Modulation of poly-N-acetylglucosamine accumulation within mature Staphylococcus epidermidis biofilms grown in excess glucose

PNAG is a major component of Staphylococcus epidermidis biofilms involved in intercellular adhesion as well as in the interaction of the biofilm with components of the host immune response. Synthesis of PNAG has been found to be regulated by several environmental factors. In the present study, the effect of glucose metabolism-dependent culture medium acidification in PNAG accumulation was evaluated. Established S. epidermidis biofilms were allowed to grow in excess glucose with or without maintained pH conditions. PNAG accumulation in these biofilms was determined by flow cytometry and fluorescence microscopy using wheat germ agglutinin as a fluorescent probe. Biofilms grown in maintained pH conditions presented significantly higher amounts of this polymer as well as higher icaA expression than biofilms grown in acidic pH conditions. Moreover, PNAG accumulation in biofilms grown in non-maintained pH conditions occurred in association with cell death. Overall, we show that glucose metabolism by decreasing the culture pH affects biofilm physiology in respect to PNAG production and cell death. The reported in vitro modulation of PNAG accumulation within S. epidermidis biofilms further highlights the role of environment on determining the biofilm physiological state.     

Effect of Batch and Fed-Batch Growth Modes on Biofilm Formation by <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> at Different Temperatures

The influence of Listeria monocytogenes (L. monocytogenes) biofilm formation feeding conditions (batch and fed-batch) at different temperatures on biofilm biomass and activity was determined. Biofilm biomass and cellular metabolic activity were assessed by Crystal Violet (CV) staining and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) colorimetric method, respectively. Live/Dead staining was also performed in order to get microscopic visualization of the different biofilms. Results revealed that at refrigeration temperature (4°C) a higher amount of biofilm was produced when batch conditions were applied, while at higher temperatures the fed-batch feeding condition was the most effective on biofilm formation. Moreover, independently of the temperature used, biofilms formed under fed-batch conditions were metabolically more active than those formed in batch mode. In conclusion, this work shows that different growth modes significantly influence L. monocytogenes biofilm formation on abiotic surfaces as well as the metabolic activity of cells within biofilms.     

Characterization of the poly-β-1,6-N-acetylglucosamine polysaccharide component of Burkholderia biofilms

We demonstrated the production of poly-β-1,6-N-acetylglucosamine (PNAG) polysaccharide in the biofilms of Burkholderia multivorans, Burkholderia vietnamiensis, Burkholderia ambifaria, Burkholderia cepacia, and Burkholderia cenocepacia using an immunoblot assay for PNAG. These results were confirmed by further studies, which showed that the PNAG hydrolase, dispersin B, eliminated immunoreactivity of extracts from the species that were tested (B. cenocepacia and B. multivorans). Dispersin B also inhibited biofilm formation and dispersed preformed biofilms of Burkholderia species. These results imply a role for PNAG in the maintenance of Burkholderia biofilm integrity. While PNAG was present in biofilms of all of the wild-type test organisms, a ΔpgaBC mutant of B. multivorans (Mu5) produced no detectable PNAG, indicating that these genes are needed for Burkholderia PNAG formation. Furthermore, restoration of PNAG production in PNAG negative E. coli TRXWMGΔC (ΔpgaC) by complementation with B. multivorans pgaBCD confirmed the involvement of these genes in Burkholderia PNAG production. While the confocal scanning laser microscopy of untreated wild-type B. multivorans showed thick, multilayered biofilm, Mu5 and dispersin B-treated wild-type biofilms were thin, poorly developed, and disrupted, confirming the involvement of PNAG in B. multivorans biofilm formation. Thus, PNAG appears to be an important component of Burkholderia biofilms, potentially contributing to its resistance to multiple antibiotics and persistence during chronic infections, including cystic fibrosis-associated infection.     

Carbohydrate-containing components of biofilms produced in vitro by some staphylococcal strains related to orthopaedic prosthesis infections

The capacity of coagulase-negative staphylococci to colonize implanted medical devices is generally attributed to their ability to produce biofilms. Biofilm of the model strain of Staphylococcus epidermidis RP62A was shown to contain two carbohydrate-containing moieties, a linear poly-beta-(1-->6)-N-acetyl-D-glucosamine (PNAG) and teichoic acid. In the present study, we investigated several biofilm-producing staphylococci isolated from infected orthopaedic implants and characterized the composition of the laboratory-grown biofilms using chemical analysis and 1H nuclear magnetic resonance spectroscopy. Extracellular teichoic acid was produced by all strains studied. Some of the clinical strains were shown to produce biofilms with compositions similar to that of the model strain, containing a varying amount of PNAG. The chemical structure of PNAG of the clinical strains was similar to that previously described for the model strains S. epidermidis RP62A and Staphylococcus aureus MN8m, differing only in the amount of charged groups. Biofilms of the strains producing a substantial amount of PNAG were detached by dispersin B, a PNAG-degrading enzyme, while being unsusceptible to proteinase K treatment. On the other hand, some strains produced biofilms without any detectable amount of PNAG. The biofilms of these strains were dispersed by proteinase K, but not by dispersin B.     

In vitro activity of xanthorrhizol against Streptococcus mutans biofilms

AIMS: We determined the effect of xanthorrhizol (XTZ) purified from the rhizome of Curcuma xanthorrhiza Roxb. on the Streptococcus mutans biofilms in vitro. METHODS AND RESULTS: The biofilms of S. mutans at different phases of growth were exposed to XTZ at different concentrations (5, 10 and 50 micromol l(-1)) and for different time exposures (1, 10, 30 and 60 min). The results demonstrated that the activity of XTZ in removing S. mutans biofilm was dependent on the concentration, exposure time and the phase growth of biofilm. A concentration of 5 micromol l(-1) of XTZ completely inhibited biofilm formation by S. mutans at adherent phases of growth, whereas 50 micromol l(-1) of XTZ removed 76% of biofilm at plateau accumulated phase when exposed to S. mutans biofilm for 60 min. CONCLUSIONS: Xanthorrhizol isolated from an edible plant (C. xanthorrhiza Roxb.) shows promise as an antibacterial agent for inhibiting and removing S. mutans biofilms in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: XTZ could be used as a potential antibacterial agent against biofilm formation by S. mutans.     

Protozoan grazing and its impact upon population dynamics in biofilm communities

AIMS: To determine the impact of protozoan grazing on the population dynamics of a multispecies bacterial biofilm community. METHODS AND RESULTS: Grazing by Acanthamoeba castellanii and the ciliate Colpoda maupasi upon biofilm and planktonic communities, composed of Klebsiella pneumoniae, Pseudomonas fluorescens and Staphylococcus epidermidis was investigated. Biofilms were formed using glass coverslips, held in a carousel device, as substrata for biofilm formation or in glass flow cells. The predatory effects of the amoeba were generally confined to the biofilm, where grazing rates corresponded to losses from the biofilm equivalent to ca 30,000 biofilm cells cm(-2) h(-1), with the amoeba becoming an integral part of the community. C. maupasi reduced the thickness of mature multispecies biofilms at steady-state from 500 to <200 microm. CONCLUSIONS: We report that the presence of the protozoa A. castellanii and C. maupasi markedly influence population dynamics within defined biofilm communities. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study dispels the popular opinion that biofilms are protected against predation by protozoa. A. castellanii clearly has the capacity to graze mixed biofilm communities and to become integrally associated with them, whereas the ciliate C. maupasi reduced biofilm thickness by up to 60%.     

In vitro activity of allicin against Staphylococcus epidermidis and influence of subinhibitory concentrations on biofilm formation

AIMS: The aim of this study is to determine the in vitro activity of allicin against Staphylococcus epidermidis and to evaluate the influence of allicin on biofilm formation. METHODS AND RESULTS: In vitro activity of allicin (diallyl thiosulphinate) against 38 strains of S. epidermidis was investigated. The activity of allicin was similar against S. epidermidis methicillin susceptible and methicillin resistant strains [minimum inhibitory concentration (MIC)90=8 mg l(-1)]. In general, subinhibitory concentrations (sub-MIC) of allicin diminished biofilm formation in the five strains analysed. CONCLUSION: The results confirm the antibacterial effect of allicin. Sub-MICs of allicin also diminished the biofilm formations by S. epidermidis. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study shows that allicin is active in vitro against S. epidermidis and that sub-MICs of allicin may play a role in the prevention of adherence of this bacteria to medical devices.     

Effect of ovotransferrin, protamine sulfate and EDTA combination on biofilm formation by catheter-associated bacteria

AIMS: To determine the effect of a composition comprising ovotransferrin (OT), protamine sulfate (PS) and ethylenediaminetetraacetic acid (EDTA) on biofilm formation by catheter-associated bacteria. METHODS AND RESULTS: The in vitro activity of OT, PS and EDTA alone and in combinations against biofilm formation by Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Enterococcus faecalis and Staphylococcus epidermidis was investigated. All the three compounds either alone or in combinations failed to inhibit the growth completely at the concentrations tested. However, the subinhibitory concentrations of three compounds in a composition showed synergistic inhibitory effect on biofilm formation by K. pneumoniae, Ps. aeruginosa and S. epidermidis. Furthermore, 79-95% reduction in Ps. aeruginosa and S. epidermidis biofilm formation was observed in a clear vinyl urinary catheter treated with the composition. CONCLUSION: The subinhibitory concentrations of OT, PS and EDTA in a composition were effective in reducing biofilm formation by catheter-associated bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that a synergistic composition-comprising non-antibiotic generally regarded as safe (GRAS) compounds such as OT, PS and EDTA may be used in the prevention of catheter-related infections.     

ica and beyond: biofilm mechanisms and regulation in Staphylococcus epidermidis and Staphylococcus aureus

Recent progress in elucidating the role of the icaADBC-encoded polysaccharide intercellular adhesin (PIA) or polymeric N-acetyl-glucosamine (PNAG) in staphylococcal biofilm development has in turn contributed significantly to our understanding of the pathogenesis of device-related infections. Nevertheless, our understanding of how the ica locus and PIA/PNAG biosynthesis are regulated is far from complete and many questions remain. Moreover, beyond ica, evidence is now emerging for the existence of ica-independent biofilm mechanisms in both Staphylococcus aureus and Staphylococcus epidermidis. Teichoic acids, which are a major carbohydrate component of the S. epidermidis biofilm matrix and the major cell wall autolysin, play an important role in the primary attachment phase of biofilm development, whereas the cell surface biofilm-associated protein and accumulation-associated protein are capable of mediating intercellular accumulation. These findings raise the exciting prospect that other surface proteins, which typically function as antigenic determinants or in binding to extracellular matrix proteins, may also act as biofilm adhesins. Given the impressive array of surface proteins expressed by S. aureus and S. epidermidis, future research into their potential role in biofilm development either independent of PIA/PNAG or in cooperation with PIA/PNAG will be of particular interest.     

Differential Roles of Poly-N-Acetylglucosamine Surface Polysaccharide and Extracellular DNA in Staphylococcus aureus and Staphylococcus epidermidis Biofilms

Staphylococcus aureus and Staphylococcus epidermidis are major human pathogens of increasing importance due to the dissemination of antibiotic-resistant strains. Evidence suggests that the ability to form matrix-encased biofilms contributes to the pathogenesis of S. aureus and S. epidermidis. In this study, we investigated the functions of two staphylococcal biofilm matrix polymers: poly-N-acetylglucosamine surface polysaccharide (PNAG) and extracellular DNA (ecDNA). We measured the ability of a PNAG-degrading enzyme (dispersin B) and DNase I to inhibit biofilm formation, detach preformed biofilms, and sensitize biofilms to killing by the cationic detergent cetylpyridinium chloride (CPC) in a 96-well microtiter plate assay. When added to growth medium, both dispersin B and DNase I inhibited biofilm formation by both S. aureus and S. epidermidis. Dispersin B detached preformed S. epidermidis biofilms but not S. aureus biofilms, whereas DNase I detached S. aureus biofilms but not S. epidermidis biofilms. Similarly, dispersin B sensitized S. epidermidis biofilms to CPC killing, whereas DNase I sensitized S. aureus biofilms to CPC killing. We concluded that PNAG and ecDNA play fundamentally different structural roles in S. aureus and S. epidermidis biofilms.     

Fluid flow induces biofilm formation in Staphylococcus epidermidis polysaccharide intracellular adhesin-positive clinical isolates

Staphylococcus epidermidis is a common cause of catheter-related bloodstream infections, resulting in significant morbidity and mortality and increased hospital costs. The ability to form biofilms plays a crucial role in pathogenesis; however, not all clinical isolates form biofilms under normal in vitro conditions. Strains containing the ica operon can display significant phenotypic variation with respect to polysaccharide intracellular adhesin (PIA)-based biofilm formation, including the induction of biofilms upon environmental stress. Using a parallel microfluidic approach to investigate flow as an environmental signal for S. epidermidis biofilm formation, we demonstrate that fluid shear alone induces PIA-positive biofilms of certain clinical isolates and influences biofilm structure. These findings suggest an important role of the catheter microenvironment, particularly fluid flow, in the establishment of S. epidermidis infections by PIA-dependent biofilm formation.     

Susceptibility of Staphylococcus epidermidis planktonic cells and biofilms to the lytic action of staphylococcus bacteriophage K

AIMS: To evaluate differences in biofilm or planktonic bacteria susceptibility to be killed by the polyvalent antistaphylococcus bacteriophage K. METHODS AND RESULTS: In this study, the ability of phage K to infect and kill several clinical isolates of Staphylococcus epidermidis was tested. Strains were grown in suspension or as biofilms to compare the susceptibility of both phenotypes to the phage lytic action. Most strains (10/11) were susceptible to phage K, and phage K was also effective in reducing biofilm biomass after 24 h of challenging. Biofilm cells were killed at a lower rate than the log-phase planktonic bacteria but at similar rate as stationary phase planktonic bacteria. CONCLUSIONS: Staphylococcus epidermidis biofilms and stationary growth phase planktonic bacteria are more resistant to phage K lysis than the exponential phase planktonic bacteria. SIGNIFICANCE OF STUDY: This study shows the differences in Staph. epidermidis susceptibility to be killed by bacteriophage K, when grown in biofilm or planktonic phenotypes.     

Air-liquid interface biofilms of Bacillus cereus: formation, sporulation, and dispersion

Biofilm formation by Bacillus cereus was assessed using 56 strains of B. cereus, including the two sequenced strains, ATCC 14579 and ATCC 10987. Biofilm production in microtiter plates was found to be strongly dependent on incubation time, temperature, and medium, as well as the strain used, with some strains showing biofilm formation within 24 h and subsequent dispersion within the next 24 h. A selection of strains was used for quantitative analysis of biofilm formation on stainless steel coupons. Thick biofilms of B. cereus developed at the air-liquid interface, while the amount of biofilm formed was much lower in submerged systems. This suggests that B. cereus biofilms may develop particularly in industrial storage and piping systems that are partly filled during operation or where residual liquid has remained after a production cycle. Moreover, depending on the strain and culture conditions, spores constituted up to 90% of the total biofilm counts. This indicates that B. cereus biofilms can act as a nidus for spore formation and subsequently can release their spores into food production environments.     

Air-Liquid Interface Biofilms of Bacillus cereus: Formation, Sporulation, and Dispersion

Biofilm formation by Bacillus cereus was assessed using 56 strains of B. cereus, including the two sequenced strains, ATCC 14579 and ATCC 10987. Biofilm production in microtiter plates was found to be strongly dependent on incubation time, temperature, and medium, as well as the strain used, with some strains showing biofilm formation within 24 h and subsequent dispersion within the next 24 h. A selection of strains was used for quantitative analysis of biofilm formation on stainless steel coupons. Thick biofilms of B. cereus developed at the air-liquid interface, while the amount of biofilm formed was much lower in submerged systems. This suggests that B. cereus biofilms may develop particularly in industrial storage and piping systems that are partly filled during operation or where residual liquid has remained after a production cycle. Moreover, depending on the strain and culture conditions, spores constituted up to 90% of the total biofilm counts. This indicates that B. cereus biofilms can act as a nidus for spore formation and subsequently can release their spores into food production environments.     

The giant extracellular matrix-binding protein of Staphylococcus epidermidis mediates biofilm accumulation and attachment to fibronectin

Virulence of nosocomial pathogen Staphylococcus epidermidis is essentially related to formation of adherent biofilms, assembled by bacterial attachment to an artificial surface and subsequent production of a matrix that mediates interbacterial adhesion. Growing evidence supports the idea that proteins are functionally involved in S. epidermidis biofilm accumulation. We found that in S. epidermidis 1585v overexpression of a 460 kDa truncated isoform of the extracellular matrix-binding protein (Embp) is necessary for biofilm formation. Embp is a giant fibronectin-binding protein harbouring 59 Found In Various Architectures (FIVAR) and 38 protein G-related albumin-binding (GA) domains. Studies using defined Embp-positive and -negative S.  epidermidis strains proved that Embp is sufficient and necessary for biofilm formation. Further data showed that the FIVAR domains of Embp mediate binding of S. epidermidis to solid-phase attached fibronectin, constituting the first step of biofilm formation on conditioned surfaces. The binding site in fibronectin was assigned to the fibronectin domain type III12. Embp-mediated biofilm formation also protected S. epidermidis from phagocytosis by macrophages. Thus, Embp is a multifunctional cell surface protein that mediates attachment to host extracellular matrix, biofilm accumulation and escape from phagocytosis, and therefore is well suited for promoting implant-associated infections.     

葡萄球菌生物膜形成机制与ica之间的关系

ica位点编码的胞外多糖(PIA/PNAG)对理解葡萄球菌生物膜相关感染病理学方面具有重要的意义.关于ica位点与PIA/PNAG之间如何调节的研究还不全面,另外一种独立于ica的生物膜形成机制存在于表皮葡萄球菌和金黄色葡萄球菌中;细胞表面相关蛋白也能调节生物膜的形成,这些发现为探究它们在生物膜形成机制的潜在作用提供了重要基础.     

连翘苷和黄芩苷对表皮葡萄球菌生物膜抑制作用的研究

目的通过中药有效成分连翘苷和黄芩苷分别对表皮葡萄球菌生物膜抑制作用的研究,为表皮葡萄球菌生物膜引起的相关感染提供新的治疗途径。方法体外构建表皮葡萄球菌生物膜,XTT减低法评价连翘苷、黄芩苷对表皮葡萄球菌初始黏附及生物膜内细菌代谢的影响,显微镜下观察用药后表皮葡萄球菌生物膜形态和结构改变。结果连翘苷和黄芩苷对表皮葡萄球菌生物膜的早期黏附均无抑制作用;连翘苷对表皮葡萄球菌生物膜菌的SMIC50为31.25μg/ml,而黄芩苷对表皮葡萄球菌生物膜菌的代谢无影响;在显微镜下观察,连翘苷使部分表皮葡萄球菌被膜的形态发生改变,而黄芩苷对其形态影响不显著。结论连翘苷对表皮葡萄球菌生物膜的初始黏附阶段无抑制作用,对生物膜菌的代谢和生物膜形态均有显著影响;黄芩苷对表皮葡萄球菌生物膜无显著作用。