Removal and Inactivation of Staphylococcus epidermidis Biofilms by Electrolysis

Bacterial biofilms were exposed to electrolysis by making the steel substratum an electrode in a circuit including a 6-V battery. These treatments resulted in killing (2.1-log reduction) and removal (4.0-log reduction) of viable cells at the anode and cathode, respectively, within a few minutes.     

Poly-N-Acetylglucosamine Matrix Polysaccharide Impedes Fluid Convection and Transport of the Cationic Surfactant Cetylpyridinium Chloride through Bacterial Biofilms

Biofilms are composed of bacterial cells encased in a self-synthesized, extracellular polymeric matrix. Poly-β(1,6)-N-acetyl-d-glucosamine (PNAG) is a major biofilm matrix component in phylogenetically diverse bacteria. In this study we investigated the physical and chemical properties of the PNAG matrix in biofilms produced in vitro by the gram-negative porcine respiratory pathogen Actinobacillus pleuropneumoniae and the gram-positive device-associated pathogen Staphylococcus epidermidis. The effect of PNAG on bulk fluid flow was determined by measuring the rate of fluid convection through biofilms cultured in centrifugal filter devices. The rate of fluid convection was significantly higher in biofilms cultured in the presence of the PNAG-degrading enzyme dispersin B than in biofilms cultured without the enzyme, indicating that PNAG decreases bulk fluid flow. PNAG also blocked transport of the quaternary ammonium compound cetylpyridinium chloride (CPC) through the biofilms. Binding of CPC to biofilms further impeded fluid convection and blocked transport of the azo dye Allura red. Bioactive CPC was efficiently eluted from biofilms by treatment with 1 M sodium chloride. Taken together, these findings suggest that CPC reacts directly with the PNAG matrix and alters its physical and chemical properties. Our results indicate that PNAG plays an important role in controlling the physiological state of biofilms and may contribute to additional biofilm-associated processes such as biocide resistance.Biofilms are composed of bacterial cells encased in a self-synthesized, extracellular polymeric matrix (7). The main function of the biofilm matrix is to provide a structural framework that holds the cells together in a mass and firmly attaches the bacterial mass to the underlying surface. In addition to having a structural role, the matrix provides biofilm cells with a protected microenvironment containing dissolved nutrients, secreted enzymes, DNA, and phages. The matrix may also contribute to the increased antimicrobial resistance exhibited by biofilm cells, either by providing a diffusion barrier or by directly binding to antimicrobial agents and preventing their penetration into the biofilm (19).Polysaccharides are a major matrix component in most bacterial biofilms (26). Poly-β(1,6)-N-acetyl-d-glucosamine (PNAG) is an extracellular polysaccharide that mediates biofilm cohesion in numerous gram-negative members of the Proteobacteria family, including Escherichia coli, Yersinia pestis, Pseudomonas fluorescens, Bordetella spp., Xenorhabdus nematophila, Aggregatibacter actinomycetemcomitans, and Actinobacillus pleuropneumoniae (4, 8, 15, 22), and in the gram-positive species Staphylococcus aureus and Staphylococcus epidermidis (3, 17). Specific biofilm-related functions ascribed to PNAG include abiotic surface attachment (1), epithelial cell attachment (23, 28), intercellular adhesion (15, 17), and resistance to killing by antibiotics, detergents, antimicrobial peptides, and mammalian phagocytic cells (9, 10, 16, 27, 29).In the present study we investigated the physical and chemical properties of the PNAG matrix in biofilms produced by the porcine respiratory pathogen A. pleuropneumoniae and the device-associated pathogen S. epidermidis. By using a novel centrifugal filter device assay, we obtained evidence that PNAG significantly inhibits fluid convection and solute transport through A. pleuropneumoniae and S. epidermidis biofilms.     

Extracellular DNA in Single- and Multiple-Species Unsaturated Biofilms

The extracellular polymeric substances (EPS) of bacterial biofilms form a hydrated barrier between cells and their external environment. Better characterization of EPS could be useful in understanding biofilm physiology. The EPS are chemically complex, changing with both bacterial strain and culture conditions. Previously, we reported that Pseudomonas aeruginosa unsaturated biofilm EPS contains large amounts of extracellular DNA (eDNA) (R. E. Steinberger, A. R. Allen, H. G. Hansma, and P. A. Holden, Microb. Ecol. 43:416-423, 2002). Here, we investigated the compositional similarity of eDNA to cellular DNA, the relative quantity of eDNA, and the terminal restriction fragment length polymorphism (TRFLP) community profile of eDNA in multiple-species biofilms. By randomly amplified polymorphic DNA analysis, cellular DNA and eDNA appear identical for P. aeruginosa biofilms. Significantly more eDNA was produced in P. aeruginosa and Pseudomonas putida biofilms than in Rhodococcus erythropolis or Variovorax paradoxus biofilms. While the amount of eDNA in dual-species biofilms was of the same order of magnitude as that of of single-species biofilms, the amounts were not predictable from single-strain measurements. By the Shannon diversity index and principle components analysis of TRFLP profiles generated from 16S rRNA genes, eDNA of four-species biofilms differed significantly from either cellular or total DNA of the same biofilm. However, total DNA- and cellular DNA-based TRFLP analyses of this biofilm community yielded identical results. We conclude that extracellular DNA production in unsaturated biofilms is species dependent and that the phylogenetic information contained in this DNA pool is quantifiable and distinct from either total or cellular DNA.     

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

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

金黄色葡萄球菌荚膜多糖研究进展

金黄色葡萄球菌作为引起人和动物发病的一种主要的致病菌,已严重影响到人们的身体健康和畜牧业的发展。致病性金黄色葡萄球菌多数含有荚膜成分,并且这种荚膜成分与金黄色葡萄球菌的毒力和抗噬菌作用有关。主要从金黄色葡萄球菌荚膜多糖的5型和8型血清学分类、分子结构、影响因素、表达调控等方面简要介绍了目前国内外关于金黄色葡萄球菌荚膜多糖的研究进展。     

Electric Current-Induced Detachment of Staphylococcus epidermidis Biofilms from Surgical Stainless Steel

Biomaterial-centered infections of orthopedic percutaneous implants are serious complications which can ultimately lead to osteomyelitis, with devastating effects on bone and surrounding tissues, especially since the biofilm mode of growth offers protection against antibiotics and since removal frequently is the only ultimate solution. Recently, it was demonstrated that as a possible pathway to prevent infections of percutaneous stainless steel implants, electric currents of 60 to 100 μA were effective at stimulating the detachment of initially adhering staphylococci from surgical stainless steel. However, initially adhering bacteria are known to adhere more reversibly than bacteria growing in the later stages of biofilm formation. Hence, the aim of this study was to examine whether a growing Staphylococcus epidermidis biofilm can be stimulated to detach from surgical stainless steel by the use of electric currents. In separate experiments, four currents, i.e., 60 and 100 μA of direct current (DC) and 60 and 100 μA of block current (50% duty cycle, 1 Hz), were applied for 360 min to stimulate the detachment of an S. epidermidis biofilm that had grown for 200 min. A 100-μA DC yielded 78% detachment, whereas a 100-μA block current under the same experimental conditions yielded only 31% detachment. The same trend was found for 60 μA, with 37% detachment for a DC and 24% for a block current. Bacteria remaining on the surface after the current application were less viable than they were prior to the current application, as demonstrated by confocal laser scanning microscopy. In conclusion, these results suggest that DCs are preferred for curing infections.     

Transposase-dependent formation of circular IS256 derivatives in Staphylococcus epidermidis and Staphylococcus aureus

IS256 is a highly active insertion sequence (IS) element of multiresistant staphylococci and enterococci. Here we show that, in a Staphylococcus epidermidis clinical isolate, as well as in recombinant Staphylococcus aureus and Escherichia coli carrying a single IS256 insertion on a plasmid, IS256 excises as an extrachromosomal circular DNA molecule. First, circles were identified that contained a complete copy of IS256. In this case, the sequence connecting the left and right ends of IS256 was derived from flanking DNA sequences of the parental genetic locus. Second, circle junctions were detected in which one end of IS256 was truncated. Nucleotide sequencing of circle junctions revealed that (i) either end of IS256 can attack the opposite terminus and (ii) the circle junctions vary significantly in size. Upon deletion of the IS256 open reading frame at the 3' end and site-directed mutageneses of the putative DDE motif, circular IS256 molecules were no longer detectable, which implicates the IS256-encoded transposase protein with the circularization of the element.     

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.     

Effects of Growth in the Presence of Subinhibitory Concentrations of Dicloxacillin on Staphylococcus epidermidis and Staphylococcus haemolyticus Biofilms

Low concentrations of antibiotics can inhibit microbial adherence to medical device surfaces. However, little is known about the changes that occur in the physiology of bacteria within biofilms formed in the presence of subinhibitory (sub-MIC) concentrations of antibiotics. In this study, the densities and matrix compositions ofbiofilms formed by two coagulase-negative Staphylococcus species in the absence and in the presence of sub-MIC concentrations of dicloxacillin were evaluated. Biofilms formed in the presence of sub-MIC concentrations of dicloxacillin contained less biomass, and there were notable changes in the composition of the biofilm matrix. Changes in the spatial structure were also verified by confocal scanning laser microscopy, indicating that biofilms grown in the presence of sub-MIC concentrations of dicloxicilln had a lower cell density. Physiological alterations in the bacteria within biofilms grown in the presence of subinhibitory concentrations of the antibiotic were also evaluated. The results showed that there were differences in bacterial surface characteristics when cultures were grown in the presence of sub-MIC concentrations of dicloxacillin, including decreased hydrophobicity and decreased expression of the exopolysaccharide poly-N-acetylglucosamine. The elemental composition of the cell surface was also analyzed, and whereas in Staphylococcus epidermidis there were decreases in the oxygen and nitrogen contents, in Staphylococcus haemolyticus there were increases in these two parameters. Additionally, increases in resistance to several antibiotics were observed for the cells within biofilms formed in the presence of dicloxacillin.     

Viscoelastic properties of Staphylococcus aureus and Staphylococcus epidermidis mono‐microbial biofilms

The viscoelastic properties of mono‐microbial biofilms produced by ocular and reference staphylococcal strains were investigated. The microorganisms were characterized for their haemolytic activity and agr typing and the biofilms, grown on stainless steel surface under static conditions, were analysed by Confocal Laser Scanning Microscopy. Static and dynamic rheometric tests were carried out to determine the steady‐flow viscosity and the elastic and viscous moduli. The analysed biofilms showed the typical time‐dependent behaviour of viscoelastic materials with considerable elasticity and mechanical stability except for Staphylococcus aureus ATCC 29213 biofilm which showed a very fragile structure. In particular, S. aureus 6ME biofilm was more compact than other staphylococcal biofilms studied with a yield stress ranging between 2 and 3 Pa. The data obtained in this work could represent a starting point for developing new therapeutic strategies against biofilm‐associated infections, such as improving the drug effect by associating an antimicrobial agent with a biofilm viscoelasticity modifier.     

Population Diversification in Staphylococcus aureus Biofilms May Promote Dissemination and Persistence

The biofilm mode of growth can lead to diversification of the bacterial population by promoting the emergence of variants. Here we report the identification and characterization of two major subpopulations of morphological variants arising in biofilms of S. aureus. One of these lacked pigmentation (termed white variants; WVs), whilst the other formed colonies on agar that were larger and paler than the parental strain (termed large pale variants; LPVs). WVs were unable to form biofilms, and exhibited increased proteolysis and haemolysis; all phenotypes attributable to loss-of-function mutations identified in the gene encoding the alternative sigma factor, sigB. For LPVs, no differences in biofilm forming capacity or proteolysis were observed compared with the parental strain. Genetic analysis of LPVs revealed that they had undergone mutation in the accessory gene regulator system (agrA), and deficiency in agr was confirmed by demonstrating loss of both colony spreading and haemolytic activity. The observation that S. aureus biofilms elaborate large subpopulations of sigB and agr mutants, both genotypes that have independently been shown to be of importance in staphylococcal disease, has implications for our understanding of staphylococcal infections involving a biofilm component.     

血流感染患者表皮葡萄球菌与金黄色葡萄球菌的分布与耐药性分析

目的比较血流感染患者表皮葡萄球菌与金黄色葡萄球菌的耐药性差异,为临床合理选用抗菌药物提供参考。方法对贵州医科大学第三附属医院2011年6月至2014年11月血流感染标本中分离出的表皮葡萄球菌与金黄色葡萄球菌共140株进行比较分析,按照《全国临床检验操作规程》进行微生物鉴定,采用K-B纸片法进行药敏试验,药敏结果按美国临床和实验室标准化协会(CLSI)的标准判断,数据采用SPSS 17.0软件进行统计分析。结果血流感染标本共分离出表皮葡萄球菌与金黄色葡萄球菌140株,其中表皮葡萄球菌分离出88株,占62.86%;金黄色葡萄球菌分离出52株,占37.14%。血流感染表皮葡萄球菌与金黄色葡萄球菌对苯唑西林、头孢唑林、头孢噻肟、克林霉素、红霉素、阿奇霉素、环丙沙星、氧氟沙星、利福平、呋喃妥因、头孢噻吩、亚胺培南、左氧氟沙星、麦迪霉素、复方新诺明等耐药率分别为81.82%,45.15%;35.23%,15.38%;20.45%,5.77%;47.73%,26.92%;76.14%,51.92%;69.32%,50.00%;64.77%,9.62%;54.55%,28.85%;14.77%,0.00%;10.23%,0.00%;39.77%,3.85%;11.36%,0.00%;54.55%,28.85%;67.05%,5.77%;79.55%,32.69%;表皮葡萄球菌耐药率明显高于金黄色葡萄球菌,差异有性统计学意义(P0.05)。结论血流感染中表皮葡萄球菌的感染率与耐药率不断上升,耐药率明显高于金黄色葡萄球菌,应引起重视。     

Comparison of three methods detection of slime production by Staphylococcus aureus and Staphylococcus epidermidis

In this article, slime production of Staphylococcus aureus and Staphylococcus epidermidis strains from infective skin lesions was evaluated by three different methods: Congo red agar method (CRA), Christensen tube method (CT) and spectrophotometric method (SC). All strains by CT method interpreted as negative (dark-claret or red colonies of the surface). 12 (37.5%) strains of S. aureus, 16 (50.0%) strains of S. epidermidis produced slime as shown by CT method, 6 (18.7%) strains of S. aureus, 8 (25,0%) strains of S. epidermidis by SC method. They also found a correlation of slime production by CT and SC method (p > 0.05).     

The molecular characteristic of Staphylococcus aureus and Staphylococcus epidermidis strains isolated from clinical sources

The aim of study was the molecular characteristic of S. aureus and S. epidermidis isolates obtained from skin surface, wounds, deep tissues of hospitalized patients and from skin surface of non-hospitalized patients. Genes encoding virulence factors were examined using PCR reaction and specific primers. Genes encoding adhesinsfnbA and cna and gene eta for epidermolytic toxin were mostly present in S. aureus isolates coming from wounds and deep tissues compared to these from skin surface. Gene atlE encoding autolysin of S. epidermidis was detected in all studied isolates, whereas gene icaAB was present in almost all isolates. Comparison of results obtained by PCR and conventional method of the resistance to methicillin estimation showed discrepances suggesting the need for using of both methods in some clinically difficult cases of S. aureus infection.