Staphylococcus aureus, an important human pathogen, is particularly adept at producing biofilms on implanted medical devices. Although antibiotic treatment of nonsusceptible bacteria will not kill these strains, the consequences should be studied. The present study focuses on investigating the effect of vancomycin on biofilm formation by vancomycin-non-susceptible S. aureus. Biofilm adherence assays and scanning electron microscopy demonstrated that biofilm formation was significantly enhanced following vancomycin treatment. Bacterial autolysis of some subpopulations was observed and was confirmed by the live/dead staining and confocal laser scanning microscopy. A significant increase in polysaccharide intercellular adhesin (PIA) production was observed by measuring icaA transcript levels and in a semi-quantitative PIA assay in one resistant strain. We show that the release of extracellular DNA (eDNA) via cidA-mediated autolysis is a major contributor to vancomycin-enhanced biofilm formation. The addition of xenogeneic DNA could also significantly enhance biofilm formation by a PIA-overproducing S. aureus strain. The magnitude of the development of the biofilm depends on a balance between the amounts of eDNA and PIA. In conclusion, sublethal doses of cell wall-active antibiotics like vancomycin induce biofilm formation through an autolysis-dependent mechanism in vancomycin-non-susceptible S. aureus.

Staphylococcus aureus is a human pathogen that forms biofilm on catheters and medical implants. The authors' earlier study established that 1,2,3,4,6-penta- O -galloyl-β-D-glucopyranose (PGG) inhibits biofilm formation by S. aureus by preventing the initial attachment of the cells to a solid surface and reducing the production of polysaccharide intercellular adhesin (PIA). Our cDNA microarray and MALDI-TOF mass spectrometric studies demonstrate that PGG treatment causes the expression of genes and proteins that are normally expressed under iron-limiting conditions. A chemical assay using ferrozine verifies that PGG is a strong iron chelator that depletes iron from the culture medium. This study finds that adding FeSO 4 to a medium that contains PGG restores the biofilm formation and the production of PIA by S. aureus SA113. The requirement of iron for biofilm formation by S. aureus SA113 can also be verified using a semi-defined medium, BM, that contains an iron chelating agent, 2, 2′-dipyridyl (2-DP). Similar to the effect of PGG, the addition of 2-DP to BM medium inhibits biofilm formation and adding FeSO 4 to BM medium that contains 2-DP restores biofilm formation. This study reveals an important mechanism of biofilm formation by S. aureus SA113.

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Serratia marcescens cells swarm at 30°C but not at 37°C, and the underlying mechanism is not characterized. Our previous studies had shown that a temperature upshift from 30 to 37°C reduced the expression levels of flhDC Sm and hag Sm in S. marcescens CH-1. Mutation in rssA or rssB, cognate genes that comprise a twocomponent system, also resulted in precocious swarming phenotypes at 37°C. To further characterize the underlying mechanism, in the present study, we report that expression of flhDC Sm and synthesis of flagella are significantly increased in the rssA mutant strain at 37°C. Primer extension analysis for determination of the transcriptional start site(s) of flhDC Sm revealed two transcriptional start sites, P1 and P2, in S. marcescens CH-1. Characterization of the phosphorylated RssB (RssBϳP) binding site by an electrophoretic mobility shift assay showed direct interaction of RssBϳP, but not unphosphorylated RssB [RssB(D51E)], with the P2 promoter region. A DNase I footprinting assay using a capillary electrophoresis approach further determined that the RssBϳP binding site is located between base pair positions ؊341 and ؊364 from the translation start codon ATG in the flhDC Sm promoter region. The binding site overlaps with the P2 "؊35" promoter region. A modified chromatin immunoprecipitation assay was subsequently performed to confirm that RssBϳP binds to the flhDC Sm promoter region in vivo. In conclusion, our results indicated that activated RssA-RssB signaling directly inhibits flhDC Sm promoter activity at 37°C. This inhibitory effect was comparatively alleviated at 30°C. This finding might explain, at least in part, the phenomenon of inhibition of S. marcescens swarming at 37°C.Swarming is a bacterial population surface translocation behavior demonstrated in a wide range of diverse bacterial genera and species (2,12,14). In Serratia spp., swarming requires close interactions between the environment and the bacterial cells, as well as among the cells, in order to develop a high degree of complex cell coordination within the swarming colony (2,9,13,26,33,35). Previous studies on the regulation of swarming showed that bacterial flagellar, quorum-sensing, and two-component systems are important for swarming (3,7,33). Among these, flagellar motility, which is one of the essential factors for bacterial swarming, is controlled by the flagellar system, comprising large and complex regulons (4, 9). Studies with the flagellar systems of Escherichia coli and Salmonella enterica serovar Typhimurium have identified around 50 genes organized into three hierarchical transcriptional classes. At the top of the hierarchical cascade is the class I master operon flhDC (4). The FlhD 2 C 2 complex is a transcriptional activator of 70 -dependent transcription from class II promoters (4). Thus, activation of the whole set of flagellar motility genes depends mainly on the expression of flhDC.Serratia marcescens cells swarm at 30°C but not at 37°C (15).In a previous study utilizing a mini-Tn5 mutagenesis approach, we had ...

Staphylococcus aureus is an important pathogen that forms biofilms on the surfaces of medical implants. Biofilm formation by S. aureus is associated with the production of poly N-acetylglucosamine (PNAG), also referred to as polysaccharide intercellular adhesin (PIA), which mediates bacterial adhesion, leading to the accumulation of bacteria on solid surfaces. This study shows that the ability of S. aureus SA113 to adhere to nasal epithelial cells is reduced after the deletion of the ica operon, which contains genes encoding PIA/PNAG synthesis. However, this ability is restored after a plasmid carrying the entire ica operon is transformed into the mutant strain, S. aureus SA113Δica, showing that the synthesis of PIA/PNAG is important for adhesion to epithelial cells. Additionally, S. carnosus TM300, which does not produce PIA/PNAG, forms a biofilm and adheres to epithelial cells after the bacteria are transformed with a PIA/PNAG-expressing plasmid, pTXicaADBC. The adhesion of S. carnosus TM300 to epithelial cells is also demonstrated by adding purified exopolysaccharide (EPS), which contains PIA/PNAG, to the bacteria. In addition, using a mouse model, we find that the abscess lesions and bacterial burden in lung tissues is higher in mice infected with S. aureus SA113 than in those infected with the mutant strain, S. aureus SA113Δica. The results indicate that PIA/PNAG promotes the adhesion of S. aureus to human nasal epithelial cells and lung infections in a mouse model. This study elucidates a mechanism that is important to the pathogenesis of S. aureus infections.

The alternative transcription factor σB is responsible for transcription in Staphylococcus aureus during the stress response. Many virulence-associated genes are directly or indirectly regulated by σB. We hypothesized that treatment with antibiotics may act as an environmental stressor that induces σB activity in antibiotic-resistant strains. Several antibiotics with distinct modes of action, including ampicillin (12 µg/ml), vancomycin (16 or 32 µg/ml), chloramphenicol (15 µg/ml), ciprofloxacin (0.25 µg/ml), and sulfamethoxazole/trimethoprim (SXT, 0.8 µg/ml), were investigated for their ability to activate this transcription factor. We were especially interested in the stress response in vancomycin-resistant S. aureus (VRSA) strains treated with vancomycin. The transcription levels of selected genes associated with virulence were also measured. Real-time quantitative reverse transcription PCR was employed to evaluate gene transcription levels. Contact hemolytic and cytotoxicity assays were used to evaluate cell damage following antibiotic treatment. Antibiotics that target the cell wall (vancomycin and ampicillin) and SXT induced σB activity in VRSA strains. Expression of σB-regulated virulence genes, including hla and fnbA, was associated with the vancomycin-induced σB activity in VRSA strains and the increase in cytotoxicity upon vancomycin treatment. These effects were not observed in the sigB-deficient strain but were observed in the complemented strain. We demonstrate that sub-minimum inhibitory concentration (sub-MIC) levels of antibiotics act as environmental stressors and activate the stress response sigma factor, σB. The improper use of antibiotics may alter the expression of virulence factors through the activation of σB in drug-resistant strains of S. aureus and lead to worse clinical outcomes.

Staphylococcus aureus is a bacterial pathogen that produces and exports many virulence factors that cause diseases in humans. PrsA, a membrane-bound foldase, is expressed ubiquitously in Gram-positive bacteria and required for the folding of exported proteins into a stable and active structure. To understand the involvement of PrsA in posttranslocational protein folding in S. aureus, a PrsA-deficient mutant of S. aureus HG001 was constructed. Using isobaric tags for relative and absolute quantification (iTRAQ)-based mass spectrometry analyses, the exoproteomes of PrsA mutant and wild type S. aureus were comparatively profiled, and 163 cell wall-associated proteins and 67 exoproteins with altered levels have been identified in the PrsA-deficient mutant. Bioinformatics analyses further reveal that prsA deletion altered the amounts of proteins that are potentially involved in the regulation of cell surface properties and bacterial pathogenesis. To determine the relevancy of our findings, we investigated the functional consequence of prsA deletion in S. aureus. PrsA deficiency can enhance bacterial autoaggregation and increase the adhesion ability of S. aureus to human lung epithelial cells. Moreover, mice infected with PrsA-deficient S. aureus had a better survival rate compared with those infected with the wild-type S. aureus. Collectively, our findings reveal that PrsA is required for the posttranslocational folding of numerous exported proteins and critically affects the cell surface properties and pathogenesis of S. aureus.

OprD polymorphisms, particularly the F170L substitution and the specific shortening in loop 7, appear to determine the potential for P. aeruginosa to develop carbapenem resistance.