Biofilm and the role of the ica operon and aap in Staphylococcus epidermidis isolates causing neurosurgical meningitis.

Fifty-five Staphylococcus epidermidis isolates, classified as contaminants or causing device-related meningitis, from external ventricular drain (EVD) and non-EVD cerebrospinal fluid specimens were characterized. Thirty-three of 42 (78.6%) meningitis isolates were PCR-positive for ica and aap, known determinants of polysaccharide- and protein-mediated biofilm production, whereas five of 13 (38.5%) contaminants were ica- and aap-negative; 71.4% of meningitis isolates and 84.6% of contaminants produced biofilm. ica+aap+ meningitis isolates produced more biofilm than ica+aap- isolates (p 0.0020). ica+aap- isolates did not produce more biofilm than ica-aap+ isolates (p 0.4368). Apparently, ica and aap are associated with biofilm production in S. epidermidis device-related meningitis isolates

Accumulation of Succinyl Coenzyme A Perturbs the Methicillin-Resistant Staphylococcus aureus (MRSA) Succinylome and Is Associated with Increased Susceptibility to Beta-Lactam Antibiotics

Penicillin binding protein 2a (PBP2a)-dependent resistance to β-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA) is regulated by the activity of the tricarboxylic acid (TCA) cycle via a poorly understood mechanism. We report that mutations in sucC and sucD, but not other TCA cycle enzymes, negatively impact β-lactam resistance without changing PBP2a expression. Increased intracellular levels of succinyl coenzyme A (succinyl-CoA) in the sucC mutant significantly perturbed lysine succinylation in the MRSA proteome. Suppressor mutations in sucA or sucB, responsible for succinyl-CoA biosynthesis, reversed sucC mutant phenotypes. The major autolysin (Atl) was the most succinylated protein in the proteome, and increased Atl succinylation in the sucC mutant was associated with loss of autolytic activity. Although PBP2a and PBP2 were also among the most succinylated proteins in the MRSA proteome, peptidoglycan architecture and cross-linking were unchanged in the sucC mutant. These data reveal that perturbation of the MRSA succinylome impacts two interconnected cell wall phenotypes, leading to repression of autolytic activity and increased susceptibility to β-lactam antibiotics. IMPORTANCE mecA-dependent methicillin resistance in MRSA is subject to regulation by numerous accessory factors involved in cell wall biosynthesis, nucleotide signaling, and central metabolism. Here, we report that mutations in the TCA cycle gene, sucC, increased susceptibility to β-lactam antibiotics and was accompanied by significant accumulation of succinyl-CoA, which in turn perturbed lysine succinylation in the proteome. Although cell wall structure and cross-linking were unchanged, significantly increased succinylation of the major autolysin Atl, which was the most succinylated protein in the proteome, was accompanied by near complete repression of autolytic activity. These findings link central metabolism and levels of succinyl-CoA to the regulation of β-lactam antibiotic resistance in MRSA through succinylome-mediated control of two interlinked cell wall phenotypes. Drug-mediated interference of the SucCD-controlled succinylome may help overcome β-lactam resistance

Oxygen-mediated regulation of biofilm development is controlled by the alternative sigma factor sigma(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.Science Foundation IrelandThe title of the submitted paper is different from the article\u27s. Copyright fine. JG suggests to amend the bibliographic details - AV 24/01/201

Fibronectin-binding proteins are required for biofilm formation by community-associated methicillin-resistantstaphylococcus aureusstrain lac

Community-associated methicillin-resistant Staphylococcus aureus of the USA300 lineage is emerging as an important cause of medical device-related infection. However, few factors required for biofilm accumulation by USA300 strains have been identified, and the processes involved are poorly understood. Here, we identify S.aureus proteins required for the USA300 isolate LAC to form biofilm. A mutant with a deletion of the fnbA and fnbB genes did not express the fibronectin-binding proteins FnBPA and FnBPB and lacked the ability to adhere to fibronectin or to form biofilm. Biofilm formation by the mutant LACfnbAfnbB could be restored by expression of FnBPA or FnBPB from a plasmid demonstrating that both of these proteins can mediate biofilm formation when expressed by LAC. Expression of FnBPA and FnBPB increased bacterial aggregation suggesting that fibronectin-binding proteins can promote the accumulation phase of biofilm. Loss of fibronectin-binding proteins reduced the initial adherence of bacteria, indicating that these proteins are also involved in primary attachment. In summary, these findings improve our understanding of biofilm formation by the USA300 strain LAC by demonstrating that the fibronectin-binding proteins are required

Methicillin resistance and the biofilm phenotype in staphylococcus aureus

Antibiotic resistance and biofilm-forming capacity contribute to the success of Staphylococcus aureus as a human pathogen in both healthcare and community settings. These virulence factors do not function independently of each other and the biofilm phenotype expressed by clinical isolates of S. aureus is influenced by acquisition of the methicillin resistance gene mecA. Methicillin-sensitive S. aureus (MSSA) strains commonly produce an icaADBC operon-encoded polysaccharide intercellular adhesin (PIA)-dependent biofilm. In contrast, the release of extracellular DNA (eDNA) and cell surface expression of a number of sortase-anchored proteins, and the major autolysin have been implicated in the biofilm phenotype of methicillin-resistant S. aureus (MRSA) isolates. Expression of high level methicillin resistance in a laboratory MSSA strain resulted in (i) repression of PIA-mediated biofilm production, (ii) down-regulation of the accessory gene regulator (Agr) system, and (iii) attenuation of virulence in murine sepsis and device infection models. Here we review the mechanisms of MSSA and MRSA biofilm production and the relationships between antibiotic resistance, biofilm and virulence gene regulation in S. aureus

The major autolysin is redundant forstaphylococcus aureususa300 lac je2 virulence in a murine device-related infection model

The major Staphylococcus aureus autolysin, Atl, has been implicated in attachment to surfaces and release of extracellular DNA during biofilm formation under laboratory conditions. Consistent with this, polyclonal antibodies to the amidase and glucosaminidase domains of Atl inhibited in vitro biofilm formation. However, in a murine model of device-related infection the community-associated S. aureus strain USA300 LAC JE2 established a successful infection in the absence of atl. These data indicate that Atl activity is not required for biofilm production in this infection model and reveal the importance of characterizing the contribution of biofilm phenotypes to virulence under in vivo conditions

Arac-type regulator rbf controls the staphylococcus epidermidis biofilm phenotype by negatively regulating theicaadbcrepressor sarr

Regulation of icaADBC-encoded polysaccharide intercellular adhesin (PIA)/poly-N-acetylglucosasmine (PNAG) production in staphylococci plays an important role in biofilm-associated medical-device-related infections. Here, we report that the AraC-type transcriptional regulator Rbf activates icaADBC operon transcription and PIA production in Staphylococcus epidermidis. Purified recombinant Rbf did not bind to the ica operon promoter region in electrophoretic mobility shift assays (EMSAs), indicating that Rbf regulates ica transcription indirectly. To identify the putative transcription factor(s) involved in Rbf-mediated icaADBC regulation, the ability of recombinant Rbf to interact with the promoter sequences of known icaADBC regulators was investigated. Recombinant Rbf bound to the sarR promoter and not the sarX, sarA, sarZ, spx, and srrA promoters. Reverse transcription (RT)-PCR demonstrated that Rbf acts as a repressor of sarR transcription. PIA expression and biofilm production were restored to wild-type levels in an rbf sarR double mutant grown in brain heart infusion (BHI) medium supplemented with NaCl, which is known to activate the ica locus, but not in BHI medium alone. RT-PCR further demonstrated that although Rbf does not bind the sarX promoter, it nevertheless exerted a negative effect on sarX expression. Apparently, direct downregulation of the SarR repressor by Rbf has a dominant effect over indirect repression of the SarX activator by Rbf in the control of S. epidermidis PIA production and biofilm formation. IMPORTANCE The importance of Staphylococcus epidermidis as an opportunistic pathogen in hospital patients with implanted medical devices derives largely from its capacity to form biofilm. Expression of the icaADBC-encoded extracellular polysaccharide is the pre-dominant biofilm mechanism in S. epidermidis clinical isolates and is tightly regulated. Here, we report that the transcriptional regulator Rbf promotes icaADBC expression by negatively regulating expression of sarR, which encodes an ica operon repressor. Furthermore, Rbf indirectly represses the ica operon activator, SarX. The data reveal complicated interplay between Rbf and two Sar family proteins in fine-tuning regulation of the biofilm phenotype and indicate that in the hierarchy of biofilm regulators, IcaR is dominant over the Rbf-SarR-SarX axis

Redeploying β-lactam antibiotics as a novel antivirulence strategy for the treatment of methicillin-resistantstaphylococcus aureusinfections

Innovative approaches to the use of existing antibiotics is an important strategy in efforts to address the escalating antimicrobial resistance crisis. We report a new approach to the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections by demonstrating that oxacillin can be used to significantly attenuate the virulence of MRSA despite the pathogen being resistant to this drug. Using mechanistic in vitro assays and in vivo models of invasive pneumonia and sepsis, we show that oxacillin-treated MRSA strains are significantly attenuated in virulence. This effect is based primarily on the oxacillin-dependent repression of the accessory gene regulator quorum-sensing system and altered cell wall architecture, which in turn lead to increased susceptibility to host killing of MRSA. Our data indicate that beta-lactam antibiotics should be included in the treatment regimen as an adjunct antivirulence therapy for patients with MRSA infections. This would represent an important change to current clinical practice for treatment of MRSA infection, with the potential to significantly improve patient outcomes in a safe, cost-effective manner

tandem amplification of the staphylococcal cassette chromosome mec element can drive high-level methicillin resistance in methicillin-resistant staphylococcus aureus

Hospital-associated methicillin-resistant Staphylococcus aureus (MRSA) strains typically express high-level, homogeneous (HoR) beta-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 mu g/ml to 130 mu 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 similar to 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 mu 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

Recombination-mediated remodelling of host–pathogen interactions during staphylococcus aureus niche adaptation

Large-scale recombination events have led to the emergence of epidemic clones of several major bacterial pathogens. However, the functional impact of the recombination on clonal success is not understood. Here, we identified a novel widespread hybrid clone (ST71) of livestock-associated Staphylococcus aureus that evolved from an ancestor belonging to the major bovine lineage CC97, through multiple large-scale recombination events with other S. aureus lineages occupying the same ruminant niche. The recombination events, affecting a 329 kb region of the chromosome spanning the origin of replication, resulted in allele replacement and loss or gain of an array of genes influencing host-pathogen interactions. Of note, molecular functional analyses revealed that the ST71 hybrid clone has acquired multiple novel pathogenic traits associated with acquired and innate immune evasion and bovine extracellular matrix adherence. These findings provide a paradigm for the impact of large-scale recombination events on the rapid evolution of bacterial pathogens within defined ecological niches