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Sample records for aureus biofilm maturation

  1. Staphylococcus aureus biofilms

    PubMed Central

    Archer, Nathan K; Mazaitis, Mark J; Costerton, J William; Leid, Jeff G; Powers, Mary Elizabeth

    2011-01-01

    Increasing attention has been focused on understanding bacterial biofilms and this growth modality's relation to human disease. In this review we explore the genetic regulation and molecular components involved in biofilm formation and maturation in the context of the Gram-positive cocci, Staphylococcus aureus. In addition, we discuss diseases and host immune responses, along with current therapies associated with S. aureus biofilm infections and prevention strategies. PMID:21921685

  2. Vancomycin displays time-dependent eradication of mature Staphylococcus aureus biofilms.

    PubMed

    Post, Virginia; Wahl, Peter; Richards, R Geoff; Moriarty, T Fintan

    2017-02-01

    This study was carried out to determine the time and concentration profile required to achieve vancomycin-mediated eradication of Staphylococcus aureus biofilm. This information is critical for the identification of performance targets for local antibiotic delivery vehicles that target biofilm infections. S. aureus UAMS-1 biofilms were grown for 7 days on titanium-aluminium-niobium discs in Mueller Hinton broth. After 7 days, the discs were then incubated in Mueller Hinton broth containing vancomycin at concentrations of 100, 200, 500, 1,000, and 2,000 mg/L. Biofilm eradication was assessed under both static and shaking conditions. Samples were retrieved at regular intervals for up to 28 days for quantification of residual biofilm. One additional disc was processed per time point for scanning electron microscopy. Progressive and significant reduction of viable bacteria was observed over time at all concentrations compared to unexposed controls. After 28 days under static conditions, the S. aureus biofilm was completely eradicated at 200 mg/L vancomycin and higher concentrations, but not at 100 mg/L. In contrast, bacterial biofilm could not be eradicated under shaking conditions at any concentration.

  3. Staphylococcus aureus biofilms: recent developments in biofilm dispersal.

    PubMed

    Lister, Jessica L; Horswill, Alexander R

    2014-01-01

    Staphylococcus aureus is a major cause of nosocomial and community-acquired infections and represents a significant burden on the healthcare system. S. aureus attachment to medical implants and host tissue, and the establishment of a mature biofilm, play an important role in the persistence of chronic infections. The formation of a biofilm, and encasement of cells in a polymer-based matrix, decreases the susceptibility to antimicrobials and immune defenses, making these infections difficult to eradicate. During infection, dispersal of cells from the biofilm can result in spread to secondary sites and worsening of the infection. In this review, we discuss the current understanding of the pathways behind biofilm dispersal in S. aureus, with a focus on enzymatic and newly described broad-spectrum dispersal mechanisms. Additionally, we explore potential applications of dispersal in the treatment of biofilm-mediated infections.

  4. Staphylococcus aureus biofilms: recent developments in biofilm dispersal

    PubMed Central

    Lister, Jessica L.; Horswill, Alexander R.

    2014-01-01

    Staphylococcus aureus is a major cause of nosocomial and community-acquired infections and represents a significant burden on the healthcare system. S. aureus attachment to medical implants and host tissue, and the establishment of a mature biofilm, play an important role in the persistence of chronic infections. The formation of a biofilm, and encasement of cells in a polymer-based matrix, decreases the susceptibility to antimicrobials and immune defenses, making these infections difficult to eradicate. During infection, dispersal of cells from the biofilm can result in spread to secondary sites and worsening of the infection. In this review, we discuss the current understanding of the pathways behind biofilm dispersal in S. aureus, with a focus on enzymatic and newly described broad-spectrum dispersal mechanisms. Additionally, we explore potential applications of dispersal in the treatment of biofilm-mediated infections. PMID:25566513

  5. Prevention and treatment of Staphylococcus aureus biofilms

    PubMed Central

    Bhattacharya, Mohini; Wozniak, Daniel J; Stoodley, Paul; Hall-Stoodley, Luanne

    2016-01-01

    S. aureus colonizes both artificial and tissue surfaces in humans causing chronic persistent infections that are difficult to cure. It is a notorious pathogen due to its antibiotic recalcitrance and phenotypic adaptability, both of which are facilitated by its ability to develop biofilms. S. aureus biofilms challenge conventional anti-infective approaches, most notably antibiotic therapy. Therefore there is an unmet need to develop and include parallel approaches that target S. aureus biofilm infections. This review discusses two broad anti-infective strategies: (1) preventative approaches (anti-biofilm surface coatings, the inclusion of biofilm-specific vaccine antigens); and (2) approaches aimed at eradicating established S. aureus biofilms, particularly those associated with implant infections. Advances in understanding the distinct nature of S. aureus biofilm development and pathogenesis have led to growing optimism in S. aureus biofilm targeted anti-infective strategies. Further research is needed however, to see the successful administration and validation of these approaches to the diverse types of infections caused by S. aureus biofilms from multiple clinical strains. PMID:26646248

  6. Hyaluronan Modulation Impacts Staphylococcus aureus Biofilm Infection

    PubMed Central

    Ibberson, Carolyn B.; Parlet, Corey P.; Kwiecinski, Jakub; Crosby, Heidi A.; Meyerholz, David K.

    2016-01-01

    Staphylococcus aureus is a leading cause of chronic biofilm infections. Hyaluronic acid (HA) is a large glycosaminoglycan abundant in mammalian tissues that has been shown to enhance biofilm formation in multiple Gram-positive pathogens. We observed that HA accumulated in an S. aureus biofilm infection using a murine implant-associated infection model and that HA levels increased in a mutant strain lacking hyaluronidase (HysA). S. aureus secretes HysA in order to cleave HA during infection. Through in vitro biofilm studies with HA, the hysA mutant was found to accumulate increased biofilm biomass compared to the wild type, and confocal microscopy showed that HA is incorporated into the biofilm matrix. Exogenous addition of purified HysA enzyme dispersed HA-containing biofilms, while catalytically inactive enzyme had no impact. Additionally, induction of hysA expression prevented biofilm formation and also dispersed an established biofilm in the presence of HA. These observations were corroborated in the implant model, where there was decreased dissemination from an hysA mutant biofilm infection compared to the S. aureus wild type. Histopathology demonstrated that infection with an hysA mutant caused significantly reduced distribution of tissue inflammation compared to wild-type infection. To extend these studies, the impact of HA and S. aureus HysA on biofilm-like aggregates found in joint infections was examined. We found that HA contributes to the formation of synovial fluid aggregates, and HysA can disrupt aggregate formation. Taken together, these studies demonstrate that HA is a relevant component of the S. aureus biofilm matrix and HysA is important for dissemination from a biofilm infection. PMID:27068096

  7. Activity of Gallidermin on Staphylococcus aureus and Staphylococcus epidermidis Biofilms

    PubMed Central

    Saising, Jongkon; Dube, Linda; Ziebandt, Anne-Kathrin; Voravuthikunchai, Supayang Piyawan; Nega, Mulugeta

    2012-01-01

    Due to their abilities to form strong biofilms, Staphylococcus aureus and Staphylococcus epidermidis are the most frequently isolated pathogens in persistent and chronic implant-associated infections. As biofilm-embedded bacteria are more resistant to antibiotics and the immune system, they are extremely difficult to treat. Therefore, biofilm-active antibiotics are a major challenge. Here we investigated the effect of the lantibiotic gallidermin on two representative biofilm-forming staphylococcal species. Gallidermin inhibits not only the growth of staphylococci in a dose-dependent manner but also efficiently prevents biofilm formation by both species. The effect on biofilm might be due to repression of biofilm-related targets, such as ica (intercellular adhesin) and atl (major autolysin). However, gallidermin's killing activity on 24-h and 5-day-old biofilms was significantly decreased. A subpopulation of 0.1 to 1.0% of cells survived, comprising “persister” cells of an unknown genetic and physiological state. Like many other antibiotics, gallidermin showed only limited activity on cells within mature biofilms. PMID:22926575

  8. Temporal and stochastic control of Staphylococcus aureus biofilm development.

    PubMed

    Moormeier, Derek E; Bose, Jeffrey L; Horswill, Alexander R; Bayles, Kenneth W

    2014-10-14

    Biofilm communities contain distinct microniches that result in metabolic heterogeneity and variability in gene expression. Previously, these niches were visualized within Staphylococcus aureus biofilms by observing differential expression of the cid and lrg operons during tower formation. In the present study, we examined early biofilm development and identified two new stages (designated "multiplication" and "exodus") that were associated with changes in matrix composition and a distinct reorganization of the cells as the biofilm matured. The initial attachment and multiplication stages were shown to be protease sensitive but independent of most cell surface-associated proteins. Interestingly, after 6 h of growth, an exodus of the biofilm population that followed the transition of the biofilm to DNase I sensitivity was demonstrated. Furthermore, disruption of the gene encoding staphylococcal nuclease (nuc) abrogated this exodus event, causing hyperproliferation of the biofilm and disrupting normal tower development. Immediately prior to the exodus event, S. aureus cells carrying a nuc::gfp promoter fusion demonstrated Sae-dependent expression but only in an apparently random subpopulation of cells. In contrast to the existing model for tower development in S. aureus, the results of this study suggest the presence of a Sae-controlled nuclease-mediated exodus of biofilm cells that is required for the development of tower structures. Furthermore, these studies indicate that the differential expression of nuc during biofilm development is subject to stochastic regulatory mechanisms that are independent of the formation of metabolic microniches. Importance: In this study, we provide a novel view of four early stages of biofilm formation by the human pathogen Staphylococcus aureus. We identified an initial nucleoprotein matrix during biofilm development that is DNase I insensitive until a critical point when a nuclease-mediated exodus of the population is induced prior

  9. Screening a repurposing library for potentiators of antibiotics against Staphylococcus aureus biofilms.

    PubMed

    Van den Driessche, Freija; Brackman, Gilles; Swimberghe, Rosalie; Rigole, Petra; Coenye, Tom

    2017-03-01

    Staphylococcus aureus biofilms are involved in a wide range of infections that are extremely difficult to treat with conventional antibiotic therapy. We aimed to identify potentiators of antibiotics against mature biofilms of S. aureus Mu50, a methicillin-resistant and vancomycin-intermediate-resistant strain. Over 700 off-patent drugs from a repurposing library were screened in combination with vancomycin in a microtitre plate (MTP)-based biofilm model system. This led to the identification of 25 hit compounds, including four phenothiazines among which thioridazine was the most potent. Their activity was evaluated in combination with other antibiotics both against planktonic and biofilm-grown S. aureus cells. The most promising combinations were subsequently tested in an in vitro chronic wound biofilm infection model. Although no synergistic activity was observed against planktonic cells, thioridazine potentiated the activity of tobramycin, linezolid and flucloxacillin against S. aureus biofilm cells. However, this effect was only observed in a general biofilm model and not in a chronic wound model of biofilm infection. Several drug compounds were identified that potentiated the activity of vancomycin against biofilms formed in a MTP-based biofilm model. A selected hit compound lost its potentiating activity in a model that mimics specific aspects of wound biofilms. This study provides a platform for discovering and evaluating potentiators against bacterial biofilms and highlights the necessity of using relevant in vitro biofilm model systems.

  10. Investigation of biofilm formation in clinical isolates of Staphylococcus aureus.

    PubMed

    Cassat, James E; Lee, Chia Y; Smeltzer, Mark S

    2007-01-01

    As with many other bacterial species, the most commonly used method to assess staphylococcal biofilm formation in vitro is the microtiter plate assay. This assay is particularly useful for comparison of multiple strains including large-scale screens of mutant libraries. When such screens are applied to the coagulase-negative staphylococci in general, and Staphylococcus epidermidis in particular, they are relatively straightforward by comparison with microtiter plate assays used to assess biofilm formation in other bacterial species. However, in the case of clinical isolates of Staphylococcus aureus, including methicillin-resistant S. aureus, we have found it necessary to employ specific modifications including precoating of the wells of the microtiter plate with plasma proteins and supplementation of the medium with both salt and glucose. In this chapter, we describe the microtiter plate assay in the specific context of clinical isolates of S. aureus and the use of these modifications. A second in vitro method, which also is generally dependent on coating with plasma proteins and supplementation of the growth medium, is the use of flow cells. In this method, bacteria are allowed to attach to a surface and then monitored with respect to their ability to remain attached to the substrate and differentiate into mature biofilms under the constant pressure of fluid shear force. Although flow cells are not applicable to large-scale screens, we have found that they provide a more reproducible and accurate assessment of the capacity of S. aureus clinical isolates to form a biofilm. They also provide a means of analyzing structural differences in biofilm architecture and isolating bacteria and/or spent media for analysis of physiological and metabolic changes associated with the adaptive response to growth in a biofilm. While a primary focus of this chapter is on the use of in vitro assays to assess biofilm formation in clinical isolates of S. aureus, it is important to

  11. Statins and Antimicrobial Effects: Simvastatin as a Potential Drug against Staphylococcus aureus Biofilm

    PubMed Central

    Franco, Gilson Cesar; Schwartz-Filho, Humberto Osvaldo; de Andrade, Eduardo Dias

    2015-01-01

    Statins are important lipid-lowering agents with other pleiotropic effects. Several studies have explored a possible protective effect of statins to reduce the morbidity and mortality of many infectious diseases. Staphylococcus aureus is one of the main pathogens implicated in nosocomial infections; its ability to form biofilms makes treatment difficult. The present study observed the MIC of atorvastatin, pravastatin and simvastatin against S. aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis. Simvastatin was the only agent with activity against clinical isolates and reference strains of methicilin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA). Thus, the effects of simvastatin on the growth, viability and biofilm formation of S. aureus were tested. In addition, a possible synergistic effect between simvastatin and vancomycin was evaluated. Simvastatin’s MIC was 15.65 µg/mL for S. aureus 29213 and 31.25 µg/mL for the other strains of S. aureus. The effect of simvastatin was bactericidal at 4xMIC and bacteriostatic at the MIC concentration. No synergistic effect was found between simvastatin and vancomycin. However, the results obtained against S. aureus biofilms showed that, in addition to inhibiting adhesion and biofilm formation at concentrations from 1/16xMIC to 4xMIC, simvastatin was also able to act against mature biofilms, reducing cell viability and extra-polysaccharide production. In conclusion, simvastatin showed pronounced antimicrobial activity against S. aureus biofilms, reducing their formation and viability. PMID:26020797

  12. Streptokinase Treatment Reverses Biofilm-Associated Antibiotic Resistance in Staphylococcus aureus

    PubMed Central

    Jørgensen, Nis Pedersen; Zobek, Natalia; Dreier, Cindy; Haaber, Jakob; Ingmer, Hanne; Larsen, Ole Halfdan; Meyer, Rikke L.

    2016-01-01

    Biofilms formed by Staphylococcus aureus is a serious complication to the use of medical implants. A central part of the pathogenesis relies on S. aureus’ ability to adhere to host extracellular matrix proteins, which adsorb to medical implants and stimulate biofilm formation. Being coagulase positive, S. aureus furthermore induces formation of fibrin fibers from fibrinogen in the blood. Consequently, we hypothesized that fibrin is a key component of the extracellular matrix of S. aureus biofilms under in vivo conditions, and that the recalcitrance of biofilm infections can be overcome 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 microscopy. Addition of human plasma stimulated biofilm formation in BHI in a dose-dependent manner, and biofilms could be partially dispersed by streptokinase. The biofilms could be eradicated with physiologically relevant concentrations of streptokinase in combination with rifampicin and vancomycin 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. aureus biofilm infections. PMID:27681928

  13. Eradication of Staphylococcus aureus Catheter-Related Biofilm Infections Using ML:8 and Citrox

    PubMed Central

    Hogan, S.; Zapotoczna, M.; Stevens, N. T.; Humphreys, H.; O'Gara, J. P.

    2016-01-01

    Staphylococci are a leading cause of catheter-related infections (CRIs) due to biofilm formation. CRIs are typically managed by either device removal or systemic antibiotics, often in combination with catheter lock solutions (CLSs). CLSs provide high concentrations of the antimicrobial agent at the site of infection. However, the most effective CLSs against staphylococcal biofilm-associated infections have yet to be determined. The purpose of this study was to evaluate the efficacy and suitability of two newly described antimicrobial agents, ML:8 and Citrox, as CLSs against Staphylococcus aureus biofilms. ML:8 (1% [vol/vol]) and Citrox (1% [vol/vol]), containing caprylic acid and flavonoids, respectively, were used to treat S. aureus biofilms grown in vitro using newly described static and flow biofilm assays. Both agents reduced biofilm viability >97% after 24 h of treatment. Using a rat model of CRI, ML:8 was shown to inactivate early-stage S. aureus biofilms in vivo, while Citrox inactivated established, mature in vivo biofilms. Cytotoxicity and hemolytic activity of ML:8 and Citrox were equivalent to those of other commercially available CLSs. Neither ML:8 nor Citrox induced a cytokine response in human whole blood, and exposure of S. aureus to either agent for 90 days was not associated with any increase in resistance. Taken together, these data reveal the therapeutic potential of these agents for the treatment of S. aureus catheter-related biofilm infections. PMID:27458213

  14. Activity of novel inhibitors of Staphylococcus aureus biofilms.

    PubMed

    Woo, Seung-Gyun; Lee, So-Yeon; Lee, So-Min; Lim, Kyoung-Hee; Ha, Eun-Ju; Eom, Yong-Bin

    2017-03-01

    Staphylococcus aureus is one of the most important pathogens causing chronic biofilm infections. These are becoming more difficult to treat owing to drug resistance, particularly because S. aureus biofilms limit the efficacy of antimicrobial agents, leading to high morbidity and mortality. In the present study, we screened for inhibitors of S. aureus biofilm formation using a natural product library from the Korea Chemical Bank (KCB). Screening by crystal violet-based biomass staining assay identified hit compounds. Further examination of antibiofilm properties of these compounds was conducted and led to the identification of celastrol and telithromycin. In vitro, both celastrol and telithromycin were toxic to planktonic S. aureus and also active against a clinical methicillin-resistant S. aureus (MRSA) isolate. The effect of the compounds on preformed biofilms of clinical MRSA isolates was evaluated by confocal laser scanning microscopy (CLSM), which revealed the absence of typical biofilm architecture. In addition, celastrol and telithromycin inhibited the production of extracellular protein at selected sub-MIC concentrations, which revealed the reduced extracellular polymeric substance (EPS) secretion. Celastrol exhibited greater cytotoxicity than telithromycin. These data suggest that the hit compounds, especially telithromycin, could be considered novel inhibitors of S. aureus biofilm. Although the mechanisms of the effects on S. aureus biofilms are not fully understood, our data suggest that telithromycin could be a useful adjuvant therapeutic agent for S. aureus biofilm-related infections.

  15. Impact of engineered surface microtopography on biofilm formation of Staphylococcus aureus.

    PubMed

    Chung, Kenneth K; Schumacher, James F; Sampson, Edith M; Burne, Robert A; Antonelli, Patrick J; Brennan, Anthony B

    2007-06-01

    The surface of an indwelling medical device can be colonized by human pathogens that can form biofilms and cause infections. In most cases, these biofilms are resistant to antimicrobial therapy and eventually necessitate removal or replacement of the device. An engineered surface microtopography based on the skin of sharks, Sharklet AF, has been designed on a poly(dimethyl siloxane) elastomer (PDMSe) to disrupt the formation of bacterial biofilms without the use of bactericidal agents. The Sharklet AF PDMSe was tested against smooth PDMSe for biofilm formation of Staphylococcus aureus over the course of 21 days. The smooth surface exhibited early-stage biofilm colonies at 7 days and mature biofilms at 14 days, while the topographical surface did not show evidence of early biofilm colonization until day 21. At 14 days, the mean value of percent area coverage of S. aureus on the smooth surface was 54% compared to 7% for the Sharklet AF surface (p<0.01). These results suggest that surface modification of indwelling medical devices and exposed sterile surfaces with the Sharklet AF engineered topography may be an effective solution in disrupting biofilm formation of S. aureus.

  16. Synergistic activity between an antimicrobial polyacrylamide and daptomycin versus Staphylococcus aureus biofilm.

    PubMed

    Siala, Wafi; Van Bambeke, Françoise; Taresco, Vincenzo; Piozzi, Antonella; Francolini, Iolanda

    2016-07-01

    Antibiotic resistance of bacteria growing in biofilms compared to their planktonic counterparts enhances the difficulty to eradicate biofilm-associated infections. In the last decade, combination antibiotic therapy has emerged as an attractive strategy for treating biofilm infections, even if in most of tolerant biofilms the optimal combinations are still unknown. In this study, an antimicrobial cationic polyacrylamide was used in combination with daptomycin or moxifloxacin against mature biofilms of Staphylococcus aureus clinical isolates to examine a possible improvement of the antibiofilm activity of the two antibiotics. The polymer did not have an effect on moxifloxacin but significantly increased the antibiofilm efficacy of daptomycin. These findings are presumably related to the different mechanism of action of the two drugs. In summary, our data highlighted the ability of polycations to increase daptomycin antibiofilm activity providing a potential strategy to eradicate biofilms in industrial or medical settings.

  17. A modified CDC biofilm reactor to produce mature biofilms on the surface of peek membranes for an in vivo animal model application.

    PubMed

    Williams, Dustin L; Woodbury, Kassie L; Haymond, Bryan S; Parker, Albert E; Bloebaum, Roy D

    2011-06-01

    Biofilm-related infections have become a major clinical concern. Typically, animal models that involve inoculation with planktonic bacteria have been used to create positive infection signals and examine antimicrobial strategies for eradicating or preventing biofilm-related infection. However, it is estimated that 99.9% of bacteria in nature dwell in established biofilms. As such, open wounds have significant potential to become contaminated with bacteria that reside in a well-established biofilm. In this study, a modified CDC biofilm reactor was developed to repeatably grow mature biofilms of Staphylococcus aureus on the surface of polyetheretherketone (PEEK) membranes for inoculation in a future animal model of orthopaedic implant biofilm-related infection. Results indicated that uniform, mature biofilms repeatably grew on the surface of the PEEK membranes.

  18. Susceptibility of Staphylococcus aureus biofilms to reactive discharge gases

    PubMed Central

    Traba, Christian; Liang, Jun F.

    2011-01-01

    Formation of bacterial biofilms at solid-liquid interfaces creates numerous problems in both industrial and biomedical sciences. In this study, the susceptibility of Staphylococcus aureus biofilms to discharge gas generated from plasma was tested. It was found that despite distinct chemical/physical properties, discharge gases from oxygen, nitrogen, and argon demonstrated very potent and almost the same anti-biofilm activity. The bacterial cells in S. aureus biofilms were killed (>99.9%) by discharge gas within minutes of exposure. Under optimal experimental conditions, no bacteria and biofilm re-growth from discharge gas treated biofilms was found. Further studies revealed that the anti-biofilm activity of the discharge gas occurred by two distinct mechanisms: 1) killing bacteria in biofilms by causing severe cell membrane damage, and 2) damaging the extracellular polymeric matrix in the architecture of the biofilm to release biofilm from the surface of the solid substratum . Information gathered from this study provides an insight into the anti-biofilm mechanisms of plasma and confirms the applications of discharge gas in the treatment of biofilms and biofilm related bacterial infections. PMID:21774615

  19. Characterization of Staphylococcus aureus Biofilm Formation in Urinary Tract Infection

    PubMed Central

    YOUSEFI, Masoud; POURMAND, Mohammad Reza; FALLAH, Fatemeh; HASHEMI, Ali; MASHHADI, Rahil; NAZARI-ALAM, Ali

    2016-01-01

    Background: The aim of this study was to investigate the antibiotic susceptibility pattern as well as the phenotypic and genotypic biofilm formation ability of Staphylococcus aureus isolates from patients with urinary tract infection (UTI). Methods: A total of 39 isolates of S. aureus were collected from patients with UTI. The antibiotic susceptibility patterns of the isolates were determined by the Kirby-Bauer disk-diffusion. We used the Modified Congo red agar (MCRA) and Microtiter plate methods to assess the ability of biofilm formation. All isolates were examined for determination of biofilm related genes, icaA, fnbA, clfA and bap using PCR method. Results: Linezolid, quinupristin/dalfopristin and chloramphenicol were the most effective agents against S. aureus isolates. Overall, 69.2% of S. aureus isolates were biofilm producers. Resistance to four antibiotics such as nitrofurantoin (71.4% vs. 28.6%, P=0.001), tetracycline (57.7% vs. 42.3%, P=0.028), erythromycin and ciprofloxacin (56% vs. 44%, P=0.017) was higher among biofilm producers than non-biofilm producers. The icaA, fnbA and clfA genes were present in all S. aureus isolates. However, bap gene was not detected in any of the isolates. Conclusion: Our findings reinforce the role of biofilm formation in resistance to antimicrobial agents. Trimethoprimsulfamethoxazole and doxycycline may be used as an effective treatment for UTI caused by biofilm producers S. aureus. Our results suggest that biofilm formation is not dependent to just icaA, fnbA, clfA and bap genes harbor in S. aureus strains. PMID:27252918

  20. Biofilm Matrix Exoproteins Induce a Protective Immune Response against Staphylococcus aureus Biofilm Infection

    PubMed Central

    Gil, Carmen; Solano, Cristina; Burgui, Saioa; Latasa, Cristina; García, Begoña; Toledo-Arana, Alejandro

    2014-01-01

    The Staphylococcus aureus biofilm mode of growth is associated with several chronic infections that are very difficult to treat due to the recalcitrant nature of biofilms to clearance by antimicrobials. Accordingly, there is an increasing interest in preventing the formation of S. aureus biofilms and developing efficient antibiofilm vaccines. Given the fact that during a biofilm-associated infection, the first primary interface between the host and the bacteria is the self-produced extracellular matrix, in this study we analyzed the potential of extracellular proteins found in the biofilm matrix to induce a protective immune response against S. aureus infections. By using proteomic approaches, we characterized the exoproteomes of exopolysaccharide-based and protein-based biofilm matrices produced by two clinical S. aureus strains. Remarkably, results showed that independently of the nature of the biofilm matrix, a common core of secreted proteins is contained in both types of exoproteomes. Intradermal administration of an exoproteome extract of an exopolysaccharide-dependent biofilm induced a humoral immune response and elicited the production of interleukin 10 (IL-10) and IL-17 in mice. Antibodies against such an extract promoted opsonophagocytosis and killing of S. aureus. Immunization with the biofilm matrix exoproteome significantly reduced the number of bacterial cells inside a biofilm and on the surrounding tissue, using an in vivo model of mesh-associated biofilm infection. Furthermore, immunized mice also showed limited organ colonization by bacteria released from the matrix at the dispersive stage of the biofilm cycle. Altogether, these data illustrate the potential of biofilm matrix exoproteins as a promising candidate multivalent vaccine against S. aureus biofilm-associated infections. PMID:24343648

  1. Effectiveness of Chitosan against Mature Biofilms Formed by Food Related Bacteria

    PubMed Central

    Orgaz, Belen; Lobete, Maria M.; Puga, Carmen H.; Jose, Carmen San

    2011-01-01

    Chitosan has proven antimicrobial properties against planktonic cell growth. Little is known, however, about its effects on already established biofilms. Oriented for application in food industry disinfection, the effectiveness of both medium molecular weight (MMW) chitosan and its enzymatically hydrolyzed product was tested against mature biofilms of four pathogenic strains, Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus and Salmonella enterica, and a food spoilage species, Pseudomonas fluorescens. Unexpectedly, log reductions were in some cases higher for biofilm than for planktonic cells. One hour exposure to MMW chitosan (1% w/v) caused a 6 log viable cell reduction on L. monocytogenes monospecies mature biofilms and reduced significantly (3–5 log reductions) the attached population of the other organisms tested, except S. aureus. Pronase-treated chitosan was more effective than MMW chitosan on all tested microorganisms, also with the exception of S. aureus, offering best results (8 log units) against the attached cells of B. cereus. These treatments open a new possibility to fight against mature biofilms in the food industry. PMID:21340015

  2. Staphylococcus aureus Biofilms Induce Macrophage Dysfunction Through Leukocidin AB and Alpha-Toxin

    PubMed Central

    Scherr, Tyler D.; Hanke, Mark L.; Huang, Ouwen; James, David B. A.; Horswill, Alexander R.; Bayles, Kenneth W.; Fey, Paul D.; Torres, Victor J.

    2015-01-01

    ABSTRACT The macrophage response to planktonic Staphylococcus aureus involves the induction of proinflammatory microbicidal activity. However, S. aureus biofilms can interfere with these responses in part by polarizing macrophages toward an anti-inflammatory profibrotic phenotype. Here we demonstrate that conditioned medium from mature S. aureus biofilms inhibited macrophage phagocytosis and induced cytotoxicity, suggesting the involvement of a secreted factor(s). Iterative testing found the active factor(s) to be proteinaceous and partially agr-dependent. Quantitative mass spectrometry identified alpha-toxin (Hla) and leukocidin AB (LukAB) as critical molecules secreted by S. aureus biofilms that inhibit murine macrophage phagocytosis and promote cytotoxicity. A role for Hla and LukAB was confirmed by using hla and lukAB mutants, and synergy between the two toxins was demonstrated with a lukAB hla double mutant and verified by complementation. Independent confirmation of the effects of Hla and LukAB on macrophage dysfunction was demonstrated by using an isogenic strain in which Hla was constitutively expressed, an Hla antibody to block toxin activity, and purified LukAB peptide. The importance of Hla and LukAB during S. aureus biofilm formation in vivo was assessed by using a murine orthopedic implant biofilm infection model in which the lukAB hla double mutant displayed significantly lower bacterial burdens and more macrophage infiltrates than each single mutant. Collectively, these findings reveal a critical synergistic role for Hla and LukAB in promoting macrophage dysfunction and facilitating S. aureus biofilm development in vivo. PMID:26307164

  3. Inhibitory effects of antibiofilm compound 1 against Staphylococcus aureus biofilms.

    PubMed

    Shrestha, Looniva; Kayama, Shizuo; Sasaki, Michiko; Kato, Fuminori; Hisatsune, Junzo; Tsuruda, Keiko; Koizumi, Kazuhisa; Tatsukawa, Nobuyuki; Yu, Liansheng; Takeda, Kei; Sugai, Motoyuki

    2016-03-01

    A novel benzimidazole molecule that was identified in a small-molecule screen and is known as antibiofilm compound 1 (ABC-1) has been found to prevent bacterial biofilm formation by multiple bacterial pathogens, including Staphylococcus aureus, without affecting bacterial growth. Here, the biofilm inhibiting ability of 156 μM ABC-1 was tested in various biofilm-forming strains of S. aureus. It was demonstrated that ABC-1 inhibits biofilm formation by these strains at micromolar concentrations regardless of the strains' dependence on Polysaccharide Intercellular Adhesin (PIA), cell wall-associated protein dependent or cell wall- associated extracellular DNA (eDNA). Of note, ABC-1 treatment primarily inhibited Protein A (SpA) expression in all strains tested. spa gene disruption showed decreased biofilm formation; however, the mutants still produced more biofilm than ABC-1 treated strains, implying that ABC-1 affects not only SpA but also other factors. Indeed, ABC-1 also attenuated the accumulation of PIA and eDNA on cell surface. Our results suggest that ABC-1 has pleotropic effects on several biofilm components and thus inhibits biofilm formation by S. aureus.

  4. Effects of bacteriocins on methicillin-resistant Staphylococcus aureus biofilm.

    PubMed

    Okuda, Ken-ichi; Zendo, Takeshi; Sugimoto, Shinya; Iwase, Tadayuki; Tajima, Akiko; Yamada, Satomi; Sonomoto, Kenji; Mizunoe, Yoshimitsu

    2013-11-01

    Control of biofilms formed by microbial pathogens is an important subject for medical researchers, since the development of biofilms on foreign-body surfaces often causes biofilm-associated infections in patients with indwelling medical devices. The present study examined the effects of different kinds of bacteriocins, which are ribosomally synthesized antimicrobial peptides produced by certain bacteria, on biofilms formed by a clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA). The activities and modes of action of three bacteriocins with different structures (nisin A, lacticin Q, and nukacin ISK-1) were evaluated. Vancomycin, a glycopeptide antibiotic used in the treatment of MRSA infections, showed bactericidal activity against planktonic cells but not against biofilm cells. Among the tested bacteriocins, nisin A showed the highest bactericidal activity against both planktonic cells and biofilm cells. Lacticin Q also showed bactericidal activity against both planktonic cells and biofilm cells, but its activity against biofilm cells was significantly lower than that of nisin A. Nukacin ISK-1 showed bacteriostatic activity against planktonic cells and did not show bactericidal activity against biofilm cells. Mode-of-action studies indicated that pore formation leading to ATP efflux is important for the bactericidal activity against biofilm cells. Our results suggest that bacteriocins that form stable pores on biofilm cells are highly potent for the treatment of MRSA biofilm infections.

  5. Nanoscale Plasma Coating Inhibits Formation of Staphylococcus aureus Biofilm

    PubMed Central

    Xu, Yuanxi; Jones, John E.; Yu, Haiqing; Yu, Qingsong; Christensen, Gordon D.

    2015-01-01

    Staphylococcus aureus commonly infects medical implants or devices, with devastating consequences for the patient. The infection begins with bacterial attachment to the device, followed by bacterial multiplication over the surface of the device, generating an adherent sheet of bacteria known as a biofilm. Biofilms resist antimicrobial therapy and promote persistent infection, making management difficult to futile. Infections might be prevented by engineering the surface of the device to discourage bacterial attachment and multiplication; however, progress in this area has been limited. We have developed a novel nanoscale plasma coating technology to inhibit the formation of Staphylococcus aureus biofilms. We used monomeric trimethylsilane (TMS) and oxygen to coat the surfaces of silicone rubber, a material often used in the fabrication of implantable medical devices. By quantitative and qualitative analysis, the TMS/O2 coating significantly decreased the in vitro formation of S. aureus biofilms; it also significantly decreased in vivo biofilm formation in a mouse model of foreign-body infection. Further analysis demonstrated TMS/O2 coating significantly changed the protein adsorption, which could lead to reduced bacterial adhesion and biofilm formation. These results suggest that TMS/O2 coating can be used to effectively prevent medical implant-related infections. PMID:26369955

  6. Bap, a Staphylococcus aureus Surface Protein Involved in Biofilm Formation

    PubMed Central

    Cucarella, Carme; Solano, Cristina; Valle, Jaione; Amorena, Beatriz; Lasa, Íñigo; Penadés, José R.

    2001-01-01

    Identification of new genes involved in biofilm formation is needed to understand the molecular basis of strain variation and the pathogenic mechanisms implicated in chronic staphylococcal infections. A biofilm-producing Staphylococcus aureus isolate was used to generate biofilm-negative transposon (Tn917) insertion mutants. Two mutants were found with a significant decrease in attachment to inert surfaces (early adherence), intercellular adhesion, and biofilm formation. The transposon was inserted at the same locus in both mutants. This locus (bap [for biofilm associated protein]) encodes a novel cell wall associated protein of 2,276 amino acids (Bap), which shows global organizational similarities to surface proteins of gram-negative (Pseudomonas aeruginosa and Salmonella enterica serovar Typhi) and gram-positive (Enteroccocus faecalis) microorganisms. Bap's core region represents 52% of the protein and consists of 13 successive nearly identical repeats, each containing 86 amino acids. bap was present in a small fraction of bovine mastitis isolates (5% of the 350 S. aureus isolates tested), but it was absent from the 75 clinical human S. aureus isolates analyzed. All staphylococcal isolates harboring bap were highly adherent and strong biofilm producers. In a mouse infection model bap was involved in pathogenesis, causing a persistent infection. PMID:11292810

  7. Development of a novel ex vivo porcine skin explant model for the assessment of mature bacterial biofilms.

    PubMed

    Yang, Qingping; Phillips, Priscilla L; Sampson, Edith M; Progulske-Fox, Ann; Jin, Shouguang; Antonelli, Patrick; Schultz, Gregory S

    2013-01-01

    Bacterial biofilms have been proposed to be a major factor contributing to the failure of chronic wounds to heal because of their increased tolerance to antimicrobial agents and the prolonged inflammation they cause. Phenotypic characteristics of bacterial biofilms vary depending on the substratum to which they attach, the nutritional environment, and the microorganisms within the biofilm community. To develop an ex vivo biofilm model that more closely mimics biofilms in chronic skin wounds, we developed an optimal procedure to grow mature biofilms on a central partial-thickness wound in 12-mm porcine skin explants. Chlorine gas produced optimal sterilization of explants while preserving histological properties of the epidermis and dermis. Pseudomonas aeruginosa and Staphylococcus aureus developed mature biofilms after 3 days that had dramatically increased tolerance to gentamicin and oxacillin (∼100× and 8,000× minimal inhibitory concentration, respectively) and to sodium hypochlorite (0.6% active chlorine). Scanning electron microscopy and confocal microscopy verified extensive exopolymeric biofilm structures on the explants. Despite a significant delay, a ΔlasI quorum-sensing mutant of P. aeruginosa developed biofilm as antibiotic-tolerant as wild-type after 3 days. This ex vivo model simulates growth of biofilms on skin wounds and provides an accurate model to assess effects of antimicrobial agents on mature biofilms.

  8. Oxidative and nitrosative stress in Staphylococcus aureus biofilm.

    PubMed

    Arce Miranda, Julio E; Sotomayor, Claudia E; Albesa, Inés; Paraje, María G

    2011-02-01

    Diverse chemical and physical agents can alter cellular functions associated with oxidative metabolism, thus stimulating the production of reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) in planktonic bacterial physiology. However, more research is necessary to determine the precise role of cellular stress in biofilm. The present study was designed to address the issues of Staphylococcus aureus biofilm formation with respect to the generation of oxidative and nitrosative stress. We studied three pathogenic S. aureus clinical strains and an ATCC strain exposed to a different range of culture conditions (time, temperature, pH, reduction and atmospheric conditions) using quantitative methods of biofilm detection. We observed that cellular stress could be produced inside biofilms, thereby affecting their growth, resulting in an increase of ROS and RNI production, and a decrease of the extracellular matrix under unfavorable conditions. These radical oxidizers could then accumulate in an extracellular medium and thus affect the matrix. These results contribute to a better understanding of the processes that enable adherent biofilms to grow on inert surfaces and lead to an improved knowledge of ROS and RNI regulation, which may help to clarify the relevance of biofilm formation in medical devices.

  9. Distribution and Inhibition of Liposomes on Staphylococcus aureus and Pseudomonas aeruginosa Biofilm

    PubMed Central

    Dong, Dong; Thomas, Nicky; Thierry, Benjamin; Vreugde, Sarah; Prestidge, Clive A.; Wormald, Peter-John

    2015-01-01

    Background Staphylococcus aureus and Pseudomonas aeruginosa are major pathogens in chronic rhinosinusitis (CRS) and their biofilms have been associated with poorer postsurgical outcomes. This study investigated the distribution and anti-biofilm effect of cationic (+) and anionic (-) phospholipid liposomes with different sizes (unilamellar and multilamellar vesicle, ULV and MLV respectively) on S. aureus and P. aeruginosa biofilms. Method Specific biofilm models for S. aureus ATCC 25923 and P. aeruginosa ATCC 15692 were established. Liposomal distribution was determined by observing SYTO9 stained biofilm exposed to DiI labeled liposomes using confocal scanning laser microscopy, followed by quantitative image analysis. The anti-biofilm efficacy study was carried out by using the alamarBlue assay to test the relative viability of biofilm treated with various liposomes for 24 hours and five minutes. Results The smaller ULVs penetrated better than larger MLVs in both S. aureus and P. aeruginosa biofilm. Except that +ULV and –ULV displayed similar distribution in S. aureus biofilm, the cationic liposomes adhered better than their anionic counterparts. Biofilm growth was inhibited at 24-hour and five-minute exposure time, although the decrease of viability for P. aeruginosa biofilm after liposomal treatment did not reach statistical significance. Conclusion The distribution and anti-biofilm effects of cationic and anionic liposomes of different sizes differed in S. aureus and P. aeruginosa biofilms. Reducing the liposome size and formulating liposomes as positively charged enhanced the penetration and inhibition of S. aureus and P. aeruginosa biofilms. PMID:26125555

  10. Innovative approaches to treat Staphylococcus aureus biofilm-related infections.

    PubMed

    Richter, Katharina; Van den Driessche, Freija; Coenye, Tom

    2017-02-28

    Many bacterial infections in humans and animals are caused by bacteria residing in biofilms, complex communities of attached organisms embedded in an extracellular matrix. One of the key properties of microorganisms residing in a biofilm is decreased susceptibility towards antimicrobial agents. This decreased susceptibility, together with conventional mechanisms leading to antimicrobial resistance, makes biofilm-related infections increasingly difficult to treat and alternative antibiofilm strategies are urgently required. In this review, we present three such strategies to combat biofilm-related infections with the important human pathogen Staphylococcus aureus: (i) targeting the bacterial communication system with quorum sensing (QS) inhibitors, (ii) a 'Trojan Horse' strategy to disturb iron metabolism by using gallium-based therapeutics and (iii) the use of 'non-antibiotics' with antibiofilm activity identified through screening of repurposing libraries.

  11. The Active Component of Aspirin, Salicylic Acid, Promotes Staphylococcus aureus Biofilm Formation in a PIA-dependent Manner.

    PubMed

    Dotto, Cristian; Lombarte Serrat, Andrea; Cattelan, Natalia; Barbagelata, María S; Yantorno, Osvaldo M; Sordelli, Daniel O; Ehling-Schulz, Monika; Grunert, Tom; Buzzola, Fernanda R

    2017-01-01

    Aspirin has provided clear benefits to human health. But salicylic acid (SAL) -the main aspirin biometabolite- exerts several effects on eukaryote and prokaryote cells. SAL can affect, for instance, the expression of Staphylococcus aureus virulence factors. SAL can also form complexes with iron cations and it has been shown that different iron chelating molecules diminished the formation of S. aureus biofilm. The aim of this study was to elucidate whether the iron content limitation caused by SAL can modify the S. aureus metabolism and/or metabolic regulators thus changing the expression of the main polysaccharides involved in biofilm formation. The exposure of biofilm to 2 mM SAL induced a 27% reduction in the intracellular free Fe(2+) concentration compared with the controls. In addition, SAL depleted 23% of the available free Fe(2+) cation in culture media. These moderate iron-limited conditions promoted an intensification of biofilms formed by strain Newman and by S. aureus clinical isolates related to the USA300 and USA100 clones. The slight decrease in iron bioavailability generated by SAL was enough to induce the increase of PIA expression in biofilms formed by methicillin-resistant as well as methicillin-sensitive S. aureus strains. S. aureus did not produce capsular polysaccharide (CP) when it was forming biofilms under any of the experimental conditions tested. Furthermore, SAL diminished aconitase activity and stimulated the lactic fermentation pathway in bacteria forming biofilms. The polysaccharide composition of S. aureus biofilms was examined and FTIR spectroscopic analysis revealed a clear impact of SAL in a codY-dependent manner. Moreover, SAL negatively affected codY transcription in mature biofilms thus relieving the CodY repression of the ica operon. Treatment of mice with SAL induced a significant increase of S aureus colonization. It is suggested that the elevated PIA expression induced by SAL might be responsible for the high nasal

  12. The Active Component of Aspirin, Salicylic Acid, Promotes Staphylococcus aureus Biofilm Formation in a PIA-dependent Manner

    PubMed Central

    Dotto, Cristian; Lombarte Serrat, Andrea; Cattelan, Natalia; Barbagelata, María S.; Yantorno, Osvaldo M.; Sordelli, Daniel O.; Ehling-Schulz, Monika; Grunert, Tom; Buzzola, Fernanda R.

    2017-01-01

    Aspirin has provided clear benefits to human health. But salicylic acid (SAL) -the main aspirin biometabolite- exerts several effects on eukaryote and prokaryote cells. SAL can affect, for instance, the expression of Staphylococcus aureus virulence factors. SAL can also form complexes with iron cations and it has been shown that different iron chelating molecules diminished the formation of S. aureus biofilm. The aim of this study was to elucidate whether the iron content limitation caused by SAL can modify the S. aureus metabolism and/or metabolic regulators thus changing the expression of the main polysaccharides involved in biofilm formation. The exposure of biofilm to 2 mM SAL induced a 27% reduction in the intracellular free Fe2+ concentration compared with the controls. In addition, SAL depleted 23% of the available free Fe2+ cation in culture media. These moderate iron-limited conditions promoted an intensification of biofilms formed by strain Newman and by S. aureus clinical isolates related to the USA300 and USA100 clones. The slight decrease in iron bioavailability generated by SAL was enough to induce the increase of PIA expression in biofilms formed by methicillin-resistant as well as methicillin-sensitive S. aureus strains. S. aureus did not produce capsular polysaccharide (CP) when it was forming biofilms under any of the experimental conditions tested. Furthermore, SAL diminished aconitase activity and stimulated the lactic fermentation pathway in bacteria forming biofilms. The polysaccharide composition of S. aureus biofilms was examined and FTIR spectroscopic analysis revealed a clear impact of SAL in a codY-dependent manner. Moreover, SAL negatively affected codY transcription in mature biofilms thus relieving the CodY repression of the ica operon. Treatment of mice with SAL induced a significant increase of S aureus colonization. It is suggested that the elevated PIA expression induced by SAL might be responsible for the high nasal colonization

  13. Antimicrobial activity of essential oils against Staphylococcus aureus biofilms.

    PubMed

    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 < 0.01) the number of viable biofilm cells, but none of them could remove biofilms completely. Thyme and patchouli 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.

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

    PubMed

    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.

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

    PubMed

    Nicholson, Tracy L; Shore, Sarah M; Smith, Tara C; Frana, Timothy S; Fraena, Timothy S

    2013-01-01

    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.

  16. Functionalized polyanilines disrupt Pseudomonas aeruginosa and Staphylococcus aureus biofilms.

    PubMed

    Gizdavic-Nikolaidis, Marija R; Pagnon, Joanne C; Ali, Naseem; Sum, Reuben; Davies, Noel; Roddam, Louise F; Ambrose, Mark

    2015-12-01

    The purpose of the present study was to investigate the antimicrobial effects of functionalized polyanilines (fPANIs) against stationary phase cells and biofilms of Pseudomonas aeruginosa and Staphylococcus aureus using homopolymer of sulfanilic acid (poly-SO3H) as a model. The chemically synthesized poly-SO3H was characterized using Fourier Transform Infra-Red (FTIR) and Ultraviolet-Visible (UV-Vis) spectroscopies. The molecular weight (Mw) and elemental analysis of homopolymer poly-SO3H were also examined. We found that poly-SO3H was bactericidal against stationary phase cells of P. aeruginosa and S. aureus at a concentration of 20 mgml(-1). Surprisingly, we discovered that the same concentration (20 mgml(-1)) of poly-SO3H significantly disrupted and killed bacterial cells present in pre-established forty-eight hour static biofilms of these organisms, as shown by crystal violet and bacterial live/dead fluorescence staining assays. In support of these data, poly-SO3H extensively diminished the expression of bacterial genes related to biofilm formation in stationary phase cells of P. aeruginosa, and seemed to greatly reduce the amount of the quorum sensing molecule N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL) able to be recovered from biofilms of this organism. Furthermore, we found that poly-SO3H was able to effectively penetrate and kill cells in biofilms formed by the P. aeruginosa (AESIII) isolate derived from the sputum of a cystic fibrosis patient. Taken together, the results of the present study emphasise the broad antimicrobial activities of fPANI, and suggest that they could be developed further and used in some novel ways to construct medical devices and/or industrial equipment that are refractory to colonization by biofilm-forming bacteria.

  17. Efficacy of Combined Vancomycin and Fosfomycin against Methicillin-Resistant Staphylococcus aureus in Biofilms In Vivo

    PubMed Central

    Wang, Li; Zhang, Han-Bo; Chen, Qian; Liu, Hua; Tang, Xun; Jin, Tao; Zhu, Chong-Tao; Li, Fu-Bing; Sun, Lin-Hui; Xu, Xin-Ming; Xu, Yong-Qing

    2014-01-01

    Infection by methicillin-resistant Staphylococcus aureus (MRSA) is a life-threatening condition, and formation of biofilms can lead to treatment failure in a clinical setting. The aim of this study was to demonstrate the in vivo bactericidal effects of a combination of vancomycin (VAN) and fosfomycin (FOS) against MRSA in a rat carboxymethyl cellulose-pouch biofilm model. The results of the time-kill assay showed that the combination therapy was capable of killing at low minimal inhibitory concentrations (MIC) (½× MIC VAN +1× MIC FOS and 1× MIC VAN + 1× MIC FOS). In the in vivo study, a synergistically bactericidal effect was observed when using the combination therapy on MRSA embedded in the mature biofilm model. In comparison with the untreated control group and the groups receiving either VAN or FOS alone, the rats treated with combination therapy had lower MRSA colony counts in exudates from the pouch, lower white blood cell and neutrophil counts, and C-reactive protein (CRP) in peripheral blood. Furthermore, histological analysis of the pouch wall indicated combination therapy resulted in disappearance of biofilm-like structures, marked decrease in necrosis, and formation of granular tissue. In conclusion, the combination of VAN with FOS had a synergistic bactericidal effect on chronic MRSA infection embedded in biofilm, providing an alternative approach to treating this condition. PMID:25551618

  18. Activity of daptomycin- and vancomycin-loaded poly-epsilon-caprolactone microparticles against mature staphylococcal biofilms.

    PubMed

    Ferreira, Inês Santos; Bettencourt, Ana F; Gonçalves, Lídia M D; Kasper, Stefanie; Bétrisey, Bertrand; Kikhney, Judith; Moter, Annette; Trampuz, Andrej; Almeida, António J

    2015-01-01

    The aim of the present study was to develop novel daptomycin-loaded poly-epsilon-caprolactone (PCL) microparticles with enhanced antibiofilm activity against mature biofilms of clinically relevant bacteria, methicillin-resistant Staphylococcus aureus (MRSA) and polysaccharide intercellular adhesin-positive Staphylococcus epidermidis. Daptomycin was encapsulated into PCL microparticles by a double emulsion-solvent evaporation method. For comparison purposes, formulations containing vancomycin were also prepared. Particle morphology, size distribution, encapsulation efficiency, surface charge, thermal behavior, and in vitro release were assessed. All formulations exhibited a spherical morphology, micrometer size, and negative surface charge. From a very early time stage, the released concentrations of daptomycin and vancomycin were higher than the minimal inhibitory concentration and continued so up to 72 hours. Daptomycin presented a sustained release profile with increasing concentrations of the drug being released up to 72 hours, whereas the release of vancomycin stabilized at 24 hours. The antibacterial activity of the microparticles was assessed by isothermal microcalorimetry against planktonic and sessile MRSA and S. epidermidis. Regarding planktonic bacteria, daptomycin-loaded PCL microparticles presented the highest antibacterial activity against both strains. Isothermal microcalorimetry also revealed that lower concentrations of daptomycin-loaded microparticles were required to completely inhibit the recovery of mature MRSA and S. epidermidis biofilms. Further characterization of the effect of daptomycin-loaded PCL microparticles on mature biofilms was performed by fluorescence in situ hybridization. Fluorescence in situ hybridization showed an important reduction in MRSA biofilm, whereas S. epidermidis biofilms, although inhibited, were not eradicated. In addition, an important attachment of the microparticles to MRSA and S. epidermidis biofilms was

  19. Ecological changes in oral microcosm biofilm during maturation

    NASA Astrophysics Data System (ADS)

    Kim, Young-Seok; Kang, Si-Mook; Lee, Eun-Song; Lee, Ji Hyun; Kim, Bo-Ra; Kim, Baek-Il

    2016-10-01

    The aim of this study was to evaluate the ecological changes in the biofilm at different stages of maturation using 16S rDNA gene amplicon sequencing and to identify correlations between red/green (R/G) fluorescence ratio and ecological changes. An oral microcosm biofilm was initiated from the saliva of a single donor and grown anaerobically for up to 10 days in basal medium mucin. Quantitative light-induced fluorescence analysis was shown that the R/G ratio of the biofilm increased consistently, but the slope rapidly decreased after six days. The bacterial compositions of 10 species also consistently changed over time. However, there was no significant correlation between each bacteria and red fluorescence. The monitoring of the maturation process of oral microcosm biofilm over 10 days revealed that the R/G ratio and the bacterial composition within biofilm consistently changed. Therefore, the R/G fluorescence ratio of biofilm may be related with its ecological change rather than specific bacteria.

  20. Temperature-dependent control of Staphylococcus aureus biofilms and virulence by thermoresponsive oligo(N-vinylcaprolactam).

    PubMed

    Lee, Jin-Hyung; Kim, Yong-Guy; Lee, Kayeon; Kim, Seong-Cheol; Lee, Jintae

    2015-04-01

    Bacterial biofilms are associated with persistent infections because they are highly tolerant of antimicrobial agents, and in the case of Staphylococcus aureus, which is a leading cause of nosocomial infections because of its resistance to diverse antibiotics, biofilm formation is a known mechanism of drug resistance. In the present study, we investigated the ability of thermoresponsive oligo (N-vinylcaprolactam) (OVCL) to control biofilm formation by and the virulence of S. aureus. One synthetic and four commercial OVCLs (MW ≤ 240,000) at 50 µg/mL were found to increase S. aureus biofilm formation 7-fold at 25 °C, but to markedly inhibit S. aureus biofilm formation at 37 °C. Confocal and scanning electron microscopy confirmed the temperature-dependent effect of OVCL on S. aureus biofilms. It was found that the addition of OVCL to S. aureus culture caused cells to become dramatically more hydrophilic at 37 °C, which partially supports the biofilm reduction. Also, transcriptional analysis showed that OVCL temperature-dependently regulated biofilm-related genes (aur, agrA, and icaA) in S. aureus. In addition, it was found surface coatings containing OVCL effectively controlled S. aureus biofilm formation on solid glass surfaces. Furthermore, OVCL inhibited the hemolysis of human red blood cells by S. aureus at 37 °C and attenuated S. aureus virulence in the nematode Caenorhabditis elegans. These results suggest that OVCL has potential use for controlling bacterial biofilm formation and virulence.

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

    PubMed

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

    2017-03-29

    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 (MBIC50 - 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 MBIC50 values than their respective glycone forms. The lowest MBIC50 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.

  2. Protease production by Staphylococcus epidermidis and its effect on Staphylococcus aureus biofilms.

    PubMed

    Vandecandelaere, Ilse; Depuydt, Pieter; Nelis, Hans J; Coenye, Tom

    2014-04-01

    Due to the resistance of Staphylococcus aureus to several antibiotics, treatment of S. aureus infections is often difficult. As an alternative to conventional antibiotics, the field of bacterial interference is investigated. Staphylococcus epidermidis produces a serine protease (Esp) which inhibits S. aureus biofilm formation and which degrades S. aureus biofilms. In this study, we investigated the protease production of 114 S. epidermidis isolates, obtained from biofilms on endotracheal tubes (ET). Most of the S. epidermidis isolates secreted a mixture of serine, cysteine and metalloproteases. We found a link between high protease production by S. epidermidis and the absence of S. aureus in ET biofilms obtained from the same patient. Treating S. aureus biofilms with the supernatant (SN) of the most active protease producing S. epidermidis isolates resulted in a significant biomass decrease compared to untreated controls, while the number of metabolically active cells was not affected. The effect on the biofilm biomass was mainly due to serine proteases. Staphylococcus aureus biofilms treated with the SN of protease producing S. epidermidis were thinner with almost no extracellular matrix. An increased survival of Caenorhabditis elegans, infected with S. aureus Mu50, was observed when the SN of protease positive S. epidermidis was added.

  3. In vitro antimicrobial activity of honokiol against Staphylococcus aureus in biofilm mode.

    PubMed

    Li, Wen-Li; Zhao, Xing-Chen; Zhao, Zi-Wen; Huang, Yan-Jun; Zhu, Xuan-Zhi; Meng, Ri-Zeng; Shi, Ce; Yu, Lu; Guo, Na

    2016-12-01

    Staphylococcus aureus (S. aureus) can attach to food, host tissues and the surfaces of medical implants and form a biofilm, which makes it difficult to eliminate. The purpose of this study was to evaluate the effect of honokiol on biofilm-grown S. aureus. In this report, honokiol showed effective antibacterial activity against S. aureus in biofilms. S. aureus isolates are capable of producing distinct types of biofilms mediated by polysaccharide intercellular adhesion (PIA) or extracellular DNA (eDNA). The biofilms' susceptibility to honokiol was evaluated using confocal laser scanning microscopy (CLSM) analysis. The transcript levels of the biofilm-related genes, the expression of PIA, and the amount of eDNA of biofilm-grown S. aureus exposed to honokiol were also investigated. The results of this study show that honokiol can detach existing biofilms, kill bacteria in biofilms, and simultaneously inhibit the transcript levels of sarA, cidA and icaA, eDNA release, and the expression of PIA.

  4. The Natural Surfactant Glycerol Monolaurate Significantly Reduces Development of Staphylococcus aureus and Enterococcus faecalis Biofilms

    PubMed Central

    Hess, Donavon J.; Henry-Stanley, Michelle J.

    2015-01-01

    Abstract Background: Bacterial biofilms are involved in a large proportion of clinical infections, including device-related infections. Unfortunately, biofilm-associated bacteria are typically less susceptible to antibiotics, and infected devices must often be removed. On the basis of a recent observation that lipid-rich biofilm matrix material is present in early biofilm formation and may protect a population of bacteria from interacting with ordinarily diffusible small molecules, we hypothesized that surfactants may be useful in preventing biofilm development. Methods: Experimental Staphylococcus aureus or Enterococcus faecalis biofilms were cultivated on surgical suture suspended in a growth medium supplemented with the natural surfactant glycerol monolaurate (GML) or with a component molecule, lauric acid. After 16 h incubation, the numbers of viable biofilm-associated bacteria were measured by standard microbiologic techniques and biofilm biomass was measured using the colorimetric crystal violet assay. Results: Both GML and lauric acid were effective in inhibiting biofilm development as measured by decreased numbers of viable biofilm-associated bacteria as well as decreased biofilm biomass. Compared with lauric acid on a molar basis, GML represented a more effective inhibitor of biofilms formed by either S. aureus or E. faecalis. Conclusions: Because the natural surfactant GML inhibited biofilm development, resulting data were consistent with the hypothesis that lipids may play an important role in biofilm growth, implying that interfering with lipid formation may help control development of clinically relevant biofilms. PMID:26110557

  5. Secreted Proteases Control Autolysin-mediated Biofilm Growth of Staphylococcus aureus*

    PubMed Central

    Chen, Chen; Krishnan, Vengadesan; Macon, Kevin; Manne, Kartik; Narayana, Sthanam V. L.; Schneewind, Olaf

    2013-01-01

    Staphylococcus epidermidis, a commensal of humans, secretes Esp protease to prevent Staphylococcus aureus biofilm formation and colonization. Blocking S. aureus colonization may reduce the incidence of invasive infectious diseases; however, the mechanism whereby Esp disrupts biofilms is unknown. We show here that Esp cleaves autolysin (Atl)-derived murein hydrolases and prevents staphylococcal release of DNA, which serves as extracellular matrix in biofilms. The three-dimensional structure of Esp was revealed by x-ray crystallography and shown to be highly similar to that of S. aureus V8 (SspA). Both atl and sspA are necessary for biofilm formation, and purified SspA cleaves Atl-derived murein hydrolases. Thus, S. aureus biofilms are formed via the controlled secretion and proteolysis of autolysin, and this developmental program appears to be perturbed by the Esp protease of S. epidermidis. PMID:23970550

  6. Biofilm formation by Staphylococcus aureus isolates from a dental clinic in Konya, Turkey.

    PubMed

    Torlak, Emrah; Korkut, Emre; Uncu, Ali T; Şener, Yağmur

    2017-02-14

    The ability of Staphylococcus aureus to form biofilm is considered to be a major virulence factor influencing its survival and persistence in both the environment and the host. Biofilm formation in S. aureus is most frequently associated with production of polysaccharide intercellular adhesion by ica operon-encoded enzymes. The present work aimed at evaluating the in vitro biofilm production and presence of the icaA and icaD genes in S. aureus isolates from a dental clinic in Konya, Turkey. The surfaces of inanimate objects were sampled over a period of six months. S. aureus isolates were subjected to Congo Red Agar (CRA) and crystal violet (CV) staining assays to evaluate their ability of biofilm production, while the presence of the icaA and icaD genes was determined by polymerase chain reaction. S. aureus contamination was detected in 13.2% of the environmental samples. All the 32 isolates were observed to be positive for both the icaA and icaD genes. Phenotypic evaluations revealed that CV staining assay is a more reliable alternative to CRA assay to determine biofilm formation ability. A high percentage of agreement (91%) was observed between the results from CV staining and ica genes' detection assays. Phenotypic and genotypic evaluations should be combined to detect biofilm formation in S. aureus. Our findings indicate that dental clinic environments should be considered as potential reservoir for biofilm-producing S. aureus and thus cross contamination.

  7. Mature biofilms of Enterococcus faecalis and Enterococcus faecium are highly resistant to antibiotics.

    PubMed

    Holmberg, Anna; Rasmussen, Magnus

    2016-01-01

    Enterococcus faecalis and Enterococcus faecium are important nosocomial pathogens that form biofilms on implanted materials. We compare the antibiotic sensitivity of bacteria in new (established during 24 hours) and mature (established during 120 hours) enterococcal biofilms. Mature biofilms contained more bacteria and were much more tolerant to antibiotics, including rifampicin-containing combinations, as judged by determination of minimal biofilm eradication concentrations and by time-kill experiments of bacteria in biofilms formed on beads of bone cement.

  8. Filaments in curved flow: Rapid formation of Staphylococcus aureus biofilm streamers

    NASA Astrophysics Data System (ADS)

    Kim, Min Young; Drescher, Knut; Pak, On Shun; Bassler, Bonnie L.; Stone, Howard A.

    2014-03-01

    Biofilms are surface-associated conglomerates of bacteria that are highly resistant to antibiotics. These bacterial communities can cause chronic infections in humans by colonizing, for example, medical implants, heart valves, or lungs. Staphylococcus aureus, a notorious human pathogen, causes some of the most common biofilm-related infections. Despite the clinical importance of S. aureus biofilms, it remains mostly unknown how physical effects, in particular flow, and surface structure influence biofilm dynamics. Here we use model microfluidic systems to investigate how environmental factors, such as surface geometry, surface chemistry, and fluid flow affect biofilm development in S. aureus.We discovered that S. aureus rapidly forms flow-induced, filamentous biofilm streamers, and furthermore if surfaces are coated with human blood plasma, streamers appear within minutes and clog the channels more rapidly than if the channels are uncoated. To understand how biofilm streamer filaments reorient in curved flow to bridge the distances between corners, we developed a mathematical model based on resistive force theory and slender filaments. Understanding physical aspects of biofilm formation in S. aureus may lead to new approaches for interrupting biofilm formation of this pathogen.

  9. Filaments in curved streamlines: rapid formation of Staphylococcus aureus biofilm streamers

    NASA Astrophysics Data System (ADS)

    Kim, Minyoung Kevin; Drescher, Knut; Pak, On Shun; Bassler, Bonnie L.; Stone, Howard A.

    2014-06-01

    Biofilms are surface-associated conglomerates of bacteria that are highly resistant to antibiotics. These bacterial communities can cause chronic infections in humans by colonizing, for example, medical implants, heart valves, or lungs. Staphylococcus aureus, a notorious human pathogen, causes some of the most common biofilm-related infections. Despite the clinical importance of S. aureus biofilms, it remains mostly unknown how physical effects, in particular flow, and surface structure influence biofilm dynamics. Here we use model microfluidic systems to investigate how environmental factors, such as surface geometry, surface chemistry, and fluid flow affect biofilm development of S. aureus. We discovered that S. aureus rapidly forms flow-induced, filamentous biofilm streamers, and furthermore if surfaces are coated with human blood plasma, streamers appear within minutes and clog the channels more rapidly than if the channels are uncoated. To understand how biofilm streamer filaments reorient in flows with curved streamlines to bridge the distances between corners, we developed a mathematical model based on resistive force theory of slender filaments. Understanding physical aspects of biofilm formation of S. aureus may lead to new approaches for interrupting biofilm formation of this pathogen.

  10. Dissecting the contribution of Staphylococcus aureus α-phenol-soluble modulins to biofilm amyloid structure

    PubMed Central

    Marinelli, Patrizia; Pallares, Irantzu; Navarro, Susanna; Ventura, Salvador

    2016-01-01

    The opportunistic pathogen Staphylococcus aureus is recognized as one of the most frequent causes of biofilm-associated infections. The recently discovered phenol soluble modulins (PSMs) are small α-helical amphipathic peptides that act as the main molecular effectors of staphylococcal biofilm maturation, promoting the formation of an extracellular fibril structure with amyloid-like properties. Here, we combine computational, biophysical and in cell analysis to address the specific contribution of individual PSMs to biofilm structure. We demonstrate that despite their highly similar sequence and structure, contrary to what it was previously thought, not all PSMs participate in amyloid fibril formation. A balance of hydrophobic/hydrophilic forces and helical propensity seems to define the aggregation propensity of PSMs and control their assembly and function. This knowledge would allow to target specifically the amyloid properties of these peptides. In this way, we show that Epigallocatechin-3-gallate (EGCG), the principal polyphenol in green tea, prevents the assembly of amyloidogenic PSMs and disentangles their preformed amyloid fibrils. PMID:27708403

  11. Inhibition of Staphylococcus aureus by antimicrobial biofilms formed by competitive exclusion microorganisms on stainless steel.

    PubMed

    Son, Hyeri; Park, Sunhyung; Beuchat, Larry R; Kim, Hoikyung; Ryu, Jee-Hoon

    2016-12-05

    The goal of this study was to develop a desiccation resistant antimicrobial surface using biofilm of competitive exclusion (CE) microorganism inhibitory to Staphylococcus aureus. We isolated 161 microorganisms from soils, foods, and food-contact surfaces that are inhibitory to S. aureus. Among them, three CE microorganisms (Streptomyces spororaveus strain Gaeunsan-18, Bacillus safensis strain Chamnamu-sup 5-25, and Pseudomonas azotoformans strain Lettuce-9) exhibiting strong antibacterial activity and high growth rates were selected for evaluation. These isolates formed biofilms within 24h on stainless steel coupons (SSCs) immersed in Bennet's broth and tryptic soy broth at 25°C. Cells in these biofilms showed significantly (P≤0.05) enhanced resistance to a desiccation (43% relative humidity [RH]) compared to those attached to SSCs but not in biofilms. The antimicrobial activities of biofilms formed by these isolates on SSCs against S. aureus at 25°C and 43% RH were determined. Compared to SSCs lacking biofilms formed by CE microorganisms, populations of S. aureus on SSCs harboring CE biofilms were significantly lower (P≤0.05). Results indicate that persistent antimicrobial activity against S. aureus on stainless steel surfaces can be achieved by the presence of biofilms of CE microorganisms. This information will be useful when developing strategies to improve the microbiological safety of foods during storage, processing, and distribution by facilitating the development of effective antimicrobial food-contact surfaces.

  12. Vancomycin and maltodextrin affect structure and activity of Staphylococcus aureus biofilms.

    PubMed

    Kiamco, Mia Mae; Atci, Erhan; Khan, Qaiser Farid; Mohamed, Abdelrhman; Renslow, Ryan S; Abu-Lail, Nehal; Fransson, Boel A; Call, Douglas R; Beyenal, Haluk

    2015-12-01

    Hyperosmotic agents such as maltodextrin negatively impact bacterial growth through osmotic stress without contributing to drug resistance. We hypothesized that a combination of maltodextrin (osmotic agent) and vancomycin (antibiotic) would be more effective against Staphylococcus aureus biofilms than either alone. To test our hypothesis, S. aureus was grown in a flat plate flow cell reactor. Confocal laser scanning microscopy images were analyzed to quantify changes in biofilm structure. We used dissolved oxygen microelectrodes to quantify how vancomycin and maltodextrin affected the respiration rate and oxygen penetration into the biofilm. We found that treatment with vancomycin or maltodextrin altered biofilm structure. The effect on the structure was significant when they were used simultaneously to treat S. aureus biofilms. In addition, vancomycin treatment increased the oxygen respiration rate, while maltodextrin treatment caused an increase and then a decrease. An increased maltodextrin concentration decreased the diffusivity of the antibiotic. Overall, we conclude that (1) an increased maltodextrin concentration decreases vancomycin diffusion but increases the osmotic effect, leading to the optimum treatment condition, and (2) the combination of vancomycin and maltodextrin is more effective against S. aureus biofilms than either alone. Vancomycin and maltodextrin act together to increase the effectiveness of treatment against S. aureus biofilm growth.

  13. Vancomycin and Maltodextrin Affect Structure and Activity of Staphylococcus aureus Biofilms

    PubMed Central

    Kiamco, Mia Mae; Atci, Erhan; Khan, Qaiser Farid; Mohamed, Abdelrhman; Renslow, Ryan S.; Abu-Lail, Nehal; Fransson, Boel A.; Call, Douglas R.; Beyenal, Haluk

    2016-01-01

    Hyperosmotic agents such as maltodextrin negatively impact bacterial growth through osmotic stress without contributing to drug resistance. We hypothesized that a combination of maltodextrin (osmotic agent) and vancomycin (antibiotic) would be more effective against Staphylococcus aureus biofilms than either alone. To test our hypothesis, S. aureus was grown in a flat plate flow cell reactor. Confocal laser scanning microscopy images were analyzed to quantify changes in biofilm structure. We used dissolved oxygen microelectrodes to quantify how vancomycin and maltodextrin affected the respiration rate and oxygen penetration into the biofilm. We found that treatment with vancomycin or maltodextrin altered biofilm structure. The effect on the structure was significant when they were used simultaneously to treat S. aureus biofilms. In addition, vancomycin treatment increased the oxygen respiration rate, while maltodextrin treatment caused an increase and then a decrease. An increased maltodextrin concentration decreased the diffusivity of the antibiotic. Overall, we conclude that (1) an increased maltodextrin concentration decreases vancomycin diffusion but increases the osmotic effect, leading to the optimum treatment condition, and (2) the combination of vancomycin and maltodextrin is more effective against S. aureus biofilms than either alone. Vancomycin and maltodextrin act together to increase the effectiveness of treatment against S. aureus biofilm growth. PMID:26084588

  14. Inhibition by EGTA of the formation of a biofilm by clinical strains of Staphylococcus aureus.

    PubMed

    Liesse Iyamba, J M; Seil, M; Nagant, C; Dulanto, S; Deplano, A; El Khattabi, C; Takaisi Kikuni, N B; Dehaye, J P

    2014-07-01

    The effect of EGTA on the adhesion and on the formation of a biofilm by two reference and eight clinical strains of Staphylococcus aureus was studied. All the clinical strains were isolated from patients from Kinshasa. Spa typing confirmed that these clinical strains were distinct. The Biofilm Ring Test (BFRT®) showed that EGTA (100 µM-10 mM) inhibited the adhesion of the four clinical methicillin-resistant (MRSA) strains and the crystal violet staining method that it inhibited the formation of a biofilm by all the strains. Divalent cations abolished the effect of EGTA on the formation of a biofilm, specially in the clinical MRSA strains. EGTA had no effect on established biofilms. Only concentrations of EGTA higher than 10 mM were toxic to eukaryotic cells. Our results establish the effectiveness and the safety of lock solutions with EGTA to prevent the formation in vitro of biofilms by S. aureus.

  15. The antifungal caspofungin increases fluoroquinolone activity against Staphylococcus aureus biofilms by inhibiting N-acetylglucosamine transferase

    PubMed Central

    Siala, Wafi; Kucharíková, Soňa; Braem, Annabel; Vleugels, Jef; Tulkens, Paul M; Mingeot-Leclercq, Marie-Paule; Van Dijck, Patrick; Van Bambeke, Françoise

    2016-01-01

    Biofilms play a major role in Staphylococcus aureus pathogenicity but respond poorly to antibiotics. Here, we show that the antifungal caspofungin improves the activity of fluoroquinolones (moxifloxacin, delafloxacin) against S. aureus biofilms grown in vitro (96-well plates or catheters) and in vivo (murine model of implanted catheters). The degree of synergy among different clinical isolates is inversely proportional to the expression level of ica operon, the products of which synthesize poly-N-acetyl-glucosamine polymers, a major constituent of biofilm matrix. In vitro, caspofungin inhibits the activity of IcaA, which shares homology with β-1-3-glucan synthase (caspofungin's pharmacological target in fungi). This inhibition destructures the matrix, reduces the concentration and polymerization of exopolysaccharides in biofilms, and increases fluoroquinolone penetration inside biofilms. Our study identifies a bacterial target for caspofungin and indicates that IcaA inhibitors could potentially be useful in the treatment of biofilm-related infections. PMID:27808087

  16. Biofilm formation in invasive Staphylococcus aureus isolates is associated with the clonal lineage.

    PubMed

    Naicker, Preneshni R; Karayem, Karayem; Hoek, Kim G P; Harvey, Justin; Wasserman, Elizabeth

    2016-01-01

    The contribution of the genetic background of Staphylococcus aureus to biofilm formation is poorly understood. We investigated the association between the genetic background and the biofilm forming ability of clinical invasive S. aureus isolates. Secondary objectives included investigating any correlation with biofilm formation and methicillin resistance or the source of bacteraemia. The study was conducted at a 1300-bed tertiary hospital in Cape Town, South Africa. S. aureus isolates obtained from blood cultures between January 2010 and January 2012 were included. Genotypic characterization was performed by PFGE, spa typing, SCCmec typing and MLST. Thirty genotypically unique strains were assessed for phenotypic biofilm formation with the microtitre plate assay. All isolates were tested in triplicate and an average optical density, measured at a wavelength of 490 nm, was determined. The biofilm forming ability of isolates with A490 ≤ 0.17 were considered non-adherent, A490 > 0.17 'weak positive' and A490 > 0.34 'strong positive'. Fifty seven percent of isolates formed biofilms. Weak biofilm formation occurred in 40% (n = 12) and strong biofilm formation in 17% (n = 5) of isolates. All 5 isolates capable of strong biofilm formation belong to one spa clonal complex (spa-CC 064). Strains from spa-CC 064 were capable of higher biofilm formation than other spa clonal complexes (p = 0.00002). These 5 strains belonged to MLST CC5 and CC8. Biofilm formation correlates with the spa clonal lineage in our population of invasive S. aureus strains. Biofilm formation did not correlate with methicillin resistance and was not related to the source of bacteraemia.

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

    PubMed Central

    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

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

    PubMed

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

    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

  19. Induction of resistance to S. aureus in an environmental marine biofilm grown in Sydney Harbor, NSW, Australia.

    PubMed

    Lafleur, John E; Rice, Scott A

    2015-02-01

    The study of environmental biofilms is complicated by the difficulty of working with them under lab conditions. Nonetheless, knowledge of cellular activity and interactions within environmental biofilms could lead to novel biomedical applications. To address this problem we previously proposed a new technique for inducing resistance to Staphylococcus aureus in an intact environmental biofilm. In the current follow-up study we applied the new technique in a biogeographically distinct environment using a different strain of S. aureus. The proposed technique for inducing resistance to S. aureus in an environmental biofilm involves growing the environmental biofilms over several days in media reflecting their natural habitat on agar that contains spent culture supernatant from S. aureus over-night culture. We found in this second study that it was possible to induce resistance to S. aureus in an environmental biofilm from a biogeographically distinct environment, though not in the same way as we had previously observed. Environmental consortia from Sydney Harbor, Australia display an ability to inhibit biofilm formation by S. aureus; only in the case where the environmental biofilms were pretreated with UV radiation was there a difference in activity between environmental consortia grown on plain agar, and that grown on S. aureus agar. Application of the new technique in the current study also differs in that significant killing of cells within an established S. aureus biofilm by environmental consortia grown on S. aureus agar was possible.

  20. In vitro effect of branch extracts of Juniperus species from Turkey on Staphylococcus aureus biofilm.

    PubMed

    Marino, Andreana; Bellinghieri, Valentina; Nostro, Antonia; Miceli, Natalizia; Taviano, Maria Fernanda; Güvenç, Ayşegül; Bisignano, Giuseppe

    2010-08-01

    Methanol and aqueous branch extracts of five Juniperus species were examined for their effects on Staphylococcus aureus ATCC 6538P and S. aureus 810 biofilm. The Turkish plant material was Juniperus communis L. var. communis, J. communis L. var. saxatilis Pall., Juniperus drupacea Labill., Juniperus oxycedrus L. ssp. oxycedrus, J. oxycedrus L. ssp. macrocarpa (Sibth. & Sm.) Ball. The Juniperus extracts were subjected to preliminary phytochemical analysis by thin-layer chromatography. The antimicrobial activity was evaluated using the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). The effects of the extracts on biofilm formation and preformed biofilm were quantified by both biomass OD and the CFU counting method. The phytochemical screening revealed the presence of polyphenols, coumarins, lignans, steroids, alkaloids and terpenes. For both strains, the MICs of all extracts were in the range of 4.88-78.12 microg mL(-1). On S. aureus ATCC 6538P, the effects of subinhibitory concentration (0.5 MIC) of the extracts were minimal on planktonic growth and on adhering cells, whereas they were greater on biofilm formation. Differently, on S. aureus 810, they showed only a rather low efficacy on biofilm formation. The extracts at 2 MIC demonstrated a good activity on a preformed biofilm of S. aureus ATCC 6538P.

  1. Early effects of Staphylococcus aureus biofilm secreted products on inflammatory responses of human epithelial keratinocytes

    PubMed Central

    2014-01-01

    Background Chronic wounds such as diabetic foot ulcers, pressure ulcers, and venous leg ulcers contribute to a considerable amount of mortality in the U.S. annually. The inability of these wounds to heal has now been associated with the presence of microbial biofilms. The aim of this study was to determine if products secreted by S. aureus biofilms play an active role in chronic wounds by promoting inflammation, which is a hallmark of chronic wounds. Methods In vitro experiments were conducted to examine changes in gene expression profiles and inflammatory response of human epithelial keratinocytes (HEKa) exposed to products secreted by S. aureus grown in biofilms or products secreted by S. aureus grown planktonically. Results After only two hours of exposure, gene expression microarray data showed marked differences in inflammatory, apoptotic, and nitric oxide responses between HEKa cells exposed to S. aureus biofilm conditioned media (BCM) and HEKa cells exposed to S. aureus planktonic conditioned media (PCM). As early as 4 hours post exposure, ELISA results showed significant increases in IL-6, IL-8, TNFα, and CXCL2 production by HEKa cells exposed to BCM compared to HEKa cells exposed to PCM or controls. Nitric oxide assay data also showed significant increases in nitric oxide production by HEKa cells treated with BCM compared to HEKa cells treated with PCM, or controls. Conclusions Taken together, these results support and extend previous findings that indicate products secreted by S. aureus biofilms directly contribute to the chronic inflammation associated with chronic wounds. PMID:24936153

  2. Susceptibility of biofilm Escherichia coli, Salmonella Enteritidis and Staphylococcus aureus to detergents and sanitizers.

    PubMed

    Ueda, Shigeko; Kuwabara, Yoshihiro

    2007-12-01

    This study was conducted to investigate the susceptibility of the biofilm cells of Escherichia coli O157, Salmonella Enteritidis, and Staphylococcus aureus to some cleaning detergents and sanitizers. No weakly acidic, neutral, and weakly alkaline detergent could remove the biofilm bacteria from stainless steel chips at commonly used concentrations recommended by manufacturers. Among sanitizers, sodium hypochlorite did not completely inactivate any biofilm bacteria at active chlorine concentrations of 25 to 200 microg/ml. Benzalkonium chloride, alkyldiaminoethyl glycine hydrochloride, chlorhexidine digluconate, and polyhexamethylenebiganide inactivated the great majority of E. coli and S. Enteritidis at commonly used concentrations, but did not inactivate S. aureus effectively enough. The biofilm S. aureus population was shown to be more tolerant than the E. coli and/or S. Enteritidis populations to the sanitizers.

  3. An Optimized Lock Solution Containing Micafungin, Ethanol and Doxycycline Inhibits Candida albicans and Mixed C. albicans – Staphyloccoccus aureus Biofilms

    PubMed Central

    Lown, Livia; Peters, Brian M.; Walraven, Carla J.; Noverr, Mairi C.; Lee, Samuel A.

    2016-01-01

    Candida albicans is a major cause of catheter-related bloodstream infections and is associated with high morbidity and mortality. Due to the propensity of C. albicans to form drug-resistant biofilms, the current standard of care includes catheter removal; however, reinsertion may be technically challenging or risky. Prolonged exposure of an antifungal lock solution within the catheter in conjunction with systemic therapy has been experimentally attempted for catheter salvage. Previously, we demonstrated excellent in vitro activity of micafungin, ethanol, and high-dose doxycycline as single agents for prevention and treatment of C. albicans biofilms. Thus, we sought to investigate optimal combinations of micafungin, ethanol, and/or doxycycline as a lock solution. We performed two- and three-drug checkerboard assays to determine the in vitro activity of pairwise or three agents in combination for prevention or treatment of C. albicans biofilms. Optimal lock solutions were tested for activity against C. albicans clinical isolates, reference strains and polymicrobial C. albicans-S. aureus biofilms. A solution containing 20% (v/v) ethanol, 0.01565 μg/mL micafungin, and 800 μg/mL doxycycline demonstrated a reduction of 98% metabolic activity and no fungal regrowth when used to prevent fungal biofilm formation; however there was no advantage over 20% ethanol alone. This solution was also successful in inhibiting the regrowth of C. albicans from mature polymicrobial biofilms, although it was not fully bactericidal. Solutions containing 5% ethanol with low concentrations of micafungin and doxycycline demonstrated synergistic activity when used to prevent monomicrobial C. albicans biofilm formation. A combined solution of micafungin, ethanol and doxycycline is highly effective for the prevention of C. albicans biofilm formation but did not demonstrate an advantage over 20% ethanol alone in these studies. PMID:27428310

  4. Compositional Analysis of Biofilms Formed by Staphylococcus aureus Isolated from Food Sources

    PubMed Central

    Oniciuc, Elena-Alexandra; Cerca, Nuno; Nicolau, Anca I.

    2016-01-01

    Sixteen Staphylococcus aureus isolates originating from foods (eight from dairy products, five from fish and fish products and three from meat and meat products) were evaluated regarding their biofilms formation ability. Six strains (E2, E6, E8, E10, E16, and E23) distinguished as strong biofilm formers, either in standard Tryptic Soy Broth or in Tryptic Soy Broth supplemented with 0.4% glucose or with 4% NaCl. The composition of the biofilms formed by these S. aureus strains on polystyrene surfaces was first inferred using enzymatic and chemical treatments. Later on, biofilms were characterized by confocal laser scanning microscope (CLSM). Our experiments proved that protein-based matrices are of prime importance for the structure of biofilms formed by S. aureus strains isolated from food sources. These biofilm matrix compositions are similar to those put into evidence for coagulase negative staphylococci. This is a new finding having in view that scientific literature mentions exopolysaccharide abundance in biofilms produced by clinical isolates and food processing environment isolates of S. aureus. PMID:27065962

  5. Staphylococcus epidermidis Esp inhibits Staphylococcus aureus biofilm formation and nasal colonization.

    PubMed

    Iwase, Tadayuki; Uehara, Yoshio; Shinji, Hitomi; Tajima, Akiko; Seo, Hiromi; Takada, Koji; Agata, Toshihiko; Mizunoe, Yoshimitsu

    2010-05-20

    Commensal bacteria are known to inhibit pathogen colonization; however, complex host-microbe and microbe-microbe interactions have made it difficult to gain a detailed understanding of the mechanisms involved in the inhibition of colonization. Here we show that the serine protease Esp secreted by a subset of Staphylococcus epidermidis, a commensal bacterium, inhibits biofilm formation and nasal colonization by Staphylococcus aureus, a human pathogen. Epidemiological studies have demonstrated that the presence of Esp-secreting S. epidermidis in the nasal cavities of human volunteers correlates with the absence of S. aureus. Purified Esp inhibits biofilm formation and destroys pre-existing S. aureus biofilms. Furthermore, Esp enhances the susceptibility of S. aureus in biofilms to immune system components. In vivo studies have shown that Esp-secreting S. epidermidis eliminates S. aureus nasal colonization. These findings indicate that Esp hinders S. aureus colonization in vivo through a novel mechanism of bacterial interference, which could lead to the development of novel therapeutics to prevent S. aureus colonization and infection.

  6. Combining biofilm matrix measurements with biomass and viability assays in susceptibility assessments of antimicrobials against Staphylococcus aureus biofilms.

    PubMed

    Skogman, Malena Elise; Vuorela, Pia Maarit; Fallarero, Adyary

    2012-09-01

    Despite that three types of assays (measuring biofilm viability, biomass, or matrix) are described to assess anti-biofilm activity, they are rarely used together. As infections can easily reappear if the matrix is not affected after antibiotic treatments, our goal was to explore the simultaneous effects of antibiotics on the viability, biomass and matrix of Staphylococcus aureus biofilms (ATCC 25923). Viability and biomass were quantified using resazurin and crystal violet staining sequentially in the same plate, while matrix staining was conducted with a wheat germ agglutinin-Alexa Fluor 488 fluorescent conjugate. Establishment of the detection limits and linearity ranges allowed concluding that all three methods were able to estimate biofilm formation in a similar fashion. In a susceptibility study with 18-h biofilms, two model compounds (penicillin G and ciprofloxacin) caused a reduction on the viability and biomass accompanied by an increase or not changed levels of the matrix, respectively. This response pattern was also proven for S. aureus Newman, S. epidermidis and E. coli biofilms. A classification of antibiotics based on five categories according to their effects on viability and matrix has been proposed earlier. Our data suggests a sixth group, represented by penicillin, causing decrease in bacterial viability but showing stimulatory effects on the matrix. Further, if effects on the matrix are not taken into account, the long-term chemotherapeutic effect of antibiotics can be jeopardized in spite of the positive effects on biofilms viability and biomass. Thus, measuring all these three endpoints simultaneously provide a more complete and accurate picture.

  7. Role of JAK-STAT signaling in maturation of phagosomes containing Staphylococcus aureus

    PubMed Central

    Zhu, Fei; Zhou, Yadong; Jiang, Chunxia; Zhang, Xiaobo

    2015-01-01

    Phagocytosis is a required mechanism for the defense against pathogens. Staphylococcus aureus, an important bacterial pathogen, can promptly escape from phagosomes and proliferate within the cytoplasm of host. However, the mechanism of phagocytosis against S. aureus has not been intensively investigated. In this study, the S. aureus was engulfed by macrophages (RAW264.7 cells) but not digested by the cells, suggesting that the phagosomes did not maturate in macrophages. Further investigation revealed that peptidoglycan (PG) induced the phagosome maturation of macrophages, resulting in the eradication of S. aureus. Genome-wide analysis and quantitative real-time PCR indicated that the JAK-STAT pathway was activated by PG during the phagosome maturation of macrophages against S. aureus. This finding presented that the PG-activated JAK-STAT pathway was required for phagosome maturation. Therefore, our study contributed evidence that revealed a novel aspect of PG-triggered JAK-STAT pathway in the phagosome maturation of macrophages. PMID:26442670

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

    PubMed Central

    Dakheel, Khulood Hamid; Abdul Rahim, Raha; Hun, Tan Geok

    2016-01-01

    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. PMID:28078291

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

    PubMed

    Dakheel, Khulood Hamid; Abdul Rahim, Raha; Neela, Vasantha Kumari; Al-Obaidi, Jameel R; Hun, Tan Geok; Yusoff, Khatijah

    2016-01-01

    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.

  10. Bdellovibrio bacteriovorus Inhibits Staphylococcus aureus Biofilm Formation and Invasion into Human Epithelial Cells

    NASA Astrophysics Data System (ADS)

    Monnappa, Ajay K.; Dwidar, Mohammed; Seo, Jeong Kon; Hur, Jin-Hoe; Mitchell, Robert J.

    2014-01-01

    Bdellovibrio bacteriovorus HD100 is a predatory bacterium that attacks many Gram-negative human pathogens. A serious drawback of this strain, however, is its ineffectiveness against Gram-positive strains, such as the human pathogen Staphylococcus aureus. Here we demonstrate that the extracellular proteases produced by a host-independent B. bacteriovorus (HIB) effectively degrade/inhibit the formation of S. aureus biofilms and reduce its virulence. A 10% addition of HIB supernatant caused a 75% or greater reduction in S. aureus biofilm formation as well as 75% dispersal of pre-formed biofilms. LC-MS-MS analyses identified various B. bacteriovorus proteases within the supernatant, including the serine proteases Bd2269 and Bd2321. Tests with AEBSF confirmed that serine proteases were active in the supernatant and that they impacted S. aureus biofilm formation. The supernatant also possessed a slight DNAse activity. Furthermore, treatment of planktonic S. aureus with the supernatant diminished its ability to invade MCF-10a epithelial cells by 5-fold but did not affect the MCF-10a viability. In conclusion, this study illustrates the hitherto unknown ability of B. bacteriovorus to disperse Gram-positive pathogenic biofilms and mitigate their virulence.

  11. Infectious Dose Dictates the Host Response during Staphylococcus aureus Orthopedic-Implant Biofilm Infection

    PubMed Central

    Vidlak, Debbie

    2016-01-01

    Staphylococcus aureus is a leading cause of prosthetic joint infections (PJIs) that are typified by biofilm formation. Given the diversity of S. aureus strains and their propensity to cause community- or hospital-acquired infections, we investigated whether the immune response and biofilm growth during PJI were conserved among distinct S. aureus clinical isolates. Three S. aureus strains representing USA200 (UAMS-1), USA300 (LAC), and USA400 (MW2) lineages were equally effective at biofilm formation in a mouse model of PJI and elicited similar leukocyte infiltrates and cytokine/chemokine profiles. Another factor that may influence the course of PJI is infectious dose. In particular, higher bacterial inocula could accelerate biofilm formation and alter the immune response, making it difficult to discern underlying pathophysiological mechanisms. To address this issue, we compared the effects of two bacterial doses (103 or 105 CFU) on inflammatory responses in interleukin-12p40 (IL-12p40) knockout mice that were previously shown to have reduced myeloid-derived suppressor cell recruitment concomitant with bacterial clearance after low-dose challenge (103 CFU). Increasing the infectious dose of LAC to 105 CFU negated these differences in IL-12p40 knockout animals, demonstrating the importance of bacterial inoculum on infection outcome. Collectively, these observations highlight the importance of considering infectious dose when assessing immune responsiveness, whereas biofilm formation during PJI is conserved among clinical isolates commonly used in mouse S. aureus infection models. PMID:27091926

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

    PubMed

    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.

  13. In vitro Activity of Linezolid in Combination with Photodynamic Inactivation Against Staphylococcus aureus Biofilms

    PubMed Central

    Kashef, Nasim; Akbarizare, Mahboobeh; Razzaghi, Mohammad Reza

    2017-01-01

    Background: Biofilm infections are a major challenge in medical practice. Bacteria that live in a biofilm phenotype are more resistant to both antimicrobial therapy and host immune responses compared to their planktonic counterparts. So, there is need for new therapeutic strategies to combat these infections. A promising approach [known as Photodynamic Inactivation (PDI)] to kill bacteria growing as biofilms uses light in combination with a photosensitizer to induce a phototoxic reaction which produces reactive oxygen species that can destroy lipids and proteins causing cell death. PDI does not always guarantee full success, so, combination of PDI with antibiotics may give increased efficiency. This study aimed to determine if PDI was effective in the eradication of Staphylococcus aureus (S. aureus) biofilms in combination with linezolid. Methods: The susceptibility of biofilm cultures of three S. aureus strains to Methylene Blue (MB) and Toluidine Blue O (TBO)-mediated PDI was determined alone and in combination with linezolid. Results: Bactericidal activity (≥3 log10 reduction in viable cell count) was not achieved with MB/TBO-PDI or antibiotic treatment alone. When antibiotic treatment was combined with TBO-PDI, a greater reduction in viable count than antibiotic alone was observed for two strains. Conclusion: This study showed that although TBO-PDI did not have good bactericidal activity against S. aureus biofilms; it increased the antimicrobial activity of linezolid against these bacteria. PMID:28090280

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

    PubMed Central

    Rodríguez-Mirones, Cristina; Acosta, Felix; Icardo, Jose M.; Martínez-Martínez, Luis; Ramos-Vivas, José

    2016-01-01

    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. PMID:26800524

  15. σB Regulates IS256-Mediated Staphylococcus aureus Biofilm Phenotypic Variation▿

    PubMed Central

    Valle, Jaione; Vergara-Irigaray, Marta; Merino, Nekane; Penadés, José R.; Lasa, Iñigo

    2007-01-01

    Biofilm formation in Staphylococcus aureus is subject to phase variation, and biofilm-negative derivatives emerge sporadically from a biofilm-positive bacterial population. To date, the only known mechanism for generating biofilm phenotypic variation in staphylococci is the reversible insertion/excision of IS256 in biofilm-essential genes. In this study, we present evidence suggesting that the absence of the σB transcription factor dramatically increases the rate of switching to the biofilm-negative phenotype in the clinical isolate S. aureus 15981, under both steady-state and flow conditions. The phenotypic switching correlates with a dramatic increase in the number of IS256 copies in the chromosomes of biofilm-negative variants, as well as with an augmented IS256 insertion frequency into the icaC and the sarA genes. IS256-mediated biofilm switching is reversible, and biofilm-positive variants could emerge from biofilm-negative σB mutants. Analysis of the chromosomal insertion frequency using a recombinant IS256 element tagged with an erythromycin marker showed an almost three-times-higher transposition frequency in a ΔσB strain. However, regulation of IS256 activity by σB appears to be indirect, since transposase transcription is not affected in the absence of σB and IS256 activity is inhibited to wild-type levels in a ΔσB strain under NaCl stress. Overall, our results identify a new role for σB as a negative regulator of insertion sequence transposition and support the idea that deregulation of IS256 activity abrogates biofilm formation capacity in S. aureus. PMID:17277051

  16. Inhibition of Biofilm Formation by Esomeprazole in Pseudomonas aeruginosa and Staphylococcus aureus

    PubMed Central

    Singh, Vandana; Arora, Vaneet; Alam, M. Jahangir

    2012-01-01

    Staphylococcus aureus and Pseudomonas aeruginosa are common nosocomial pathogens responsible for biofilm-associated infections. Proton pump inhibitors (PPI), such as esomeprazole, may have novel antimicrobial properties. The objective of this study was to assess whether esomeprazole prevents sessile bacterial growth and biofilm formation and whether it may have synergistic killing effects with standard antibiotics. The antibiofilm activity of esomeprazole at 0.25 mM was tested against two strains each of S. aureus and P. aeruginosa. Bacterial biofilms were prepared using a commercially available 96-peg-plate Calgary biofilm device. Sessile bacterial CFU counts and biomass were assessed during 72 hours of esomeprazole exposure. The killing activities after an additional 24 hours of vancomycin (against S. aureus) and meropenem (against P. aeruginosa) treatment with or without preexposure to esomeprazole were also assessed by CFU and biomass analyses. P. aeruginosa and S. aureus strains exposed to esomeprazole displayed decreased sessile bacterial growth and biomass (P < 0.001, each parameter). After 72 h of exposure, there was a 1-log10 decrease in the CFU/ml of esomeprazole-exposed P. aeruginosa and S. aureus strains compared to controls (P < 0.001). After 72 h of exposure, measured absorbance was 100% greater in P. aeruginosa control strains than in esomeprazole-exposed strains (P < 0.001). Increased killing and decreased biomass were observed for esomeprazole-treated bacteria compared to untreated controls exposed to conventional antibiotics (P < 0.001, each parameter). Reduced biofilm growth after 24 h was visibly apparent by light micrographs for P. aeruginosa and S. aureus isolates exposed to esomeprazole compared to untreated controls. In conclusion, esomeprazole demonstrated an antibiofilm effect against biofilm-producing S. aureus and P. aeruginosa. PMID:22664967

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

    PubMed

    Chen, Yan; Liu, Tangjuan; Wang, Ke; Hou, Changchun; Cai, Shuangqi; Huang, Yingying; Du, Zhongye; Huang, Hong; Kong, Jinliang; Chen, Yiqiang

    2016-01-01

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

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

    PubMed Central

    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

  19. Phevalin (aureusimine B) production by Staphylococcus aureus biofilm and impacts on human keratinocyte gene expression.

    PubMed

    Secor, Patrick R; Jennings, Laura K; James, Garth A; Kirker, Kelly R; Pulcini, Elinor Delancey; McInnerney, Kate; Gerlach, Robin; Livinghouse, Tom; Hilmer, Jonathan K; Bothner, Brian; Fleckman, Philip; Olerud, John E; Stewart, Philip S

    2012-01-01

    Staphylococcus aureus biofilms are associated with chronic skin infections and are orders of magnitude more resistant to antimicrobials and host responses. S. aureus contains conserved nonribosomal peptide synthetases that produce the cyclic dipeptides tyrvalin and phevalin (aureusimine A and B, respectively). The biological function of these compounds has been speculated to be involved in virulence factor gene expression in S. aureus, protease inhibition in eukaryotic cells, and interspecies bacterial communication. However, the exact biological role of these compounds is unknown. Here, we report that S. aureus biofilms produce greater amounts of phevalin than their planktonic counterparts. Phevalin had no obvious impact on the extracellular metabolome of S. aureus as measured by high-performance liquid chromatography-mass spectrometry and nuclear magnetic resonance. When administered to human keratinocytes, phevalin had a modest effect on gene expression. However, conditioned medium from S. aureus spiked with phevalin amplified differences in keratinocyte gene expression compared to conditioned medium alone. Phevalin may be exploited as potential biomarker and/or therapeutic target for chronic, S. aureus biofilm-based infections.

  20. Increased biofilm formation ability and accelerated transport of Staphylococcus aureus along a catheter during reciprocal movements.

    PubMed

    Haraga, Isao; Abe, Shintaro; Jimi, Shiro; Kiyomi, Fumiaki; Yamaura, Ken

    2017-01-01

    Staphylococcus spp. is a major cause of device-related infections. However, the mechanisms of deep-tissue infection by staphylococci from the skin surface remain unclear. We performed in vitro experiments to determine how staphylococci are transferred from the surface to the deeper layers of agar along the catheter for different strains of Staphylococcus aureus with respect to bacterial concentrations, catheter movements, and biofilm formation. We found that when 5-mm reciprocal movements of the catheter were repeated every 8h, all catheter samples of S. aureus penetrated the typical distance of 50mm from the skin to the epidural space. The number of reciprocal catheter movements and the depth of bacterial growth were correlated. A greater regression coefficient for different strains implied faster bacterial growth. Enhanced biofilm formation by different strains implied larger regression coefficients. Increased biofilm formation ability may accelerate S. aureus transport along a catheter due to physical movements by patients.

  1. SaeRS-Dependent Inhibition of Biofilm Formation in Staphylococcus aureus Newman

    PubMed Central

    Lei, Mei G.; Blevins, Jon S.; Smeltzer, Mark S.; Lee, Chia Y.

    2015-01-01

    The SaeRS two-component regulatory system of Staphylococcus aureus is known to affect the expression of many genes. The SaeS protein is the histidine kinase responsible for phosphorylation of the response regulator SaeR. In S. aureus Newman, the sae system is constitutively expressed due to a point mutation in saeS, relative to other S. aureus strains, which results in substitution of proline for leucine at amino acid 18. Strain Newman is unable to form a robust biofilm and we report here that the biofilm-deficient phenotype is due to the saeSP allele. Replacement of the Newman saeSP with saeSL, or deletion of saeRS, resulted in a biofilm-proficient phenotype. Newman culture supernatants were observed to inhibit biofilm formation by other S. aureus strains, but did not affect biofilm formation by S. epidermidis. Culture supernatants of Newman saeSL or Newman ΔsaeRS had no significant effect on biofilm formation. The inhibitory factor was inactivated by incubation with proteinase K, but survived heating, indicating that the inhibitory protein is heat-stable. The inhibitory protein was found to affect the attachment step in biofilm formation, but had no effect on preformed biofilms. Replacement of saeSL with saeSP in the biofilm-proficient S. aureus USA300 FPR3757 resulted in the loss of biofilm formation. Culture supernatants of USA300 FPR3757 saeSP, did not inhibit biofilm formation by other staphylococci, suggesting that the inhibitory factor is produced but not secreted in the mutant strain. A number of biochemical methods were utilized to isolate the inhibitory protein. Although a number of candidate proteins were identified, none were found to be the actual inhibitor. In an effort to reduce the number of potential inhibitory genes, RNA-Seq analyses were done with wild-type strain Newman and the saeSL and ΔsaeRS mutants. RNA-Seq results indicated that sae regulates many genes that may affect biofilm formation by Newman. PMID:25853849

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

    PubMed

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

    2009-01-01

    Bacteria colonizing chronic wounds are believed to exist as polymicrobial, biofilm communities; however, there are few studies demonstrating the role of biofilms in chronic wound pathogenesis. This study establishes a novel method for studying the effect of biofilms on the cell types involved in wound healing. Cocultures of Staphylococcus aureus biofilms and human keratinocytes (HK) were created by initially growing S. aureus biofilms on tissue culture inserts then transferring the inserts to existing HK cultures. Biofilm-conditioned medium (BCM) was prepared by culturing the insert-supported biofilm in cell culture medium. As a control planktonic-conditioned medium (PCM) was also prepared. Biofilm, BCM, and PCM were used in migration, cell viability, and apoptosis assays. Changes in HK morphology were followed by brightfield and confocal microscopy. After only 3 hours exposure to BCM, but not PCM, HK formed dendrite-like extensions and displayed reduced viability. After 9 hours, there was an increase in apoptosis (p< or =0.0004). At 24 hours, biofilm-, BCM-, and PCM-exposed HK all exhibited reduced scratch closure (p< or =0.0001). The results demonstrated that soluble products of both S. aureus planktonic cells and biofilms inhibit scratch closure. Furthermore, S. aureus biofilms significantly reduced HK viability and significantly increased HK apoptosis compared with planktonic S. aureus.

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

    PubMed Central

    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

  4. Reduced Staphylococcus aureus biofilm formation in the presence of chitosan-coated iron oxide nanoparticles

    PubMed Central

    Shi, Si-feng; Jia, Jing-fu; Guo, Xiao-kui; Zhao, Ya-ping; Chen, De-sheng; Guo, Yong-yuan; Zhang, Xian-long

    2016-01-01

    Staphylococcus aureus can adhere to most foreign materials and form biofilm on the surface of medical devices. Biofilm infections are difficult to resolve. The goal of this in vitro study was to explore the use of chitosan-coated nanoparticles to prevent biofilm formation. For this purpose, S. aureus was seeded in 96-well plates to incubate with chitosan-coated iron oxide nanoparticles in order to study the efficiency of biofilm formation inhibition. The biofilm bacteria count was determined using the spread plate method; biomass formation was measured using the crystal violet staining method. Confocal laser scanning microscopy and scanning electron microscopy were used to study the biofilm formation. The results showed decreased viable bacteria numbers and biomass formation when incubated with chitosan-coated iron oxide nanoparticles at all test concentrations. Confocal laser scanning microscopy showed increased dead bacteria and thinner biofilm when incubated with nanoparticles at a concentration of 500 µg/mL. Scanning electron microscopy revealed that chitosan-coated iron oxide nanoparticles inhibited biofilm formation in polystyrene plates. Future studies should be performed to study these nanoparticles for anti-infective use. PMID:27994455

  5. Reduced Staphylococcus aureus biofilm formation in the presence of chitosan-coated iron oxide nanoparticles.

    PubMed

    Shi, Si-Feng; Jia, Jing-Fu; Guo, Xiao-Kui; Zhao, Ya-Ping; Chen, De-Sheng; Guo, Yong-Yuan; Zhang, Xian-Long

    Staphylococcus aureus can adhere to most foreign materials and form biofilm on the surface of medical devices. Biofilm infections are difficult to resolve. The goal of this in vitro study was to explore the use of chitosan-coated nanoparticles to prevent biofilm formation. For this purpose, S. aureus was seeded in 96-well plates to incubate with chitosan-coated iron oxide nanoparticles in order to study the efficiency of biofilm formation inhibition. The biofilm bacteria count was determined using the spread plate method; biomass formation was measured using the crystal violet staining method. Confocal laser scanning microscopy and scanning electron microscopy were used to study the biofilm formation. The results showed decreased viable bacteria numbers and biomass formation when incubated with chitosan-coated iron oxide nanoparticles at all test concentrations. Confocal laser scanning microscopy showed increased dead bacteria and thinner biofilm when incubated with nanoparticles at a concentration of 500 µg/mL. Scanning electron microscopy revealed that chitosan-coated iron oxide nanoparticles inhibited biofilm formation in polystyrene plates. Future studies should be performed to study these nanoparticles for anti-infective use.

  6. Role of Biofilm-Associated Protein Bap in the Pathogenesis of Bovine Staphylococcus aureus

    PubMed Central

    Cucarella, Carme; Tormo, M. Ángeles; Úbeda, Carles; Trotonda, M. Pilar; Monzón, Marta; Peris, Critòfol; Amorena, Beatriz; Lasa, Íñigo; Penadés, José R.

    2004-01-01

    Staphylococcus aureus is a common cause of intramammary infections, which frequently become chronic, associated with the ability of the bacteria to produce biofilm. Here, we report a relationship between the ability to produce chronic bovine mastitis and biofilm formation. We have classified bovine mastitis S. aureus isolates into three groups based on the presence of particular genetic elements required for biofilm formation: group 1 (ica+ bap+), group 2 (ica+, bap negative), and group 3 (ica negative, bap negative). Overall, animals naturally infected with group 1 and 2 isolates had a lower milk somatic cell count than those infected with isolates of group 3. In addition, Bap-positive isolates were significantly more able to colonize and persist in the bovine mammary gland in vivo and were less susceptible to antibiotic treatments when forming biofilms in vitro. Analysis of the structural bap gene revealed the existence of alternate forms of expression of the Bap protein in S. aureus isolates obtained under field conditions throughout the animal's life. The presence of anti-Bap antibodies in serum samples taken from animals with confirmed S. aureus infections indicated the production of Bap during infection. Furthermore, disruption of the ica operon in a bap-positive strain had no effect on in vitro biofilm formation, a finding which strongly suggested that Bap could compensate for the deficiency of the PIA/PNAG product (a biofilm matrix polysaccharide). Altogether, these results demonstrate that, in the bovine intramammary gland, the presence of Bap may facilitate a biofilm formation connected with the persistence of S. aureus. PMID:15039341

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

    PubMed Central

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

    2017-01-01

    Background: 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. Methods: 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. Results: 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. Conclusion: 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 the progress of infection. PMID:27695030

  8. Effects of tea tree (Melaleuca alternifolia) oil on Staphylococcus aureus in biofilms and stationary growth phase.

    PubMed

    Kwieciński, Jakub; Eick, Sigrun; Wójcik, Kinga

    2009-04-01

    Tea tree oil (TTO) is known for its antimicrobial activity. In this study, we determined whether TTO is effective against Staphylococcus aureus in biofilms and how TTO activity is affected by the S. aureus growth phase. All clinical strains tested were killed by TTO both as planktonic cells and as biofilms. The minimum biofilm eradication concentration was usually two times higher than the minimum bactericidal concentration, yet it was never higher than 1% v/v. The fastest killing of biofilm occurred during the first 15min of contact with TTO and was not influenced by increasing TTO concentration above 1% v/v. Planktonic stationary phase cells exhibited decreased susceptibility to TTO compared with exponential phase cells. The killing rate for stationary phase cells was also less affected by increasing TTO concentration than that for exponential phase cells. These data show that TTO efficiently kills S. aureus in the stationary growth phase and within biofilms and is therefore a promising tool for S. aureus eradication.

  9. Differential Protection from Tobramycin by Extracellular Polymeric Substances from Acinetobacter baumannii and Staphylococcus aureus Biofilms

    PubMed Central

    Davenport, Emily K.; Call, Douglas R.

    2014-01-01

    We investigated biofilms of two pathogens, Acinetobacter baumannii and Staphylococcus aureus, to characterize mechanisms by which the extracellular polymeric substance (EPS) found in biofilms can protect bacteria against tobramycin exposure. To do so, it is critical to study EPS-antibiotic interactions in a homogeneous environment without mass transfer limitations. Consequently, we developed a method to grow biofilms, harvest EPS, and then augment planktonic cultures with isolated EPS and tobramycin. We demonstrated that planktonic cultures respond differently to being treated with different types of EPS (A. baumannii versus S. aureus) in the presence of tobramycin. By harvesting EPS from the biofilms, we found that A. baumannii EPS acts as a “universal protector” by inhibiting tobramycin activity against bacterial cells regardless of species; S. aureus EPS did not show any protective ability in cell cultures. Adding Mg2+ or Ca2+ reduced the protective effect of A. baumannii EPS. Finally, when we selectively digested the proteins or DNA of the EPS, we found that the protective ability did not change, suggesting that neither has a significant role in protection. To the best of our knowledge, this is the first study that demonstrates how EPS protects pathogens against antibiotics in a homogeneous system without mass transfer limitations. Our results suggest that EPS protects biofilm communities, in part, by adsorbing antibiotics near the surface. This may limit antibiotic diffusion to the bottom of the biofilms but is not likely to be the only mechanism of protection. PMID:24913166

  10. Differential protection from tobramycin by extracellular polymeric substances from Acinetobacter baumannii and Staphylococcus aureus biofilms.

    PubMed

    Davenport, Emily K; Call, Douglas R; Beyenal, Haluk

    2014-08-01

    We investigated biofilms of two pathogens, Acinetobacter baumannii and Staphylococcus aureus, to characterize mechanisms by which the extracellular polymeric substance (EPS) found in biofilms can protect bacteria against tobramycin exposure. To do so, it is critical to study EPS-antibiotic interactions in a homogeneous environment without mass transfer limitations. Consequently, we developed a method to grow biofilms, harvest EPS, and then augment planktonic cultures with isolated EPS and tobramycin. We demonstrated that planktonic cultures respond differently to being treated with different types of EPS (A. baumannii versus S. aureus) in the presence of tobramycin. By harvesting EPS from the biofilms, we found that A. baumannii EPS acts as a "universal protector" by inhibiting tobramycin activity against bacterial cells regardless of species; S. aureus EPS did not show any protective ability in cell cultures. Adding Mg(2+) or Ca(2+) reduced the protective effect of A. baumannii EPS. Finally, when we selectively digested the proteins or DNA of the EPS, we found that the protective ability did not change, suggesting that neither has a significant role in protection. To the best of our knowledge, this is the first study that demonstrates how EPS protects pathogens against antibiotics in a homogeneous system without mass transfer limitations. Our results suggest that EPS protects biofilm communities, in part, by adsorbing antibiotics near the surface. This may limit antibiotic diffusion to the bottom of the biofilms but is not likely to be the only mechanism of protection.

  11. Mechanical properties of a mature biofilm from a wastewater system: from microscale to macroscale level.

    PubMed

    Safari, Ashkan; Tukovic, Zeljko; Walter, Maik; Casey, Eoin; Ivankovic, Alojz

    2015-01-01

    A fundamental understanding of biofilm mechanical stability is critical in order to describe detachment and develop biofouling control strategies. It is thus important to characterise the elastic deformation and flow behaviour of the biofilm under different modes of applied force. In this study, the mechanical properties of a mature wastewater biofilm were investigated with methods including macroscale compression and microscale indentation using atomic force microscopy (AFM). The mature biofilm was found to be mechanically isotropic at the macroscale level as its mechanical properties did not depend on the scales and modes of loading. However, the biofilm showed a tendency for mechanical inhomogeneity at the microscale level as indentation progressed deeper into the matrix. Moreover, it was observed that the adhesion force had a significant influence on the elastic properties of the biofilm at the surface, subjected to microscale tensile loading. These results are expected to inform a damage-based model for biofilm detachment.

  12. Effect of silver nanoparticles on Pseudomonas putida biofilms at different stages of maturity.

    PubMed

    Thuptimdang, Pumis; Limpiyakorn, Tawan; McEvoy, John; Prüß, Birgit M; Khan, Eakalak

    2015-06-15

    This study determined the effect of silver nanoparticles (AgNPs) on Pseudomonas putida KT2440 biofilms at different stages of maturity. Three biofilm stages (1-3, representing early to late stages of development) were identified from bacterial adenosine triphosphate (ATP) activity under static (96-well plate) and dynamic conditions (Center for Disease Control and Prevention biofilm reactor). Extracellular polymeric substance (EPS) levels, measured using crystal violet and total carbohydrate assays, and expression of the EPS-associated genes, csgA and alg8, supported the conclusion that biofilms at later stages were older than those at earlier stages. More mature biofilms (stages 2 and 3) showed little to no reduction in ATP activity following exposure to AgNPs. In contrast, the same treatment reduced ATP activity by more than 90% in the less mature stage 1 biofilms. Regardless of maturity, biofilms with EPS stripped off were more susceptible to AgNPs than controls with intact EPS, demonstrating that EPS is critical for biofilm tolerance of AgNPs. The findings from this study show that stage of maturity is an important factor to consider when studying effect of AgNPs on biofilms.

  13. Molecular typing of nosocomial Staphylococcus aureus strains associated to biofilm based on the coagulase and protein A gene polymorphisms

    PubMed Central

    Salehzadeh, Ali; Zamani, Hojjatolah; Langeroudi, Maedeh Keshtkar; Mirzaie, Amir

    2016-01-01

    Objective(s): Staphylococcus aureus is an important bacterial pathogen responsible for a variety numbers of nosocomial and community acquired infections. Biofilm formation is regarded as an important factor in the establishment of S. aureus infection. The contribution of the genetic background of S. aureus to biofilm formation is poorly understood. The aim of the present work was to genotype S. aureus strains associated to biofilm based on the coagulase and protein A genes and to evaluate the association between the genetic background and the biofilm forming ability of clinical S. aureus isolates. Materials and Methods: A total number of 100 S. aureus were isolated from nosocomial infections and biofilm formation capability was investigated using phenotypic assay and molecular detection of biofilm associated genes. The strains were genotyped based on coagulase (coa) and protein A (spa) gene polymorphisms using restriction fragments length polymorphism-polymerase chain reaction (RFLP-PCR). Results: RFLP-PCR of coa gene generated two types and three subtypes. Amplification of spa gene resulted in two banding patterns and their restriction digestion generated three subtypes. The combined coa and spa RFLP patterns generated nine genotypes (G1-G9). The genotypes G4 and G1 were the most prevalent (32.1% and 24.3%, respectively). Conclusion: High clonal diversity of S. aureus strains able to produce biofilm was observed. Biofilm formation correlates with the spa and coa clonal lineage in our population and testing for multiple gene polymorphisms could be employed for local epidemiologic purposes. PMID:28096965

  14. Streptococcus pneumoniae Eradicates Preformed Staphylococcus aureus Biofilms through a Mechanism Requiring Physical Contact

    PubMed Central

    Khan, Faidad; Wu, Xueqing; Matzkin, Gideon L.; Khan, Mohsin A.; Sakai, Fuminori; Vidal, Jorge E.

    2016-01-01

    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 TIGR4ΔspxB 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 hydrogen peroxide

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

    PubMed Central

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

    2016-01-01

    Enzymatic debridement is a therapeutic strategy used clinically to remove necrotic tissue from wounds. Some of the enzymes utilized for debridement have been tested against bacterial pathogens, but the effectiveness of these agents in dispersing clinically relevant biofilms has not been fully characterized. Here, we developed an in vitro Staphylococcus aureus biofilm model that mimics wound-like conditions and employed this model to investigate the antibiofilm activity of four enzymatic compounds. Human plasma at concentrations of 0%–50% was supplemented into growth media and used to evaluate biofilm biomass accumulation over 24 hours and 48 hours in one methicillin-sensitive and five methicillin-resistant strains of S. aureus. Supplementation of media with 10% human plasma resulted in the most robust biofilms in all six strains. The enzymes α-amylase, bromelain, lysostaphin, and papain were then tested against S. aureus biofilms cultured in 10% human plasma. Quantification of biofilms after 2 hours and 24 hours of treatment using the crystal violet assay revealed that lysostaphin decreased biomass by up to 76%, whereas α-amylase, bromelain, and papain reduced biomass by up to 97%, 98%, and 98%, respectively. Scanning electron microscopy confirmed that the dispersal agents detached the biofilm exopolysaccharide matrix and bacteria from the growth surface. Lysostaphin caused less visible dispersal of the biofilms, but unlike the other enzymes, induced morphological changes indicative of bacterial cell damage. Overall, our results indicate that use of enzymes may be an effective means of eradicating biofilms and a promising strategy to improve treatment of multidrug-resistant bacterial infections. PMID:27175088

  16. Streptococcus pneumoniae Eradicates Preformed Staphylococcus aureus Biofilms through a Mechanism Requiring Physical Contact.

    PubMed

    Khan, Faidad; Wu, Xueqing; Matzkin, Gideon L; Khan, Mohsin A; Sakai, Fuminori; Vidal, Jorge E

    2016-01-01

    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 TIGR4ΔspxB 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 hydrogen peroxide.

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

    NASA Astrophysics Data System (ADS)

    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

  18. Red wines and flavonoids diminish Staphylococcus aureus virulence with anti-biofilm and anti-hemolytic activities.

    PubMed

    Cho, Hyun Seob; Lee, Jin-Hyung; Cho, Moo Hwan; Lee, Jintae

    2015-01-01

    The emergence of antibiotic resistant Staphylococcus aureus presents a worldwide problem that requires non-antibiotic strategies. This study investigated the anti-biofilm and anti-hemolytic activities of four red wines and two white wines against three S. aureus strains. All red wines at 0.5-2% significantly inhibited S. aureus biofilm formation and hemolysis by S. aureus, whereas the two white wines had no effect. Furthermore, at these concentrations, red wines did not affect bacterial growth. Analyses of hemolysis and active component identification in red wines revealed that the anti-biofilm compounds and anti-hemolytic compounds largely responsible were tannic acid, trans-resveratrol, and several flavonoids. In addition, red wines attenuated S. aureus virulence in vivo in the nematode Caenorhabditis elegans, which is killed by S. aureus. These findings show that red wines and their compounds warrant further attention in antivirulence strategies against persistent S. aureus infection.

  19. Methicillin-Resistant Staphylococcus aureus Grown on Vancomycin-Supplemented Screening Agar Displays Enhanced Biofilm Formation

    PubMed Central

    Chang, Wenjiao; Ding, Ding; Zhang, Shanshan; Dai, Yuanyuan; Pan, Qing; Lu, Huaiwei; Luo, Qingli; Shen, Jilong

    2015-01-01

    Brain heart infusion agar containing 3 mg/liter vancomycin (BHI-V3) was used to screen for heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA). There was markedly greater biofilm formation by isolates that grew on BHI-V3 than by strains that did not grow on BHI-V3. Increased biofilm formation by hVISA may be mediated by FnbA- and polysaccharide intercellular adhesin-dependent pathways, and upregulation of atlA and sarA may also contribute to enhanced biofilm formation by hVISA upon prolonged exposure to vancomycin. PMID:26459889

  20. Antibacterial synergy of glycerol monolaurate and aminoglycosides in Staphylococcus aureus biofilms.

    PubMed

    Hess, Donavon J; Henry-Stanley, Michelle J; Wells, Carol L

    2014-11-01

    Glycerol monolaurate (GML) is a natural surfactant with antimicrobial properties. At ∼0.3 mM, both GML and its component lauric acid were bactericidal for antibiotic-resistant Staphylococcus aureus biofilms. With the use of MICs of antibiotics obtained from planktonic cells, GML and lauric acid acted synergistically with gentamicin and streptomycin, but not ampicillin or vancomycin, to eliminate detectable viable biofilm bacteria. Images of GML-treated biofilms suggested that GML may facilitate antibiotic interaction with matrix-embedded bacteria.

  1. Antibacterial Synergy of Glycerol Monolaurate and Aminoglycosides in Staphylococcus aureus Biofilms

    PubMed Central

    Hess, Donavon J.; Henry-Stanley, Michelle J.

    2014-01-01

    Glycerol monolaurate (GML) is a natural surfactant with antimicrobial properties. At ∼0.3 mM, both GML and its component lauric acid were bactericidal for antibiotic-resistant Staphylococcus aureus biofilms. With the use of MICs of antibiotics obtained from planktonic cells, GML and lauric acid acted synergistically with gentamicin and streptomycin, but not ampicillin or vancomycin, to eliminate detectable viable biofilm bacteria. Images of GML-treated biofilms suggested that GML may facilitate antibiotic interaction with matrix-embedded bacteria. PMID:25182634

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

    PubMed

    de Oliveira, Adilson; Cataneli Pereira, Valéria; Pinheiro, Luiza; Moraes Riboli, Danilo Flávio; Benini Martins, Katheryne; Ribeiro de Souza da Cunha, Maria de Lourdes

    2016-09-01

    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 CoNS species

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

    PubMed Central

    de Oliveira, Adilson; Cataneli Pereira, Valéria; Pinheiro, Luiza; Moraes Riboli, Danilo Flávio; Benini Martins, Katheryne; Ribeiro de Souza da Cunha, Maria de Lourdes

    2016-01-01

    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 CoNS species

  4. SarA Positively Controls Bap-Dependent Biofilm Formation in Staphylococcus aureus

    PubMed Central

    Trotonda, María Pilar; Manna, Adhar C.; Cheung, Ambrose L.; Lasa, Iñigo; Penadés, José R.

    2005-01-01

    The biofilm-associated protein Bap is a staphylococcal surface protein involved in biofilm formation. We investigated the influence of the global regulatory locus sarA on bap expression and Bap-dependent biofilm formation in three unrelated Staphylococcus aureus strains. The results showed that Bap-dependent biofilm formation was diminished in the sarA mutants by an agr-independent mechanism. Complementation studies using a sarA clone confirmed that the defect in biofilm formation was due to the sarA mutation. As expected, the diminished capacity to form biofilms in the sarA mutants correlated with the decreased presence of Bap in the bacterial surface. Using transcriptional fusion and Northern analysis data, we demonstrated that the sarA gene product acts as an activator of bap expression. Finally, the bap promoter was characterized and the transcriptional start point was mapped by the rapid amplification of cDNA ends technique. As expected, we showed that purified SarA protein binds specifically to the bap promoter, as determined by gel shift and DNase I footprinting assays. Based on the previous studies of others as well as our work demonstrating the role for SarA in icaADBC and bap expression (J. Valle, A. Toledo-Arana, C. Berasain, J. M. Ghigo, B. Amorena, J. R. Penades, and I. Lasa, Mol. Microbiol. 48:1075-1087), we propose that SarA is an essential regulator controlling biofilm formation in S. aureus. PMID:16077127

  5. Liposome containing cinnamon oil with antibacterial activity against methicillin-resistant Staphylococcus aureus biofilm.

    PubMed

    Cui, Haiying; Li, Wei; Li, Changzhu; Vittayapadung, Saritporn; Lin, Lin

    2016-01-01

    The global burden of bacterial disease remains high and is set against a backdrop of increasing antimicrobial resistance. There is a pressing need for highly effective and natural antibacterial agents. In this work, the anti-biofilm effect of cinnamon oil on methicillin-resistant Staphylococcus aureus was evaluated. Then, cinnamon oil was encapsulated in liposomes to enhance its chemical stability. The anti-biofilm activities of the liposome-encapsulated cinnamon oil against MRSA biofilms on stainless steel, gauze, nylon membrane and non-woven fabrics were evaluated by colony forming unit determination. Scanning electron microscopy and laser scanning confocal microscopy analyses were employed to observe the morphological changes in MRSA biofilms treated with the encapsulated cinnamon oil. As a natural and safe spice, the cinnamon oil exhibited a satisfactory antibacterial performance on MRSA and its biofilms. The application of liposomes further improves the stability of antimicrobial agents and extends the action time.

  6. Small colony variants have a major role in stability and persistence of Staphylococcus aureus biofilms.

    PubMed

    Mirani, Zulfiqar Ali; Aziz, Mubashir; Khan, Seema Ismat

    2015-02-01

    The present study was conducted to investigate the significance of small colony variants (SCVs) in biofilm life cycle of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA). All of these MRSA and MSSA isolates were recovered from different food commodities. Molecular typing showed that 21 MRSA isolates carry SCCmecA type IV and belong to agr type II. Out of 15 MSSA isolates, 7 were found to carry agr type II, 5 agr type I and 2 agr type III. All of the MRSA isolates studied adopted biofilm mode of growth after exposure to sublethal doses of oxacillin. MSSA isolates, on the other hand, were biofilm producers by nature, that is, without exposure to any stress. The biomass of the biofilm reaches its maximum thickness after 48 h of incubation at 35 °C. It was noticed that biofilm population consists of wild type and SCVs. Moreover, the number of SCVs increases with the age of biofilm. The SCVs of MRSA were unable to readopt biofilm mode of growth independently, irrespective of the presence or absence of oxacillin. The SCVs of MSSA, on the other hand, quickly revert to normal life just after a single subculture and show biofilm formation without any stress. Molecular studies showed a parallel reduction in the expression of the genes icaA, sigβ and sarA, and also in the extracellular matrix production in SCVs of MRSA. This might be due to oxacillin as it seems to be a stress factor responsible for induction of biofilm formation in MRSA isolates. Contrary to the wild type, SCVs are metabolically inactive and do not respond to oxacillin, which is only active against the growing cells. Therefore, stress-responsive genes, that is, sigβ and sarA, are not induced. Conversely, MSSA isolates are natural biofilm producers without induction through any known factors.

  7. New Insight into Daptomycin Bioavailability and Localization in Staphylococcus aureus Biofilms by Dynamic Fluorescence Imaging

    PubMed Central

    Briandet, Romain; Revest, Matthieu; Jacqueline, Cédric; Caillon, Jocelyne; Fontaine-Aupart, Marie-Pierre; Steenkeste, Karine

    2016-01-01

    Staphylococcus aureus is one of the most frequent pathogens responsible for biofilm-associated infections (BAI), and the choice of antibiotics to treat these infections remains a challenge for the medical community. In particular, daptomycin has been reported to fail against implant-associated S. aureus infections in clinical practice, while its association with rifampin remains a good candidate for BAI treatment. To improve our understanding of such resistance/tolerance toward daptomycin, we took advantage of the dynamic fluorescence imaging tools (time-lapse imaging and fluorescence recovery after photobleaching [FRAP]) to locally and accurately assess the antibiotic diffusion reaction in methicillin-susceptible and methicillin-resistant S. aureus biofilms. To provide a realistic representation of daptomycin action, we optimized an in vitro model built on the basis of our recently published in vivo mouse model of prosthetic vascular graft infections. We demonstrated that at therapeutic concentrations, daptomycin was inefficient in eradicating biofilms, while the matrix was not a shield to antibiotic diffusion and to its interaction with its bacterial target. In the presence of rifampin, daptomycin was still present in the vicinity of the bacterial cells, allowing prevention of the emergence of rifampin-resistant mutants. Conclusions derived from this study strongly suggest that S. aureus biofilm resistance/tolerance toward daptomycin may be more likely to be related to a physiological change involving structural modifications of the membrane, which is a strain-dependent process. PMID:27297479

  8. Cefuroxime axetil loaded solid lipid nanoparticles for enhanced activity against S. aureus biofilm.

    PubMed

    Singh, Bhupender; Vuddanda, Parameswara Rao; M R, Vijayakumar; Kumar, Vinod; Saxena, Preeti S; Singh, Sanjay

    2014-09-01

    The present research work is focused on the development of solid lipid nanoparticles of cefuroxime axetil (CA-SLN) for its enhanced inhibitory activity against Staphylococcus aureus produced biofilm. CA-SLN was prepared by solvent emulsification/evaporation method using single lipid (stearic acid (SA)) and binary lipids (SA and tristearin (TS)). Process variables such as volume of dispersion medium, concentration of surfactant, homogenization speed and time were optimized. The prepared SLN were characterized for encapsulation efficiency, drug polymer interaction studies (DSC and FT-IR), shape and surface morphology (SEM and AFM), in vitro drug release, stability studies and in vitro anti biofilm activity against S. aureus biofilm. Among the process variables, increased volume of dispersion medium, homogenization speed and time led to increase in particle size whereas increase in surfactant concentration decreased the particle size. SLN prepared using binary lipids exhibited higher entrapment efficiency than the single lipid. DSC and FT-IR studies showed no incompatible interaction between drug and excipients. CA-SLN showed two folds higher anti-biofilm activity in vitro than pristine CA against S. aureus biofilm.

  9. Experimental toxicity and bioaccumulation of cadmium in freshwater periphytic diatoms in relation with biofilm maturity.

    PubMed

    Duong, Thi Thuy; Morin, Soizic; Coste, Michel; Herlory, Olivier; Feurtet-Mazel, Agnès; Boudou, Alain

    2010-01-01

    A study was undertaken to examine cadmium accumulation in freshwater biofilm, its effects on biofilm development and on diatom community structure in laboratory experimental conditions. A suspension of a biofilm originated from the Riou-Mort River (South West France) was inoculated into three experimental units containing clean glass substrates under laboratory conditions. Settling and already developed biofilms were exposed to a Cd concentration of 100 microg L(-1). Metal accumulation (total and intracellular metal content) in biofilms, dry weight and ash-free dry mass, diatom cell density and diatom community composition were analyzed. Both total and intracellular Cd accumulated by the biofilm throughout the experiment increased with duration of metal exposure. Biofilms in the course of maturation were showed higher Cd content and less effective development than settled biofilms. However diatom communities in younger biofilms exposed to Cd increased their tolerance to Cd by a highly significant development of Nitzschia palea. In contrast, Cd exposure had different effect in installed biofilm and taxonomic composition. These results indicate that mature biofilm may limit Cd accumulation into its architecture and protect diatom communities from the effects of metals.

  10. Staphylococcus epidermidis Esp Degrades Specific Proteins Associated with Staphylococcus aureus Biofilm Formation and Host-Pathogen Interaction

    PubMed Central

    Iwamoto, Takeo; Takada, Koji; Okuda, Ken-ichi; Tajima, Akiko; Iwase, Tadayuki

    2013-01-01

    Staphylococcus aureus exhibits a strong capacity to attach to abiotic or biotic surfaces and form biofilms, which lead to chronic infections. We have recently shown that Esp, a serine protease secreted by commensal Staphylococcus epidermidis, disassembles preformed biofilms of S. aureus and inhibits its colonization. Esp was expected to degrade protein determinants of the adhesive and cohesive strength of S. aureus biofilms. The aim of this study was to elucidate the substrate specificity and target proteins of Esp and thereby determine the mechanism by which Esp disassembles S. aureus biofilms. We used a mutant Esp protein (EspS235A) with defective proteolytic activity; this protein did not disassemble the biofilm formed by a clinically isolated methicillin-resistant S. aureus (MRSA) strain, thereby indicating that the proteolytic activity of Esp is essential for biofilm disassembly. Esp degraded specific proteins in the biofilm matrix and cell wall fractions, in contrast to proteinase K, which is frequently used for testing biofilm robustness and showed no preference for proteolysis. Proteomic and immunological analyses showed that Esp degrades at least 75 proteins, including 11 biofilm formation- and colonization-associated proteins, such as the extracellular adherence protein, the extracellular matrix protein-binding protein, fibronectin-binding protein A, and protein A. In addition, Esp selectively degraded several human receptor proteins of S. aureus (e.g., fibronectin, fibrinogen, and vitronectin) that are involved in its colonization or infection. These results suggest that Esp inhibits S. aureus colonization and biofilm formation by degrading specific proteins that are crucial for biofilm construction and host-pathogen interaction. PMID:23316041

  11. Staphylococcus epidermidis Esp degrades specific proteins associated with Staphylococcus aureus biofilm formation and host-pathogen interaction.

    PubMed

    Sugimoto, Shinya; Iwamoto, Takeo; Takada, Koji; Okuda, Ken-Ichi; Tajima, Akiko; Iwase, Tadayuki; Mizunoe, Yoshimitsu

    2013-04-01

    Staphylococcus aureus exhibits a strong capacity to attach to abiotic or biotic surfaces and form biofilms, which lead to chronic infections. We have recently shown that Esp, a serine protease secreted by commensal Staphylococcus epidermidis, disassembles preformed biofilms of S. aureus and inhibits its colonization. Esp was expected to degrade protein determinants of the adhesive and cohesive strength of S. aureus biofilms. The aim of this study was to elucidate the substrate specificity and target proteins of Esp and thereby determine the mechanism by which Esp disassembles S. aureus biofilms. We used a mutant Esp protein (Esp(S235A)) with defective proteolytic activity; this protein did not disassemble the biofilm formed by a clinically isolated methicillin-resistant S. aureus (MRSA) strain, thereby indicating that the proteolytic activity of Esp is essential for biofilm disassembly. Esp degraded specific proteins in the biofilm matrix and cell wall fractions, in contrast to proteinase K, which is frequently used for testing biofilm robustness and showed no preference for proteolysis. Proteomic and immunological analyses showed that Esp degrades at least 75 proteins, including 11 biofilm formation- and colonization-associated proteins, such as the extracellular adherence protein, the extracellular matrix protein-binding protein, fibronectin-binding protein A, and protein A. In addition, Esp selectively degraded several human receptor proteins of S. aureus (e.g., fibronectin, fibrinogen, and vitronectin) that are involved in its colonization or infection. These results suggest that Esp inhibits S. aureus colonization and biofilm formation by degrading specific proteins that are crucial for biofilm construction and host-pathogen interaction.

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

    PubMed

    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.

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

    PubMed Central

    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

  14. Systematic exploration of natural and synthetic flavonoids for the inhibition of Staphylococcus aureus biofilms.

    PubMed

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

    2013-09-25

    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.

  15. Activity of ozonated water and ozone against Staphylococcus aureus and Pseudomonas aeruginosa biofilms

    PubMed Central

    Bialoszewski, Dariusz; Pietruczuk-Padzik, Anna; Kalicinska, Agnieszka; Bocian, Ewa; Czajkowska, Magdalena; Bukowska, Bozena; Tyski, Stefan

    2011-01-01

    Summary Background The known bactericidal properties of ozone have not been checked in relation to its action on bacterial biofilms. This is especially true of ozonated fluids. The aim of this study was to investigate the bactericidal activity of ozonated water and that of a mixture of ozone and oxygen against biofilms. Material/Methods Eighteen clinical strains of Staphylococcus aureus and Pseudomonas aeruginosa exhibiting various levels of antibiotic sensitivity were investigated. Bacteria were cultured in biofilm form on polystyrene titration plates for periods of 2 to 72 hours. The biofilms formed in this way were exposed to in statu nascendi ozonated water produced in a prototype device that had been tested in clinical conditions, or to a mixture of oxygen and ozone generated in the same device. Live cells in the biofilm were stained with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) bromide solution. The degree of reduction of viable bacteria following ozone exposure was determined. Results Ozonated water was found to be an effective bactericidal agent against biofilms after as little as 30 seconds of exposure, while the bactericidal activity of the ozone-oxygen solution was much lower. Prolongation of the duration of biofilm exposure to the gaseous disinfectant to 40 minutes led to a reduction in the viable cell count, which nevertheless remained high. Conclusions Unlike the ozone-oxygen mixture, ozonated water effectively destroys bacterial biofilms in vitro. PMID:22037737

  16. Human methicillin-sensitive Staphylococcus aureus biofilms: potential associations with antibiotic resistance persistence and surface polysaccharide antigens.

    PubMed

    Babra, Charlene; Tiwari, Jully; Costantino, Paul; Sunagar, Raju; Isloor, Shrikrishna; Hegde, Nagendra; Mukkur, Trilochan

    2014-07-01

    The development of persistent antibiotic resistance by human methicillin-sensitive Staphylococcus aureus (MSSA) strains and substantial association with poly-N-acetyl glucosamine (PNAG) in biofilms is reported in this investigation. Sixteen of 31 MSSA strains under study were found to have developed resistance to one or more antibiotics, with four strains, two of which did not produce biofilms, showing resistance to cefoxitin, undetectable by mecA amplification. Antibiotic resistance displayed by 13/14 biofilm-forming S. aureus isolates remained persistent for 4 weeks prior to reverting back to the original antibiotic susceptibility, prompting a suggestion of determining antibiograms for clinical S. aureus isolates subcultured from biofilms developed in vitro as well as planktonic subcultures prepared from the site of infection. While there was correlation of antibiotic resistance with biofilm formation confirming previous reports, this is the first time that persistence of the biofilm-associated antibiotic resistance by S. aureus as planktonic cells is reported. Among the two methods used for assessment of biofilm formation, the tissue culture plate (TCP) method revealed that almost all strains were strong or moderate biofilm producers whereas only 19/31 strains were biofilm producers using the Congo Red agar (CRA) method indicating the superiority of the TCP method in detecting biofilm producers. We also observed no association between biofilm formation and major capsule types. However, substantial, although not absolute, association of biofilm formation with PNAG was observed, warranting continued identification of additional surface-associated polysaccharide and/or protein antigens associated with biofilm formation for development of an effective vaccine against S. aureus infections regardless of capsular phenotype.

  17. 5-aminolevulinic acid-mediated photodynamic therapy and its strain-dependent combined effect with antibiotics on Staphylococcus aureus biofilm

    PubMed Central

    Li, Xian-Hui; Yang, Chen; Guo, Li-Min; Liu, Chun-Hong; Qu, Di; Zheng, Chun-Quan

    2017-01-01

    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. PMID:28358851

  18. 5-aminolevulinic acid-mediated photodynamic therapy and its strain-dependent combined effect with antibiotics on Staphylococcus aureus biofilm.

    PubMed

    Zhang, Qing-Zhao; Zhao, Ke-Qing; Wu, Yang; Li, Xian-Hui; Yang, Chen; Guo, Li-Min; Liu, Chun-Hong; Qu, Di; Zheng, Chun-Quan

    2017-01-01

    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.

  19. Impact of growth temperature and surface type on the resistance of Pseudomonas aeruginosa and Staphylococcus aureus biofilms to disinfectants.

    PubMed

    Abdallah, Marwan; Khelissa, Oussama; Ibrahim, Ali; Benoliel, Corinne; Heliot, Laurent; Dhulster, Pascal; Chihib, Nour-Eddine

    2015-12-02

    Biofilm formation of Pseudomonas aeruginosa and Staphylococcus aureus on food-contact-surfaces represents a significant risk for the public health. In this context, the present study investigates the relationship between the environmental conditions of biofilm formation and the resistance to disinfectants. Therefore, a static biofilm reactor, called NEC-Biofilm System, was established in order to study the effect of growth temperature (20, 30 and 37°C), and of the surface type (stainless steel and polycarbonate), on biofilm resistance to disinfectants. These conditions were selected to mimic the biofilm formation on abiotic surfaces of food processing industries. The antibiofilm assays were performed on biofilms grown during 24 h. The results showed that the growth temperature influenced significantly the biofilm resistance to disinfectants. These data also revealed that the growth temperature has a significant effect on the biofilm structure of both bacteria. Furthermore, the increase of the biofilm growth temperature increased significantly the algD transcript level in sessile P. aeruginosa cells, whereas the icaA one was not affected in S. aureus cells. Overall, our findings show that the biofilm structure and matrix cannot fully explain the biofilm resistance to disinfectant agents. Nevertheless, it underlines the intimate link between environmental conditions, commonly met in food sectors, and the biofilm resistance to disinfectants.

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

    PubMed

    Marques, Viviane Figueira; Motta, Cássia Couto da; Soares, Bianca da Silva; Melo, Dayanne Araújo de; Coelho, Shana de Mattos de Oliveira; Coelho, Irene da Silva; Barbosa, Helene Santos; Souza, Miliane Moreira Soares de

    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.

  1. Staphylococcus aureus and MRSA Growth and Biofilm Formation after Treatment with Antibiotics and SeNPs.

    PubMed

    Cihalova, Kristyna; Chudobova, Dagmar; Michalek, Petr; Moulick, Amitava; Guran, Roman; Kopel, Pavel; Adam, Vojtech; Kizek, Rene

    2015-10-16

    Methicillin-resistant Staphylococcus aureus (MRSA) is a dangerous pathogen resistant to β-lactam antibiotics. Due to its resistance, it is difficult to manage the infections caused by this strain. We examined this issue in terms of observation of the growth properties and ability to form biofilms in sensitive S. aureus and MRSA after the application of antibiotics (ATBs)-ampicillin, oxacillin and penicillin-and complexes of selenium nanoparticles (SeNPs) with these ATBs. The results suggest the strong inhibition effect of SeNPs in complexes with conventional ATBs. Using the impedance method, a higher disruption of biofilms was observed after the application of ATB complexes with SeNPs compared to the group exposed to ATBs without SeNPs. The biofilm formation was intensely inhibited (up to 99%±7% for S. aureus and up to 94%±4% for MRSA) after application of SeNPs in comparison with bacteria without antibacterial compounds whereas ATBs without SeNPs inhibited S. aureus up to 79%±5% and MRSA up to 16%±2% only. The obtained results provide a basis for the use of SeNPs as a tool for the treatment of bacterial infections, which can be complicated because of increasing resistance of bacteria to conventional ATB drugs.

  2. Staphylococcus aureus and MRSA Growth and Biofilm Formation after Treatment with Antibiotics and SeNPs

    PubMed Central

    Cihalova, Kristyna; Chudobova, Dagmar; Michalek, Petr; Moulick, Amitava; Guran, Roman; Kopel, Pavel; Adam, Vojtech; Kizek, Rene

    2015-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a dangerous pathogen resistant to β-lactam antibiotics. Due to its resistance, it is difficult to manage the infections caused by this strain. We examined this issue in terms of observation of the growth properties and ability to form biofilms in sensitive S. aureus and MRSA after the application of antibiotics (ATBs)—ampicillin, oxacillin and penicillin—and complexes of selenium nanoparticles (SeNPs) with these ATBs. The results suggest the strong inhibition effect of SeNPs in complexes with conventional ATBs. Using the impedance method, a higher disruption of biofilms was observed after the application of ATB complexes with SeNPs compared to the group exposed to ATBs without SeNPs. The biofilm formation was intensely inhibited (up to 99% ± 7% for S. aureus and up to 94% ± 4% for MRSA) after application of SeNPs in comparison with bacteria without antibacterial compounds whereas ATBs without SeNPs inhibited S. aureus up to 79% ± 5% and MRSA up to 16% ± 2% only. The obtained results provide a basis for the use of SeNPs as a tool for the treatment of bacterial infections, which can be complicated because of increasing resistance of bacteria to conventional ATB drugs. PMID:26501270

  3. Synthesis and biofilm formation reduction of pyrazole-4-carboxamide derivatives in some Staphylococcus aureus strains.

    PubMed

    Cascioferro, Stella; Maggio, Benedetta; Raffa, Demetrio; Raimondi, Maria Valeria; Cusimano, Maria Grazia; Schillaci, Domenico; Manachini, Barbara; Plescia, Fabiana; Daidone, Giuseppe

    2016-11-10

    The ability of several N-phenyl-1H-pyrazole-4-carboxamide derivatives and other pyrazoles opportunely modified at the positions 3, 4 and 5, to reduce the formation of the biofilm in some Staphylococcus aureus strains (ATCC 29213, ATCC 25923 and ATCC 6538) were investigated. All the tested compounds were able, although to a different extent, to reduce the biofilm formation of the three bacterial strains considered. Among these, the 1-(2,5-dichlorophenyl)-5-methyl-N-phenyl-1H-pyrazole-4-carboxamide 14 resulted as the best inhibitor of biofilm formation showing an IC50 ranging from 2.3 to 32 μM, against all the three strains of S. aureus. Compound 14 also shows a good protective effect in vivo by improving the survival of wax moth larva (Galleria mellonella) infected with S. aureus ATCC 29213. These findings indicate that 14d is a potential lead compound for the development of new anti-virulence agents against S. aureus infections.

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

    PubMed

    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

  5. Bactericidal Effect of a Photoresponsive Carbon Monoxide-Releasing Nonwoven against Staphylococcus aureus Biofilms

    PubMed Central

    Klinger-Strobel, Mareike; Gläser, Steve; Makarewicz, Oliwia; Wyrwa, Ralf; Weisser, Jürgen

    2016-01-01

    Staphylococcus aureus is a leading pathogen in skin and skin structure infections, including surgical and traumatic infections that are associated with biofilm formation. Because biofilm formation is accompanied by high phenotypic resistance of the embedded bacteria, they are almost impossible to eradicate by conventional antibiotics. Therefore, alternative therapeutic strategies are of high interest. We generated nanostructured hybrid nonwovens via the electrospinning of a photoresponsive carbon monoxide (CO)-releasing molecule [CORM-1, Mn2(CO)10] and the polymer polylactide. This nonwoven showed a CO-induced antimicrobial activity that was sufficient to reduce the biofilm-embedded bacteria by 70% after photostimulation at 405 nm. The released CO increased the concentration of reactive oxygen species (ROS) in the biofilms, suggesting that in addition to inhibiting the electron transport chain, ROS might play a role in the antimicrobial activity of CORMs on S. aureus. The nonwoven showed increased cytotoxicity on eukaryotic cells after longer exposure, most probably due to the released lactic acid, that might be acceptable for local and short-time treatments. Therefore, CO-releasing nonwovens might be a promising local antimicrobial therapy against biofilm-associated skin wound infections. PMID:27114272

  6. Eugenol: A Phyto-Compound Effective against Methicillin-Resistant and Methicillin-Sensitive Staphylococcus aureus Clinical Strain Biofilms

    PubMed Central

    Yadav, Mukesh Kumar; Chae, Sung-Won; Im, Gi Jung; Chung, Jae-Woo; Song, Jae-Jun

    2015-01-01

    Background Inhibition and eradication of Staphylococcus aureus biofilms with conventional antibiotic is difficult, and the treatment is further complicated by the rise of antibiotic resistance among staphylococci. Consequently, there is a need for novel antimicrobials that can treat biofilm-related infections and decrease antibiotics burden. Natural compounds such as eugenol with anti-microbial properties are attractive agents that could reduce the use of conventional antibiotics. In this study we evaluated the effect of eugenol on MRSA and MSSA biofilms in vitro and bacterial colonization in vivo. Methods and Results Effect of eugenol on in vitro biofilm and in vivo colonization were studied using microtiter plate assay and otitis media-rat model respectively. The architecture of in vitro biofilms and in vivo colonization of bacteria was viewed with SEM. Real-time RT-PCR was used to study gene expression. Check board method was used to study the synergistic effects of eugenol and carvacrol on established biofilms. Eugenol significantly inhibited biofilms growth of MRSA and MSSA in vitro in a concentration-dependent manner. Eugenol at MIC or 2×MIC effectively eradicated the pre-established biofilms of MRSA and MSSA clinical strains. In vivo, sub-MIC of eugenol significantly decreased 88% S. aureus colonization in rat middle ear. Eugenol was observed to damage the cell-membrane and cause a leakage of the cell contents. At sub-inhibitory concentration, it decreases the expression of biofilm-and enterotoxin-related genes. Eugenol showed a synergistic effect with carvacrol on the eradication of pre-established biofilms. Conclusion/Major Finding This study demonstrated that eugenol exhibits notable activity against MRSA and MSSA clinical strains biofilms. Eugenol inhibited biofilm formation, disrupted the cell-to-cell connections, detached the existing biofilms, and killed the bacteria in biofilms of both MRSA and MSSA with equal effectiveness. Therefore, eugenol may

  7. Comparison of the In vitro Activity of Five Antimicrobial Drugs against Staphylococcus pseudintermedius and Staphylococcus aureus Biofilms

    PubMed Central

    Ferran, Aude A.; Liu, JingJing; Toutain, Pierre-Louis; Bousquet-Mélou, Alain

    2016-01-01

    Resistance in canine pathogenic staphylococci is necessitating re-evaluation of the current antimicrobial treatments especially for biofilm-associated infections. Long, repeated treatments are often required to control such infections due to the tolerance of bacteria within the biofilm. To comply with the goal of better antibiotic stewardship in veterinary medicine, the efficacies of the available drugs need to be directly assessed on bacterial biofilms. We compared the activities of amoxicillin, cefalexin, clindamycin, doxycycline, and marbofloxacin on in vitro biofilms of Staphylococcus pseudintermedius and Staphylococcus aureus. Exposure of biofilms for 15 h to maximum concentrations of the antibiotics achievable in canine plasma only reduced biofilm bacteria by 0.5–2.0 log10 CFU, compared to the control, except for marbofloxacin which reduced S. aureus biofilms by 5.4 log10 CFU. Two-antibiotic combinations did not improve, and even decreased, bacterial killing. In comparison, 5 min-exposure to 2% chlorhexidine reduced biofilms of the two tested strains by 4 log10 CFU. Our results showed that S. pseudintermedius and S. aureus biofilms were highly tolerant to all the drugs tested, consistent with the treatment failures observed in practice. Under our in vitro conditions, the use of chlorhexidine was more efficacious than antimicrobials to reduce S. pseudintermedius biofilm. PMID:27531995

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

    NASA Astrophysics Data System (ADS)

    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

  9. Ozonated saline shows activity against planktonic and biofilm growing Staphylococcus aureus in vitro: a potential irrigant for infected wounds.

    PubMed

    Al-Saadi, Hayder; Potapova, Inga; Rochford, Edward Tj; Moriarty, Thomas F; Messmer, Peter

    2016-10-01

    Infections associated with deep wounds require extensive surgical and medical care. New adjunctive treatments are required to aid in the eradication of the bacterial biofilms found on infected wounds and, in particular, any underlying hardware. Ozone has been used as a safe and efficient disinfectant in water treatment plants for many years. The purpose of this study is to investigate the anti-biofilm potential of ozonated saline against biofilms of Staphylococcus aureus, a microorganism commonly implicated in wound infections. A custom-made bacterial biofilm bioreactor was used to grow S. aureus biofilms on discs of medical grade titanium alloy. An ozone generator was connected in-line and biofilms and planktonic bacteria were exposed to ozone in saline. Cytotoxicity was assessed against primary ovine osteoblasts in the same system. In tests against planktonic S. aureus, a 99% reduction in bacterial numbers was detected within 15 minutes of exposure. S. aureus biofilms were significantly more resistant to ozone, although complete eradication of the biofilm was eventually achieved within 5 hours. Ozonated saline was not found to be cytotoxic to primary ovine osteoblasts. Ozonated saline may be suitable as an adjuvant therapy to treat patients as an instillation fluid for wound irrigation and sterilisation.

  10. Image-based fluorescence recovery after photobleaching (FRAP) to dissect vancomycin diffusion-reaction processes in Staphylococcus aureus biofilms

    NASA Astrophysics Data System (ADS)

    Daddi Oubekka, S.; Briandet, R.; Waharte, F.; Fontaine-Aupart, M.-P.; Steenkeste, K.

    2011-07-01

    The diffusion capabilities of free fluorophores inside the heterogeneous three dimensional structure of Staphylococcus aureus biofilm were studied by an original image-based Fluorescence Recovery After Photobleaching method. The study was extended to BODIPY-vancomycin in order to better understand the mechanisms involved in the high tolerance of the bacteria embedded in a biofilm to the antibiotic.

  11. Antimicrobial Activity of Selected Phytochemicals against Escherichia coli and Staphylococcus aureus and Their Biofilms

    PubMed Central

    Monte, Joana; Abreu, Ana C.; Borges, Anabela; Simões, Lúcia Chaves; Simões, Manuel

    2014-01-01

    Abstract Bacteria can be resistant to multiple antibiotics and we are fast approaching a time when antibiotics will not work on some bacterial infections. New antimicrobial compounds are urgently necessary. Plants are considered the greatest source to obtain new antimicrobials. This study aimed to assess the antimicrobial activity of four phytochemicals—7-hydroxycoumarin (7-HC), indole-3-carbinol (I3C), salicylic acid (SA) and saponin (SP)—against Escherichia coli and Staphylococcus aureus, either as planktonic cells or as biofilms. These bacteria are commonly found in hospital-acquired infections. Some aspects on the phytochemicals mode of action, including surface charge, hydrophobicity, motility and quorum-sensing inhibition (QSI) were investigated. In addition, the phytochemicals were combined with three antibiotics in order to assess any synergistic effect. 7-HC and I3C were the most effective phytochemicals against E. coli and S. aureus. Both phytochemicals affected the motility and quorum-sensing (QS) activity, which means that they can play an important role in the interference of cell-cell interactions and in biofilm formation and control. However, total biofilm removal was not achieved with any of the selected phytochemicals. Dual combinations between tetracycline (TET), erythromycin (ERY) and ciprofloxacin (CIP) and I3C produced synergistic effects against S. aureus resistant strains. The overall results demonstrates the potential of phytochemicals to control the growth of E. coli and S. aureus in both planktonic and biofilm states. In addition, the phytochemicals demonstrated the potential to act synergistically with antibiotics, contributing to the recycling of old antibiotics that were once considered ineffective due to resistance problems. PMID:25437810

  12. Effect of Bacoside A on growth and biofilm formation by Staphylococcus aureus and Pseudomonas aeruginosa.

    PubMed

    Parai, Debaprasad; Islam, Ekramul; Mitra, Jayati; Mukherjee, Samir Kumar

    2017-02-01

    The goal of this study was to evaluate the antibiofilm and antimicrobial activities of Bacoside A, a formulation of phytochemicals from Bacopa monnieri, against Staphylococcus aureus and Pseudomonas aeruginosa, which are known to form biofilms as one of their virulence traits. The antimicrobial effects of Bacoside A were tested using the minimum inhibitory concentration and minimum bactericidal concentration assays. A cell membrane disruption assay was performed to find its possible target site. MTT assay, crystal violet assay, and microscopic studies were performed to assess the antibiofilm activity. Bacoside A showed antimicrobial activity against both test organisms in their planktonic and biofilm states. At a subminimum inhibitory concentration of 200 μg·mL(-1), Bacoside A significantly removed ∼88%-93% of bacterial biofilm developed on microtiter plates. Biochemical and microscopic studies suggested that the eradication of biofilm might be due to the loss of extracellular polymeric substances and to a change in cell membrane integrity of the selected bacterial strains treated with Bacoside A. These results indicate that Bacoside A might be considered as an antimicrobial having the ability to disrupt biofilms. Thus, either alone or in combination with other therapeutics, Bacoside A could be useful to treat biofilm-related infections caused by opportunistic bacterial pathogens.

  13. Resistance to benzalkonium chloride, peracetic acid and nisin during formation of mature biofilms by Listeria monocytogenes.

    PubMed

    Saá Ibusquiza, P; Herrera, J J R; Cabo, M L

    2011-05-01

    Increase of resistance to the application of benzalkonium chloride (BAC), peracetic acid (PA) and nisin during biofilm formation at 25 °C by three strains of Listeria monocytogenes (CECT 911, CECT 4032, CECT 5873 and BAC-adapted CECT 5873) in different scenarios was compared. For this purpose, resistance after 4 and 11-days of biofilm formation was quantified in terms of lethal dose 90% values (LD(90)), determined according with a dose-response logistic mathematical model. Microscopic analyses after 4 and 11-days of L. monocytogenes biofilm formation were also carried out. Results demonstrated a relation between the microscopic structure and the resistance to the assayed biocides in matured biofilms. The worst cases being biofilms formed by the strain 4032 (in both stainless steel and polypropylene), which showed a complex "cloud-type" structure that correlates with the highest resistance of this strain against the three biocides during biofilm maturation. However, that increase in resistance and complexity appeared not to be dependent on initial bacterial adherence, thus indicating mature biofilms rather than planctonic cells or early-stage biofilms must be considered when disinfection protocols have to be optimized. PA seemed to be the most effective of the three disinfectants used for biofilms. We hypothesized both its high oxidizing capacity and low molecular size could suppose an advantage for its penetration inside the biofilm. We also demonstrated that organic material counteract with the biocides, thus indicating the importance of improving cleaning protocols. Finally, by comparing strains 5873 and 5873 adapted to BAC, several adaptative cross-responses between BAC and nisin or peracetic acid were identified.

  14. Screening a Commercial Library of Pharmacologically Active Small Molecules against Staphylococcus aureus Biofilms

    PubMed Central

    Torres, Nelson S.; Abercrombie, Johnathan J.; Srinivasan, Anand; Lopez-Ribot, Jose L.

    2016-01-01

    It is now well established that bacterial infections are often associated with biofilm phenotypes that demonstrate increased resistance to common antimicrobials. Further, due to the collective attrition of new antibiotic development programs by the pharmaceutical industries, drug repurposing is an attractive alternative. In this work, we screened 1,280 existing commercially available drugs in the Prestwick Chemical Library, some with previously unknown antimicrobial activity, against Staphylococcus aureus, one of the commonly encountered causative pathogens of burn and wound infections. From the primary screen of the entire Prestwick Chemical Library at a fixed concentration of 10 μM, 104 drugs were found to be effective against planktonic S. aureus strains, and not surprisingly, these were mostly antimicrobials and antiseptics. The activity of 18 selected repurposing candidates, that is, drugs that show antimicrobial activity that are not already considered antimicrobials, observed in the primary screen was confirmed in dose-response experiments. Finally, a subset of nine of these drug candidates was tested against preformed biofilms of S. aureus. We found that three of these drugs, niclosamide, carmofur, and auranofin, possessed antimicrobial activity against preformed biofilms, making them attractive candidates for repurposing as novel antibiofilm therapies. PMID:27401577

  15. Regulatory Mutations Impacting Antibiotic Susceptibility in an Established Staphylococcus aureus Biofilm.

    PubMed

    Atwood, Danielle N; Beenken, Karen E; Lantz, Tamara L; Meeker, Daniel G; Lynn, William B; Mills, Weston B; Spencer, Horace J; Smeltzer, Mark S

    2016-01-11

    We previously determined the extent to which mutations of different Staphylococcus aureus regulatory loci impact biofilm formation as assessed under in vitro conditions. Here we extend these studies to determine the extent to which those regulatory loci that had the greatest effect on biofilm formation also impact antibiotic susceptibility. The experiments were done under in vitro and in vivo conditions using two clinical isolates of S. aureus (LAC and UAMS-1) and two functionally diverse antibiotics (daptomycin and ceftaroline). Mutation of the staphylococcal accessory regulator (sarA) or sigB was found to significantly increase susceptibilities to both antibiotics and in both strains in a manner that could not be explained by changes in the MICs. The impact of a mutation in sarA was comparable to that of a mutation in sigB and greater than the impact observed with any other mutant. These results suggest that therapeutic strategies targeting sarA and/or sigB have the greatest potential to facilitate the ability to overcome the intrinsic antibiotic resistance that defines S. aureus biofilm-associated infections.

  16. Significance of rpoS during maturation of Escherichia coli biofilms.

    PubMed

    Ito, Akinobu; May, Thithiwat; Kawata, Koji; Okabe, Satoshi

    2008-04-15

    Presence of starved, stationary phase-like zones in biofilms seems to be an important factor for biofilm formation. In this study, roles of rpoS gene in the formation of Escherichia coli biofilms were investigated. E. coli MG1655 wild type (WT) and rpoS mutant (DeltarpoS) strains were used to compare biofilm formation capacity and global gene expression. Even though the DeltarpoS strain could attach and form microcolonies on glass surfaces, it could not establish mature biofilms. DNA microarray analysis revealed that WT biofilms (WBF) showed similar pattern of gene expression with WT planktonic stationary phase, whereas DeltarpoS biofilms (MBF) showed similar pattern of gene expression with WT planktonic exponential phase. Genes involved in energy metabolism (atpIBEFHAG, atpC, cydAB) and flagella synthesis (flgB, flgC, flhD, fliA, fliC, fliY) showed increased expression in the MBF, but not in the WBF. Moreover, genes involved in stress responses (blc, cspG, dinD poxB, wcaF, wcaI, and yfcF) showed increased expression in the WBF compared to the MBF. These results suggested that the rpoS gene contributed in maturation of E. coli biofilms through regulation of global gene expression including energy metabolism, motility, and stress responses.

  17. Chelating agents exert distinct effects on biofilm formation in Staphylococcus aureus depending on strain background: role for clumping factor B

    PubMed Central

    Abraham, Nabil M.; Lamlertthon, Supaporn; Fowler, Vance G.

    2012-01-01

    Staphylococcus aureus is a leading cause of catheter infections, and biofilm formation plays a key role in the pathogenesis. Metal ion chelators inhibit bacterial biofilm formation and viability, making them attractive candidates as components in catheter lock solutions. The goal of this study was to characterize further the effect of chelators on biofilm formation. The effect of the calcium chelators ethylene glycol tetraacetic acid (EGTA) and trisodium citrate (TSC) on biofilm formation by 30 S. aureus strains was tested. The response to subinhibitory doses of EGTA and TSC varied dramatically depending on strain variation. In some strains, the chelators prevented biofilm formation, in others they had no effect, and they actually enhanced biofilm formation in others. The molecular basis for this phenotypic variability was investigated using two related strains: Newman, in which biofilm formation was inhibited by chelators, and 10833, which formed strong biofilms in the presence of chelators. It was found that deletion of the gene encoding the surface adhesin clumping factor B (clfB) completely eliminated chelator-induced biofilm formation in strain 10833. The role of ClfB in biofilm formation activity in chelators was confirmed in additional strains. It was concluded that biofilm-forming ability varies strikingly depending on strain background, and that ClfB is involved in biofilm formation in the presence EGTA and citrate. These results suggest that subinhibitory doses of chelating agents in catheter lock solutions may actually augment biofilm formation in certain strains of S. aureus, and emphasize the importance of using these agents appropriately so that inhibitory doses are achieved consistently. PMID:22516131

  18. Infection mechanism of biofilm-forming Staphylococcus aureus on indwelling foreign materials in mice.

    PubMed

    Makino, Taro; Jimi, Shiro; Oyama, Takuto; Nakano, Yuki; Hamamoto, Kouichi; Mamishin, Kanako; Yahiro, Tomoko; Hara, Shuuji; Takata, Tohru; Ohjimi, Hiroyuki

    2015-04-01

    Indwelling foreign-body infections are a critical medical problem, especially in immunocompromised patients. To examine the pathogenicity of biofilm-forming bacteria settling on foreign materials, mice implanted with plastic discs were infected with Staphylococcus aureus. After opening a wide subcutaneous pocket on the dorsal side of mice with or without temporal leukocytopenia, a plastic sheet was placed in the left subcutaneous space; subsequently, bacteria in a planktonic state were dispersed over the subcutaneous space. Bacterial numbers were examined 7 days after inoculation. In subcutaneous tissue on the right, S. aureus was found only in leukocytopenic mice. Meanwhile, bacteria were detected on the plastic and neighbouring tissue in both leukocytopenic and normal mice; however, colony-forming analysis indicated that leukocytopenic mice possessed significantly more bacteria. Tissue reaction against bacteria was pathologically examined. Invading S. aureus induced severe inflammation. In transient leukocytopenic mice, bacterial microcolonies formed on the plastic as well as in the developed necrotic tissue - both of which were shielded from inflammatory cell infiltration - result in bacteraemia. These results indicate that biofilm-forming S. aureus settling on indwelling foreign material are tolerant against host immunity and assault neighbouring tissue, which may lead to chronic wound infection.

  19. Role of Phenol-Soluble Modulins in Formation of Staphylococcus aureus Biofilms in Synovial Fluid

    PubMed Central

    Dastgheyb, Sana S.; Villaruz, Amer E.; Le, Katherine Y.; Tan, Vee Y.; Duong, Anthony C.; Chatterjee, Som S.; Cheung, Gordon Y. C.; Joo, Hwang-Soo; Hickok, Noreen J.

    2015-01-01

    Staphylococcus aureus is a leading cause of prosthetic joint infections, which, as we recently showed, proceed with the involvement of biofilm-like clusters that cause recalcitrance to antibiotic treatment. Here we analyzed why these clusters grow extraordinarily large, reaching macroscopically visible extensions (>1 mm). We found that while specific S. aureus surface proteins are a prerequisite for agglomeration in synovial fluid, low activity of the Agr regulatory system and subsequent low production of the phenol-soluble modulin (PSM) surfactant peptides cause agglomerates to grow to exceptional dimensions. Our results indicate that PSMs function by disrupting interactions of biofilm matrix molecules, such as the polysaccharide intercellular adhesin (PIA), with the bacterial cell surface. Together, our findings support a two-step model of staphylococcal prosthetic joint infection: As we previously reported, interaction of S. aureus surface proteins with host matrix proteins such as fibrin initiates agglomeration; our present results show that, thereafter, the bacterial agglomerates grow to extremely large sizes owing to the lack of PSM expression under the specific conditions present in joints. Our findings provide a mechanistic explanation for the reported extreme resistance of joint infection to antibiotic treatment, lend support to the notions that Agr functionality and PSM production play a major role in defining different forms of S. aureus infection, and have important implications for antistaphylococcal therapeutic strategies. PMID:25964472

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

    PubMed Central

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

    2016-01-01

    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 Ca2+ 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. PMID:26763935

  1. Impact of food-related environmental factors on the adherence and biofilm formation of natural Staphylococcus aureus isolates.

    PubMed

    Vázquez-Sánchez, Daniel; Habimana, Olivier; Holck, Askild

    2013-02-01

    Staphylococcus aureus is a pathogenic bacterium capable of developing biofilms on food-processing surfaces, a pathway leading to cross contamination of foods. The purpose of this study was to investigate the influence of environmental stress factors found during seafood production on the adhesion and biofilm-forming properties of S. aureus. Adhesion and biofilm assays were performed on 26 S. aureus isolated from seafood and two S. aureus reference strains (ATCC 6538 and ATCC 43300). Cell surface properties were evaluated by affinity measurements to solvents in a partitioning test, while adhesion and biofilm assays were performed in polystyrene microplates under different stress conditions of temperature, osmolarity, and nutrient content. The expression of genes implicated in the regulation of biofilm formation (icaA, rbf and σ( B )) was analyzed by reverse transcription and quantitative real time PCR. In general, S. aureus isolates showed moderate hydrophobic properties and a marked Lewis-base character. Initial adhesion to polystyrene was positively correlated with the ionic strength of the growth medium. Most of the strains had a higher biofilm production at 37 °C than at 25 °C, promoted by the addition of glucose, whereas NaCl and MgCl(2) had a lower impact markedly affected by incubation temperatures. Principal Component Analysis revealed a considerable variability in adhesion and biofilm-forming properties between S. aureus isolates. Transcriptional analysis also indicated variations in gene expression between three characteristic isolates under different environmental conditions. These results suggested that the prevalence of S. aureus strains on food-processing surfaces is above all conditioned by the ability to adapt to the environmental stress conditions present during food production. These findings are relevant for food safety and may be of importance when choosing the safest environmental conditions and material during processing, packaging, and

  2. Integration of Posttranscriptional Gene Networks into Metabolic Adaptation and Biofilm Maturation in Candida albicans

    PubMed Central

    Harrison, Paul F.; Lo, Tricia L.; Quenault, Tara; Dagley, Michael J.; Bellousoff, Matthew; Powell, David R.; Beilharz, Traude H.; Traven, Ana

    2015-01-01

    The yeast Candida albicans is a human commensal and opportunistic pathogen. Although both commensalism and pathogenesis depend on metabolic adaptation, the regulatory pathways that mediate metabolic processes in C. albicans are incompletely defined. For example, metabolic change is a major feature that distinguishes community growth of C. albicans in biofilms compared to suspension cultures, but how metabolic adaptation is functionally interfaced with the structural and gene regulatory changes that drive biofilm maturation remains to be fully understood. We show here that the RNA binding protein Puf3 regulates a posttranscriptional mRNA network in C. albicans that impacts on mitochondrial biogenesis, and provide the first functional data suggesting evolutionary rewiring of posttranscriptional gene regulation between the model yeast Saccharomyces cerevisiae and C. albicans. A proportion of the Puf3 mRNA network is differentially expressed in biofilms, and by using a mutant in the mRNA deadenylase CCR4 (the enzyme recruited to mRNAs by Puf3 to control transcript stability) we show that posttranscriptional regulation is important for mitochondrial regulation in biofilms. Inactivation of CCR4 or dis-regulation of mitochondrial activity led to altered biofilm structure and over-production of extracellular matrix material. The extracellular matrix is critical for antifungal resistance and immune evasion, and yet of all biofilm maturation pathways extracellular matrix biogenesis is the least understood. We propose a model in which the hypoxic biofilm environment is sensed by regulators such as Ccr4 to orchestrate metabolic adaptation, as well as the regulation of extracellular matrix production by impacting on the expression of matrix-related cell wall genes. Therefore metabolic changes in biofilms might be intimately linked to a key biofilm maturation mechanism that ultimately results in untreatable fungal disease. PMID:26474309

  3. Norlichexanthone Reduces Virulence Gene Expression and Biofilm Formation in Staphylococcus aureus

    PubMed Central

    Bojer, Martin S.; Zhao, Yu; Friberg, Cathrine; Ifrah, Dan; Glasser Heede, Nina; Larsen, Thomas O.; Frøkiær, Hanne; Frees, Dorte; Zhang, Lixin; Dai, Huanqin

    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 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 regulator, AgrA, which controls expression of RNAIII and the phenol soluble modulins responsible for human neutrophil killing. We show that norlichexanthone reduces S. aureus toxicity towards human neutrophils and interferes directly with AgrA binding to its DNA target. In contrast to ω-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. PMID:28005941

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

    PubMed

    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.

  5. Resolution of Staphylococcus aureus biofilm infection using vaccination and antibiotic treatment.

    PubMed

    Brady, Rebecca A; O'May, Graeme A; Leid, Jeff G; Prior, Megan L; Costerton, J William; Shirtliff, Mark E

    2011-04-01

    Staphylococcus aureus infections, particularly those from methicillin-resistant strains (i.e., MRSA), are reaching epidemic proportions, with no effective vaccine available. The vast number and transient expression of virulence factors in the infectious course of this pathogen have made the discovery of protective antigens particularly difficult. In addition, the divergent planktonic and biofilm modes of growth with their accompanying proteomic changes also demonstrate significant hindrances to vaccine development. In this study, a multicomponent vaccine was evaluated for its ability to clear a staphylococcal biofilm infection. Antigens (glucosaminidase, an ABC transporter lipoprotein, a conserved hypothetical protein, and a conserved lipoprotein) were chosen since they were found in previous studies to have upregulated and sustained expression in a biofilm, both in vitro and in vivo. Antibodies against these antigens were first used in microscopy studies to localize their expression in in vitro biofilms. Each of the four antigens showed heterogeneous production in various locations within the complex biofilm community in the biofilm. Based upon these studies, the four antigens were delivered simultaneously as a quadrivalent vaccine in order to compensate for this varied production. In addition, antibiotic treatment was also administered to clear the remaining nonattached planktonic cells since the vaccine antigens may have been biofilm specific. The results demonstrated that when vaccination was coupled with vancomycin treatment in a biofilm model of chronic osteomyelitis in rabbits, clinical and radiographic signs of infection significantly reduced by 67 and 82%, respectively, compared to infected animals that were either treated with vancomycin or left untreated. In contrast, vaccination alone resulted in a modest, and nonsignificant, decrease in clinical (34% reduction) and radiographic signs (9% reduction) of infection, compared to nonvaccinated animal groups

  6. Effects of antimicrobial peptides on Staphylococcus aureus growth and biofilm formation in vitro following isolation from implant-associated infections

    PubMed Central

    Zhao, Guangfeng; Zhong, Huiming; Zhang, Mao; Hong, Yucai

    2015-01-01

    To prevent biomaterial-associated infections, antibiotic agents are recommended for various medical conditions requiring biomaterial implants, but resistance often appears after the introduction of antibiotics into clinical use. Therefore, new strategies for the prevention or treatment for biomaterial-associated infections are required. The purpose of this study was to evaluate the effects of antimicrobial peptides on growth and biofilm formation of Staphylococcus aureus isolated from implant-associated infections. A total of 20 patients with culture-proven staphylococcal infection associated with stable orthopedic implants were selected as the experimental group. S. aureus were isolated from tissue biopsies for identification, the isolated strains were mixed with Tet213 incubated at 37°C and viable bactrial number of S. aureus was counted. For the biofilm formation, the broad spectrum AMP Tet213 was selected and loaded onto the Ti coating first. At the same time Ti coated with Tet213 were mixed with S. aureus in vitro to form biofilm. After 30 min, 2 h, 4 h, 6 h, 8 h, the population of S. aureus in the biofilm was counted. Tet213 showed significant antibacterial effect on 16 strains (P < 0.05, Table 1). The inhibition rate reached above 80% among 12 strains of the clinically isolated strain. In biofilm experiments, counts of the NO. 1, 2, 3, 4 strains in biofilms decreased significantly after 2 h (P < 0.05), while there was no obvious difference in counts of NO. 5 strain (P > 0.05). The broad spectrum AMP Tet213 could strongly reduce the growth and biofilm formation of S. aureus in vitro, and the use of this might be an important new approach to target implant-associated infections. PMID:25785171

  7. Bap, a Biofilm Matrix Protein of Staphylococcus aureus Prevents Cellular Internalization through Binding to GP96 Host Receptor

    PubMed Central

    Valle, Jaione; Latasa, Cristina; Gil, Carmen; Toledo-Arana, Alejandro; Solano, Cristina; Penadés, José R.; Lasa, Iñigo

    2012-01-01

    The biofilm matrix, composed of exopolysaccharides, proteins, nucleic acids and lipids, plays a well-known role as a defence structure, protecting bacteria from the host immune system and antimicrobial therapy. However, little is known about its responsibility in the interaction of biofilm cells with host tissues. Staphylococcus aureus, a leading cause of biofilm-associated chronic infections, is able to develop a biofilm built on a proteinaceous Bap-mediated matrix. Here, we used the Bap protein as a model to investigate the role that components of the biofilm matrix play in the interaction of S. aureus with host cells. The results show that Bap promotes the adhesion but prevents the entry of S. aureus into epithelial cells. A broad analysis of potential interaction partners for Bap using ligand overlayer immunoblotting, immunoprecipitation with purified Bap and pull down with intact bacteria, identified a direct binding between Bap and Gp96/GRP94/Hsp90 protein. The interaction of Bap with Gp96 provokes a significant reduction in the capacity of S. aureus to invade epithelial cells by interfering with the fibronectin binding protein invasion pathway. Consistent with these results, Bap deficient bacteria displayed an enhanced capacity to invade mammary gland epithelial cells in a lactating mice mastitis model. Our observations begin to elucidate the mechanisms by which components of the biofilm matrix can facilitate the colonization of host tissues and the establishment of persistent infections. PMID:22876182

  8. Relevant role of fibronectin-binding proteins in Staphylococcus aureus biofilm-associated foreign-body infections.

    PubMed

    Vergara-Irigaray, Marta; Valle, Jaione; Merino, Nekane; Latasa, Cristina; García, Begoña; Ruiz de Los Mozos, Igor; Solano, Cristina; Toledo-Arana, Alejandro; Penadés, José R; Lasa, Iñigo

    2009-09-01

    Staphylococcus aureus can establish chronic infections on implanted medical devices due to its capacity to form biofilms. Analysis of the factors that assemble cells into a biofilm has revealed the occurrence of strains that produce either a polysaccharide intercellular adhesin/poly-N-acetylglucosamine (PIA/PNAG) exopolysaccharide- or a protein-dependent biofilm. Examination of the influence of matrix nature on the biofilm capacities of embedded bacteria has remained elusive, because a natural strain that readily converts between a polysaccharide- and a protein-based biofilm has not been studied. Here, we have investigated the clinical methicillin (meticillin)-resistant Staphylococcus aureus strain 132, which is able to alternate between a proteinaceous and an exopolysaccharidic biofilm matrix, depending on environmental conditions. Systematic disruption of each member of the LPXTG surface protein family identified fibronectin-binding proteins (FnBPs) as components of a proteinaceous biofilm formed in Trypticase soy broth-glucose, whereas a PIA/PNAG-dependent biofilm was produced under osmotic stress conditions. The induction of FnBP levels due to a spontaneous agr deficiency present in strain 132 and the activation of a LexA-dependent SOS response or FnBP overexpression from a multicopy plasmid enhanced biofilm development, suggesting a direct relationship between the FnBP levels and the strength of the multicellular phenotype. Scanning electron microscopy revealed that cells growing in the FnBP-mediated biofilm formed highly dense aggregates without any detectable extracellular matrix, whereas cells in a PIA/PNAG-dependent biofilm were embedded in an abundant extracellular material. Finally, studies of the contribution of each type of biofilm matrix to subcutaneous catheter colonization revealed that an FnBP mutant displayed a significantly lower capacity to develop biofilm on implanted catheters than the isogenic PIA/PNAG-deficient mutant.

  9. A Novel Repressor of the ica Locus Discovered in Clinically Isolated Super-Biofilm-Elaborating Staphylococcus aureus

    PubMed Central

    Yu, Liansheng; Hisatsune, Junzo; Hayashi, Ikue; Tatsukawa, Nobuyuki; Sato’o, Yusuke; Mizumachi, Emiri; Kato, Fuminori; Hirakawa, Hideki; Pier, Gerald B.

    2017-01-01

    ABSTRACT Staphylococcus aureus TF2758 is a clinical isolate from an atheroma and a super-biofilm-elaborating/polysaccharide intercellular adhesin (PIA)/poly-N-acetylglucosamine (PNAG)-overproducing strain (L. Shrestha et al., Microbiol Immunol 60:148–159, 2016, https://doi.org/10.1111/1348-0421.12359). A microarray analysis and DNA genome sequencing were performed to identify the mechanism underlying biofilm overproduction by TF2758. We found high transcriptional expression levels of a 7-gene cluster (satf2580 to satf2586) and the ica operon in TF2758. Within the 7-gene cluster, a putative transcriptional regulator gene designated rob had a nonsense mutation that caused the truncation of the protein. The complementation of TF2758 with rob from FK300, an rsbU-repaired derivative of S. aureus strain NCTC8325-4, significantly decreased biofilm elaboration, suggesting a role for rob in this process. The deletion of rob in non-biofilm-producing FK300 significantly increased biofilm elaboration and PIA/PNAG production. In the search for a gene(s) in the 7-gene cluster for biofilm elaboration controlled by rob, we identified open reading frame (ORF) SAOUHSC_2898 (satf2584). Our results suggest that ORF SAOUHSC_2898 (satf2584) and icaADBC are required for enhanced biofilm elaboration and PIA/PNAG production in the rob deletion mutant. Rob bound to a palindromic sequence within its own promoter region. Furthermore, Rob recognized the TATTT motif within the icaR-icaA intergenic region and bound to a 25-bp DNA stretch containing this motif, which is a critically important short sequence regulating biofilm elaboration in S. aureus. Our results strongly suggest that Rob is a long-sought repressor that recognizes and binds to the TATTT motif and is an important regulator of biofilm elaboration through its control of SAOUHSC_2898 (SATF2584) and Ica protein expression in S. aureus. PMID:28143981

  10. Methicillin Resistance Alters the Biofilm Phenotype and Attenuates Virulence in Staphylococcus aureus Device-Associated Infections

    PubMed Central

    Rudkin, Justine K.; Schaeffer, Carolyn R.; Lohan, Amanda J.; Tong, Pin; Loftus, Brendan J.; Pier, Gerald B.; Fey, Paul D.; Massey, Ruth C.; O'Gara, James P.

    2012-01-01

    Clinical isolates of Staphylococcus aureus can express biofilm phenotypes promoted by the major cell wall autolysin and the fibronectin-binding proteins or the icaADBC-encoded polysaccharide intercellular adhesin/poly-N-acetylglucosamine (PIA/PNAG). Biofilm production in methicillin-susceptible S. aureus (MSSA) strains is typically dependent on PIA/PNAG whereas methicillin-resistant isolates express an Atl/FnBP-mediated biofilm phenotype suggesting a relationship between susceptibility to β-lactam antibiotics and biofilm. By introducing the methicillin resistance gene mecA into the PNAG-producing laboratory strain 8325-4 we generated a heterogeneously resistant (HeR) strain, from which a homogeneous, high-level resistant (HoR) derivative was isolated following exposure to oxacillin. The HoR phenotype was associated with a R602H substitution in the DHHA1 domain of GdpP, a recently identified c-di-AMP phosphodiesterase with roles in resistance/tolerance to β-lactam antibiotics and cell envelope stress. Transcription of icaADBC and PNAG production were impaired in the 8325-4 HoR derivative, which instead produced a proteinaceous biofilm that was significantly inhibited by antibodies against the mecA-encoded penicillin binding protein 2a (PBP2a). Conversely excision of the SCCmec element in the MRSA strain BH1CC resulted in oxacillin susceptibility and reduced biofilm production, both of which were complemented by mecA alone. Transcriptional activity of the accessory gene regulator locus was also repressed in the 8325-4 HoR strain, which in turn was accompanied by reduced protease production and significantly reduced virulence in a mouse model of device infection. Thus, homogeneous methicillin resistance has the potential to affect agr- and icaADBC-mediated phenotypes, including altered biofilm expression and virulence, which together are consistent with the adaptation of healthcare-associated MRSA strains to the antibiotic-rich hospital environment in which they are

  11. Major components of orange oil inhibit Staphylococcus aureus growth and biofilm formation, and alter its virulence factors.

    PubMed

    Federman, Cassandra; Ma, Christopher; Biswas, Debabrata

    2016-07-01

    Bovine mastitis is a costly disease in the dairy industry and does not always respond to antibiotic treatment. The major components of terpeneless, cold-pressed Valencia orange oil - citral, linalool, decanal and valencene - were examined as potential alternative treatments for Staphylococcus aureus-associated mastitis. The minimum inhibitory concentration (MIC) of all four components against S. aureus was determined after incubation for 24 h. Growth inhibition assays were performed for all effective components on S. aureus for either a 3 h or 72 h treatment. These components were tested for the ability to disrupt pre-formed S. aureus biofilms after 24 h of treatment by measuring absorbance at 540 nm. Cytotoxicity against immortalized bovine mammary epithelial (MAC-T) cells was measured using an MTT assay following a 1 h exposure. Only concentrations below the 50 % cytostatic concentration (CC50) were used in an adherence and invasion assay of S. aureus on MAC-T cells, and for measurements of virulence and biofilm gene expression via qPCR. The MICs of citral and linalool were 0.02 % and 0.12 %, respectively, but decanal and valencene were ineffective. Citral and linalool were capable of inhibiting growth of S. aureus after 24 h at their MIC values and inhibited pre-formed biofilms of S. aureus . The concentrations below the CC50 were 0.02 % for citral and 0.12 % for linalool. These concentrations inhibited the adhesion and invasion ability of S. aureus and downregulated virulence genes. Only 0.12 % linalool downregulated the expression of S. aureus biofilm-forming genes. These components should be considered for further in vivo study.

  12. Effect of growth temperature, surface type and incubation time on the resistance of Staphylococcus aureus biofilms to disinfectants.

    PubMed

    Abdallah, Marwan; Chataigne, Gabrielle; Ferreira-Theret, Pauline; Benoliel, Corinne; Drider, Djamel; Dhulster, Pascal; Chihib, Nour-Eddine

    2014-03-01

    The goal of this study was to investigate the effect of the environmental conditions such as the temperature change, incubation time and surface type on the resistance of Staphylococcus aureus biofilms to disinfectants. The antibiofilm assays were performed against biofilms grown at 20 °C, 30 °C and 37 °C, on the stainless steel and polycarbonate, during 24 and 48 h. The involvement of the biofilm matrix and the bacterial membrane fluidity in the resistance of sessile cells were investigated. Our results show that the efficiency of disinfectants was dependent on the growth temperature, the surface type and the disinfectant product. The increase of growth temperature from 20 °C to 37 °C, with an incubation time of 24 h, increased the resistance of biofilms to cationic antimicrobials. This change of growth temperature did not affect the major content of the biofilm matrix, but it decreased the membrane fluidity of sessile cells through the increase of the anteiso-C19 relative amount. The increase of the biofilm resistance to disinfectants, with the rise of the incubation time, was dependent on both growth temperature and disinfectant product. The increase of the biofilm age also promoted increases in the matrix production and the membrane fluidity of sessile cells. The resistance of S. aureus biofilm seems to depend on the environment of the biofilm formation and involves both extracellular matrix and membrane fluidity of sessile cells. Our study represents the first report describing the impact of environmental conditions on the matrix production, sessile cells membrane fluidity and resistance of S. aureus biofilms to disinfectants.

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

    PubMed Central

    Grzywacz, Daria; Kamysz, Wojciech; Lourenço, Anália; Pereira, Maria Olívia

    2017-01-01

    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. PMID:28355248

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

    PubMed Central

    Gowrishankar, Shanmugaraj; Duncun Mosioma, Nyagwencha; Karutha Pandian, Shunmugiah

    2012-01-01

    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. PMID:22988476

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

    NASA Astrophysics Data System (ADS)

    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.

  16. Outer membrane protein OmpQ of Bordetella bronchiseptica is required for mature biofilm formation.

    PubMed

    Cattelan, Natalia; Villalba, María Inés; Parisi, Gustavo; Arnal, Laura; Serra, Diego Omar; Aguilar, Mario; Yantorno, Osvaldo

    2016-02-01

    Bordetella bronchiseptica, an aerobic Gram-negative bacterium, is capable of colonizing the respiratory tract of diverse animals and chronically persists inside the hosts by forming biofilm. Most known virulence factors in Bordetella species are regulated by the BvgAS two-component transduction system. The Bvg-activated proteins play a critical role during host infection. OmpQ is an outer membrane porin protein which is expressed under BvgAS control. Here, we studied the contribution of OmpQ to the biofilm formation process by B. bronchiseptica. We found that the lack of expression of OmpQ did not affect the growth kinetics and final biomass of B. bronchiseptica under planktonic growth conditions. The ΔompQ mutant strain displayed no differences in attachment level and in early steps of biofilm formation. However, deletion of the ompQ gene attenuated the ability of B. bronchiseptica to form a mature biofilm. Analysis of ompQ gene expression during the biofilm formation process by B. bronchiseptica showed a dynamic expression pattern, with an increase of biofilm culture at 48 h. Moreover, we demonstrated that the addition of serum anti-OmpQ had the potential to reduce the biofilm biomass formation in a dose-dependent manner. In conclusion, we showed for the first time, to the best of our knowledge, evidence of the contribution of OmpQ to a process of importance for B. bronchiseptica pathobiology. Our results indicate that OmpQ plays a role during the biofilm development process, particularly at later stages of development, and that this porin could be a potential target for strategies of biofilm formation inhibition.

  17. Antimicrobial efficacy of combined clarithromycin plus daptomycin against biofilms-formed methicillin-resistant Staphylococcus aureus on titanium medical devices.

    PubMed

    Fujimura, Shigeru; Sato, Tetsuro; Hayakawa, Sachiko; Kawamura, Masato; Furukawa, Emiko; Watanabe, Akira

    2015-10-01

    In vitro efficacy of combined eradication therapy with clarithromycin and daptomycin against biofilm-formed methicillin-resistant Staphylococcus aureus on the orthopedic titanium devices was evaluated. The bactericidal effect of this antibiotic was investigated by a re-culture test, the scanning electron microscopy, and fluorescence microscopy using a double-staining dyes. Clarithromycin decreased the amount to half in 24 h. Although MRSA biofilms were not eradicated with clarithromycin or daptomycin alone, clarithromycin combined with daptomycin was useful to sterilize titanium devices within 72 h. This in vitro study showed that combined treatment with clarithromycin plus daptomycin is useful to eradicate staphylococcal biofilms formed on orthopedic devices.

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

    PubMed

    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.

  19. Evaluation of Multidrug Resistant Staphylococcus aureus and their Association with Biofilm Production in a Tertiary Care Hospital, Tripura, Northeast India

    PubMed Central

    Bir, Raunak; Majumdar, Tapan

    2015-01-01

    Background High morbidity and mortality rates are associated with Methicillin-resistant Staphylococcus aureus (MRSA) because of development of multidrug resistance. Staphylococcus aureus (S. aureus) has the ability to colonize and form biofilms on biomaterials which is causing resistance towards antimicrobials and thus making them difficult to eradicate from the infected hosts. Materials and Methods Culture isolation, identification was done following standard protocol and antibiogram of the isolates were done. The detection of MRSA, Macrolide-Lincosamide-Streptogramin B resistance (MLSB), vancomycin resistance phenotypes were done by using cefoxitin disc diffusion test, D zone test and vancomycin E test. Biofilm was detected by Congo red agar method. Results A total of 100 (31.7%) S. aureus strains were isolated from 315 clinical specimens. The prevalence of MRSA was 47% (47/100) with 85.1% were homogeneous MRSA and 14.9% were heterogeneous. Out of 47 MRSA strains, 63.8% were Hospital acquired-MRSA (HA-MRSA) infections whereas rests 36.2% were caused by Community acquired-MRSA (CA-MRSA) strains. Maximum number of MRSA isolates belonged to group A biotype (34%). A 14.9% isolates were of nontypeable group. Out of 100 S. aureus isolates, the prevalence of Vancomycin resistant S. aureus (VRSA) was found to be 3%. The MLSB phenotypes showed that the rates of inducible MLSB (iMLSB), constitutive MLSB (cMLSB) and Macrolide-Streptogramin B (MSB) in case of MRSA to be 19.1%, 31.9% and 12.8%. Prevalence of low-level (MUPL) and high-level mupirocin resistance (MUPH) among MRSA was 19.1% and 6.4%. Biofilm production was found in 55% strains of S. aureus. Out of 47 MRSA strains 76.6%were producing biofilm in comparison to 38.8% in methicillin-sensitive S. aureus (MSSA). Higher degree of antibiotic resistance in biofilm producers was seen especially in case of ciprofloxacin, co-trimoxazole, rifampicin, kanamycin, erythromycin and clindamycin whereas gentamycin, tetracycline and

  20. The inactivation of Staphylococcus aureus biofilms using low-power argon plasma in a layer-by-layer approach.

    PubMed

    Traba, Christian; Liang, Jun F

    2015-01-01

    The direct application of low power argon plasma for the decontamination of pre-formed Staphylococcus aureus biofilms on various surfaces was examined. Distinct chemical/physical properties of reactive species found in argon plasmas generated at different wattages all demonstrated very potent but very different anti-biofilm mechanisms of action. An in-depth analysis of the results showed that: (1) the different reactive species produced in each plasma demonstrated specific antibacterial and/or anti-biofilm activity; and (2) the commonly associated etching effect could be manipulated and even controlled, depending on the experimental conditions. Under optimal experimental parameters, bacterial cells in S. aureus biofilms were killed (> 99.9%) by plasmas within 10 min of exposure and no bacteria nor biofilm regrowth from argon discharge gas treated biofilms was observed for 150 h. The decontamination ability of plasmas for the treatment of biofilm related contaminations on various materials was confirmed and an entirely novel layer-by-layer decontamination approach was designed and examined.

  1. Role of extra-cellular fatty acids in vancomycin induced biofilm formation by vancomycin resistant Staphylococcus aureus.

    PubMed

    Mirani, Zulfiqar Ali; Jamil, Nusrat

    2013-03-01

    In the present study a vancomycin resistant Staphylococcus aureus (S. aureus) (VRSA) (Labeled as CP2) was isolated from the blood of a post-operative cardiac patient is described. It harbors a plasmid which carry vanA gene and exhibited low-level vancomycin resistance (MIC 16μg/mL), was sensitive to teicoplanin. It has been observed that sub-lethal dose of vancomycin induced biofilm formation by CP2 on nylon and silicon indwelling. The results divulge new insights into associations between vancomycin induced biofilms and extra-cellular fatty acids. Gas chromatography coupled with mass spectrometry (GC-MS) revealed that biofilm matrix of CP2 contains a variety of saturated and un-saturated fatty acids, especially, diverse species of octadecanoic (C18:0) and octadecenoic acids (C18:1). A large difference in fatty acids composition was noticed in biofilms, isolated from hydrophobic and hydrophilic surfaces. CP2 produced thicker layer of biofilms on hydrophobic silicon and nylon surfaces which contains variety of saturated, un-saturated and cyclic fatty acids. Contrary to this on hydrophilic glass surfaces it produced thinner layer of biofilm which contains only straight chain saturated fatty acids. These fatty acid components seem to play a crucial role in cell-cell communication and in the establishment of biofilms, consequently, advantageous for pathogens to survive in hospital environment under enormous antibiotics pressure.

  2. Caspofungin at catheter lock concentrations eradicates mature biofilms of Candida lusitaniae and Candida guilliermondii.

    PubMed

    Simitsopoulou, Maria; Kyrpitzi, Daniela; Velegraki, Aristea; Walsh, Thomas J; Roilides, Emmanuel

    2014-08-01

    The antibiofilm activities of caspofungin, anidulafungin, micafungin, and liposomal amphotericin B were studied against Candida lusitaniae, Candida guilliermondii, and a Candida albicans control strain. While anidulafungin and micafungin (0.007 to 2,048 mg/liter) showed reduced activity against biofilms of both test species, caspofungin displayed concentration-dependent antibiofilm activity, reaching complete and persistent eradication at concentrations achievable during lock therapy (512 to 2,048 mg/liter, P < 0.05). Although liposomal amphotericin B strongly inhibited mature biofilms, it possessed lower antibiofilm activity than caspofungin (P < 0.05).

  3. inhibitory effects of citral, cinnamaldehyde, and tea polyphenols on mixed biofilm formation by foodborne Staphylococcus aureus and Salmonella enteritidis.

    PubMed

    Zhang, Hongmei; Zhou, Wenyuan; Zhang, Wenyan; Yang, Anlin; Liu, Yanlan; Jiang, Yan; Huang, Shaosong; Su, Jianyu

    2014-06-01

    Biofilms are significant hazards in the food industry. In this study, we investigated the effects of food additive such as citral, cinnamaldehyde, and tea polyphenols on mixed biofilm formation by foodborne Staphylococcus aureus and Salmonella serotype Enteritidis. The adhesion rates of mixed strains in sub-MIC of additives were determined by a microtiter plate assay and bacterial communication signal autoinducer 2 (AI-2) production via a bioluminescence reporter Vibrio harveyi BB170. The structure of mixed biofilm was analyzed using scanning electron microscopy. The effect of the disinfectants hydrogen peroxide, sodium hypochlorite, and peracetic acid was tested on the mixed biofilm. Our results demonstrated that citral, cinnamaldehyde, and tea polyphenols were able to significantly inhibit mixed biofilm formation, while citral could reduce the synthesis of AI-2. Conversely, we observed a significant increase in AI-2 mediated by cinnamaldehyde. Tea polyphenols at lower concentrations induced AI-2 synthesis; however, AI-2 synthesis was significantly inhibited at higher concentrations (300 m g/ml). Food additives inhibited the adhesion of mixed bacteria on stainless steel chips and increased the sensitivity of the mixed biofilm to disinfectants. In conclusion, citral, cinnamaldehyde, and tea polyphenols had strong inhibitory effects on mixed biofilm formation and also enhanced the effect of disinfectant on mixed biofilm formation. This study provides a scientific basis for the application of natural food additives to control biofilm formation of foodborne bacteria.

  4. Injections through skin colonized with Staphylococcus aureus biofilm introduce contamination despite standard antimicrobial preparation procedures

    PubMed Central

    Wang, Yi; Leng, Valery; Patel, Viraj; Phillips, K. Scott

    2017-01-01

    While surgical site preparation has been extensively studied, there is little information about resistance of skin microbiota in the biofilm form to antimicrobial decontamination, and there are no quantitative models to study how biofilm might be transferred into sterile tissue/implant materials during injections for joint spine and tendon, aspiration biopsies and dermal fillers (DF). In this work, we develop two in vitro models to simulate the process of skin preparation and DF injection using pig skin and SimSkin (silicone) materials, respectively. Using the pig skin model, we tested three of the most common skin preparation wipes (alcohol, chlorhexidine and povidone iodine) and found that during wiping they reduced the biofilm bacterial burden of S. aureus (CFU cm−2) by three logs with no statistically significant differences between wipes. Using the SimSkin model, we found that transfer of viable bacteria increased with needle diameter for 30G, 25G and 18G needles. Transfer incidence decreased as injection depth was increased from 1 mm to 3 mm. Serial puncture and linear threading injection styles had similar transfer incidence, whereas fanning significantly increased transfer incidence. The results show that contamination of DF during injection is a risk that can be reduced by modifying skin prep and injection practices. PMID:28332593

  5. Air pollution alters Staphylococcus aureus and Streptococcus pneumoniae biofilms, antibiotic tolerance and colonisation.

    PubMed

    Hussey, Shane J K; Purves, Joanne; Allcock, Natalie; Fernandes, Vitor E; Monks, Paul S; Ketley, Julian M; Andrew, Peter W; Morrissey, Julie A

    2017-02-14

    Air pollution is the world's largest single environmental health risk (WHO). Particulate matter such as black carbon is one of the main components of air pollution. The effects of particulate matter on human health are well established however the effects on bacteria, organisms central to ecosystems in humans and in the natural environment, are poorly understood. We report here for the first time that black carbon drastically changes the development of bacterial biofilms, key aspects of bacterial colonisation and survival. Our data show that exposure to black carbon induces structural, compositional and functional changes in the biofilms of both S. pneumoniae and S. aureus. Importantly, the tolerance of the biofilms to multiple antibiotics and proteolytic degradation is significantly affected. Additionally, our results show that black carbon impacts bacterial colonisation in vivo. In a mouse nasopharyngeal colonisation model, black carbon caused S. pneumoniae to spread from the nasopharynx to the lungs, which is essential for subsequent infection. Therefore our study highlights that air pollution has a significant effect on bacteria that has been largely overlooked. Consequently these findings have important implications concerning the impact of air pollution on human health and bacterial ecosystems worldwide.

  6. Injections through skin colonized with Staphylococcus aureus biofilm introduce contamination despite standard antimicrobial preparation procedures.

    PubMed

    Wang, Yi; Leng, Valery; Patel, Viraj; Phillips, K Scott

    2017-03-23

    While surgical site preparation has been extensively studied, there is little information about resistance of skin microbiota in the biofilm form to antimicrobial decontamination, and there are no quantitative models to study how biofilm might be transferred into sterile tissue/implant materials during injections for joint spine and tendon, aspiration biopsies and dermal fillers (DF). In this work, we develop two in vitro models to simulate the process of skin preparation and DF injection using pig skin and SimSkin (silicone) materials, respectively. Using the pig skin model, we tested three of the most common skin preparation wipes (alcohol, chlorhexidine and povidone iodine) and found that during wiping they reduced the biofilm bacterial burden of S. aureus (CFU cm(-2)) by three logs with no statistically significant differences between wipes. Using the SimSkin model, we found that transfer of viable bacteria increased with needle diameter for 30G, 25G and 18G needles. Transfer incidence decreased as injection depth was increased from 1 mm to 3 mm. Serial puncture and linear threading injection styles had similar transfer incidence, whereas fanning significantly increased transfer incidence. The results show that contamination of DF during injection is a risk that can be reduced by modifying skin prep and injection practices.

  7. Comparison of virulence factors and biofilm formation among Staphylococcus aureus strains isolated from human and bovine infections.

    PubMed

    Khoramian, Babak; Jabalameli, Fereshteh; Niasari-Naslaji, Amir; Taherikalani, Morovat; Emaneini, Mohammad

    2015-11-01

    The aim of this study was to find different prevalence of genes involved in the biofilm formation process and to assess the phenotypic and genotypic markers of biofilm formation among Staphylococcus aureus strains isolated from human and bovine infections. In this study, 215 S. aureus strains were collected from human and dairy cow's infections. The biofilm forming capacity of the strains was evaluated using a colorimetric microtiter plate assay. The genes encoding microbial surface components, recognizing adhesive matrix molecules (MSCRAMMs) (ebpS, eno, fib, fnbA, fnbB, cna and bap), and the intracellular adhesion (ica) genes (icaA, and icaD) were targeted by polymerase chain reaction (PCR)-based method. Approximately 70% of the isolates produced biofilm. Among these, 59.3% were producers of weakly adherent biofilms while 34.8% and 5.8% produced moderate and strong biofilms, respectively. The most prevalent gene was icaD found in 88.4% of the isolates, followed by icaA, fib and eno found in 87.9%, 75.8% and 75.3% of the isolates, respectively. The bap gene was not detected in any of the isolates. The prevalence of ebpS and fnbA genes among bovine isolates were significantly higher than those in human isolates, whilst the prevalence of cna gene was significantly higher in the human isolates. In this study, a high prevalence of biofilm production was found among S. aureus strains isolated from human and bovine infections. Most biofilm producing isolates were positive for MSCRAMM, icaA, and icaD genes.

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

    NASA Astrophysics Data System (ADS)

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

    2017-03-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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%. One hypothesis to explain the high prevalence of MRSA in swine herds is the ability of these organisms to exist as biofilms. To invest...

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

    PubMed

    de Souza, Evandro Leite; Meira, Quênia Gramile Silva; de Medeiros Barbosa, Isabella; Athayde, Ana Júlia Alves Aguiar; da Conceição, Maria Lúcia; de Siqueira Júnior, José Pinto

    2014-01-01

    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 × 2 cm) when cultivated in a meat-based broth at 28 and 7 °C. 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.

  11. Composition of Microbial Oral Biofilms during Maturation in Young Healthy Adults

    PubMed Central

    Langfeldt, Daniela; Neulinger, Sven C.; Heuer, Wieland; Staufenbiel, Ingmar; Künzel, Sven; Baines, John F.; Eberhard, Jörg; Schmitz, Ruth A.

    2014-01-01

    In the present study we aimed to analyze the bacterial community structure of oral biofilms at different maturation stages in young healthy adults. Oral biofilms established on membrane filters were collected from 32 human subjects after 5 different maturation intervals (1, 3, 5, 9 and 14 days) and the respective phylogenetic diversity was analyzed by 16S rDNA amplicon sequencing. Our analyses revealed highly diverse entire colonization profiles, spread into 8 phyla/candidate divisions and in 15 different bacterial classes. A large inter-individual difference in the subjects’ microbiota was observed, comprising 35% of the total variance, but lacking conspicuous general temporal trends in both alpha and beta diversity. We further obtained strong evidence that subjects can be categorized into three clusters based on three differently occurring and mutually exclusive species clusters. PMID:24503584

  12. Staphylococcus aureus Develops an Alternative, ica-Independent Biofilm in the Absence of the arlRS Two-Component System†

    PubMed Central

    Toledo-Arana, Alejandro; Merino, Nekane; Vergara-Irigaray, Marta; Débarbouillé, Michel; Penadés, José R.; Lasa, Iñigo

    2005-01-01

    The biofilm formation capacity of Staphylococcus aureus clinical isolates is considered an important virulence factor for the establishment of chronic infections. Environmental conditions affect the biofilm formation capacity of S. aureus, indicating the existence of positive and negative regulators of the process. The majority of the screening procedures for identifying genes involved in biofilm development have been focused on genes whose presence is essential for the process. In this report, we have used random transposon mutagenesis and systematic disruption of all S. aureus two-component systems to identify negative regulators of S. aureus biofilm development in a chemically defined medium (Hussain-Hastings-White modified medium [HHWm]). The results of both approaches coincided in that they identified arlRS as a repressor of biofilm development under both steady-state and flow conditions. The arlRS mutant exhibited an increased initial attachment as well as increased accumulation of poly-N-acetylglucosamine (PNAG). However, the biofilm formation of the arlRS mutant was not affected when the icaADBC operon was deleted, indicating that PNAG is not an essential compound of the biofilm matrix produced in HHWm. Disruption of the major autolysin gene, atl, did not produce any effect on the biofilm phenotype of an arlRS mutant. Epistatic experiments with global regulators involved in staphylococcal-biofilm formation indicated that sarA deletion abolished, whereas agr deletion reinforced, the biofilm development promoted by the arlRS mutation. PMID:16030226

  13. Staphylococcus aureus and Escherichia coli dual-species biofilms on nanohydroxyapatite loaded with CHX or ZnO nanoparticles.

    PubMed

    Barros, Joana; Grenho, Liliana; Fontenente, Sílvia; Manuel, Cândida M; Nunes, Olga C; Melo, Luís F; Monteiro, Fernando J; Ferraz, Maria P

    2017-02-01

    Implant-associated infections are caused by surface-adhering microorganisms persisting as biofilms, resistant to host defense and antimicrobial agents. Given the limited efficacy of traditional antibiotics, novel strategies may rely on the prevention of such infections through the design of new biomaterials. In this work, two antimicrobial agents applied to nanohydroxyapatite materials-namely, chlorhexidine digluconate (CHX) and zinc oxide (ZnO) nanoparticles-were compared concerning their ability to avoid single- or dual-species biofilms of Staphylococcus aureus and Escherichia coli. The resulting biofilms were quantified by the enumeration of colony-forming units and examined by confocal microscopy using both Live/Dead staining and bacterial-specific fluorescent in situ hybridization. The sessile population arrangement was also observed by scanning electron microscopy. Both biomaterials showed to be effective in impairing bacterial adhesion and proliferation for either single- or dual-species biofilms. Furthermore, a competitive interaction was observed for dual-species biofilms wherein E. coli exhibited higher proliferative capacity than S. aureus, an inverse behavior from the one observed in single-species biofilms. Therefore, either nanoHA-CHX or nanoHA-ZnO surfaces appear as promising alternatives to antibiotics for the prevention of devices-related infections avoiding the critical risk of antibiotic-resistant strains emergence. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 491-497, 2017.

  14. Nfu facilitates the maturation of iron-sulfur proteins and participates in virulence in Staphylococcus aureus

    PubMed Central

    Mashruwala, Ameya A.; Pang, Yun Y.; Rosario-Cruz, Zuelay; Chahal, Harsimranjit K.; Benson, Meredith A.; Anzaldi-Mike, Laura L.; Skaar, Eric P.; Torres, Victor J.; Nauseef, William M.; Boyd, Jeffrey M.

    2015-01-01

    Summary The acquisition and metabolism of iron (Fe) by the human pathogen Staphylococcus aureus is critical for disease progression. S. aureus requires Fe to synthesize inorganic cofactors called iron-sulfur (Fe-S) clusters, which are required for functional Fe-S proteins. In this study we investigated the mechanisms utilized by S. aureus to metabolize Fe-S clusters. We identified that S. aureus utilizes the Suf biosynthetic system to synthesize Fe-S clusters and we provide genetic evidence suggesting that the sufU and sufB gene products are essential. Additional biochemical and genetic analyses identified Nfu as a Fe-S cluster carrier, which aids in the maturation of Fe-S proteins. We find that deletion of the nfu gene negatively impacts staphylococcal physiology and pathogenicity. A nfu mutant accumulates both increased intracellular non-incorporated Fe and endogenous reactive oxygen species (ROS) resulting in DNA damage. In addition, a strain lacking Nfu is sensitive to exogenously supplied ROS and reactive nitrogen species. Congruous with ex vivo findings, a nfu mutant strain is more susceptible to oxidative killing by human polymorphonuclear leukocytes and displays decreased tissue colonization in a murine model of infection. We conclude that Nfu is necessary for staphylococcal pathogenesis and establish Fe-S cluster metabolism as an attractive antimicrobial target. PMID:25388433

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

    PubMed

    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.

  16. Detection of Staphylococcus aureus adhesion and biofilm-producing genes and their expression during internalization in bovine mammary epithelial cells.

    PubMed

    Pereyra, Elizabet A L; Picech, Florencia; Renna, María S; Baravalle, Celina; Andreotti, Carolina S; Russi, Romina; Calvinho, Luis F; Diez, Cristina; Dallard, Bibiana E

    2016-02-01

    Staphylococcus aureus is one of the most prevalent pathogens isolated from bovine mastitis, causing chronic intramammary infections (IMI) that limit profitable dairying. The course of infection is often associated with factors both related to the host and the bacterium. Aims of this study were to select S. aureus isolates from bovine IMI with different genotypic profiles harboring genes involved in adherence and biofilm production, to determine the behavior of these strains in contact with bovine mammary epithelial cells (MAC-T) and the expression of those genes during bacterial-cell early interactions. The genetic diversity of 20 S. aureus strains that were isolated from milk samples taken from cows with persistent-P and non-persistent-NP IMI was high, discriminated into 13 fingerprint groups. The occurrence of genes coding for S. aureus surface proteins (clfA, clfB, fnbA, fnbB, fib, cna) and biofilm formation (icaA, icaD, icaC, bap) and in vitro biofilm-forming ability was not related to strain clinical origin (NP or P). Internalization of S. aureus into MAC-T cells was strain-dependent and internalized bacteria overexpressed adherence and biofilm-forming genes compared with those that remained in the supernatant of co-cultures; particularly those genes encoding FnBPs and IcaD. Strains yielding highest invasion percentages were those able to overexpress fnBP, irrespectively of the presence of other evaluated genes. Strains from NP IMI showed a greater multiplication capacity in vitro compared with strains from P IMI. These results provide new insights about S. aureus differential gene expression of adhesion-internalization factors during early interaction with mammary epithelial cells.

  17. The Xanthomonas axonopodis pv. citri flagellum is required for mature biofilm and canker development.

    PubMed

    Malamud, Florencia; Torres, Pablo S; Roeschlin, Roxana; Rigano, Luciano A; Enrique, Ramón; Bonomi, Hernán R; Castagnaro, Atilio P; Marano, María Rosa; Vojnov, Adrián A

    2011-03-01

    Xanthomonas axonopodis pv. citri (Xac) is the causative agent of citrus canker. This bacterium develops a characteristic biofilm on both biotic and abiotic surfaces. To evaluate the participation of the single flagellum of Xac in biofilm formation, mutants in the fliC (flagellin) and the flgE (hook) genes were generated. Swimming motility, assessed on 0.25 % agar plates, was markedly reduced in fliC and flgE mutants. However, the fliC and flgE mutants exhibited a flagellar-independent surface translocation on 0.5 % agar plates. Mutation of either the rpfF or the rpfC gene, which both encode proteins involved in cell-cell signalling mediated by diffusible signal factor (DSF), led to a reduction in both flagellar-dependent and flagellar-independent surface translocation, indicating a regulatory role for DSF in both types of motility. Confocal laser scanning microscopy of biofilms produced in static culture demonstrated that the flagellum is also involved in the formation of mushroom-shaped structures and water channels, and in the dispersion of biofilms. The presence of the flagellum was required for mature biofilm development on lemon leaf surfaces. The absence of flagellin produced a slight reduction in Xac pathogenicity and this reduction was more severe when the complete flagellum structure was absent.

  18. Influence of tigecycline on expression of virulence factors in biofilm-associated cells of methicillin-resistant Staphylococcus aureus.

    PubMed

    Smith, Karen; Gould, Katherine A; Ramage, Gordon; Gemmell, Curtis G; Hinds, Jason; Lang, Sue

    2010-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) infections are complicated by the ability of the organism to grow in surface-adhered biofilms on a multitude of abiotic and biological surfaces. These multicellular communities are notoriously difficult to eradicate with antimicrobial therapy. Cells within the biofilm may be exposed to a sublethal concentration of the antimicrobial due to the metabolic and phenotypic diversity of the biofilm-associated cells or the protection offered by the biofilm structure. In the present study, the influence of a sublethal concentration of tigecycline on biofilms formed by an epidemic MRSA-16 isolate was investigated by transcriptome analysis. In the presence of the drug, 309 genes were upregulated and 213 genes were downregulated by more than twofold in comparison to the levels of gene regulation detected for the controls not grown in the presence of the drug. Microarray data were validated by real-time reverse transcription-PCR and phenotypic assays. Tigecycline altered the expression of a number of genes encoding proteins considered to be crucial for the virulence of S. aureus. These included the reduced expression of icaC, which is involved in polysaccharide intercellular adhesin production and biofilm development; the upregulation of fnbA, clfB, and cna, which encode adhesins which attach to human proteins; and the downregulation of the cap genes, which mediate the synthesis of the capsule polysaccharide. The expression of tst, which encodes toxic shock syndrome toxin 1 (TSST-1), was also significantly reduced; and an assay performed to quantify TSST-1 showed that the level of toxin production by cells treated with tigecycline decreased by 10-fold (P < 0.001) compared to the level of production by untreated control cells. This study suggests that tigecycline may reduce the expression of important virulence factors in S. aureus and supports further investigation to determine whether it could be a useful adjunct to therapy

  19. Comparison of methods for the detection of biofilm formation by Staphylococcus aureus isolated from bovine subclinical mastitis

    PubMed Central

    de Castro Melo, Poliana; Ferreira, Luciano Menezes; Filho, Antônio Nader; Zafalon, Luiz Francisco; Vicente, Hinig Isa Godoy; de Souza, Viviane

    2013-01-01

    Biofilm formation is considered to be a selective advantage for Staphylococcus aureus mastitis isolates by facilitating bacterial persistence in the udder. It requires attachment to mammary epithelium, proliferation and accumulation of cells in multilayers. The objective of this study was to determine the sensitivity and specificity of three techniques for the detection of S. aureus biofilm-positive strains. Two phenotypic tests, including growth on microtitre plates and Congo red agar, were compared with a PCR technique using 94 S. aureus strains obtained from cows with subclinical mastitis from two farms in the state of São Paulo. These strains were characterised by in vitro slime production on Congo red agar, biofilm formation on microtitre plates and the presence of the icaA and icaD genes. The results revealed that 85% of the isolates tested produced slime on the Congo red agar, 98.9% of the isolates produced biofilms in vitro by adhering to sterile 96-well “U” bottom polystyrene tissue culture plates, and 95.7% of the isolates carried the icaA and icaD genes. The results of the phenotypic tests for biofilm formation were compared with those of the molecular analysis, and the sensitivity and specificity of the Congo red agar test were 88.9% and 100%, respectively, while those of the microtitre plate test were 100% and 25%, respectively. When the phenotypic methods for the detection of biofilm producers, namely growth on microtitre plates and Congo red agar, were compared, the sensitivity and specificity were 86% and 100%, respectively. Therefore, growth on Congo red agar and the microtitre plate test are methods that could be used to determine whether an isolate has the potential for biofilm production. PMID:24159293

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

    NASA Technical Reports Server (NTRS)

    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.

  1. 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

    PubMed Central

    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

    Background 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. Methods 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. Results 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. Conclusion 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

  2. A novel chimeric lysin with robust antibacterial activity against planktonic and biofilm methicillin-resistant Staphylococcus aureus

    PubMed Central

    Yang, Hang; Zhang, Huaidong; Wang, Jing; Yu, Junping; Wei, Hongping

    2017-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most threatening pathogens due to its multi-drug resistance (MDR) and strong biofilm-forming capacity. Here, we described the screening of a novel chimeolysin (ClyF) that was active against planktonic and biofilm MRSA. Biochemical tests showed that ClyF was active against all S. aureus clinical isolates tested under planktonic and biofilm conditions. Structure analysis revealed that ClyF has an enhanced thermostability and pH tolerance than its parental lysin Pc by forming a hydrophobic cleft in the catalytic domain and an Ig-like structure in the cell-wall binding domain. A single intraperitoneally or topically administration of ClyF showed good MRSA removing efficacy in mouse models of bacteremia and burn wound infection, respectively. Our data collectively demonstrated that ClyF has good bactericidal activity against planktonic and biofilm MRSA both in vitro and in vivo, and therefore represents a useful antibacterial to combat MDR S. aureus. PMID:28067286

  3. Vaccine development in Staphylococcus aureus: taking the biofilm phenotype into consideration

    PubMed Central

    Harro, Janette M; Peters, Brian M; O'May, Graeme A; Archer, Nathan; Kerns, Patrick; Prabhakara, Ranjani; Shirtliff, Mark E

    2010-01-01

    Vaccine development against pathogenic bacteria is an imperative initiative as bacteria are gaining resistance to current antimicrobial therapies and few novel antibiotics are being developed. Candidate antigens for vaccine development can be identified by a multitude of high-throughput technologies that were accelerated by access to complete genomes. While considerable success has been achieved in vaccine development against bacterial pathogens, many species with multiple virulence factors and modes of infection have provided reasonable challenges in identifying protective antigens. In particular, vaccine candidates should be evaluated in the context of the complex disease properties, whether planktonic (e.g. sepsis and pneumonia) and/or biofilm associated (e.g. indwelling medical device infections). Because of the phenotypic differences between these modes of growth, those vaccine candidates chosen only for their efficacy in one disease state may fail against other infections. This review will summarize the history and types of bacterial vaccines and adjuvants as well as present an overview of modern antigen discovery and complications brought about by polymicrobial infections. Finally, we will also use one of the better studied microbial species that uses differential, multifactorial protein profiles to mediate an array of diseases, Staphylococcus aureus, to outline some of the more recently identified problematic issues in vaccine development in this biofilm-forming species. PMID:20602638

  4. Reduced Staphylococcus aureus proliferation and biofilm formation on zinc oxide nanoparticle PVC composite surfaces.

    PubMed

    Seil, Justin T; Webster, Thomas J

    2011-06-01

    Conventional particulate zinc oxide (ZnO) is a known antibacterial agent. Studies have shown that reducing the size of ZnO particles to nanoscale dimensions further enhances their antibacterial properties. Polymers, like all biomaterials, run the risk of harboring bacteria which may produce an antibiotic-resistant biofilm. The addition of ZnO nanoparticles to form a polymer composite material may thus reduce undesirable bacteria activity. The purpose of the present in vitro study was to investigate the antibacterial properties of ZnO nanoparticles when incorporated into a traditional polymeric biomaterial. For this purpose, Staphylococcus aureus were seeded at a known cell density onto coverslips coated with a film of polyvinyl chloride (PVC) with varying concentrations of ZnO nanoparticles. Samples were cultured for 24 or 72 h. Methods of analysis, including optical density readings and crystal violet staining, indicated a reduced presence of a biofilm on ZnO nanoparticle polymer composites compared to pure polymer controls. Live/dead bacteria assays provided images to confirm the reduced presence of active bacteria on samples with zinc oxide nanoparticles. Conditioning of the cell culture medium by the composites was also investigated by measuring concentrations of elemental zinc (Zn(2+)) and bacteria growth in the presence of conditioned medium. This study demonstrated that the development of ZnO polymer composites may improve biomaterial effectiveness for numerous applications, such as endotracheal tubes, catheter and implanted biomaterials, which are prone to bacterial infection.

  5. Zinc-dependent mechanical properties of Staphylococcus aureus biofilm-forming surface protein SasG.

    PubMed

    Formosa-Dague, Cécile; Speziale, Pietro; Foster, Timothy J; Geoghegan, Joan A; Dufrêne, Yves F

    2016-01-12

    Staphylococcus aureus surface protein SasG promotes cell-cell adhesion during the accumulation phase of biofilm formation, but the molecular basis of this interaction remains poorly understood. Here, we unravel the mechanical properties of SasG on the surface of living bacteria, that is, in its native cellular environment. Nanoscale multiparametric imaging of living bacteria reveals that Zn(2+) strongly increases cell wall rigidity and activates the adhesive function of SasG. Single-cell force measurements show that SasG mediates cell-cell adhesion via specific Zn(2+)-dependent homophilic bonds between β-sheet-rich G5-E domains on neighboring cells. The force required to unfold individual domains is remarkably strong, up to ∼500 pN, thus explaining how SasG can withstand physiological shear forces. We also observe that SasG forms homophilic bonds with the structurally related accumulation-associated protein of Staphylococcus epidermidis, suggesting the possibility of multispecies biofilms during host colonization and infection. Collectively, our findings support a model in which zinc plays a dual role in activating cell-cell adhesion: adsorption of zinc ions to the bacterial cell surface increases cell wall cohesion and favors the projection of elongated SasG proteins away from the cell surface, thereby enabling zinc-dependent homophilic bonds between opposing cells. This work demonstrates an unexpected relationship between mechanics and adhesion in a staphylococcal surface protein, which may represent a general mechanism among bacterial pathogens for activating cell association.

  6. Copper stress induces a global stress response in Staphylococcus aureus and represses sae and agr expression and biofilm formation.

    PubMed

    Baker, Jonathan; Sitthisak, Sutthirat; Sengupta, Mrittika; Johnson, Miranda; Jayaswal, R K; Morrissey, Julie A

    2010-01-01

    Copper is an important cofactor for many enzymes; however, high levels of copper are toxic. Therefore, bacteria must ensure there is sufficient copper for use as a cofactor but, more importantly, must limit free intracellular levels to prevent toxicity. In this study, we have used DNA microarray to identify Staphylococcus aureus copper-responsive genes. Transcriptional profiling of S. aureus SH1000 grown in excess copper identified a number of genes which fall into four groups, suggesting that S. aureus has four main mechanisms for adapting to high levels of environmental copper, as follows: (i) induction of direct copper homeostasis mechanisms; (ii) increased oxidative stress resistance; (iii) expression of the misfolded protein response; and (iv) repression of a number of transporters and global regulators such as Agr and Sae. Our experimental data confirm that resistance to oxidative stress and particularly to H2O2 scavenging is an important S. aureus copper resistance mechanism. Our previous studies have demonstrated that Eap and Emp proteins, which are positively regulated by Agr and Sae, are required for biofilm formation under low-iron growth conditions. Our transcriptional analysis has confirmed that sae, agr, and eap are repressed under high-copper conditions and that biofilm formation is indeed repressed under high-copper conditions. Therefore, our results may provide an explanation for how copper films can prevent biofilm formation on catheters.

  7. Dynamics of Biofilm Formation and the Interaction between Candida albicans and Methicillin-Susceptible (MSSA) and -Resistant Staphylococcus aureus (MRSA)

    PubMed Central

    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. PMID:25875834

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

    PubMed Central

    Oyama, Takuto; Miyazaki, Motoyasu; Yoshimura, Michinobu; Takata, Tohru; Ohjimi, Hiroyuki; Jimi, Shiro

    2016-01-01

    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. PMID:27376326

  9. Efficient Eradication of Mature Pseudomonas aeruginosa Biofilm via Controlled Delivery of Nitric Oxide Combined with Antimicrobial Peptide and Antibiotics.

    PubMed

    Ren, Hang; Wu, Jianfeng; Colletta, Alessandro; Meyerhoff, Mark E; Xi, Chuanwu

    2016-01-01

    Fast eradication of mature biofilms is the 'holy grail' in the clinical management of device-related infections. Endogenous nitric oxide (NO) produced by macrophages plays an important role in host defense against intracellular pathogens, and NO is a promising agent in preventing biofilms formation in vitro. However, the rate of delivery of NO by various NO donors (e.g., diazeniumdiolates, S-nitrosothiols, etc.) is difficult to control, which hinders fundamental studies aimed at understanding the role of NO in biofilm control. In this study, by using a novel precisely controlled electrochemical NO releasing catheter device, we examine the effect of physiological levels of NO on eradicating mature Pseudomonas aeruginosa biofilm (7 days), as well as the potential application of the combination of NO with antimicrobial agents. It is shown that physiological levels of NO exhibit mixed effects of killing bacteria and dispersing ambient biofilm. The overall biofilm-eradicating effect of NO is quite efficient in a dose-dependent manner over a 3 h period of NO treatment. Moreover, NO also greatly enhances the efficacy of antimicrobial agents, including human beta-defensin 2 (BD-2) and several antibiotics, in eradicating biofilm and its detached cells, which otherwise exhibited high recalcitrance to these antimicrobial agents. The electrochemical NO release technology offers a powerful tool in evaluating the role of NO in biofilm control as well as a promising approach when combined with antimicrobial agents to treat biofilm-associated infections in hospital settings, especially infections resulting from intravascular catheters.

  10. Effects of extracts from Italian medicinal plants on planktonic growth, biofilm formation and adherence of methicillin-resistant Staphylococcus aureus

    PubMed Central

    Quave, Cassandra L.; Plano, Lisa R.W.; Pantuso, Traci; Bennett, Bradley C.

    2008-01-01

    One-third of botanical remedies from southern Italy are used to treat skin and soft tissue infection (SSTI). Staphylococcus aureus, a common cause of SSTI, has generated increasing concern due to drug resistance. Many plants possess antimicrobial agents and provide effective remedies for SSTI. Our aim was to investigate plants from different ethnobotanical usage groups for inhibition of growth and biofilms in methicillin-resistant S. aureus (MRSA). Three groups were assessed: plant remedies for SSTI, plant remedies not involving the skin, and plants with no ethnomedical application. We screened 168 extracts, representing 104 botanical species, for activity against MRSA (ATCC 33593). We employed broth dilution methods to determine the MIC after 18 hours growth using an optical density (OD600nm) reading. Anti-biofilm effects were assessed by growing biofilms for 40 hours, then fixing and staining with crystal violet. After washing, 10% Tween 80 was added and OD570nm readings were taken. Extracts from 10 plants exhibited an IC50 ≤32 μg/ml for biofilm inhibition: Lonicera alpigena, Castanea sativa, Juglans regia, Ballota nigra, Rosmarinus officinalis, Leopoldia comosa, Malva sylvestris, Cyclamen hederifolium, Rosa canina, and Rubus ulmifolius. Limited bacteriostatic activity was evident. The anti-biofilm activity of medicinal plants was significantly greater than plants without any ethnomedical applications. PMID:18556162

  11. D-amino acids enhance the activity of antimicrobials against biofilms of clinical wound isolates of Staphylococcus aureus and Pseudomonas aeruginosa.

    PubMed

    Sanchez, Carlos J; Akers, Kevin S; Romano, Desiree R; Woodbury, Ronald L; Hardy, Sharanda K; Murray, Clinton K; Wenke, Joseph C

    2014-08-01

    Within wounds, microorganisms predominantly exist as biofilms. Biofilms are associated with chronic infections and represent a tremendous clinical challenge. As antibiotics are often ineffective against biofilms, use of dispersal agents as adjunctive, topical therapies for the treatment of wound infections involving biofilms has gained interest. We evaluated in vitro the dispersive activity of D-amino acids (D-AAs) on biofilms from clinical wound isolates of Staphylococcus aureus and Pseudomonas aeruginosa; moreover, we determined whether combinations of D-AAs and antibiotics (clindamycin, cefazolin, oxacillin, rifampin, and vancomycin for S. aureus and amikacin, colistin, ciprofloxacin, imipenem, and ceftazidime for P. aeruginosa) enhance activity against biofilms. D-Met, D-Phe, and D-Trp at concentrations of ≥ 5 mM effectively dispersed preformed biofilms of S. aureus and P. aeruginosa clinical isolates, an effect that was enhanced when they were combined as an equimolar mixture (D-Met/D-Phe/D-Trp). When combined with D-AAs, the activity of rifampin was significantly enhanced against biofilms of clinical isolates of S. aureus, as indicated by a reduction in the minimum biofilm inhibitory concentration (MBIC) (from 32 to 8 μg/ml) and a >2-log reduction of viable biofilm bacteria compared to treatment with antibiotic alone. The addition of D-AAs was also observed to enhance the activity of colistin and ciprofloxacin against biofilms of P. aeruginosa, reducing the observed MBIC and the number of viable bacteria by >2 logs and 1 log at 64 and 32 μg/ml in contrast to antibiotics alone. These findings indicate that the biofilm dispersal activity of D-AAs may represent an effective strategy, in combination with antimicrobials, to release bacteria from biofilms, subsequently enhancing antimicrobial activity.

  12. d-Amino Acids Enhance the Activity of Antimicrobials against Biofilms of Clinical Wound Isolates of Staphylococcus aureus and Pseudomonas aeruginosa

    PubMed Central

    Akers, Kevin S.; Romano, Desiree R.; Woodbury, Ronald L.; Hardy, Sharanda K.; Murray, Clinton K.; Wenke, Joseph C.

    2014-01-01

    Within wounds, microorganisms predominantly exist as biofilms. Biofilms are associated with chronic infections and represent a tremendous clinical challenge. As antibiotics are often ineffective against biofilms, use of dispersal agents as adjunctive, topical therapies for the treatment of wound infections involving biofilms has gained interest. We evaluated in vitro the dispersive activity of d-amino acids (d-AAs) on biofilms from clinical wound isolates of Staphylococcus aureus and Pseudomonas aeruginosa; moreover, we determined whether combinations of d-AAs and antibiotics (clindamycin, cefazolin, oxacillin, rifampin, and vancomycin for S. aureus and amikacin, colistin, ciprofloxacin, imipenem, and ceftazidime for P. aeruginosa) enhance activity against biofilms. d-Met, d-Phe, and d-Trp at concentrations of ≥5 mM effectively dispersed preformed biofilms of S. aureus and P. aeruginosa clinical isolates, an effect that was enhanced when they were combined as an equimolar mixture (d-Met/d-Phe/d-Trp). When combined with d-AAs, the activity of rifampin was significantly enhanced against biofilms of clinical isolates of S. aureus, as indicated by a reduction in the minimum biofilm inhibitory concentration (MBIC) (from 32 to 8 μg/ml) and a >2-log reduction of viable biofilm bacteria compared to treatment with antibiotic alone. The addition of d-AAs was also observed to enhance the activity of colistin and ciprofloxacin against biofilms of P. aeruginosa, reducing the observed MBIC and the number of viable bacteria by >2 logs and 1 log at 64 and 32 μg/ml in contrast to antibiotics alone. These findings indicate that the biofilm dispersal activity of d-AAs may represent an effective strategy, in combination with antimicrobials, to release bacteria from biofilms, subsequently enhancing antimicrobial activity. PMID:24841260

  13. Investigation of Biofilm Formation and its Association with the Molecular and Clinical Characteristics of Methicillin-resistant Staphylococcus aureus

    PubMed Central

    Cha, Jeong-Ok; Yoo, Jae Il; Yoo, Jung Sik; Chung, Hae-Sun; Park, Sun-Hee; Kim, Hwa Su; Lee, Yeong Seon; Chung, Gyung Tae

    2013-01-01

    Objectives To investigate the biofilm-forming related factors against MRSA bloodstream isolates and evaluates their clinical features and treatment outcomes by biofilm production. Methods We collected 126 consecutive methicillin-resistant Staphylococcus aureus (MRSA) causing blood stream infections (BSIs) at 10 tertiary hospitals from 2007 to 2009. We investigated biofilm-forming ability using a microtiter plate assay, and molecular characteristics including multilocus sequence typing, staphylococcal cassette chromosome mec and accessory gene regulator types. We compared the clinical characteristics and outcomes of patients infected with biofilm-forming and non-biofilm-forming MRSA isolates. Results Of the 126 samples, 86 (68.3%), including 5 strong level (OD570 ≥ 1.0) and 81 weak level (0.2 ≤ OD570 < 1.0), had biofilm-forming capacity. Detection of fibronectinbinding protein in biofilm-forming strains was significantly higher than biofilm non-forming ones (p = 0.001) and three enterotoxin genes (sec-seg-sei) islands had a high frequency regardless of biofilm production. However, biofilm-forming strains were more likely to be multidrug resistant (three or more non-β-lactam antibiotics) than biofilm non-forming ones [79.2% vs. 59.2%, p = 0.015, odds ratio (OR) 2.629, 95% confidence interval (CI) 1.92–5.81]. Clinical features of patients with BSIs caused by biofilm-forming MRSA strains were more likely to be hospital onset [77.9% vs. 60.0%, p = 0.024, OR 2.434, 95% CI 1.11–5.33) and more frequently occurred in patients with use of invasive devices [85.7% vs. 61.2%, p = 0.002, OR 3.879, 95% CI 1.61–8.97]. The other clinical features were compared with the clinical outcomes of the two groups and were not significant (p > 0.05). Conclusion Biofilm-forming MRSA strains showed higher frequency of fnbB gene than biofilm non-forming ones and more incidence rates on particular genotypes. And, their patient's features were not significantly different between two

  14. Hyperosmotic Agents and Antibiotics Affect Dissolved Oxygen and pH Concentration Gradients in Staphylococcus aureus Biofilms.

    PubMed

    Kiamco, Mia Mae; Atci, Erhan; Mohamed, Abdelrhman; Call, Douglas R; Beyenal, Haluk

    2017-03-15

    Biofilms on wound surfaces are treated topically with hyperosmotic agents, such as medical-grade honey and cadexomer iodine; in some cases, these treatments are combined with antibiotics. Tissue repair requires oxygen, and a low pH is conducive to oxygen release from red blood cells and epithelialization. We investigated the variation of dissolved oxygen concentration and pH with biofilm depth and the variation in oxygen consumption rates when biofilms are challenged with medical-grade honey or cadexomer iodine combined with vancomycin or ciprofloxacin. Dissolved oxygen and pH depth profiles in Staphylococcus aureus biofilms were measured using microelectrodes. The presence of cadexomer iodine with vancomycin or ciprofloxacin on the surface of the biofilm permitted a measurable concentration of oxygen at greater biofilm depths (101.6 ± 27.3 μm, P = 0.02; and 155.5 ± 27.9 μm, P = 0.016, respectively) than in untreated controls (30.1 μm). Decreases in pH of ∼0.6 and ∼0.4 units were observed in biofilms challenged with medical-grade honey alone and combined with ciprofloxacin, respectively (P < 0.001 and 0.01, respectively); the number of bacteria recovered from biofilms was significantly reduced (1.26 log) by treatment with cadexomer iodine and ciprofloxacin (P = 0.002) compared to the untreated control. Combining cadexomer iodine and ciprofloxacin improved dissolved oxygen concentration and penetration depth into the biofilm, while medical-grade honey was associated with a lower pH; not all treatments established a bactericidal effect in the time frame used in the experiments.IMPORTANCE Reports about using hyperosmotic agents and antibiotics against wound biofilms focus mostly on killing bacteria, but the results of these treatments should additionally be considered in the context of how they affect physiologically important parameters, such as oxygen concentration and pH. We confirmed that the combination of a hyperosmotic agent and an antibiotic results

  15. MyD88-Dependent Signaling Influences Fibrosis and Alternative Macrophage Activation during Staphylococcus aureus Biofilm Infection

    PubMed Central

    Hanke, Mark L.; Angle, Amanda; Kielian, Tammy

    2012-01-01

    Bacterial biofilms represent a significant therapeutic challenge based on their ability to evade host immune and antibiotic-mediated clearance. Recent studies have implicated IL-1β in biofilm containment, whereas Toll-like receptors (TLRs) had no effect. This is intriguing, since both the IL-1 receptor (IL-1R) and most TLRs impinge on MyD88-dependent signaling pathways, yet the role of this key adaptor in modulating the host response to biofilm growth is unknown. Therefore, we examined the course of S. aureus catheter-associated biofilm infection in MyD88 knockout (KO) mice. MyD88 KO animals displayed significantly increased bacterial burdens on catheters and surrounding tissues during early infection, which coincided with enhanced dissemination to the heart and kidney compared to wild type (WT) mice. The expression of several proinflammatory mediators, including IL-6, IFN-γ, and CXCL1 was significantly reduced in MyD88 KO mice, primarily at the later stages of infection. Interestingly, immunofluorescence staining of biofilm-infected tissues revealed increased fibrosis in MyD88 KO mice concomitant with enhanced recruitment of alternatively activated M2 macrophages. Taken in the context of previous studies with IL-1β, TLR2, and TLR9 KO mice, the current report reveals that MyD88 signaling is a major effector pathway regulating fibrosis and macrophage polarization during biofilm formation. Together these findings represent a novel example of the divergence between TLR and MyD88 action in the context of S. aureus biofilm infection. PMID:22879997

  16. Evaluation of Antibiotics Active against Methicillin-Resistant Staphylococcus aureus Based on Activity in an Established Biofilm

    PubMed Central

    Meeker, Daniel G.; Beenken, Karen E.; Mills, Weston B.; Loughran, Allister J.; Spencer, Horace J.; Lynn, William B.

    2016-01-01

    We used in vitro and in vivo models of catheter-associated biofilm formation to compare the relative activity of antibiotics effective against methicillin-resistant Staphylococcus aureus (MRSA) in the specific context of an established biofilm. The results demonstrated that, under in vitro conditions, daptomycin and ceftaroline exhibited comparable activity relative to each other and greater activity than vancomycin, telavancin, oritavancin, dalbavancin, or tigecycline. This was true when assessed using established biofilms formed by the USA300 methicillin-resistant strain LAC and the USA200 methicillin-sensitive strain UAMS-1. Oxacillin exhibited greater activity against UAMS-1 than LAC, as would be expected, since LAC is an MRSA strain. However, the activity of oxacillin was less than that of daptomycin and ceftaroline even against UAMS-1. Among the lipoglycopeptides, telavancin exhibited the greatest overall activity. Specifically, telavancin exhibited greater activity than oritavancin or dalbavancin when tested against biofilms formed by LAC and was the only lipoglycopeptide capable of reducing the number of viable bacteria below the limit of detection. With biofilms formed by UAMS-1, telavancin and dalbavancin exhibited comparable activity relative to each other and greater activity than oritavancin. Importantly, ceftaroline was the only antibiotic that exhibited greater activity than vancomycin when tested in vivo in a murine model of catheter-associated biofilm formation. These results emphasize the need to consider antibiotics other than vancomycin, most notably, ceftaroline, for the treatment of biofilm-associated S. aureus infections, including by the matrix-based antibiotic delivery methods often employed for local antibiotic delivery in the treatment of these infections. PMID:27401574

  17. Effect of sub-lethal doses of vancomycin and oxacillin on biofilm formation by vancomycin intermediate resistant Staphylococcus aureus.

    PubMed

    Mirani, Zulfiqar Ali; Jamil, Nusrat

    2011-04-01

    Biofilms are means of protection to bacteria against antibiotics and antibodies. Catheters and others tube devices used by patients are prone to accumulation of thick layers of biofilms as hiding place for etiologic agents, resulting in substantial morbidity and mortality. Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-acquired infections. Vancomycin remains the only treatment of choice for MRSA infections. In the present study a vancomycin resistant S. aureus (VRSA) (Labeled as CP2) was isolated from the blood of a post-operative cardiac patient. It harbors a plasmid which carry vanA gene and exhibited low-level vancomycin resistance (MIC 16 μg/ml), high level of oxacillin/methicillin resistance (MIC 500 μg/ml) and was sensitive to teicoplanin. CP2 also found to carry icaA gene on its chromosome. This strain exhibited resistance to triton-X100 induced autolysis under sub-inhibitory concentration of vancomycin and produced some extracellular matrix material that surrounding the cells. These characteristic features have warranted us to study the biofilm formation by CP2 on biomedical indwellings in presence of vancomycin and oxacillin. Our findings suggest that sub-lethal dose of vancomycin induced the biofilm formation by CP2 on nylon and silicon indwellings whereas oxacillin facilitated the biofilm formation on glass surfaces exclusively. This implicates that not only the antibiotics but also the indwelling material influences biofilm formation. Therefore, these implants serve as potential surfaces for bacterial adhesion that lead to biofilm formation, thus provide hiding places for pathogens from the actions of antimicrobials.

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

    PubMed

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

    2011-05-01

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

  19. Ginkgolic acids and Ginkgo biloba extract inhibit Escherichia coli O157:H7 and Staphylococcus aureus biofilm formation.

    PubMed

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

    2014-03-17

    Infection by enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a worldwide problem, and there is no effective therapy. Biofilm formation is closely related to EHEC infection and is also a mechanism of antimicrobial resistance. Antibiofilm screening of 560 purified phytochemicals against EHEC showed that ginkgolic acids C15:1 and C17:1 at 5μg/ml and Ginkgo biloba extract at 100μg/ml significantly inhibited EHEC biofilm formation on the surfaces of polystyrene and glass, and on nylon membranes. Importantly, at their working concentrations, ginkgolic acids and G. biloba extract did not affect bacterial growth. Transcriptional analyses showed that ginkgolic acid C15:1 repressed curli genes and prophage genes in EHEC, and these findings were in-line with reduced fimbriae production and biofilm reductions. Interestingly, ginkgolic acids and G. biloba extract did not inhibit the biofilm formation of a commensal E. coli K-12 strain. In addition, ginkgolic acids and G. biloba extract inhibited the biofilm formation of three Staphylococcus aureus strains. The findings of this study suggest that plant secondary metabolites represent an important resource for biofilm inhibitors.

  20. Antibacterial effect of antibiotic-loaded SBA-15 on biofilm formation by Staphylococcus aureus and Staphylococcus epidermidis.

    PubMed

    Aguilar-Colomer, Anna; Doadrio, Juan Carlos; Pérez-Jorge, Concepción; Manzano, Miguel; Vallet-Regí, Maria; Esteban, Jaime

    2017-03-01

    Staphylococcus aureus and Staphylococcus epidermidis are human pathogens involved in implant-related infections. During those diseases, they are able to form biofilms showing resistance to the effect of many different antibiotics. Drug delivery systems allow a local and effective delivery of antibiotics at high concentrations in the infected tissue without causing the cytotoxic effects commonly linked to systemic administration. We report the use of a porous ceramic biomaterial, such as SBA-15 loaded with antibiotics, to deliver them directly to the infected tissue. SBA-15 discs were loaded with Vancomycin, Rifampin and a combination of both, introduced in a suspension of S. aureus 15981 and S. epidermidis ATCC 35984 and incubated during 6 and 24 h. A statistically significant decrease in the biofilm density and the number of viable bacteria was detected for all antibiotics at 6 h in both bacteria. Rifampin showed an increase in the biofilm density and the number of viable bacteria at 24 h. No differences were detected between Vancomycin and the combination of antibiotics. S. epidermidis was more sensitive to the effect of the antibiotics than S. aureus. Here we have demonstrated that SBA-15 is able to act as an effective drug delivery system not only from a pharmaceutical point of view, but also from a biological one.

  1. Different Phenotypes of Mature Biofilm in Flavobacterium psychrophilum Share a Potential for Virulence That Differs from Planktonic State

    PubMed Central

    Levipan, Héctor A.; Avendaño-Herrera, Ruben

    2017-01-01

    Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease and the rainbow trout fry syndrome in salmonid aquaculture worldwide. However, there have been few studies into the capacity of F. psychrophilum to form biofilms and how these cellular accretions differ from planktonic cells or how they affect potential virulence. We evaluated the biofilm formation by three Chilean isolates of F. psychrophilum (LM-02-Fp, LM-06-Fp, and LM-13-Fp) and two non-Chilean strains (JIP02/86 and NCMB1947T), and compared biofilm and planktonic states to obtain insights into expression differences of virulence- and biofilm-related genes (VBRGs). Our findings are based on scanning confocal laser microscopy (SCLM) and LIVE/DEAD staining, enzymatic reactions, reverse transcription-quantitative PCR (RT-qPCR) of genes encoding putative virulence factors, and transcriptomes (RNA-Seq). The LM-02-Fp and NCMB1947T strains were the strongest and weakest biofilm producers, respectively. The strong-biofilm producer showed different physiological cell states distributed in different layers of mature biofilms, whereas the NCMB1947T biofilms consisted of cells arranged in a monolayer. WGA-binding exopolysaccharides would be the main components of their corresponding extracellular matrices. Transcriptomes of F. psychrophilum NCMB1947T and LM-02-Fp were clustered by state (biofilm vs. planktonic) rather than by strain, indicating important state-dependent differences in gene expression. Analysis of differentially expressed genes between states identified putative VBRGs involved in polysaccharide biosynthesis, lateral gene transfer, membrane transport (e.g., for drugs and Fe3+), sensory mechanisms, and adhesion, and indicated that about 60–100% of VBRGs involved in these processes was significantly upregulated in the biofilm state. Conversely, upregulated motility-related genes in the biofilm state were not identified, whereas a lower fraction of proteolysis-related genes (33

  2. Activation of sarX by Rbf Is Required for Biofilm Formation and icaADBC Expression in Staphylococcus aureus

    PubMed Central

    Cue, David; Lei, Mei G.

    2013-01-01

    A major constituent of many Staphylococcus aureus biofilms is a polysaccharide known as the polysaccharide intercellular adhesin, or poly N-acetylglucosamine (PIA/PNAG). PIA/PNAG is synthesized by the 4 gene products of the icaADBC operon, which is negatively regulated by the divergently transcribed icaR gene. We previously reported the identification of a gene, rbf, involved in the positive transcriptional regulation of icaADBC transcription by repressing icaR in S. aureus strain 8325-4. However, we were unable to show binding of Rbf to DNA upstream of icaR or icaA, suggesting that Rbf may control expression of an unknown factor(s) that, in turn, regulates ica expression. Here we report that the unknown factor is SarX protein. Results from epistasis assays and genetic complementation analyses suggest that Rbf upregulates SarX, which then downregulates IcaR, thereby activating icaADBC. Electrophoretic mobility shift assays revealed that SarX protein bound to a sequence upstream of icaR within the icaA coding region. Cross-linking and immunoprecipitation experiments further suggested that Rbf binds to the sarX promoter in S. aureus. These results demonstrate that Rbf and SarX represent a regulatory cascade that promotes PIA-dependent biofilm formation in S. aureus. PMID:23354746

  3. Synergistic Photothermal and Antibiotic Killing of Biofilm-Associated Staphylococcus aureus Using Targeted Antibiotic-Loaded Gold Nanoconstructs

    PubMed Central

    2016-01-01

    Resistance to conventional antibiotics is a growing public health concern that is quickly outpacing the development of new antibiotics. This has led the Infectious Diseases Society of America (IDSA) to designate Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species as “ESKAPE pathogens” on the basis of the rapidly decreasing availability of useful antibiotics. This emphasizes the urgent need for alternative therapeutic strategies to combat infections caused by these and other bacterial pathogens. In this study, we used Staphylococcus aureus (S. aureus) as a proof-of-principle ESKAPE pathogen to demonstrate that an appropriate antibiotic (daptomycin) can be incorporated into polydopamine-coated gold nanocages (AuNC@PDA) and that daptomycin-loaded AuNC@PDA can be conjugated to antibodies targeting a species-specific surface protein (staphylococcal protein A; Spa) as a means of achieving selective delivery of the nanoconstructs directly to the bacterial cell surface. Targeting specificity was confirmed by demonstrating a lack of binding to mammalian cells, reduced photothermal and antibiotic killing of the Spa-negative species Staphylococcus epidermidis, and reduced killing of S. aureus in the presence of unconjugated anti-Spa antibodies. We demonstrate that laser irradiation at levels within the current safety standard for use in humans can be used to achieve both a lethal photothermal effect and controlled release of the antibiotic, thus resulting in a degree of therapeutic synergy capable of eradicating viable S. aureus cells. The system was validated using planktonic bacterial cultures of both methicillin-sensitive and methicillin-resistant S. aureus strains and subsequently shown to be effective in the context of an established biofilm, thus indicating that this approach could be used to facilitate the effective treatment of intrinsically resistant biofilm infections. PMID

  4. The Quorum Sensing Inhibitor Hamamelitannin Increases Antibiotic Susceptibility of Staphylococcus aureus Biofilms by Affecting Peptidoglycan Biosynthesis and eDNA Release

    PubMed Central

    Brackman, Gilles; Breyne, Koen; De Rycke, Riet; Vermote, Arno; Van Nieuwerburgh, Filip; Meyer, Evelyne; Van Calenbergh, Serge; Coenye, Tom

    2016-01-01

    Treatment of Staphylococcus aureus infections has become increasingly challenging due to the rapid emergence and dissemination of methicillin-resistant strains. In addition, S. aureus reside within biofilms at the site of infection. Few novel antibacterial agents have been developed in recent years and their bacteriostatic or bactericidal activity results in selective pressure, inevitably inducing antimicrobial resistance. Consequently, innovative antimicrobials with other modes of action are urgently needed. One alternative approach is targeting the bacterial quorum sensing (QS) system. Hamamelitannin (2′,5-di-O-galloyl-d-hamamelose; HAM) was previously suggested to block QS through the TraP QS system and was shown to increase S. aureus biofilm susceptibility towards vancomycin (VAN) although mechanistic insights are still lacking. In the present study we provide evidence that HAM specifically affects S. aureus biofilm susceptibility through the TraP receptor by affecting cell wall synthesis and extracellular DNA release of S. aureus. We further provide evidence that HAM can increase the susceptibility of S. aureus biofilms towards different classes of antibiotics in vitro. Finally, we show that HAM increases the susceptibility of S. aureus to antibiotic treatment in in vivo Caenorhabditis elegans and mouse mammary gland infection models. PMID:26828772

  5. Antibacterial Activity of Blue Light against Nosocomial Wound Pathogens Growing Planktonically and as Mature Biofilms

    PubMed Central

    Thwaite, Joanne E.; Burt, Rebecca; Laws, Thomas R.; Raguse, Marina; Moeller, Ralf; Webber, Mark A.; Oppenheim, Beryl A.

    2016-01-01

    ABSTRACT The blue wavelengths within the visible light spectrum are intrinisically antimicrobial and can photodynamically inactivate the cells of a wide spectrum of bacteria (Gram positive and negative) and fungi. Furthermore, blue light is equally effective against both drug-sensitive and -resistant members of target species and is less detrimental to mammalian cells than is UV radiation. Blue light is currently used for treating acnes vulgaris and Helicobacter pylori infections; the utility for decontamination and treatment of wound infections is in its infancy. Furthermore, limited studies have been performed on bacterial biofilms, the key growth mode of bacteria involved in clinical infections. Here we report the findings of a multicenter in vitro study performed to assess the antimicrobial activity of 400-nm blue light against bacteria in both planktonic and biofilm growth modes. Blue light was tested against a panel of 34 bacterial isolates (clinical and type strains) comprising Acinetobacter baumannii, Enterobacter cloacae, Stenotrophomonas maltophilia, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus faecium, Klebsiella pneumoniae, and Elizabethkingia meningoseptica. All planktonic-phase bacteria were susceptible to blue light treatment, with the majority (71%) demonstrating a ≥5-log10 decrease in viability after 15 to 30 min of exposure (54 J/cm2 to 108 J/cm2). Bacterial biofilms were also highly susceptible to blue light, with significant reduction in seeding observed for all isolates at all levels of exposure. These results warrant further investigation of blue light as a novel decontamination strategy for the nosocomial environment, as well as additional wider decontamination applications. IMPORTANCE Blue light shows great promise as a novel decontamination strategy for the nosocomial environment, as well as additional wider decontamination applications (e.g., wound closure during surgery). This warrants further

  6. The Behavior of Staphylococcus aureus Dual-Species Biofilms Treated with Bacteriophage phiIPLA-RODI Depends on the Accompanying Microorganism.

    PubMed

    González, Silvia; Fernández, Lucía; Campelo, Ana Belén; Gutiérrez, Diana; Martínez, Beatriz; Rodríguez, Ana; García, Pilar

    2017-02-01

    The use of bacteriophages as antimicrobials against pathogenic bacteria offers a promising alternative to traditional antibiotics and disinfectants. Significantly, phages may help to remove biofilms, which are notoriously resistant to commonly used eradication methods. However, the successful development of novel antibiofilm strategies must take into account that real-life biofilms usually consist of mixed-species populations. Within this context, this study aimed to explore the effectiveness of bacteriophage-based sanitation procedures for removing polymicrobial biofilms from food industry surfaces. We treated dual-species biofilms formed by the food pathogenic bacterium Staphylococcus aureus in combination with Lactobacillus plantarum, Enterococcus faecium, or Lactobacillus pentosus with the staphylococcal phage phiIPLA-RODI. Our results suggest that the impact of bacteriophage treatment on S. aureus mixed-species biofilms varies depending on the accompanying species and the infection conditions. For instance, short treatments (4 h) with a phage suspension under nutrient-limiting conditions reduced the number of S. aureus cells in 5-h biofilms by ∼1 log unit without releasing the nonsusceptible species. In contrast, longer infection periods (18 h) with no nutrient limitation increased the killing of S. aureus cells by the phage (decrease of up to 2.9 log units). However, in some cases, these conditions promoted the growth of the accompanying species. For example, the L. plantarum cell count in the treated sample was up to 2.3 log units higher than that in the untreated control. Furthermore, phage propagation inside dual-species biofilms also depended greatly on the accompanying species, with the highest rate detected in biofilms formed by S. aureus-L. pentosus Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) also showed changes in the three-dimensional structures of the mixed-species biofilms after phage treatment. Altogether

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

    PubMed

    Cincarova, Lenka; Polansky, Ondrej; Babak, Vladimir; Kulich, Pavel; Kralik, Petr

    2016-01-01

    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.

  8. Effects of nisin and lysozyme on growth inhibition and biofilm formation capacity of Staphylococcus aureus strains isolated from raw milk and cheese samples.

    PubMed

    Sudagidan, Mert; Yemenicioğlu, Ahmet

    2012-09-01

    Effects of nisin and lysozyme on growth inhibition and biofilm formation capacity of 25 Staphylococcus aureus strains isolated from raw milk (13 strains) and cheese (12 strains) were studied. Nisin was tested at concentrations between 0.5 and 25 μg/ml; the growth of all strains was inhibited at 25 μg/ml, but the resistances of strains showed a great variation at lower nisin concentrations. In contrast, lysozyme tested at concentrations up to 5.0 mg/ml showed no inhibition on the growth of strains. Nisin used at the growth inhibitory concentration prevented the biofilm formation of strains, but strains continued biofilm formation at subinhibitory nisin concentrations. Lysozyme did not affect the biofilm formation of 19 of the strains, but it caused a considerable activation in the biofilm formation capacity of six strains. Twelve of the strains contained both biofilm-related protease genes (sspA, sspB, and aur) and active proteases; eight of these strains were nisin resistant. These results suggest a potential risk of S. aureus growth and biofilm formation when lysozyme is used in the biopreservation of dairy products. Nisin can be used to control growth and biofilm formation of foodborne S. aureus, unless resistance against this biopreservative develops.

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

    PubMed Central

    Miyamoto, Hiroshi; Shobuike, Takeo; Kobatake, Tomoki; Mawatari, Masaaki

    2016-01-01

    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. PMID:28105433

  10. Newly-synthesized chalcones-inhibition of adherence and biofilm formation of methicillin-resistant Staphylococcus aureus

    PubMed Central

    Bozic, Dragana D.; Milenkovic, Marina; Ivkovic, Branka; Cirkovic, Ivana

    2014-01-01

    Biofilm formation and adherence of bacteria to host tissue are one of the most important virulence factors of methicillin-resistant strains of Staphylococcus aureus (MRSA). The number of resistant strains is seriously increasing during the past years and bacteria have become resistant, not only to methicillin, but also to other commonly used antistaphylococcal antibiotics. There is a great need for discovering a novel antimicrobial agent for the treatment of staphylococcal infections. One of the most promising groups of compounds appears to be chalcones. In present study we evaluated the in vitro effect of three newly synthesized chalcones: 1,3- Bis-(2-hydroxy-phenyl)-propenone, 3-(3-Hydroxy-phenyl)-1-(2-hydroxy-phenyl)-propenone and 3-(4-Hydroxy-phenyl)-1-(2-hydroxy-phenyl)-propenone on glycocalyx production, biofilm formation and adherence to human fibronectin of clinical isolates and laboratory control strain of MRSA (ATCC 43300). Subinhibitory concentrations of the tested compounds reduced the production of glycocalyx, biofilm formation and adherence to human fibronectin of all MRSA strains. Inhibition of biofilm formation was dose dependent and the most effective was 1,3- Bis-(2-hydroxy-phenyl)-propenone. In our study we demonstrated that three newly-synthesized chalcones exhibited significant effect on adherence and biofilm formation of MRSA strains. Chalcones may be considered as promising new antimicrobial agents that can be used for prevention of staphylococcal infections or as adjunct to antibiotics in conventional therapy. PMID:24948943

  11. Efficient Eradication of Mature Pseudomonas aeruginosa Biofilm via Controlled Delivery of Nitric Oxide Combined with Antimicrobial Peptide and Antibiotics

    PubMed Central

    Ren, Hang; Wu, Jianfeng; Colletta, Alessandro; Meyerhoff, Mark E.; Xi, Chuanwu

    2016-01-01

    Fast eradication of mature biofilms is the ‘holy grail’ in the clinical management of device-related infections. Endogenous nitric oxide (NO) produced by macrophages plays an important role in host defense against intracellular pathogens, and NO is a promising agent in preventing biofilms formation in vitro. However, the rate of delivery of NO by various NO donors (e.g., diazeniumdiolates, S-nitrosothiols, etc.) is difficult to control, which hinders fundamental studies aimed at understanding the role of NO in biofilm control. In this study, by using a novel precisely controlled electrochemical NO releasing catheter device, we examine the effect of physiological levels of NO on eradicating mature Pseudomonas aeruginosa biofilm (7 days), as well as the potential application of the combination of NO with antimicrobial agents. It is shown that physiological levels of NO exhibit mixed effects of killing bacteria and dispersing ambient biofilm. The overall biofilm-eradicating effect of NO is quite efficient in a dose-dependent manner over a 3 h period of NO treatment. Moreover, NO also greatly enhances the efficacy of antimicrobial agents, including human beta-defensin 2 (BD-2) and several antibiotics, in eradicating biofilm and its detached cells, which otherwise exhibited high recalcitrance to these antimicrobial agents. The electrochemical NO release technology offers a powerful tool in evaluating the role of NO in biofilm control as well as a promising approach when combined with antimicrobial agents to treat biofilm-associated infections in hospital settings, especially infections resulting from intravascular catheters. PMID:27582732

  12. Effectiveness of a polyhexanide irrigation solution on methicillin-resistant Staphylococcus aureus biofilms in a porcine wound model.

    PubMed

    Davis, Stephen C; Harding, Andrew; Gil, Joel; Parajon, Fernando; Valdes, Jose; Solis, Michael; Higa, Alex

    2017-03-07

    Irrigation and removal of necrotic debris can be beneficial for proper healing. It is becoming increasingly evident that wounds colonized with biofilm forming bacteria, such as Staphylococcus aureus (SA), can be more difficult to eradicate. Here we report our findings of the effects of an irrigation solution containing propyl-betaine and polyhexanide (PHMB) on methicillin-resistant Staphylococcus aureus (MRSA) biofilms in a porcine wound model. Thirty-nine deep partial thickness wounds were created with six wounds assigned to one of six treatment groups: (i) PHMB, (ii) Ringer's solution, (iii) hypochlorous acid/sodium hypochlorite, (iv) sterile water, (v) octenidine dihydrochloride, and (vi) octenilin. Wounds were inoculated with MRSA and covered with a polyurethane dressing for 24 hours to allow biofilm formation. The dressings were then removed and the wounds were irrigated twice daily for 3 days with the appropriate solution. MRSA from four wounds were recovered from each treatment group at 3 days and 6 days hours after initial treatment. Irrigation of wounds with the PHMB solution resulted in 97·85% and 99·64% reductions of MRSA at the respective 3 days and 6 days assessment times when compared to the untreated group. Both of these reductions were statistically significant compared to all other treatment groups (P values <0·05).

  13. Comparative Transcriptome Analysis of Desulfovibrio Vulgaris Grown in Planktonic Culture and Mature Biofilm on a Steel Surface

    SciTech Connect

    Zhang, Weiwen; Culley, David E.; Nie, Lei; Scholten, Johannes C.

    2007-08-01

    The build-up of biofilms of sulphate -reducing bacteria (SRB) on metals surfaces may lead to severe corrosion of iron. To understand the processes at molecular level, in this study, a whole-genome oligonucleotide microarray was used to examine differential expression patterns between planktonic populations and mature biofilm of model SRB species Desulfovibrio vulgaris. Statistical analysis revealed that 472 genes were differentially expressed (1.5 fold or more with a p value less than 0.025) when comparing biofilm to planktonic cells. Among the differentially expressed genes were several that corresponded to biofilm formation genes identified in many aerobic bacterial biofilms (i.e., Pseudomonas species and Escherichia coli), such as down-regulation of genes encoding flagellin, flagellar motor switch protein and chemotaxis proteins involved in cell motility and induction of genes encoding sugar transferase and glycogen synthase involved in exopolysaccharide biosynthesis. In addition, D. vulgaris biofilm-bound cells exhibited decreased transcription of genes involved in protein synthesis, energy metabolism and sulfate reduction, as well as genes involved in general stress responses. These findings were all consistent with early suggestion that the average physiology of biofilm cells were similar to planktonic cells of stationary phases. Most notably, up-regulation of large number of outer membrane proteins was observed in D. vulgaris biofilm. Although their function is still unknown, the higher expression of these genes in D. vulgaris biofilm could implicate important roles formation and maintenance of multi-cellular consortium on metal surface. The study provided insights into the metabolic networks associated with D. vulgaris biofilm formation and maintenance on an iron surface.

  14. The role of the globin-coupled sensor YddV in a mature E. coli biofilm population.

    PubMed

    Donné, Joke; Van Kerckhoven, Marian; Maes, Louis; Cos, Paul; Dewilde, Sylvia

    2016-07-01

    Biofilm-associated infections are hard to treat because of their high antibiotic resistance and the presence of a very persistent subpopulation of bacteria. The second messenger molecule cyclic di-guanosine monophosphate (c-di-GMP) plays a very important role in this biofilm physiology. Here, we evaluated the role of YddV, an enzyme with a c-di-GMP synthesis function, in the formation and maturation of Escherichia coli biofilms. Our results suggest that YddV stimulates biofilm growth via its role in the production of c-di-GMP and this likely by influencing the production of matrix (e.g. poly-N-acetylglucosamine (PGA)). However, lowering the YddV expression did not alter the biofilm formation since there was no significant difference between the biofilm phenotypes of WT E. coli and YddV-knockout bacteria. Additionally, YddV expression had no significant influence on the amount of persister cells within the biofilm population, questioning the use of YddV as therapeutic target.

  15. Methicillin Resistant Staphylococcus Aureus Biofilm Formation Over A Separated Flow Region Under Steady And Pulsatile Flow Conditions

    NASA Astrophysics Data System (ADS)

    Salek, M. Mehdi; Martinuzzi, Robert

    2012-02-01

    Several researchers have observed that the formation, morphology and susceptibility of bacterial biofilms are affected by the local hydrodynamic condition and, in particular, shear stresses acting on the fluid-biofilm interface. A backwards facing step (BFS) experimental model has been widely utilized as an in vitro model to examine and characterize the effect of flow separation and recirculation zones comparable to those present within various medical devices as well as those observed in vivo. The specific geometry of BFS covers a vide range of flow features observed in physiological or environmental conditions. The hypothesis of this study is that the flow behavior and structures can effectively contribute to the transport and attachment of cells and affecting the morphology of adhered colonies as well as suspended structures (i.e. biofilm streamers). Hence, the formation of the recirculation region occurring within a backward facing step (BFS) under steady and pulsatile conditions as well as three-dimensional flow structures arising close to the side walls are investigated to correlate to biofilms behavior. This hypothesis is investigated using a backward facing step incorporated into a flow cell under steady and pulsatile flow regimes to study the growth of methicillin resistant Staphylococcus aureus (MRSA) UC18 as the study microorganism.

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

    PubMed

    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.

  17. A comparison of Staphylococcus aureus biofilm formation on cobalt-chrome and titanium-alloy spinal implants.

    PubMed

    Patel, Shalin S; Aruni, Wilson; Inceoglu, Serkan; Akpolat, Yusuf T; Botimer, Gary D; Cheng, Wayne K; Danisa, Olumide A

    2016-09-01

    The use of cobalt chrome (CoCr) implants in spinal surgery has become increasingly popular. However, there have been no studies specifically comparing biofilm formation on CoCr with that of titanium-alloy spinal implants. The objective of this study was to compare the difference in propensity for biofilm formation between these two materials, as it specifically relates to spinal rods. Staphylococcus aureus subsp. Aureus (ATCC 6538) were incubated with two different types of spinal rods composed of either CoCr or titanium-alloy. The spinal rods were then subject to a trypsin wash to allow for isolation of the colonized organism and associated biofilms. The associated optical density values (OD) from the bacterial isolates were obtained and the bacterial solutions were plated on brain-heart infusion agar plates and the resultant colony-forming units (CFU) were counted. The OD values for the titanium-alloy rods were 1.105±0.096nm (mean±SD) and 1.040±0.026nm at 48hours and 96hours, respectively. In contrast, the OD values for the CoCr rods were 1.332±0.161nm and 1.115±0.207nm at 48 and 96hours, respectively (p<0.05). The CFU values were 1481±417/100mm(2) and 745±159/100mm(2) at 48 and 96hours, respectively for the titanium-alloy group. These values were significantly lower than the CFU values obtained from the CoCr group which were 2721±605/100mm(2) and 928±88/100mm(2) (p<0.001) at both 48 and 96hours respectively. Our findings, evaluating both the OD and CFU values, indicate that implants composed of CoCr had a higher proclivity towards biofilm formation compared to titanium-alloy implants.

  18. Methicillin-resistant food-related Staphylococcus aureus: a review of current knowledge and biofilm formation for future studies and applications.

    PubMed

    Doulgeraki, Agapi I; Di Ciccio, Pierluigi; Ianieri, Adriana; Nychas, George-John E

    2017-01-01

    There is increasing concern about the public health impact of methicillin-resistant Staphylococcus aureus. Food and animal are vectors of transmission, but the contribution of a contaminated environment is not well characterized. With regard to this, staphylococcal biofilms serve as a virulence factor, allowing MRSA strains to adhere to surfaces and other materials used in the food industry. Methicillin resistance and biofilm-forming capacity may contribute to the success of S. aureus as a human pathogen in both health care and community settings and the food production chain. This review summarizes current knowledge about the significance of food- and animal-derived MRSA strains and provides data on attachment and biofilm formation of MRSA. In addition, the impact of quorum sensing on MRSA gene expression and biofilm formation is examined.

  19. Sustained Nitric Oxide-Releasing Nanoparticles Interfere with Methicillin-Resistant Staphylococcus aureus Adhesion and Biofilm Formation in a Rat Central Venous Catheter Model.

    PubMed

    Mihu, Mircea Radu; Cabral, Vitor; Pattabhi, Rodney; Tar, Moses T; Davies, Kelvin P; Friedman, Adam J; Martinez, Luis R; Nosanchuk, Joshua D

    2017-01-01

    Staphylococcus aureus is frequently isolated in the setting of infections of indwelling medical devices, which are mediated by the microbe's ability to form biofilms on a variety of surfaces. Biofilm-embedded bacteria are more resistant to antimicrobial agents than their planktonic counterparts and often cause chronic infections and sepsis, particularly in patients with prolonged hospitalizations. In this study, we demonstrate that sustained nitric oxide-releasing nanoparticles (NO-np) interfere with S. aureus adhesion and prevent biofilm formation on a rat central venous catheter (CVC) model of infection. Confocal and scanning electron microscopy showed that NO-np-treated staphylococcal biofilms displayed considerably reduced thicknesses and bacterial numbers compared to those of control biofilms in vitro and in vivo, respectively. Although both phenotypes, planktonic and biofilm-associated staphylococci, of multiple clinical strains were susceptible to NO-np, bacteria within biofilms were more resistant to killing than their planktonic counterparts. Furthermore, chitosan, a biopolymer found in the exoskeleton of crustaceans and structurally integrated into the nanoparticles, seems to add considerable antimicrobial activity to the technology. Our findings suggest promising development and translational potential of NO-np for use as a prophylactic or therapeutic against bacterial biofilms on CVCs and other medical devices.

  20. Identification of ypqP as a New Bacillus subtilis Biofilm Determinant That Mediates the Protection of Staphylococcus aureus against Antimicrobial Agents in Mixed-Species Communities

    PubMed Central

    Sanchez-Vizuete, Pilar; Le Coq, Dominique; Bridier, Arnaud; Herry, Jean-Marie; Aymerich, Stéphane

    2014-01-01

    In most habitats, microbial life is organized in biofilms, three-dimensional edifices sustained by extracellular polymeric substances that enable bacteria to resist harsh and changing environments. Under multispecies conditions, bacteria can benefit from the polymers produced by other species (“public goods”), thus improving their survival under toxic conditions. A recent study showed that a Bacillus subtilis hospital isolate (NDmed) was able to protect Staphylococcus aureus from biocide action in multispecies biofilms. In this work, we identified ypqP, a gene whose product is required in NDmed for thick-biofilm formation on submerged surfaces and for resistance to two biocides widely used in hospitals. NDmed and S. aureus formed mixed biofilms, and both their spatial arrangement and pathogen protection were mediated by YpqP. Functional ypqP is present in other natural B. subtilis biofilm-forming isolates. However, the gene is disrupted by the SPβ prophage in the weak submerged-biofilm-forming strains NCIB3610 and 168, which are both less resistant than NDmed to the biocides tested. Furthermore, in a 168 laboratory strain cured of the SPβ prophage, the reestablishment of a functional ypqP gene led to increased thickness and resistance to biocides of the associated biofilms. We therefore propose that YpqP is a new and important determinant of B. subtilis surface biofilm architecture, protection against exposure to toxic compounds, and social behavior in bacterial communities. PMID:25326298

  1. Identification of ypqP as a New Bacillus subtilis biofilm determinant that mediates the protection of Staphylococcus aureus against antimicrobial agents in mixed-species communities.

    PubMed

    Sanchez-Vizuete, Pilar; Le Coq, Dominique; Bridier, Arnaud; Herry, Jean-Marie; Aymerich, Stéphane; Briandet, Romain

    2015-01-01

    In most habitats, microbial life is organized in biofilms, three-dimensional edifices sustained by extracellular polymeric substances that enable bacteria to resist harsh and changing environments. Under multispecies conditions, bacteria can benefit from the polymers produced by other species ("public goods"), thus improving their survival under toxic conditions. A recent study showed that a Bacillus subtilis hospital isolate (NDmed) was able to protect Staphylococcus aureus from biocide action in multispecies biofilms. In this work, we identified ypqP, a gene whose product is required in NDmed for thick-biofilm formation on submerged surfaces and for resistance to two biocides widely used in hospitals. NDmed and S. aureus formed mixed biofilms, and both their spatial arrangement and pathogen protection were mediated by YpqP. Functional ypqP is present in other natural B. subtilis biofilm-forming isolates. However, the gene is disrupted by the SPβ prophage in the weak submerged-biofilm-forming strains NCIB3610 and 168, which are both less resistant than NDmed to the biocides tested. Furthermore, in a 168 laboratory strain cured of the SPβ prophage, the reestablishment of a functional ypqP gene led to increased thickness and resistance to biocides of the associated biofilms. We therefore propose that YpqP is a new and important determinant of B. subtilis surface biofilm architecture, protection against exposure to toxic compounds, and social behavior in bacterial communities.

  2. The DUF59 Containing Protein SufT Is Involved in the Maturation of Iron-Sulfur (FeS) Proteins during Conditions of High FeS Cofactor Demand in Staphylococcus aureus

    PubMed Central

    Bhatt, Shiven; Poudel, Saroj; Boyd, Eric S.; Boyd, Jeffrey M.

    2016-01-01

    Proteins containing DUF59 domains have roles in iron-sulfur (FeS) cluster assembly and are widespread throughout Eukarya, Bacteria, and Archaea. However, the function(s) of this domain is unknown. Staphylococcus aureus SufT is composed solely of a DUF59 domain. We noted that sufT is often co-localized with sufBC, which encode for the Suf FeS cluster biosynthetic machinery. Phylogenetic analyses indicated that sufT was recruited to the suf operon, suggesting a role for SufT in FeS cluster assembly. A S. aureus ΔsufT mutant was defective in the assembly of FeS proteins. The DUF59 protein Rv1466 from Mycobacterium tuberculosis partially corrected the phenotypes of a ΔsufT mutant, consistent with a widespread role for DUF59 in FeS protein maturation. SufT was dispensable for FeS protein maturation during conditions that imposed a low cellular demand for FeS cluster assembly. In contrast, the role of SufT was maximal during conditions imposing a high demand for FeS cluster assembly. SufT was not involved in the repair of FeS clusters damaged by reactive oxygen species or in the physical protection of FeS clusters from oxidants. Nfu is a FeS cluster carrier and nfu displayed synergy with sufT. Furthermore, introduction of nfu upon a multicopy plasmid partially corrected the phenotypes of the ΔsufT mutant. Biofilm formation and exoprotein production are critical for S. aureus pathogenesis and vancomycin is a drug of last-resort to treat staphylococcal infections. Defective FeS protein maturation resulted in increased biofilm formation, decreased production of exoproteins, increased resistance to vancomycin, and the appearance of phenotypes consistent with vancomycin-intermediate resistant S. aureus. We propose that SufT, and by extension the DUF59 domain, is an accessory factor that functions in the maturation of FeS proteins. In S. aureus, the involvement of SufT is maximal during conditions of high demand for FeS proteins. PMID:27517714

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

    PubMed

    Raffaella, Campana; 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.

  4. Impact of individual extracellular proteases on Staphylococcus aureus biofilm formation in diverse clinical isolates and their isogenic sarA mutants.

    PubMed

    Loughran, Allister J; Atwood, Danielle N; Anthony, Allison C; Harik, Nada S; Spencer, Horace J; Beenken, Karen E; Smeltzer, Mark S

    2014-12-01

    We demonstrate that the purified Staphylococcus aureus extracellular proteases aureolysin, ScpA, SspA, and SspB limit biofilm formation, with aureolysin having the greatest impact. Using protease-deficient derivatives of LAC, we confirmed that this is due to the individual proteases themselves. Purified aureolysin, and to a lesser extent ScpA and SspB, also promoted dispersal of an established biofilm. Mutation of the genes encoding these proteases also only partially restored biofilm formation in an FPR3757 sarA mutant and had little impact on restoring virulence in a murine bacteremia model. In contrast, eliminating the production of all of these proteases fully restored both biofilm formation and virulence in a sarA mutant generated in the closely related USA300 strain LAC. These results confirm an important role for multiple extracellular proteases in S. aureus pathogenesis and the importance of sarA in repressing their production. Moreover, purified aureolysin limited biofilm formation in 14 of 15 methicillin-resistant isolates and 11 of 15 methicillin-susceptible isolates, while dispersin B had little impact in UAMS-1, LAC, or 29 of 30 contemporary isolates of S. aureus. This suggests that the role of sarA and its impact on protease production is important in diverse strains of S. aureus irrespective of their methicillin resistance status.

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

    PubMed

    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.

  6. Prevalence of Panton-Valentine leucocidin and phenotypic and genotypic characterization of biofilm formation among Staphylococcus aureus strains isolated from children with adenoid hypertrophy.

    PubMed

    Emaneini, Mohammad; Khoramrooz, Seyed Sajjad; Shahsavan, Shadi; Dabiri, Hossein; Jabalameli, Fereshteh

    2015-12-01

    Adenoids as a first line of host defense against respiratory microbes play an important role in majority of upper airway infectious and noninfectious illnesses. Bacterial pathogen can colonize on the adenoid tissue and probably act as a reservoir for them. To determine phenotypic and genotypic characterization of biofilm forming capacity of Staphylococcus aureus isolates from children with adenoid hypertrophy and prevalence of Panton-Valentine leukocidin (PVL) gene we collected 17 consecutive, clinically significant S. aureus isolates from children with adenoid hypertrophy undergoing adenoidectomy with one or more of the upper airway obstruction symptoms, nasal obstruction, mouth breathing, snoring, or sleep apnea. Biofilm formation was evaluated by colorimetric microtiter plate's assay. Gene encoding PVL and adhesion- or biofilm formation-encoding genes were targeted by polymerase chain reaction (PCR) assay. According to the results, all strains produced biofilm. Seven (41.2%) isolates produced strong biofilm whereas 7 (41.2%) isolates produced week and 3 (17.6%) isolates produced medium biofilm. Regarding the adhesion- or biofilm formation-encoding genes, 16 (94.1%) isolates were positive for the gene eno, 13(76.4%) for icaA, 13 (76.4%) for icaD, 10 (58.8%) for fib, 10 (58.8%) for fnbB, 4(23.5%) for can, and 1(5.8%) for fnbA. The high prevalence of genes encoding biofilms and adhesins and phenotypic ability to form a biofilm by S. aureus strains emphasizes the pathogenic character of strains isolated from children with adenoid hypertrophy.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed Central

    Polansky, Ondrej; Babak, Vladimir; Kulich, Pavel

    2016-01-01

    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. PMID:27868063

  9. [The biological kinetics of biofilms of clinical strains of Staphylococcus aureus and Pseudomonas aeruginosa separated from patients with bronchopulmonary complications under traumatic disease of spinal cord].

    PubMed

    Ul'ianov, V Iu; Opredelentseva, S V; Shvidenko, I G; Norkin, I A; Korshunov, G V; Gladkova, E V

    2014-08-01

    The capacity and intensity of formation of microbial biofilms was analyzed in 24 strains of Staphylococcus aureus and Pseudomonas aeruginosa in static conditions of cultivation during 24, 48, 72 and 96 yours. The microorganisms were separated from patients with bronchopulmonary infectious complications in acute and early periods of traumatic disease of spinal cord.

  10. Bacteriophage exploitation of bacterial biofilms: phage preference for less mature targets?

    PubMed

    Abedon, Stephen T

    2016-02-01

    Robust evidence is somewhat lacking for biofilm susceptibility to bacteriophages in nature, contrasting often substantial laboratory biofilm vulnerability to phages. To help bridge this divide, I review a two-part scenario for 'heterogeneous' phage interaction even with phage-permissive single-species biofilms. First, through various mechanisms, those bacteria which are both more newly formed and located at biofilm surfaces may be particularly vulnerable to phage adsorption, rather than biofilm matrix being homogeneously resistant to phage penetration. Second, though phage infection of older, less metabolically active bacteria may still be virion productive, nevertheless the majority of phage population growth in association with biofilm bacteria could involve infection particularly of those bacteria which are more metabolically active and thereby better able to support larger phage bursts, versus clonally related biofilm bacteria equivalently supporting phage production. To the extent that biofilms are physiologically or structurally heterogeneous, with phages exploiting particularly relatively newly divided biofilm-surface bacteria, then even effective phage predation of natural biofilms could result in less than complete overall biofilm clearance. Phage tendencies toward only partial exploitation of even single-species biofilms could be consistent with observations that chronic bacterial infections in the clinic can require more aggressive or extensive phage therapy to eradicate.

  11. Comparative impact of diverse regulatory loci on Staphylococcus aureus biofilm formation

    PubMed Central

    Atwood, Danielle N; Loughran, Allister J; Courtney, Ashleah P; Anthony, Allison C; Meeker, Daniel G; Spencer, Horace J; Gupta, Ravi Kr; Lee, Chia Y; Beenken, Karen E; Smeltzer, Mark S

    2015-01-01

    The relative impact of 23 mutations on biofilm formation was evaluated in the USA300, methicillin-resistant strain LAC. Mutation of sarA, atl, codY, rsbU, and sigB limited biofilm formation in comparison to the parent strain, but the limitation imposed by mutation of sarA was greater than that imposed by mutation of any of these other genes. The reduced biofilm formation of all mutants other than the atl mutant was correlated with increased levels of extracellular proteases. Mutation of fur- and mgrA-enhanced biofilm formation but in LAC had no impact on protease activity, nuclease activity, or accumulation of the polysaccharide intercellular adhesin (PIA). The increased capacity of these mutants to form a biofilm was reversed by mutation of sarA, and this was correlated with increased protease production. Mutation of sarA, mgrA, and sigB had the same phenotypic effect in the methicillin-sensitive strain UAMS-1, but mutation of codY increased rather than decreased biofilm formation. As with the UAMS-1 mgrA mutant, this was correlated with increased production of PIA. Examination of four additional clinical isolates suggests that the differential impact of codY on biofilm formation may be a conserved characteristic of methicillin-resistant versus methicillin-sensitive strains. PMID:25810138

  12. Comparative impact of diverse regulatory loci on Staphylococcus aureus biofilm formation.

    PubMed

    Atwood, Danielle N; Loughran, Allister J; Courtney, Ashleah P; Anthony, Allison C; Meeker, Daniel G; Spencer, Horace J; Gupta, Ravi Kr; Lee, Chia Y; Beenken, Karen E; Smeltzer, Mark S

    2015-06-01

    The relative impact of 23 mutations on biofilm formation was evaluated in the USA300, methicillin-resistant strain LAC. Mutation of sarA, atl, codY, rsbU, and sigB limited biofilm formation in comparison to the parent strain, but the limitation imposed by mutation of sarA was greater than that imposed by mutation of any of these other genes. The reduced biofilm formation of all mutants other than the atl mutant was correlated with increased levels of extracellular proteases. Mutation of fur- and mgrA-enhanced biofilm formation but in LAC had no impact on protease activity, nuclease activity, or accumulation of the polysaccharide intercellular adhesin (PIA). The increased capacity of these mutants to form a biofilm was reversed by mutation of sarA, and this was correlated with increased protease production. Mutation of sarA, mgrA, and sigB had the same phenotypic effect in the methicillin-sensitive strain UAMS-1, but mutation of codY increased rather than decreased biofilm formation. As with the UAMS-1 mgrA mutant, this was correlated with increased production of PIA. Examination of four additional clinical isolates suggests that the differential impact of codY on biofilm formation may be a conserved characteristic of methicillin-resistant versus methicillin-sensitive strains.

  13. [Comparison of tigecycline and vancomycin activities in an in vitro biofilm model generated with methicillin-resistant Staphylococcus aureus].

    PubMed

    Aslan, Halil; Yapar, Nur

    2015-10-01

    Today, the most common cause of bloodstream infections, which led to high mortality, prolonged hospitalization and increased costs are the intravenous catheters. Among the microorganisms associated with catheter infections, staphylococci took the first place and because of their biofilm-forming properties they cause serious problems in treatment and management of the patients. Although the drug of choice in the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infection is vancomycin, its effect on the bacterial biofilm is known to be low. Tigecycline, newly used in our country is a well tolerated glycylcycline antibiotic. In this study, we aimed to compare the efficacy of tigecycline and vancomycin in an in vitro MRSA biofilm model. The study consisted of 10 MRSA strains, which were detected as causative agents of catheter-related infections in our hospital. The methicillin resistance of the strains were performed by disk diffusion test with oxacillin (1 μg) disks and the biofilm forming capacity of the strains was evaluated using the Congo red agar method. The silicone disks with created biofilm layer were exposed to tigecycline (2 mg/ml) and vancomycin (2 mg/ml) for 24 hours and for 5 days 4-hours per day in a model of antibiotic lock therapy. The present study showed that, after incubating the silicon discs in antibiotic solution for 24 hours, colony forming unit counts of MRSA decreased from 10(5) cfu/ml to 510 cfu/ml in the tigecycline group and from 105 cfu/ml to 3.800 cfu/ml in the vancomycin group and remained the same in the control (10(5) cfu/ml) group (p< 0.001). In the antibiotic lock therapy model, incubation with antibiotics for 4 hours per day, yielded that the average growth was 1.800 cfu/ml in the tigecycline group and 8.700 cfu/ml in the vancomycin group, which was statistically significant (p< 0.001). No growth was detected in the tigecycline group (0 cfu/ml) while in vancomycin group number of colonies in second, thirth and

  14. Prevention of Staphylococcus aureus biofilm formation by antibiotics in 96-Microtiter Well Plates and Drip Flow Reactors: critical factors influencing outcomes

    PubMed Central

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

    2017-01-01

    Biofilm formation leads to the failure of antimicrobial therapy. Thus, biofilm prevention is a desirable goal of antimicrobial research. In this study, the efficacy of antibiotics (doxycycline, oxacillin and rifampicin) in preventing Staphylococcus aureus biofilms was investigated using Microtiter Well Plates (MWP) and Drip Flow Reactors (DFR), two models characterized by the absence and the presence of a continuous flow of nutrients, respectively. Planktonic culture of S. aureus was exposed to antibiotics for one hour followed by 24 hours incubation with fresh nutrients in MWP or continuous flow of nutrients in DFR. The DFR grown biofilms were significantly more tolerant to the antibiotics than those grown in MWP without the continuous flow. The differences in log reductions (LR) between the two models could not be attributed to differences in the cell density, the planktonic inoculum concentration or the surface-area-to-volume ratios. However, eliminating the flow in the DFR significantly restored the antibiotic susceptibility. These findings demonstrate the importance of considering differences between experimental conditions in different model systems, particularly the flow of nutrients, when performing anti-biofilm efficacy evaluations. Biofilm antibiotic efficacy studies should be assessed using various models and more importantly, in a model mimicking conditions of its clinical application. PMID:28252025

  15. [In vitro activity of Eucalyptus smithii and Juniperus communis essential oils against bacterial biofilms and efficacy perspectives of complementary inhalation therapy in chronic and recurrent upper respiratory tract infections].

    PubMed

    Camporese, Alessandro

    2013-06-01

    Staphylococcus aureus and Pseudomonas aeruginosa have a high propensity to develop biofilms that are resistant to antimicrobial agents. Eucalyptus smithii and Juniperus communis essential oils are credited with a series of traditional therapeutical properties, including mucolytic effect. As S. aureus and P. aeruginosa biofilms are known to be important factors underlying their virulence and pathogenicity, the aim of this study was to investigate whether E. smithii and J. communis essential oils can interfere with biofilm formation as well as acting on mature biofilms. Tests of two S. aureus and P. aeruginosa clinical strains and two ATCC strains (S. aureus ATCC 25923 and P. aeruginosa ATCC 27853) showed that both E. smithii and J. communis essential oils interfere with the starting phases of biofilm production, as well as with mature biofilms. The results of this study reveal new relevant perspectives for a complementary inhalatory treatment of chronic and/or recurrent upper respiratory tract infections.

  16. Role of initial contamination levels, biofilm maturity and presence of salt and fat on desiccation survival of Listeria monocytogenes on stainless steel surfaces.

    PubMed

    Hingston, Patricia A; Stea, Emma C; Knøchel, Susanne; Hansen, Truelstrup

    2013-10-01

    This study investigated the effect of initial contamination levels, biofilm maturity and presence of salt and fatty food soils on desiccation survival of Listeria monocytogenes on stainless steel (SS) coupons. L. monocytogenes cultures grown (at 15 °C for 48 h) in Tryptic Soy Broth with 1% glucose (TSB-glu) containing either 0.5 or 5% (w/v) NaCl were re-suspended in TSB-glu containing either 0.5 or 5% NaCl and used to contaminate SS coupons at levels of 3.5, 5.5, and 7.5 log CFU/cm². Desiccation (at 15 °C for 20 days, 43% RH) commenced immediately (non-biofilm) or following biofilm formation (at 15 °C for 48 h, 100% RH). To study the impact of food lipids, non-biofilm L. monocytogenes cells were suspended in TSB-glu containing either canola oil (5-10%) or lard (20-60%) and desiccated as above on SS coupons. Following desiccation for 20 days, survivors decreased by 1.4-3.7 log CFU/cm² for non-biofilm L. monocytogenes cells. The contamination level had no significant (p > 0.05) effect on survival kinetics. SEM micrographs showed mature biofilms on coupons initially contaminated with 5.5 and 7.5 log CFU/cm². Mature biofilm cells were significantly (p < 0.05) more desiccation resistant than cells in immature biofilms formed by the lowest contamination level. Besides biofilm maturity/formation, previous osmoadaptation, exposure to lard (20-60%) or salt (5%) during desiccation significantly (p < 0.05) increased the bacterium's survival. In conclusion, L. monocytogenes desiccation survival can be greatly reduced by preventing presence of mature biofilms and salty or fatty soils on food contact surfaces.

  17. Impact of the Maturation of Human Primary Bone-Forming Cells on Their Behavior in Acute or Persistent Staphylococcus aureus Infection Models

    PubMed Central

    Josse, Jérôme; Guillaume, Christine; Bour, Camille; Lemaire, Flora; Mongaret, Céline; Draux, Florence; Velard, Frédéric; Gangloff, Sophie C.

    2016-01-01

    Staphylococcus aureus is one of the most frequently involved pathogens in bacterial infections such as skin abscess, pneumonia, endocarditis, osteomyelitis, and implant-associated infection. As for bone homeostasis, it is partly altered during infections by S. aureus by the induction of various responses from osteoblasts, which are the bone-forming cells responsible for extracellular matrix synthesis and its mineralization. Nevertheless, bone-forming cells are a heterogeneous population with different stages of maturation and the impact of the latter on their responses toward bacteria remains unclear. We describe the impact of S. aureus on two populations of human primary bone-forming cells (HPBCs) which have distinct maturation characteristics in both acute and persistent models of interaction. Cell maturation did not influence the internalization and survival of S. aureus inside bone-forming cells or the cell death related to the infection. By studying the expression of chemokines, cytokines, and osteoclastogenic regulators by HPBCs, we observed different profiles of chemokine expression according to the degree of cell maturation. However, there was no statistical difference in the amounts of proteins released by both populations in the presence of S. aureus compared to the non-infected counterparts. Our findings show that cell maturation does not impact the behavior of HPBCs infected with S. aureus and suggest that the role of bone-forming cells may not be pivotal for the inflammatory response in osteomyelitis. PMID:27446812

  18. Mature Biofilm Degradation by Potential Probiotics: Aggregatibacter actinomycetemcomitans versus Lactobacillus spp.

    PubMed Central

    Mizuno, Kouhei; Okinaga, Toshinori

    2016-01-01

    The biofilm degradation of Aggregatibacter actinomycetemcomitans is essential as a complete periodontal disease therapy, and here we show the effects of potential probiotic bacteria such as Lactobacillus spp. for the biofilm of several serotypes of A. actinomycetemcomitans strains. Eight of the 13 species showed the competent biofilm degradation of ≥ 90% reduction in biofilm values in A. actinomycetemcomitans Y4 (serotype b) as well as four of the seven species for the biofilm of A. actinomycetemcomitans OMZ 534 (serotype e). In contrast, the probiotic bacteria did not have a big impact for the degradation of A. actinomycetemcomitans SUNY 75 (serotype a) biofilm. The dispersed A. actinomycetemcomitans Y4 cells through the biofilm detachment were still viable and plausible factors for the biofilm degradation were not due to the lactic acid and low pH conditions. The three enzymes, protease, lipase, and amylase may be responsible for the biofilm degradation; in particular, lipase was the most effective enzyme for the biofilm degradation of A. actinomycetemcomitans Y4 along with the protease activity which should be also important for the other serotypes. Remarkable lipase enzyme activities were detected from some of the potential probiotics and a supporting result using a lipase inhibitor presented corroborating evidence that lipase activity is one of the contributing factors for biofilm degradation outside of the protease which is also another possible factor for the biofilm of the other serotype of A. actinomycetemcomitans strains. On the other hand, the biofilm of A. actinomycetemcomitans SUNY 75 (serotype a) was not powerfully degraded by the lipase enzyme because the lipase inhibitor was slightly functional for only two of potential probiotics. PMID:27438340

  19. Biofilms.

    PubMed

    Callow, J A; Callow, M E

    2006-01-01

    Biofilms of bacteria, frequently in association with algae, protozoa and fungi, are found on all submerged structures in the marine environment. Although it is likely that for the majority of organisms a biofilmed surface is not a pre-requisite for settlement, in practice, colonization by spores and larvae of fouling organisms almost always takes place via a biofilmed surface. Therefore, the properties of the latter may be expected to influence colonization, positively or negatively. Biofilms are responsible for a range of surface-associated and diffusible signals, which may moderate the settling behaviour of cells, spores and larvae. However, there is no consensus view regarding either cause and effect or the mechanism(s) by which biofilms moderate settlement. Studies with mixed biofilms, especially field experiments, are difficult to interpret because of the conflicting signals produced by different members of the biofilm community as well as their spatial organisation. Molecular techniques highlight the deficiencies of culture methods in identifying biofilm bacteria; hence, the strains with the most impact on settlement of spores and larvae may not yet have been isolated and cultured. Furthermore, secondary products isolated from cultured organisms may not reflect the situation that pertains in nature. The evidence that bacterial quorum sensing signal molecules stimulate settlement of spores of the green macroalga, Ulva, is discussed in some detail. New molecular and analytical tools should provide the opportunity to improve our fundamental understanding of the interactions between fouling organisms and biofilms, which in turn may inform novel strategies to control biofouling.

  20. 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.

    PubMed

    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.

  1. In Vivo Monitoring of Staphylococcus aureus Biofilm Infections and Antimicrobial Therapy by [18F]Fluoro-Deoxyglucose–MicroPET in a Mouse Model

    PubMed Central

    Garrido, Victoria; Collantes, María; Barberán, Montserrat; Peñuelas, Iván; Arbizu, Javier; Amorena, Beatriz

    2014-01-01

    A mouse model was developed for in vivo monitoring of infection and the effect of antimicrobial treatment against Staphylococcus aureus biofilms, using the [18F]fluoro-deoxyglucose–MicroPET ([18F]FDG-MicroPET) image technique. In the model, sealed Vialon catheters were briefly precolonized with S. aureus strains ATCC 15981 or V329, which differ in cytotoxic properties and biofilm matrix composition. After subcutaneous implantation of catheters in mice, the S. aureus strain differences found in bacterial counts and the inflammatory reaction triggered were detected by the regular bacteriological and histological procedures and also by [18F]FDG-MicroPET image signal intensity determinations in the infection area and regional lymph node. Moreover, [18F]FDG-MicroPET imaging allowed the monitoring of the rifampin treatment effect, identifying the periods of controlled infection and those of reactivated infection due to the appearance of bacteria naturally resistant to rifampin. Overall, the mouse model developed may be useful for noninvasive in vivo determinations in studies on S. aureus biofilm infections and assessment of new therapeutic approaches. PMID:25155589

  2. In vitro effectiveness of 455-nm blue LED to reduce the load of Staphylococcus aureus and Candida albicans biofilms in compact bone tissue.

    PubMed

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

    2016-01-01

    The aim of this study was to evaluate the effectiveness of a 455-nm blue light-emitting diode (LED), at different application times, to reduce the load of Staphylococcus aureus and Candida albicans biofilms applied to compact bone tissue. The microorganisms S. aureus (ATCC 25923) and C. albicans (ATCC 18804) were used to form biofilms on 160 specimens of compact bones that had been divided into eight experimental groups (n = 10) for each microorganism, according to the times of application of the 455-nm blue LED (1, 2, 3, 4, 5, 7, and 10 min) with an irradiance of 75 mW/cm2. After LED application, decimal dilutions of microorganisms were performed, plated on BHI or Sabouraud agar and incubated for 24 h/35 °C to obtain CFU/mL counts. The findings were statistically analyzed using a ANOVA 5 %. For the group of S. aureus biofilms, all groups of 455-nm LED application differ compared with the control group (p < 0.05), in which no treatment was given. The largest reduction was obtained in the group receiving LED for 10 min (p = 0.00); within this group, a 3.2 log reduction was observed. For the C. albicans biofilms, only those samples receiving 3, 7, and 10 min of LED application presented a significant difference compared with the control group (p < 0.00), indicating that longer application times are required to achieve efficacy. The results of this study show that 455-nm LED light was effective to reduce the load of S. aureus and C. albicans biofilms, especially during 10 min of application.

  3. Cadexomer iodine provides superior efficacy against bacterial wound biofilms in vitro and in vivo.

    PubMed

    Fitzgerald, Daniel J; Renick, Paul J; Forrest, Emma C; Tetens, Shannon P; Earnest, David N; McMillan, Jillian; Kiedaisch, Brett M; Shi, Lei; Roche, Eric D

    2017-01-01

    Examination of clinical samples indicates bacterial biofilms are present in the majority of chronic wounds, and substantial evidence suggests biofilms contribute significantly to delayed healing. Bacteria in biofilms are highly tolerant of antimicrobials, and little data exist to guide the choice of anti-biofilm wound therapy. Cadexomer iodine (CI) was recently reported to have superior efficacy compared to diverse wound dressings against Pseudomonas aeruginosa biofilms in an ex vivo model. In the current study, the strong performance of CI vs. P. aeruginosa biofilm was confirmed using colony and colony drip-flow in vitro wound biofilm models. Similar in vitro efficacy of CI was also demonstrated against mature Staphylococcus aureus biofilms using the same models. Additionally, the rapid kill of mature S. aureus and P. aeruginosa colony biofilms was visualized by confocal microscopy using Live/Dead fluorescent stains. Superior in vitro efficacy of CI vs. staphylococcal biofilms was further demonstrated against methicillin-resistant S. aureus (MRSA) using multiple biofilm models with log reduction, Live/Dead, and metabolic endpoints. Comparator antimicrobial dressings, including silver-based dressings used throughout and other active agents used in individual models, elucidated only limited effects against the mature biofilms. Given the promising in vitro activity, CI was tested in an established mouse model of MRSA wound biofilm. CI had significantly greater impact on MRSA biofilm in mouse wounds than silver dressings or mupirocin based on Gram-stained histology sections and quantitative microbiology from biopsy samples (>4 log reduction in CFU/g vs. 0.7-1.6, p < 0.0001). The superior efficacy for CI in these in vitro and in vivo models suggests CI topical products may represent a better choice to address established bacterial biofilm in chronic wounds.

  4. Synthetic amphibian peptides and short amino-acids derivatives against planktonic cells and mature biofilm of Providencia stuartii clinical strains.

    PubMed

    Ostrowska, Kinga; Kamysz, Wojciech; Dawgul, Małgorzata; Różalski, Antoni

    2014-01-01

    Over the last decade, the growing number of multidrug resistant strains limits the use of many of the currently available chemotherapeutic agents. Furthermore, bacterial biofilm, due to its complex structure, constitutes an effective barrier to conventional antibiotics. The in vitro activities of naturally occurring peptide (Citropin 1.1), chemically engineered analogue (Pexiganan), newly-designed, short amino-acid derivatives (Pal-KK-NH2, Pal-KKK-NH2, Pal-RRR-NH2) and six clinically used antimicrobial agents (Gatifloxacin, Ampicilin, Cefotaxime, Ceftriaxone, Cefuroxime and Cefalexin) were investigated against planktonic cells and mature biofilm of multidrug-resistant Providencia stuartii strains, isolated from urological catheters. The MICs, MBCs values were determined by broth microdilution technique. Inhibition of biofilm formation by antimicrobial agents as well as biofilm susceptibility assay were tested using a surrogate model based on the Crystal Violet method. The antimicrobial activity of amino-acids derivatives and synthetic peptides was compared to that of clinically used antibiotics. For planktonic cells, MICs of peptides and antibiotics ranged between 1 and 256 μg/ml and 256 and ≥ 2048 μg/ml, respectively. The MBCs values of Pexiganan, Citropin 1.1 and amino-acids derivatives were between 16 and 256 μg/ml, 64 and 256 μg/ml and 16 and 512 μg/ml, respectively. For clinically used antibiotics the MBCs values were above 2048 μg/ml. All of the tested peptides and amino-acids derivatives, showed inhibitory activity against P. stuartii biofilm formation, in relation to their concentrations. Pexiganan and Citropin 1.1 in concentration range 32 and 256 μg/ml caused both strong and complete suppression of biofilm formation. None of the antibiotics caused complete inhibition of biofilm formation process. The biofilm susceptibility assay verified the extremely poor antibiofilm activity of conventional antibiotics compared to synthetic peptides. The

  5. Proteome Analyses of Staphylococcus aureus Biofilm at Elevated Levels of NaCl.

    PubMed

    Islam, Nazrul; Ross, Julia M; Marten, Mark R

    2015-10-01

    Our studies demonstrate that sodium chloride (NaCl) induces changes in biofilm, mediated by increased production of polysaccharides intercellular adhesion (PIA). We identified 12 proteins that showed higher abundance in increased level of NaCl. This includes one important protein (IsaA) known to be associated with biofilm stability. In addition, we also found higher abundance of a cold shock protein, CspA, at higher NaCl. We have also identified several other proteins that are differentially expressed to the elevated levels of NaCl and mapped them in the regulatory pathways of PIA. The majority of proteins are involved with various aspects bacterial metabolic function. Our results demonstrated that NaCl influences gene regulatory networks controlling exopolysaccharide expression.

  6. Disruption of Methicillin-resistant Staphylococcus aureus Biofilms with Enzymatic Therapeutics

    DTIC Science & Technology

    2015-04-29

    concentrations of α- amylase , lysostaphin, bromelain, or papain for 2 or 24 hours (n=3 to 4). Biofilm biomass was quantified using the crystal violet...polysaccharide matrix and bacteria from the growth surface. α- Amylase , bromelain, and papain caused removal of most of the polysaccharide matrix...clinically or can easily transition to the clinical setting. These enzymes included an anti-polysaccharide agent, α- amylase , an anti-peptidoglycan agent

  7. Gel-Entrapped Staphylococcus aureus Bacteria as Models of Biofilm Infection Exhibit Growth in Dense Aggregates, Oxygen Limitation, Antibiotic Tolerance, and Heterogeneous Gene Expression

    PubMed Central

    Pabst, Breana; Pitts, Betsey; Lauchnor, Ellen

    2016-01-01

    An experimental model that mimicked the structure and characteristics of in vivo biofilm infections, such as those occurring in the lung or in dermal wounds where no biomaterial surface is present, was developed. In these infections, microbial biofilm forms as cell aggregates interspersed in a layer of mucus or host matrix material. This structure was modeled by filling glass capillary tubes with an agarose gel that had been seeded with Staphylococcus aureus bacteria and then incubating the gel biofilm in medium for up to 30 h. Confocal microscopy showed that the bacteria formed in discrete pockets distributed throughout the gel matrix. These aggregates enlarged over time and also developed a size gradient, with the clusters being larger near the nutrient- and oxygen-supplied interface and smaller at greater depths. Bacteria entrapped in gels for 24 h grew slowly (specific growth rate, 0.06 h−1) and were much less susceptible to oxacillin, minocycline, or ciprofloxacin than planktonic cells. Microelectrode measurements showed that the oxygen concentration decreased with depth into the gel biofilm, falling to values less than 3% of air saturation at depths of 500 μm. An anaerobiosis-responsive green fluorescent protein reporter gene for lactate dehydrogenase was induced in the region of the gel where the measured oxygen concentrations were low, confirming biologically relevant hypoxia. These results show that the gel biofilm model captures key features of biofilm infection in mucus or compromised tissue: formation of dense, distinct aggregates, reduced specific growth rates, local hypoxia, and antibiotic tolerance. PMID:27503656

  8. Plectranthus amboinicus leaf extract mediated synthesis of zinc oxide nanoparticles and its control of methicillin resistant Staphylococcus aureus biofilm and blood sucking mosquito larvae

    NASA Astrophysics Data System (ADS)

    Vijayakumar, S.; Vinoj, G.; Malaikozhundan, B.; Shanthi, S.; Vaseeharan, B.

    2015-02-01

    In this study, zinc oxide nanoparticles were biologically synthesized using the leaf extract of Plectranthus amboinicus (Pam-ZnO NPs). The synthesized Pam-ZnO NPs were characterized by UV-Vis spectrophotometer, FTIR, TEM and XRD analysis. TEM analysis of Pam-ZnO NPs showed the average size of about 20-50 nm. Pam-ZnO NPs control the growth of methicillin-resistant Staphylococcus aureus biofilms (MRSA ATCC 33591) at the concentration of 8-10 μg/ml. Confocal laser scanning microscope (CLSM) images revealed that Pam-ZnO NPs strongly inhibited the biofilm forming ability of S. aureus. In addition, Pam-ZnO NPs showed 100% mortality of fourth instar mosquito larvae of Anopheles stephensi, Culex quinquefasciatus and Culex tritaeniorhynchus at the concentration of 8 and 10 μg/ml. The histopathological studies of Pam-ZnO NPs treated A. stephensi and C. quinquefasciatus larvae revealed the presence of damaged cells and tissues in the mid-gut. The damaged tissues suffered major changes including rupture and disintegration of epithelial layer and cellular vacuolization. The present study conclude that Pam-ZnO NPs showed effective control of S. aureus biofilms and mosquito larvae by damaging the mid gut cells.

  9. Plectranthus amboinicus leaf extract mediated synthesis of zinc oxide nanoparticles and its control of methicillin resistant Staphylococcus aureus biofilm and blood sucking mosquito larvae.

    PubMed

    Vijayakumar, S; Vinoj, G; Malaikozhundan, B; Shanthi, S; Vaseeharan, B

    2015-02-25

    In this study, zinc oxide nanoparticles were biologically synthesized using the leaf extract of Plectranthus amboinicus (Pam-ZnO NPs). The synthesized Pam-ZnO NPs were characterized by UV-Vis spectrophotometer, FTIR, TEM and XRD analysis. TEM analysis of Pam-ZnO NPs showed the average size of about 20-50 nm. Pam-ZnO NPs control the growth of methicillin-resistant Staphylococcus aureus biofilms (MRSA ATCC 33591) at the concentration of 8-10 μg/ml. Confocal laser scanning microscope (CLSM) images revealed that Pam-ZnO NPs strongly inhibited the biofilm forming ability of S. aureus. In addition, Pam-ZnO NPs showed 100% mortality of fourth instar mosquito larvae of Anopheles stephensi, Culex quinquefasciatus and Culex tritaeniorhynchus at the concentration of 8 and 10 μg/ml. The histopathological studies of Pam-ZnO NPs treated A. stephensi and C. quinquefasciatus larvae revealed the presence of damaged cells and tissues in the mid-gut. The damaged tissues suffered major changes including rupture and disintegration of epithelial layer and cellular vacuolization. The present study conclude that Pam-ZnO NPs showed effective control of S. aureus biofilms and mosquito larvae by damaging the mid gut cells.

  10. Activity of daptomycin on biofilms produced on a plastic support by Staphylococcus spp.

    PubMed

    Roveta, S; Marchese, A; Schito, G C

    2008-04-01

    The aim of this study was to assess whether the novel lipopeptide daptomycin might be capable of disrupting or inhibiting the synthesis of biofilms produced by staphylococci. Fourteen recently isolated slime-producing methicillin-susceptible (MET-S) and methicillin-resistant (MET-R) strains (three MET-S Staphylococcus aureus, three MET-R S. aureus, three MET-S Staphylococcus epidermidis, three MET-R S. epidermidis and two vancomycin-intermediate S. aureus (VISA)) were tested. Slime formation on polystyrene plates was quantified spectrophotometrically. Daptomycin (2-64 mg/L) inhibited slime synthesis by > or =80% in MET-S strains, by 60-80% in MET-R S. aureus and by 70-95% in MET-R S. epidermidis. At 64 mg/L, biofilm synthesis decreased by 80% in the VISA isolates. Daptomycin also disrupted pre-formed biofilm: >50% breakdown of initial biofilm (5h) was observed in all strains. Disruption of mature biofilms (48 h), in terms of percentage, was more variable depending on the strain, ranging from ca. 20% in a MET-R S. epidermidis strain to almost 70% in two MET-S strains (one S. aureus and one S. epidermidis). Daptomycin at concentrations achievable during therapy promoted a statistically significant inhibition of slime synthesis (preventing biofilm building) and induced slime disruption (disaggregating its structure) both in initial and mature biofilms on a plastic support in all staphylococcal strains studied.

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

    DTIC Science & Technology

    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...inoculated with wild-type or mutant S. aureus on postoperative day 3. Bac- terial solutions were diluted such that each wound was inoculated with a...antibiotics (Mupirocin 2% oint- ment; Teva Pharmaceuticals, Sellersville, Pa.) were applied on postoperative day 4 to eliminate free-floating, planktonic

  12. Candida albicans Mycofilms Support Staphylococcus aureus Colonization and Enhances Miconazole Resistance in Dual-Species Interactions

    PubMed Central

    Kean, Ryan; Rajendran, Ranjith; Haggarty, Jennifer; Townsend, Eleanor M.; Short, Bryn; Burgess, Karl E.; Lang, Sue; Millington, Owain; Mackay, William G.; Williams, Craig; Ramage, Gordon

    2017-01-01

    Polymicrobial inter-kingdom biofilm infections represent a clinical management conundrum. The presence of co-isolation of bacteria and fungi complicates the ability to routinely administer single antimicrobial regimens, and synergy between the microorganisms influences infection severity. We therefore investigated the nosocomial pathogens Staphylococcus aureus and Candida albicans with respect to antimicrobial intervention. We characterized the interaction using biofilm assays and evaluated the effect of miconazole treatment using in vitro and in vivo assays. Finally, we assessed the impact of biofilm extracellular matrix (ECM) on these interactions. Data indicated that the C. albicans mycofilms supported adhesion and colonization by S. aureus through close interactions with hyphal elements, significantly increasing S. aureus biofilm formation throughout biofilm maturation. Miconazole sensitivity was shown to be reduced in both mono- and dual-species biofilms compared to planktonic cells. Within a three-dimensional biofilm model sensitivity was also hindered. Galleria mellonella survival analysis showed both enhanced pathogenicity of the dual-species infection, which was concomitantly desensitized to miconazole treatment. Analysis of the ECM revealed the importance of extracellular DNA, which supported the adhesion of S. aureus and the development of the dual-species biofilm structures. Collectively, these data highlight the clinical importance of dual-species inter-kingdom biofilm infections, though also provides translational opportunities to manage them more effectively. PMID:28280487

  13. Decreased Staphylococcus aureus biofilm formation on nanomodified endotracheal tubes: a dynamic airway model.

    PubMed

    Machado, Mary C; Tarquinio, Keiko M; Webster, Thomas J

    2012-01-01

    Ventilator-associated pneumonia (VAP) is a serious and costly clinical problem. Specifically, receiving mechanical ventilation for over 24 hours increases the risk of VAP and is associated with high morbidity, mortality, and medical costs. Cost-effective endotracheal tubes (ETTs) that are resistant to bacterial infections could help prevent this problem. The objective of this study was to determine differences in the growth of Staphylococcus aureus on nanomodified and unmodified polyvinyl chloride (PVC) ETTs under dynamic airway conditions simulating a ventilated patient. PVC ETTs were modified to have nanometer surface features by soaking them in Rhizopus arrhisus, a fungal lipase. Twenty-four-hour experiments (supported by computational models) showed that airflow conditions within the ETT influenced both the location and the concentration of bacterial growth on the ETTs, especially within areas of tube curvature. More importantly, experiments revealed a 1.5 log reduction in the total number of S. aureus on the novel nanomodified ETTs compared with the conventional ETTs after 24 hours of airflow. This dynamic study showed that lipase etching can create nanorough surface features on PVC ETTs that suppress S. aureus growth, and thus may provide clinicians with an effective and inexpensive tool to combat VAP.

  14. Decreased Staphylococcus aureus biofilm formation on nanomodified endotracheal tubes: a dynamic airway model

    PubMed Central

    Machado, Mary C; Tarquinio, Keiko M; Webster, Thomas J

    2012-01-01

    Ventilator-associated pneumonia (VAP) is a serious and costly clinical problem. Specifically, receiving mechanical ventilation for over 24 hours increases the risk of VAP and is associated with high morbidity, mortality, and medical costs. Cost-effective endotracheal tubes (ETTs) that are resistant to bacterial infections could help prevent this problem. The objective of this study was to determine differences in the growth of Staphylococcus aureus on nanomodified and unmodified polyvinyl chloride (PVC) ETTs under dynamic airway conditions simulating a ventilated patient. PVC ETTs were modified to have nanometer surface features by soaking them in Rhizopus arrhisus, a fungal lipase. Twenty-four-hour experiments (supported by computational models) showed that airflow conditions within the ETT influenced both the location and the concentration of bacterial growth on the ETTs, especially within areas of tube curvature. More importantly, experiments revealed a 1.5 log reduction in the total number of S. aureus on the novel nanomodified ETTs compared with the conventional ETTs after 24 hours of airflow. This dynamic study showed that lipase etching can create nanorough surface features on PVC ETTs that suppress S. aureus growth, and thus may provide clinicians with an effective and inexpensive tool to combat VAP. PMID:22904622

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

    PubMed

    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%).

  16. Relationship between multiple drug resistance and biofilm formation in Staphylococcus aureus isolated from medical and non-medical personnel in Yaounde, Cameroon

    PubMed Central

    Eyoh, Agnes Bedie; Toukam, Michel; Atashili, Julius; Fokunang, Charles; Gonsu, Hortense; Lyonga, Emilia Enjema; Mandi, Henshaw; Ikomey, George; Mukwele, Bertha; Mesembe, Martha; Assoumou, Marie Claire Okomo

    2014-01-01

    Introduction Monitoring the prevalence of nasal carriage of multiple drug resistance (MDR) Staphylococcus aureus (SA) strains in hospital personnel is essential. These strains when transmitted from hospital personnel to patients with already weakened immune states or in-built medical devices, may limit the latter's treatment options. This study aimed at assessing the potential exposure of patients to these MDR SA in a resource-limited hospital setting by assessing the prevalence and relationship between antimicrobial susceptibility and biofilm forming capacity of SA isolates from hospital personnel. Methods A total of 59 bacteria isolates phenotypically identified as Staphylococcus aureus obtained from medical (39) and non-medical personnel (20) in Yaounde were used in the study. Multiple drug resistance defined as resistance to four or more of twelve locally used antibiotics were determined by Kirby Bauer disc diffusion technique whereas quantification of biofilm production was by the microtitre plate method. Results Among the 59 SA isolates, the prevalence of MDR was 50.9%. Among medical personnel 48.7% had MDR as against 55.9% for non-medical personnel (p-value=0.648). The overall percentage of weak biofilm producers was 35.6%. Although the prevalence of weak biofilm formers was higher in isolates from non-medical personnel (40%) than medical personnel (33.3%) the difference was not statistically significant (p-value= 0.246). Slightly less than half (42.9%) of the weak biofilm producers were MDR. Conclusion Considering the high rates of MDR and that slightly less than half of biofilm formers were MDR, these trends need to be monitored regularly among hospital personnel in Yaounde. PMID:25396012

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

    NASA Astrophysics Data System (ADS)

    Montes Aguirre, Melissa Mariluz

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

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

    PubMed Central

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

    2017-01-01

    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. PMID:28071679

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

    PubMed

    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.

  20. Low-level predation by lytic phage phiIPLA-RODI promotes biofilm formation and triggers the stringent response in Staphylococcus aureus.

    PubMed

    Fernández, Lucía; González, Silvia; Campelo, Ana Belén; Martínez, Beatriz; Rodríguez, Ana; García, Pilar

    2017-01-19

    An important lesson from the war on pathogenic bacteria has been the need to understand the physiological responses and evolution of natural microbial communities. Bacterial populations in the environment are generally forming biofilms subject to some level of phage predation. These multicellular communities are notoriously resistant to antimicrobials and, consequently, very difficult to eradicate. This has sparked the search for new therapeutic alternatives, including phage therapy. This study demonstrates that S. aureus biofilms formed in the presence of a non-lethal dose of phage phiIPLA-RODI exhibit a unique physiological state that could potentially benefit both the host and the predator. Thus, biofilms formed under phage pressure are thicker and have a greater DNA content. Also, the virus-infected biofilm displayed major transcriptional differences compared to an untreated control. Significantly, RNA-seq data revealed activation of the stringent response, which could slow down the advance of the bacteriophage within the biofilm. The end result would be an equilibrium that would help bacterial cells to withstand environmental challenges, while maintaining a reservoir of sensitive bacterial cells available to the phage upon reactivation of the dormant carrier population.

  1. Low-level predation by lytic phage phiIPLA-RODI promotes biofilm formation and triggers the stringent response in Staphylococcus aureus

    PubMed Central

    Fernández, Lucía; González, Silvia; Campelo, Ana Belén; Martínez, Beatriz; Rodríguez, Ana; García, Pilar

    2017-01-01

    An important lesson from the war on pathogenic bacteria has been the need to understand the physiological responses and evolution of natural microbial communities. Bacterial populations in the environment are generally forming biofilms subject to some level of phage predation. These multicellular communities are notoriously resistant to antimicrobials and, consequently, very difficult to eradicate. This has sparked the search for new therapeutic alternatives, including phage therapy. This study demonstrates that S. aureus biofilms formed in the presence of a non-lethal dose of phage phiIPLA-RODI exhibit a unique physiological state that could potentially benefit both the host and the predator. Thus, biofilms formed under phage pressure are thicker and have a greater DNA content. Also, the virus-infected biofilm displayed major transcriptional differences compared to an untreated control. Significantly, RNA-seq data revealed activation of the stringent response, which could slow down the advance of the bacteriophage within the biofilm. The end result would be an equilibrium that would help bacterial cells to withstand environmental challenges, while maintaining a reservoir of sensitive bacterial cells available to the phage upon reactivation of the dormant carrier population. PMID:28102347

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

    PubMed Central

    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. PMID:26089817

  3. Rhodomyrtus tomentosa (Aiton) Hassk. ethanol extract and rhodomyrtone: a potential strategy for the treatment of biofilm-forming staphylococci.

    PubMed

    Saising, Jongkon; Ongsakul, Metta; Voravuthikunchai, Supayang Piyawan

    2011-12-01

    The anti-staphylococcal activity of an ethanol extract of Rhodomyrtus tomentosa and its pure compound, rhodomyrtone, as well as their effects on staphylococcal biofilm formation and biofilm-grown cells were assessed. MIC and minimal bactericidal concentration values of the ethanol extract and rhodomyrtone against planktonic cultures and biofilms of five clinical strains each of Staphylococcus aureus and Staphylococcus epidermidis, and American Type Culture Collection (ATCC) strains of both species, were 32-512 and 0.25-2 µg ml(-1), respectively. Results from time-kill studies indicated that rhodomyrtone at a concentration of 4× MIC could reduce the number of Staphylococcus aureus ATCC 25923 and Staphylococcus epidermidis ATCC 35984 cells by 99.9% within 3 and 13 h, respectively. The ability of rhodomyrtone and the ethanol extract to prevent biofilm formation and kill mature biofilms was assessed: both demonstrated better activity than vancomycin at inhibiting staphylococcal biofilm formation. In addition, the viability of 24 h and 5-day staphylococcal biofilm-grown cells decreased after treatment with the ethanol extract and rhodomyrtone. The ability to reduce biofilm formation and kill mature biofilms occurred in a dose-dependent manner. Scanning electron microscopy clearly confirmed that treatment with rhodomyrtone at 16× MIC could reduce 24 h biofilm formation and the numbers of staphylococci, whilst at 64× MIC this compound destroyed the organisms in the 5-day established biofilm. These results suggest that rhodomyrtone has the potential for further drug development for the treatment of biofilm-forming staphylococcal infections.

  4. Influence of Adhesion Force on icaA and cidA Gene Expression and Production of Matrix Components in Staphylococcus aureus Biofilms

    PubMed Central

    Harapanahalli, Akshay K.; Chen, Yun; Li, Jiuyi; Busscher, Henk J.

    2015-01-01

    The majority of human infections are caused by biofilms. The biofilm mode of growth enhances the pathogenicity of Staphylococcus spp. considerably, because once they adhere, staphylococci embed themselves in a protective, self-produced matrix of extracellular polymeric substances (EPSs). The aim of this study was to investigate the influence of forces of staphylococcal adhesion to different biomaterials on icaA (which regulates the production of EPS matrix components) and cidA (which is associated with cell lysis and extracellular DNA [eDNA] release) gene expression in Staphylococcus aureus biofilms. Experiments were performed with S. aureus ATCC 12600 and its isogenic mutant, S. aureus ATCC 12600 Δpbp4, deficient in peptidoglycan cross-linking. Deletion of pbp4 was associated with greater cell wall deformability, while it did not affect the planktonic growth rate, biofilm formation, cell surface hydrophobicity, or zeta potential of the strains. The adhesion forces of S. aureus ATCC 12600 were the strongest on polyethylene (4.9 ± 0.5 nN), intermediate on polymethylmethacrylate (3.1 ± 0.7 nN), and the weakest on stainless steel (1.3 ± 0.2 nN). The production of poly-N-acetylglucosamine, eDNA presence, and expression of icaA genes decreased with increasing adhesion forces. However, no relation between adhesion forces and cidA expression was observed. The adhesion forces of the isogenic mutant S. aureus ATCC 12600 Δpbp4 (deficient in peptidoglycan cross-linking) were much weaker than those of the parent strain and did not show any correlation with the production of poly-N-acetylglucosamine, eDNA presence, or expression of the icaA and cidA genes. This suggests that adhesion forces modulate the production of the matrix molecule poly-N-acetylglucosamine, eDNA presence, and icaA gene expression by inducing nanoscale cell wall deformation, with cross-linked peptidoglycan layers playing a pivotal role in this adhesion force sensing. PMID:25746995

  5. In vitro study of biofilm growth on biologic prosthetics.

    PubMed

    Bellows, Charles; Smith, Alison

    2014-01-01

    Biologic prosthetics are increasingly used for the repair of abdominal wall hernia defects but can become infected as a result of peri- or early post-operative bacterial contamination. Data evaluating biofilm formation on biologic prosthetics is lacking. The aim of this study was to investigate the influence of different biologic prosthetics on the growth behavior of two different bacterial species and their ability to form biofilms. Methicillin resistant Staphylococcus aureus (MRSA) or Pseudomrnonas aeruginosa were incubated on disks of two biologic prosthetics-human acellular dermis (ADM), and porcine small intestinal submucosa (SIS). The bacteria were allowed to attach to the prosthetics and propagate into mature biofilms for 24 hours at 370C. Images of biofilms were obtained using confocal microscopy and scanning electron microscopy (SEM). The number of viable cells and the biofilm biomass were quantified by colony forming units (CFUs) and crystal violet staining respectively. Analysis of variance was performed to compare the mean values for the different prosthetics. Each biologic matrix had a distinct surface characteristic. SEM visualized mature biofilms characterized by highly organized multi-cellular structures on surface of both biologic prosthetics. Quantification of bacterial growth over time showed that ADM had the lowest CFUs and biofilm biomass at 24 hours post-inoculation compared to SIS for both bacterial strains. MRSA and P. aeruginosa can form mature biofilms on biologic prosthetics but the relative abundance of the biofilm varies on different prosthetic constructs. Biologic material composition and manufacturing methods may influence bacterial adherence.

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

    PubMed

    de la Fuente-Núñez, César; Mansour, Sarah C; Wang, Zhejun; Jiang, Lucy; Breidenstein, Elena B M; Elliott, Melissa; Reffuveille, Fany; Speert, David P; Reckseidler-Zenteno, Shauna L; Shen, Ya; Haapasalo, Markus; Hancock, Robert E W

    2014-01-01

    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.

  7. Evaluation of Antimicrobial Effects of Different Concentrations of Triple Antibiotic Paste on Mature Biofilm of Enterococcus faecalis

    PubMed Central

    Frough Reyhani, Mohammad; Rahimi, Saeed; Fathi, Zahra; Shakouie, Sahar; Salem Milani, Amin; Soroush Barhaghi, Mohammad Hossein; Shokri, Javad

    2015-01-01

    Background and aims. Triple antibiotic paste (TAP) is widely used in endodontics for root canal disinfection, particularly in regenerative procedures. The aim of this in vitro study was to evaluate the antimicrobial effects of different concentrations of TAP at 1-, 2-, 3-, and 4-week intervals on mature Enterococcus faecalis biofilm. Materials and methods. A total of 287 extracted one-rooted human central incisors were infected with E. faecalis ATCC 29212 after removing the crown and preparation. The root canal space was filled with one of the 0.01-, 0.1-, 1-, 10-, 100-, and 1000-mg/mL concentrations of TAP or normal saline (control). The root canal dentin was sampled after 1, 2, 3, and 4 weeks. The dentinal shavings were cultured on Mueller-Hinton agar plates after serial dilutions. The classic colony-forming unit (CFU) counting technique was used to determine remaining bacterial counts. Data were analyzed by using the two-way ANOVA, post hoc Tukey tests and one-way ANOVA (P<0.05). Results. TAP completely eliminated E. faecalis biofilms at all the intervals at concentrations of 1000, 100, and 10 mg/mL, whereas 1-, 0.1-, and 0.01-mg/mL TAP resulted in significant reduction of CFU means compared with the control group. There were no statistically significant differences between the four time intervals. Conclusion. Use of lower concentrations of TAP at short term could eradicate E. faecalis biofilm and decrease high-concentration side effects. PMID:26697145

  8. A High-Affinity Native Human Antibody Disrupts Biofilm from Staphylococcus aureus Bacteria and Potentiates Antibiotic Efficacy in a Mouse Implant Infection Model

    PubMed Central

    Estellés, Angeles; Woischnig, Anne-Kathrin; Liu, Keyi; Stephenson, Robert; Lomongsod, Evelene; Nguyen, Da; Zhang, Jianzhong; Heidecker, Manfred; Yang, Yifan; Simon, Reyna J.; Tenorio, Edgar; Ellsworth, Stote; Leighton, Anton; Ryser, Stefan; Gremmelmaier, Nina Khanna

    2016-01-01

    Many serious bacterial infections are difficult to treat due to biofilm formation, which provides physical protection and induces a sessile phenotype refractory to antibiotic treatment compared to the planktonic state. A key structural component of biofilm is extracellular DNA, which is held in place by secreted bacterial proteins from the DNABII family: integration host factor (IHF) and histone-like (HU) proteins. A native human monoclonal antibody, TRL1068, has been discovered using single B-lymphocyte screening technology. It has low-picomolar affinity against DNABII homologs from important Gram-positive and Gram-negative bacterial pathogens. The disruption of established biofilm was observed in vitro at an antibody concentration of 1.2 μg/ml over 12 h. The effect of TRL1068 in vivo was evaluated in a murine tissue cage infection model in which a biofilm is formed by infection with methicillin-resistant Staphylococcus aureus (MRSA; ATCC 43300). Treatment of the established biofilm by combination therapy of TRL1068 (15 mg/kg of body weight, intraperitoneal [i.p.] administration) with daptomycin (50 mg/kg, i.p.) significantly reduced adherent bacterial count compared to that after daptomycin treatment alone, accompanied by significant reduction in planktonic bacterial numbers. The quantification of TRL1068 in sample matrices showed substantial penetration of TRL1068 from serum into the cage interior. TRL1068 is a clinical candidate for combination treatment with standard-of-care antibiotics to overcome the drug-refractory state associated with biofilm formation, with potential utility for a broad spectrum of difficult-to-treat bacterial infections. PMID:26833157

  9. The anti-biofilm effect of macrolides in a rat model of S. aureus foreign-body infection: Might it be of clinical relevance?

    PubMed

    El Haj, Cristina; Murillo, Oscar; Ribera, Alba; Garcia-Somoza, Dolors; Tubau, Fe; Cabellos, Carmen; Cabo, Javier; Ariza, Javier

    2017-02-01

    Using a tissue cage infection rat model, we test the anti-biofilm effect of clarithromycin on the efficacy of daptomycin and a daptomycin + rifampicin combination against methicillin-susceptible (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA). In vitro: kill curves, daptomycin exposure studies and clarithromycin activity against biofilm were studied. In vivo: the efficacies of clarithromycin, daptomycin or daptomycin + clarithromycin, daptomycin + rifampicin and daptomycin + rifampicin + clarithromycin combinations were evaluated. In vitro: the addition of clarithromycin to daptomycin improved its activity only against one MRSA strain. Changes in daptomycin MIC values appeared more quickly in MSSA than in MRSA strain, and this was not modified by clarithromycin. Clarithromycin prevented biofilm formation but did not eradicate it. In vivo: the daptomycin + rifampicin combination was the most effective treatment and was not improved by the addition of clarithromycin. Daptomycin and daptomycin + clarithromycin had similar effectiveness; the combination protected against the appearance of daptomycin resistance only in one MRSA strain. Using a staphylococcal foreign-body infection model, we observed a slight effect with the addition of clarithromycin to daptomycin, which resulted in protection against the appearance of daptomycin-resistant strains. However, efficacy was not improved. Overall, our findings do not support a relevant clinical role for macrolides in treating device-related staphylococcal infections based on their anti-biofilm effect.

  10. 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.

    PubMed

    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.

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

    PubMed

    Luther, Megan K; LaPlante, Kerry L

    2015-12-01

    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.

  12. Herpes Simplex Virus (HSV) Modulation of Staphylococcus aureus and Candida albicans Initiation of HeLa 299 Cell-Associated Biofilm.

    PubMed

    Plotkin, Balbina J; Sigar, Ira M; Tiwari, Vaibhav; Halkyard, Scott

    2016-05-01

    Although herpes simplex virus type-1 (HSV-1), and type-2 (HSV-2), Staphylococcus aureus and Candida albicans co-habit the oral and genital mucosa, their interaction is poorly understood. We determined the effect HSV has on bacterial and/or fungal adherence, the initial step in biofilm formation. HeLa229 cells were infected with HSV-1 (KOS) gL86 or HSV-2 (KOS) 333gJ (-) at a multiplicity of infection (MOI) of 50 and 10. S. aureus (ATCC 25923) and/or C. albicans (yeast forms or germ tube forms) were co-incubated for 30 min (37 °C; 5 % CO2; 5:1 organism: HeLa cell ratio; n = 16) with virus-infected HeLa cells or uninfected HeLa cell controls. Post-incubation, the monolayers were washed (3x; PBS), lysed (RIPA), and the lysate plated onto Fungisel and/or mannitol salts agar for standard colony count. The level of HeLa-associated S. aureus was significantly decreased (P < 0.05) for both HSV-1- and HSV-2-infected cells, as compared to virus-free HeLa cell controls (38 and 59 % of control, respectively). In contrast, HSV-1 and HSV-2 significantly (P < 0.05) enhanced HeLa cell association of C. albicans yeast forms and germ tube approximately two-fold, respectively. The effect of S. aureus on germ tube and yeast form adherence to HSV-1- and HSV-2-infected cells was specific for the Candida phenotype tested. Our study suggests that HSV, while antagonist towards S. aureus adherence enhances Candida adherence. Furthermore, the combination of the three pathogens results in S. aureus adherence that is either unaffected, or partially restored depending on both the herpes viral species and the fungal phenotype present.

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

    PubMed Central

    Reddinger, Ryan M.; Luke-Marshall, Nicole R.

    2016-01-01

    ABSTRACT 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. PMID:27507829

  14. Uniformity of glycyl bridge lengths in the mature cell walls of fem mutants of methicillin-resistant Staphylococcus aureus.

    PubMed

    Sharif, Shasad; Kim, Sung Joon; Labischinski, Harald; Chen, Jiawei; Schaefer, Jacob

    2013-04-01

    Peptidoglycan (PG) composition in intact cells of methicillin-resistant Staphylococcus aureus (MRSA) and its isogenic Fem mutants has been characterized by measuring the glycine content of PG bridge structures by solid-state nuclear magnetic resonance (NMR). The glycine content estimated from integrated intensities (rather than peak heights) in the cell walls of whole cells was increased by approximately 30% for the FemA mutant and was reduced by 25% for the FemB mutant relative to expected values for homogeneous structures. In contrast, the expected compositions were observed in isolated cell walls of the same mutants. For FemA mutant whole cells, the increase was due to the presence of triglycyl bridge PG units (confirmed directly by mass spectrometric analysis), which constituted 10% of the total PG. These species were coalesced in some sort of a lattice or aggregate with spatial proximity to other PG bridges. This result suggests that the triglycyl-bridged PG units form a PG-like structure that is not incorporated into the mature cell wall.

  15. Escherichia coli harboring a natural IncF conjugative F plasmid develops complex mature biofilms by stimulating synthesis of colanic acid and Curli.

    PubMed

    May, Thithiwat; Okabe, Satoshi

    2008-11-01

    It has been shown that Escherichia coli harboring the derepressed IncFI and IncFII conjugative F plasmids form complex mature biofilms by using their F-pilus connections, whereas a plasmid-free strain forms only patchy biofilms. Therefore, in this study we investigated the contribution of a natural IncF conjugative F plasmid to the formation of E. coli biofilms. Unlike the presence of a derepressed F plasmid, the presence of a natural IncF F plasmid promoted biofilm formation by generating the cell-to-cell mating F pili between pairs of F(+) cells (approximately two to four pili per cell) and by stimulating the formation of colanic acid and curli meshwork. Formation of colanic acid and curli was required after the initial deposition of F-pilus connections to generate a three-dimensional mushroom-type biofilm. In addition, we demonstrated that the conjugative factor of F plasmid, rather than a pilus synthesis function, was involved in curli production during biofilm formation, which promoted cell-surface interactions. Curli played an important role in the maturation process. Microarray experiments were performed to identify the genes involved in curli biosynthesis and regulation. The results suggested that a natural F plasmid was more likely an external activator that indirectly promoted curli production via bacterial regulatory systems (the EnvZ/OmpR two-component regulators and the RpoS and HN-S global regulators). These data provided new insights into the role of a natural F plasmid during the development of E. coli biofilms.

  16. Arginine deiminase in Staphylococcus epidermidis functions to augment biofilm maturation through pH homeostasis.

    PubMed

    Lindgren, J K; Thomas, V C; Olson, M E; Chaudhari, S S; Nuxoll, A S; Schaeffer, C R; Lindgren, K E; Jones, J; Zimmerman, M C; Dunman, P M; Bayles, K W; Fey, P D

    2014-06-01

    Allelic replacement mutants were constructed within arginine deiminase (arcA1 and arcA2) to assess the function of the arginine deiminase (ADI) pathway in organic acid resistance and biofilm formation of Staphylococcus epidermidis 1457. A growth-dependent acidification assay (pH ∼5.0 to ∼5.2) determined that strain 1457 devoid of arginine deiminase activity (1457 ΔADI) was significantly less viable than the wild type following depletion of glucose and in the presence of arginine. However, no difference in viability was noted for individual 1457 ΔarcA1 (native) or ΔarcA2 (arginine catabolic mobile element [ACME]-derived) mutants, suggesting that the native and ACME-derived ADIs are compensatory in S. epidermidis. Furthermore, flow cytometry and electron paramagnetic resonance spectroscopy results suggested that organic acid stress resulted in oxidative stress that could be partially rescued by the iron chelator dipyridyl. Collectively, these results suggest that formation of hydroxyl radicals is partially responsible for cell death via organic acid stress and that ADI-derived ammonia functions to counteract this acid stress. Finally, static biofilm assays determined that viability, ammonia synthesis, and pH were reduced in strain 1457 ΔADI following 120 h of growth in comparison to strain 1457 and the arcA1 and arcA2 single mutants. It is hypothesized that ammonia synthesis via the ADI pathway is important to reduce pH stress in specific microniches that contain high concentrations of organic acids.

  17. Antimicrobial Resistance, Biofilm Formation and mecA Characterization of Methicillin-Susceptible S. aureus and Non-S. aureus of Beef Meat Origin in Egypt.

    PubMed

    Osman, Kamelia M; Amer, Aziza M; Badr, Jihan M; Helmy, Nashwa M; Elhelw, Rehab A; Orabi, Ahmed; Bakry, Magdy; Saad, Aalaa S A

    2016-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) have been found in various farm animal species throughout the world. Yet, methicillin-susceptible S. aureus (MSSA), methicillin-susceptible non-S. aureus (MS-NSA), and methicillin-resistant non-S. aureus (MR-NSA) were not investigated. Therefore, we persued to determine the diversity in their phenotypic virulence assay, phenotypic antimicrobial resistance profile and molecular characterization in one of the food chains in Egypt. Samples were collected during 2013 from beef meat at retail. Twenty seven isolates comprising five species (S. hyicus, S. aureus, S. schleiferi subsp. coagulans, S. intermedius, and S. lentus) were characterized for their antibiotic resistance phenotypic profile and antibiotic resistance genes (mecA, cfr, gyrA, gyrB, and grlA). Out of the 27 Staphylococcus isolates only one isolate was resistant to the 12 antibiotics representing nine classes. Raw beef meat sold across the Great Cairo zone, contains 66.7% of MRS, with highest prevalence was reported in S. aureus (66.7%), while the MRS non-S. aureus strains constituted 66.7% from which S. hyicus (60%), S. intermedius (33.3%), S. schleiferi subsp. coagulans (100%), and S. lentus (100%) were MRS. Seven S. aureus, six S. hyicus, four S. schleiferi subsp. coagulans, three S. intermedius, and one S. lentus isolates although being resistant to oxacillin yet, 11/27 (40.7%) carried the mecA gene. At the same time, the cfr gene was present in 2 of the nine S. aureus isolates, and totally undetectable in S. hyicus, S. schleiferi subsp. coagulans, S. intermedius, and S. lentus. Although, global researches largely focused into MRSA and MR-NSA in animals on pigs, the analysis of our results stipulates, that buffaloes and cattle could be MRSA dispersers and that this theme is not specific to pigs. Detection of MSSA virulence determinants is a must, as although oxacillin resistance may be absent yet, the MSSA may carry the virulence determinants which

  18. Antimicrobial Resistance, Biofilm Formation and mecA Characterization of Methicillin-Susceptible S. aureus and Non-S. aureus of Beef Meat Origin in Egypt

    PubMed Central

    Osman, Kamelia M.; Amer, Aziza M.; Badr, Jihan M.; Helmy, Nashwa M.; Elhelw, Rehab A.; Orabi, Ahmed; Bakry, Magdy; Saad, Aalaa S. A.

    2016-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) have been found in various farm animal species throughout the world. Yet, methicillin-susceptible S. aureus (MSSA), methicillin-susceptible non-S. aureus (MS-NSA), and methicillin-resistant non-S. aureus (MR-NSA) were not investigated. Therefore, we persued to determine the diversity in their phenotypic virulence assay, phenotypic antimicrobial resistance profile and molecular characterization in one of the food chains in Egypt. Samples were collected during 2013 from beef meat at retail. Twenty seven isolates comprising five species (S. hyicus, S. aureus, S. schleiferi subsp. coagulans, S. intermedius, and S. lentus) were characterized for their antibiotic resistance phenotypic profile and antibiotic resistance genes (mecA, cfr, gyrA, gyrB, and grlA). Out of the 27 Staphylococcus isolates only one isolate was resistant to the 12 antibiotics representing nine classes. Raw beef meat sold across the Great Cairo zone, contains 66.7% of MRS, with highest prevalence was reported in S. aureus (66.7%), while the MRS non-S. aureus strains constituted 66.7% from which S. hyicus (60%), S. intermedius (33.3%), S. schleiferi subsp. coagulans (100%), and S. lentus (100%) were MRS. Seven S. aureus, six S. hyicus, four S. schleiferi subsp. coagulans, three S. intermedius, and one S. lentus isolates although being resistant to oxacillin yet, 11/27 (40.7%) carried the mecA gene. At the same time, the cfr gene was present in 2 of the nine S. aureus isolates, and totally undetectable in S. hyicus, S. schleiferi subsp. coagulans, S. intermedius, and S. lentus. Although, global researches largely focused into MRSA and MR-NSA in animals on pigs, the analysis of our results stipulates, that buffaloes and cattle could be MRSA dispersers and that this theme is not specific to pigs. Detection of MSSA virulence determinants is a must, as although oxacillin resistance may be absent yet, the MSSA may carry the virulence determinants which

  19. Effects of photodynamic laser and violet-blue led irradiation on Staphylococcus aureus biofilm and Escherichia coli lipopolysaccharide attached to moderately rough titanium surface: in vitro study.

    PubMed

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

    2017-03-10

    Effective decontamination of biofilm and bacterial toxins from the surface of dental implants is a yet unresolved issue. This study investigates the in vitro efficacy of photodynamic treatment (PDT) with methylene blue (MB) photoactivated with λ 635 nm diode laser and of λ 405 nm violet-blue LED phototreatment for the reduction of bacterial biofilm and lipopolysaccharide (LPS) adherent to titanium surface mimicking the bone-implant interface. Staphylococcus aureus biofilm grown on titanium discs with a moderately rough surface was subjected to either PDT (0.1% MB and λ 635 nm diode laser) or λ 405 nm LED phototreatment for 1 and 5 min. Bactericidal effect was evaluated by vital staining and residual colony-forming unit count. Biofilm and titanium surface morphology were analyzed by scanning electron microscopy (SEM). In parallel experiments, discs coated with Escherichia coli LPS were treated as above before seeding with RAW 264.7 macrophages to quantify LPS-driven inflammatory cell activation by measuring the enhanced generation of nitric oxide (NO). Both PDT and LED phototreatment induced a statistically significant (p < 0.05 or higher) reduction of viable bacteria, up to -99 and -98% (5 min), respectively. Moreover, besides bactericidal effect, PDT and LED phototreatment also inhibited LPS bioactivity, assayed as nitrite formation, up to -42%, thereby blunting host inflammatory response. Non-invasive phototherapy emerges as an attractive alternative in the treatment of peri-implantitis to reduce bacteria and LPS adherent to titanium implant surface without causing damage of surface microstructure. Its efficacy in the clinical setting remains to be investigated.

  20. Temporal expression of agrB, cidA, and alsS in the early development of Staphylococcus aureus UAMS-1 biofilm formation and the structural role of extracellular DNA and carbohydrates.

    PubMed

    Grande, Rossella; Nistico, Laura; Sambanthamoorthy, Karthik; Longwell, Mark; Iannitelli, Antonio; Cellini, Luigina; Di Stefano, Antonio; Hall Stoodley, Luanne; Stoodley, Paul

    2014-04-01

    Extracellular DNA (eDNA) is an important component of the extracellular polymeric substance matrix and is important in the establishment and persistence of Staphylococcus aureus UAMS-1 biofilms. The aim of the study was to determine the temporal expression of genes involved in early biofilm formation and eDNA production. We used qPCR to investigate expression of agrB, which is associated with secreted virulence factors and biofilm dispersal, cidA, which is associated with biofilm adherence and genomic DNA release, and alsS, which is associated with cell lysis, eDNA release and acid tolerance. The contribution of eDNA to the stability of the biofilm matrix was assessed by digesting with DNase I (Pulmozyme) and quantifying structure by confocal microscopy and comstat image analysis. AgrB expression initially increased at 24 h but then dramatically decreased at 72 h in an inverse relationship to biomass, supporting its role in regulating biofilm dispersal. cidA and alsS expression steadily increased over 72 h, suggesting that eDNA was an important component of early biofilm development. DNase I had no effect on biomass, but did cause the biofilms to become more heterogeneous. Carbohydrates in the matrix appeared to play an important role in structural stability.

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

    PubMed

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

    2014-03-01

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

  2. 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.

    PubMed

    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.

  3. Dynamic remodeling of microbial biofilms by functionally distinct exopolysaccharides.

    PubMed

    Chew, Su Chuen; Kundukad, Binu; Seviour, Thomas; van der Maarel, Johan R C; Yang, Liang; Rice, Scott A; Doyle, Patrick; Kjelleberg, Staffan

    2014-08-05

    Biofilms are densely populated communities of microbial cells protected and held together by a matrix of extracellular polymeric substances. The structure and rheological properties of the matrix at the microscale influence the retention and transport of molecules and cells in the biofilm, thereby dictating population and community behavior. Despite its importance, quantitative descriptions of the matrix microstructure and microrheology are limited. Here, particle-tracking microrheology in combination with genetic approaches was used to spatially and temporally study the rheological contributions of the major exopolysaccharides Pel and Psl in Pseudomonas aeruginosa biofilms. Psl increased the elasticity and effective cross-linking within the matrix, which strengthened its scaffold and appeared to facilitate the formation of microcolonies. Conversely, Pel reduced effective cross-linking within the matrix. Without Psl, the matrix becomes more viscous, which facilitates biofilm spreading. The wild-type biofilm decreased in effective cross-linking over time, which would be advantageous for the spreading and colonization of new surfaces. This suggests that there are regulatory mechanisms to control production of the exopolysaccharides that serve to remodel the matrix of developing biofilms. The exopolysaccharides were also found to have profound effects on the spatial organization and integration of P. aeruginosa in a mixed-species biofilm model of P. aeruginosa-Staphylococcus aureus. Pel was required for close association of the two species in mixed-species microcolonies. In contrast, Psl was important for P. aeruginosa to form single-species biofilms on top of S. aureus biofilms. Our results demonstrate that Pel and Psl have distinct physical properties and functional roles during biofilm formation. Importance: Most bacteria grow as biofilms in the environment or in association with eukaryotic hosts. Removal of biofilms that form on surfaces is a challenge in clinical

  4. Potential novel therapeutic strategies in cystic fibrosis: antimicrobial and anti-biofilm activity of natural and designed α-helical peptides against Staphylococcus aureus, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia

    PubMed Central

    2012-01-01

    Background Treatment of cystic fibrosis-associated lung infections is hampered by the presence of multi-drug resistant pathogens, many of which are also strong biofilm producers. Antimicrobial peptides, essential components of innate immunity in humans and animals, exhibit relevant in vitro antimicrobial activity although they tend not to select for resistant strains. Results Three α-helical antimicrobial peptides, BMAP-27 and BMAP-28 of bovine origin, and the artificial P19(9/B) peptide were tested, comparatively to Tobramycin, for their in vitro antibacterial and anti-biofilm activity against 15 Staphylococcus aureus, 25 Pseudomonas aeruginosa, and 27 Stenotrophomonas maltophilia strains from cystic fibrosis patients. All assays were carried out in physical-chemical experimental conditions simulating a cystic fibrosis lung. All peptides showed a potent and rapid bactericidal activity against most P. aeruginosa, S. maltophilia and S. aureus strains tested, at levels generally higher than those exhibited by Tobramycin and significantly reduced biofilm formation of all the bacterial species tested, although less effectively than Tobramycin did. On the contrary, the viability-reducing activity of antimicrobial peptides against preformed P. aeruginosa biofilms was comparable to and, in some cases, higher than that showed by Tobramycin. Conclusions The activity shown by α-helical peptides against planktonic and biofilm cells makes them promising “lead compounds” for future development of novel drugs for therapeutic treatment of cystic fibrosis lung disease. PMID:22823964

  5. Quantifying the natural history of biofilm formation in vivo during the establishment of chronic implant-associated Staphylococcus aureus osteomyelitis in mice to identify critical pathogen and host factors

    PubMed Central

    Nishitani, Kohei; Sutipornpalangkul, Werasak; de Mesy Bentley, Karen L.; Varrone, John J.; Bello-Irizarry, Sheila N.; Ito, Hiromu; Matsuda, Shuichi; Kates, Stephen L.; Daiss, John L.; Schwarz, Edward M.

    2015-01-01

    While it is well known that Staphylococcus aureus establishes chronic implant-associated osteomyelitis by generating and persisting in biofilm, research to elucidate pathogen and host specific factors controlling this process has been limited due to the absence of a quantitative in vivo model. To address this, we developed a murine tibia implant model with ex vivo region of interest (ROI) imaging analysis by scanning electron microscopy (SEM). Implants were coated with Staphylococcus aureus strains (SH1000, UAMS-1, USA300LAC) with distinct in vitro biofilm phenotypes, were used to infect C57BL/6 or Balb/c mice. In contrast to their in vitro biofilm phenotype, results from all bacteria strains in vivo were similar, and demonstrated that biofilm on the implant is established within the first day, followed by a robust proliferation phase peaking on Day 3 in Balb/c mice, and persisting until Day 7 in C57BL/6 mice, as detected by SEM and bioluminescent imaging. Biofilm formation peaked at Day 14, covering ~40% of the ROI coincident with massive agr-dependent bacterial emigration, as evidenced by large numbers of empty lacunae with few residual bacteria, which were largely culture negative (80%) and PCR positive (87.5%), supporting the clinical relevance of this implant model. PMID:25820925

  6. 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

    PubMed Central

    Beltrame, Cristiana Ossaille; Côrtes, Marina Farrel; Bonelli, Raquel Regina; Côrrea, Ana Beatriz de Almeida; Botelho, Ana Maria Nunes; Américo, Marco Antônio; Fracalanzza, Sérgio Eduardo Longo; Figueiredo, Agnes Marie Sá

    2015-01-01

    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. PMID:26406329

  7. Preferential colonization and release of Legionella pneumophila from mature drinking water biofilms grown on copper versus unplasticized polyvinylchloride coupons

    EPA Science Inventory

    Legionella persistence and amplification in premise drinking water systems is a known contributor to legionellosis outbreaks, especially in the presence of suitable eukaryotic hosts. Here we examined Legionella pneumophila behavior within drinking water biofilms grown on copper ...

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

    PubMed

    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.

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

    PubMed Central

    Gowrishankar, Shanmugaraj; Kamaladevi, Arumugam; Balamurugan, Krishnaswamy

    2016-01-01

    The present investigation was deliberately aimed at evaluating the biofilm-forming ability of 63 clinical MRSA isolates recovered from pharyngitis patients through different phenotypic assays. The molecular detection of adhesion (icaA/icaD/icaB/icaC), adhesins (fnbA/fnbB, clfA, and cna), staphylococcal accessory regulator (sarA), and α-toxin (hla) genes was done by employing polymerase chain reaction (PCR). Out of 63 isolates, 49 (77.8%) were found slime positive by the Congo red agar (CRA) method and 44 (69.8%) as biofilm positive by the quantitative microtitre plate assays. The results of MATH assay showed that most of the test pathogens are hydrophilic in nature. The molecular investigation of biofilm-associated genes revealed that 84.13% (n = 53) of isolates were found positive for icaADBC genes. The fnbA and fnbB genes were present in 49 (77.8%) and 51 (81%) MRSA isolates, respectively. In addition, 58.7% (n = 37), 73% (n = 46), and 69.8% (n = 44) of the isolates harboured the clfA, cna, and hla genes, respectively. Further, nearly 81% (n = 51) of the isolates were found positive for the gene sarA and all the ica negative isolates were also negative for the gene. Furthermore, the results of in vivo adherence assay unveiled the factual commonness in the in vitro adherence method. PMID:27761465

  10. Internalisation potential of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica subsp. enterica serovar Typhimurium and Staphylococcus aureus in lettuce seedlings and mature plants.

    PubMed

    Standing, Taryn-Ann; du Plessis, Erika; Duvenage, Stacey; Korsten, Lise

    2013-06-01

    The internalisation potential of Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7 and Salmonella enterica subsp. enterica serovar Typhimurium in lettuce was evaluated using seedlings grown in vermiculite in seedling trays as well as hydroponically grown lettuce. Sterile distilled water was spiked with one of the four human pathogenic bacteria (10(5) CFU/mL) and used to irrigate the plants. The potential for pathogen internalisation was investigated over time using light microscopy, transmission electron microscopy and viable plate counts. Additionally, the identities of the pathogens isolated from internal lettuce plant tissues were confirmed using polymerase chain reaction with pathogen-specific oligonucleotides. Internalisation of each of the human pathogens was evident in both lettuce seedlings and hydroponically grown mature lettuce plants. To our knowledge, this is the first report of S. aureus internalisation in lettuce plants. In addition, the levels of background microflora in the lettuce plants were determined by plate counting and the isolates identified using matrix-assisted laser ionisation-time of flight (MALDI-TOF). Background microflora assessments confirmed the absence of the four pathogens evaluated in this study. A low titre of previously described endophytes and soil inhabitants, i.e., Enterobacter cloacae, Enterococcus faecalis, Lysinibacillus fusiformis, Rhodococcus rhodochrous, Staphylococcus epidermidis and Staphylococcus hominis were identified.

  11. Existence of two groups of Staphylococcus aureus strains isolated from bovine mastitis based on biofilm formation, intracellular survival, capsular profile and agr-typing.

    PubMed

    Bardiau, Marjorie; Caplin, Jonathan; Detilleux, Johann; Graber, Hans; Moroni, Paolo; Taminiau, Bernard; Mainil, Jacques G

    2016-03-15

    Staphylococcus (S.) aureus is recognised worldwide as an important pathogen causing contagious acute and chronic bovine mastitis. Chronic mastitis account for a significant part of all bovine cases and represent an important economic problem for dairy producers. Several properties (biofilm formation, intracellular survival, capsular expression and group agr) are thought to be associated with this chronic status. In a previous study, we found the existence of two groups of strains based on the association of these features. The aim of the present work was to confirm on a large international and non-related collection of strains the existence of these clusters and to associate them with case history records. In addition, the genomes of eight strains were sequenced to study the genomic differences between strains of each cluster. The results confirmed the existence of both groups based on capsular typing, intracellular survival and agr-typing: strains cap8-positive, belonging to agr group II, showing a low invasion rate and strains cap5-positive, belonging to agr group I, showing a high invasion rate. None of the two clusters were associated with the chronic status of the cow. When comparing the genomes of strains belonging to both clusters, the genes specific to the group "cap5-agrI" would suggest that these strains are better adapted to live in hostile environment. The existence of these two groups is highly important as they may represent two clusters that are adapted differently to the host and/or the surrounding environment.

  12. Inhibition of Staphylococcus aureus biofilm by a copper-bearing 317L-Cu stainless steel and its corrosion resistance.

    PubMed

    Sun, Da; Xu, Dake; Yang, Chunguang; Chen, Jia; Shahzad, M Babar; Sun, Ziqing; Zhao, Jinlong; Gu, Tingyue; Yang, Ke; Wang, Guixue

    2016-12-01

    The present study investigated the antibacterial performance, corrosion resistance and surface properties of antibacterial austenitic 317L-Cu stainless steel (317L-Cu SS). After 4.5wt% copper was added to 317L stainless steel (317L SS), the new alloy underwent solid solution and aging heat treatment. Fluorescent staining using 4',6-diamidino-2-phenylindole (DAPI) revealed that the 317L-Cu SS showed strong antibacterial efficacy, achieving a 99% inhibition rate of sessile Staphylococcus aureus cells after 5days. The corrosion data obtained by potentiodynamic polarization curves indicated that in comparison with 317L SS, the pitting potential and corrosion current density of 317L-Cu slightly decreased due to the addition of Cu. The 317L-Cu SS exhibited no cytotoxicity against zebrafish (Danio rerio) embryos. The experimental results in this study demonstrated that the new alloy has potential applications in medical and daily uses.

  13. Screening of Escherichia coli Species Biodiversity Reveals New Biofilm-Associated Antiadhesion Polysaccharides

    PubMed Central

    Rendueles, Olaya; Travier, Laetitia; Latour-Lambert, Patricia; Fontaine, Thierry; Magnus, Julie; Denamur, Erick; Ghigo, Jean-Marc

    2011-01-01

    ABSTRACT Bacterial biofilms often form multispecies communities in which complex but ill-understood competition and cooperation interactions occur. In light of the profound physiological modifications associated with this lifestyle, we hypothesized that the biofilm environment might represent an untapped source of natural bioactive molecules interfering with bacterial adhesion or biofilm formation. We produced cell-free solutions extracted from in vitro mature biofilms formed by 122 natural Escherichia coli isolates, and we screened these biofilm extracts for antiadhesion molecules active on a panel of Gram-positive and Gram-negative bacteria. Using this approach, we showed that 20% of the tested biofilm extracts contained molecules that antagonize bacterial growth or adhesion. We characterized a compound, produced by a commensal animal E. coli strain, for which activity is detected only in biofilm extract. Biochemical and genetic analyses showed that this compound corresponds to a new type of released high-molecular-weight polysaccharide whose biofilm-associated production is regulated by the RfaH protein. We demonstrated that the antiadhesion activity of this polysaccharide was restricted to Gram-positive bacteria and that its production reduced susceptibility to invasion and provided rapid exclusion of Staphylococcus aureus from mixed E. coli and S. aureus biofilms. Our results therefore demonstrate that biofilms contain molecules that contribute to the dynamics of mixed bacterial communities and that are not or only poorly detected in unconcentrated planktonic supernatants. Systematic identification of these compounds could lead to strategies that limit pathogen surface colonization and reduce the burden associated with the development of bacterial biofilms on medical devices. PMID:21558434

  14. Evaluation of Ceftaroline Alone and in Combination against Biofilm-Producing Methicillin-Resistant Staphylococcus aureus with Reduced Susceptibility to Daptomycin and Vancomycin in an In Vitro Pharmacokinetic/Pharmacodynamic Model

    PubMed Central

    Barber, Katie E.; Smith, Jordan R.; Ireland, Cortney E.; Boles, Blaise R.; Rose, Warren E.

    2015-01-01

    Annually, medical device infections are associated with >250,000 catheter-associated bloodstream infections (CLABSI), with up to 25% mortality. Staphylococcus aureus, a primary pathogen in these infections, is capable of biofilm production, allowing organism persistence in harsh environments, offering antimicrobial protection. With increases in S. aureus isolates with reduced susceptibility to current agents, ceftaroline (CPT) offers a therapeutic alternative. Therefore, we evaluated whether CPT would have a role against biofilm-producing methicillin-resistant S. aureus (MRSA), including those with decreased susceptibilities to alternative agents. In this study, we investigated CPT activity alone or combined with daptomycin (DAP) or rifampin (RIF) against 3 clinical biofilm-producing MRSA strains in an in vitro biofilm pharmacokinetic/pharmacodynamic (PK/PD) model. Simulated antimicrobial regimens were as follows: 600 mg of CPT every 8 h (q8h) (free maximum concentration of drug [fCmax], 17.04 mg/liter; elimination half-life [t1/2], 2.66 h), 12 mg/kg of body weight/day of DAP (fCmax, 14.7 mg/liter; t1/2, 8 h), and 450 mg of RIF q12h (fCmax, 3.5 mg/liter; t1/2, 3.4 h), CPT plus DAP, and CPT plus RIF. Samples were obtained and plated to determine colony counts. Differences in log10 CFU/cm2 were evaluated by analysis of variance with Tukey's post hoc test. The strains were CPT and vancomycin susceptible and DAP nonsusceptible (DNS). CPT displayed activity throughout the experiment. DAP demonstrated initial activity with regrowth at 24 h in all strains. RIF was comparable to the drug-free control, and little benefit was observed when combined with CPT. CPT plus DAP displayed potent activity, with an average log10 CFU/cm2 reduction of 3.33 ± 1.01 from baseline. CPT demonstrated activity against biofilm-producing DNS MRSA. CPT plus DAP displayed therapeutic enhancement over monotherapy, providing a potential option for difficult-to-treat medical device infections. PMID

  15. Inhibitory effects of Lactobacillus fermentum on microbial growth and biofilm formation.

    PubMed

    Rybalchenko, Oxana V; Bondarenko, Viktor M; Orlova, Olga G; Markov, Alexander G; Amasheh, S

    2015-10-01

    Beneficial effects of Lactobacilli have been reported, and lactic bacteria are employed for conservation of foods. Therefore, the effects of a Lactobacillus fermentum strain were analyzed regarding inhibitory effects on staphylococci, Candida albicans and enterotoxigenic enterobacteria by transmission electron microscopy (TEM). TEM of bacterial biofilms was performed using cocultures of bacteriocin-producing L. fermentum 97 with different enterotoxigenic strains: Staphylococcus epidermidis expressing the ica gene responsible for biofilm formation, Staphylococcus aureus producing enterotoxin type A, Citrobacter freundii, Enterobacter cloaceae, Klebsiella oxytoca, Proteus mirabilis producing thermolabile and thermostable enterotoxins determined by elt or est genes, and Candida albicans. L. fermentum 97 changed morphological features and suppressed biofilm formation of staphylococci, enterotoxigenic enterobacteria and Candida albicans; a marked transition to resting states, a degradation of the cell walls and cytoplasm, and a disruption of mature bacterial biofilms were observed, the latter indicating efficiency even in the phase of higher cell density.

  16. In vitro activity of tea-tree oil against clinical skin isolates of meticillin-resistant and -sensitive Staphylococcus aureus and coagulase-negative staphylococci growing planktonically and as biofilms.

    PubMed

    Brady, Aaron; Loughlin, Ryan; Gilpin, Deirdre; Kearney, Paddy; Tunney, Michael

    2006-10-01

    The susceptibility of Staphylococcus aureus [meticillin-resistant (MRSA) and meticillin-sensitive (MSSA)] and coagulase-negative staphylococci (CoNS), which respectively form part of the transient and commensal skin flora, to tea-tree oil (TTO) was compared using broth microdilution and quantitative in vitro time-kill test methods. MRSA and MSSA isolates were significantly less susceptible than CoNS isolates, as measured by both MIC and minimum bactericidal concentration. A significant decrease in the mean viable count of all isolates in comparison with the control was seen at each time interval in time-kill assays. However, the only significant difference in the overall mean log10 reduction in viable count between the groups of isolates was between CoNS and MSSA at 3 h, with CoNS isolates demonstrating a significantly lower mean reduction. To provide a better simulation of in vivo conditions on the skin, where bacteria are reported to grow as microcolonies encased in glycocalyx, the bactericidal activity of TTO against isolates grown as biofilms was also compared. Biofilms formed by MSSA and MRSA isolates were completely eradicated following exposure to 5 % TTO for 1 h. In contrast, of the biofilms formed by the nine CoNS isolates tested, only five were completely killed, although a reduction in viable count was apparent for the other four isolates. These results suggest that TTO exerts a greater bactericidal activity against biofilm-grown MRSA and MSSA isolates than against some biofilm-grown CoNS isolates.

  17. Efficacy of antiseptics containing povidone-iodine, octenidine dihydrochloride and ethacridine lactate against biofilm formed by Pseudomonas aeruginosa and Staphylococcus aureus measured with the novel biofilm-oriented antiseptics test.

    PubMed

    Junka, Adam; Bartoszewicz, Marzenna; Smutnicka, Danuta; Secewicz, Anna; Szymczyk, Patrycja

    2014-12-01

    Increasing data suggesting that microorganisms in the biofilm form are among the leading agents of persistent infections of chronic wounds require the development of new approaches to treatment. The aim of this article was to compare the efficacy of three commonly used antiseptics using a biofilm-oriented approach. Biofilm-oriented antiseptics test (BOAT), the innovative method, allows to estimate, in a quick and reliable manner, the in vitro activity of working solutions of antiseptics in real contact times against bacteria in the biofilm form and to use the results in the selection of an appropriate antiseptic to treat local infections in the clinical practice.

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

    PubMed

    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.

  19. Bacterial biofilms: prokaryotic adventures in multicellularity.

    PubMed

    Webb, Jeremy S; Givskov, Michael; Kjelleberg, Staffan

    2003-12-01

    The development of bacterial biofilms includes both the initial social behavior of undifferentiated cells, as well as cell death and differentiation in the mature biofilm, and displays several striking similarities with higher organisms. Recent advances in the field provide new insight into differentiation and cell death events in bacterial biofilm development and propose that biofilms have an unexpected level of multicellularity.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2017-01-01

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

  3. Glycerol Monolaurate Antibacterial Activity in Broth and Biofilm Cultures

    PubMed Central

    Schlievert, Patrick M.; Peterson, Marnie L.

    2012-01-01

    Background Glycerol monolaurate (GML) is an antimicrobial agent that has potent activity against gram-positive bacteria. This study examines GML antibacterial activity in comparison to lauric acid, in broth cultures compared to biofilm cultures, and against a wide range of gram-positive, gram-negative, and non-gram staining bacteria. Methodology/Principal Findings GML is ≥200 times more effective than lauric acid in bactericidal activity, defined as a ≥3 log reduction in colony-forming units (CFU)/ml, against Staphylococcus aureus and Streptococcus pyogenes in broth cultures. Both molecules inhibit superantigen production by these organisms at concentrations that are not bactericidal. GML prevents biofilm formation by Staphylococcus aureus and Haemophilus influenzae, as representative gram-positive and gram-negative organisms, tested in 96 well microtiter plates, and simultaneously is bactericidal for both organisms in mature biofilms. GML is bactericidal for a wide range of potential bacterial pathogens, except for Pseudomonas aeruginosa and Enterobacteriaceae. In the presence of acidic pH and the cation chelator ethylene diamine tetraacetic acid, GML has greatly enhanced bactericidal activity for Pseudomonas aeruginosa and Enterobacteriaceae. Solubilization of GML in a nonaqueous delivery vehicle (related to K-Y Warming®) enhances its bactericidal activity against S. aureus. Both R and S, and 1 and 2 position lauric acid derivatives of GML exhibit bactericidal activity. Despite year-long passage of Staphylococcus aureus on sub-growth inhibitory concentrations of GML (0.5 x minimum bactericidal concentration), resistance to GML did not develop. Conclusions/Significance GML may be useful as a broad-spectrum human or animal topical microbicide and may be useful as an environmental surface microbicide for management of bacterial infections and contamination. PMID:22808139

  4. Analysis of structure and composition of bacterial core communities in mature drinking water biofilms and bulk water of a citywide network in Germany.

    PubMed

    Henne, Karsten; Kahlisch, Leila; Brettar, Ingrid; Höfle, Manfred G

    2012-05-01

    The bacterial core communities of bulk water and corresponding biofilms of a more than 20-year-old drinking water network were compared using 16S rRNA single-strand confirmation polymorphism (SSCP) fingerprints based on extracted DNA and RNA. The structure and composition of the bacterial core community in the bulk water was highly similar (>70%) across the city of Braunschweig, Germany, whereas all biofilm samples contained a unique community with no overlapping phylotypes from bulk water. Biofilm samples consisted mainly of Alphaproteobacteria (26% of all phylotypes), Gammaproteobacteria (11%), candidate division TM6 (11%), Chlamydiales (9%), and Betaproteobacteria (9%). The bulk water community consisted primarily of Bacteroidetes (25%), Betaproteobacteria (20%), Actinobacteria (16%), and Alphaproteobacteria (11%). All biofilm communities showed higher relative abundances of single phylotypes and a reduced richness compared to bulk water. Only biofilm communities sampled at nearby sampling points showed similar communities irrespective of support materials. In all of our bulk water studies, the community composition determined from 16S rRNA was completely different from the 16S rRNA gene-based community composition, whereas in biofilms both molecular fractions resulted in community compositions that were similar to each other. We hypothesize that a higher fraction of active bacterial phylotypes and a better protection from oxidative stress in drinking water biofilms are responsible for this higher similarity.

  5. Analysis of Structure and Composition of Bacterial Core Communities in Mature Drinking Water Biofilms and Bulk Water of a Citywide Network in Germany

    PubMed Central

    Henne, Karsten; Kahlisch, Leila; Brettar, Ingrid

    2012-01-01

    The bacterial core communities of bulk water and corresponding biofilms of a more than 20-year-old drinking water network were compared using 16S rRNA single-strand confirmation polymorphism (SSCP) fingerprints based on extracted DNA and RNA. The structure and composition of the bacterial core community in the bulk water was highly similar (>70%) across the city of Braunschweig, Germany, whereas all biofilm samples contained a unique community with no overlapping phylotypes from bulk water. Biofilm samples consisted mainly of Alphaproteobacteria (26% of all phylotypes), Gammaproteobacteria (11%), candidate division TM6 (11%), Chlamydiales (9%), and Betaproteobacteria (9%). The bulk water community consisted primarily of Bacteroidetes (25%), Betaproteobacteria (20%), Actinobacteria (16%), and Alphaproteobacteria (11%). All biofilm communities showed higher relative abundances of single phylotypes and a reduced richness compared to bulk water. Only biofilm communities sampled at nearby sampling points showed similar communities irrespective of support materials. In all of our bulk water studies, the community composition determined from 16S rRNA was completely different from the 16S rRNA gene-based community composition, whereas in biofilms both molecular fractions resulted in community compositions that were similar to each other. We hypothesize that a higher fraction of active bacterial phylotypes and a better protection from oxidative stress in drinking water biofilms are responsible for this higher similarity. PMID:22389373

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

    NASA Astrophysics Data System (ADS)

    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.

  7. Anti-bacterial and Anti-biofilm Evaluation of Thiazolopyrimidinone Derivatives Targeting the Histidine Kinase YycG Protein of Staphylococcus epidermidis

    PubMed Central

    Lv, Zhihui; Zhao, Dan; Chang, Jun; Liu, Huayong; Wang, Xiaofei; Zheng, Jinxin; Huang, Renzheng; Lin, Zhiwei; Shang, Yongpeng; Ye, Lina; Wu, Yang; Han, Shiqing; Qu, Di

    2017-01-01

    Staphylococcus epidermidis is one of the most important opportunistic pathogens in nosocomial infections. The main pathogenicity associated with S. epidermidis involves the formation of biofilms on implanted medical devices, biofilms dramatically decrease the efficacy of conventional antibiotics and the host immune system. This emphasizes the urgent need for designing novel anti-staphylococcal biofilm agents. Based on the findings that compound 5, targeting the histidine kinase domain of S. epidermidis YycG, possessed bactericidal activity against staphylococci, 39 derivatives of compound 5 with intact thiazolopyrimidinone core structures were newly designed, 7 derivatives were further screened to explore their anti-bacterial and anti-biofilm activities. The seven derivatives strongly inhibited the growth of S. epidermidis and Staphylococcus aureus in the minimal inhibitory concentration range of 1.56–6.25 μM. All the derivatives reduced the proportion of viable cells in mature biofilms. They all displayed low cytotoxicity on mammalian cells and were not hemolytic to human erythrocytes. The biofilm inhibition activities of four derivatives (H5-32, H5-33, H5-34, and H5-35) were further investigated under shearing forces, they all led to significant decreases in the biofilm formation of S. epidermidis. These results were suggestive that the seven derivatives of compound 5 have the potential to be developed into agents for eradicating biofilm-associated infections.

  8. D-Amino Acids Enhance the Activity of Antimicrobials against Biofilms of Clinical Wound Isolates of Staphylococcus aureus and Pseudomonas aeruginosa

    DTIC Science & Technology

    2014-05-19

    Wagner EH. 1999. Incidence, outcomes, and cost of foot ulcers in patients with diabetes . Diabetes Care 22:382–387. http://dx.doi.org/10 .2337/diacare...McKeehan T, Smith E, Rhoads D. 2008. Polymicrobial nature of chronic diabetic foot ulcer biofilm infections de- termined using bacterial tag encoded...wounds are common in individuals with underlyingmedical conditions, such as diabetes mellitus, as well as in wounds resulting from traumatic injury, and

  9. A blue fluorescent labeling technique utilizing micro- and nanoparticles for tracking in LIVE/DEAD® stained pathogenic biofilms of Staphylococcus aureus and Burkholderia cepacia.

    PubMed

    Klinger-Strobel, Mareike; Ernst, Julia; Lautenschläger, Christian; Pletz, Mathias W; Fischer, Dagmar; Makarewicz, Oliwia

    2016-01-01

    Strategies that target and treat biofilms are widely applied to bacterial cultures using popular live/dead staining techniques with mostly red or green fluorescent markers (eg, with SYTO(®) 9, propidium iodide, fluorescein). Therefore, visualizing drugs or micro- and nanoparticulate delivery systems to analyze their distribution and effects in biofilms requires a third fluorescent dye that does not interfere with the properties of the live/dead markers. The present study establishes and evaluates a model for tracking polymeric particles in fluorescently stained biological material. To this end, poly(D,L-lactide-co-glycolide) (PLGA)-based micro- and nanoparticles were used as well-established model systems, which, because of their favorable safety profiles, are expected to play important future roles with regard to drug delivery via inhalation. PLGA was covalently and stably labeled with 7-amino-4-methyl-3-coumarinylacetic acid (AMCA), after which blue fluorescent poly(ethylene glycol)-block-PLGA (PEG-PLGA) particles were prepared using a mixture of fluorescent AMCA-PLGA and PEG-PLGA. Because chitosan is known to reduce negative surface charge, blue fluorescent PEG-PLGA-particles with chitosan were also prepared. These micro- and nanoparticles were physicochemically characterized and could be clearly distinguished from live/dead stained bacteria in biofilms using confocal laser scanning microscopy.

  10. A blue fluorescent labeling technique utilizing micro- and nanoparticles for tracking in LIVE/DEAD® stained pathogenic biofilms of Staphylococcus aureus and Burkholderia cepacia

    PubMed Central

    Klinger-Strobel, Mareike; Ernst, Julia; Lautenschläger, Christian; Pletz, Mathias W; Fischer, Dagmar; Makarewicz, Oliwia

    2016-01-01

    Strategies that target and treat biofilms are widely applied to bacterial cultures using popular live/dead staining techniques with mostly red or green fluorescent markers (eg, with SYTO® 9, propidium iodide, fluorescein). Therefore, visualizing drugs or micro- and nanoparticulate delivery systems to analyze their distribution and effects in biofilms requires a third fluorescent dye that does not interfere with the properties of the live/dead markers. The present study establishes and evaluates a model for tracking polymeric particles in fluorescently stained biological material. To this end, poly(d,l-lactide-co-glycolide) (PLGA)-based micro- and nanoparticles were used as well-established model systems, which, because of their favorable safety profiles, are expected to play important future roles with regard to drug delivery via inhalation. PLGA was covalently and stably labeled with 7-amino-4-methyl-3-coumarinylacetic acid (AMCA), after which blue fluorescent poly(ethylene glycol)-block-PLGA (PEG-PLGA) particles were prepared using a mixture of fluorescent AMCA-PLGA and PEG-PLGA. Because chitosan is known to reduce negative surface charge, blue fluorescent PEG-PLGA-particles with chitosan were also prepared. These micro- and nanoparticles were physicochemically characterized and could be clearly distinguished from live/dead stained bacteria in biofilms using confocal laser scanning microscopy. PMID:26917959

  11. Antifungal activity against Candida biofilms.

    PubMed

    Iñigo, Melania; Pemán, Javier; Del Pozo, Jose L

    2012-10-01

    Candida species have two distinct lifestyles: planktonic, and surface-attached communities called biofilms. Mature C. albicans biofilms show a complex three-dimensional architecture with extensive spatial heterogeneity, and consist of a dense network of yeast, hyphae, and pseudohyphae encased within a matrix of exopolymeric material. Several key processes are likely to play vital roles at the different stages of biofilm development, such as cell-substrate and cell-cell adherence, hyphal development, and quorum sensing. Biofilm formation is a survival strategy, since biofilm yeasts are more resistant to antifungals and environmental stress. Antifungal resistance is a multifactorial process that includes multidrug efflux pumps, target proteins of the ergosterol biosynthetic pathway. Most studies agree in presenting azoles as agents with poor activity against Candida spp. biofilms. However, recent studies have demonstrated that echinocandins and amphotericin B exhibit remarkable activity against C. albicans and Candida non-albicans biofilms. The association of Candida species with biofilm formation increases the therapeutic complexity of foreign body-related yeast infections. The traditional approach to the management of these infections has been to explant the affected device. There is a strong medical but also economical motivation for the development of novel anti-fungal biofilm strategies due to the constantly increasing resistance of Candida biofilms to conventional antifungals, and the high mortality caused by related infections. A better description of the extent and role of yeast in biofilms may be critical for developing novel therapeutic strategies in the clinical setting.

  12. Intra- and inter-species interactions within biofilms of important foodborne bacterial pathogens

    PubMed Central

    Giaouris, Efstathios; Heir, Even; Desvaux, Mickaël; Hébraud, Michel; Møretrø, Trond; Langsrud, Solveig; Doulgeraki, Agapi; Nychas, George-John; Kačániová, Miroslava; Czaczyk, Katarzyna; Ölmez, Hülya; Simões, Manuel

    2015-01-01

    A community-based sessile life style is the normal mode of growth and survival for many bacterial species. Under such conditions, cell-to-cell interactions are inevitable and ultimately lead to the establishment of dense, complex and highly structured biofilm populations encapsulated in a self-produced extracellular matrix and capable of coordinated and collective behavior. Remarkably, in food processing environments, a variety of different bacteria may attach to surfaces, survive, grow, and form biofilms. Salmonella enterica, Listeria monocytogenes, Escherichia coli, and Staphylococcus aureus are important bacterial pathogens commonly implicated in outbreaks of foodborne diseases, while all are known to be able to create biofilms on both abiotic and biotic surfaces. Particularly challenging is the attempt to understand the complexity of inter-bacterial interactions that can be encountered in such unwanted consortia, such as competitive and cooperative ones, together with their impact on the final outcome of these communities (e.g., maturation, physiology, antimicrobial resistance, virulence, dispersal). In this review, up-to-date data on both the intra- and inter-species interactions encountered in biofilms of these pathogens are presented. A better understanding of these interactions, both at molecular and biophysical levels, could lead to novel intervention strategies for controlling pathogenic biofilm formation in food processing environments and thus improve food safety. PMID:26347727

  13. Acoustic vibration can enhance bacterial biofilm formation.

    PubMed

    Murphy, Mark F; Edwards, Thomas; Hobbs, Glyn; Shepherd, Joanna; Bezombes, Frederic

    2016-12-01

    This paper explores the use of low-frequency-low-amplitude acoustic vibration on biofilm formation. Biofilm development is thought to be governed by a diverse range of environmental signals and much effort has gone into researching the effects of environmental factors including; nutrient availability, pH and temperature on the growth of biofilms. Many biofilm-forming organisms have evolved to thrive in mechanically challenging environments, for example soil yet, the effects of the physical environment on biofilm formation has been largely ignored. Exposure of Pseudomonas aeruginosa to vibration at 100, 800 and 1600 Hz for 48 h, resulted in a significant increase in biofilm formation compared with the control, with the greatest growth seen at 800 Hz vibration. The results also show that this increase in biofilm formation is accompanied with an increase in P. aeruginosa cell number. Acoustic vibration was also found to regulate the spatial distribution of biofilm formation in a frequency-dependent manner. Exposure of Staphylococcus aureus to acoustic vibration also resulted in enhanced biofilm formation with the greatest level of biofilm being formed following 48 h exposure at 1600 Hz. These results show that acoustic vibration can be used to control biofilm formation and therefore presents a novel and potentially cost effective means to manipulate the development and yield of biofilms in a range of important industrial and medical processes.

  14. Tannic acid inhibits Staphylococcus aureus surface colonization in an IsaA-dependent manner.

    PubMed

    Payne, David E; Martin, Nicholas R; Parzych, Katherine R; Rickard, Alex H; Underwood, Adam; Boles, Blaise R

    2013-02-01

    Staphylococcus aureus is a human commensal and pathogen that is capable of forming biofilms on a variety of host tissues and implanted medical devices. Biofilm-associated infections resist antimicrobial chemotherapy and attack from the host immune system, making these infections particularly difficult to treat. In order to gain insight into environmental conditions that influence S. aureus biofilm development, we screened a library of small molecules for the ability to inhibit S. aureus biofilm formation. This led to the finding that the polyphenolic compound tannic acid inhibits S. aureus biofilm formation in multiple biofilm models without inhibiting bacterial growth. We present evidence that tannic acid inhibits S. aureus biofilm formation via a mechanism dependent upon the putative transglycosylase IsaA. Tannic acid did not inhibit biofilm formation of an isaA mutant. Overexpression of wild-type IsaA inhibited biofilm formation, whereas overexpression of a catalytically dead IsaA had no effect. Tannin-containing drinks like tea have been found to reduce methicillin-resistant S. aureus nasal colonization. We found that black tea inhibited S. aureus biofilm development and that an isaA mutant resisted this inhibition. Antibiofilm activity was eliminated from tea when milk was added to precipitate the tannic acid. Finally, we developed a rodent model for S. aureus throat colonization and found that tea consumption reduced S. aureus throat colonization via an isaA-dependent mechanism. These findings provide insight into a molecular mechanism by which commonly consumed polyphenolic compounds, such as tannins, influence S. aureus surface colonization.

  15. Tannic Acid Inhibits Staphylococcus aureus Surface Colonization in an IsaA-Dependent Manner

    PubMed Central

    Payne, David E.; Martin, Nicholas R.; Parzych, Katherine R.; Rickard, Alex H.; Underwood, Adam

    2013-01-01

    Staphylococcus aureus is a human commensal and pathogen that is capable of forming biofilms on a variety of host tissues and implanted medical devices. Biofilm-associated infections resist antimicrobial chemotherapy and attack from the host immune system, making these infections particularly difficult to treat. In order to gain insight into environmental conditions that influence S. aureus biofilm development, we screened a library of small molecules for the ability to inhibit S. aureus biofilm formation. This led to the finding that the polyphenolic compound tannic acid inhibits S. aureus biofilm formation in multiple biofilm models without inhibiting bacterial growth. We present evidence that tannic acid inhibits S. aureus biofilm formation via a mechanism dependent upon the putative transglycosylase IsaA. Tannic acid did not inhibit biofilm formation of an isaA mutant. Overexpression of wild-type IsaA inhibited biofilm formation, whereas overexpression of a catalytically dead IsaA had no effect. Tannin-containing drinks like tea have been found to reduce methicillin-resistant S. aureus nasal colonization. We found that black tea inhibited S. aureus biofilm development and that an isaA mutant resisted this inhibition. Antibiofilm activity was eliminated from tea when milk was added to precipitate the tannic acid. Finally, we developed a rodent model for S. aureus throat colonization and found that tea consumption reduced S. aureus throat colonization via an isaA-dependent mechanism. These findings provide insight into a molecular mechanism by which commonly consumed polyphenolic compounds, such as tannins, influence S. aureus surface colonization. PMID:23208606

  16. Staphylococcus aureus induces hypoxia and cellular damage in porcine dermal explants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methicillin-resistant Staphylococcus aureus (MRSA) can infect wounds and produce difficult-to- treat biofilms. To determine the extent that MRSA biofilms can deplete oxygen, change pH and damage host tissue, we developed a porcine dermal explant model on which we cultured GFP-labeled MRSA biofilms. ...

  17. Presence of extracellular DNA in the Candida albicans biofilm matrix and its contribution to biofilms

    PubMed Central

    Martins, Margarida; Uppuluri, Priya; Thomas, Derek P.; Cleary, Ian A.; Henriques, Mariana; Lopez-Ribot, José L.; Oliveira, Rosário

    2014-01-01

    DNA has been described as a structural component of the extracellular matrix (ECM) in bacterial biofilms. In Candida albicans there is a scarce knowledge concerning the contribution of extracellular DNA (eDNA) to biofilm matrix and overall structure. This work examined the presence and quantified the amount of eDNA in C.albicans biofilm ECM and the effect of DNase treatment and the addition of exogenous DNA on C. albicans biofilm development as indicators of a role for eDNA in biofilm development. We were able to detect the accumulation of eDNA in biofilm ECM extracted from C. albicans biofilms formed under conditions of flow, although the quantity of eDNA detected differed according to growth conditions, in particular with regards to the medium used to grow the biofilms. Experiments with C. albicans biofilms formed statically using a microtiter plate model indicated that the addition of exogenous DNA (>160 ng/ml) increases biofilm biomass and, conversely, DNase treatment (>0.03 mg/ml) decreases biofilm biomass at later time points of biofilm development. We present evidence for the role of eDNA in C. albicans biofilm structure and formation, consistent with eDNA being a key element of the ECM in mature C. albicans biofilms and playing a predominant role in biofilm structural integrity and maintenance. PMID:20012895

  18. Presence of extracellular DNA in the Candida albicans biofilm matrix and its contribution to biofilms.

    PubMed

    Martins, Margarida; Uppuluri, Priya; Thomas, Derek P; Cleary, Ian A; Henriques, Mariana; Lopez-Ribot, José L; Oliveira, Rosário

    2010-05-01

    DNA has been described as a structural component of the extracellular matrix (ECM) in bacterial biofilms. In Candida albicans, there is a scarce knowledge concerning the contribution of extracellular DNA (eDNA) to biofilm matrix and overall structure. This work examined the presence and quantified the amount of eDNA in C. albicans biofilm ECM and the effect of DNase treatment and the addition of exogenous DNA on C. albicans biofilm development as indicators of a role for eDNA in biofilm development. We were able to detect the accumulation of eDNA in biofilm ECM extracted from C. albicans biofilms formed under conditions of flow, although the quantity of eDNA detected differed according to growth conditions, in particular with regards to the medium used to grow the biofilms. Experiments with C. albicans biofilms formed statically using a microtiter plate model indicated that the addition of exogenous DNA (>160 ng/ml) increases biofilm biomass and, conversely, DNase treatment (>0.03 mg/ml) decreases biofilm biomass at later time points of biofilm development. We present evidence for the role of eDNA in C. albicans biofilm structure and formation, consistent with eDNA being a key element of the ECM in mature C. albicans biofilms and playing a predominant role in biofilm structural integrity and maintenance.

  19. Escherichia coli biofilms

    PubMed Central

    Beloin, Christophe; Roux, Agnès; Ghigo, Jean-Marc

    2008-01-01

    Escherichia coli is a predominant species among facultative anaerobic bacteria of the gastrointestinal tract. Both its frequent community lifestyle and the availability of a wide array of genetic tools contributed to establish E. coli as a relevant model organism for the study of surface colonization. Several key factors, including different extracellular appendages, are implicated in E. coli surface colonization and their expression and activity are finely regulated, both in space and time, to ensure productive events leading to mature biofilm formation. This chapter will present known molecular mechanisms underlying biofilm development in both commensal and pathogenic E. coli. PMID:18453280

  20. Differential effects of planktonic and biofilm MRSA on human fibroblasts.

    PubMed

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

    2012-01-01

    Bacteria colonizing chronic wounds often exist as biofilms, yet their role in chronic wound pathogenesis remains unclear. Staphylococcus aureus biofilms induce apoptosis in dermal keratinocytes, and given that chronic wound biofilms also colonize dermal tissue, it is important to investigate the effects of bacterial biofilms on dermal fibroblasts. The effects of a predominant wound pathogen, methicillin-resistant S. aureus, on normal, human, dermal fibroblasts were examined in vitro. Cell-culture medium was conditioned with equivalent numbers of either planktonic or biofilm methicillin-resistant S. aureus and then fed to fibroblast cultures. Fibroblast response was evaluated using scratch, viability, and apoptosis assays. The results suggested that fibroblasts experience the same fate when exposed to the soluble products of either planktonic or biofilm methicillin-resistant S. aureus, namely limited migration followed by death. Enzyme-linked immunosorbent assays demonstrated that fibroblast production of cytokines, growth factors, and proteases were differentially affected by planktonic and biofilm-conditioned medium. Planktonic-conditioned medium induced more interleukin-6, interleukin-8, vascular endothelial growth factor, transforming growth factor-β1, heparin-bound epidermal growth factor, matrix metalloproteinase-1, and metalloproteinase-3 production in fibroblasts than the biofilm-conditioned medium. Biofilm-conditioned medium induced more tumor necrosis factor-α production in fibroblasts compared with planktonic-conditioned medium, and suppressed metalloproteinase-3 production compared with controls.

  1. Physicochemical characteristics and microbial community evolution of biofilms during the start-up period in a moving bed biofilm reactor.

    PubMed

    Zhu, Yan; Zhang, Yan; Ren, Hong-Qiang; Geng, Jin-Ju; Xu, Ke; Huang, Hui; Ding, Li-Li

    2015-03-01

    This study aimed to investigate biofilm properties evolution coupled with different ages during the start-up period in a moving bed biofilm reactor system. Physicochemical characteristics including adhesion force, extracellular polymeric substances (EPS), morphology as well as volatile solid and microbial community were studied. Results showed that the formation and development of biofilms exhibited four stages, including (I) initial attachment and young biofilm formation, (II) biofilms accumulation, (III) biofilm sloughing and updating, and (IV) biofilm maturation. During the whole start-up period, adhesion force was positively and significantly correlated with the contents of EPS, especially the content of polysaccharide. In addition, increased adhesion force and EPS were beneficial for biofilm retention. Gram-negative bacteria mainly including Sphaerotilus, Zoogloea and Haliscomenobacter were predominant in the initial stage. Actinobacteria was beneficial to resist sloughing. Furthermore, filamentous bacteria were dominant in maturation biofilm.

  2. Synthesis and Activity of Biomimetic Biofilm Disruptors

    PubMed Central

    2013-01-01

    Biofilms are often associated with human bacterial infections, and the natural tolerance of biofilms to antibiotics challenges treatment. Compounds with antibiofilm activity could become useful adjuncts to antibiotic therapy. We used norspermidine, a natural trigger for biofilm disassembly in the developmental cycle of Bacillus subtilis, to develop guanidine and biguanide compounds with up to 20-fold increased potency in preventing biofilm formation and breaking down existing biofilms. These compounds also were active against pathogenic Staphylococcus aureus. An integrated approach involving structure–activity relationships, protonation constants, and crystal structure data on a focused synthetic library revealed that precise spacing of positively charged groups and the total charge at physiological pH distinguish potent biofilm inhibitors. PMID:23406351

  3. Maltodextrin enhances biofilm elimination by electrochemical scaffold

    PubMed Central

    Sultana, Sujala T.; Call, Douglas R.; Beyenal, Haluk

    2016-01-01

    Electrochemical scaffolds (e-scaffolds) continuously generate low concentrations of H2O2 suitable for damaging wound biofilms without damaging host tissue. Nevertheless, retarded diffusion combined with H2O2 degradation can limit the efficacy of this potentially important clinical tool. H2O2 diffusion into biofilms and bacterial cells can be increased by damaging the biofilm structure or by activating membrane transportation channels by exposure to hyperosmotic agents. We hypothesized that e-scaffolds would be more effective against Acinetobacter baumannii and Staphylococcus aureus biofilms in the presence of a hyperosmotic agent. E-scaffolds polarized at −600 mVAg/AgCl were overlaid onto preformed biofilms in media containing various maltodextrin concentrations. E-scaffold alone decreased A. baumannii and S. aureus biofilm cell densities by (3.92 ± 0.15) log and (2.31 ± 0.12) log, respectively. Compared to untreated biofilms, the efficacy of the e-scaffold increased to a maximum (8.27 ± 0.05) log reduction in A. baumannii and (4.71 ± 0.12) log reduction in S. aureus biofilm cell densities upon 10 mM and 30 mM maltodextrin addition, respectively. Overall ~55% decrease in relative biofilm surface coverage was achieved for both species. We conclude that combined treatment with electrochemically generated H2O2 from an e-scaffold and maltodextrin is more effective in decreasing viable biofilm cell density. PMID:27782161

  4. Maltodextrin enhances biofilm elimination by electrochemical scaffold.

    PubMed

    Sultana, Sujala T; Call, Douglas R; Beyenal, Haluk

    2016-10-26

    Electrochemical scaffolds (e-scaffolds) continuously generate low concentrations of H2O2 suitable for damaging wound biofilms without damaging host tissue. Nevertheless, retarded diffusion combined with H2O2 degradation can limit the efficacy of this potentially important clinical tool. H2O2 diffusion into biofilms and bacterial cells can be increased by damaging the biofilm structure or by activating membrane transportation channels by exposure to hyperosmotic agents. We hypothesized that e-scaffolds would be more effective against Acinetobacter baumannii and Staphylococcus aureus biofilms in the presence of a hyperosmotic agent. E-scaffolds polarized at -600 mVAg/AgCl were overlaid onto preformed biofilms in media containing various maltodextrin concentrations. E-scaffold alone decreased A. baumannii and S. aureus biofilm cell densities by (3.92 ± 0.15) log and (2.31 ± 0.12) log, respectively. Compared to untreated biofilms, the efficacy of the e-scaffold increased to a maximum (8.27 ± 0.05) log reduction in A. baumannii and (4.71 ± 0.12) log reduction in S. aureus biofilm cell densities upon 10 mM and 30 mM maltodextrin addition, respectively. Overall ~55% decrease in relative biofilm surface coverage was achieved for both species. We conclude that combined treatment with electrochemically generated H2O2 from an e-scaffold and maltodextrin is more effective in decreasing viable biofilm cell density.

  5. Permeabilizing biofilms

    DOEpatents

    Soukos, Nikolaos S.; Lee, Shun; Doukas,; Apostolos G.

    2008-02-19

    Methods for permeabilizing biofilms using stress waves are described. The methods involve applying one or more stress waves to a biofilm, e.g., on a surface of a device or food item, or on a tissue surface in a patient, and then inducing stress waves to create transient increases in the permeability of the biofilm. The increased permeability facilitates delivery of compounds, such as antimicrobial or therapeutic agents into and through the biofilm.

  6. Increased Mutability of Staphylococci in Biofilms as a Consequence of Oxidative Stress

    PubMed Central

    Ryder, Victoria J.; Chopra, Ian; O’Neill, Alex J.

    2012-01-01

    Objectives To investigate the development of mutational resistance to antibiotics in staphylococcal biofilms. Methods Mutation frequencies to resistance against mupirocin and rifampicin were determined for planktonic cultures and for biofilms generated using either a novel static biofilm model or by continuous flow. DNA microarray analysis was performed to detect differences in transcriptional profiles between planktonic and biofilm cultures. Results The mutability of biofilm cultures increased up to 60-fold and 4-fold for S. aureus and S. epidermidis, respectively, compared with planktonic cultures. Incorporation of antioxidants into S. aureus biofilms reduced mutation frequencies, indicating that increased oxidative stress underlies the heightened mutability. Transcriptional profiling of early biofilm cultures revealed up-regulation of the superoxide dismutase gene, sodA, also suggestive of enhanced oxidative stress in these cultures. The addition of catalase to biofilms of S. aureus SH1000 reduced mutation frequencies, a finding which implicated hydrogen peroxide in increased biofilm mutability. However, catalase had no effect on biofilm mutability in S. aureus UAMS-1, suggesting that there is more than one mechanism by which the mutability of staphylococci may increase during the biofilm mode of growth. Conclusion Our findings suggest that biofilms represent an enriched source of mutational resistance to antibiotics in the staphylococci. PMID:23110091

  7. Potent in vitro synergism of fusidic acid (FA) and berberine chloride (BBR) against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA).

    PubMed

    Liang, Rong-mei; Yong, Xiao-lan; Duan, Yu-qin; Tan, Yong-hong; Zeng, Ping; Zhou, Zi-ying; Jiang, Yan; Wang, Shi-hua; Jiang, Yun-ping; Huang, Xiao-chun; Dong, Zhao-hui; Hu, Ting-ting; Shi, Hui-qing; Li, Nan

    2014-11-01

    It was found in the present study that combined use of fusidic acid (FA) and berberine chloride (BBR) offered an in vitro synergistic action against 7 of the 30 clinical methicillin-resistant Staphylococcus aureus (MRSA) strains, with a fractional inhibitory concentration (FIC) index ranging from 0.5 to 0.19. This synergistic effect was most pronounced on MRSA 4806, an FA-resistant isolate, with a minimum inhibitory concentration (MIC) value of 1,024 μg/ml. The time-kill curve experiment showed that FA plus BBR yielded a 4.2 log10 c.f.u./ml reduction in the number of MRSA 4806 bacteria after 24-h incubation as compared with BBR alone. Viable count analysis showed that FA plus BBR produced a 3.0 log10 c.f.u./ml decrease in biofilm formation and a 1.5 log10 c.f.u./ml decrease in mature biofilm in viable cell density as compared with BBR alone. In addition, phase contrast micrographs confirmed that biofilm formation was significantly inhibited and mature biofilm was obviously destructed when FA was used in combination with BBR. These results provide evidence that combined use of FA and BBR may prove to be a promising clinical therapeutic strategy against MRSA.

  8. Medical Biofilms

    PubMed Central

    2009-01-01

    For more than two decades, Biotechnology and Bioengineering has documented research focused on natural and engineered microbial biofilms within aquatic and subterranean ecosystems, wastewater and waste-gas treatment systems, marine vessels and structures, and industrial bioprocesses. Compared to suspended culture systems, intentionally engineered biofilms are heterogeneous reaction systems that can increase reactor productivity, system stability, and provide inherent cell: product separation. Unwanted biofilms can create enormous increases in fluid frictional resistances, unacceptable reductions in heat transfer efficiency, product contamination, enhanced material deterioration, and accelerated corrosion. Missing from B&B has been an equivalent research dialogue regarding the basic molecular microbiology, immunology, and biotechnological aspects of medical biofilms. Presented here are the current problems related to medical biofilms; current concepts of biofilm formation, persistence, and interactions with the host immune system; and emerging technologies for controlling medical biofilms. PMID:18366134

  9. Inhibition of methicillin resistant Staphylococcus aureus by a plasma needle

    NASA Astrophysics Data System (ADS)

    Miletić, Maja; Vuković, Dragana; Živanović, Irena; Dakić, Ivana; Soldatović, Ivan; Maletić, Dejan; Lazović, Saša; Malović, Gordana; Petrović, Zoran; Puač, Nevena

    2014-03-01

    In numerous recent papers plasma chemistry of non equilibrium plasma sources operating at atmospheric pressure has been linked to plasma medical effects including sterilization. In this paper we present a study of the effectiveness of an atmospheric pressure plasma source, known as plasma needle, in inhibition of the growth of biofilm produced by methicillin resistant Staphylococcus aureus (MRSA). Even at the lowest powers the biofilms formed by inoculi of MRSA of 104 and 105 CFU have been strongly affected by plasma and growth in biofilms was inhibited. The eradication of the already formed biofilm was not achieved and it is required to go to more effective sources.

  10. Green synthesis of silk fibroin-silver nanoparticle composites with effective antibacterial and biofilm-disrupting properties.

    PubMed

    Fei, Xiang; Jia, Minghui; Du, Xin; Yang, Yuhong; Zhang, Ren; Shao, Zhengzhong; Zhao, Xia; Chen, Xin

    2013-12-09

    Natural polymer Bombyx mori silk fibroin is used as a biotemplate to produce silver nanoparticles in situ under light (both incandescent light and sunlight) at room temperature. Silk fibroin provides multiple functions in the whole reaction system, serving as the reducing agent of silver, and the dispersing and stabilizing agent of the resulted silver nanoparticles. As the reaction needs not any other chemicals and only uses light as power source, the synthetic route of silver nanoparticles reported here is rather environment-friendly and energy-saving. The silk fibroin-silver nanoparticle composite prepared by this method can be stably stored in a usual environment (room temperature, exposure to light, and so forth) for at least one month. Such a silk fibroin-silver nanoparticle composite shows an effective antibacterial activity against the methicillin-resistant Staphylococcus aureus (S. aureus) and subsequently inhibits the biofilm formation caused by the same bacterium. Moreover, a maturely formed biofilm created by methicillin-resistant S. aureus can be destroyed by the silk fibroin-silver nanoparticle composite, which meets the demand of clinical application. Therefore, the silk fibroin-silver nanoparticle composite prepared by this clean and facile method is expected to be an effective and economical antimicrobial material in biomedical fields.

  11. Colonisation and interaction between S. epidermidis and S. aureus in the nose and throat of healthy adolescents.

    PubMed

    Fredheim, E G A; Flægstad, T; Askarian, F; Klingenberg, C

    2015-01-01

    Nasal colonisation with Staphylococcus aureus is a risk factor for developing nosocomial infections. It has been reported that S. epidermidis may produce a serine protease (Esp) inhibiting S. aureus biofilm formation and nasal colonisation. We aimed to analyse the correlation between S. aureus nasal and/or throat carriage and co-colonisation with S. epidermidis strains carrying esp, and the inhibitory effects of S. epidermidis culture supernatants on S. aureus biofilm formation and growth. We obtained 114 S. epidermidis isolates from the nose and 74 S. aureus from the nose and/or throat of healthy adolescents. S. aureus biofilm formation was analysed in a microtitre plate assay and the prevalence of ica, encoding biofilm formation, and esp was analysed with polymerase chain reaction (PCR). Inhibitory effects of S. epidermidis culture supernatants on S. aureus biofilm formation and growth was analysed in vitro. esp prevalence and expression was correlated with inhibitory effects. We detected biofilm formation in 45/74 (61%) S. aureus strains. The ica operon was more prevalent in isolates colonising the nose (12/15; 80%) versus isolates colonising the throat only (8/46; 17%). Almost two-thirds of S. epidermidis culture supernatants displayed high (≥ 50%) S. aureus biofilm inhibitory activity, without affecting growth. We found no correlation between the level of inhibitory activity and S. aureus colonisation. esp was ubiquitous in S. epidermidis, but esp expression did not correlate with biofilm inhibitory activity. S. epidermidis culture supernatants inhibit S. aureus biofilm formation, but do not affect bacterial growth. esp expression was not correlated with the inhibitory effects observed.

  12. Fluorescent Reporters for Staphylococcus aureus

    PubMed Central

    Malone, Cheryl L.; Boles, Blaise R.; Lauderdale, Katherine J.; Thoendel, Matthew; Kavanaugh, Jeffrey S.; Horswill, Alexander R.

    2009-01-01

    With the emergence of Staphylococcus aureus as a prominent pathogen in community and healthcare settings, there is a growing need for effective reporter tools to facilitate physiology and pathogenesis studies. Fluorescent proteins are ideal as reporters for their convenience in monitoring gene expression, performing host interaction studies, and monitoring biofilm growth. We have developed a suite of fluorescent reporter plasmids for labeling S. aureus cells. These plasmids encode either green fluorescent protein (GFP) or higher wavelength reporter variants for yellow (YFP) and red (mCherry) labeling. The reporters were placed under control of characterized promoters to enable constitutive or inducible expression. Additionally, plasmids were assembled with fluorescent reporters under control of the agr quorum-sensing and Sigma factor B promoters, and the fluorescent response with wildtype and relevant mutant strains was characterized. Interestingly, reporter expression displayed a strong dependence on ribosome binding site (RBS) sequence, with the superoxide dismutase RBS displaying the strongest expression kinetics of the sequences examined. To test the robustness of the reporter plasmids, cell imaging was performed with fluorescence microscopy and cell populations were separated using florescence activated cell sorting (FACS), demonstrating the possibilities of simultaneous monitoring of multiple S. aureus properties. Finally, a constitutive YFP reporter displayed stable, robust labeling of biofilm growth in a flow cell apparatus. This toolbox of fluorescent reporter plasmids will facilitate cell labeling for a variety of different experimental applications. PMID:19264102

  13. Biofilm induced tolerance towards antimicrobial peptides.

    PubMed

    Folkesson, Anders; Haagensen, Janus A J; Zampaloni, Claudia; Sternberg, Claus; Molin, Søren

    2008-04-02

    Increased tolerance to antimicrobial agents is thought to be an important feature of microbes growing in biofilms. We address the question of how biofilm organization affects antibiotic susceptibility. We established Escherichia coli biofilms with differential structural organization due to the presence of IncF plasmids expressing altered forms of the transfer pili in two different biofilm model systems. The mature biofilms were subsequently treated with two antibiotics with different molecular targets, the peptide antibiotic colistin and the fluoroquinolone ciprofloxacin. The dynamics of microbial killing were monitored by viable count determination, and confocal laser microscopy. Strains forming structurally organized biofilms show an increased bacterial survival when challenged with colistin, compared to strains forming unstructured biofilms. The increased survival is due to genetically regulated tolerant subpopulation formation and not caused by a general biofilm property. No significant difference in survival was detected when the strains were challenged with ciprofloxacin. Our data show that biofilm formation confers increased colistin tolerance to cells within the biofilm structure, but the protection is conditional being dependent on the structural organization of the biofilm, and the induction of specific tolerance mechanisms.

  14. Biofilm Induced Tolerance towards Antimicrobial Peptides

    PubMed Central

    Folkesson, Anders; Haagensen, Janus A. J.; Zampaloni, Claudia; Sternberg, Claus; Molin, Søren

    2008-01-01

    Increased tolerance to antimicrobial agents is thought to be an important feature of microbes growing in biofilms. We address the question of how biofilm organization affects antibiotic susceptibility. We established Escherichia coli biofilms with differential structural organization due to the presence of IncF plasmids expressing altered forms of the transfer pili in two different biofilm model systems. The mature biofilms were subsequently treated with two antibiotics with different molecular targets, the peptide antibiotic colistin and the fluoroquinolone ciprofloxacin. The dynamics of microbial killing were monitored by viable count determination, and confocal laser microscopy. Strains forming structurally organized biofilms show an increased bacterial survival when challenged with colistin, compared to strains forming unstructured biofilms. The increased survival is due to genetically regulated tolerant subpopulation formation and not caused by a general biofilm property. No significant difference in survival was detected when the strains were challenged with ciprofloxacin. Our data show that biofilm formation confers increased colistin tolerance to cells within the biofilm structure, but the protection is conditional being dependent on the structural organization of the biofilm, and the induction of specific tolerance mechanisms. PMID:18382672

  15. Formation of biofilms under phage predation: considerations concerning a biofilm increase.

    PubMed

    Hosseinidoust, Zeinab; Tufenkji, Nathalie; van de Ven, Theo G M

    2013-01-01

    Bacteriophages are emerging as strong candidates for combating bacterial biofilms. However, reports indicating that host populations can, in some cases, respond to phage predation by an increase in biofilm formation are of concern. This study investigates whether phage predation can enhance the formation of biofilm and if so, if this phenomenon is governed by the emergence of phage-resistance or by non-evolutionary mechanisms (eg spatial refuge). Single-species biofilms of three bacterial pathogens (Pseudomonas aeruginosa, Salmonella enterica serotype Typhimurium, and Staphylococcus aureus) were pretreated and post-treated with species-specific phages. Some of the phage treatments resulted in an increase in the levels of biofilm of their host. It is proposed that the phenotypic change brought about by acquiring phage resistance is the main reason for the increase in the level of biofilm of P. aeruginosa. For biofilms of S. aureus and S. enterica Typhimurium, although resistance was detected, increased formation of biofilm appeared to be a result of non-evolutionary mechanisms.

  16. Commonly used disinfectants fail to eradicate Salmonella enterica biofilms from food contact surface materials.

    PubMed

    Corcoran, M; Morris, D; De Lappe, N; O'Connor, J; Lalor, P; Dockery, P; Cormican, M

    2014-02-01

    Salmonellosis is the second most common cause of food-borne illness worldwide. Contamination of surfaces in food processing environments may result in biofilm formation with a risk of food contamination. Effective decontamination of biofilm-contaminated surfaces is challenging. Using the CDC biofilm reactor, the activities of sodium hypochlorite, sodium hydroxide, and benzalkonium chloride were examined against an early (48-h) and relatively mature (168-h) Salmonella biofilm. All 3 agents result in reduction in viable counts of Salmonella; however, only sodium hydroxide resulted in eradication of the early biofilm. None of the agents achieved eradication of mature biofilm, even at the 90-min contact time. Studies of activity of chemical disinfection against biofilm should include assessment of activity against mature biofilm. The difficulty of eradication of established Salmonella biofilm serves to emphasize the priority of preventing access of Salmonella to postcook areas of food production facilities.

  17. AFM Structural Characterization of Drinking Water Biofilm ...

    EPA Pesticide Factsheets

    Due to the complexity of mixed culture drinking water biofilm, direct visual observation under in situ conditions has been challenging. In this study, atomic force microscopy (AFM) revealed the three dimensional morphology and arrangement of drinking water relevant biofilm in air and aqueous solution. Operating parameters were optimized to improve imaging of structural details for a mature biofilm in liquid. By using a soft cantilever (0.03 N/m) and slow scan rate (0.5 Hz), biofilm and individual bacterial cell’s structural topography were resolved and continuously imaged in liquid without loss of spatial resolution or sample damage. The developed methodology will allow future in situ investigations to temporally monitor mixed culture drinking water biofilm structural changes during disinfection treatments. Due to the complexity of mixed culture drinking water biofilm, direct visual observation under in situ conditions has been challenging. In this study, atomic force microscopy (AFM) revealed the three dimensional morphology and arrangement of drinking water relevant biofilm in air and aqueous solution. Operating parameters were optimized to improve imaging of structural details for a mature biofilm in liquid. By using a soft cantilever (0.03 N/m) and slow scan rate (0.5 Hz), biofilm and individual bacterial cell’s structural topography were resolved and continuously imaged in liquid without loss of spatial resolution or sample damage. The developed methodo

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

    PubMed

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

    2015-01-01

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

  19. A mucosal model to study microbial biofilm development and anti-biofilm therapeutics

    PubMed Central

    Anderson, Michele J.; Parks, Patrick J.; Peterson, Marnie L.

    2013-01-01

    Biofilms are a sessile colony of bacteria which adhere to and persist on surfaces. The ability of bacteria to form biofilms is considered a virulence factor, and in fact is central to the pathogenesis of some organisms. Biofilms are inherently resistant to chemotherapy and host immune responses. Clinically, biofilms are considered a primary cause of a majority of infections, such as otitis media, pneumonia in cystic fibrosis patients and endocarditis. However, the vast majority of the data on biofilm formation comes from traditional microtiter-based or flow displacement assays with no consideration given to host factors. These assays, which have been a valuable tool in high-throughput screening for biofilm-related factors, do not mimic a host-pathogen interaction and may contribute to an inappropriate estimation of the role of some factors in clinical biofilm formation. We describe the development of a novel ex vivo model of biofilm formation on a mucosal surface by an important mucosal pathogen, methicillin resistant S. aureus (MRSA). This model is being used for the identification of microbial virulence factors important in mucosal biofilm formation and novel anti-biofilm therapies. PMID:23246911

  20. [THE FORMATION OF BIOFILM IN OPPORTUNISTIC MICROORGANISMS IN BLOOD PLASMA DEPENDING ON CONTENT OF IRON].

    PubMed

    Leonov, V V; Mironov, A Yu

    2016-01-01

    The article considers results of analysis offormation of biofilm of priority opportunistic pathogens in blood plasma and LB-broth. As compared with LB-broth, bloodplasma stimulates formation of biofilm of microorganisms in the following sequence: Staphylococcus aureus > Pseudomonas aeruginosa > Escherichia coli. The application oftechnique of infra-redspectroscopy of bio-films established that blood plasma promotes formation of external exopolysaccharides of S.aureus. The cultivation of bio-films in plasma depending on content of iron demonstrated that the analyzed strains of S. aureus, P. aeruginosa, E. coli form bio-films in a better way in plasma with normal content of iron and iron-deficient and iron-loaded plasma decreases their activity of formation of biofilm.

  1. On-Demand Removal of Bacterial Biofilms via Shape Memory Activation

    PubMed Central

    2016-01-01

    Bacterial biofilms are a major cause of chronic infections and biofouling; however, effective removal of established biofilms remains challenging. Here we report a new strategy for biofilm control using biocompatible shape memory polymers with defined surface topography. These surfaces can both prevent bacterial adhesion and remove established biofilms upon rapid shape change with moderate increase of temperature, thereby offering more prolonged antifouling properties. We demonstrate that this strategy can achieve a total reduction of Pseudomonas aeruginosa biofilms by 99.9% compared to the static flat control. It was also found effective against biofilms of Staphylococcus aureus and an uropathogenic strain of Escherichia coli. PMID:27517738

  2. A flow cytometric approach to quantify biofilms.

    PubMed

    Kerstens, Monique; Boulet, Gaëlle; Van Kerckhoven, Marian; Clais, Sofie; Lanckacker, Ellen; Delputte, Peter; Maes, Louis; Cos, Paul

    2015-07-01

    Since biofilms are important in many clinical, industrial, and environmental settings, reliable methods to quantify these sessile microbial populations are crucial. Most of the currently available techniques do not allow the enumeration of the viable cell fraction within the biofilm and are often time consuming. This paper proposes flow cytometry (FCM) using the single-stain viability dye TO-PRO(®)-3 iodide as a fast and precise alternative. Mature biofilms of Candida albicans and Escherichia coli were used to optimize biofilm removal and dissociation, as a single-cell suspension is needed for accurate FCM enumeration. To assess the feasibility of FCM quantification of biofilms, E. coli and C. albicans biofilms were analyzed using FCM and crystal violet staining at different time points. A combination of scraping and rinsing proved to be the most efficient technique for biofilm removal. Sonicating for 10 min eliminated the remaining aggregates, resulting in a single-cell suspension. Repeated FCM measurements of biofilm samples revealed a good intraday precision of approximately 5 %. FCM quantification and the crystal violet assay yielded similar biofilm growth curves for both microorganisms, confirming the applicability of our technique. These results show that FCM using TO-PRO(®)-3 iodide as a single-stain viability dye is a valid fast alternative for the quantification of viable cells in a biofilm.

  3. Characterization of temporal protein production in Pseudomonas aeruginosa biofilms.

    PubMed

    Southey-Pillig, Christopher J; Davies, David G; Sauer, Karin

    2005-12-01

    Phenotypic and genetic evidence supporting the notion of biofilm formation as a developmental process is growing. In the present work, we provide additional support for this hypothesis by identifying the onset of accumulation of biofilm-stage specific proteins during Pseudomonas aeruginosa biofilm maturation and by tracking the abundance of these proteins in planktonic and three biofilm developmental stages. The onset of protein production was found to correlate with the progression of biofilms in developmental stages. Protein identification revealed that proteins with similar function grouped within similar protein abundance patterns. Metabolic and housekeeping proteins were found to group within a pattern separate from virulence, antibiotic resistance, and quorum-sensing-related proteins. The latter were produced in a progressive manner, indicating that attendant features that are characteristic of biofilms such as antibiotic resistance and virulence may be part of the biofilm developmental process. Mutations in genes for selected proteins from several protein production patterns were made, and the impact of these mutations on biofilm development was evaluated. The proteins cytochrome c oxidase, a probable chemotaxis transducer, a two-component response regulator, and MexH were produced only in mature and late-stage biofilms. Mutations in the genes encoding these proteins did not confer defects in growth, initial attachment, early biofilm formation, or twitching motility but were observed to arrest biofilm development at the stage of cell cluster formation we call the maturation-1 stage. The results indicated that expression of theses genes was required for the progression of biofilms into three-dimensional structures on abiotic surfaces and the completion of the biofilm developmental cycle. Reverse transcription-PCR analysis confirmed the detectable change in expression of the respective genes ccoO, PA4101, and PA4208. We propose a possible mechanism for the

  4. Human plasma enhances the expression of Staphylococcal microbial surface components recognizing adhesive matrix molecules promoting biofilm formation and increases antimicrobial tolerance In Vitro

    PubMed Central

    2014-01-01

    Background Microbial biofilms have been associated with the development of chronic human infections and represent a clinical challenge given their increased antimicrobial tolerance. Staphylococcus aureus is a major human pathogen causing a diverse range of diseases, of which biofilms are often involved. Staphylococcal attachment and the formation of biofilms have been shown to be facilitated by host factors that accumulate on surfaces. To better understand how host factors enhance staphylococcal biofilm formation, we evaluated the effect of whole human plasma on biofilm formation in clinical isolates of S. aureus and the expression of seven microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) known to be involved in biofilm formation by quantitative real-time PCR. We also evaluated whether plasma augmented changes in S. aureus biofilm morphology and antimicrobial resistance. Results Exposure of clinical isolates of S. aureus to human plasma (10%) within media, and to a lesser extent when coated onto plates, significantly enhanced biofilm formation in all of the clinical isolates tested. Compared to biofilms grown under non-supplemented conditions, plasma-augmented biofilms displayed significant changes in both the biofilm phenotype and cell morphology as determined by confocal scanning laser microscopy (CLSM) and scanning electron microscopy (SEM), respectively. Exposure of bacteria to plasma resulted in a significant fold-increase in MSCRAMM expression in both a time and isolate-dependent manner. Additionally, plasma-augmented biofilms displayed an increased tolerance to vancomycin compared to biofilms grown in non-supplemented media. Conclusions Collectively, these studies support previous findings demonstrating a role for host factors in biofilm formation and provide further insight into how plasma, a preferred growth medium for staphylococcal biofilm formation enhances as well as augments other intrinsic properties of S. aureus biofilms

  5. Differential effects of antifungal agents on expression of genes related to formation of Candida albicans biofilms.

    PubMed

    Chatzimoschou, Athanasios; Simitsopoulou, Maria; Antachopoulos, Charalampos; Walsh, Thomas J; Roilides, Emmanuel

    2016-01-01

    The purpose of this study was to analyse specific molecular mechanisms involved in the intrinsic resistance of C. albicans biofilms to antifungals. We investigated the transcriptional profile of three genes (BGL2, SUN41, ECE1) involved in Candida cell wall formation in response to voriconazole or anidulafungin after the production of intermediate and mature biofilms. C. albicans M61, a well-documented biofilm producer strain, was used for the development of intermediate (12 h and 18 h) and completely mature biofilms (48 h). After exposure of cells from each biofilm growth mode to voriconazole (128 and 512 mg l(-1)) or anidulafungin (0.25 and 1 mg l(-1)) for 12-24 h, total RNA samples extracted from biofilm cells were analysed by RT-PCR. The voriconazole and anidulafungin biofilm MIC was 512 and 0.5 mg l(-1) respectively. Anidulafungin caused significant up-regulation of SUN41 (3.7-9.3-fold) and BGL2 (2.2-2.8 fold) in intermediately mature biofilms; whereas, voriconazole increased gene expression in completely mature biofilms (SUN41 2.3-fold, BGL2 2.1-fold). Gene expression was primarily down-regulated by voriconazole in intermediately, but not completely mature biofilms. Both antifungals caused down-regulation of ECE1 in intermediately mature biofilms.

  6. Effects of the Selected Iminosugar Derivatives on Pseudomonas aeruginosa Biofilm Formation.

    PubMed

    Strus, Magdalena; Mikołajczyk, Diana; Machul, Agnieszka; Heczko, Piotr B; Chronowska, Aleksandra; Stochel, Grażyna; Gallienne, Estelle; Nicolas, Cyril; Martin, Olivier R; Kyzioł, Agnieszka

    2016-12-01

    A lack of an effective way to eliminate pathogenic bacteria hidden in the biofilm is a major problem in the treatment of chronic bacterial infections. Iminosugar derivatives are potential candidates for inhibitors of enzymes taking part in the biosynthesis of exopolysaccharides, which are forming bacterial biofilm. Investigated iminosugars were studied either at an early stage of biofilm formation or later on when the mature biofilm of Pseudomonas aeruginosa was already formed. A series of diverse iminosugar structures significantly inhibited biofilm formation, whereas they showed no influence on already formed biofilm. This indicates a possible mechanism of their action based on inhibition of exopolysaccharide backbone synthesis in the early stages of biofilm formation. Moreover, iminosugar derivatives did not show significant effect on the viable bacterial numbers in both early and mature biofilm forms. Importantly, they were not cytotoxic against human Caco-2 cells in vitro, which may be to their advantage in case of their medical application in preventing P. aeruginosa biofilm formation.

  7. Plant Biofilm Inhibitors to Discover Biofilm Genes

    DTIC Science & Technology

    2011-04-08

    REPORT Final Report for Plant Biofilm Inhibitors to Discover Biofilm Genes 14. ABSTRACT 16. SECURITY CLASSIFICATION OF: To control biofilms , we have...synthesized the natural biofilm inhibitor (5Z)-4-bromo-5-(bromomethylene) -3-butyl-2(5H)-furanone from the red alga Delisea pulchra and determined that...Research Office P.O. Box 12211 Research Triangle Park, NC 27709-2211 15. SUBJECT TERMS biofilms , biofilm inhibitors Thomas K. Wood Texas Engineering

  8. Antibiotic resistance in Pseudomonas aeruginosa biofilms: towards the development of novel anti-biofilm therapies.

    PubMed

    Taylor, Patrick K; Yeung, Amy T Y; Hancock, Robert E W

    2014-12-10

    The growth of bacteria as structured aggregates termed biofilms leads to their protection from harsh environmental conditions such as physical and chemical stresses, shearing forces, and limited nutrient availability. Because of this highly adapted ability to survive adverse environmental conditions, bacterial biofilms are recalcitrant to antibiotic therapies and immune clearance. This is particularly problematic in hospital settings where biofilms are a frequent cause of chronic and device-related infections and constitute a significant burden on the health-care system. The major therapeutic strategy against infections is the use of antibiotics, which, due to adaptive resistance, are often insufficient to clear biofilm infections. Thus, novel biofilm-specific therapies are required. Specific features of biofilm development, such as surface adherence, extracellular matrix formation, quorum sensing, and highly regulated biofilm maturation and dispersal are currently being studied as targets to be exploited in the development of novel biofilm-specific treatments. Using Pseudomonas aeruginosa for illustrative purposes, this review highlights the antibiotic resistance mechanisms of biofilms, and discusses current research into novel biofilm-specific therapies.

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

    PubMed Central

    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

  10. Wound biofilms: lessons learned from oral biofilms

    PubMed Central

    Mancl, Kimberly A.; Kirsner, Robert S.; Ajdic, Dragana

    2013-01-01

    Biofilms play an important role in the development and pathogenesis of many chronic infections. Oral biofilms, more commonly known as dental plaque,are a primary cause of oral diseases including caries, gingivitis and periodontitis. Oral biofilms are commonly studied as model biofilm systems as they are easily accessible, thus biofilm research in oral diseases is advanced with details of biofilm formation and bacterial interactions being well-elucidated. In contrast, wound research has relatively recently directed attentionto the role biofilms have in chronic wounds. This review discusses the biofilms in periodontal disease and chronic wounds with comparisons focusing on biofilm detection, biofilm formation, the immune response to biofilms, bacterial interaction and quorum sensing. Current treatment modalities used by both fields as well as future therapies are also discussed. PMID:23551419

  11. Fractal analysis of Xylella fastidiosa biofilm formation

    NASA Astrophysics Data System (ADS)

    Moreau, A. L. D.; Lorite, G. S.; Rodrigues, C. M.; Souza, A. A.; Cotta, M. A.

    2009-07-01

    We have investigated the growth process of Xylella fastidiosa biofilms inoculated on a glass. The size and the distance between biofilms were analyzed by optical images; a fractal analysis was carried out using scaling concepts and atomic force microscopy images. We observed that different biofilms show similar fractal characteristics, although morphological variations can be identified for different biofilm stages. Two types of structural patterns are suggested from the observed fractal dimensions Df. In the initial and final stages of biofilm formation, Df is 2.73±0.06 and 2.68±0.06, respectively, while in the maturation stage, Df=2.57±0.08. These values suggest that the biofilm growth can be understood as an Eden model in the former case, while diffusion-limited aggregation (DLA) seems to dominate the maturation stage. Changes in the correlation length parallel to the surface were also observed; these results were correlated with the biofilm matrix formation, which can hinder nutrient diffusion and thus create conditions to drive DLA growth.

  12. Portrait of Candida Species Biofilm Regulatory Network Genes.

    PubMed

    Araújo, Daniela; Henriques, Mariana; Silva, Sónia

    2017-01-01

    Most cases of candidiasis have been attributed to Candida albicans, but Candida glabrata, Candida parapsilosis and Candida tropicalis, designated as non-C. albicans Candida (NCAC), have been identified as frequent human pathogens. Moreover, Candida biofilms are an escalating clinical problem associated with significant rates of mortality. Biofilms have distinct developmental phases, including adhesion/colonisation, maturation and dispersal, controlled by complex regulatory networks. This review discusses recent advances regarding Candida species biofilm regulatory network genes, which are key components for candidiasis.

  13. Influence of attached bacteria and biofilm on double-layer capacitance during biofilm monitoring by electrochemical impedance spectroscopy.

    PubMed

    Kim, Taeyoung; Kang, Junil; Lee, Joon-Hee; Yoon, Jeyong

    2011-10-01

    Development of an effective strategy for biofilm control in water-related system has become a matter of significant concern nowadays. Electrochemical monitoring, especially electrochemical impedance spectroscopy (EIS), is one of the efficient approaches to dealing with biofilm-related issues. However, currently used EIS methods without a redox probe intend to detect all effects generated from media components, bacteria, and bacterial metabolites, which used to make the signals from the attached bacteria and biofilm weakened. In this study, we tried improved EIS measurement to monitor bacterial adhesion and biofilm maturation using a double-layer capacitance. In this improved method, we minimized background signal by subtracting the interference of electrolyte caused by bacterial metabolism. Pseudomonas aeruginosa PA14 wild type and wspF mutant that form the biofilm of distinct nature were used for the model strains to test our method. During bacterial adhesion and biofilm maturation, EIS data were collected and equivalent circuit analysis was carried out to obtain constant phase element (CPE) values representing double-layer capacitance. Since the influence by the bacterial growth-related culture media condition was eliminated by adopting fresh electrolyte at the measurement, the contribution of attached bacteria and biofilm was exclusively measured. As a result, the bacterial adhesion at the early stage of biofilm development was specifically monitored from reduction in double-layer capacitance. Particularly, the plateau in double-layer capacitance appeared upon biofilm maturation, indicating that biofilm maturation could be expected beyond this point. In conclusion, this study found that measurement of double-layer capacitance based on EIS could provide a monitoring parameter suggesting bacterial adhesion and the initiation point of biofilm maturation.

  14. Microscale Confinement features in microfluidic devices can affect biofilm

    SciTech Connect

    Kumar, Aloke; Karig, David K; Neethirajan, Suresh; Acharya, Rajesh K; Mukherjee, Partha P; Retterer, Scott T; Doktycz, Mitchel John

    2013-01-01

    Biofilms are aggregations of microbes that are encased by extra-cellular polymeric substances (EPS) and adhere to surfaces and interfaces. Biofilm development on abiotic surfaces is a dynamic process, which typically proceeds through an initial phase of adhesion of plankntonic microbes to the substrate, followed by events such as growth, maturation and EPS secretion. However, the coupling of hydrodynamics, microbial adhesion and biofilm growth remain poorly understood. Here, we investigate the effect of semiconfined features on biofilm formation. Using a microfluidic device and fluorescent time-lapse microscopy, we establish that confinement features can significantly affect biofilm formation. Biofilm dynamics change not only as a function of confinement features, but also of the total fluid flow rate, and our combination of experimental results and numerical simulations reveal insights into the link between hydrodynamics and biofilm formation.

  15. Targeting microbial biofilms using Ficin, a nonspecific plant protease

    PubMed Central

    Baidamshina, Diana R.; Trizna, Elena Y.; Holyavka, Marina G.; Bogachev, Mikhail I.; Artyukhov, Valeriy G.; Akhatova, Farida S.; Rozhina, Elvira V.; Fakhrullin, Rawil F.; Kayumov, Airat R.

    2017-01-01

    Biofilms, the communities of surface-attached bacteria embedded into extracellular matrix, are ubiquitous microbial consortia securing the effective resistance of constituent cells to environmental impacts and host immune responses. Biofilm-embedded bacteria are generally inaccessible for antimicrobials, therefore the disruption of biofilm matrix is the potent approach to eradicate microbial biofilms. We demonstrate here the destruction of Staphylococcus aureus and Staphylococcus epidermidis biofilms with Ficin, a nonspecific plant protease. The biofilm thickness decreased two-fold after 24 hours treatment with Ficin at 10 μg/ml and six-fold at 1000 μg/ml concentration. We confirmed the successful destruction of biofilm structures and the significant decrease of non-specific bacterial adhesion to the surfaces after Ficin treatment using confocal laser scanning and atomic force microscopy. Importantly, Ficin treatment enhanced the effects of antibiotics on biofilms-embedded cells via disruption of biofilm matrices. Pre-treatment with Ficin (1000 μg/ml) considerably reduced the concentrations of ciprofloxacin and bezalkonium chloride required to suppress the viable Staphylococci by 3 orders of magnitude. We also demonstrated that Ficin is not cytotoxic towards human breast adenocarcinoma cells (MCF7) and dog adipose derived stem cells. Overall, Ficin is a potent tool for staphylococcal biofilm treatment and fabrication of novel antimicrobial therapeutics for medical and veterinary applications. PMID:28387349

  16. Spatiometabolic stratification of Shewanella oneidensis biofilms.

    PubMed

    Teal, Tracy K; Lies, Douglas P; Wold, Barbara J; Newman, Dianne K

    2006-11-01

    Biofilms, or surface-attached microbial communities, are both ubiquitous and resilient in the environment. Although much is known about how biofilms form, develop, and detach, very little is understood about how these events are related to metabolism and its dynamics. It is commonly thought that large subpopulations of cells within biofilms are not actively producing proteins or generating energy and are therefore dead. An alternative hypothesis is that within the growth-inactive domains of biofilms, significant populations of living cells persist and retain the capacity to dynamically regulate their metabolism. To test this, we employed unstable fluorescent reporters to measure growth activity and protein synthesis in vivo over the course of biofilm development and created a quantitative routine to compare domains of activity in independently grown biofilms. Here we report that Shewanella oneidensis biofilm structures reproducibly stratify with respect to growth activity and metabolism as a function of size. Within domains of growth-inactive cells, genes typically upregulated under anaerobic conditions are expressed well after growth has ceased. These findings reveal that, far from being dead, the majority of cells in mature S. oneidensis biofilms have actively turned-on metabolic programs appropriate to their local microenvironment and developmental stage.

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

    PubMed

    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.

  18. Bacillus cereus Biofilms-Same, Only Different.

    PubMed

    Majed, Racha; Faille, Christine; Kallassy, Mireille; Gohar, Michel

    2016-01-01

    Bacillus cereus displays a high diversity of lifestyles and ecological niches and include beneficial as well as pathogenic strains. These strains are widespread in the environment, are found on inert as well as on living surfaces and contaminate persistently the production lines of the food industry. Biofilms are suspected to play a key role in this ubiquitous distribution and in this persistency. Indeed, B. cereus produces a variety of biofilms which differ in their architecture and mechanism of formation, possibly reflecting an adaptation to various environments. Depending on the strain, B. cereus has the ability to grow as immersed or floating biofilms, and to secrete within the biofilm a vast array of metabolites, surfactants, bacteriocins, enzymes, and toxins, all compounds susceptible to act on the biofilm itself and/or on its environment. Within the biofilm, B. cereus exists in different physiological states and is able to generate highly resistant and adhesive spores, which themselves will increase the resistance of the bacterium to antimicrobials or to cleaning procedures. Current researches show that, despite similarities with the regulation processes and effector molecules involved in the initiation and maturation of the extensively studied Bacillus subtilis biofilm, important differences exists between the two species. The present review summarizes the up to date knowledge on biofilms produced by B. cereus and by two closely related pathogens, Bacillus thuringiensis and Bacillus anthracis. Economic issues caused by B. cereus biofilms and management strategies implemented to control these biofilms are included in this review, which also discuss the ecological and functional roles of biofilms in the lifecycle of these bacterial species and explore future developments in this important research area.

  19. Laser Microbial Killing and Biofilm Disruption

    NASA Astrophysics Data System (ADS)

    Krespi, Yosef P.; Kizhner, Victor

    2009-06-01

    Objectives: To analyze the ability of NIR lasers to reduce bacterial load and demonstrate the capability of fiber-based Q-switched Nd:YAG laser disrupting biofilm. Study Design: NIR diode laser was tested in vitro and in vivo using pathogenic microorganisms (S. aureus, S. pneumoniae, P. aeruginosa). In addition biofilms were grown from clinical Pseudomonas isolates and placed in culture plates, screws, tympanostomy tubes and PET sutures. Methods: In the animal experiments acute rhinosinusitis model was created by packing the rabbit nose with bacteria soaked solution. The nasal pack was removed in two days and nose was exposed to laser irradiation. A 940 nm diode laser with fiber diffuser was used. Nasal cultures were obtained before and after the laser treatments. Animals were sacrificed fifteen days following laser treatment and bacteriologic/histologic results analyzed. Q-switched Nd:YAG laser generated shockwave pulses were delivered on biofilm using special probes over culture plates, screws, tubes, and PET sutures for the biofilm experiments. Results: Average of two log bacteria reduction was achieved with NIR laser compared to controls. Histologic studies demonstrated preservation of tissue integrity without significant damage to mucosa. Biofilms were imaged before, during and after treatment using a confocal microscope. During laser-generated shockwave application, biofilm was initially seen to oscillate and eventually break off. Large and small pieces of biofilm were totally and instantly removed from the surface to which they were attached in seconds. Conclusions: Significant bacterial reduction was achieved with NIR laser therapy in this experimental in vitro and animal study. In addition we disrupted Pseudomonas aeruginosa biofilms using Q-switched Nd:YAG laser and special probes generating plasma and shockwave. This new and innovative method of bacteria killing and biofilm disruption without injuring host tissue may have clinical application in the

  20. Successional development of biofilms in moving bed biofilm reactor (MBBR) systems treating municipal wastewater.

    PubMed

    Biswas, Kristi; Taylor, Michael W; Turner, Susan J

    2014-02-01

    Biofilm-based technologies, such as moving bed biofilm reactor (MBBR) systems, are widely used to treat wastewater. Biofilm development is important for MBBR systems as much of the microbial biomass is retained within reactors as biofilm on suspended carriers. Little is known about this process of biofilm development and the microorganisms upon which MBBRs rely. We documented successional changes in microbial communities as biofilms established in two full-scale MBBR systems treating municipal wastewater over two seasons. 16S rRNA gene-targeted pyrosequencing and clone libraries were used to describe microbial communities. These data indicate a successional process that commences with the establishment of an aerobic community dominated by Gammaproteobacteria (up to 52 % of sequences). Over time, this community shifts towards dominance by putatively anaerobic organisms including Deltaproteobacteria and Clostridiales. Significant differences were observed between the two wastewater treatment plants (WWTPs), mostly due to a large number of sequences (up to 55 %) representing Epsilonproteobacteria (mostly Arcobacter) at one site. Archaea in young biofilms included several lineages of Euryarchaeota and Crenarchaeota. In contrast, the mature biofilm consisted entirely of Methanosarcinaceae (Euryarchaeota). This study provides new insights into the community structure of developing biofilms at full-scale WWTPs and provides the basis for optimizing MBBR start-up and operational parameters.

  1. Biofilm formation of Achromobacter xylosoxidans on contact lens.

    PubMed

    Konstantinović, Neda; Ćirković, Ivana; Đukić, Slobodanka; Marić, Vesna; Božić, Dragana D

    2017-02-20

    Achromobacter spp. may contaminate lenses, lens cases, and contact lens solutions and cause ocular infections. The aim of this study was to investigate the possibility of isolated strain of Achromobacter xylosoxidans to form biofilm on the surface of soft contact lenses (CL), to quantify the production of the formed biofilm, and compare it with the reference strains (Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae). Bacterial strain isolated from one contact lens case was identified as A. xylosoxidans using Vitek2 Automated System. Biofilm forming capacity of isolated strain of A. xylosoxidans and reference strains of P. aeruginosa, S. aureus, and H. influenzae on soft CL were analyzed by commonly used microtitre plate method. Our results showed that isolated strain of A. xylosoxidans was capable to form biofilm on the surface of soft contact lens. A. xylosoxidans was strong biofilm producer while all examined reference strains were moderate biofilm producers. A. xylosoxidans appears to be superior biofilm producer on soft CL compared to reference strains.

  2. Garcinia xanthochymus Benzophenones Promote Hyphal Apoptosis and Potentiate Activity of Fluconazole against Candida albicans Biofilms.

    PubMed

    Jackson, Desmond N; Yang, Lin; Wu, ShiBiao; Kennelly, Edward J; Lipke, Peter N

    2015-10-01

    Xanthochymol and garcinol, isoprenylated benzophenones purified from Garcinia xanthochymus fruits, showed multiple activities against Candida albicans biofilms. Both compounds effectively prevented emergence of fungal germ tubes and were also cytostatic, with MICs of 1 to 3 μM. The compounds therefore inhibited development of hyphae and subsequent biofilm maturation. Xanthochymol treatment of developing and mature biofilms induced cell death. In early biofilm development, killing had the characteristics of apoptosis, including externalization of phosphatidyl serine and DNA fragmentation, as evidenced by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) fluorescence. These activities resulted in failure of biofilm maturation and hyphal death in mature biofilms. In mature biofilms, xanthochymol and garcinol caused the death of biofilm hyphae, with 50% effective concentrations (EC50s) of 30 to 50 μM. Additionally, xanthochymol-mediated killing was complementary with fluconazole against mature biofilms, reducing the fluconazole EC50 from >1,024 μg/ml to 13 μg/ml. Therefore, xanthochymol has potential as an adjuvant for antifungal treatments as well as in studies of fungal apoptosis.

  3. Colony spreading in Staphylococcus aureus.

    PubMed

    Kaito, Chikara; Sekimizu, Kazuhisa

    2007-03-01

    Wild-type Staphylococcus aureus rapidly expands on the surface of soft agar plates. The rates of expansion and the shapes of the resultant giant colonies were distinct for different strains of laboratory stocks and clinical isolates. The colony spreading abilities did not correlate with the biofilm-forming abilities in these strains. Insertional disruption of the dltABCD operon, which functions at the step of D-alanine addition to teichoic acids, and of the tagO gene, which is responsible for the synthesis of wall teichoic acids, decreased the colony spreading ability. The results indicate that wall teichoic acids and D-alanylation of teichoic acids are required for colony spreading.

  4. Inhibitory Effect of Biocides on the Viable Masses and Matrices of Staphylococcus aureus and Pseudomonas aeruginosa Biofilms▿

    PubMed Central

    Toté, K.; Horemans, T.; Berghe, D. Vanden; Maes, L.; Cos, P.

    2010-01-01

    Bacteria and matrix are essential for the development of biofilms, and assays should therefore target both components. The current European guidelines for biocidal efficacy testing are not adequate for sessile microorganisms; hence, alternative discriminatory test protocols should be used. The activities of a broad range of biocides on Staphylococcus aureus and Pseudomonas aeruginosa biofilms were evaluated using such in vitro assays. Nearly all selected biocides showed a significant decrease in S. aureus biofilm viability, with sodium hypochlorite and peracetic acid as the most active biocides. Only hydrogen peroxide and sodium hypochlorite showed some inhibitory effect on the matrix. Treatment of P. aeruginosa biofilms was roughly comparable to that of S. aureus biofilms. Peracetic acid was the most active on viable mass within 1 min of contact. Isopropanol ensured a greater than 99.999% reduction of P. aeruginosa viability after at least 30 min of contact. Comparable to results with S. aureus, sodium hypochlorite and hydrogen peroxide markedly reduced the P. aeruginosa matrix. This study clearly demonstrated that despite their aspecific mechanisms of action, most biocides were active only against biofilm bacteria, leaving the matrix undisturbed. Only hydrogen peroxide and sodium hypochlorite were active on both the biofilm matrix and the viable mass, making them the better antibiofilm agents. In addition, this study emphasizes the need for updated and standardized guidelines for biofilm susceptibility testing of biocides. PMID:20363795

  5. Polysaccharide intercellular adhesin in biofilm: structural and regulatory aspects

    PubMed Central

    Arciola, Carla Renata; Campoccia, Davide; Ravaioli, Stefano; Montanaro, Lucio

    2015-01-01

    Staphylococcus aureus and Staphylococcus epidermidis are the leading etiologic agents of implant-related infections. Biofilm formation is the main pathogenetic mechanism leading to the chronicity and irreducibility of infections. The extracellular polymeric substances of staphylococcal biofilms are the polysaccharide intercellular adhesin (PIA), extracellular-DNA, proteins, and amyloid fibrils. PIA is a poly-β(1-6)-N-acetylglucosamine (PNAG), partially deacetylated, positively charged, whose synthesis is mediated by the icaADBC locus. DNA sequences homologous to ica locus are present in many coagulase-negative staphylococcal species, among which S. lugdunensis, however, produces a biofilm prevalently consisting of proteins. The product of icaA is an N-acetylglucosaminyltransferase that synthetizes PIA oligomers from UDP-N-acetylglucosamine. The product of icaD gives optimal efficiency to IcaA. The product of icaC is involved in the externalization of the nascent polysaccharide. The product of icaB is an N-deacetylase responsible for the partial deacetylation of PIA. The expression of ica locus is affected by environmental conditions. In S. aureus and S. epidermidis ica-independent alternative mechanisms of biofilm production have been described. S. epidermidis and S. aureus undergo to a phase variation for the biofilm production that has been ascribed, in turn, to the transposition of an insertion sequence in the icaC gene or to the expansion/contraction of a tandem repeat naturally harbored within icaC. A role is played by the quorum sensing system, which negatively regulates biofilm formation, favoring the dispersal phase that disseminates bacteria to new infection sites. Interfering with the QS system is a much debated strategy to combat biofilm-related infections. In the search of vaccines against staphylococcal infections deacetylated PNAG retained on the surface of S. aureus favors opsonophagocytosis and is a potential candidate for immune-protection. PMID

  6. Kinetics of biofilm formation by drinking water isolated Penicillium expansum.

    PubMed

    Simões, Lúcia Chaves; Simões, Manuel; Lima, Nelson

    2015-01-01

    Current knowledge on drinking water (DW) biofilms has been obtained mainly from studies on bacterial biofilms. Very few reports on filamentous fungi (ff) biofilms are available, although they can contribute to the reduction in DW quality. This study aimed to assess the dynamics of biofilm formation by Penicillium expansum using microtiter plates under static conditions, mimicking water flow behaviour in stagnant regions of drinking water distribution systems. Biofilms were analysed in terms of biomass (crystal violet staining), metabolic activity (resazurin, fluorescein diacetate and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide [MTT]) and morphology (epifluorescence [calcofluor white M2R, FUN-1, FDA and acridine orange] and bright-field microscopies). Biofilm development over time showed the typical sigmoidal curve with noticeable different phases in biofilm formation (induction, exponential, stationary, and sloughing off). The methods used to assess metabolic activity provided similar results. The microscope analysis allowed identification of the involvement of conidia in initial adhesion (4 h), germlings (8 h), initial monolayers (12 h), a monolayer of intertwined hyphae (24 h), mycelial development, hyphal layering and bundling, and development of the mature biofilms (≥48 h). P. expansum grows as a complex, multicellular biofilm in 48 h. The metabolic activity and biomass of the fungal biofilms were shown to increase over time and a correlation between metabolism, biofilm mass and hyphal development was found.

  7. Purpurin triggers caspase-independent apoptosis in Candida dubliniensis biofilms.

    PubMed

    Tsang, Paul Wai-Kei; Wong, Alan Pak-Kin; Yang, Hai-Ping; Li, Ngai-For

    2013-01-01

    Candida dubliniensis is an important human fungal pathogen that causes oral infections in patients with AIDS and diabetes mellitus. However, C. Dubliniensis has been frequently reported in bloodstream infections in clinical settings. Like its phylogenetically related virulent species C. albicans, C. Dubliniensis is able to grow and switch between yeast form and filamentous form (hyphae) and develops biofilms on both abiotic and biotic surfaces. Biofilms are recalcitrant to antifungal therapies and C. Dubliniensis readily turns drug resistant upon repeated exposure. More than 80% of infections are associated with biofilms. Suppression of fungal biofilms may therefore represent a viable antifungal strategy with clinical relevance. Here, we report that C. dubliniensis biofilms were inhibited by purpurin, a natural anthraquinone pigment isolated from madder root. Purpurin inhibited C. dubliniensis biofilm formation in a concentration-dependent manner; while mature biofilms were less susceptible to purpurin. Scanning electron microscopy (SEM) analysis revealed scanty structure consisting of yeast cells in purpurin-treated C. dubliniensis biofilms. We sought to delineate the mechanisms of the anti-biofilm activity of purpurin on C. Dubliniensis. Intracellular ROS levels were significantly elevated in fungal biofilms and depolarization of MMP was evident upon purpurin treatment in a concentration-dependent manner. DNA degradation was evident. However, no activated metacaspase could be detected. Together, purpurin triggered metacaspase-independent apoptosis in C. dubliniensis biofilms.

  8. Esp-independent biofilm formation by Enterococcus faecalis.

    PubMed

    Kristich, Christopher J; Li, Yung-Hua; Cvitkovitch, Dennis G; Dunny, Gary M

    2004-01-01

    Enterococcus faecalis is a gram-positive opportunistic pathogen known to form biofilms in vitro. In addition, this organism is often isolated from biofilms on the surfaces of various indwelling medical devices. However, the molecular mechanisms regulating biofilm formation in these clinical isolates are largely unknown. Recent work has suggested that a specific cell surface protein (Esp) of E. faecalis is critical for biofilm formation by this organism. However, in the same study, esp-deficient strains of E. faecalis were found to be capable of biofilm formation. To test the hypothesis that Esp is dispensable for biofilm formation by E. faecalis, we used microtiter plate assays and a chemostat-based biofilm fermentor assay to examine biofilm formation by genetically well-defined, non-Esp-expressing strains. Our results demonstrate that in vitro biofilm formation occurs, not only in the absence of esp, but also in the absence of the entire pathogenicity island that harbors the esp coding sequence. Using scanning electron microscopy to evaluate biofilms of E. faecalis OG1RF grown in the fermentor system, biofilm development was observed to progress through multiple stages, including attachment of individual cells to the substratum, microcolony formation, and maturation into complex multilayered structures apparently containing water channels. Microtiter plate biofilm analyses indicated that biofilm formation or maintenance was modulated by environmental conditions. Furthermore, our results demonstrate that expression of a secreted metalloprotease, GelE, enhances biofilm formation by E. faecalis. In summary, E. faecalis forms complex biofilms by a process that is sensitive to environmental conditions and does not require the Esp surface protein.

  9. Ultrastructural morphologic changes in mycobacterial biofilm in different extreme condition.

    PubMed

    Kumar, Virendra; Sachan, Tarun Kumar; Sharma, Pragya; Rawat, Krishna Dutta

    2015-02-01

    The aim of this study was to investigate the morphologic and ultrastructural features of biofilms of slow and fast-growing mycobacteria in different stress conditions, presence and absence of oleic acid albumin dextrose catalase (OADC) enrichment and at different temperatures: 30, 37 and 42 °C. Four hundred mycobacterial isolates were taken. The biomass of each biofilm was quantified using a modified microtiter plate assay method. Isolates were divided into those that formed fully established biofilms, moderately attached biofilms and weakly adherent biofilms by comparison with a known biofilm-forming strain. The large quantity of biofilm was produced by Mycobacterium smegmatis at temperature 37 and 42 °C as compared to 30 °C. Mycobacterium fortuitum and M. avium developed large amount of biofilm at 30 °C as compared to 37 and 42 °C. Mycobacterium tuberculosis developed strong biofilm at 37 °C and no biofilm at 30 and 42 °C in Sauton's media. The selected non-tuberculous mycobacteria and H37Rv developed strong biofilm in the presence of OADC enrichment in Sauton's medium. Microscopic examination of biofilms by scanning electron microscopy revealed that poorly adherent biofilm formers failed to colonize the entire surface of the microtiter well. While moderately adherent biofilm formers grew in uniform monolayers but failed to develop a mature three-dimensional structure. SEM analysis of an isolate representative of the group formed fully established biofilms with a textured, multi-layered, three-dimensional structure.

  10. Plasma is the main regulator of Staphylococcus epidermidis biofilms virulence genes transcription in human blood.

    PubMed

    França, Angela; Cerca, Nuno

    2016-03-01

    Staphylococcus epidermidis is frequently associated with the emergence of medical-device-associated bloodstream infections, due to its ability to form biofilms on the surface of vascular catheters. Although these biofilms may be in continuous contact with human blood, how S. epidermidis biofilm cells interact with blood and its cellular and soluble components is poorly understood. Herein, we evaluated biofilm structure, biofilm cells culturability and viability, and the transcription of a panel of genes associated with S. epidermidis biofilms virulence, upon interaction with whole human blood or plasma. Our results showed that although whole human blood caused significant alterations in biofilm structure and in the number of culturable and viable cells, plasma was the main regulator of the transcription of genes with central role in biofilm formation, maturation and immune evasion. These findings highlight the urgent need to intensify studies aiming to evaluate the impact of host soluble factors on S. epidermidis biofilms fitness and persistence.

  11. Bacterial swimmers that infiltrate and take over the biofilm matrix.

    PubMed

    Houry, Ali; Gohar, Michel; Deschamps, Julien; Tischenko, Ekaterina; Aymerich, Stéphane; Gruss, Alexandra; Briandet, Romain

    2012-08-07

    Bacteria grow in either planktonic form or as biofilms, which are attached to either inert or biological surfaces. Both growth forms are highly relevant states in nature and of paramount scientific focus. However, interchanges between bacteria in these two states have been little explored. We discovered that a subpopulation of planktonic bacilli is propelled by flagella to tunnel deep within a biofilm structure. Swimmers create transient pores that increase macromolecular transfer within the biofilm. Irrigation of the biofilm by swimmer bacteria may improve biofilm bacterial fitness by increasing nutrient flow in the matrix. However, we show that the opposite may also occur (i.e., swimmers can exacerbate killing of biofilm bacteria by facilitating penetration of toxic substances from the environment). We combined these observations with the fact that numerous bacteria produce antimicrobial substances in nature. We hypothesized and proved that motile bacilli expressing a bactericide can also kill a heterologous biofilm population, Staphylococcus aureus in this case, and then occupy the newly created space. These findings identify microbial motility as a determinant of the biofilm landscape and add motility to the complement of traits contributing to rapid alterations in biofilm populations.

  12. Control of marine biofouling and medical biofilm formation with engineered topography

    NASA Astrophysics Data System (ADS)

    Schumacher, James Frederick

    friendly coating for antifouling applications in the ocean. In addition, a biomaterial-grade silicone modified with a tailored engineered topography significantly inhibited the bacterial biofilm growth from Staphylococcus aureus for up to 14 days exposure without the use of bactericidal agents. Mature biofilms were present on equivalently exposed smooth silicone surfaces. Engineered surface topographies present a promising means of blocking biofilm development on medical surfaces and reducing the rate of related infections.

  13. Adhesiveness of opportunistic Staphylococcus aureus to materials used in dental office: In vitro study.

    PubMed

    Merghni, Abderrahmen; Bekir, Karima; Kadmi, Yassine; Dallel, Ines; Janel, Sébastien; Bovio, Simone; Barois, Nicolas; Lafont, Frank; Mastouri, Maha

    2017-02-01

    Staphylococcus aureus (S. aureus) is one of several opportunistic microbial pathogens associated with many healthcare problems. In the present study, S. aureus was assessed for its biofilm-forming ability on materials routinely used in dental offices, including stainless steel (SS), polyethylene (PE), and polyvinyl chloride (PVC). Materials that were tested were characterized for roughness (Ra) and surface free energy (SFE). The adhesion forces exerted by S. aureus to each substratum were investigated using atomic force microscopy (AFM), and biofilm formation was quantitatively assessed by crystal violet staining assay. AFM measurements demonstrated that the strongest adhesion forces (20 nN) were exerted on the PE surfaces (P < 0.05) and depended more on Ra. In addition, the results of biofilm formation capability indicated that S. aureus exhibited more affinity to SS materials when compared to the other materials (P < 0.05). This ability of biofilm formation seems to be more correlated to SFE (R = 0.65). Hence, control of the surface properties of materials used in dental practices is of crucial importance for preventing biofilm formation on dental materials to be used for patients' dental care.

  14. Clay-Bacteria Systems and Biofilm Production

    NASA Astrophysics Data System (ADS)

    Steiner, J.; Alimova, A.; Katz, A.; Steiner, N.; Rudolph, E.; Gottlieb, P.

    2007-12-01

    Soil clots and the aerosol transport of bacteria and spores are promoted by the formation of biofilms (bacteria cells in an extracellular polymeric matrix). Biofilms protect microorganisms by promoting adhesion to both organic and inorganic surfaces. Time series experiments on bacteria-clay suspensions demonstrate that biofilm growth is catalyzed by the presence of hectorite in minimal growth media for the studied species: Gram negatives (Pseudomonas syringae and Escherichia coli,) and Gram positives (Staphylococcus aureus and Bacillus subtilis). Soil organisms (P. syringae, B. subtilis) and organisms found in the human population (E. coli, S. aureus) are both used to demonstrate the general applicability of clay involvement. Fluorescent images of the biofilms are acquired by staining with propidium iodide, a component of the BacLightTM Live/Dead bacterial viability staining kit (Molecular Probes, Eugene, OR). The evolving polysaccharide-rich biofilm reacts with the clay interlayer site causing a complex substitution of the two-water hectorite interlayer with polysaccharide. The result is often a three-peak composite of the (001) x-ray diffraction maxima resulting from polysaccharide-expanded clays and an organic-driven contraction of a subset of the clays in the reaction medium. X-ray diffractograms reveal that the expanded set creates a broad maximum with clay subsets at 1.84 nm and 1.41 nm interlayer spacings as approximated by a least squares double Lorentzian fit, and a smaller shoulder at larger 2q, deriving from a contraction of the interlayer spacing. Washing with chlorox removes organic material from the contracted clay and creates a 1-water hectorite single peak in place of the double peak. The clay response can be used as an indirect indicator of biofilm in an environmental system.

  15. Colonization of epidermal tissue by Staphylococcus aureus produces localized hypoxia and stimulates secretion of antioxidant and caspase-14 proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A partial-thickness epidermal explant model was colonized with GFP-expressing S. aureus and the pattern of S. aureus biofilm growth was characterized using electron and confocal laser scanning microscopy. Oxygen concentration in explants and H2O2 in media was quantified using microelectrodes. The re...

  16. Incidence of Staphylococcus aureus and Analysis of Associated Bacterial Communities on Food Industry Surfaces

    PubMed Central

    Gutiérrez, Diana; Delgado, Susana; Vázquez-Sánchez, Daniel; Martínez, Beatriz; Cabo, Marta López; Rodríguez, Ana; Herrera, Juan J.

    2012-01-01

    Biofilms are a common cause of food contamination with undesirable bacteria, such as pathogenic bacteria. Staphylococcus aureus is one of the major bacteria causing food-borne diseases in humans. A study designed to determine the presence of S. aureus on food contact surfaces in dairy, meat, and seafood environments and to identify coexisting microbiota has therefore been carried out. A total of 442 samples were collected, and the presence of S. aureus was confirmed in 6.1% of samples. Sixty-three S. aureus isolates were recovered and typed by random amplification of polymorphic DNA (RAPD). Profiles were clustered into four groups which were related to specific food environments. All isolates harbored some potential virulence factors such as enterotoxin production genes, biofilm formation-associated genes, antibiotic resistance, or lysogeny. PCR-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprints of bacterial communities coexisting with S. aureus revealed the presence of bacteria either involved in food spoilage or of concern for food safety in all food environments. Food industry surfaces could thus be a reservoir for S. aureus forming complex communities with undesirable bacteria in multispecies biofilms. Uneven microbiological conditions were found in each food sector, which indicates the need to improve hygienic conditions in food processing facilities, particularly the removal of bacterial biofilms, to enhance the safety of food products. PMID:23023749

  17. Incidence of Staphylococcus aureus and analysis of associated bacterial communities on food industry surfaces.

    PubMed

    Gutiérrez, Diana; Delgado, Susana; Vázquez-Sánchez, Daniel; Martínez, Beatriz; Cabo, Marta López; Rodríguez, Ana; Herrera, Juan J; García, Pilar

    2012-12-01

    Biofilms are a common cause of food contamination with undesirable bacteria, such as pathogenic bacteria. Staphylococcus aureus is one of the major bacteria causing food-borne diseases in humans. A study designed to determine the presence of S. aureus on food contact surfaces in dairy, meat, and seafood environments and to identify coexisting microbiota has therefore been carried out. A total of 442 samples were collected, and the presence of S. aureus was confirmed in 6.1% of samples. Sixty-three S. aureus isolates were recovered and typed by random amplification of polymorphic DNA (RAPD). Profiles were clustered into four groups which were related to specific food environments. All isolates harbored some potential virulence factors such as enterotoxin production genes, biofilm formation-associated genes, antibiotic resistance, or lysogeny. PCR-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprints of bacterial communities coexisting with S. aureus revealed the presence of bacteria either involved in food spoilage or of concern for food safety in all food environments. Food industry surfaces could thus be a reservoir for S. aureus forming complex communities with undesirable bacteria in multispecies biofilms. Uneven microbiological conditions were found in each food sector, which indicates the need to improve hygienic conditions in food processing facilities, particularly the removal of bacterial biofilms, to enhance the safety of food products.

  18. Biofilm formation and persistence on abiotic surfaces in the context of food and medical environments.

    PubMed

    Abdallah, Marwan; Benoliel, Corinne; Drider, Djamel; Dhulster, Pascal; Chihib, Nour-Eddine

    2014-07-01

    The biofilm formation on abiotic surfaces in food and medical sectors constitutes a great public health concerns. In fact, biofilms present a persistent source for pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus, which lead to severe infections such as foodborne and nosocomial infections. Such biofilms are also a source of material deterioration and failure. The environmental conditions, commonly met in food and medical area, seem also to enhance the biofilm formation and their resistance to disinfectant agents. In this regard, this review highlights the effect of environmental conditions on bacterial adhesion and biofilm formation on abiotic surfaces in the context of food and medical environment. It also describes the current and emergent strategies used to study the biofilm formation and its eradication. The mechanisms of biofilm resistance to commercialized disinfectants are also discussed, since this phenomenon remains unclear to date.

  19. A Self-produced Trigger for Biofilm Disassembly that Targets Exopolysaccharide

    PubMed Central

    Kolodkin-Gal, Ilana; Cao, Shugeng; Chai, Liraz; Böttcher, Thomas; Kolter, Roberto; Clardy, Jon; Losick, Richard

    2012-01-01

    Biofilms are structured communities of bacteria that are held together by an extracellular matrix consisting of protein and exopolysaccharide. Biofilms often have a limited lifespan, disassembling as nutrients become exhausted and waste products accumulate. D-amino acids were previously identified as a self-produced factor that mediates biofilm disassembly by causing the release of the protein component of the matrix in Bacillus subtilis. Here we report that B. subtilis produces an additional biofilm-disassembly factor, norspermidine. Dynamic light scattering and scanning electron microscopy experiments indicated that norspermidine interacts directly and specifically with exopolysaccharide. D-amino acids and norspermidine acted together to break down existing biofilms and mutants blocked in the production of both formed long-lived biofilms. Norspermidine, but not closely related polyamines, prevented biofilm formation by B. subtilis, Escherichia coli and Staphylococcus aureus. PMID:22541437

  20. Novel strategies against Candida biofilms: interest of synthetic compounds.

    PubMed

    Girardot, Marion; Imbert, Christine

    2016-01-01

    A biofilm is a consortium of microbial cells that are attached to a substratum or an interface. It should be considered a reservoir that may induce serious infections. Indeed, Candidaspp. biofilms may be involved in the persistence or worsening of some chronic inflammatory diseases as well as in systemic infections, which may lead to high morbidity and mortality rates. New strategies are currently being explored, utilizing several synthetic compounds to prevent or fight these Candida biofilms. This article focuses on active synthetic compounds classified with regards to their modes of action: inhibition of early adherence phase, inhibition or control of biofilm maturation and finally elimination of already formed biofilms. Some of them show promise in fighting biofilm.

  1. Photodynamic inactivation of bacterial and yeast biofilms with a cationic porphyrin.

    PubMed

    Beirão, Sandra; Fernandes, Sara; Coelho, Joel; Faustino, Maria A F; Tomé, João P C; Neves, Maria G P M S; Tomé, Augusto C; Almeida, Adelaide; Cunha, Angela

    2014-01-01

    The efficiency of 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin tetra-iodide (Tetra-Py(+)-Me) in the photodynamic inactivation of single-species biofilms of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans and mixed biofilms of S. aureus and C. albicans was evaluated. The effect on the extracellular matrix of P. aeruginosa was also assessed. Irradiation with white light up to an energy dose of 64.8 J cm(-2) in the presence of 20 μm of Tetra-Py(+)-Me caused significant inactivation in all single-species biofilms (3-6 log reductions), although the susceptibility was attenuated in relation to planktonic cells. In mixed biofilms, the inactivation of S. aureus was as efficient as in single-species biofilms but the susceptibility of C. albicans decreased. In P. aeruginosa biofilms, a reduction of 81% in the polysaccharide content of the matrix was observed after treatment with a 20 μm PS concentration and a total light dose of 64.8 J cm(-2). The results show that the Tetra-Py(+)-Me causes significant inactivation of the microorganisms, either in biofilms or in the planktonic form, and demonstrate that polysaccharides of the biofilm matrix may be a primary target of photodynamic damage.

  2. Marine Sponge-Derived Streptomyces sp. SBT343 Extract Inhibits Staphylococcal Biofilm Formation

    PubMed Central

    Balasubramanian, Srikkanth; Othman, Eman M.; Kampik, Daniel; Stopper, Helga; Hentschel, Ute; Ziebuhr, Wilma; Oelschlaeger, Tobias A.; Abdelmohsen, Usama R.

    2017-01-01

    Staphylococcus epidermidis and Staphylococcus aureus are opportunistic pathogens that cause nosocomial and chronic biofilm-associated infections. Indwelling medical devices and contact lenses are ideal ecological niches for formation of staphylococcal biofilms. Bacteria within biofilms are known to display reduced susceptibilities to antimicrobials and are protected from the host immune system. High rates of acquired antibiotic resistances in staphylococci and other biofilm-forming bacteria further hamper treatment options and highlight the need for new anti-biofilm strategies. Here, we aimed to evaluate the potential of marine sponge-derived actinomycetes in inhibiting biofilm formation of several strains of S. epidermidis, S. aureus, and Pseudomonas aeruginosa. Results from in vitro biofilm-formation assays, as well as scanning electron and confocal microscopy, revealed that an organic extract derived from the marine sponge-associated bacterium Streptomyces sp. SBT343 significantly inhibited staphylococcal biofilm formation on polystyrene, glass and contact lens surfaces, without affecting bacterial growth. The extract also displayed similar antagonistic effects towards the biofilm formation of other S. epidermidis and S. aureus strains tested but had no inhibitory effects towards Pseudomonas biofilms. Interestingly the extract, at lower effective concentrations, did not exhibit cytotoxic effects on mouse fibroblast, macrophage and human corneal epithelial cell lines. Chemical analysis by High Resolution Fourier Transform Mass Spectrometry (HRMS) of the Streptomyces sp. SBT343 extract proportion revealed its chemical richness and complexity. Preliminary physico-chemical characterization of the extract highlighted the heat-stable and non-proteinaceous nature of the active component(s). The combined data suggest that the Streptomyces sp. SBT343 extract selectively inhibits staphylococcal biofilm formation without interfering with bacterial cell viability. Due to

  3. Marine Sponge-Derived Streptomyces sp. SBT343 Extract Inhibits Staphylococcal Biofilm Formation.

    PubMed

    Balasubramanian, Srikkanth; Othman, Eman M; Kampik, Daniel; Stopper, Helga; Hentschel, Ute; Ziebuhr, Wilma; Oelschlaeger, Tobias A; Abdelmohsen, Usama R

    2017-01-01

    Staphylococcus epidermidis and Staphylococcus aureus are opportunistic pathogens that cause nosocomial and chronic biofilm-associated infections. Indwelling medical devices and contact lenses are ideal ecological niches for formation of staphylococcal biofilms. Bacteria within biofilms are known to display reduced susceptibilities to antimicrobials and are protected from the host immune system. High rates of acquired antibiotic resistances in staphylococci and other biofilm-forming bacteria further hamper treatment options and highlight the need for new anti-biofilm strategies. Here, we aimed to evaluate the potential of marine sponge-derived actinomycetes in inhibiting biofilm formation of several strains of S. epidermidis, S. aureus, and Pseudomonas aeruginosa. Results from in vitro biofilm-formation assays, as well as scanning electron and confocal microscopy, revealed that an organic extract derived from the marine sponge-associated bacterium Streptomyces sp. SBT343 significantly inhibited staphylococcal biofilm formation on polystyrene, glass and contact lens surfaces, without affecting bacterial growth. The extract also displayed similar antagonistic effects towards the biofilm formation of other S. epidermidis and S. aureus strains tested but had no inhibitory effects towards Pseudomonas biofilms. Interestingly the extract, at lower effective concentrations, did not exhibit cytotoxic effects on mouse fibroblast, macrophage and human corneal epithelial cell lines. Chemical analysis by High Resolution Fourier Transform Mass Spectrometry (HRMS) of the Streptomyces sp. SBT343 extract proportion revealed its chemical richness and complexity. Preliminary physico-chemical characterization of the extract highlighted the heat-stable and non-proteinaceous nature of the active component(s). The combined data suggest that the Streptomyces sp. SBT343 extract selectively inhibits staphylococcal biofilm formation without interfering with bacterial cell viability. Due to

  4. Continuous Drip Flow System to Develop Biofilm of E. faecalis under Anaerobic Conditions

    PubMed Central

    Gonzalez, Ana Maria; Corpus, Erika; Silva-Herzog, Daniel; Aragon-Piña, Antonio; Cohenca, Nestor

    2014-01-01

    Purpose. To evaluate a structurally mature E. faecalis biofilm developed under anaerobic/dynamic conditions in an in vitro system. Methods. An experimental device was developed using a continuous drip flow system designed to develop biofilm under anaerobic conditions. The inoculum was replaced every 24 hours with a fresh growth medium for up to 10 days to feed the system. Gram staining was done every 24 hours to control the microorganism purity. Biofilms developed under the system were evaluated under the scanning electron microscope (SEM). Results. SEM micrographs demonstrated mushroom-shaped structures, corresponding to a mature E. faecalis biofilm. In the mature biofilm bacterial cells are totally encased in a polymeric extracellular matrix. Conclusions. The proposed in vitro system model provides an additional useful tool to study the biofilm concept in endodontic microbiology, allowing for a better understanding of persistent root canal infections. PMID:25371913

  5. Destruction of single-species biofilms of Escherichia coli or Klebsiella pneumoniae subsp. pneumoniae by dextranase, lactoferrin, and lysozyme

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The activity of dextranase, lactoferrin, lysozyme, and nisin against biofilms composed of either Klebsiella pneumonia or Escherichia coli was examined using the MBEC Assay™. Mature biofilms were treated and then sonicated to remove the adherent biofilm. This material was quantified using a lumines...

  6. Microrheology of growing Escherichia coli biofilms investigated by using magnetic force modulation atomic force microscopy.

    PubMed

    Gan, Tiansheng; Gong, Xiangjun; Schönherr, Holger; Zhang, Guangzhao

    2016-12-01

    Microrheology of growing biofilms provides insightful information about its structural evolution and properties. In this study, the authors have investigated the microrheology of Escherichia coli (strain HCB1) biofilms at different indentation depth (δ) by using magnetic force modulation atomic force microscopy as a function of disturbing frequency (f). As δ increases, the dynamic stiffness (ks) for the biofilms in the early stage significantly increases. However, it levels off when the biofilms are matured. The facts indicate that the biofilms change from inhomogeneous to homogeneous in structure. Moreover, ks is scaled to f, which coincides with the rheology of soft glasses. The exponent increases with the incubation time, indicating the fluidization of biofilms. In contrast, the upper layer of the matured biofilms is solidlike in that the storage modulus is always larger than the loss modulus, and its viscoelasticity is slightly influenced by the shear stress.

  7. Aryl Rhodanines Specifically Inhibit Staphylococcal and Enterococcal Biofilm Formation▿ †

    PubMed Central

    Opperman, Timothy J.; Kwasny, Steven M.; Williams, John D.; Khan, Atiyya R.; Peet, Norton P.; Moir, Donald T.; Bowlin, Terry L.

    2009-01-01

    Staphylococcus epidermidis and Staphylococcus aureus are the leading causative agents of indwelling medical device infections because of their ability to form biofilms on artificial surfaces. Here we describe the antibiofilm activity of a class of small molecules, the aryl rhodanines, which specifically inhibit biofilm formation of S. aureus, S. epidermidis, Enterococcus faecalis, E. faecium, and E. gallinarum but not the gram-negative species Pseudomonas aeruginosa or Escherichia coli. The aryl rhodanines do not exhibit antibacterial activity against any of the bacterial strains tested and are not cytotoxic against HeLa cells. Preliminary mechanism-of-action studies revealed that the aryl rhodanines specifically inhibit the early stages of biofilm development by preventing attachment of the bacteria to surfaces. PMID:19651903

  8. Exposure of conjugative plasmid carrying Escherichia coli biofilms to male-specific bacteriophages

    PubMed Central

    May, Thithiwat; Tsuruta, Kenji; Okabe, Satoshi

    2011-01-01

    Escherichia coli carrying a natural conjugative F-plasmid generates F-pili mating pairs, which is important for early biofilm formation. In this study, we investigated the effect of male-specific filamentous single stranded DNA bacteriophage (f1) and RNA bacteriophage (MS2) on the formation of biofilms by E. coli carrying a natural conjugative F-plasmid. We showed that the early biofilm formation was completely inhibited by addition of the f1 phage, but not the MS2 phage. This suggests that the tip of F-pili is the specific attachment site for mating pairs formation and the side of F-pili has a non-obligatory role during biofilm formation. The inhibitory effect of the f1 phage was dependent on the time of addition during the biofilm formation. No inhibitory effect was observed when the f1 phages were added to the mature biofilms. This resistant mechanism of the mature biofilms could be attributed to the biofilm-specific phenotypes representing that the F-pili mating pairs were already formed and then the curli production commenced during the biofilm maturation. The pre-formed mating pairs seemed to resist the f1 phages. Altogether, our results indicate a close relationship between the presence of conjugative plasmid and male-specific bacteriophages within sessile biofilm communities, as well as the possibility of using the male-specific bacteriophages to control biofilm formation. PMID:20962879

  9. The Role of Microbial Biofilms as Ecosystem Engineers in Streams

    NASA Astrophysics Data System (ADS)

    Battin, T. I.; Battin, T. I.; Kaplan, L. A.; Newbold, J. D.

    2001-12-01

    Microbial biofilms growing on and through the surface of streambeds physically alter the interface between the water column and benthic zone and influence the biogeochemistry within the steambed and hyporheic zone. We monitored the development of biofilms within stream-side flumes, and were able to relate changes in biofilm structure to concomitant changes in hydrodynamics, particle deposition, and dissolved organic carbon (DOC) uptake. Biofilm development was assessed by measurements of ash free dry mass, bacterial density, concentrations of chlorophyll a and exopolysaccharides, and confocal microscopy of fluorescent-stained biotic and abiotic assemblages. The microbial biofilms were followed through an initial colonization period, the development of mm-thick mats that included streamers undulating in the current, and the eventual erosion and sloughing of these structural features. As the biofilms matured, hydrologic exchange rate, transient storage capacity, and particle deposition rates increased, reached a plateau, and eventually declined. The uptake of glucose and arabinose, added in nM concentrations to the flumes, showed a preferential uptake of glucose over arabinose. However, as the biofilms grew, the differences between the uptake of these two saccharides declined. This change is consistent with a shift in the rate-limiting step for DOC uptake from internal biofilm processes to greater diffusion-limitation as biofilm thickness, and thus the diffusion barrier, increased. We suggest that microscale processes, which alter biofilm structure, in turn alter large-scale physical and biogeochemical processes, including streamwater/subsurface hydrodynamics and organic matter fluxes.

  10. Role of the nuclease of nontypeable Haemophilus influenzae in dispersal of organisms from biofilms.

    PubMed

    Cho, Christine; Chande, Aroon; Gakhar, Lokesh; Bakaletz, Lauren O; Jurcisek, Joseph A; Ketterer, Margaret; Shao, Jian; Gotoh, Kenji; Foster, Eric; Hunt, Jason; O'Brien, Erin; Apicella, Michael A

    2015-03-01

    Nontypeable Haemophilus influenzae (NTHI) forms biofilms in the middle ear during human infection. The biofilm matrix of NTHI contains extracellular DNA. We show that NTHI possesses a potent nuclease, which is a homolog of the thermonuclease of Staphylococcus aureus. Using a biofilm dispersal assay, studies showed a biofilm dispersal pattern in the parent strain, no evidence of dispersal in the nuclease mutant, and a partial return of dispersion in the complemented mutant. Quantitative PCR of mRNA from biofilms from a 24-h continuous flow system demonstrated a significantly increased expression of the nuclease from planktonic organisms compared to those in the biofilm phase of growth (P < 0.042). Microscopic analysis of biofilms grown in vitro showed that in the nuclease mutant the nucleic acid matrix was increased compared to the wild-type and complemented strains. Organisms were typically found in large aggregates, unlike the wild-type and complement biofilms in which the organisms were evenly dispersed throughout the biofilm. At 48 h, the majority of the organisms in the mutant biofilm were dead. The nuclease mutant formed a biofilm in the chinchilla model of otitis media and demonstrated a propensity to also form similar large aggregates of organisms. These studies indicate that NTHI nuclease is involved in biofilm remodeling and organism dispersal.

  11. Sensitization of Candida albicans biofilms to fluconazole by terpenoids of plant origin.

    PubMed

    Doke, Sonali Kashinath; Raut, Jayant Shankar; Dhawale, Shashikant; Karuppayil, Sankunny Mohan

    2014-01-01

    Infections associated with the biofilms of Candida albicans are a challenge to antifungal treatment. Combinatorial therapy involving plant molecules with antifungal drugs would be an effective complementary approach against drug-resistant Candida biofilms. The aim of this study was to evaluate the efficacy of three bioactive terpenoids (carvacrol, eugenol and thymol) in combination with fluconazole against planktonic cells, biofilm development and mature biofilms of C. albicans. Activities of the selected molecules were tested using a microplate-based methodology, while their combinations with fluconazole were performed in a checkerboard format. Biofilms were quantitated by XTT-metabolic assay and confirmed by microscopic observations. Combinations of carvacrol and eugenol with fluconazole were found synergistic against planktonic growth of C. albicans, while that of thymol with fluconazole did not have any interaction. Biofilm development and mature biofilms were highly resistant to fluconazole, but susceptible to three terpenoids. Sensitization of cells by sub-inhibitory concentrations of carvacrol and eugenol resulted in prevention of biofilm formation at low fluconazole concentrations, i.e. 0.032 and 0.002 mg ml(-1), respectively. Addition of thymol could not potentiate activity of fluconazole against biofilm formation by C. albicans. Fractional inhibitory concentration indices (FICI) for carvacrol-fluconazole and eugenol-fluconazole combinations for biofilm formation were 0.311 and 0.25, respectively. The FICI value of 1.003 indicated a status of indifference for the combination of thymol and fluconazole against biofilm formation. Eugenol and thymol combinations with fluconazole did not have useful interaction against mature biofilms of C. albicans, but the presence of 0.5 mg ml(-1) of carvacrol caused inhibition of mature biofilms at a significantly low concentration (i.e. 0.032 mg ml(-1)) of fluconazole. The study indicated that carvacrol and eugenol

  12. Fluid dynamic effects on staphylococci bacteria biofilms

    NASA Astrophysics Data System (ADS)

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

    2016-11-01

    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. It is known that flow conditions play a role in the development, dispersion and propagation of biofilms in general. The influence of flow on tower formation, however, is not at all understood. This work is focused on the effect of applied shear on tower development. The hypothesis being examined is that tower structures form within a specific range of shear stresses and that there is an as yet ill defined fluid dynamic phenomenon that occurs hours before a tower forms. In this study, a range of shear stresses is examined that brackets 0.6 dynes/cm2, the nominal shear stress where towers seem most likely to form. This talk will include µPTV measurements and cell density data indicating variations in flow and biofilm evolution as a function of the applied shear. Causal relations between flow and biofilm development will be discussed.

  13. Enzymes Enhance Biofilm Removal Efficiency of Cleaners.

    PubMed

    Stiefel, Philipp; Mauerhofer, Stefan; Schneider, Jana; Maniura-Weber, Katharina; Rosenberg, Urs; Ren, Qun

    2016-06-01

    Efficient removal of biofilms from medical devices is a big challenge in health care to avoid hospital-acquired infections, especially from delicate devices like flexible endoscopes, which cannot be reprocessed using harsh chemicals or high temperatures. Therefore, milder solutions such as enzymatic cleaners have to be used, which need to be carefully developed to ensure efficacious performance. In vitro biofilm in a 96-well-plate system was used to select and optimize the formulation of novel enzymatic cleaners. Removal of the biofilm was quantified by crystal violet staining, while the disinfecting properties were evaluated by a BacTiter-Glo assay. The biofilm removal efficacy of the selected cleaner was further tested by using European standard (EN) for endoscope cleaning EN ISO 15883, and removal of artificial blood soil was investigated by treating TOSI (Test Object Surgical Instrument) cleaning indicators. Using the process described here, a novel enzymatic endoscope cleaner was developed, which removed 95% of Staphylococcus aureus and 90% of Pseudomonas aeruginosa biofilms in the 96-well plate system. With a >99% reduction of CFU and a >90% reduction of extracellular polymeric substances, this cleaner enabled subsequent complete disinfection and fulfilled acceptance criteria of EN ISO 15883. Furthermore, it efficiently removed blood soil and significantly outperformed comparable commercial products. The cleaning performance was stable even after storage of the cleaner for 6 months. It was demonstrated that incorporation of appropriate enzymes into the cleaner enhanced performance significantly.

  14. Removal of pathogenic bacterial biofilms by combinations of oxidizing compounds.

    PubMed

    Olmedo, Gabriela María; Grillo-Puertas, Mariana; Cerioni, Luciana; Rapisarda, Viviana Andrea; Volentini, Sabrina Inés

    2015-05-01

    Bacterial biofilms are commonly formed on medical devices and food processing surfaces. The antimicrobials used have limited efficacy against the biofilms; therefore, new strategies to prevent and remove these structures are needed. Here, the effectiveness of brief oxidative treatments, based on the combination of sodium hypochlorite (NaClO) and hydrogen peroxide (H2O2) in the presence of copper sulfate (CuSO4), were evaluated against bacterial laboratory strains and clinical isolates, both in planktonic and biofilm states. Simultaneous application of oxidants synergistically inactivated planktonic cells and prevented biofilm formation of laboratory Escherichia coli, Salmonella enterica serovar Typhimurium, Klebsiella pneumoniae, and Staphylococcus aureus strains, as well as clinical isolates of Salmonella enterica subsp. enterica, Klebsiella oxytoca, and uropathogenic E. coli. In addition, preformed biofilms of E. coli C, Salmonella Typhimurium, K. pneumoniae, and Salmonella enterica exposed to treatments were removed by applying 12 mg/L NaClO, 0.1 mmol/L CuSO4, and 350 mmol/L H2O2 for 5 min. Klebsiella oxytoca and Staphylococcus aureus required a 5-fold increase in NaClO concentration, and the E. coli clinical isolate remained unremovable unless treatments were applied on biofilms formed within 24 h instead of 48 h. The application of treatments that last a few minutes using oxidizing compounds at low concentrations represents an interesting disinfection strategy against pathogens associated with medical and industrial settings.

  15. In vitro model of bacterial biofilm formation on polyvinyl chloride biomaterial.

    PubMed

    Zhao, Guang-qiang; Ye, Lian-hua; Huang, Yun-chao; Yang, Da-kuan; Li, Li; Xu, Geng; Lei, Yu-jie

    2011-11-01

    The aim of the study was to establish an in vitro model of Staphylococcus epidermidis biofilms on polyvinyl chloride (PVC) material, and to investigate bacterial biofilm formation and its structure using the combined approach of confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM). Staphylococcus epidermidis bacteria (stain RP62A) were incubated with PVC pieces in Tris buffered saline to form biofilms. Biofilm formation was examined at 6, 12, 18, 24, 30, and 48 h. Thicknesses of these biofilms and the number, and percentage of viable cells in biofilms were measured. CT scan images of biofilms were obtained using CLSM and environmental SEM. The results of this study showed that Staphylococcus epidermidis biofilm is a highly organized multi-cellular structure. The biofilm is constituted of large number of viable and dead bacterial cells. Bacterial biofilm formation on the surface of PVC material was found to be a dynamic process with maximal thickness being attained at 12-18 h. These biofilms became mature by 24 h. There was significant difference in the percentage of viable cells along with interior, middle, and outer layers of biofilms (P < 0.05). Staphylococcus epidermidis biofilm is sophisticated in structure and the combination method involving CLSM and SEM was ideal for investigation of biofilms on PVC material.

  16. Staphylococcus aureus Induces Hypoxia and Cellular Damage in Porcine Dermal Explants

    PubMed Central

    Lone, Abdul G.; Atci, Erhan; Renslow, Ryan; Beyenal, Haluk; Noh, Susan; Fransson, Boel; Abu-Lail, Nehal; Park, Jeong-Jin; Gang, David R.

    2015-01-01

    We developed a porcine dermal explant model to determine the extent to which Staphylococcus aureus biofilm communities deplete oxygen, change pH, and produce damage in underlying tissue. Microelectrode measurements demonstrated that dissolved oxygen (DO) in biofilm-free dermal tissue was 4.45 ± 1.17 mg/liter, while DO levels for biofilm-infected tissue declined sharply from the surface, with no measurable oxygen detectable in the underlying dermal tissue. Magnetic resonance imaging demonstrated that biofilm-free dermal tissue had a significantly lower relative effective diffusion coefficient (0.26 ± 0.09 to 0.30 ± 0.12) than biofilm-infected dermal tissue (0.40 ± 0.12 to 0.48 ± 0.12; P < 0.0001). Thus, the difference in DO level was attributable to biofilm-induced oxygen demand rather than changes in oxygen diffusivity. Microelectrode measures showed that pH within biofilm-infected explants was more alkaline than in biofilm-free explants (8.0 ± 0.17 versus 7.5 ± 0.15, respectively; P < 0.002). Cellular and nuclear details were lost in the infected explants, consistent with cell death. Quantitative label-free shotgun proteomics demonstrated that both proapoptotic programmed cell death protein 5 and antiapoptotic macrophage migration inhibitory factor accumulated in the infected-explant spent medium, compared with uninfected-explant spent media (1,351-fold and 58-fold, respectively), consistent with the cooccurrence of apoptosis and necrosis in the explants. Biofilm-origin proteins reflected an extracellular matrix-adapted lifestyle of S. aureus. S. aureus biofilms deplete oxygen, increase pH, and induce cell death, all factors that contribute to impede wound healing. PMID:25847960

  17. Bacteriophages as an alternative strategy for fighting biofilm development.

    PubMed

    Parasion, Sylwia; Kwiatek, Magdalena; Gryko, Romuald; Mizak, Lidia; Malm, Anna

    2014-01-01

    The ability of microbes to form biofilms is an important element of their pathogenicity, and biofilm formation is a serious challenge for today's medicine. Fighting the clinical complications associated with biofilm formation is very difficult and linked to a high risk of failure, especially in a time of increasing bacterial resistance to antibiotics. Bacterial species most commonly isolated from biofilms include coagulase-negative staphylococci, Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. The frequent failure of antibiotic therapy led researchers to look for alternative methods and experiment with the use of antibacterial factors with a mechanism of action different from that of antibiotics. Experimental studies with bacteriophages and mixtures thereof, expressing lytic properties against numerous biofilm-forming bacterial species showed that bacteriophages may both prevent biofilm formation and contribute to eradication of biofilm bacteria. A specific role is played here by phage depolymerases, which facilitate the degradation of extracellular polymeric substances (EPS) and thus the permeation of bacteriophages into deeper biofilm layers and lysis of the susceptible bacterial cells. Much hope is placed in genetic modifications of bacteriophages that would allow the equipping bacteriophages with the function of depolymerase synthesis. The use of phage cocktails prevents the development of phage-resistant bacteria.

  18. Biophysics of Biofilm Infection

    PubMed Central

    Stewart, Philip S.

    2014-01-01

    This article examines a likely basis of the tenacity of biofilm infections that has received relatively little attention: the resistance of biofilms to mechanical clearance. One way that a biofilm infection persists is by withstanding the flow of fluid or other mechanical forces that work to wash or sweep microorganisms out of the body. The fundamental criterion for mechanical persistence is that the biofilm failure strength exceeds the external applied stress. Mechanical failure of the biofilm and release of planktonic microbial cells is also important in vivo because it can result in dissemination of infection. The fundamental criterion for detachment and dissemination is that the applied stress exceeds the biofilm failure strength. The apparent contradiction for a biofilm to both persist and disseminate is resolved by recognizing that biofilm material properties are inherently heterogeneous. There are also mechanical aspects to the ways that infectious biofilms evade leukocyte phagocytosis. The possibility of alternative therapies for treating biofilm infections that work by reducing biofilm cohesion could: 1) allow prevailing hydrodynamic shear to remove biofilm, 2) increase the efficacy of designed interventions for removing biofilms, 3) enable phagocytic engulfment of softened biofilm aggregates, and 4) improve phagocyte mobility and access to biofilm. PMID:24376149

  19. The innate immune protein calprotectin promotes Pseudomonas aeruginosa and Staphylococcus aureus interaction

    PubMed Central

    Wakeman, Catherine A.; Moore, Jessica L.; Noto, Michael J.; Zhang, Yaofang; Singleton, Marc D.; Prentice, Boone M.; Gilston, Benjamin A.; Doster, Ryan S.; Gaddy, Jennifer A.; Chazin, Walter J.; Caprioli, Richard M.; Skaar, Eric P.

    2016-01-01

    Microorganisms form biofilms containing differentiated cell populations. To determine factors driving differentiation, we herein visualize protein and metal distributions within Pseudomonas aeruginosa biofilms using imaging mass spectrometry. These in vitro experiments reveal correlations between differential protein distribution and metal abundance. Notably, zinc- and manganese-depleted portions of the biofilm repress the production of anti-staphylococcal molecules. Exposure to calprotectin (a host protein known to sequester metal ions at infectious foci) recapitulates responses occurring within metal-deplete portions of the biofilm and promotes interaction between P. aeruginosa and Staphylococcus aureus. Consistent with these results, the presence of calprotectin promotes co-colonization of the murine lung, and polymicrobial communities are found to co-exist in calprotectin-enriched airspaces of a cystic fibrosis lung explant. These findings, which demonstrate that metal fluctuations are a driving force of microbial community structure, have clinical implications because of the frequent occurrence of P. aeruginosa and S. aureus co-infections. PMID:27301800

  20. Nitroxoline: a broad-spectrum biofilm-eradicating agent against pathogenic bacteria.

    PubMed

    Abouelhassan, Yasmeen; Yang, Qingping; Yousaf, Hussain; Nguyen, Minh Thu; Rolfe, Melanie; Schultz, Gregory S; Huigens, Robert W

    2017-02-01

    Bacterial biofilms are surface-attached communities of slow-growing or non-replicating bacteria tolerant to conventional antibiotic therapies. Although biofilms are known to occur in ca. 80% of all bacterial infections, no therapeutic agent has been developed to eradicate bacteria housed within biofilms. We have discovered that nitroxoline, an antibacterial agent used to treat urinary tract infections, displays broad-spectrum biofilm-eradicating activities against major human pathogens, including drug-resistant Staphylococcus aureus and Acinetobacter baumannii strains. In this study, the effectiveness of nitroxoline to eradicate biofilms was determined using an in vitro [minimum biofilm eradication concentration (MBEC) = 46.9 µM against A. baumannii] and ex vivo porcine skin model (2-3 log reduction in viable biofilm cells). Nitroxoline was also found to eradicate methicillin-resistant S. aureus (MRSA) persister cells in non-biofilm (stationary) cultures, whereas vancomycin and daptomycin were found to be inactive. These findings could lead to effective, nitroxoline-based therapies for biofilm-associated infections.

  1. Integration of non-oral bacteria into in vitro oral biofilms.

    PubMed

    Thurnheer, Thomas; Belibasakis, Georgios N

    2015-01-01

    Biofilms are polymicrobial communities that grow on surfaces in nature. Oral bacteria can spontaneously form biofilms on the surface of teeth, which may compromise the health of the teeth, or their surrounding (periodontal) tissues. While the oral bacteria exhibit high tropism for their specialized ecological niche, it is not clear if bacteria that are not part of the normal oral microbiota can efficiently colonize and grow within oral biofilms. By using an in vitro "supragingival" biofilm model of 6 oral species, this study aimed to investigate if 3 individual bacterial species that are not part of the normal oral microbiota (Eschericia coli, Staphylococcus aureus, Enterococcus faecails) and one not previously tested oral species (Aggregatibacter actinomycetemcomitans) can be incorporated into this established supragingival biofilm model. Staphylococcus aureus and A. actinomycetemcomitans were able to grow efficiently in the biofilm, without disrupting the growth of the remaining species. They localized in sparse small aggregates within the biofilm mass. Enterococcus faecalis and E. coli were both able to populate the biofilm at high numbers, and suppressed the growth of A. oris and S. mutants. Enterococcus faecalis was arranged in a chain-like conformation, whereas E. coli was densely and evenly spread throughout the biofilm mass. In conclusion, it is possible for selected species that are not part of the normal oral microbiota to be introduced into an oral biofilm, under the given experimental micro-environmental conditions. Moreover, the equilibrated incorporation of A. actinomycetemcomitans and S. aureus in this oral biofilm model could be a useful tool in the study of aggressive periodontitis and peri-implantitis, in which these organisms are involved, respectively.

  2. Dynamics of biofilm formation during anaerobic digestion of organic waste.

    PubMed

    Langer, Susanne; Schropp, Daniel; Bengelsdorf, Frank R; Othman, Maazuza; Kazda, Marian

    2014-10-01

    Biofilm-based reactors are effectively used for wastewater treatment but are not common in biogas production. This study investigated biofilm dynamics on biofilm carriers incubated in batch biogas reactors at high and low organic loading rates for sludge from meat industry dissolved air flotation units. Biofilm formation and dynamics were studied using various microscopic techniques. Resulting micrographs were analysed for total cell numbers, thickness of biofilms, biofilm-covered surface area, and the area covered by extracellular polymeric substances (EPS). Cell numbers within biofilms (10(11) cells ml(-1)) were up to one order of magnitude higher compared to the numbers of cells in the fluid reactor content. Further, biofilm formation and structure mainly correlated with the numbers of microorganisms present in the fluid reactor content and the organic loading. At high organic loading (45 kg VS m(-3)), the thickness of the continuous biofilm layer ranged from 5 to 160 μm with an average of 51 μm and a median of 26 μm. Conversely, at lower organic loading (15 kg VS m(-3)), only microcolonies were detectable. Those microcolonies increased in their frequency of occurrence during ongoing fermentation. Independently from the organic loading rate, biofilms were embedded completely in EPS within seven days. The maturation and maintenance of biofilms changed during the batch fermentation due to decreasing substrate availability. Concomitant, detachment of microorganisms within biofilms was observed simultaneously with the decrease of biogas formation. This study demonstrates that biofilms of high cell densities can enhance digestion of organic waste and have positive effects on biogas production.

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

    PubMed Central

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

    2017-01-01

    Background/Aims 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. Methods 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. Results 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. Conclusions 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. PMID:27653617

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

    PubMed

    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.

  5. Bdellovibrio bacteriovorus directly attacks Pseudomonas aeruginosa and Staphylococcus aureus Cystic fibrosis isolates.

    PubMed

    Iebba, Valerio; Totino, Valentina; Santangelo, Floriana; Gagliardi, Antonella; Ciotoli, Luana; Virga, Alessandra; Ambrosi, Cecilia; Pompili, Monica; De Biase, Riccardo V; Selan, Laura; Artini, Marco; Pantanella, Fabrizio; Mura, Francesco; Passariello, Claudio; Nicoletti, Mauro; Nencioni, Lucia; Trancassini, Maria; Quattrucci, Serena; Schippa, Serena

    2014-01-01

    Bdellovibrio bacteriovorus is a predator bacterial species found in the environment and within the human gut, able to attack Gram-negative prey. Cystic fibrosis (CF) is a genetic disease which usually presents lung colonization by Pseudomonas aeruginosa or Staphylococcus aureus biofilms. Here, we investigated the predatory behavior of B. bacteriovorus against these two pathogenic species with: (1) broth culture; (2) "static" biofilms; (3) field emission scanning electron microscope (FESEM); (4) "flow" biofilms; (5) zymographic technique. We had the first evidence of B. bacteriovorus survival with a Gram-positive prey, revealing a direct cell-to-cell contact with S. aureus and a new "epibiotic" foraging strategy imaged with FESEM. Mean attaching time of HD100 to S. aureus cells was 185 s, while "static" and "flow" S. aureus biofilms were reduced by 74 (at 24 h) and 46% (at 20 h), respectively. Furthermore, zymograms showed a differential bacteriolytic activity exerted by the B. bacteriovorus lysates on P. aeruginosa and S. aureus. The dual foraging system against Gram-negative (periplasmic) and Gram-positive (epibiotic) prey could suggest the use of B. bacteriovorus as a "living antibiotic" in CF, even if further studies are required to simulate its in vivo predatory behavior.

  6. In Vitro Approach for Identification of the Most Effective Agents for Antimicrobial Lock Therapy in the Treatment of Intravascular Catheter-Related Infections Caused by Staphylococcus aureus

    PubMed Central

    Hogan, S.; Zapotoczna, M.; Stevens, N. T.; Humphreys, H.; O'Gara, J. P.

    2016-01-01

    Infection of intravascular catheters by Staphylococcus aureus is a significant risk factor within the health care setting. To treat these infections and attempt salvage of an intravascular catheter, antimicrobial lock solutions (ALSs) are being increasingly used. However, the most effective ALSs against these biofilm-mediated infections have yet to be determined, and clinical practice varies greatly. The purpose of this study was to evaluate and compare the efficacies of antibiotics and antiseptics in current clinical use against biofilms produced by reference and clinical isolates of S. aureus. Static and flow biofilm assays were developed using newly described in vivo-relevant conditions to examine the effect of each agent on S. aureus within the biofilm matrix. The antibiotics daptomycin, tigecycline, and rifampin and the antiseptics ethanol and Taurolock inactivated established S. aureus biofilms, while other commonly used antistaphylococcal antibiotics and antiseptic agents were less effective. These findings were confirmed by live/dead staining of S. aureus biofilms formed and treated within a flow cell model. The results from this study demonstrate the most effective clinically used agents and their concentrations which should be used within an ALS to treat S. aureus-mediated intravascular catheter-related infections. PMID:26926633

  7. Micron-scale Fe 2+/Fe 3+, intermediate sulfur species and O 2 gradients across the biofilm-solution-sediment interface control biofilm organization

    NASA Astrophysics Data System (ADS)

    Ma, Shufen; Banfield, Jillian F.

    2011-06-01

    We measured micron-scale Fe 2+/Fe 3+ and intermediate sulfur species gradients across the biofilm-solution interface and defined the microbial community composition in natural and bioreactor-cultivated acid mine drainage biofilms to investigate how community organization correlated with geochemical conditions. Intermediate sulfur species concentrations were also measured in associated sediments. Under initial conditions of high Fe 2+ and O 2 concentrations, the first biofilm colonists were Leptospirillum Group II, UBA genotype, and a few Archaea. Cytochrome 579 concentration in early formed biofilms was high, correlating with rapid Fe 2+ oxidation. As biofilm thickness increased, O 2 concentrations in the middle of biofilms decreased, indicating that diffusion limitation of O 2 may control activity levels of aerobic organisms. Calculated low O 2 and high Fe 3+ concentrations in the interior regions of biofilms may explain the previously reported suppression of the UBA genotype in mature biofilms. Instead, Leptospirillum Group II, 5-way CG genotype, dominated under these conditions. Leptospirillum Group III and eukaryotes appeared in the community as the biofilm thickened and Fe 3+/Fe 2+ increased. In mature biofilms, the architecture changed from planar to crenulated, perhaps to increase the surface area of biofilms and decrease O 2 diffusion limitation. In thick, mature biofilms, layering is associated with segregation of Leptospirillum Group II and Archaea and the concentration of cytochrome 579 is lower. The accumulation of Archaea close to the biofilm-air interface may facilitate their aerobic metabolism of waste carbon compounds. Sulfite, thiosulfate and polysulfides were detected in AMD sediments and thiosulfate was detected in solution. These compounds indicate the redox status of the system and represent potential energy sources. Temporal and spatial heterogeneity in community structure correlate with heterogeneity in geochemical conditions, implying active

  8. New Derivatives of Pyridoxine Exhibit High Antibacterial Activity against Biofilm-Embedded Staphylococcus Cells.

    PubMed

    Kayumov, Airat R; Nureeva, Aliya A; Trizna, Elena Yu; Gazizova, Guzel R; Bogachev, Mikhail I; Shtyrlin, Nikita V; Pugachev, Mikhail V; Sapozhnikov, Sergey V; Shtyrlin, Yurii G

    2015-01-01

    Opportunistic bacteria Staphylococcus aureus and Staphylococcus epidermidis often form rigid biofilms on tissues and inorganic surfaces. In the biofilm bacterial cells are embedded in a self-produced polysaccharide matrix and thereby are inaccessible to biocides, antibiotics, or host immune system. Here we show the antibacterial activity of newly synthesized cationic biocides, the quaternary ammonium, and bisphosphonium salts of pyridoxine (vitamin B6) against biofilm-embedded Staphylococci. The derivatives of 6-hydroxymethylpyridoxine were ineffective against biofilm-embedded S. aureus and S. epidermidis at concentrations up to 64 μg/mL, although all compounds tested exhibited low MICs (2 μg/mL) against planktonic cells. In contrast, the quaternary ammonium salt of pyridoxine (N,N-dimethyl-N-((2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-yl)methyl)octadecan-1-aminium chloride (3)) demonstrated high biocidal activity against both planktonic and biofilm-embedded bacteria. Thus, the complete death of biofilm-embedded S. aureus and S. epidermidis cells was obtained at concentrations of 64 and 16 μg/mL, respectively. We suggest that the quaternary ammonium salts of pyridoxine are perspective to design new synthetic antibiotics and disinfectants for external application against biofilm-embedded cells.

  9. The Extracellular Matrix Component Psl Provides Fast-Acting Antibiotic Defense in Pseudomonas aeruginosa Biofilms

    PubMed Central

    Billings, Nicole; Ramirez Millan, Maria; Caldara, Marina; Rusconi, Roberto; Tarasova, Yekaterina; Stocker, Roman; Ribbeck, Katharina

    2013-01-01

    Bacteria within biofilms secrete and surround themselves with an extracellular matrix, which serves as a first line of defense against antibiotic attack. Polysaccharides constitute major elements of the biofilm matrix and are implied in surface adhesion and biofilm organization, but their contributions to the resistance properties of biofilms remain largely elusive. Using a combination of static and continuous-flow biofilm experiments we show that Psl, one major polysaccharide in the Pseudomonas aeruginosa biofilm matrix, provides a generic first line of defense toward antibiotics with diverse biochemical properties during the initial stages of biofilm development. Furthermore, we show with mixed-strain experiments that antibiotic-sensitive “non-producing” cells lacking Psl can gain tolerance by integrating into Psl-containing biofilms. However, non-producers dilute the protective capacity of the matrix and hence, excessive incorporation can result in the collapse of resistance of the entire community. Our data also reveal that Psl mediated protection is extendible to E. coli and S. aureus in co-culture biofilms. Together, our study shows that Psl represents a critical first bottleneck to the antibiotic attack of a biofilm community early in biofilm development. PMID:23950711

  10. Role of Multicellular Aggregates in Biofilm Formation

    PubMed Central

    Kragh, Kasper N.; Hutchison, Jaime B.; Melaugh, Gavin; Rodesney, Chris; Roberts, Aled E. L.; Irie, Yasuhiko; Jensen, Peter Ø.; Diggle, Stephen P.; Allen, Rosalind J.

    2016-01-01

    ABSTRACT In traditional models of in vitro biofilm development, individual bacterial cells seed a surface, multiply, and mature into multicellular, three-dimensional structures. Much research has been devoted to elucidating the mechanisms governing the initial attachment of single cells to surfaces. However, in natural environments and during infection, bacterial cells tend to clump as multicellular aggregates, and biofilms can also slough off aggregates as a part of the dispersal process. This makes it likely that biofilms are often seeded by aggregates and single cells, yet how these aggregates impact biofilm initiation and development is not known. Here we use a combination of experimental and computational approaches to determine the relative fitness of single cells and preformed aggregates during early development of Pseudomonas aeruginosa biofilms. We find that the relative fitness of aggregates depends markedly on the density of surrounding single cells, i.e., the level of competition for growth resources. When competition between aggregates and single cells is low, an aggregate has a growth disadvantage because the aggregate interior has poor access to growth resources. However, if competition is high, aggregates exhibit higher fitness, because extending vertically above the surface gives cells at the top of aggregates better access to growth resources. Other advantages of seeding by aggregates, such as earlier switching to a biofilm-like phenotype and enhanced resilience toward antibiotics and immune response, may add to this ecological benefit. Our findings suggest that current models of biofilm formation should be reconsidered to incorporate the role of aggregates in biofilm initiation. PMID:27006463

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

    PubMed

    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.

  12. Identification of a Novel Benzimidazole That Inhibits Bacterial Biofilm Formation in a Broad-Spectrum Manner▿

    PubMed Central

    Sambanthamoorthy, Karthik; Gokhale, Ankush A.; Lao, Weiwei; Parashar, Vijay; Neiditch, Matthew B.; Semmelhack, Martin F.; Lee, Ilsoon; Waters, Christopher M.

    2011-01-01

    Bacterial biofilm formation causes significant industrial economic loss and high morbidity and mortality in medical settings. Biofilms are defined as multicellular communities of bacteria encased in a matrix of protective extracellular polymers. Because biofilms have a high tolerance for treatment with antimicrobials, protect bacteria from immune defense, and resist clearance with standard sanitation protocols, it is critical to develop new approaches to prevent biofilm formation. Here, a novel benzimidazole molecule, named antibiofilm compound 1 (ABC-1), identified in a small-molecule screen, was found to prevent bacterial biofilm formation in multiple Gram-negative and Gram-positive bacterial pathogens, including Pseudomonas aeruginosa and Staphylococcus aureus, on a variety of different surface types. Importantly, ABC-1 itself does not inhibit the growth of bacteria, and it is effective at nanomolar concentrations. Also, coating a polystyrene surface with ABC-1 reduces biofilm formation. These data suggest ABC-1 is a new chemical scaffold for the development of antibiofilm compounds. PMID:21709104

  13. Detailed comparison of Candida albicans and Candida glabrata biofilms under different conditions and their susceptibility to caspofungin and anidulafungin.

    PubMed

    Kucharíková, Sona; Tournu, Hélène; Lagrou, Katrien; Van Dijck, Patrick; Bujdáková, Helena

    2011-09-01

    Candida biofilm development can be influenced by diverse factors such as substrate, culture medium, carbohydrate source and pH. We have analysed biofilm formation of Candida albicans SC5314 and Candida glabrata ATCC 2001 wild-type strains in the presence of different media (RPMI 1640 versus YNB) and using different pH values (pH 5.6 or 7.0). We determined adhesion and biofilm formation on polystyrene, changes in the expression of adhesin genes during these processes and the susceptibility of mature biofilms to echinocandins. Biofilms formed on polystyrene by both Candida species proved to be influenced strongly by the composition of the medium rather than pH. C. albicans and C. glabrata formed thicker biofilms in RPMI 1640 medium, whereas in YNB medium, both species manifested adhesion rather than characteristic multilayer biofilm architecture. The stimulated biofilm formation in RPMI 1640 medium at pH 7.0 corroborated positively with increased expression of adhesin genes, essential to biofilm formation in vitro, including ALS3 and EAP1 in C. albicans and EPA6 in C. glabrata. The thicker biofilms grown in RPMI 1640 medium were more tolerant to caspofungin and anidulafungin than YNB-grown biofilms. We also observed that mature C. glabrata biofilms were less susceptible in RPMI 1640 medium to echinocandins than C. albicans biofilms. Environmental conditions, i.e. medium and pH, can significantly affect not only biofilm architecture, but also the expression profile of several genes involved during the different stages of biofilm development. In addition, growth conditions may also influence the antifungal-susceptibility profile of fungal populations within biofilm structures. Therefore, before designing any experimental biofilm set-up, it is important to consider the potential influence of external environmental factors on Candida biofilm development.

  14. Histones from Avian Erythrocytes Exhibit Antibiofilm activity against methicillin-sensitive and methicillin-resistant Staphylococcus aureus

    PubMed Central

    Rose-Martel, Megan; Kulshreshtha, Garima; Ahferom Berhane, Nahom; Jodoin, Joelle; Hincke, Maxwell T.

    2017-01-01

    Staphylococcus aureus, a human pathogen associated with many illnesses and post-surgical infections, can resist treatment due to the emergence of antibiotic-resistant strains and through biofilm formation. The current treatments for chronic biofilm infections are antibiotics and/or surgical removal of the contaminated medical device. Due to higher morbidity and mortality rates associated with overuse/misuse of antibiotics, alternate treatments are essential. This study reports the antibiofilm activity of avian erythrocyte histones against methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA). Fluorescence and scanning electron microscopy revealed membrane damage to bacteria in histone-treated biofilms. Histones and indolicidin (positive control) increased the expression of apsS and apsR, which are associated with the Antimicrobial Peptide (AMP) sensor/regulator system in S. aureus. The expression of dltB, and vraF, associated with AMP resistance mechanisms, were under histone inducible control in the biofilm-embedded bacterial cells. The time kill kinetics for histones against S. aureus revealed a rapid biocidal activity (<5 min). Purified erythrocyte-specific histone H5 possessed 3–4 fold enhanced antimicrobial activity against planktonic cells compared to the histone mixture (H1, H2A, H2B, H3, H4, H5). These results demonstrate the promise of histones and histone-like derivatives as novel antibiotics against pathogens in their planktonic and biofilm forms. PMID:28378802

  15. Biofilms of Clostridium species.

    PubMed

    Pantaléon, Véronique; Bouttier, Sylvie; Soavelomandroso, Anna Philibertine; Janoir, Claire; Candela, Thomas

    2014-12-01

    The biofilm is a microbial community embedded in a synthesized matrix and is the main bacterial way of life. A biofilm adheres on surfaces or is found on interfaces. It protects bacteria from the environment, toxic molecules and may have a role in virulence. Clostridium species are spread throughout both environments and hosts, but their biofilms have not been extensively described in comparison with other bacterial species. In this review we describe all biofilms formed by Clostridium species during both industrial processes and in mammals where biofilms may be formed either during infections or associated to microbiota in the gut. We have specifically focussed on Clostridium difficile and Clostridium perfringens biofilms, which have been studied in vitro. Regulatory processes including sporulation and germination highlight how these Clostridium species live in biofilms. Furthermore, biofilms may have a role in the survival and spreading of Clostridium species.

  16. Quantification of confocal images of biofilms grown on irregular surfaces

    PubMed Central

    Ross, Stacy Sommerfeld; Tu, Mai Han; Falsetta, Megan L.; Ketterer, Margaret R.; Kiedrowski, Megan R.; Horswill, Alexander R.; Apicella, Michael A.; Reinhardt, Joseph M.; Fiegel, Jennifer

    2014-01-01

    Bacterial biofilms grow on many types of surfaces, including flat surfaces such as glass and metal and irregular surfaces such as rocks, biological tissues and polymers. While laser scanning confocal microscopy can provide high-resolution images of biofilms grown on any surface, quantification of biofilm-associated bacteria is currently limited to bacteria grown on flat surfaces. This can limit researchers studying irregular surfaces to qualitative analysis or quantification of only the total bacteria in an image. In this work, we introduce a new algorithm called modified connected volume filtration (MCVF) to quantify bacteria grown on top of an irregular surface that is fluorescently labeled or reflective. Using the MCVF algorithm, two new quantification parameters are introduced. The modified substratum coverage parameter enables quantification of the connected-biofilm bacteria on top of the surface and on the imaging substratum. The utility of MCVF and the modified substratum coverage parameter were shown with Pseudomonas aeruginosa and Staphylococcus aureus biofilms grown on human airway epithelial cells. A second parameter, the percent association, provides quantified data on the colocalization of the bacteria with a labeled component, including bacteria within a labeled tissue. The utility of quantifying the bacteria associated with the cell cytoplasm was demonstrated with Neisseria gonorrhoeae biofilms grown on cervical epithelial cells. This algorithm provides more flexibility and quantitative ability to researchers studying biofilms grown on a variety of irregular substrata. PMID:24632515

  17. Printed paper-based arrays as substrates for biofilm formation

    PubMed Central

    2014-01-01

    The suitability of paper-based arrays for biofilm formation studies by Staphylococcus aureus is demonstrated. Laboratory-coated papers with different physicochemical properties were used as substrates. The array platform was fabricated by patterning the coated papers with vinyl-substituted polydimethylsiloxane (PDMS) -based ink. The affinity of bacteria onto the flexographically printed hydrophobic and smooth PDMS film was very low whereas bacterial adhesion and biofilm formation occurred preferentially on the unprinted areas, i.e. in the reaction arrays. The concentration of the attached bacteria was quantified by determining the viable colony forming unit (CFU/cm2) numbers. The distribution and the extent of surface coverage of the biofilms were determined by atomic force microscopy. In static conditions, the highest bacterial concentration and most highly organized biofilms were observed on substrates with high polarity. On a rough paper surface with low polarity, the biofilm formation was most hindered. Biofilms were effectively removed from a polar substrate upon exposure to (+)-dehydroabietic acid, an anti-biofilm compound. PMID:25006538

  18. IMPACTS OF BIOFILM FORMATION ON CELLULOSE FERMENTATION

    SciTech Connect

    Leschine, Susan

    2009-10-31

    This project addressed four major areas of investigation: i) characterization of formation of Cellulomonas uda biofilms on cellulose; ii) characterization of Clostridium phytofermentans biofilm development; colonization of cellulose and its regulation; iii) characterization of Thermobifida fusca biofilm development; colonization of cellulose and its regulation; and iii) description of the architecture of mature C. uda, C. phytofermentans, and T. fusca biofilms. This research is aimed at advancing understanding of biofilm formation and other complex processes involved in the degradation of the abundant cellulosic biomass, and the biology of the microbes involved. Information obtained from these studies is invaluable in the development of practical applications, such as the single-step bioconversion of cellulose-containing residues to fuels and other bioproducts. Our results have clearly shown that cellulose-decomposing microbes rapidly colonize cellulose and form complex structures typical of biofilms. Furthermore, our observations suggest that, as cells multiply on nutritive surfaces during biofilms formation, dramatic cell morphological changes occur. We speculated that morphological changes, which involve a transition from rod-shaped cells to more rounded forms, might be more apparent in a filamentous microbe. In order to test this hypothesis, we included in our research a study of biofilm formation by T. fusca, a thermophilic cellulolytic actinomycete commonly found in compost. The cellulase system of T. fusca has been extensively detailed through the work of David Wilson and colleagues at Cornell, and also, genome sequence of a T. fusca strain has been determine by the DOE Joint Genome Institute. Thus, T. fusca is an excellent subject for studies of biofilm development and its potential impacts on cellulose degradation. We also completed a study of the chitinase system of C. uda. This work provided essential background information for understanding how C. uda

  19. Staphylococcal biofilm formation on the surface of three different calcium phosphate bone grafts: a qualitative and quantitative in vivo analysis.

    PubMed

    Furustrand Tafin, Ulrika; Betrisey, Bertrand; Bohner, Marc; Ilchmann, Thomas; Trampuz, Andrej; Clauss, Martin

    2015-03-01

    Differences in physico-chemical characteristics of bone grafts to fill bone defects have been demonstrated to influence in vitro bacterial biofilm formation. Aim of the study was to investigate in vivo staphylococcal biofilm formation on different calcium phosphate bone substitutes. A foreign-body guinea-pig infection model was used. Teflon cages prefilled with β-tricalcium phosphate, calcium-deficient hydroxyapatite, or dicalcium phosphate (DCP) scaffold were implanted subcutaneously. Scaffolds were infected with 2 × 10(3) colony-forming unit of Staphylococcus aureus (two strains) or S. epidermidis and explanted after 3, 24 or 72 h of biofilm formation. Quantitative and qualitative biofilm analysis was performed by sonication followed by viable counts, and microcalorimetry, respectively. Independently of the material, S. aureus formed increasing amounts of biofilm on the surface of all scaffolds over time as determined by both methods. For S. epidermidis, the biofilm amount decreased over time, and no biofilm was detected by microcalorimetry on the DCP scaffolds after 72 h of infection. However, when using a higher S. epidermidis inoculum, increasing amounts of biofilm were formed on all scaffolds as determined by microcalorimetry. No significant variation in staphylococcal in vivo biofilm formation was observed between the different materials tested. This study highlights the importance of in vivo studies, in addition to in vitro studies, when investigating biofilm formation of bone grafts.

  20. Biofilms: A microbial home

    PubMed Central

    Chandki, Rita; Banthia, Priyank; Banthia, Ruchi

    2011-01-01

    Microbial biofilms are mainly implicated in etiopathogenesis of caries and periodontal disease. Owing to its properties, these pose great challenges. Continuous and regular disruption of these biofilms is imperative for prevention and management of oral diseases. This essay provides a detailed insight into properties, mechanisms of etiopathogenesis, detection and removal of these microbial biofilms. PMID:21976832

  1. Mechanisms of post-transcriptional gene regulation in bacterial biofilms

    PubMed Central

    Martínez, Luary C.; Vadyvaloo, Viveka

    2014-01-01

    Biofilms are characterized by a dense multicellular community of microorganisms that can be formed by the attachment of bacteria to an inert surface and to each other. The development of biofilm involves the initial attachment of planktonic bacteria to a surface, followed by replication, cell-to-cell adhesion to form microcolonies, maturation, and detachment. Mature biofilms are embedded in a self-produced extracellular polymeric matrix composed primarily of bacterial-derived exopolysaccharides, specialized proteins, adhesins, and occasionally DNA. Because the synthesis and assembly of biofilm matrix components is an exceptionally complex process, the transition between its different phases requires the coordinate expression and simultaneous regulation of many genes by complex genetic networks involving all levels of gene regulation. The finely controlled intracellular level of the chemical second messenger molecule, cyclic-di-GMP is central to the post-transcriptional mechanisms governing the switch between the motile planktonic lifestyle and the sessile biofilm forming state in many bacteria. Several other post-transcriptional regulatory mechanisms are known to dictate biofilm development and assembly and these include RNA-binding proteins, small non-coding RNAs, toxin-antitoxin systems, riboswitches, and RNases. Post-transcriptional regulation is therefore a powerful molecular mechanism employed by bacteria to rapidly adjust to the changing environment and to fine tune gene expression to the developmental needs of the cell. In this review, we discuss post-transcriptional mechanisms that influence the biofilm developmental cycle in a variety of pathogenic bacteria. PMID:24724055

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

    PubMed

    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.

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

    PubMed Central

    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</