Methicillin resistance alters the biofilm phenotype and attenuates virulence in Staphylococcus aureus device-associated infections.
ABSTRACT: Clinical isolates of Staphylococcus aureus can express biofilm phenotypes promoted by the major cell wall autolysin and the fibronectin-binding proteins or the icaADBC-encoded polysaccharide intercellular adhesin/poly-N-acetylglucosamine (PIA/PNAG). Biofilm production in methicillin-susceptible S. aureus (MSSA) strains is typically dependent on PIA/PNAG whereas methicillin-resistant isolates express an Atl/FnBP-mediated biofilm phenotype suggesting a relationship between susceptibility to ?-lactam antibiotics and biofilm. By introducing the methicillin resistance gene mecA into the PNAG-producing laboratory strain 8325-4 we generated a heterogeneously resistant (HeR) strain, from which a homogeneous, high-level resistant (HoR) derivative was isolated following exposure to oxacillin. The HoR phenotype was associated with a R???H substitution in the DHHA1 domain of GdpP, a recently identified c-di-AMP phosphodiesterase with roles in resistance/tolerance to ?-lactam antibiotics and cell envelope stress. Transcription of icaADBC and PNAG production were impaired in the 8325-4 HoR derivative, which instead produced a proteinaceous biofilm that was significantly inhibited by antibodies against the mecA-encoded penicillin binding protein 2a (PBP2a). Conversely excision of the SCCmec element in the MRSA strain BH1CC resulted in oxacillin susceptibility and reduced biofilm production, both of which were complemented by mecA alone. Transcriptional activity of the accessory gene regulator locus was also repressed in the 8325-4 HoR strain, which in turn was accompanied by reduced protease production and significantly reduced virulence in a mouse model of device infection. Thus, homogeneous methicillin resistance has the potential to affect agr- and icaADBC-mediated phenotypes, including altered biofilm expression and virulence, which together are consistent with the adaptation of healthcare-associated MRSA strains to the antibiotic-rich hospital environment in which they are frequently responsible for device-related infections in immuno-compromised patients.
Project description:A major constituent of many Staphylococcus aureus biofilms is a polysaccharide known as the polysaccharide intercellular adhesin, or poly N-acetylglucosamine (PIA/PNAG). PIA/PNAG is synthesized by the 4 gene products of the icaADBC operon, which is negatively regulated by the divergently transcribed icaR gene. We previously reported the identification of a gene, rbf, involved in the positive transcriptional regulation of icaADBC transcription by repressing icaR in S. aureus strain 8325-4. However, we were unable to show binding of Rbf to DNA upstream of icaR or icaA, suggesting that Rbf may control expression of an unknown factor(s) that, in turn, regulates ica expression. Here we report that the unknown factor is SarX protein. Results from epistasis assays and genetic complementation analyses suggest that Rbf upregulates SarX, which then downregulates IcaR, thereby activating icaADBC. Electrophoretic mobility shift assays revealed that SarX protein bound to a sequence upstream of icaR within the icaA coding region. Cross-linking and immunoprecipitation experiments further suggested that Rbf binds to the sarX promoter in S. aureus. These results demonstrate that Rbf and SarX represent a regulatory cascade that promotes PIA-dependent biofilm formation in S. aureus.
Project description:Production of icaADBC-encoded polysaccharide intercellular adhesin, or poly-N-acetylglucosamine (PIA/PNAG), represents an important biofilm mechanism in staphylococci. We previously described a glucose-induced, ica-independent biofilm mechanism in four methicillin-resistant Staphylococcus aureus (MRSA) isolates. Here, biofilm regulation by NaCl and glucose was characterized in 114 MRSA and 98 methicillin-sensitive S. aureus (MSSA) isolates from diagnosed device-related infections. NaCl-induced biofilm development was significantly more prevalent among MSSA than MRSA isolates, and this association was independent of the isolate's genetic background as assessed by spa sequence typing. Among MSSA isolates, PIA/PNAG production correlated with biofilm development in NaCl, whereas in MRSA isolates grown in NaCl or glucose, PIA/PNAG production was not detected even though icaADBC was transcribed and regulated. Glucose-induced biofilm in MRSA was ica independent and apparently mediated by a protein adhesin(s). Experiments performed with strains that were amenable to genetic manipulation revealed that deletion of icaADBC had no effect on biofilm in a further six MRSA isolates but abolished biofilm in four MSSA isolates. Mutation of sarA abolished biofilm in seven MRSA and eight MSSA isolates. In contrast, mutation of agr in 13 MRSA and 8 MSSA isolates substantially increased biofilm (more than twofold) in only 5 of 21 (23%) isolates and had no significant impact on biofilm in the remaining 16 isolates. We conclude that biofilm development in MRSA is ica independent and involves a protein adhesin(s) regulated by SarA and Agr, whereas SarA-regulated PIA/PNAG plays a more important role in MSSA biofilm development.
Project description:<h4>Objectives</h4>One of the main characteristics of methicillin-resistant Staphylococcus aureus (MRSA) from both hospitals and community is their heterogeneous expression of resistance. Recently, we reported new heterogeneous MRSA isolates phenotypically susceptible to oxacillin despite being mecA positive. These low-level mecA-mediated resistance MRSA strains are very heterogeneous in expression (HeR) and are likely to be clinically relevant since exposure of such isolates to beta-lactams can result in high-level homotypic resistance (HoR). We hypothesized that HeR to HoR selection in these clinically relevant strains may be determined by the pre-existence of a hypermutable population that favours its selection in the presence of oxacillin.<h4>Methods</h4>Using established procedures, SA13011 HeR to HoR selection was performed by using subinhibitory concentrations of oxacillin and examined for mutability. Real-time RT-PCR and transcriptional profiling by DNA microarray were used to compare gene expression between both populations and related genetically modified SA13011 strain.<h4>Results</h4>We found that HeR/HoR selection by oxacillin was associated with increased mutation rate and oxacillin-mediated SOS response. We determined increased expression of both mecA and SOS response lexA/recA regulators. Mutational inactivation of lexA repressor resulted in a significant decrease in both mutation rate and oxacillin resistance in the HoR cells. Complementation of the lexA mutant strain restored oxacillin resistance to the high levels observed in the corresponding HoR wild-type strain.<h4>Conclusions</h4>The present results support the notion that SOS response is mechanistically involved in generating mutations that, in addition to mecA induction, allow the selection of a highly oxacillin-resistant population.
Project description:<i>Staphylococcus aureus</i> clinical strains are able to produce at least two distinct types of biofilm matrixes: biofilm matrixes made of the polysaccharide intercellular adhesin (PIA) or poly-<i>N</i>-acetylglucosamine (PNAG), whose synthesis is mediated by the <i>icaADBC</i> locus, and biofilm matrixes built of proteins (polysaccharide independent). ?<sup>B</sup> is a conserved alternative sigma factor that regulates the expression of more than 100 genes in response to changes in environmental conditions. While numerous studies agree that ?<sup>B</sup> is required for polysaccharide-independent biofilms, controversy persists over the role of ?<sup>B</sup> in the regulation of PIA/PNAG-dependent biofilm development. Here, we show that genetically unrelated <i>S. aureus</i> ?<sup>B</sup>-deficient strains produced stronger biofilms under both static and flow conditions and accumulated higher levels of PIA/PNAG exopolysaccharide than their corresponding wild-type strains. The increased accumulation of PIA/PNAG in the ?<sup>B</sup> mutants correlated with a greater accumulation of the IcaC protein showed that it was not due to adjustments in <i>icaADBC</i> operon transcription and/or <i>icaADBC</i> mRNA stability. Overall, our results reveal that in the presence of active ?<sup>B</sup>, the turnover of Ica proteins is accelerated, reducing the synthesis of PIA/PNAG exopolysaccharide and consequently the PIA/PNAG-dependent biofilm formation capacity.<b>IMPORTANCE</b> Due to its multifaceted lifestyle, <i>Staphylococcus aureus</i> needs a complex regulatory network to connect environmental signals with cellular physiology. One particular transcription factor, named ?<sup>B</sup> (SigB), is involved in the general stress response and the expression of virulence factors. For many years, great confusion has existed about the role of ?<sup>B</sup> in the regulation of the biofilm lifestyle in <i>S. aureus</i> Our study demonstrated that ?<sup>B</sup> is not necessary for exopolysaccharide-dependent biofilms and, even more, that <i>S. aureus</i> produces stronger biofilms in the absence of ?<sup>B</sup> The increased accumulation of exopolysaccharide correlates with higher stability of the proteins responsible for its synthesis. The present findings reveal an additional regulatory layer to control biofilm exopolysaccharide synthesis under stress conditions.
Project description:Staphylococcus epidermidis biofilm formation is associated with the production of the polysaccharide intercellular adhesin (PIA)--poly-N-acetylglucosamine polysaccharide (PNAG) by the products of the icaADBC operon. Recent evidence indicates that SarA, a central regulatory element that controls the production of Staphylococcus aureus virulence factors, is essential for the synthesis of PIA/PNAG and the ensuing biofilm development in this species. Based on the presence of a sarA homolog, we hypothesized that SarA could also be involved in the regulation of the biofilm formation process in S. epidermidis. To investigate this, we constructed nonpolar sarA deletions in two genetically unrelated S. epidermidis clinical strains, O-47 and CH845. The SarA mutants were completely defective in biofilm formation, both in the steady-state conditions of a microtiter dish assay and in the flow conditions of microfermentors. Reverse transcription-PCR experiments showed that the mutation in the sarA gene resulted in downregulation of the icaADBC operon transcription in an IcaR-independent manner. Purified SarA protein showed high-affinity binding to the icaA promoter region by electrophoretic mobility shift assays. Consequently, mutation in sarA provoked a significant decrease in the amount of PIA/PNAG on the cell surface. Furthermore, heterologous complementation of S. aureus sarA mutants with the sarA gene of S. epidermidis completely restored biofilm formation. In summary, SarA appeared to be a positive regulator of transcription of the ica locus, and in its absence, PIA/PNAG production and biofilm formation were diminished. Additionally, we present experimental evidence showing that SarA may be an important regulatory element that controls S. epidermidis virulence factors other than biofilm formation.
Project description:The formation of biofilms by Staphylococcus aureus and Staphylococcus epidermidis is an important aspect of many staphylococcal infections, most notably endocarditis, osteomyelitis and infections associated with indwelling medical devices. The major constituents of staphylococcal biofilms are polysaccharides, such as poly N-acetyl glucosamine (PIA/PNAG), cell surface and secreted bacterial proteins, and extracellular DNA. The exact composition of biofilms often varies considerably between different strains of staphylococci and between different sites of infection by the same strain. PIA/PNAG is synthesized by the products of four genes, icaADBC, that are encoded in a single operon. A fifth gene, icaR, is a negative regulator of icaADBC. Expression of icaADBC is tightly regulated, but can often be induced in vitro by growing staphylococci in the presence of high salt, high glucose, or ethanol. Regulation of icaADBC is complex and numerous regulatory factors have been implicated in control of icaADBC. Many of these are well known global transcriptional regulatory factors like SarA and sigmaB, whereas other regulators, such as IcaR, seem to affect expression of relatively few genes. Here, we will summarize how various regulatory factors affect the production of PIA/PNAG in staphylococci.
Project description:Hospital-associated methicillin-resistant Staphylococcus aureus (MRSA) strains typically express high-level, homogeneous (HoR) ?-lactam resistance, whereas community-associated MRSA (CA-MRSA) more commonly express low-level heterogeneous (HeR) resistance. Expression of the HoR phenotype typically requires both increased expression of the mecA gene, carried on the staphylococcal cassette chromosome mec element (SCCmec), and additional mutational event(s) elsewhere on the chromosome. Here the oxacillin concentration in a chemostat culture of the CA-MRSA strain USA300 was increased from 8 ?g/ml to 130 ?g/ml over 13 days to isolate highly oxacillin-resistant derivatives. A stable, small-colony variant, designated HoR34, which had become established in the chemostat culture was found to have acquired mutations in gdpP, clpX, guaA, and camS Closer inspection of the genome sequence data further revealed that reads covering SCCmec were ?10 times overrepresented compared to other parts of the chromosome. Quantitative PCR (qPCR) confirmed >10-fold-higher levels of mecA DNA on the HoR34 chromosome, and MinION genome sequencing verified the presence of 10 tandem repeats of the SCCmec element. qPCR further demonstrated that subculture of HoR34 in various concentrations of oxacillin (0 to 100 ?g/ml) was accompanied by accordion-like contraction and amplification of the SCCmec element. Although slower growing than strain USA300, HoR34 outcompeted the parent strain in the presence of subinhibitory oxacillin. These data identify tandem amplification of the SCCmec element as a new mechanism of high-level methicillin resistance in MRSA, which may provide a competitive advantage for MRSA under antibiotic selection.
Project description:Staphylococcus aureus can establish chronic infections on implanted medical devices due to its capacity to form biofilms. Analysis of the factors that assemble cells into a biofilm has revealed the occurrence of strains that produce either a polysaccharide intercellular adhesin/poly-N-acetylglucosamine (PIA/PNAG) exopolysaccharide- or a protein-dependent biofilm. Examination of the influence of matrix nature on the biofilm capacities of embedded bacteria has remained elusive, because a natural strain that readily converts between a polysaccharide- and a protein-based biofilm has not been studied. Here, we have investigated the clinical methicillin (meticillin)-resistant Staphylococcus aureus strain 132, which is able to alternate between a proteinaceous and an exopolysaccharidic biofilm matrix, depending on environmental conditions. Systematic disruption of each member of the LPXTG surface protein family identified fibronectin-binding proteins (FnBPs) as components of a proteinaceous biofilm formed in Trypticase soy broth-glucose, whereas a PIA/PNAG-dependent biofilm was produced under osmotic stress conditions. The induction of FnBP levels due to a spontaneous agr deficiency present in strain 132 and the activation of a LexA-dependent SOS response or FnBP overexpression from a multicopy plasmid enhanced biofilm development, suggesting a direct relationship between the FnBP levels and the strength of the multicellular phenotype. Scanning electron microscopy revealed that cells growing in the FnBP-mediated biofilm formed highly dense aggregates without any detectable extracellular matrix, whereas cells in a PIA/PNAG-dependent biofilm were embedded in an abundant extracellular material. Finally, studies of the contribution of each type of biofilm matrix to subcutaneous catheter colonization revealed that an FnBP mutant displayed a significantly lower capacity to develop biofilm on implanted catheters than the isogenic PIA/PNAG-deficient mutant.
Project description:<h4>Objectives</h4>We aimed to characterise the staphylococcal cassette chromosome mec (SCCmec) type, genetic relatedness, biofilm formation and composition, icaADBC genes detection, icaD expression, and antibiotic susceptibility of planktonic and biofilm cells of Staphylococcus hominis isolates from blood.<h4>Methods</h4>The study included 67 S. hominis blood isolates. Methicillin resistance was evaluated with the cefoxitin disk test. mecA gene and SCCmec were detected by multiplex PCR. Genetic relatedness was determined by pulsed-field gel electrophoresis. Biofilm formation and composition were evaluated by staining with crystal violet and by detachment assay, respectively; and the biofilm index (BI) was determined. Detection and expression of icaADBC genes were performed by multiplex PCR and real-time PCR, respectively. Antibiotic susceptibilities of planktonic cells (minimum inhibitory concentration, MIC) and biofilm cells (minimum biofilm eradication concentration, MBEC) were determined by the broth dilution method.<h4>Results</h4>Eighty-five percent (57/67) of isolates were methicillin resistant and mecA positive. Of the mecA-positive isolates, 66.7% (38/57) carried a new putative SCCmec type. Four clones were detected, with two to five isolates each. Among all isolates, 91% (61/67) were categorised as strong biofilm producers. Biofilm biomass composition was heterogeneous (polysaccharides, proteins and DNA). All isolates presented the icaD gene, and 6.66% (1/15) isolates expressed icaD. This isolate presented the five genes of ica operon. Higher BI and MBEC values than the MIC values were observed for amikacin, vancomycin, linezolid, oxacillin, ciprofloxacin, and chloramphenicol.<h4>Conclusions</h4>S. hominis isolates were highly resistant to methicillin and other antimicrobials. Most of the detected SCCmec types were different than those described for S. aureus. Isolates indicated low clonality. The results indicate that S. hominis is a strong biofilm producer with an extracellular matrix with similar composition of proteins, DNA and N-acetylglucosamine; and presents high frequency and low expression of icaD gene. Biofilm production is associated with increased antibiotic resistance.
Project description:Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as one of the most important pathogens both in health care and community-onset infections. The prerequisite for methicillin resistance is mecA, which encodes a ?-lactam-insensitive penicillin binding protein PBP2a. A characteristic of MRSA strains from hospital and community associated infections is their heterogeneous expression of resistance to ?-lactam (HeR) in which only a small portion (? 0.1%) of the population expresses resistance to oxacillin (OXA) ? 10 µg/ml, while in other isolates, most of the population expresses resistance to a high level (homotypic resistance, HoR). The mechanism associated with heterogeneous expression requires both increase expression of mecA and a mutational event that involved the triggering of a ?-lactam-mediated SOS response and related lexA and recA genes. In the present study we investigated the cellular physiology of HeR-MRSA strains during the process of ?-lactam-mediated HeR/HoR selection at sub-inhibitory concentrations by using a combinatorial approach of microarray analyses and global biochemical profiling employing gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) to investigate changes in metabolic pathways and the metabolome associated with ?-lactam-mediated HeR/HoR selection in clinically relevant heterogeneous MRSA. We found unique features present in the oxacillin-selected SA13011-HoR derivative when compared to the corresponding SA13011-HeR parental strain that included significant increases in tricarboxyl citric acid (TCA) cycle intermediates and a concomitant decrease in fermentative pathways. Inactivation of the TCA cycle enzyme cis-aconitase gene in the SA13011-HeR strain abolished ?-lactam-mediated HeR/HoR selection demonstrating the significance of altered TCA cycle activity during the HeR/HoR selection. These results provide evidence of both the metabolic cost and the adaptation that HeR-MRSA clinical strains undergo when exposed to ?-lactam pressure, indicating that the energy production is redirected to supply the cell wall synthesis/metabolism, which in turn contributes to the survival response in the presence of ?-lactam antibiotics.