm formation quantified and the variation compared among the host. A Welch two-sample t -test found to be statistically significant, t=2.3179, df=28.103, and p=0.02795. Chicken embryo model found effective to test the pathogenicity of the strains. The study helped to conclude healthy bovines can act as S. aureus reservoirs. Bovine origin S. aureus strains are more virulent than ovine origin strains. Bovine origin strains have high probability to become zoonotic pathogen. Further, gene knock out studies may be conducted to conclude zoonocity of the bovine origin strains.

Host-pathogen interactions in bovine mammary epithelial cells and HeLa cells by Staphylococcus aureus isolated from subclinical bovine mastitis.

Science.gov (United States)

Castilho, Ivana G; Dantas, Stéfani Thais Alves; Langoni, Hélio; Araújo, João P; Fernandes, Ary; Alvarenga, Fernanda C L; Maia, Leandro; Cagnini, Didier Q; Rall, Vera L M

2017-08-01

Staphylococcus aureus is a common pathogen that causes subclinical bovine mastitis due to several virulence factors. In this study, we analyzed S. aureus isolates collected from the milk of cows with subclinical mastitis that had 8 possible combinations of bap, icaA, and icaD genes, to determine their capacity to produce biofilm on biotic (bovine primary mammary epithelial cells and HeLa cells) and abiotic (polystyrene microplates) surfaces, and their ability to adhere to and invade these cells. We also characterized isolates for microbial surface components recognizing adhesive matrix molecules (MSCRAMM) and agr genes, and for their susceptibility to cefquinome sulfate in the presence of biofilm. All isolates adhered to and invaded both cell types, but invasion indexes were higher in bovine primary mammary epithelial cells. Using tryptic soy broth + 1% glucose on abiotic surfaces, 5 out of 8 isolates were biofilm producers, but only the bap + icaA + icaD + isolate was positive in Dulbecco's Modified Eagle's medium. The production of biofilm on biotic surfaces occurred only with this isolate and only on HeLa cells, because the invasion index for bovine primary mammary epithelial cells was too high, making it impossible to use these cells in this assay. Of the 5 biofilm producers in tryptic soy broth + 1% glucose, 4 presented with the bap/fnbA/clfA/clfB/eno/fib/ebpS combination, and all were protected from cefquinome sulfate. We found no predominance of any agr group. The high invasive potential of S. aureus made it impossible to observe biofilm in bovine primary mammary epithelial cells, and we concluded that cells with lower invasion rates, such as HeLa cells, were more appropriate for this assay. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Elasticity and physico-chemical properties during drinking water biofilm formation.

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Abe, Yumiko; Polyakov, Pavel; Skali-Lami, Salaheddine; Francius, Grégory

2011-08-01

Atomic force microscope techniques and multi-staining fluorescence microscopy were employed to study the steps in drinking water biofilm formation. During the formation of a conditioning layer, surface hydrophobic forces increased and the range of characteristic hydrophobic forces diversified with time, becoming progressively complex in macromolecular composition, which in return triggered irreversible cellular adhesion. AFM visualization of 1 to 8 week drinking water biofilms showed a spatially discontinuous and heterogeneous distribution comprising an extensive network of filamentous fungi in which biofilm aggregates were embedded. The elastic modulus of 40-day-old biofilms ranged from 200 to 9000 kPa, and the biofilm deposits with a height >0.5 μm had an elastic modulus water biofilms were composed of a soft top layer and a basal layer with significantly higher elastic modulus values falling in the range of fungal elasticity.

Fluorescence lifetime imaging of oxygen in dental biofilm

Science.gov (United States)

Gerritsen, Hans C.; de Grauw, Cees J.

2000-12-01

Dental biofilm consists of micro-colonies of bacteria embedded in a matrix of polysaccharides and salivary proteins. pH and oxygen concentration are of great importance in dental biofilm. Both can be measured using fluorescence techniques. The imaging of dental biofilm is complicated by the thickness of the biofilms that can be up to several hundred micrometers thick. Here, we employed a combination of two-photon excitation microscopy with fluorescence lifetime imaging to quantify the oxygen concentration in dental biofilm. Collisional quenching of fluorescent probes by molecular oxygen leads to a reduction of the fluorescence lifetime of the probe. We employed this mechanism to measure the oxygen concentration distribution in dental biofilm by means of fluorescence lifetime imaging. Here, TRIS Ruthenium chloride hydrate was used as an oxygen probe. A calibration procedure on buffers was use to measure the lifetime response of this Ruthenium probe. The results are in agreement with the Stern-Volmer equation. A linear relation was found between the ratio of the unquenched and the quenched lifetime and the oxygen concentration. The biofilm fluorescence lifetime imaging results show a strong oxygen gradient at the buffer - biofilm interface and the average oxygen concentration in the biofilm amounted to 50 μM.

Komodo dragon-inspired synthetic peptide DRGN-1 promotes wound-healing of a mixed-biofilm infected wound.

Science.gov (United States)

M C Chung, Ezra; Dean, Scott N; Propst, Crystal N; Bishop, Barney M; van Hoek, Monique L

2017-01-01

Cationic antimicrobial peptides are multifunctional molecules that have a high potential as therapeutic agents. We have identified a histone H1-derived peptide from the Komodo dragon ( Varanus komodoensis) , called VK25. Using this peptide as inspiration, we designed a synthetic peptide called DRGN-1. We evaluated the antimicrobial and anti-biofilm activity of both peptides against Pseudomonas aeruginosa and Staphylococcus aureus . DRGN-1, more than VK25, exhibited potent antimicrobial and anti-biofilm activity, and permeabilized bacterial membranes. Wound healing was significantly enhanced by DRGN-1 in both uninfected and mixed biofilm ( Pseudomonas aeruginosa and Staphylococcus aureus )-infected murine wounds. In a scratch wound closure assay used to elucidate the wound healing mechanism, the peptide promoted the migration of HEKa keratinocyte cells, which was inhibited by mitomycin C (proliferation inhibitor) and AG1478 (epidermal growth factor receptor inhibitor). DRGN-1 also activated the EGFR-STAT1/3 pathway. Thus, DRGN-1 is a candidate for use as a topical wound treatment. Wound infections are a major concern; made increasingly complicated by the emerging, rapid spread of bacterial resistance. The novel synthetic peptide DRGN-1 (inspired by a peptide identified from Komodo dragon) exhibits pathogen-directed and host-directed activities in promoting the clearance and healing of polymicrobial ( Pseudomonas aeruginosa & Staphylococcus aureus ) biofilm infected wounds. The effectiveness of this peptide cannot be attributed solely to its ability to act upon the bacteria and disrupt the biofilm, but also reflects the peptide's ability to promsote keratinocyte migration. When applied in a murine model, infected wounds treated with DRGN-1 healed significantly faster than did untreated wounds, or wounds treated with other peptides. The host-directed mechanism of action was determined to be via the EGFR-STAT1/3 pathway. The pathogen-directed mechanism of action was

Evaluation of CAMP-Like Effect, Biofilm Formation, and Discrimination of Candida africana from Vaginal Candida albicans Species

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Keyvan Pakshir

2017-01-01

Full Text Available Candida africana as a species recovered from female genital specimens is highly close to C. albicans. The present study was conducted to discriminate C. africana from presumptive vaginal C. albicans strains by molecular assay and evaluate their hemolysin activity, biofilm formation, and cohemolytic effect (CAMP with vaginal bacterial flora. A total of 110 stock vaginal C. albicans isolates were examined by HWP1 gene amplification. Hemolysin activity and the ability of biofilm formation were evaluated by blood plate assay and visual detection methods, respectively. Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus agalactiae were used to evaluate the CAMP-like effects in Sabouraud blood agar media. Based on the size of the amplicons (941 bp, all isolates were identified as C. albicans. All samples were able to produce beta-hemolysin. Moreover, 69 out of 110 of the isolates (62.7% were biofilm-positive, 54 out of 110 Candida isolates (49% demonstrated cohemolytic effects with S. agalactiae, and 48 out of 110 showed this effect with S. aureus (43.6%. All isolates were CAMP-negative with S. epidermidis. We detected all isolates as Candida albicans and almost half of the isolates were CAMP-positive with S. aureus and S. agalactiae, suggesting that these bacteria increase the pathogenicity of Candida in vaginal candidiasis.

Evaluation of CAMP-Like Effect, Biofilm Formation, and Discrimination of Candida africana from Vaginal Candida albicans Species

Science.gov (United States)

Bordbar, Mahboubeh; Nouraei, Hasti; Khodadadi, Hossein

2017-01-01

Candida africana as a species recovered from female genital specimens is highly close to C. albicans. The present study was conducted to discriminate C. africana from presumptive vaginal C. albicans strains by molecular assay and evaluate their hemolysin activity, biofilm formation, and cohemolytic effect (CAMP) with vaginal bacterial flora. A total of 110 stock vaginal C. albicans isolates were examined by HWP1 gene amplification. Hemolysin activity and the ability of biofilm formation were evaluated by blood plate assay and visual detection methods, respectively. Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus agalactiae were used to evaluate the CAMP-like effects in Sabouraud blood agar media. Based on the size of the amplicons (941 bp), all isolates were identified as C. albicans. All samples were able to produce beta-hemolysin. Moreover, 69 out of 110 of the isolates (62.7%) were biofilm-positive, 54 out of 110 Candida isolates (49%) demonstrated cohemolytic effects with S. agalactiae, and 48 out of 110 showed this effect with S. aureus (43.6%). All isolates were CAMP-negative with S. epidermidis. We detected all isolates as Candida albicans and almost half of the isolates were CAMP-positive with S. aureus and S. agalactiae, suggesting that these bacteria increase the pathogenicity of Candida in vaginal candidiasis. PMID:29318048

Effects of Local Delivery of d-amino Acids from Biofilm-dispersive Scaffolds on Infection in Contaminated Rat Segmental Defects

Science.gov (United States)

2013-07-05

2655e61. [26] Kolodkin-Gal I, Romero D, Cao S, Clardy J, Kolter R, Losick R. D-amino acids trigger biofilm disassembly. Science 2010;328:627e9. [27...Hochbaum AI, Kolodkin-Gal I, Foulston L, Kolter R, Aizenberg J, Losick R. Inhibitory effects of D-amino acids on Staphylococcus aureus biofilm devel

Antibacterial activity of Thymoquinone, an active principle of Nigella sativa and its potency to prevent bacterial biofilm formation

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Bakhrouf Amina

2011-04-01

Full Text Available Abstract Background Thymoquinone is an active principle of Nigella sativa seed known as "Habbah Al-Sauda" in Arabic countries and "Sinouj" in Tunisia. Bacterial biofilms tend to exhibit significant tolerance to antimicrobials drugs during infections. Methods The antibacterial activity of Thymoquinone (TQ and its biofilm inhibition potencies were investigated on 11 human pathogenic bacteria. The growth and development of the biofilm were assessed using the crystal violet (CV and the 2, 3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT reduction assay. Results TQ exhibited a significant bactericidal activity against the majority of the tested bacteria (MICs values ranged from 8 to 32 μg/ml especially Gram positive cocci (Staphylococcus aureus ATCC 25923 and Staphylococcus epidermidis CIP 106510. Crystal violet assay demonstrated that the minimum biofilm inhibition concentration (BIC50 was reached with 22 and 60 μg/ml for Staphylococcus aureus ATCC 25923 and Staphylococcus epidermidis CIP 106510 respectively. In addition our data revealed that cells oxidative activity was influenced by TQ supplementation. In the same way, TQ prevented cell adhesion to glass slides surface. Conclusion The ability of TQ to prevent biofilm formation warrants further investigation to explore its use as bioactive substances with antibiofilm potential.

Evaluación de estrategias de control frente a biopelículas de "Staphylococcus aureus" potencialmente presentes en la industria pesquera

OpenAIRE

Vázquez Sánchez, Daniel

2014-01-01

Staphylococcus aureus is one of the major bacterial agents causing foodborne diseases in humans worldwide, due to the ingestion of food containing staphylococcal enterotoxins. Spain is one of the largest producers and consumers of fishery products in the European Union. However, S. aureus is repeatedly detected in these products as a consequence of cross-contamination from food handlers and food contact surfaces. Biofilm formation also provides S. aureus a high tolerance to biocides allowing ...

Sinus biofilms in patients with cystic fibrosis: is adjusted eradication therapy needed?

DEFF Research Database (Denmark)

Aanaes, Kasper; Eickhardt, Steffen; Johansen, Helle Krogh

2015-01-01

The paranasal sinuses can be a focus for colonisation of the cystic fibrosis (CF) lungs with pathogens. In the sinuses, bacteria can adapt to the lung environment and enhance their antibiotic resistance, with biofilm formation thought to be the most important adaptive mechanism, causing recalcitr......The paranasal sinuses can be a focus for colonisation of the cystic fibrosis (CF) lungs with pathogens. In the sinuses, bacteria can adapt to the lung environment and enhance their antibiotic resistance, with biofilm formation thought to be the most important adaptive mechanism, causing...... recalcitrant disease. The presence of biofilms in CF sinuses is sparsely described. In this descriptive cross-sectional study, the sinus mucosa from 16 CF patients were analysed by fluorescence in situ hybridization using specific peptide nucleic acid (PNA-FISH) probes for Pseudomonas aeruginosa...... and Staphylococcus aureus to demonstrate the presence of biofilms. Small clusters of biofilm were visualised lining the sinus mucosa of CF patients. Biofilms were found in 10 out of 18 cases; 7 with intermittent lung colonisation, 2 chronically infected, and one lung transplanted patient. Finding P. aeruginosa...

Nanostructured coatings for controlling bacterial biofilms and antibiotic resistance

OpenAIRE

Ivanova, Kristina Dimitrova

2017-01-01

The accelerated emergence of drug resistant bacteria is one of the most serious problems in healthcare and the difficulties in finding new antibiotics make it even more challenging. To overcome the action of antibiotics bacteria develop effective resistance mechanisms including the formation of biofilms. Biofilms are bacterial communities of cells embedded in a self-produced polymeric matrix commonly found on medical devices such as indwelling catheters. When pathogens adopt this mode of grow...

Anaerobic Pseudomonas aeruginosa and other obligately anaerobic bacterial biofilms growing in the thick airway mucus of chronically infected cystic fibrosis patients: an emerging paradigm or "Old Hat"?

Science.gov (United States)

Su, Shengchang; Hassett, Daniel J

2012-09-01

The cystic fibrosis (CF) airway mucus is an ideal niche in which many bacteria can develop antibiotic- and phagocyte-resistance in unique structures known as "mode II biofilms" where bacteria are embedded within the mucus, yet unattached to airway epithelial cells. Pseudomonas aeruginosa is the dominant CF pathogen, yet herein the authors provide burgeoning evidence that obligate anaerobic bacteria (e.g., Prevotella) actually thrive within the CF mucus, a paradigmatic shift that chronic CF is an "aerobic" disease. Interestingly, CF organisms repress virulence factor production (e.g., P. aeruginosa) while others (e.g., S. aureus) increase them under anaerobic conditions. The authors shed additional light on (i) the anoxic nature of the CF airway mucus, (ii) the relative commonality of anaerobic bacteria isolated from CF sputum, (iii) virulence factor production and cross-talk between obligate anaerobes and P. aeruginosa relative to disease progression/remission, (iv) the role of mucoidy in CF, and (v) the role of nitrosative stress in activation of bacteriophage and pyocins within biofilms. The authors conclude with insight as to how we might treat some CF bacteria during mode II biofilm infections that utilizes a metabolite of bacterial anaerobic respiration and an aerobic oxidation product of airway-generated NO, acidified NO(2)(-).

Biofilm architecture of Phanerozoic cryptic carbonate marine veneers

Science.gov (United States)

Riding, Robert

2002-01-01

Thin (mushrooms, and plumes. All can be interpreted as characteristics of attached bacterial communities, i.e., aggregates as microcolonies, originally embedded in a matrix of extracellular polymeric substances; channels as water conduits and/or uncolonized nutrient-poor spaces; external protuberances as localized growths; and plumes as surface streamers. Cryptic habitat favored pristine biofilm preservation by precluding disturbance and overgrowth, and suggests aphotic and anoxic conditions. These examples provide diagnostic morphologic criteria for wider recognition of biofilm in Phanerozoic and older carbonates.

Methods and Guidance for Testing the Efficacy of Antimicrobials against Biofilm Bacteria on Hard, Non-Porous Surfaces

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EPA is announcing the availability of two test methods (MB-19 and MB-20) for evaluating the efficacy of antimicrobial pesticides against two biofilm bacteria, Pseudomonas aeruginosa and Staphylococcus aureus.

Antimicrobial effects of Piper hispidum extract, fractions and chalcones against Candida albicans and Staphylococcus aureus.

Science.gov (United States)

Costa, G M; Endo, E H; Cortez, D A G; Nakamura, T U; Nakamura, C V; Dias Filho, B P

2016-09-01

Three chalcones, 2'-hydroxy-4,4',6'-trimethoxychalcone, 2'-hydroxy-4,4',6'-tetramethoxychalcone, and 3,2'-dihydroxy-4,4',6'-trimethoxychalcone, were isolated from the leaves of Piper hispidum in a bioguided fractionation of crude extract. The antimicrobial activity of crude extract of P. hispidum leaves was determined against bacteria Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus and yeasts Candida albicans, C. parapsilosis and C. tropicalis. Fractions and chalcones were tested against C. albicans and S. aureus. The checkerboard assay was performed to assess synergic interactions between extract and antifungal drugs, and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay was used to evaluate anti-biofilm effects of extract. The extract was active against yeasts, S. aureus and B. subtilis with MIC values between 15.6 and 62.5μg/mL. Synergistic effects of extract associated with fluconazole and nystatin were observed against C. albicans, with fractional inhibitory concentration indices of 0.37 and 0.24, respectively. The extract was also effective against C. albicans and S. aureus biofilm cells at concentrations of 62.5 and 200μg/mL, respectively. Thus, P. hispidum may be a possible source of bioactive substances with antimicrobial properties. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Application of micro-PIV to the study of staphylococci bacteria biofilm dynamics

Science.gov (United States)

Sherman, Erica; Moormeier, Derek; Bayles, Kenneth; Wei, Timothy

2014-11-01

Staphylococci bacteria are recognized as the most frequent cause of biofilm-associated infections. A localized staph infection has the potential to enter the bloodstream and lead to serious infections such as endocarditis, pneumonia, or toxic shock syndrome. Changes in flow conditions, such as shear stress, can lead to stable biofilm growth or the dispersion of portions of the biofilm downstream. Exploration of biofilm physiology indicates a link between production of a specific enzyme called nuclease and biofilm architecture -; however the physical impact of this enzyme in directing the location and behavior of biofilm growth remains unclear. This talk investigates the link between sites of nuclease production and the development of biofilm tower structures using the application of micro-PIV and fluorescently labeled bacterial cells producing nuclease. Staphylococcus aureus bacteria were cultured in a BioFlux1000 square microchannel of a 65 by 65 um cross section, and subjected to a steady shear rate of 0.6 dynes. Micro-PIV and nuclease production measurements were taken to quantify the flow over a biofilm tower structure prior and during development. Data were recorded around the structure at a series of two dimensional planes, which when stacked vertically show a two dimensional flow field as a function of tower height.

Atomic Force Microscopy (AFM) for In-Situ Biofilm Surface Characterization during Free Chlorine and Monochloramine Exposure

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Drinking water distribution system biofilm are attached to pipe walls and found in sediments. These biofilms are complex and contain a variety of microorganisms embedded in a matrix with extracellular polymeric substances (EPS), providing protection from disinfection. Without pro...

Comparison of agglutinating and neutralizing antibodies to serovar hardjo in sows immunized with two commercial whole culture polivalent anti-leptospira bacterins

Science.gov (United States)

Soto, Francisco Rafael Martins; Pinheiro, Sônia Regina; Morais, Zenaide Maria; Gonçales, Amane Paldês; de Azevedo, Sérgio Santos; Bernardi, Fernanda; Camargo, Sebastião Rodrigues; Vasconcellos, Silvio Arruda

2008-01-01

It was performed the comparison of the intensity and duration of agglutinating and neutralizing antibodies to serovar Hardjo in swines vaccinated with two commercial anti-leptospira bacterins. Sows no reactive to 24 Leptospira sp serovars in the microscopic agglutination test (MAT) were divided in three groups: Group A (n=08): received two vaccine A doses with 30 days interval, Group B (n=08) two vaccine B doses with 30 days interval and Group C (n=08): control no vaccinated against leptospirosis.Blood samples were collected each 30 days during six months following the first vaccination. The sera were tested by MAT and growth inhibition test (GIT) to serovar Hardjo in order to evaluate respectively agglutinating and neutralizing antibodies. It was found that neutralizing antibodies persisted for a longer time than the agglutinating ones and that the absence of agglutinating antibodies does not means in the absence of the neutralizing. The peaks of agglutinating antibodies was obtained at least 30 days earlier than that produced by neutralizing. The duration of both kinds of antibodies measured differed between the two bacterines tested. The period for inducing neutralizing antibodies against serovar Hardjo indicated that gilts must be immunized with two doses of whole culture anti-leptospira bacterines applied 30 days each other at least 90 days before the first mating. For the maintenance of hight levels of neutralizing antibodies the revaccinations must be performed every six months after the first vaccination. PMID:24031250

Heterogeneous corrosion of mild steel under SRB-biofilm characterised by electrochemical mapping technique

International Nuclear Information System (INIS)

Dong Zehua; Shi Wei; Ruan Hongmei; Zhang Guoan

2011-01-01

Highlights: → Highly conductive SRB-biofilm can shield the potential differences of mild steel. → Potential maps fail to indicate the localised corrosion of steel under SRB-biofilm. → Galvanic current maps can detect the location of localised corrosion under biofilm. → SRB-biofilm is super-capacitive due to the conductive sulphide micropores. - Abstract: Heterogeneous corrosion of mild steel under sulphate reducing bacteria (SRB)-biofilm was characterised by wire beam electrode (WBE) technique and electrochemical impedance spectrum. The potential/current distributions of the WBE under SRB-biofilm showed that the potential maps could not indicate the localised corrosion of steels beneath biofilm due to the fact that all wire electrodes were short-circuited by the highly conductive sulphide precipitates embedded in SRB-biofilm. Instead, the galvanic current maps may give a good indication. The characteristic of super-capacitance (0.21 F/cm 2 ) of SRB-biofilm was attributed to the huge specific surface area of conductive pore walls inside biofilm.

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

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Enea Gino Di Domenico

2016-09-01

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

Investigation of Aspergillus fumigatus biofilm formation by various omics approaches

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Laetitia eMuszkieta

2013-02-01

Full Text Available In the lung, Aspergillus fumigatus usually forms a dense colony of filaments embedded in a polymeric extracellular matrix called biofilm (BF. This extracellular matrix embeds and glues hyphae together and protects the fungus from an outside hostile environment. This extracellular matrix is absent in fungal colonies grown under classical liquid shake conditions (PL which were historically used to understand A. fumigatus pathobiology. Recent works have shown that the fungus in this aerial grown biofilm-like state exhibits reduced susceptibility to antifungal drugs and undergoes major metabolic changes that are thought to be associated to virulence. These differences in pathological and physiological characteristics between biofilm and liquid shake conditions suggest that the PL condition is a poor in vitro disease model. In the laboratory, A. fumigatus mycelium embedded by the extracellular matrix can be produced in vitro in aerial condition using an agar-based medium. To provide a global and accurate understanding of A. fumigatus in vitro biofilm growth, we utilized microarray, RNA-sequencing and proteomic analysis to compare the global gene and protein expression profiles of A. fumigatus grown under BF and PL conditions. In this review, we will present the different signatures obtained with these three omics methods. We will discuss the advantages and limitations of each method and their complementarity.

Bronchoalveolar lavage is an ideal tool in evaluation of local immune response of pigs vaccinated with Pasteurella multocida bacterin vaccine

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Shiney George

2015-04-01

Full Text Available Aim: The aim was to study the bronchoalveolar lavage (BAL technique in evaluating the local immune response of pig immunized with Pasteurella multocida bacterin vaccine. Materials and Methods: Weaned piglets were immunized with formalin-inactivated P52 strain of P. multocida bacterin and evaluated for pulmonary immune response in BAL fluid. BAL was performed before vaccination and at different post vaccination days. The BAL fluid was assayed using enzyme-linked immunosorbent assay to study the development of P. multocida specific antibody isotypes and also evaluated for different cell populations using standard protocol. Results: The average recovery percentage of BAL fluid varies from 58.33 to 61.33 in vaccinated and control group of piglets. The BAL fluid of vaccinated pigs showed increase in antibody titer up to 60th days post vaccination (8.98±0.33, IgG being the predominant isotype reached maximum titer of 6.12±0.20 on 45th days post vaccination, followed by IgM and a meager concentration of IgA could be detected. An increased concentration of the lymphocyte population and induction of plasma cells was detected in the BAL fluid of vaccinated pigs. Conclusion: Though intranasal vaccination with P. multocida plain bacterin vaccine could not provoke a strong immune response, but is promising as lymphocyte population was increased and plasma cells were detected. BAL can be performed repeatedly up to 3/4 months of age in pigs to study pulmonary immune response without affecting their health.

Efficacy of antibiotic treatment of implant-associated Staphylococcus aureus infections with moxifloxacin, flucloxacillin, rifampin, and combination therapy: an animal study.

Science.gov (United States)

Greimel, Felix; Scheuerer, Christine; Gessner, Andre; Simon, Michaela; Kalteis, Thomas; Grifka, Joachim; Benditz, Achim; Springorum, Hans-Robert; Schaumburger, Jens

2017-01-01

The efficacy of antibiotic monotherapy and combination therapy in the treatment of implant-associated infection by Staphylococcus aureus was evaluated in an animal study. The femoral medullary cavity of 66 male Wistar rats was contaminated with S. aureus (ATCC 29213) and a metal device was implanted, of which 61 could be evaluated. Six treatment groups were studied: flucloxacillin, flucloxacillin in combination with rifampin, moxifloxacin, moxifloxacin in combination with rifampin, rifampin, and a control group with aqua. The treatment was applied for 14 days. After euthanasia, the bacterial counts in the periprosthetic bone, the soft tissue, and the implant-associated biofilm were measured. Both antibiotic combination treatments (moxifloxacin plus rifampin and flucloxacillin plus rifampin) achieved a highly significant decrease in microbial counts in the bone and soft tissue and in the biofilm. Mono-antibiotic treatments with either moxifloxacin or flucloxacillin were unable to achieve a significant decrease in microbial counts in bone and soft tissue or the biofilm, whilst rifampin was able to reduce the counts significantly only in the biofilm. Antibiotic resistance was measured in 1/3 of the cases in the rifampin group, whereas no resistance was measured in all other groups. The results show that combinations of both moxifloxacin and flucloxacillin plus rifampin are adequate for the treatment of periprosthetic infections due to infections with S. aureus , whereas monotherapies are not effective or not applicable due to the rapid development of antibiotic resistance. Therefore, moxifloxacin is an effective alternative in combination with rifampin for the treatment of implant-associated infections.

[Detection of biofilm formation by selected pathogens relevant to the food industry].

Science.gov (United States)

Šilhová-Hrušková, L; Moťková, P; Šilha, D; Vytřasová, J

2015-09-01

Detection of biofilm formation by microbial pathogens relevant to the food industry and comparison of biofilm formation under different conditions of culture. The following microorganisms were selected for the study: Staphylococcus aureus, Listeria innocua, Listeria ivanovii, Cronobacter sakazakii, Cronobacter muytjensii, Arcobacter butzleri, Arcobacter cryaerophilus, Campylobacter jejuni, and Campylobacter coli. To detect biofilm formation the microtiter plate assay, as described by Christensen and culture on stainless steel coupons were used. The biofilm forming capacity was confirmed in all microorganisms tested, both on the microtiter plates and stainless steel coupons. Biofilm formation was influenced by the culture medium, material used, and culture duration as well as by the test microorganism. It was found that different species and strains of the same genus differ in biofilm formation. Differences were also found between the collection strains and isolates from the environment. Some bacteria tended to form biofilm more readily on the surface of the polyethylene microtiter plates and less readily on stainless steel coupons while others appeared to have an opposite tendency. Some pathogens were able to increase the planktonic cell density in the initial suspension even by three orders of magnitude within 72 hours while producing plenty of biofilm. The study of biofilm formation by high risk pathogens is of utmost importance, not only to the food industry. From the obtained results, it is evident that bacterial biofilms form rapidly (within 24 hours in the present study). Due to their architecture, these biofilms are difficult to eradicate, and therefore, it is crucial to prevent biofilm formation.

In Vitro Antibacterial and Antibiofilm Activity of Lippia alba Essential Oil, Citral, and Carvone against Staphylococcus aureus

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Emanuela Mesquita Porfírio

2017-01-01

Full Text Available In vitro antimicrobial and antibiofilm activities of the Lippia alba essential oil and its major components (citral and carvone against Staphylococcus aureus were investigated. Essential oils (LA1EO, LA2EO, and LA3EO were extracted from the aerial parts of three L. alba specimens by hydrodistillation and analyzed by gas chromatography coupled to a mass spectrometer. Minimum Inhibitory Concentrations (MIC and Minimum Bacterial Concentration (MBC were determined by the microdilution method. For the antibiofilm assays, the biomass formation in the biofilm was evaluated by the microtiter-plate technique with the crystal violet (CV assay and the viability of the bacterial cells was analyzed. All oils and their major components presented antibacterial activity, and the lowest MIC and MBC values were 0.5 mg mL−1 when LA1EO and citral were used. Potential inhibition (100% of S. aureus biofilm formation at the concentration of 0.5 mg mL−1 of all EOs was observed. However, the elimination of biofilm cells was confirmed at concentrations of 1 mg mL−1, 2 mg mL−1, 2 mg mL−1, and 0.5 mg mL−1 for LA1EO, LA2EO, LA3EO, and citral, respectively. The results obtained in the present research point to the promising antibacterial and antibiofilm potential of L. alba EOs against S. aureus, a species of recognized clinical interest.

The effects of diode laser on Staphylococcus aureus biofilm and Escherichia coli lipopolysaccharide adherent to titanium oxide surface of dental implants. An in vitro study.

Science.gov (United States)

Giannelli, Marco; Landini, Giulia; Materassi, Fabrizio; Chellini, Flaminia; Antonelli, Alberto; Tani, Alessia; Zecchi-Orlandini, Sandra; Rossolini, Gian Maria; Bani, Daniele

2016-11-01

Effective decontamination of biofilm and bacterial toxins from the surface of dental implants is a yet unresolved issue. This in vitro study aims at providing the experimental basis for possible use of diode laser (λ 808 nm) in the treatment of peri-implantitis. Staphylococcus aureus biofilm was grown for 48 h on titanium discs with porous surface corresponding to the bone-implant interface and then irradiated with a diode laser (λ 808 nm) in noncontact mode with airflow cooling for 1 min using a Ø 600-μm fiber. Setting parameters were 2 W (400 J/cm 2 ) for continuous wave mode; 22 μJ, 20 kHz, 7 μs (88 J/cm 2 ) for pulsed wave mode. Bactericidal effect was evaluated using fluorescence microscopy and counting the residual colony-forming units. Biofilm and titanium surface morphology were analyzed by scanning electron microscopy (SEM). In parallel experiments, the titanium discs were coated with Escherichia coli lipopolysaccharide (LPS), laser-irradiated and seeded with RAW 264.7 macrophages to quantify LPS-driven inflammatory cell activation by measuring the enhanced generation of nitric oxide (NO). Diode laser irradiation in both continuous and pulsed modes induced a statistically significant reduction of viable bacteria and nitrite levels. These results indicate that in addition to its bactericidal effect laser irradiation can also inhibit LPS-induced macrophage activation and thus blunt the inflammatory response. The λ 808-nm diode laser emerges as a valuable tool for decontamination/detoxification of the titanium implant surface and may be used in the treatment of peri-implantitis.

Removal of Foodborne Pathogen Biofilms by Acidic Electrolyzed Water

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Qiao Han

2017-06-01

Full Text Available Biofilms, which are complex microbial communities embedded in the protective extracellular polymeric substances (EPS, are difficult to remove in food production facilities. In this study, the use of acidic electrolyzed water (AEW to remove foodborne pathogen biofilms was evaluated. We used a green fluorescent protein-tagged Escherichia coli for monitoring the efficiency of AEW for removing biofilms, where under the optimal treatment conditions, the fluorescent signal of cells in the biofilm disappeared rapidly and the population of biofilm cells was reduced by more than 67%. Additionally, AEW triggered EPS disruption, as indicated by the deformation of the carbohydrate C-O-C bond and deformation of the aromatic rings in the amino acids tyrosine and phenylalanine. These deformations were identified by EPS chemical analysis and Raman spectroscopic analysis. Scanning electron microscopy (SEM images confirmed that the breakup and detachment of biofilm were enhanced after AEW treatment. Further, AEW also eradicated biofilms formed by both Gram-negative bacteria (Vibrio parahaemolyticus and Gram-positive bacteria (Listeria monocytogenes and was observed to inactivate the detached cells which are a potential source of secondary pollution. This study demonstrates that AEW could be a reliable foodborne pathogen biofilm disrupter and an eco-friendly alternative to sanitizers traditionally used in the food industry.

Screening of medicinal plants for antibacterial activities on Staphylococcus aureus strains isolated from bovine mastitis Screening de plantas medicinais com atividade antimicrobiana contra cepas de Staphylococcus aureus isoladas de mastite bovina

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Marisa A. N. Diaz

2010-11-01

Full Text Available Staphylococcus aureus is the main causative agent of bovine mastitis. The activity of several extracts from ten medicinal plants traditionally used in Brazil as antiseptic was investigated against fifteen strains of Staphylococcus aureus isolated from animals with mastitis manifestation by the disc diffusion method and broth microdilution assay. The interference of the extracts on cell in the form of adherent colonies was also evaluated. MIC values ranged from 0.5 mg/mL to 1.0 mg/mL and biofilm inhibitory concentration (BIC were between 0.25 mg/mL and 0.8 mg/mL. Results revealed the potential of extracts of Senna macranthera, Artemisia absinthium, Cymbopogon nardus and Baccharis dracunculifolia as antibacterial agents against S. aureus strains isolated from bovine mastitis and support the possible use of these phytotherapic agents in the clinical management of the disease.Staphylococcus aureus é o principal agente causador de mastite bovina. A atividade de diversos extratos de dez plantas medicinais tradicionalmente usadas no Brasil como anti-sépticas foi investigada contra quinze cepas de Staphylococcus aureus isoladas de animais com manifestação de mastite pelo método de difusão em ágar e ensaio de microdiluição. A interferência dos extratos na célula bacteriana em forma de colônias aderidas também foi avaliada. Os valores de MIC variaram de 0.5 mg/mL a 1.0 mg/mL e a concentração inibitória de biofilme (BIC variou de 0.25 mg/mL a 0.8 mg/mL. Os resultados revelaram o potencial dos extratos de Senna macranthera, Artemisia absinthium, Cymbopogon nardus e Baccharis dracunculifolia como agentes antibacterianos contra cepas de S. aureus isolados de mastite bovina e suportam o possível uso destas plantas no manejo clínico da doença.

Sustained prevention of biofilm formation on a novel silicone matrix suitable for medical devices

DEFF Research Database (Denmark)

Steffensen, Søren Langer; Merete H., Vestergaard,; Jensen, Minna Grønning

2015-01-01

Bacterial colonization and biofilm formation on medical devices constitute major challenges in clinical long-term use of e.g. catheters due to the risk of (re)infection of patients, which would result in additional use of antibiotics risking bacterial resistance development. The aim of the present...... in the range of 1–20 mg/mL. Devices containing 25% (w/w) hydrogel and loaded with ciprofloxacin displayed a strong antibacterial effect against Staphylococcus aureus bacterial colonization and subsequent biofilm formation on the device material was inhibited for 29 days. In conclusion, the hydrogel...

Bovine Leukemia Virus Infection in Dairy Cattle: Effect on Serological Response to Immunization against J5 Escherichia coli Bacterin

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Ronald J. Erskine

2011-01-01

Full Text Available Thirteen bovine leukemia virus- (BLV- negative and 22 BLV-positive Holstein cows were immunized with J5 Escherichia coli bacterin at dry off, three weeks before calving, during the second week after calving, and three weeks after the third immunization. Serum was collected before the initial immunization, immediately before the third and fourth immunizations, and 21 days after the fourth immunization. Anti-J5 E. coli IgM, IgG1, and IgG2 titers were determined by ELISA. Anti-J5 E. coli IgM titers did not differ significantly (P=.98 between groups. Increases in anti-J5 E. coli IgG1 titers were higher in the BLV-negative cows (P=.057. Geometric mean anti-J5 E. coli IgG2 titers increased fourfold in the BLV-negative cows, which was significantly higher (P=.007 than the twofold increase in the BLV-positive cows. Cattle infected with BLV may have impaired serologic responses following immunization with J5 bacterin, and response may differ according to antibody isotype.

Transfer of bacteria between stainless steel and chicken meat: A CLSM and DGGE study of biofilms

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Christine C. Gaylarde

2016-08-01

Full Text Available This study aimed to assess the interaction between bacteria and food processing surfaces using novel methods. Microbial cross contamination between stainless steel, a common food processing material, and raw chicken was studied using microbiological culture, specialized microscope and molecular techniques. Confocal laser scanning microscopy (CLSM allowed the visualization of biofilms containing single or dual species of Escherichia coli O157:H7, Salmonella typhimurium, Bacillus cereus, Staphylococcus aureus and Pseudomonas aeruginosa, formed after 6 days’ incubation on stainless steel or 4h on raw chicken. The results provided information on intra-biofilm location and stratification of species within dual species biofilms. Top-to-bottom Z-stack images revealed that, on both materials, S. typhimurium and E. coli attached concurrently, the former in greater numbers. E. coli and B. cereus segregated on steel, E. coli more frequent near the metal surface, B. cereus almost the only species in outer layers. Few cells of S. aureus, found at all depths, were seen in the 2.9 µm thick biofilm on steel with E. coli. Greatest attachment was shown by P. aeruginosa, followed by S. typhimurium, E. coli and finally Gram positive species. Large amounts of EPS in P. aeruginosa biofilms made visualization difficult on both materials, but especially on chicken meat, a limitation of this technique. Nevertheless, CLSM was useful for determining time sequence of adhesion and species makeup of thin biofilms. The technique showed that five min contact between bacterially-contaminated chicken and sterile steel resulted in greatest transfer of P. aeruginosa, followed by S. typhimurium. This was confirmed using DGGE. Gram positive bacteria transferred poorly. A biofilm containing 2.3 × 105  cfu·cm−2 B. cereus on steel transferred an undetectable number of cells to chicken after 5 min contact. This species was unable to form biofilm on chicken when incubated for 4 h

Biofilms and Persistent Wound Infections in United States Military Trauma Patients: a Case-control Analysis

Science.gov (United States)

2014-04-08

Infect Dis. 2014, 21:21-23. 51. Hochbaum AI, Kolodkin-Gal I, Foulston L, Kolter R, Aizenberg J, Losick R: Inhibitory effects of D-amino acids on...Staphylococcus aureus biofilm development. J Bacteriol 2011, 193(20):5616–5622. 52. Kolodkin-Gal I, Romero D, Cao S, Clardy J, Kolter R, Losick R: D-amino...acids trigger biofilm dissambly. Science 2010, 328(5978):627–629. 53. Kolodkin-Gal I, Cao S, Chai L, Bottcher T, Kolter R, Clardy J, Losick R: A self

Antibacterial activity of different types of snake venom from the Viperidae family against Staphylococcus aureus

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Isabela Nascimento Canhas

2017-09-01

Full Text Available Toxins and venoms produced by living organisms have exhibited a variety of biological activities against microorganisms. In this study, we tested seven snake venoms from the family Viperidae for antibacterial activity and the activities of reversal of antibiotic resistance and inhibition of biofilm formation against 22 clinical isolates of Staphylococcus aureus. Bothrops moojeni venom exhibited anti staphylococcal activity with the lowest mean value of minimum inhibitory concentration (MIC. Moreover, reversal of antibiotic resistance was observed for combinations of B. moojeni venom (½ x MIC and norfloxacin or ampicillin (both ½ x MIC for 86.4% and 50% of the isolates, respectively. B. moojeni venom alone at ½ MIC inhibited 90% of biofilm formation, whereas in combination with ciprofloxacin, both at ½ MIC, a reduction on the NorA efflux pump activity was observed. The detection of in vitro mutants colonies of S. aureus resistant to B. moojeni venom was low and they did not survive. A phospholipase A2 was purified from the venom of B. moojeni and displayed anti-staphylococcal activity when tested alone or in combination with ciprofloxacin. The results presented here will contribute to the search for new antimicrobial agents against resistant S. aureus.

Efficacy of antibiotic treatment of implant-associated Staphylococcus aureus infections with moxifloxacin, flucloxacillin, rifampin, and combination therapy: an animal study

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Greimel F

2017-06-01

Full Text Available Felix Greimel,1 Christine Scheuerer,1 Andre Gessner,2 Michaela Simon,2 Thomas Kalteis,1 Joachim Grifka,1 Achim Benditz,1 Hans-Robert Springorum,1 Jens Schaumburger1 1Department of Orthopedics, University Medical Center Regensburg, Asklepios Klinikum Bad Abbach, Bad Abbach, 2Institute of Clinical Microbiology and Hygiene, University Medical Center Regensburg, Regensburg, Bavaria, Germany Abstract: The efficacy of antibiotic monotherapy and combination therapy in the treatment of implant-associated infection by Staphylococcus aureus was evaluated in an animal study. The femoral medullary cavity of 66 male Wistar rats was contaminated with S. aureus (ATCC 29213 and a metal device was implanted, of which 61 could be evaluated. Six treatment groups were studied: flucloxacillin, flucloxacillin in combination with rifampin, moxifloxacin, moxifloxacin in combination with rifampin, rifampin, and a control group with aqua. The treatment was applied for 14 days. After euthanasia, the bacterial counts in the periprosthetic bone, the soft tissue, and the implant-associated biofilm were measured. Both antibiotic combination treatments (moxifloxacin plus rifampin and flucloxacillin plus rifampin achieved a highly significant decrease in microbial counts in the bone and soft tissue and in the biofilm. Mono-antibiotic treatments with either moxifloxacin or flucloxacillin were unable to achieve a significant decrease in microbial counts in bone and soft tissue or the biofilm, whilst rifampin was able to reduce the counts significantly only in the biofilm. Antibiotic resistance was measured in 1/3 of the cases in the rifampin group, whereas no resistance was measured in all other groups. The results show that combinations of both moxifloxacin and flucloxacillin plus rifampin are adequate for the treatment of periprosthetic infections due to infections with S. aureus, whereas monotherapies are not effective or not applicable due to the rapid development of

A 3-year long study of Staphylococcus aureus isolates from subclinical mastitis in three Azawak zebu herds at the Sahelian experimental farm of Toukounous, Niger.

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Issa, Abdoulkarim Ibrahim; Duprez, Jean-Noël; Bada-Alambedji, Rianatou; Djika, Mamane; Mainil, Jacques Georges; Bardiau, Marjorie

2016-02-01

Staphylococcus (S.) aureus is one of the most important pathogens causing bovine mastitis. The aim of the present work was to follow in three herds and during the 3 years the clonality of S. aureus isolated from California Mastitis Test (CMT)-positive cows at the experimental station of Toukounous (Niger) by (i) comparing their pulsed field gel electrophoresis (PFGE) fingerprints, (ii) identifying their virulotypes by PCR amplification and (iii) assessing the production of capsule and the formation of biofilm. The 88 S. aureus isolates belonged to 14 different pulsotypes, 3 of them being predominant: A (30 %), D (27 %), B (15 %). A and B pulsotypes had the highest profile similarity coefficient (94 %), while others had similarity coefficients under 60 %. Seventy-five S. aureus isolates were further studied for their virulotypes, capsular antigens and biofilm production. Most surface factor-, leukocidin- and haemolysin-, but not the enterotoxin-encoding genes were detected in the majority (>75 %) of the isolates and were evenly distributed between the A, B and D pulsotype isolates. The majority of the 72 S. aureus positive with the cap5H or cap8H PCR produced the CP5 (82 %) or the CP8 (88 %) capsular antigen, respectively. Biofilm production by the 57 icaA-positive isolates was strong for 8 isolates, moderate for 31 isolates but weak for 18 isolates, implying that the icaA gene may not be expressed in vitro by one third of the positive isolates. Similar to other studies, those results confirm that a restricted number of S. aureus clones circulate within the three herds at Toukounous and that their specific virulence-associated properties must still be further studied.

Adenoid Reservoir for Pathogenic Biofilm Bacteria▿

Science.gov (United States)

Nistico, L.; Kreft, R.; Gieseke, A.; Coticchia, J. M.; Burrows, A.; Khampang, P.; Liu, Y.; Kerschner, J. E.; Post, J. C.; Lonergan, S.; Sampath, R.; Hu, F. Z.; Ehrlich, G. D.; Stoodley, P.; Hall-Stoodley, L.

2011-01-01

Biofilms of pathogenic bacteria are present on the middle ear mucosa of children with chronic otitis media (COM) and may contribute to the persistence of pathogens and the recalcitrance of COM to antibiotic treatment. Controlled studies indicate that adenoidectomy is effective in the treatment of COM, suggesting that the adenoids may act as a reservoir for COM pathogens. To investigate the bacterial community in the adenoid, samples were obtained from 35 children undergoing adenoidectomy for chronic OM or obstructive sleep apnea. We used a novel, culture-independent molecular diagnostic methodology, followed by confocal microscopy, to investigate the in situ distribution and organization of pathogens in the adenoids to determine whether pathogenic bacteria exhibited criteria characteristic of biofilms. The Ibis T5000 Universal Biosensor System was used to interrogate the extent of the microbial diversity within adenoid biopsy specimens. Using a suite of 16 broad-range bacterial primers, we demonstrated that adenoids from both diagnostic groups were colonized with polymicrobial biofilms. Haemophilus influenzae was present in more adenoids from the COM group (P = 0.005), but there was no significant difference between the two patient groups for Streptococcus pneumoniae or Staphylococcus aureus. Fluorescence in situ hybridization, lectin binding, and the use of antibodies specific for host epithelial cells demonstrated that pathogens were aggregated, surrounded by a carbohydrate matrix, and localized on and within the epithelial cell surface, which is consistent with criteria for bacterial biofilms. PMID:21307211

Label-free in situ SERS imaging of biofilms.

Science.gov (United States)

Ivleva, Natalia P; Wagner, Michael; Szkola, Agathe; Horn, Harald; Niessner, Reinhard; Haisch, Christoph

2010-08-12

Surface-enhanced Raman scattering (SERS) is a promising technique for the chemical characterization of biological systems. It yields highly informative spectra, can be applied directly in aqueous environment, and has high sensitivity in comparison with normal Raman spectroscopy. Moreover, SERS imaging can provide chemical information with spatial resolution in the micrometer range (chemical imaging). In this paper, we report for the first time on the application of SERS for in situ, label-free imaging of biofilms and demonstrate the suitability of this technique for the characterization of the complex biomatrix. Biofilms, being communities of microorganisms embedded in a matrix of extracellular polymeric substances (EPS), represent the predominant mode of microbial life. Knowledge of the chemical composition and the structure of the biofilm matrix is important in different fields, e.g., medicine, biology, and industrial processes. We used colloidal silver nanoparticles for the in situ SERS analysis. Good SERS measurement reproducibility, along with a significant enhancement of Raman signals by SERS (>10(4)) and highly informative SERS signature, enables rapid SERS imaging (1 s for a single spectrum) of the biofilm matrix. Altogether, this work illustrates the potential of SERS for biofilm analysis, including the detection of different constituents and the determination of their distribution in a biofilm even at low biomass concentration.

Time-Resolved Analysis of Cytosolic and Surface-Associated Proteins of Staphylococcus aureus HG001 under Planktonic and Biofilm Conditions.

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Moche, Martin; Schlüter, Rabea; Bernhardt, Jörg; Plate, Kristina; Riedel, Katharina; Hecker, Michael; Becher, Dörte

2015-09-04

Staphylococcal biofilms are associated with persistent infections due to their capacity to protect bacteria against the host's immune system and antibiotics. Cell-surface-associated proteins are of great importance during biofilm formation. In the present study, an optimized biotinylation approach for quantitative GeLC-MS-based analysis of the staphylococcal cell-surface proteome was applied and the cytoplasmic protein fraction was analyzed to elucidate proteomic differences between colony biofilms and planktonic cells. The experimental setup enabled a time-resolved monitoring of the proteome under both culture conditions and the comparison of biofilm cells to planktonic cells at several time points. This allowed discrimination of differences attributed to delayed growth phases from responses provoked by biofilm conditions. Biofilm cells expressed CcpA-dependent catabolic proteins earlier than planktonic cells and strongly accumulated proteins that belong to the SigB stress regulon. The amount of the cell-surface protein and virulence gene regulator Rot decreased within biofilms and MgrA-dependent regulations appeared more pronounced. Biofilm cells simultaneously up-regulated activators (e.g., SarZ) as well as repressors (e.g., SarX) of RNAIII. A decreased amount of high-affinity iron uptake systems and an increased amount of the iron-storage protein FtnA possibly indicated a lower demand of iron in biofilms.

Clinical implications of microbial biofilms in chronic rhinosinusitis and orbital cellulitis.

Science.gov (United States)

Nayak, Niranjan; Satpathy, Gita; Prasad, Sujata; Thakar, Alok; Chandra, Mahesh; Nag, T C

2016-09-21

Discovery of sessile mode of microbial existence (Biofilm state) focussed much interest, during the recent years, on the study of biofilms in many recurring and chronic infections. However, the exact role of microbial biofilms in chronic rhinosinusitis and orbital cellulitis were not elucidated earlier. The purpose of the present study was to look for the adherent property and biofilm producing ability of the clinical isolates in chronic rhinosinusitis and orbital cellulitis, and to look for the effects of antimicrobial agents on these biofilms by colorimetric assay and ultrastructural analysis. Organisms were isolated and identified from various clinical samples in patients with chronic sinusitis and orbital cellulitis. Antimicrobial sensitivity testing was carried out by the standard protocol. Biofilms were developed; quantified and antimicrobial drug perfusion through the biofilm model was evaluated by the earlier devised procedure. Electronmicroscopic study of the biofilm was performed by the recommended technique. Of the total of 70 clinical samples processed, 48 i.e. 68.5 % grew bacteria and 13 i.e.(18.6 %) fungi. Staphylococcus aureus (20), S epidermidis (16) and Pseudomonas aeruginosa (6) accounted for the majority of the bacterial isolates. Aspergillus flavus (8), however was the commonest amongst the fungi. A total of 40 bacteria and 8 fungi could be tested for biofilm production. Eighteen (45 %) of the 40 bacterial isolates and 4(50 %) out of the 8 A flavus isolates were found to be biofilm producers. In vitro adherence testing revealed that majority i.e. 16 (88.8 %) of the 18 biofilm positive bacteria were adherent to artificial surfaces. Antimicrobial drug perfusion through the biofilm model was poor. Antimicrobial treatment was totally ineffective against strong biofilm producers, whose electron microscopic picture was quite similar to that observed for biofilm producers without any antimicrobial pre-treatment. Filamentous fungi, like bacteria

Development of active biofilms of quinoa (Chenopodium quinoa W.) starch containing gold nanoparticles and evaluation of antimicrobial activity.

Science.gov (United States)

Pagno, Carlos H; Costa, Tania M H; de Menezes, Eliana W; Benvenutti, Edilson V; Hertz, Plinho F; Matte, Carla R; Tosati, Juliano V; Monteiro, Alcilene R; Rios, Alessandro O; Flôres, Simone H

2015-04-15

Active biofilms of quinoa (Chenopodium quinoa, W.) starch were prepared by incorporating gold nanoparticles stabilised by an ionic silsesquioxane that contains the 1,4-diazoniabicyclo[2.2.2]octane chloride group. The biofilms were characterised and their antimicrobial activity was evaluated against Escherichiacoli and Staphylococcusaureus. The presence of gold nanoparticles produces an improvement in the mechanical, optical and morphological properties, maintaining the thermal and barrier properties unchanged when compared to the standard biofilm. The active biofilms exhibited strong antibacterial activity against food-borne pathogens with inhibition percentages of 99% against E. coli and 98% against S. aureus. These quinoa starch biofilms containing gold nanoparticles are very promising to be used as active food packaging for the maintenance of food safety and extension of the shelf life of packaged foods. Copyright © 2014 Elsevier Ltd. All rights reserved.

Biofilm on artificial pacemaker: fiction or reality?

Science.gov (United States)

Santos, Ana Paula Azevedo; Watanabe, Evandro; Andrade, Denise de

2011-11-01

Cardiac pacing through cardiac pacemaker is one of the most promising alternatives in the treatment of arrhythmias, but it can cause reactions natural or complex reactions, either early or late. This study aimed to describe the scientific evidence on the risk of infection and biofilm formation associated with cardiac pacemaker. This is a study of integrative literature review. It included 14 publications classified into three thematic categories: diagnosis (microbiological and/or clinical), complications and therapy of infections. Staphylococcus epidermidis and Staphylococcus aureus were the microorganisms most frequently isolated. It was not possible to determine the incidence of infection associated with pacemakers, since the studies were generally of prevalence. In terms of therapy, the complete removal of pacemakers stood out, especially in cases of suspected biofilm. Still controversial is the use of systemic antibiotic prophylaxis in reducing the incidence of infection associated with implantation of a pacemaker.

Biofilm attachment reduction on bioinspired, dynamic, micro-wrinkling surfaces

International Nuclear Information System (INIS)

Epstein, Alexander K; Hong, Donggyoon; Kim, Philseok; Aizenberg, Joanna

2013-01-01

Most bacteria live in multicellular communities known as biofilms that are adherent to surfaces in our environment, from sea beds to plumbing systems. Biofilms are often associated with clinical infections, nosocomial deaths and industrial damage such as bio-corrosion and clogging of pipes. As mature biofilms are extremely challenging to eradicate once formed, prevention is advantageous over treatment. However, conventional surface chemistry strategies are either generally transient, due to chemical masking, or toxic, as in the case of leaching marine antifouling paints. Inspired by the nonfouling skins of echinoderms and other marine organisms, which possess highly dynamic surface structures that mechanically frustrate bio-attachment, we have developed and tested a synthetic platform based on both uniaxial mechanical strain and buckling-induced elastomer microtopography. Bacterial biofilm attachment to the dynamic substrates was studied under an array of parameters, including strain amplitude and timescale (1–100 mm s −1 ), surface wrinkle length scale, bacterial species and cell geometry, and growth time. The optimal conditions for achieving up to ∼ 80% Pseudomonas aeruginosa biofilm reduction after 24 h growth and ∼ 60% reduction after 48 h were combinatorially elucidated to occur at 20% strain amplitude, a timescale of less than ∼ 5 min between strain cycles and a topography length scale corresponding to the cell dimension of ∼ 1 μm. Divergent effects on the attachment of P. aeruginosa, Staphylococcus aureus and Escherichia coli biofilms showed that the dynamic substrate also provides a new means of species-specific biofilm inhibition, or inversely, selection for a desired type of bacteria, without reliance on any toxic or transient surface chemical treatments. (paper)

Biofilm attachment reduction on bioinspired, dynamic, micro-wrinkling surfaces

Science.gov (United States)

Epstein, Alexander K.; Hong, Donggyoon; Kim, Philseok; Aizenberg, Joanna

2013-09-01

Most bacteria live in multicellular communities known as biofilms that are adherent to surfaces in our environment, from sea beds to plumbing systems. Biofilms are often associated with clinical infections, nosocomial deaths and industrial damage such as bio-corrosion and clogging of pipes. As mature biofilms are extremely challenging to eradicate once formed, prevention is advantageous over treatment. However, conventional surface chemistry strategies are either generally transient, due to chemical masking, or toxic, as in the case of leaching marine antifouling paints. Inspired by the nonfouling skins of echinoderms and other marine organisms, which possess highly dynamic surface structures that mechanically frustrate bio-attachment, we have developed and tested a synthetic platform based on both uniaxial mechanical strain and buckling-induced elastomer microtopography. Bacterial biofilm attachment to the dynamic substrates was studied under an array of parameters, including strain amplitude and timescale (1-100 mm s-1), surface wrinkle length scale, bacterial species and cell geometry, and growth time. The optimal conditions for achieving up to ˜ 80% Pseudomonas aeruginosa biofilm reduction after 24 h growth and ˜ 60% reduction after 48 h were combinatorially elucidated to occur at 20% strain amplitude, a timescale of less than ˜ 5 min between strain cycles and a topography length scale corresponding to the cell dimension of ˜ 1 μm. Divergent effects on the attachment of P. aeruginosa, Staphylococcus aureus and Escherichia coli biofilms showed that the dynamic substrate also provides a new means of species-specific biofilm inhibition, or inversely, selection for a desired type of bacteria, without reliance on any toxic or transient surface chemical treatments.

Giving structure to the biofilm matrix: an overview of individual strategies and emerging common themes.

Science.gov (United States)

Hobley, Laura; Harkins, Catriona; MacPhee, Cait E; Stanley-Wall, Nicola R

2015-09-01

Biofilms are communities of microbial cells that underpin diverse processes including sewage bioremediation, plant growth promotion, chronic infections and industrial biofouling. The cells resident in the biofilm are encased within a self-produced exopolymeric matrix that commonly comprises lipids, proteins that frequently exhibit amyloid-like properties, eDNA and exopolysaccharides. This matrix fulfils a variety of functions for the community, from providing structural rigidity and protection from the external environment to controlling gene regulation and nutrient adsorption. Critical to the development of novel strategies to control biofilm infections, or the capability to capitalize on the power of biofilm formation for industrial and biotechnological uses, is an in-depth knowledge of the biofilm matrix. This is with respect to the structure of the individual components, the nature of the interactions between the molecules and the three-dimensional spatial organization. We highlight recent advances in the understanding of the structural and functional role that carbohydrates and proteins play within the biofilm matrix to provide three-dimensional architectural integrity and functionality to the biofilm community. We highlight, where relevant, experimental techniques that are allowing the boundaries of our understanding of the biofilm matrix to be extended using Escherichia coli, Staphylococcus aureus, Vibrio cholerae, and Bacillus subtilis as exemplars. © FEMS 2015.

The camelliagenin from defatted seeds of Camellia oleifera as antibiotic substitute to treat chicken against infection of Escherichia coli and Staphylococcus aureus.

Science.gov (United States)

Ye, Yong; Yang, Qian; Fang, Fei; Li, Yue

2015-08-18

Escherichia coli and Staphylococcus aureus are the main pathogens infectious to poultry, and their resistances against antibiotics have become troublesome currently. Biofilm formation is an important reason for drug resistance. Our previous research has found that the extract of Camellia oleifera seeds has lots of pharmacological effects. In order to find the substitute for antibiotics, the saponin was isolated from the defatted C. oleifera seeds with structural identification. Its efficacy was evaluated by the inhibition on amoxicillin-resistant E. coli and erythromycin-resistant S. aureus and therapeutic effect on chicks infected by the two bacteria. The bacterial growth inhibition rate increased and the bacterial count in vivo decreased significantly in dose dependence after administration of the saponin and its combination with amoxicillin or erythromycin, suggesting its antibacterial effect. The saponin identified as camelliagenin shows significant inhibition on the biofilm of E. coli and S. aureus, and it is related to the decrease of mannitol dehydrogenase (MDH) activity and extracellular DNA (eDNA) content. Molecular simulation reveals the strong interaction existing between the saponin and MDH or eDNA. The mechanism of camelliagenin's improvement on antibiotic effects is its interaction with MDH and eDNA in biofilm. The saponin is a prospective substitute of antibiotics, and molecular simulation is a convenient alternative method to find out hopeful candidates of antibiotics substitute.

A full genomic characterization of the development of a stable Small Colony Variant cell-type by a clinical Staphylococcus aureus strain.

Science.gov (United States)

Bui, Long M G; Kidd, Stephen P

2015-12-01

A key to persistent and recurrent Staphylococcus aureus infections is its ability to adapt to diverse and toxic conditions. This ability includes a switch into a biofilm or to the quasi-dormant Small Colony Variant (SCV). The development and molecular attributes of SCVs have been difficult to study due to their rapid reversion to their parental cell-type. We recently described the unique induction of a matrix-embedded and stable SCV cell-type in a clinical S. aureus strain (WCH-SK2) by growing the cells with limiting conditions for a prolonged timeframe. Here we further study their characteristics. They possessed an increased viability in the presence of antibiotics compared to their non-SCV form. Their stability implied that there had been genetic changes; we therefore determined both the genome sequence of WCH-SK2 and its stable SCV form at a single base resolution, employing Single Molecular Real-Time (SMRT) sequencing that enabled the methylome to also be determined. The genetic features of WCH-SK2 have been identified; the SCCmec type, the pathogenicity and genetic islands and virulence factors. The genetic changes that had occurred in the stable SCV form were identified; most notably being in MgrA, a global regulator, and RsbU, a phosphoserine phosphatase within the regulatory pathway of the sigma factor SigB. There was a shift in the methylomes of the non-SCV and stable SCV forms. We have also shown a similar induction of this cell-type in other S. aureus strains and performed a genetic comparison to these and other S. aureus genomes. We additionally map RNAseq data to the WCH-SK2 genome in a transcriptomic analysis of the parental, SCV and stable SCV cells. The results from this study represent the unique identification of a suite of epigenetic, genetic and transcriptional factors that are implicated in the switch in S. aureus to its persistent SCV form. Copyright © 2015 Elsevier B.V. All rights reserved.

Effects of Tween 80 on growth and biofilm formation in laboratory media

Directory of Open Access Journals (Sweden)

Christina Krogsård Nielsen

2016-11-01

Full Text Available Tween 80 is a widely used nonionic emulsifier that is added to cosmetics, pharmaceuticals and foods. Because of its widespread use we need to understand how it affects bacteria on our skin, in our gut, and in food products. The aim of this study is to investigate how Tween 80 affects the growth and antimicrobial susceptibility of Staphylococcus aureus, Listeria monocytogenes and Pseudomonas fluorescens, which are common causes of spoilage and foodborne illnesses. Addition of 0.1% Tween 80 to laboratory growth media increased the growth rate of planktonic S. aureus batch cultures, and it also increased the total biomass when S. aureus was grown as biofilms. In contrast, Tween 80 had no effect on batch cultures of L. monocytogenes, it slowed the growth rate of P. fluorescens, and it led to formation of less biofilm by both L. monocytogenes and P. fluorescens. Furthermore, Tween 80 lowered the antibacterial efficacy of two hydrophobic antimicrobials: rifampicin and the essential oil isoeugenol. Our findings underline the importance of documenting indirect effects of emulsifiers when studying the efficacy of hydrophobic antimicrobials that are dispersed in solution by emulsification, or when antimicrobials are applied in food matrixes that include emulsifiers. Furthermore, the species-specific effects on microbial growth suggests that Tween 80 in cosmetics and food products could affect the composition of skin and gut microbiota, and the effect of emulsifiers on the human microbiome should therefore be explored to uncover potential health effects.

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

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

2016-11-01

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

Diffusion Retardation by Binding of Tobramycin in an Alginate Biofilm Model

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Cao, Bao; Christophersen, Lars; Kolpen, Mette

2016-01-01

Microbial cells embedded in a self-produced extracellular biofilm matrix cause chronic infections, e. g. by Pseudomonas aeruginosa in the lungs of cystic fibrosis patients. The antibiotic killing of bacteria in biofilms is generally known to be reduced by 100–1000 times relative to planktonic...... bacteria. This makes such infections difficult to treat. We have therefore proposed that biofilms can be regarded as an independent compartment with distinct pharmacokinetics. To elucidate this pharmacokinetics we have measured the penetration of the tobramycin into seaweed alginate beads which serve...... to be uniformly distributed throughout the volume of the alginate bead. The power-law appears to be a consequence of binding to a multitude of different binding sites. In a diffusion model these results are shown to produce pronounced retardation of the penetration of tobramycin into the biofilm. This filtering...

Disassembly of Bacterial Biofilms by the Self-Assembled Glycolipids Derived from Renewable Resources.

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Prasad, Yadavali Siva; Miryala, Sandeep; Lalitha, Krishnamoorthy; Ranjitha, K; Barbhaiwala, Shehnaz; Sridharan, Vellaisamy; Maheswari, C Uma; Srinandan, C S; Nagarajan, Subbiah

2017-11-22

More than 80% of chronic infections of bacteria are caused by biofilms. It is also a long-term survival strategy of the pathogens in a nonhost environment. Several amphiphilic molecules have been used in the past to potentially disrupt biofilms; however, the involvement of multistep synthesis, complicated purification and poor yield still remains a major problem. Herein, we report a facile synthesis of glycolipid based surfactant from renewable feedstocks in good yield. The nature of carbohydrate unit present in glycolipid influence the ring chain tautomerism, which resulted in the existence of either cyclic structure or both cyclic and acyclic structures. Interestingly, these glycolipids self-assemble into gel in highly hydrophobic solvents and vegetable oils, and displayed foam formation in water. The potential application of these self-assembled glycolipids to disrupt preformed biofilm was examined against various pathogens. It was observed that glycolipid 6a disrupts Staphylococcus aureus and Listeria monocytogenes biofilm, while the compound 6c was effective in disassembling uropathogenic E. coli and Salmonella enterica Typhimurium biofilms. Altogether, the supramolecular self-assembled materials, either as gel or as surfactant solution could be potentially used for surface cleansing in hospital environments or the food processing industries to effectively reduce pathogenic biofilms.

Staphylococcus aureus adherence to Candida albicans hyphae is mediated by the hyphal adhesin Als3p.

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Peters, Brian M; Ovchinnikova, Ekaterina S; Krom, Bastiaan P; Schlecht, Lisa Marie; Zhou, Han; Hoyer, Lois L; Busscher, Henk J; van der Mei, Henny C; Jabra-Rizk, Mary Ann; Shirtliff, Mark E

2012-12-01

The bacterium Staphylococcus (St.) aureus and the opportunistic fungus Candida albicans are currently among the leading nosocomial pathogens, often co-infecting critically ill patients, with high morbidity and mortality. Previous investigations have demonstrated preferential adherence of St. aureus to C. albicans hyphae during mixed biofilm growth. In this study, we aimed to characterize the mechanism behind this observed interaction. C. albicans adhesin-deficient mutant strains were screened by microscopy to identify the specific receptor on C. albicans hyphae recognized by St. aureus. Furthermore, an immunoassay was developed to validate and quantify staphylococcal binding to fungal biofilms. The findings from these experiments implicated the C. albicans adhesin agglutinin-like sequence 3 (Als3p) in playing a major role in the adherence process. This association was quantitatively established using atomic force microscopy, in which the adhesion force between single cells of the two species was significantly reduced for a C. albicans mutant strain lacking als3. Confocal microscopy further confirmed these observations, as St. aureus overlaid with a purified recombinant Als3 N-terminal domain fragment (rAls3p) exhibited robust binding. Importantly, a strain of Saccharomyces cerevisiae heterologously expressing Als3p was utilized to further confirm this adhesin as a receptor for St. aureus. Although the parental strain does not bind bacteria, expression of Als3p on the cell surface conferred upon the yeast the ability to strongly bind St. aureus. To elucidate the implications of these in vitro findings in a clinically relevant setting, an ex vivo murine model of co-infection was designed using murine tongue explants. Fluorescent microscopic images revealed extensive hyphal penetration of the epithelium typical of C. albicans mucosal infection. Interestingly, St. aureus bacterial cells were only seen within the epithelial tissue when associated with the invasive

Interactions of methicillin resistant Staphylococcus aureus USA300 and Pseudomonas aeruginosa in polymicrobial wound infection.

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Irena Pastar

Full Text Available Understanding the pathology resulting from Staphylococcus aureus and Pseudomonas aeruginosa polymicrobial wound infections is of great importance due to their ubiquitous nature, increasing prevalence, growing resistance to antimicrobial agents, and ability to delay healing. Methicillin-resistant S. aureus USA300 is the leading cause of community-associated bacterial infections resulting in increased morbidity and mortality. We utilized a well-established porcine partial thickness wound healing model to study the synergistic effects of USA300 and P. aeruginosa on wound healing. Wound re-epithelialization was significantly delayed by mixed-species biofilms through suppression of keratinocyte growth factor 1. Pseudomonas showed an inhibitory effect on USA300 growth in vitro while both species co-existed in cutaneous wounds in vivo. Polymicrobial wound infection in the presence of P. aeruginosa resulted in induced expression of USA300 virulence factors Panton-Valentine leukocidin and α-hemolysin. These results provide evidence for the interaction of bacterial species within mixed-species biofilms in vivo and for the first time, the contribution of virulence factors to the severity of polymicrobial wound infections.

Antibacterial isoeugenol coating on stainless steel and polyethylene surfaces prevents biofilm growth.

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Nielsen, C K; Subbiahdoss, G; Zeng, G; Salmi, Z; Kjems, J; Mygind, T; Snabe, T; Meyer, R L

2018-01-01

Pathogenic bacteria can spread between individuals or between food items via the surfaces they share. Limiting the survival of pathogens on surfaces, therefore, presents an opportunity to limit at least one route of how pathogens spread. In this study, we propose that a simple coating with the essential oil isoeugenol can be used to circumvent the problem of bacterial transfer via surfaces. Two commonly used materials, stainless steel and polyethylene, were coated by physical adsorption, and the coatings were characterized by Raman spectroscopy, atomic force microscopy and water contact angle measurements. We quantified and visualized the colonization of coated and uncoated surfaces by three bacteria: Staphylococcus aureus, Listeria monocytogenes and Pseudomonas fluorescens. No viable cells were detected on surfaces coated with isoeugenol. The isoeugenol coating prepared with simple adsorption proved effective in preventing biofilm formation on stainless steel and polyethylene surfaces. The result was caused by the antibacterial effect of isoeugenol, as the coating did not diminish the adhesive properties of the surface. Our study demonstrates that a simple isoeugenol coating can prevent biofilm formation of S. aureus, L. monocytogenes and P. fluorescens on two commonly used surfaces. © 2017 The Society for Applied Microbiology.

Toxicity and transformation of graphene oxide and reduced graphene oxide in bacteria biofilm.

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Guo, Zhiling; Xie, Changjian; Zhang, Peng; Zhang, Junzhe; Wang, Guohua; He, Xiao; Ma, Yuhui; Zhao, Bin; Zhang, Zhiyong

2017-02-15

Impact of graphene based material (GNMs) on bacteria biofilm has not been well understood yet. In this study, we compared the impact of graphene oxide (GO) and reduced graphene oxide (rGO) on biofilm formation and development in Luria-Bertani (LB) medium using Escherichia coli and Staphylococcus aureus as models. GO significantly enhanced the cell growth, biofilm formation, and biofilm development even up to a concentration of 500mg/L. In contrast, rGO (≥50mg/L) strongly inhibited cell growth and biofilm formation. However, the inhibitory effects of rGO (50mg/L and 100mg/L) were attenuated in the mature phase (>24h) and eliminated at 48h. GO at 250mg/L decreased the reactive oxygen species (ROS) levels in biofilm and extracellular region at mature phase. ROS levels were significantly increased by rGO at early phase, while they returned to the same levels as control at mature phase. These results suggest that oxidative stress contributed to the inhibitory effect of rGO on bacterial biofilm. We further found that supplement of extracellular polymeric substances (EPS) in the growth medium attenuated the inhibitory effect of rGO on the growth of developed biofilm. XPS results showed that rGO were oxidized to GO which can enhance the bacterial growth. We deduced that the elimination of the toxicity of rGO at mature phase was contributed by EPS protection and the oxidation of rGO. This study provides new insights into the interaction of GNMs with bacteria biofilm. Copyright © 2016 Elsevier B.V. All rights reserved.

Anti-biofilm activities from marine cold adapted bacteria against staphylococci and Pseudomonas aeruginosa

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Rosanna ePapa

2015-12-01

Full Text Available Microbial biofilms have great negative impacts on the world’s economy and pose serious problems to industry, public health and medicine. The interest in the development of new approaches for the prevention and treatment of bacterial adhesion and biofilm formation has increased. Since, bacterial pathogens living in biofilm induce persistent chronic infections due to the resistance to antibiotics and host immune system. A viable approach should target adhesive properties without affecting bacterial vitality in order to avoid the appearance of resistant mutants. Many bacteria secrete anti-biofilm molecules that function in regulating biofilm architecture or mediating the release of cells from it during the dispersal stage of biofilm life cycle. Cold-adapted marine bacteria represent an untapped reservoir of biodiversity able to synthesize a broad range of bioactive compounds, including anti-biofilm molecules.The anti-biofilm activity of cell-free supernatants derived from sessile and planktonic cultures of cold-adapted bacteria belonging to Pseudoalteromonas, Psychrobacter and Psychromonas species were tested against Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa strains. Reported results demonstrate that we have selected supernatants, from cold-adapted marine bacteria, containing non-biocidal agents able to destabilize biofilm matrix of all tested pathogens without killing cells. A preliminary physico-chemical characterization of supernatants was also performed, and these analyses highlighted the presence of molecules of different nature that act by inhibiting biofilm formation. Some of them are also able to impair the initial attachment of the bacterial cells to the surface, thus likely containing molecules acting as anti-biofilm surfactant molecules.The described ability of cold-adapted bacteria to produce effective anti-biofilm molecules paves the way to further characterization of the most promising molecules

Novel long-chain compounds with both immunomodulatory and MenA inhibitory activities against Staphylococcus aureus and its biofilm.

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Choi, Seoung-Ryoung; Frandsen, Joel; Narayanasamy, Prabagaran

2017-01-10

Menaquinone (MK) biosynthesis pathway is a potential target for evaluating antimicrobials in gram-positive bacteria. Here, 1,4-dihydroxy-2-naphthoate prenyltransferase (MenA) was targeted to reduce methicillin-resistant Staphylococcus aureus (MRSA) growth. MenA inhibiting, long chain-based compounds were designed, synthesized and evaluated against MRSA and menaquinone utilizing bacteria in aerobic conditions. The results showed that these bacteria were susceptible to most of the compounds. Menaquinone (MK-4) supplementation rescued MRSA growth, suggesting these compounds inhibit MK biosynthesis. 3a and 7c exhibited promising inhibitory activities with MICs ranging 1-8 μg/mL against MRSA strains. The compounds did not facilitate small colony variant formation. These compounds also inhibited the biofilm growth by MRSA at high concentration. Compounds 3a, 6b and 7c displayed a promising extracellular bactericidal activity against MRSA at concentrations equal to and four-fold less than their respective MICs. We also observed cytokines released from THP-1 macrophages treated with compounds 3a, 6b and 7c and found decreases in TNF-α and IL-6 release and increase in IL-1β. These data provide evidence that MenA inhibitors act as TNF-α and IL-6 inhibitors, raising the potential for development and application of these compounds as potential immunomodulatory agents.

Biofilm is a Major Virulence Determinant in Bacterial Colonization of Chronic Skin Ulcers Independently from the Multidrug Resistant Phenotype

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Enea Gino Di Domenico

2017-05-01

Full Text Available Bacterial biofilm is a major factor in delayed wound healing and high levels of biofilm production have been repeatedly described in multidrug resistant organisms (MDROs. Nevertheless, a quantitative correlation between biofilm production and the profile of antimicrobial drug resistance in delayed wound healing remains to be determined. Microbial identification, antibiotic susceptibility and biofilm production were assessed in 135 clinical isolates from 87 patients. Gram-negative bacteria were the most represented microorganisms (60.8% with MDROs accounting for 31.8% of the total isolates. Assessment of biofilm production revealed that 80% of the strains were able to form biofilm. A comparable level of biofilm production was found with both MDRO and not-MDRO with no significant differences between groups. All the methicillin-resistant Staphylococcus aureus (MRSA and 80% of Pseudomonas aeruginosa MDR strains were found as moderate/high biofilm producers. Conversely, less than 17% of Klebsiella pneumoniae extended-spectrum beta-lactamase (ESBL, Escherichia coli-ESBL and Acinetobacter baumannii were moderate/high biofilm producers. Notably, those strains classified as non-biofilm producers, were always associated with biofilm producer bacteria in polymicrobial colonization. This study shows that biofilm producers were present in all chronic skin ulcers, suggesting that biofilm represents a key virulence determinant in promoting bacterial persistence and chronicity of ulcerative lesions independently from the MDRO phenotype.

BIOFILM FORMATION OF Vibrio cholerae ON STAINLESS STEEL USED IN FOOD PROCESSING

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Milagro FERNÁNDEZ-DELGADO

2016-01-01

Full Text Available Vibrio cholerae represents a significant threat to human health in developing countries. This pathogen forms biofilms which favors its attachment to surfaces and its survival and transmission by water or food. This work evaluated the in vitro biofilm formation of V. cholerae isolated from clinical and environmental sources on stainless steel of the type used in food processing by using the environmental scanning electron microscopy (ESEM. Results showed no cell adhesion at 4 h and scarce surface colonization at 24 h. Biofilms from the environmental strain were observed at 48 h with high cellular aggregations embedded in Vibrio exopolysaccharide (VPS, while less confluence and VPS production with microcolonies of elongated cells were observed in biofilms produced by the clinical strain. At 96 h the biofilms of the environmental strain were released from the surface leaving coccoid cells and residual structures, whereas biofilms of the clinical strain formed highly organized structures such as channels, mushroom-like and pillars. This is the first study that has shown the in vitro ability of V. cholerae to colonize and form biofilms on stainless steel used in food processing.

BIOFILM FORMATION OF Vibrio cholerae ON STAINLESS STEEL USED IN FOOD PROCESSING.

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Fernández-Delgado, Milagro; Rojas, Héctor; Duque, Zoilabet; Suárez, Paula; Contreras, Monica; García-Amado, M Alexandra; Alciaturi, Carlos

2016-01-01

Vibrio cholerae represents a significant threat to human health in developing countries. This pathogen forms biofilms which favors its attachment to surfaces and its survival and transmission by water or food. This work evaluated the in vitro biofilm formation of V. cholerae isolated from clinical and environmental sources on stainless steel of the type used in food processing by using the environmental scanning electron microscopy (ESEM). Results showed no cell adhesion at 4 h and scarce surface colonization at 24 h. Biofilms from the environmental strain were observed at 48 h with high cellular aggregations embedded in Vibrio exopolysaccharide (VPS), while less confluence and VPS production with microcolonies of elongated cells were observed in biofilms produced by the clinical strain. At 96 h the biofilms of the environmental strain were released from the surface leaving coccoid cells and residual structures, whereas biofilms of the clinical strain formed highly organized structures such as channels, mushroom-like and pillars. This is the first study that has shown the in vitro ability of V. cholerae to colonize and form biofilms on stainless steel used in food processing.

Should we stay or should we go: mechanisms and ecological consequences for biofilm dispersal.

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McDougald, Diane; Rice, Scott A; Barraud, Nicolas; Steinberg, Peter D; Kjelleberg, Staffan

2011-11-28

In most environments, bacteria reside primarily in biofilms, which are social consortia of cells that are embedded in an extracellular matrix and undergo developmental programmes resulting in a predictable biofilm 'life cycle'. Recent research on many different bacterial species has now shown that the final stage in this life cycle includes the production and release of differentiated dispersal cells. The formation of these cells and their eventual dispersal is initiated through diverse and remarkably sophisticated mechanisms, suggesting that there are strong evolutionary pressures for dispersal from an otherwise largely sessile biofilm. The evolutionary aspect of biofilm dispersal is now being explored through the integration of molecular microbiology with eukaryotic ecological and evolutionary theory, which provides a broad conceptual framework for the diversity of specific mechanisms underlying biofilm dispersal. Here, we review recent progress in this emerging field and suggest that the merging of detailed molecular mechanisms with ecological theory will significantly advance our understanding of biofilm biology and ecology.

Influence of femtosecond laser produced nanostructures on biofilm growth on steel

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Epperlein, Nadja; Menzel, Friederike; Schwibbert, Karin; Koter, Robert; Bonse, Jörn; Sameith, Janin; Krüger, Jörg; Toepel, Jörg

2017-10-01

Biofilm formation poses high risks in multiple industrial and medical settings. However, the robust nature of biofilms makes them also attractive for industrial applications where cell biocatalysts are increasingly in use. Since tailoring material properties that affect bacterial growth or its inhibition is gaining attention, here we focus on the effects of femtosecond laser produced nanostructures on bacterial adhesion. Large area periodic surface structures were generated on steel surfaces using 30-fs laser pulses at 790 nm wavelength. Two types of steel exhibiting a different corrosion resistance were used, i.e., a plain structural steel (corrodible) and a stainless steel (resistant to corrosion). Homogeneous fields of laser-induced periodic surface structures (LIPSS) were realized utilizing laser fluences close to the ablation threshold while scanning the sample under the focused laser beam in a multi-pulse regime. The nanostructures were characterized with optical and scanning electron microscopy. For each type of steel, more than ten identical samples were laser-processed. Subsequently, the samples were subjected to microbial adhesion tests. Bacteria of different shape and adhesion behavior (Escherichia coli and Staphylococcus aureus) were exposed to laser structures and to polished reference surfaces. Our results indicate that E. coli preferentially avoids adhesion to the LIPSS-covered areas, whereas S. aureus favors these areas for colonization.

The exopolysaccharide matrix: a virulence determinant of cariogenic biofilm.

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Koo, H; Falsetta, M L; Klein, M I

2013-12-01

Many infectious diseases in humans are caused or exacerbated by biofilms. Dental caries is a prime example of a biofilm-dependent disease, resulting from interactions of microorganisms, host factors, and diet (sugars), which modulate the dynamic formation of biofilms on tooth surfaces. All biofilms have a microbial-derived extracellular matrix as an essential constituent. The exopolysaccharides formed through interactions between sucrose- (and starch-) and Streptococcus mutans-derived exoenzymes present in the pellicle and on microbial surfaces (including non-mutans) provide binding sites for cariogenic and other organisms. The polymers formed in situ enmesh the microorganisms while forming a matrix facilitating the assembly of three-dimensional (3D) multicellular structures that encompass a series of microenvironments and are firmly attached to teeth. The metabolic activity of microbes embedded in this exopolysaccharide-rich and diffusion-limiting matrix leads to acidification of the milieu and, eventually, acid-dissolution of enamel. Here, we discuss recent advances concerning spatio-temporal development of the exopolysaccharide matrix and its essential role in the pathogenesis of dental caries. We focus on how the matrix serves as a 3D scaffold for biofilm assembly while creating spatial heterogeneities and low-pH microenvironments/niches. Further understanding on how the matrix modulates microbial activity and virulence expression could lead to new approaches to control cariogenic biofilms.

Screening of medicinal plants for antibacterial activities on Staphylococcus aureus strains isolated from bovine mastitis

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Marisa A. N. Diaz

2010-08-01

Full Text Available Staphylococcus aureus is the main causative agent of bovine mastitis. The activity of several extracts from ten medicinal plants traditionally used in Brazil as antiseptic was investigated against fifteen strains of Staphylococcus aureus isolated from animals with mastitis manifestation by the disc diffusion method and broth microdilution assay. The interference of the extracts on cell in the form of adherent colonies was also evaluated. MIC values ranged from 0.5 mg/mL to 1.0 mg/mL and biofilm inhibitory concentration (BIC were between 0.25 mg/mL and 0.8 mg/mL. Results revealed the potential of extracts of Senna macranthera, Artemisia absinthium, Cymbopogon nardus and Baccharis dracunculifolia as antibacterial agents against S. aureus strains isolated from bovine mastitis and support the possible use of these phytotherapic agents in the clinical management of the disease.

Chlorhexidine whole-body washing of patients reduces methicillin-resistant Staphylococcus aureus and has a direct effect on the distribution of the ST5-MRSA-II (New York/Japan) clone.

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Velázquez-Meza, Maria Elena; Mendoza-Olazarán, Soraya; Echániz-Aviles, Gabriela; Camacho-Ortiz, Adrián; Martínez-Reséndez, Michel Fernando; Valero-Moreno, Vanessa; Garza-González, Elvira

2017-06-01

Methicillin-resistant Staphylococcus aureus (MRSA) colonizes the skin of hospitalized patients and is associated with high morbidity and mortality. To prevent colonization and infection by S. aureus, better disinfection practices are required. Therefore, we evaluated the effect of chlorhexidine whole-body washing on hospital-acquired S. aureus infections among intensive care unit (ICU) patients in a tertiary hospital in Mexico. The study was conducted over 18 months to evaluate the effect of 2 % chlorhexidine gluconate (CXG) whole-body washing of ICU adult patients on chlorhexidine and antibiotic resistance, biofilm production and clonal distribution of S. aureus in a tertiary care hospital. Minimum inhibitory concentrations for CXG, antibiotic susceptibility and biofilm production by S. aureus isolates were determined. Pulsed-field gel electrophoresis, multilocus sequence typing (MLST) and PCR for Panton-Valentine leucocidin (PVL) were used for molecular typing of MRSA isolates.Results/Key findings. We included 158 isolates. A reduction in antibiotic resistance in the study period was observed for clindamycin, levofloxacin, norfloxacin, oxacillin and trimethoprim/sulfamethoxazole. None of the isolates showed reduced susceptibility to CXG. Most of the isolates were non-biofilm producers (147/158). The most commonly identified clone was a descendant of the ST5-MRSA-II (New York/Japan) clone. This clone decreased during the intervention period and reappeared markedly in the post-intervention period. During the post-intervention period, two isolates were related with the clone ST8-MRSA-IV (also known as USA300). Our findings suggest that the CXG bathing favored the reduction of healthcare-associated MRSA isolates and a temporary reduction of the predominant ST5-MRSA-II (New York/Japan) clone.

Photodynamic therapy with water-soluble phtalocyanines against bacterial biofilms in teeth root canals

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Gergova, Raina; Georgieva, Tzvetelina; Angelov, Ivan; Mantareva, Vanya; Valkanov, Serjoga; Mitov, Ivan; Dimitrov, Slavcho

2012-06-01

The study presents the PDT with metal phthalocyanines on biofilms grown in root canals of ten representatives of the Gram-positive and the Gram-negative bacterial species and a fungus Candida albicans which cause aqute teeth infections in root canals.. The extracted human single-root teeth infected for 48 h with microorganisms in conditions to form biofilms of the above pathogens were PDT treated. The stage of biofilm formation and PDT effect of the samples of the teeth were determined by the scaning electron microscopy and with standard microbial tests. The PDT treating procedure included 10 min incubation with the respected phthalocyanine and irradiated with 660 nm Diode laser for 10 min. The most strongly antibacterial activity was achieved with zinc(II) phthalocyanine (ZnPc) against Enterococcus faecalis, Staphylococcus aureus and Moraxella catarrhalis. The other Gram-negative bacteria and Candida albicans were 10-100 times more resistant than the Gram-positive species. The Gram-negative Moraxella catarrhalis and Acinetobacter baumannii were more sensitive than the enterobacteria, but eradication of Pseudomonas aeruginosa in biofilm was insignificant. The influence of the stage of biofilm formation and the initial conditions (bacterial density, photosensitizer concentration and energy fluence of radiation) to the obtained level of inactivation of biofilms was investigated. The PDT with ZnPc photosensitizers show a powerful antimicrobial activity against the most frequent pathogens in endodontic infections and this method for inactivation of pathogens may be used with sucsses for treatment of the bacterial biofilms in the root canals.

The Effect of Cryopreserved Human Placental Tissues on Biofilm Formation of Wound-Associated Pathogens.

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Mao, Yong; Singh-Varma, Anya; Hoffman, Tyler; Dhall, Sandeep; Danilkovitch, Alla; Kohn, Joachim

2018-01-08

Biofilm, a community of bacteria, is tolerant to antimicrobial agents and ubiquitous in chronic wounds. In a chronic DFU (Diabetic Foot Ulcers) clinical trial, the use of a human cryopreserved viable amniotic membrane (CVAM) resulted in a high rate of wound closure and reduction of wound-related infections. Our previous study demonstrated that CVAM possesses intrinsic antimicrobial activity against a spectrum of wound-associated bacteria under planktonic culture conditions. In this study, we evaluated the effect of CVAM and cryopreserved viable umbilical tissue (CVUT) on biofilm formation of S. aureus and P. aeruginosa , the two most prominent pathogens associated with chronic wounds. Firstly, we showed that, like CVAM, CVUT released antibacterial activity against multiple bacterial pathogens and the devitalization of CVUT reduced its antibacterial activity. The biofilm formation was then measured using a high throughput method and an ex vivo porcine dermal tissue model. We demonstrate that the formation of biofilm was significantly reduced in the presence of CVAM- or CVUT-derived conditioned media compared to control assay medium. The formation of P. aeruginosa biofilm on CVAM-conditioned medium saturated porcine dermal tissues was reduced 97% compared with the biofilm formation on the control medium saturated dermal tissues. The formation of S. auerus biofilm on CVUT-conditioned medium saturated dermal tissues was reduced 72% compared with the biofilm formation on the control tissues. This study is the first to show that human cryopreserved viable placental tissues release factors that inhibit biofilm formation. Our results provide an explanation for the in vivo observation of their ability to support wound healing.

The Effect of Cryopreserved Human Placental Tissues on Biofilm Formation of Wound-Associated Pathogens

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Yong Mao

2018-01-01

Full Text Available Biofilm, a community of bacteria, is tolerant to antimicrobial agents and ubiquitous in chronic wounds. In a chronic DFU (Diabetic Foot Ulcers clinical trial, the use of a human cryopreserved viable amniotic membrane (CVAM resulted in a high rate of wound closure and reduction of wound-related infections. Our previous study demonstrated that CVAM possesses intrinsic antimicrobial activity against a spectrum of wound-associated bacteria under planktonic culture conditions. In this study, we evaluated the effect of CVAM and cryopreserved viable umbilical tissue (CVUT on biofilm formation of S. aureus and P. aeruginosa, the two most prominent pathogens associated with chronic wounds. Firstly, we showed that, like CVAM, CVUT released antibacterial activity against multiple bacterial pathogens and the devitalization of CVUT reduced its antibacterial activity. The biofilm formation was then measured using a high throughput method and an ex vivo porcine dermal tissue model. We demonstrate that the formation of biofilm was significantly reduced in the presence of CVAM- or CVUT-derived conditioned media compared to control assay medium. The formation of P. aeruginosa biofilm on CVAM-conditioned medium saturated porcine dermal tissues was reduced 97% compared with the biofilm formation on the control medium saturated dermal tissues. The formation of S. auerus biofilm on CVUT-conditioned medium saturated dermal tissues was reduced 72% compared with the biofilm formation on the control tissues. This study is the first to show that human cryopreserved viable placental tissues release factors that inhibit biofilm formation. Our results provide an explanation for the in vivo observation of their ability to support wound healing.

[The bacterial biofilm and the possibilities of chemical plaque control. Literature review].

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Gera, István

2008-06-01

Most microorganisms in the oral cavity attach to surfaces and form matrix-embedded biofilms. Biofilms are structured and spatially organized, composed of consortia of interacting microorganisms. The properties of the mass of biofilm are different from that of the simple sum of the component species. The older the plaque (biofilm) is the more structurally organized and become more resistant to environmental attacks. The bacterial community favors the growth of obligatory anaerobic microorganisms. The most effective means of the elimination of matured biofilm is the mechanical disruption of the interbacterial protective matrix and removal of bacterial colonies. The antiseptic agents are primarily effective in the prevention of biofilm formation and anticipation of the maturation of the bacterial plaque. Bacteria in matured biofilms are less susceptible to antimicrobial agents because several physical and biological factors protect the bacterial consortia. To kill bacteria in a matured, well organized biofilm, significantly higher concentration and longer exposition are required. Antiseptic mouthrinses in a conventional dose and time can only reach the superficial bacteria while the bacteria in the depth of the biofilm remains intact. Therefore, the efficacy of any antiseptic mouthwash depends not just on its microbicidal properties demonstrated in vitro, but also on its ability to penetrate the organized biofilm on the teeth. Recent studies have demonstrated that both bisbiguanid compounds and essential oils are capable of penetrating the biofilm, and reduce established plaque and gingivitis. The essential oils showed high penetrability and were more effective on organized biofilm than stannous fluorides or triclosan copolymer antiplaque agents.

The role of biofilms as environmental reservoirs of antibiotic resistance

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Jose Luis eBalcazar

2015-10-01

Full Text Available Antibiotic resistance has become a significant and growing threat to public and environmental health. To face this problem both at local and global scales, a better understanding of the sources and mechanisms that contribute to the emergence and spread of antibiotic resistance is required. Recent studies demonstrate that aquatic ecosystems are reservoirs of resistant bacteria and antibiotic resistance genes as well as potential conduits for their transmission to human pathogens. Despite the wealth of information about antibiotic pollution and its effect on the aquatic microbial resistome, the contribution of environmental biofilms to the acquisition and spread of antibiotic resistance has not been fully explored in aquatic systems. Biofilms are structured multicellular communities embedded in a self-produced extracellular matrix that acts as a barrier to antibiotic diffusion. High population densities and proximity of cells in biofilms also increases the chances for genetic exchange among bacterial species converting biofilms in hot spots of antibiotic resistance. This review focuses on the potential effect of antibiotic pollution on biofilm microbial communities, with special emphasis on ecological and evolutionary processes underlying acquired resistance to these compounds.

Characterization of Acinetobacter baumannii biofilm associated components