Polysaccharide Intercellular Adhesin Synthesis Research Articles

Ultrasonic-assisted extraction, anti-biofilm activity, and mechanism of action of Ku Shen (Sophorae Flavescentis Radix) extracts against Vibrio parahaemolyticus.

The objective of this study is to optimize the ultrasonic-assisted extraction process of Ku Shen (Sophorae Flavescentis Radix) extracts (KSE) against Vibrio parahaemolyticus and explore their anti-biofilm activity and mechanism of action. The ultrasonic-assisted extraction process of KSE optimized by single factor experiment, Box-Behnken design and response surface methodology was as follows: 93% ethanol as solvent, liquid/material ratio of 30 mL/g, ultrasonic power of 500 W, extraction temperature of 80°C and time of 30 min. Under these conditions, the diameter of inhibition circle of KSE was 15.60 ± 0.17 mm, which had no significant difference with the predicted value. The yield of dried KSE is 32.32 ± 0.57% and the content of total flavonoids in KSE was 57.02 ± 5.54%. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of KSE against V. parahaemolyticus were 0.25 and 0.5 mg/mL, respectively. Crystal violet staining, Congo red plate, spectrophotometry, CCK-8 and scanning electron microscopy were used to investigate the activity and mechanism of action of KSE against V. parahaemolyticus biofilm. The results showed that the sub-MIC of KSE could significantly inhibit biofilm formation, reduce the synthesis of polysaccharide intercellular adhesin (PIA) and the secretion of extracellular DNA. In addition, the inhibition rate of biofilm formation and clearance rate of mature biofilm of 1.0 mg/mL KSE were 85.32 and 74.04%, and the reduction rate of metabolic activity of developing and mature biofilm were 77.98 and 74.46%, respectively. These results were confirmed by visual images obtained by scanning electron microscopy. Therefore, KSE has the potential to further isolate the anti-biofilm agent and evaluate it for the preservation process of aquatic products.

Inhibitory properties of Chinese Herbal Formula SanHuang decoction on biofilm formation by antibiotic-resistant Staphylococcal strains

The aim of this study was to explore the effect of Chinese herbal SanHuang decoction (SH) on biofilm formation of antibiotic-resistant Staphylococci on titanium surface, and to explore its mechanism. Biofilm-forming ATCC 35984, ATCC 43300 and MRSE 287 were used in this study. The MICs of SH and vancomycin against Staphylococci were determined by the broth microdilution method. Six groups were designed, namely control group (bacteria cultured with medium), 1/8MIC SH group (1MIC SH was diluted by 1/8 using TSB or saline), 1/4MIC SH group, 1/2MIC SH group, 1MIC SH group and vancomycin group (bacteria cultured with 1MIC vancomycin). The inhibitory effect on bacterial adhesion and biofilm formation were observed by the spread plate method, CV staining, SEM, and CLSM. Real-time PCR was performed to determine the effect of SH on the expression levels of ica AD and ica R gene in ATCC 35984 during the biofilm formation. The strains were found to be susceptible to SH and vancomycin with MIC of 38.75 mg/ml and 2.5 μg/ml, respectively. SH with 1 MIC and 1/2 MIC could inhibit the bacteria adhesion, showing only scattered adhesion from SEM. CLSM showed that SH with 1 MIC and 1/2 MIC inhibited the biofilm formation. The quantitative results of the spread plate method and CV staining showed that there was significant differences between the SH groups (P < 0.05). Further, with an increase in SH concentration, the inhibitory effect became more obvious when compared with control group. Among the groups, vancomycin had the strongest inhibitory effect on bacterial adhesion and biofilm formation (P < 0.01). With an increase in SH concentration, the expression levels of ica AD decreased, and the expression of ica R increased correspondingly (P < 0.05). In conclusions, SH can inhibit the biofilm formation of antibiotic-resistant Staphylococci. Its probable mechanistic activity may be through the inhibition of polysaccharide intercellular adhesin synthesis by down-regulating the expression of ica AD gene.

Monoclonal Antibodies Specific to the Extracellular Domain of Histidine Kinase YycG of Staphylococcus epidermidis Inhibit Biofilm Formation.

Staphylococcus epidermidis is frequently associated with biofilm-related infections. Biofilms drastically reduce the efficacy of conventional antibiotics and the host immune system. In S. epidermidis biofilm formation, a major role is played by the YycG/YycF two-component system, and previous findings have indicated that inhibitors targeting the cytoplasmic HATPase_c domain of YycG kinase in S. epidermidis exhibit bactericidal and biofilm-killing activities. Therefore, we hypothesized that monoclonal antibodies (mAbs) against YycG extracellular (YycGex) domain would block the signal transduction and influence the biofilm formation of S. epidermidis. In this study, we screened out two YycGex-specific mAbs showing the highest affinity for the target, mAbs 2F3 and 1H1. These mAbs inhibited S. epidermidis biofilm formation in a dose-dependent manner, and at a concentration of 160 μg/mL, mAbs 2F3 and 1H1 caused 78.3 and 93.1% biofilm reduction, respectively, relative to normal mouse IgG control. When co-cultivated with YycGex mAbs, S. epidermidis cells showed diminished initial-adherence capacity, and the antibody treatment further led to a marked decrease in the synthesis of polysaccharide intercellular adhesin and in the transcriptional level of genes encoding proteins involved in biofilm formation. Lastly, we determined that the epitopes recognized by the two YycGex mAbs are located within aa 59–70 of the YycGex domain. It indicates that the YycGex domain may be a potential candidate as a vaccine for the prevention of S. epidermidis biofilm infections.

Sticky Matrix: Adhesion Mechanism of the Staphylococcal Polysaccharide Intercellular Adhesin.

The development of bacterial biofilms on surfaces leads to hospital-acquired infections that are difficult to fight. In Staphylococci, the cationic polysaccharide intercellular adhesin (PIA) forms an extracellular matrix that connects the cells together during biofilm formation, but the molecular forces involved are unknown. Here, we use advanced force nanoscopy techniques to unravel the mechanism of PIA-mediated adhesion in a clinically relevant methicillin-resistant Staphylococcus aureus (MRSA) strain. Nanoscale multiparametric imaging of the structure, adhesion, and elasticity of bacteria expressing PIA shows that the cells are surrounded by a soft and adhesive matrix of extracellular polymers. Cell surface softness and adhesion are dramatically reduced in mutant cells deficient for the synthesis of PIA or under unfavorable growth conditions. Single-cell force spectroscopy demonstrates that PIA promotes cell-cell adhesion via the multivalent electrostatic interaction with polyanionic teichoic acids on the S. aureus cell surface. This binding mechanism rationalizes, at the nanoscale, the well-known ability of PIA to strengthen intercellular adhesion in staphylococcal biofilms. Force nanoscopy offers promising prospects for understanding the fundamental forces in antibiotic-resistant biofilms and for designing anti-adhesion compounds targeting matrix polymers.

Draft Genome Sequence of Bacillus pumilus CCMA-560, Isolated from an Oil-Contaminated Mangrove Swamp

Bacillus pumilus strain CCMA-560 was isolated from an oil-contaminated mangrove swamp and was shown to produce biosurfactants. The strain appears to be capable of degrading some plant cell wall-related compounds, including hemicelluose and pectin. Genes for biopolymer export and polysaccharide intercellular adhesin synthesis were also annotated.

An extracellular Staphylococcus epidermidis polysaccharide: relation to Polysaccharide Intercellular Adhesin and its implication in phagocytosis.

BackgroundThe skin commensal and opportunistic pathogen Staphylococcus epidermidis is a leading cause of hospital-acquired and biomaterial-associated infections. The polysaccharide intercellular adhesin (PIA), a homoglycan composed of β-1,6-linked N-acetylglucosamine residues, synthesized by enzymes encoded in icaADBC is a major functional factor in biofilm accumulation, promoting virulence in experimental biomaterial-associated S. epidermidis infection. Extracellular mucous layer extracts of S. epidermidis contain another major polysaccharide, referred to as 20-kDa polysaccharide (20-kDaPS), composed mainly out of glucose, N-acetylglucosamine, and being partially sulfated. 20-kDaPS antiserum prevents adhesion of S. epidermidis on endothelial cells and development of experimental keratitis in rabbits. Here we provide experimental evidence that 20-kDaPS and PIA represent distinct molecules and that 20-kDaPS is implicated in endocytosis of S. epidermidis bacterial cells by human monocyte-derived macrophages.ResultsAnalysis of 75 clinical coagulase-negative staphylococci from blood-cultures and central venous catheter tips indicated that 20-kDaPS is expressed exclusively in S. epidermidis but not in other coagulase-negative staphylococcal species. Tn917-insertion in various locations in icaADBC in mutants M10, M22, M23, and M24 of S. epidermidis 1457 are abolished for PIA synthesis, while 20-kDaPS expression appears unaltered as compared to wild-type strains using specific anti-PIA and anti-20-kDaPS antisera. While periodate oxidation and dispersin B treatments abolish immuno-reactivity and intercellular adhesive properties of PIA, no abrogative activity is exerted towards 20-kDaPS immunochemical reactivity following these treatments. PIA polysaccharide I-containing fractions eluting from Q-Sepharose were devoid of detectable 20-kDaPS using specific ELISA. Preincubation of non-20-kDaPS-producing clinical strain with increasing amounts of 20-kDaPS inhibits endocytosis by human macrophages, whereas, preincubation of 20-kDaPS-producing strain ATCC35983 with 20-kDaPS antiserum enhances bacterial endocytosis by human macrophages.ConclusionsIn conclusion, icaADBC is not involved in 20-kDaPS synthesis, while the chemical and chromatographic properties of PIA and 20-kDaPS are distinct. 20-kDaPS exhibits anti-phagocytic properties, whereas, 20-kDaPS antiserum may have a beneficial effect on combating infection by 20-kDaPS-producing S. epidermidis.

Strong biofilm production but not adhesion virulence factors can discriminate between invasive and commensal Staphylococcus epidermidis strains

Staphylococcus epidermidis is a leading cause of hospital-acquired infections, mostly associated with the use of medical devices in immunocompromised patients. It originates from the patient's own skin flora, which is subject to severe changes as a result of selective pressure exerted by the hospital environment. This notion led us to compare S. epidermidis isolates from catheter related infections (CRI), non-catheter related bacteremia (NCRB) and catheter hub cultures (commensal isolates). The collection comprised 47 CRI strains from the Bone Marrow Transplant Centre of Tunis, 25 NCRB strains and 25 commensal isolates from patients hospitalized in the same center. Antimicrobial resistance and virulence-associated genes (icaABC, aap, atlE, bhp, fbe, embp, and IS256), polysaccharide intercellular adhesin synthesis, and biofilm formation were investigated. The clonal relationship of strains was investigated by pulsed field gel electrophoresis. Whereas bhp, atlE, fbe, embp, and aap were almost ubiquitously amplified, resistance to oxacillin, kanamycin, tobramycin, gentamicin, cotrimoxazole, and fosfomycin, biofilm production, ica genes, and IS256 were significantly more frequent in invasive (CRI and NCRB strains) than in commensal strains. Moreover, strong biofilm production was significantly more frequent among CRI strains than in NCRB strains. In conclusion, when S. epidermidis is isolated from blood cultures, the detection of strong biofilm production may be significant with regard to judging whether the detected strain is an etiologic agent of CRI.

Staphylococcus epidermidis Uses Distinct Mechanisms of Biofilm Formation To Interfere with Phagocytosis and Activation of Mouse Macrophage-Like Cells 774A.1

Assembly of adherent biofilms is the key mechanism involved in Staphylococcus epidermidis virulence during device-associated infections. Aside from polysaccharide intercellular adhesin (PIA), the accumulation-associated protein Aap and the extracellular matrix binding protein Embp act as intercellular adhesins, mediating S. epidermidis cell aggregation and biofilm accumulation. The aim of this study was to investigate structural features of PIA-, Aap-, and Embp-mediated S. epidermidis biofilms in more detail and to evaluate their specific contributions to biofilm-related S. epidermidis immune escape. PIA-, Embp-, and Aap-mediated biofilms exhibited substantial morphological differences. Basically, PIA synthesis induced formation of macroscopically visible, rough cell clusters, whereas Aap- and Embp-dependent biofilms preferentially displayed a smooth layer of aggregated bacteria. On the microscopic level, PIA was found to form a string-like organized extracellular matrix connecting the bacteria, while Embp produced small deposits of intercellular matrix and Aap was strictly localized to the bacterial surface. Despite marked differences, S. epidermidis strains using PIA, Aap, or Embp for biofilm formation were protected from uptake by J774A.1 macrophages, with similarly efficiencies. In addition, compared to biofilm-negative S. epidermidis strains, isogenic biofilm-forming S. epidermidis induced only a diminished inflammatory J774A.1 macrophage response, leading to significantly (88.2 to 88.7%) reduced NF-κB activation and 68.8 to 83% reduced interleukin-1β (IL-1β) production. Mechanical biofilm dispersal partially restored induction of NF-κB activation, although bacterial cell surfaces remained decorated with the respective intercellular adhesins. Our results demonstrate that distinct S. epidermidis biofilm morphotypes are similarly effective at protecting S. epidermidis from phagocytic uptake and at counteracting macrophage activation, providing novel insights into mechanisms that could contribute to the chronic and persistent course of biofilm-related S. epidermidis foreign material infections.

Inhibitory Effects of 1,2,3,4,6-Penta- O -Galloyl-β- d -Glucopyranose on Biofilm Formation by Staphylococcus aureus

1,2,3,4,6-Penta-O-galloyl-β-D-glucopyranose (PGG) is an active ingredient in plants that are commonly used in Chinese medicine to treat inflammation. We demonstrate here that PGG, at 6.25 μM, does not inhibit the growth of Staphylococcus aureus, and yet it prevents biofilm formation on polystyrene and polycarbonate surfaces. At the same concentration, PGG is not toxic to human epithelial and fibroblast cells. PGG has an IB₅₀ value, i.e., the PGG concentration that inhibits 50% biofilm formation, of 3.6 μM. The value is substantially lower than that of N-acetylcysteine, iodoacetamide, and N-phenyl maleimide, which are known to inhibit biofilm formation by S. aureus. Biochemical and scanning electron microscopy results also reveal that PGG inhibits initial attachment of the bacteria to solid surface and the synthesis of polysaccharide intercellular adhesin, explaining how PGG inhibits biofilm formation. The results of this study demonstrate that coating PGG on polystyrene and silicon rubber surfaces with polyaniline prevents biofilm formation, indicating that PGG is highly promising for clinical use in preventing biofilm formation by S. aureus.

Ygs Is a Novel Gene That Influences Biofilm Formation and the General Stress Response of Staphylococcus epidermidis

Infections caused by the nosocomial pathogen Staphylococcus epidermidis frequently develop on implanted medical devices and involve biofilm formation. Biofilms are surface-attached microbial communities that show increased resistance to drug treatment and mechanisms of innate host defense. In this study, a mutant library of the clinical isolate S. epidermidis 1457 was constructed using mariner-based transposon mutagenesis. About a thousand mutants were screened, and 12 mutants were identified as significantly defective in biofilm formation. We focused on a mutant in which the transposon had inserted in a gene with unknown function, SERP0541, which is annotated as a gene encoding a GSP13-like general stress response protein. The gene was named ygs (encoding an unknown general stress protein). Various stresses, including heat, pH, high osmolarity, and ethanol affected the survival of the ygs mutant to a significantly higher degree than the wild-type strain and led to increased expression of ygs. Furthermore, synthesis of polysaccharide intercellular adhesin (PIA) and transcription of the PIA biosynthetic operon were significantly decreased in the ygs mutant. These results are in accordance with the putative involvement of ygs in stress-response gene regulation and indicate that ygs influences biofilm development by controlling PIA-dependent biofilm accumulation. Moreover, ygs had a significant impact on the formation of biofilms and metastatic disease in two catheter-related rat infection models. Our study shows that the ygs gene controls S. epidermidis biofilm accumulation and stress resistance, representing a key regulator of both structural and physiological biofilm characteristics with a significant impact on biofilm-associated infection.

Using NMR Metabolomics to Investigate Tricarboxylic Acid Cycle-dependent Signal Transduction in Staphylococcus epidermidis

Staphylococcus epidermidis is a skin-resident bacterium and a major cause of biomaterial-associated infections. The transition from residing on the skin to residing on an implanted biomaterial is accompanied by regulatory changes that facilitate bacterial survival in the new environment. These regulatory changes are dependent upon the ability of bacteria to "sense" environmental changes. In S. epidermidis, disparate environmental signals can affect synthesis of the biofilm matrix polysaccharide intercellular adhesin (PIA). Previously, we demonstrated that PIA biosynthesis is regulated by tricarboxylic acid (TCA) cycle activity. The observations that very different environmental signals result in a common phenotype (i.e. increased PIA synthesis) and that TCA cycle activity regulates PIA biosynthesis led us to hypothesize that S. epidermidis is "sensing" disparate environmental signals through the modulation of TCA cycle activity. In this study, we used NMR metabolomics to demonstrate that divergent environmental signals are transduced into common metabolomic changes that are "sensed" by metabolite-responsive regulators, such as CcpA, to affect PIA biosynthesis. These data clarify one mechanism by which very different environmental signals cause common phenotypic changes. In addition, due to the frequency of the TCA cycle in diverse genera of bacteria and the intrinsic properties of TCA cycle enzymes, it is likely the TCA cycle acts as a signal transduction pathway in many bacteria.

Structure, function and contribution of polysaccharide intercellular adhesin (PIA) to Staphylococcus epidermidis biofilm formation and pathogenesis of biomaterial-associated infections

Staphylococcus epidermidis is of major importance in infections associated with indwelling medical devices. The tight pathogenic association is essentially linked to the species ability to form adherent biofilms on artificial surfaces. Aiming at identifying novel targets for vaccination or therapy much effort has been made to unravel the molecular mechanisms leading to S. epidermidis biofilm formation. At present, polysaccharide intercellular adhesin (PIA) is the best studied factor involved in S. epidermidis biofilm accumulation. PIA is a glycan of β-1,6-linked 2-acetamido-2-deoxy- D-glucopyranosyl residues of which 15 % are non- N-acetylated. PIA-producing S. epidermidis are widespread in clinical strain collections and PIA synthesis has been shown to be essential for S. epidermidis virulence. Moreover, PIA homologues have been identified in many other staphylococcal species, including the major human pathogen Staphylococcus aureus, and also Gram-negative human pathogens, suggesting that it might represent a more general pathogenicity principle in biofilm-related infections. In this review the current knowledge about the structure and biosynthesis of PIA is summarized. Additionally, information on its role in pathogenesis of biomaterial-related and other type of infections and the potential use of PIA and related compounds for prevention of infection is discussed.

Tricarboxylic Acid Cycle-Dependent Attenuation of Staphylococcus aureus In Vivo Virulence by Selective Inhibition of Amino Acid Transport

Staphylococci are the leading causes of endovascular infections worldwide. Commonly, these infections involve the formation of biofilms on the surface of biomaterials. Biofilms are a complex aggregation of bacteria commonly encapsulated by an adhesive exopolysaccharide matrix. In staphylococci, this exopolysaccharide matrix is composed of polysaccharide intercellular adhesin (PIA). PIA is synthesized when the tricarboxylic acid (TCA) cycle is repressed. The inverse correlation between PIA synthesis and TCA cycle activity led us to hypothesize that increasing TCA cycle activity would decrease PIA synthesis and biofilm formation and reduce virulence in a rabbit catheter-induced model of biofilm infection. TCA cycle activity can be induced by preventing staphylococci from exogenously acquiring a TCA cycle-derived amino acid necessary for growth. To determine if TCA cycle induction would decrease PIA synthesis in Staphylococcus aureus, the glutamine permease gene (glnP) was inactivated and TCA cycle activity, PIA accumulation, biofilm forming ability, and virulence in an experimental catheter-induced endovascular biofilm (endocarditis) model were determined. Inactivation of this major glutamine transporter increased TCA cycle activity, transiently decreased PIA synthesis, and significantly reduced in vivo virulence in the endocarditis model in terms of achievable bacterial densities in biofilm-associated cardiac vegetations, kidneys, and spleen. These data confirm the close linkage of TCA cycle activity and virulence factor production and establish that this metabolic linkage can be manipulated to alter infectious outcomes.

Oxygen-Mediated Regulation of Biofilm Development Is Controlled by the Alternative Sigma Factor σ B in Staphylococcus epidermidis

Using a modified rotating-disk reactor to sparge oxygen to Staphylococcus epidermidis cultures, we found that oxygen negatively regulates biofilm development by influencing the activity of sigma(B). Under anaerobic conditions, increased sigma(B) activity activates icaADBC, which encodes enzymes responsible for polysaccharide intercellular adhesin synthesis, by repressing transcription of the negative regulator icaR.

Tricarboxylic acid cycle-dependent regulation of Staphylococcus epidermidis polysaccharide intercellular adhesin synthesis.

Staphylococcus epidermidis is a major nosocomial pathogen primarily infecting immunocompromised individuals or those with implanted biomaterials (e.g., catheters). Biomaterial-associated infections often involve the formation of a biofilm on the surface of the medical device. In S. epidermidis, polysaccharide intercellular adhesin (PIA) is an important mediator of biofilm formation and pathogenesis. Synthesis of PIA is regulated by at least three DNA binding proteins (IcaR, SarA, and sigma(B)) and several environmental and nutritional conditions. Previously, we observed the environmental conditions that increased PIA synthesis decreased tricarboxylic acid (TCA) cycle activity. In this study, S. epidermidis TCA cycle mutants were constructed, and the function of central metabolism in PIA biosynthesis was examined. TCA cycle inactivation altered the metabolic status of S. epidermidis, resulting in a massive derepression of PIA biosynthetic genes and a redirection of carbon from growth into PIA biosynthesis. These data demonstrate that the bacterial metabolic status is a critical regulatory determinant of PIA synthesis. In addition, these data lead us to propose that the TCA cycle acts as a signal transduction pathway to translate external environmental cues into intracellular metabolic signals that modulate the activity of transcriptional regulators.

Inhibition of Staphylococcal Biofilm Formation by Nitrite

Several environmental stresses have been demonstrated to increase polysaccharide intercellular adhesin (PIA) synthesis and biofilm formation by the human pathogens Staphylococcus aureus and Staphylococcus epidermidis. In this study we characterized an adaptive response of S. aureus SA113 to nitrite-induced stress and show that it involves concomitant impairment of PIA synthesis and biofilm formation. Transcriptional analysis provided evidence that nitrite, either as the endogenous product of respiratory nitrate reduction or after external addition, causes repression of the icaADBC gene cluster, mediated likely by IcaR. Comparative microarray analysis revealed a global change in gene expression during growth in the presence of 5 mM sodium nitrite and indicated a response to oxidative and nitrosative stress. Many nitrite-induced genes are involved in DNA repair, detoxification of reactive oxygen and nitrogen species, and iron homeostasis. Moreover, preformed biofilms could be eradicated by the addition of nitrite, likely the result of the formation of toxic acidified nitrite derivatives. Nitrite-mediated inhibition of S. aureus biofilm formation was abrogated by the addition of nitric oxide (NO) scavengers, suggesting that NO is directly or indirectly involved. Nitrite also repressed biofilm formation of S. epidermidis RP62A.

Vancomycin-Intermediate Staphylococcus aureus Strains Have Impaired Acetate Catabolism: Implications for Polysaccharide Intercellular Adhesin Synthesis and Autolysis

The most common mechanism by which Staphylococcus aureus gains resistance to vancomycin is by adapting its physiology and metabolism to permit growth in the presence of vancomycin. Several studies have examined the adaptive changes occurring during the transition to vancomycin-intermediate resistance, leading to a model of vancomycin resistance in which decreased cell wall turnover and autolysis result in increased cell wall thickness and resistance to vancomycin. In the present study, we identified metabolic changes common to vancomycin-intermediate S. aureus (VISA) strains by assessing the metabolic and growth characteristics of two VISA strains (vancomycin MICs of 8 microg/ml) and two isogenic derivative strains with vancomycin MICs of 32 microg/ml. Interestingly, we observed the parental strains had impaired catabolism of nonpreferred carbon sources (i.e., acetate), and this impairment became more pronounced as vancomycin resistance increased. To determine if acetate catabolism impairment is common to VISA strains, we assessed the ability of VISA and vancomycin-sensitive S. aureus (VSSA) clinical isolates to catabolize acetate. As expected, a significantly greater percentage of VISA strains (71%) had impaired acetate catabolism relative to VSSA (8%). This is an important observation because staphylococcal acetate catabolism is implicated in growth yield and antibiotic tolerance and in regulating cell death and polysaccharide intercellular adhesin synthesis.

Correlation between biofilm formation and production of polysaccharide intercellular adhesin in clinical isolates of coagulase-negative staphylococci

The ability to form a biofilm seems to play an essential role in the virulence of coagulase-negative staphylococci (CoNS) by permitting them to cause persistent prosthetic device-related infections. The most clearly characterized component of staphylococcal biofilms is the polysaccharide intercellular adhesin (PIA) encoded by the icaADBC operon. In the present paper, we assess the link between the ability to form a biofilm (Bf+/−), to synthesize PIA (PIA+/−) and the presence of the ica locus (ica+/−). For this purpose, 66 CoNS strains were tested in vitro. Seventy three percent of all strains revealed presence of the ica locus (ica+), and therefore were potentially able to produce PIA and to form a biofilm. However, the characteristics observed indicated that 15% of all strains were biofilm forming without PIA production (Bf+, PIA−, ica+/−) while 8% were PIA producers without biofilm formation (Bf−, PIA+, ica+). On the basis of the obtained data we conclude that (i) PIA synthesis alone is not sufficient to produce a biofilm and (ii) staphylococci can also form a biofilm without producing PIA.

Biofilm Formation, icaADBC Transcription, and Polysaccharide Intercellular Adhesin Synthesis by Staphylococci in a Device-Related Infection Model

Biofilm formation of Staphylococcus epidermidis and S. aureus is mediated by the polysaccharide intercellular adhesin (PIA) encoded by the ica operon. We used a device-related animal model to investigate biofilm formation, PIA expression (immunofluorescence), and ica transcription (quantitative transcript analysis) throughout the course of infection by using two prototypic S. aureus strains and one S. epidermidis strain as well as corresponding ica mutants. During infection, the ica mutants were growth attenuated when inoculated in competition with the corresponding wild-type strains but not when grown singly. A typical biofilm was observed at the late course of infection. Only in S. aureus RN6390, not in S. aureus Newman, were PIA and ica-specific transcripts detectable after anaerobic growth in vitro. However, both S. aureus strains were PIA positive in vivo by day 8 of infection. ica transcription preceded PIA expression and biofilm formation in vivo. In S. epidermidis, both PIA and ica expression levels were elevated compared to those in the S. aureus strains in vitro as well as in vivo and were detectable throughout the course of infection. In conclusion, in S. aureus, PIA expression is dependent on the genetic background of the strain as well as on strong inducing conditions, such as those dominating in vivo. In S. epidermidis, PIA expression is elevated and less vulnerable to environmental conditions.

Detection of Virulence-Associated Genes Not Useful for Discriminating between Invasive and Commensal Staphylococcus epidermidis Strains from a Bone Marrow Transplant Unit

Because of their biofilm-forming capacity, invasive Staphylococcus epidermidis isolates, which cause the majority of nosocomial catheter-related bloodstream infections (BSIs), are thought to be selected at the time of catheter insertion from a population of less virulent commensal strains. This fact allows the prediction that invasive and contaminating strains can be differentiated via detection of virulence-associated genes. However, the hospital environment may pave the way for catheter-related infections by promoting a shift in the commensal bacterial population toward strains with enhanced virulence. The distribution of virulence-associated genes (icaADBC, aap, atlE, bhp, fbe, embp, mecA, IS256, and IS257), polysaccharide intercellular adhesin synthesis, and biofilm formation were investigated in S. epidermidis strains from independent episodes of catheter-related BSIs in individuals who have received bone marrow transplantation (BMT). The results were compared with those obtained for commensal S. epidermidis isolates from hospitalized patients after BMT and from healthy individuals, respectively. The clonal relationships of the strains were investigated by pulsed-field gel electrophoresis. icaADBC, mecA, and IS256 were significantly more prevalent in BSI isolates than in commensal isolates from healthy individuals. However, the prevalence of any of the genes in clonally independent, endogenous commensal strains from BMT patients did not differ from that in invasive BSI strains. icaADBC and methicillin resistance, factors important for the establishment of catheter-related infections, already ensure survival of the organisms in their physiological habitat in the hospital environment, resulting in a higher probability of contamination of indwelling medical devices with virulent S. epidermidis strains. The dynamics of S. epidermidis populations reveal that detection of icaADBC and mecA is not suitable for discriminating invasive from contaminating S. epidermidis strains.