Strain RP62A Research Articles

Assembly landscape of the complete B-repeat superdomain from Staphylococcus epidermidis strain 1457.

The accumulation-associated protein (Aap) is the primary determinant of Staphylococcus epidermidis device-related infections. The B-repeat superdomain is responsible for intercellular adhesion that leads to the development of biofilms occurring in such infections. It was recently demonstrated that Zn-induced B-repeat assembly leads to formation of functional amyloid fibrils, which offer strength and stability to the biofilm. Rigorous biophysical studies of Aap B-repeats from S. epidermidis strain RP62A revealed Zn-induced assembly into stable, reversible dimers and tetramers, prior to aggregation into amyloid fibrils. Genetic manipulation is not tractable for many S. epidermidis strains, including RP62A; instead, many genetic studies have used strain 1457. Therefore, to better connect findings from biophysical and structural studies of B-repeats to in vivo studies, the B-repeat superdomain from strain 1457 was examined. Differences between the B-repeats from strain RP62A and 1457 include the number of B-repeats, which has been shown to play a critical role in assembly into amyloid fibrils, as well as the distribution of consensus and variant B-repeat subtypes, which differ in assembly competency and thermal stability. Detailed investigation of the Zn-induced assembly of the full B-repeat supderdomain from strain 1457 was conducted using analytical ultracentrifugation. Whereas the previous construct from RP62A (Brpt5.5) formed a stable tetramer prior to aggregation, Brpt6.5 from 1457 forms extremely large stable species on the order of ∼28-mers, prior to aggregation into similar amyloid fibrils. Our data suggest that both assembly pathways may proceed through the same mechanism of dimerization and tetramerization, and both conclude with the formation of amyloid-like fibrils. Discussion of assembly behavior of B-repeats from different strains and of different length is provided with considerations of biological implications.

Host Soluble Factors Cause Changes in Staphylococcus epidermidis Antibiotic Susceptibility and Biofilm Formation Ability.

Staphylococcus epidermidis is a major nosocomial pathogen with a remarkable ability to adhere to the surfaces of indwelling medical devices and form biofilms. Unlike other nosocomial pathogens, the interaction of S. epidermidis with host factors has not been the focus of substantial research. This study aimed to assess the alterations in the antibiotic susceptibility and biofilm formation ability of S. epidermidis in the presence of host serum factors. S. epidermidis strain RP62A was cultured in a laboratory culture medium with or without human serum/plasma, and changes in antibiotic susceptibility, biofilm formation, and gene expression were evaluated. The data obtained revealed that exposure to host serum factors increased the susceptibility of S. epidermidis to glycopeptide antibiotics and was also detrimental to biofilm formation. Gene expression analysis revealed downregulation of both dltA and fmtC genes shortly after human serum/plasma exposure. The importance of transferrin-mediated iron sequestration as a host anti-biofilm strategy against S. epidermidis was also emphasized. We have demonstrated that serum factors play a pivotal role as part of the host's anti-infective strategy against S. epidermidis infections, highlighting the importance of incorporating such factors during in vitro studies with this pathogen.

Staphylococcus epidermidis biofilms undergo metabolic and matrix remodeling under nitrosative stress.

Staphylococcus epidermidis is a commensal skin bacterium that forms host- and antibiotic-resistant biofilms that are a major cause of implant-associated infections. Most research has focused on studying the responses to host-imposed stresses on planktonic bacteria. In this work, we addressed the open question of how S. epidermidis thrives on toxic concentrations of nitric oxide (NO) produced by host innate immune cells during biofilm assembly. We analyzed alterations of gene expression, metabolism, and matrix structure of biofilms of two clinical isolates of S. epidermidis, namely, 1457 and RP62A, formed under NO stress conditions. In both strains, NO lowers the amount of biofilm mass and causes increased production of lactate and decreased acetate excretion from biofilm glucose metabolism. Transcriptional analysis revealed that NO induces icaA, which is directly involved in polysaccharide intercellular adhesion (PIA) production, and genes encoding proteins of the amino sugar pathway (glmM and glmU) that link glycolysis to PIA synthesis. However, the strains seem to have distinct regulatory mechanisms to boost lactate production, as NO causes a substantial upregulation of ldh gene in strain RP62A but not in strain 1457. The analysis of the matrix components of the staphylococcal biofilms, assessed by confocal laser scanning microscopy (CLSM), showed that NO stimulates PIA and protein production and interferes with biofilm structure in a strain-dependent manner, but independently of the Ldh level. Thus, NO resistance is attained by remodeling the staphylococcal matrix architecture and adaptation of main metabolic processes, likely providing in vivo fitness of S. epidermidis biofilms contacting NO-proficient macrophages.

Concentration-Dependent Global Quantitative Proteome Response of Staphylococcus epidermidis RP62A Biofilms to Subinhibitory Tigecycline.

Staphylococcus epidermidis is a leading cause of biofilm-associated infections on implanted medical devices. During the treatment of an infection, bacterial cells inside biofilms may be exposed to sublethal concentrations of the antimicrobial agents. In the present study, the effect of subinhibitory concentrations of tigecycline (TC) on biofilms formed by S. epidermidis strain RP62A was investigated using a quantitative global proteomic technique. Sublethal concentrations of TC [1/8 (T1) and 1/4 minimum inhibitory concentration (MIC) (T2)] promoted biofilm production in strain RP62A, but 1/2 MIC TC (T3) significantly inhibited biofilm production. Overall, 413, 429, and 518 proteins were differentially expressed in biofilms grown with 1/8 (T1), 1/4 (T2), and 1/2 (T3) MIC of TC, respectively. As the TC concentration increased, the number of induced proteins in each Cluster of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway increased. The TC concentration dependence of the proteome response highlights the diverse mechanisms of adaptive responses in strain RP62A biofilms. In both COG and KEGG functional analyses, most upregulated proteins belong to the metabolism pathway, suggesting that it may play an important role in the defense of strain RP62A biofilm cells against TC stress. Sub-MIC TC treatment of strain RP62A biofilms led to significant changes of protein expression related to biofilm formation, antimicrobial resistance, virulence, quorum sensing, ABC transporters, protein export, purine/pyrimidine biosynthesis, ribosomes, and essential proteins. Interestingly, in addition to tetracycline resistance, proteins involved in resistance of various antibiotics, including aminoglycosides, antimicrobial peptides, β-lactams, erythromycin, fluoroquinolones, fusidic acid, glycopeptides, lipopeptides, mupirocin, rifampicin and trimethoprim were differentially expressed. Our study demonstrates that global protein expression profiling of biofilm cells to antibiotic pressure may improve our understanding of the mechanisms of antibiotic resistance in biofilms.

ShsA: A novel orthologous of sasX/sesI virulence genes is detected in Staphylococcus haemolyticus Brazilian strains.

The surface protein SasX, has a key role in methicillin-resistant Staphylococcus aureus (MRSA) colonization and pathogenesis, and has been associated with the epidemic success of some MRSA clones. To date, only one SasX homologous protein, named SesI, has been described in Staphylococcus epidermidis. In this work, we analyze the occurrence of the sasX gene and its genetic environment in Staphylococcus haemolyticus S. haemolyticus clinical strains (n=62) were screened for the presence of the sasX gene and its carrier, the prophage Φ SPβ-like. A deep characterization was done in one strain (MD43), through which we determined the complete nucleotide sequence for the S. haemolitycus sasX-like gene. Whole genome sequencing of strain MD43 was performed, and the gene, termed here because of its unique attributes, shsA, was mapped to the Φ SPβ-like prophage sequence. The shsA gene was detected in 33 out of 62 strains showing an average identity of 92 and 96% with the sasX and sesI genes and at the amino acid level, 88% identity with SasX and 92% identity with SesI. The ~124Kb Φ SPβ-like prophage sequence showed a largely intact prophage compared to its counterpart in S. epidermidis strain RP62A, including the sesI insertion site. In conclusion, we identified a new sasX ortholog in S. haemolyticus (shsA). Its horizontal spread from this reservoir could represent an emergent threat in healthcare facilities since so far, no S. aureus sasX+ strains have been reported in Brazil.

1323. Substantial Doses of Daptomycin and Rifampin Eradicate S. epidermidis Biofilm in an In Vitro Pharmacodynamic (IVPD) Model

BackgroundThe concentration of antibiotics at the site of action needed to eradicate biofilm is currently unknown. Studies have previously suggested that bacteria in biofilms are 1000-fold more resistant to antibiotics than free-floating planktonic bacteria. We sought to describe concentrations of daptomycin alone and in combination with rifampin in relation to the pharmacodynamic exposures for biofilm eradication.MethodsWe utilized a methicillin-resistant high biofilm-forming S. epidermidis strain RP62a (ATCC® 35984) over a 48-hour in vitro PD biofilm model. The Centers for Disease Control (CDC) Biofilm Reactor model was used with chromium cobalt materials to simulate an orthopedic device infection. The reactor was inoculated and underwent a 24-hr growth phase and 16-hr conditioning phase to form biofilm on the chromium cobalt coupons, and then a 48-hr PK-PD phase was run. The daptomycin MIC for RP62a was 0.5 mg/L and the rifampin MIC was 0.015 mg/L. We modeled a growth control of the isolate alone, a 12 mg/kg regimen of daptomycin (fCmax: 14.7 mg/L, Ke 0.09), a daptomycin concentration of 1000 mg/L (2000x MIC), and a combination model of daptomycin 1000 mg/L with rifampin 15 mg/L (1000x MIC). Coupons with bacteria embedded in biofilm were sonicated, vortexed, and plated on Tryptic Soy Agar for colony counts read at 24 hrs. Bactericidal activity was defined as ≥ 3-log10 CFU/mL reduction from the initial inoculum.ResultsThe simulated humanized dosing regimen of daptomycin 12 mg/kg (fAUC0-24/MIC: 204) was similar to the growth control model. Bactericidal kill was demonstrated at 24hr and 48hr in the daptomycin 1000 mg/L model (fAUC0-24/MIC: 20,248) but did not fall beneath the limit of detection. The daptomycin and rifampin combination model demonstrated bactericidal kill at 24hr and 48hr and went below the limit of detection.ConclusionThis study demonstrated that significantly higher concentrations of antibiotics are needed at the site of action to eradicate biofilm than what maximum systemic dosing can provide. Identifying these concentrations provides a foundation for localized antibiotic therapy and further studies are needed to elucidate these concentrations for a variety of antibiotics and biofilm-forming organisms.Disclosures Kerry LaPlante, PharmD, Merck (Advisor or Review Panel member, Research Grant or Support)Ocean Spray Cranberries, Inc. (Research Grant or Support)Pfizer Pharmaceuticals (Research Grant or Support)Shionogi, Inc. (Research Grant or Support)

An invasive marker Staphylococcus epidermidis surface protein I (SesI) harboured by a ST239 methicillin-resistant Staphylococcus aureus.

Recently, the virulence factor sasX was described on the mobile genetic element φSPβ prophage in a ST239-SCCmecIII methicillin-resistant Staphylococcus aureus (MRSA) clone. The aim of this study was to identify sesI, an sasX homologue, in an MRSA ST239 strain. MRSA strain VB1490 was isolated from a patient with MRSA bacteraemia in India. Staphylococcal cassette chromosomemec (SCCmec) typing and whole-genome shotgun sequencing were performed. Phylogenetic analysis of VB1490 and ST239 reference genomes from the NCBI database was performed. Amplification and sequencing of the sasX gene was performed to establish allele homology. The sasX gene identified in isolate VB1490 was more similar to the sesI gene of Staphylococcus epidermidis RP62A than the sasX gene of ST239 MRSA strain. Whole-genome analysis revealed the presence of the sasX gene on a φSPβ-like prophage that exhibited high sequence identity to that of S. epidermidis strain RP62A. These finding suggests the dissemination of the invasion-determining virulence factorsesI from S. epidermidis to a ST239 MRSA strain.

The methicillin-resistant S. epidermidis strain RP62A genome mining for potential novel drug targets identification

In unique biofilm-forming methicillin-resistant (MRSE) strains of S. epidermidis have become a serious medical problem due to the emergence of antibiotic resistance strains. There is an essential need to develop novel drug targets to address the new intervention challenge for such multidrug-resistant bacteria. Here we utilized the available genomes and proteome datasets resources of MRSE S. epidermidis RP62A for comparative and subtractive genome analyses to identify novel drug targets for future drug discovery approaches. The comparative genome based molecular biology database resources scanning identified 255 proteins as essential human non-homologs targets for epidermidis RP62A that involve in essential metabolic pathways. Among these 12 targets were found to be involved in pathogen unique metabolic pathways. Druggability potential of each of these targets by Drug Bank database further identified 5 proteins as druggable essential proteins. As antimicrobial agents inflict beneficial gut microbiome and the antibiotics causes less agitation in gut flora are considered as best candidates. We pursued additional analysis and screen our lead targets proteins against metagenomes databases holding whole genome sequences of human gut microbiome. The negligible biological similarity with gut microbiota genomes datasets predicting the future implementation of our lead identified proteins as druggable targets for S. epidermidis RP62A that may not affect the essential humans gut microbiota. Based on these evidences, we are speculating that the putative druggable proteins addressed in this study are potent enough for their future evaluation as therapeutic targets to combat the S. epidermidis RP62A infections.

The Staphylococcus epidermidis gdpS regulates biofilm formation independently of its protein-coding function.

The second messenger cyclic di-guanylate (c-di-GMP) plays an important role in controlling the switch between planktonic and biofilm lifestyles. The synthesis of c-di-GMP is catalyzed by di-guanylate cyclases (DGCs) and the enzymes are characterized by the presence of a conserved GGDEF domain. In the sequenced staphylococcal genomes, gdpS is the only gene encoding a GGDEF domain-containing protein. Previous studies have shown that gdpS contributes to staphylococcal biofilm formation, but its effect remains under debate. In the present study, we deleted gdpS in Staphylococcus epidermidis strain RP62A. Disruption of gdpS in this strain impaired biofilm formation under both static and dynamic flow conditions, suggesting that gdpS act as a positive regulator of biofilm development in this high-biofilm-forming isolate. The predicted translational start site of gdpS in S.epidermidis differs between the Refseq database and the Genbank database. By using site-directed mutagenesis and Western blot analysis, we determined GdpS is translated from the start codon annotated in the Refseq database. In addition, mutation in the GGDEF domain did not affect the ability of gdpS to complement the biofilm defect of the gdpS mutant. Heterologous di-guanylate cyclases expressed in trans failed to complement the gdpS mutant. These results confirmed that gdpS modulates staphylococcal biofilm independently of c-di-GMP signaling pathway. Furthermore, mutations of the start codon did not abolish the capacity of gdpS to enhance biofilm formation. Taken together, these findings indicated that the S.epidermidis gdpS regulates biofilm formation independently of its protein-coding function.

Construction of mutant strains of methicillin resistant Staphylococcus epidermidis with psm-mec gene deletion

Objective To construct mutant strains of methicillin resistant Staphylococcus epidermidis (MRSE) with psm-mec gene deletion and to investigate the function of psm-mec gene. Methods The drug sensitivity test and DNA sequence analysis were performed to screen out the tetracycline and chloramphenicol sensitive clinical strains of MRSE, whose upstream and downstream sequences of psm-mec gene were identical to those of the Staphylococcus epidermidis reference strain RP62A. The recombinant plasmid pBT2-Δpsm-mec was constructed by using the fusion PCR and a temperature sensitive shuttle plasmid. After being identified, the plasmid was transformed into the Staphylococcus aureus RN4220 strain by electroporation, and then transformed into the selected clinical isolates of MRSE. The mutant strains of MRSE with psm-mec deletion were screened out and identified after homologous recombination. The differences in biofilm formation between the mutant and wild-type strains were analyzed for further elucidation the relationships between the psm-mec gene and biofilm formation in MRSE strains. Results Three clinical MRSE isolates for the construction of mutant strains with psm-mec gene deletion were screened out and identified by using drug sensitivity test and sequence alignment analysis. The mutants constructed via homogenous recombination were screened out and identified. Compared with the corresponding wild-type strains, the three mutants with psm-mec gene deletion showed significantly decreased ability of biofilm formation, demonstrating that the psm-mec genes strains induced the biofilm formation of MRSE. Conclusion The Δpsm-mec mutant strains were successfully constructed. The psm-mec gene played an important role in the biofilm formation of Staphylococcus epidermdis. Key words: Staphylococcus epidermidis; psm-mec; Homologous recombination; Mutant; Biofilm formation

Early staphylococcal biofilm formation on solid orthopaedic implant materials: in vitro study.

Biofilms forming on the surface of biomaterials can cause intractable implant-related infections. Bacterial adherence and early biofilm formation are influenced by the type of biomaterial used and the physical characteristics of implant surface. In this in vitro research, we evaluated the ability of Staphylococcus epidermidis, the main pathogen in implant-related infections, to form biofilms on the surface of the solid orthopaedic biomaterials, oxidized zirconium-niobium alloy, cobalt-chromium-molybdenum alloy (Co-Cr-Mo), titanium alloy (Ti-6Al-4V), commercially pure titanium (cp-Ti) and stainless steel. A bacterial suspension of Staphylococcus epidermidis strain RP62A (ATCC35984) was added to the surface of specimens and incubated. The stained biofilms were imaged with a digital optical microscope and the biofilm coverage rate (BCR) was calculated. The total amount of biofilm was determined with the crystal violet assay and the number of viable cells in the biofilm was counted using the plate count method. The BCR of all the biomaterials rose in proportion to culture duration. After culturing for 2–4 hours, the BCR was similar for all materials. However, after culturing for 6 hours, the BCR for Co-Cr-Mo alloy was significantly lower than for Ti-6Al-4V, cp-Ti and stainless steel (P<0.05). The absorbance value determined in the crystal violet assay and the number of viable cells on Co-Cr-Mo were not significantly lower than for the other materials (P>0.05). These results suggest that surface properties, such as hydrophobicity or the low surface free energy of Co-Cr-Mo, may have some influence in inhibiting or delaying the two-dimensional expansion of biofilm on surfaces with a similar degree of smoothness.

Role of the two-component regulatory system arlRS in ica operon and aap positive but non-biofilm-forming Staphylococcus epidermidis isolates from hospitalized patients

The ica operon and aap gene are important factors for Staphylococcus epidermidis biofilm formation. However, we found 15 out of 101 S. epidermidis strains isolated from patients had both the ica operon and the aap gene in the genome but could not form biofilms (ica+aap+/BF− isolates). Compared with standard strain RP62A, the 15 ica+aap+/BF− isolates had similar growth curves and initial attachment abilities, but had much lower apparent transcription levels of the icaA gene and significantly less production of polysaccharide intercellular adhesion (PIA). Furthermore, the expression of accumulation-associated protein in ica+aap+/BF− isolates was much weaker than in RP62A. The mRNA levels of icaADBC transcription-related regulatory genes, including icaR, sarA, rsbU, srrA, arlRS and luxS, were measured in the 15 ica+aap+/BF− clinical isolates. The mRNA levels of arlR and rsbU in all of the ica+aap+/BF− isolates were lower than in RP62A at 4 h. At 10 h, 14/15 of the isolates showed lower mRNA levels of arlR and rsbU than shown by RP62A. However, expression of sarA, luxS, srrA and icaR varied in different ica+aap+/BF− isolates.To further investigate the role of arlRS in biofilm formation, we analyzed icaA, sarA and rsbU transcription, PIA synthesis, Aap expression and biofilm formation in an arlRS deletion mutant of S. epidermidis strain 1457 and all were much less than in the wild type strain. This is consistent with the hypothesis that ArlRS may play an important role in regulating biofilm formation by the ica+aap+/BF−S. epidermidis clinical isolates and operate via both ica-dependent and Aap-dependent pathways.

Monoclonal Antibody Raised against PNAG Has Variable Effects on Static S. epidermidis Biofilm Accumulation In Vitro

Staphylococcus epidermidis causes infections commonly associated with patients with indwelling medical devices due to its ability to form biofilms, bacterial structures attached to a surface and embedded in a protective matrix. Cells within biofilms are known to be more resistant than free-floating planktonic cells to the host immune response and also to antibiotic therapy often leading to relapsing infections 1. Frequently, surgical removal of an infected device is required to resolve these infections, resulting in significant effects in a patient's quality of life. Hence, preventative approaches are clearly needed to overcome this challenge. Antibodies have been shown to be one promising alternative to target surface-attached molecules and inhibit biofilm formation 2. We have previously shown that human monoclonal antibodies (mAb) specific for poly-β-1,6-N-acetylglucosamine (PNAG) were effective in killing S. epidermidis in opsonophagocytic in vitro assays, and in protecting the murine host against infection by PNAG-producing pathogens 3,4. Since S. epidermidis biofilm accumulation is mainly mediated by PNAG, it was hypothesized that the binding of this molecule by a specific mAb could impact biofilm accumulation, a process that has not previously been investigated. Here we tested the previously characterized mAb F598 3, for inhibition of S. epidermidis biofilm accumulation in vitro. Several concentrations of mAb F598 and the isotype control human mAb F429 (specific for the Pseudomonas aeruginosa alginate capsule) 5, were co-cultured with bacteria in static conditions for 1h at 37oC to allow antibody binding. Thereafter, cultures were incubated for 24h at 37oC with shaking at 250 rpm. Biofilm accumulation was then quantified by the standard crystal violet staining 6. Depending on the S. epidermidis strain used, the presence of mAb F598 had a differential effect on biofilm accumulation. In the case of the ATCC strain RP62A we observed a 42% reduction in biofilm accumulation at the highest mAb concentration tested, while the clinical strains 1457 and M184 grown in the presence of mAb F598 had a dose-dependent increase of the biofilm accumulation. In the case of the PNAG-deficient, ica-mutant strain 1457-M10, as expected, no significant effect was found on the biofilm biomass as no PNAG is produced. Furthermore, control mAb F429 had no significant effect on S. epidermidis biofilm accumulation. The results with the ica-mutant and mAb controls suggest altogether that the inhibitory or enhancing effect of the mAb is PNAG-dependent. As observed in other studies that have used antibodies specific for S. epidermidis surface molecules, the observed enhancement of biofilm formation could be a result of increased PNAG expression caused by the early blockage of the synthesis of this molecule 7. On the other hand, the specificity of mAb F598 for epitopes on PNAG that do not require the N-acetyl groups on the glucosamine monomers may have contributed to the differential effects in biofilm accumulation. These would thus depend on the level of PNAG acetylation of individual strains, ultimately, controlled by the IcaB extracellular deacetylase. Therefore, mAbs directed to other epitopes might be better suited for inhibition of in vitro biofilm accumulation. Additionally, the results presented here suggest that a difference between the effect of mAb F598 against PNAG-producing bacteria in animal models 3,4 and it efficiency at inhibiting in vitro static biofilm accumulation among different S. epidermidis strains. Notably, many biofilms are formed under flow conditions and it is not clear to what extent shear stress from flow over in vivo biofilms contributes to biofilm formation, and whether under those conditions the effect of mAb F598 might be different. While the stimulation of biofilm formation by S. epidermidis grown in vitro may raise questions regarding the usage of mAb F598 in vivo, the results do not necessarily exclude that mAb F598 could be effective in vivo against biofilm infections. The majority of studies that reported strong biofilm inhibition by monoclonal or polyclonal antibodies used only a few strains in the assays 2,8, which could result in misleading interpretations. The findings presented here further stress the necessity to use more than a few strains when testing the efficacy of new biofilm-inhibition strategies in order to ensure that the desired effect is observed in a representative number of clones of the species under study.

Adherence ability of Staphylococcus epidermidis on prosthetic biomaterials: an in vitro study

Bacterial adhesion to the surface of biomaterials is an essential step in the pathogenesis of implant-related infections. In this in vitro research, we evaluated the ability of Staphylococcus epidermidis to adhere to the surface of solid biomaterials, including oxidized zirconium-niobium alloy (Oxinium), cobalt-chromium-molybdenum alloy, titanium alloy, commercially pure titanium, and stainless steel, and performed a biomaterial-to-biomaterial comparison. The test specimens were physically analyzed to quantitatively determine the viable adherent density of the S. epidermidis strain RP62A (American Type Culture Collection [ATCC] 35984). Field emission scanning electron microscope and laser microscope examination revealed a featureless, smooth surface in all specimens (average roughness <10 nm). The amounts of S. epidermidis that adhered to the biomaterial were significantly lower for Oxinium and the cobalt-chromium-molybdenum alloy than for commercially pure titanium. These results suggest that Oxinium and cobalt-chromium-molybdenum alloy are less susceptible to bacterial adherence and are less inclined to infection than other materials of a similar degree of smoothness.

Extracellular DNA-dependent biofilm formation by Staphylococcus epidermidis RP62A in response to subminimal inhibitory concentrations of antibiotics

We measured the ability of Staphylococcus epidermidis to form biofilms in the presence of subminimal inhibitory concentrations (sub-MICs) of vancomycin, tigecycline, linezolid and novobiocin. Six strains that produce different amounts of biofilm were tested. The three strains that produced the highest amounts of biofilm exhibited steady-state or decreased biofilm formation in the presence of sub-MIC antibiotics, whereas the three strains that produced lower amounts of biofilm exhibited up to 10-fold-increased biofilm formation in the presence of sub-MIC antibiotics. In two of the inducible strains (9142 and 456a), antibiotic-induced biofilm formation was inhibited by dispersin B, an enzyme that degrades poly- N-acetylglucosamine (PNAG) biofilm polysaccharide. In the third inducible strain (RP62A), dispersin B inhibited biofilm formation in response to sub-MIC vancomycin, but not to sub-MIC tigecycline. In contrast, DNase I efficiently inhibited biofilm formation by strain RP62A in response to sub-MIC tigecycline and vancomycin. DNase I had no effect on antibiotic-induced biofilm formation in strains 9142 and 456a. Our findings indicate that antibiotic-induced biofilm formation in S. epidermidis is both strain- and antibiotic-dependent and that S. epidermidis RP62A utilizes an extracellular DNA-dependent mechanism to form biofilms in response to sub-MIC antibiotics.

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.

Conserved genes in a path from commensalism to pathogenicity: comparative phylogenetic profiles of Staphylococcus epidermidis RP62A and ATCC12228

BackgroundStaphylococcus epidermidis, long regarded as an innocuous commensal bacterium of the human skin, is the most frequent cause of nosocomial infections associated with implanted medical devices. This conditional pathogen provides a model of choice to study genome landmarks correlated with the transition between commensalism and pathogenicity. Traditional investigations stress differences in gene content. We focused on conserved genes that have accumulated small mutation differences during the transition.ResultsA comparison of strain ATCC12228, a non-biofilm forming, non-infection associated strain and strain RP62A, a methicillin-resistant biofilm clinical isolate, revealed consistent variation, mostly single-nucleotide polymorphisms (SNPs), in orthologous genes in addition to the previously investigated global changes in gene clusters. This polymorphism, scattered throughout the genome, may reveal genes that contribute to adaptation of the bacteria to different environmental stimuli, allowing them to shift from commensalism to pathogenicity. SNPs were detected in 931 pairs of orthologs with identical gene length, accounting for approximately 45% of the total pairs of orthologs. Assuming that non-synonymous mutations would mark recent evolution, and hence be associated to the onset of the pathogenic process, analysis of ratios of non-synonymous SNPs vs synonymous SNPs suggested hypotheses about possible pathogenicity determinants. The N/S ratios for virulence factors and surface proteins differed significantly from that of average SNPs. Of those gene pairs, 40 showed a disproportionate distribution of dN vs dS. Among those, the presence of the gene encoding methionine sulfoxide reductase suggested a possible involvement of reactive oxygen species. This led us to uncover that the infection associated strain was significantly more resistant to hydrogen peroxide and paraquat than the environmental strain. Some 16 genes of the list were of unknown function. We could suggest however that they were likely to belong to surface proteins or considered in priority as important for pathogenicity.ConclusionOur study proposed a novel approach to identify genes involved in pathogenic processes and provided some insight about the molecular mechanisms leading a commensal inhabitant to become an invasive pathogen.

Structural elucidation of the extracellular and cell-wall teichoic acids of Staphylococcus aureus MN8m, a biofilm forming strain

Extracellular teichoic acid, an essential constituent of the biofilm produced by Staphylococcus epidermidis strain RP62A, is also an important constituent of the extracellular matrix of another biofilm producing strain, Staphylococcus aureus MN8m. The structure of the extracellular and cell wall teichoic acids of the latter strain was studied by NMR spectroscopy and capillary electrophoresis–mass spectrometry. Both teichoic acids were found to be a mixture of two polymers, a (1→5)-linked poly(ribitol phosphate), substituted at the 4-position of ribitol residues with β-GlcNAc, and a (1→3)-linked poly(glycerol phosphate), partially substituted with the d-Ala at 2-position of glycerol residue. Such mixture is unusual for S. aureus.

IcaR encodes a transcriptional repressor involved in environmental regulation of ica operon expression and biofilm formation in Staphylococcus epidermidis.

Biofilm formation in Staphylococcus epidermidis is dependent upon the ica operon-encoded polysaccharide intercellular adhesin, which is subject to phase-variable and environmental regulation. The icaR gene, located adjacent to the ica operon, appears to be a member of the tetR family of transcriptional regulators. In the reference strain RP62A, reversible inactivation of the ica operon by IS256 accounts for 25 to 33% of phase variants. In this study, icaA and icaR regulation were compared in RP62A and a biofilm-forming clinical isolate, CSF41498, in which IS256 is absent. Predictably, ica operon expression was detected only in wild-type CSF41498 and RP62A but not in non-IS256-generated phase variants. In contrast, the icaR gene was not expressed in RP62A phase variants but was expressed in CSF41498 variants. An icaR::Em(r) insertion mutation in CSF41498 resulted in an at least a 5.8-fold increase in ica operon expression but did not significantly alter regulation of the icaR gene itself. Activation of ica operon transcription by ethanol in CSF41498 was icaR dependent. In contrast, a small but significant induction of ica by NaCl and glucose (NaCl-glucose) was observed in the icaR::Em(r) mutant. In addition, transcription of the icaR gene itself was not significantly affected by NaCl-glucose but was repressed by ethanol. Expression of the ica operon was induced by ethanol or NaCl-glucose in phase variants of CSF41498 (icaR+) but not in RP62A variants (icaR deficient). These data indicate that icaR encodes a repressor of ica operon transcription required for ethanol but not NaCl-glucose activation of ica operon expression and biofilm formation.

Mixed species biofilms of Candida albicans and Staphylococcus epidermidis.

A simple catheter disk model system was used to study the development in vitro of mixed species biofilms of Candida albicans and Staphylococcus epidermidis, two organisms commonly found in catheter-associated infections. Two strains of S. epidermidis were used: a slime-producing wild type (strain RP62A) and a slime-negative mutant (strain M7). In mixed fungal-bacterial biofilms, both staphylococcal strains showed extensive interactions with C. albicans. The susceptibility of 48-h biofilms to fluconazole, vancomycin and mixtures of the drugs was determined colorimetrically. The results indicated that the extracellular polymer produced by S. epidermidis RP62A could inhibit fluconazole penetration in mixed fungal-bacterial biofilms. Conversely, the presence of C. albicans in a biofilm appeared to protect the slime-negative staphylococcus against vancomycin. Overall, the findings suggest that fungal cells can modulate the action of antibiotics, and that bacteria can affect antifungal activity in mixed fungal-bacterial biofilms.