Mutational analyses of open reading frames within the vraSR operon and their roles in the cell wall stress response of Staphylococcus aureus.

The exposure of Staphylococcus aureus to a broad range of cell wall-damaging agents triggers the induction of a cell wall stress stimulon (CWSS) controlled by the VraSR two-component system. The vraSR genes form part of the four-cistron autoregulatory operon orf1-yvqF-vraS-vraR. The markerless inactivation of each of the genes within this operon revealed that orf1 played no observable role in CWSS induction and had no influence on resistance phenotypes for any of the cell envelope stress-inducing agents tested. The remaining three genes were all essential for the induction of the CWSS, and mutants showed various degrees of increased susceptibility to cell wall-active antibiotics. Therefore, the role of YvqF in S. aureus appears to be opposite that in other Gram-positive bacteria, where YvqF homologs have all been shown to inhibit signal transduction. This role, as an activator rather than repressor of signal transduction, corresponds well with resistance phenotypes of ΔYvqF mutants, which were similar to those of ΔVraR mutants in which CWSS induction also was completely abolished. Resistance profiles of ΔVraS mutants differed phenotypically from those of ΔYvqF and ΔVraR mutants on many non-ß-lactam antibiotics. ΔVraS mutants still became more susceptible than wild-type strains at low antibiotic concentrations, but they retained larger subpopulations that were able to grow on higher antibiotic concentrations than ΔYvqF and ΔVraR mutants. Subpopulations of ΔVraS mutants could grow on even higher glycopeptide concentrations than wild-type strains. The expression of a highly sensitive CWSS-luciferase reporter gene fusion was up to 2.6-fold higher in a ΔVraS than a ΔVraR mutant, which could be linked to differences in their respective antibiotic resistance phenotypes. Bacterial two-hybrid analysis indicated that the integral membrane protein YvqF interacted directly with VraS but not VraR, suggesting that it plays an essential role in sensing the as-yet unknown trigger of CWSS induction.

Transcriptional profiling of XdrA, a new regulator of spa transcription in Staphylococcus aureus.

Transcription of spa, encoding the virulence factor protein A in Staphylococcus aureus, is tightly controlled by a complex regulatory network, ensuring its temporal expression over growth and at appropriate stages of the infection process. Transcriptomic profiling of XdrA, a DNA-binding protein that is conserved in all S. aureus genomes and shares similarity with the XRE family of helix-turn-helix, antitoxin-like proteins, revealed it to be a previously unidentified activator of spa transcription. To assess how XdrA fits into the complex web of spa regulation, a series of regulatory mutants were constructed; consisting of single, double, triple, and quadruple mutants lacking XdrA and/or the three key regulators previously shown to influence spa transcription directly (SarS, SarA, and RNAIII). A series of lacZ reporter gene fusions containing nested deletions of the spa promoter identified regions influenced by XdrA and the other three regulators. XdrA had almost as strong an activating effect on spa as SarS and acted on the same spa operator regions as SarS, or closely overlapping regions. All data from microarrays, Northern and Western blot analyses, and reporter gene fusion experiments indicated that XdrA is a major activator of spa expression that appears to act directly on the spa promoter and not through previously characterized regulators.

Variability of SCCmec in the Zurich area.

A periodic survey of methicillin-resistant Staphylococcus aureus (MRSA) in Zurich in 2004 and 2006 revealed a consistently low prevalence of MRSA. SCCmec and ccr typing showed fluctuations in the proportions of SCCmec types and in the carriage of mobile virulence determinants. Together with the presence of variant SCCmecs these findings suggest a high clonal diversity and level of SCCmec recombination. The prevalence of a local "drug clone", associated with low-level methicillin resistance and rapid growth, significantly decreased. This clone had spread among intraveneous drug users, steadily increasing from 1994 to 2001 and was dominant in 2001. Apparently, changes in the management of the Zurich drug scene have restricted the spread of this clone.

Comparative genomics and DNA array-based genotyping of pandemic Staphylococcus aureus strains encoding Panton-Valentine leukocidin.

Within the last few years, methicillin-resistant Staphylococcus aureus (MRSA) strains encoding Panton-Valentine leukocidin (PVL) have emerged and spread worldwide. This epidemic can be attributed to a small number of distinct clones. The present study used a novel assay, based on multiplex linear DNA amplification and subsequent microarray hybridisation, to simultaneously detect all relevant exotoxins, antimicrobial resistance determinants and the allelic variants of agr. The genes of the staphylococcal exotoxin-like (set) locus were also included for typing purposes. This assay, together with multilocus sequence typing (MLST) and spa typing, was applied to 56 clinical isolates and reference strains representing all major pandemic PVL-MRSA lineages, as well as to phylogenetically-related strains and putative ancestors. Array hybridisation results allowed the assignment of isolates to clonal groups, which were in accordance with MLST and spa typing data. Ten distinct clonal groups of PVL-MRSA (ST1, ST5, ST8, ST22, ST30, ST59/359, ST80/583, ST88, ST93 and ST152), including 12 MLST types, were identified and analysed with regard to resistance determinants and genes coding for exotoxins. The array hybridisation data confirmed that pandemic PVL-positive strains originate from very diverse genetic backgrounds, and provided insights into the evolution of some lineages. The DNA microarray technique provides a valuable epidemiological tool for the detailed characterisation of clinical isolates and comparison of strains at a global level.

Mosaic staphylococcal cassette chromosome mec containing two recombinase loci and a new mec complex, B2.

A novel staphylococcal cassette chromosome (SCC) mec from a clinical methicillin-resistant Staphylococcus aureus isolate (ST100/CC5) had a mosaic structure, composed of SCC DNA from several different backgrounds. It harbored two complete ccr loci and a new variant of mec complex B, with DeltamecR1 interrupted by the aminoglycoside resistance transposon Tn4001.

Strain dependence of the cell wall-damage induced stimulon in Staphylococcus aureus.

The vancomycin stress induced transcriptome of the methicillin susceptible Staphylococcus aureus (MSSA) strain Newman was determined by microarray analysis. Subsets of the induced ORFs corresponded to those previously reported to be induced by vancomycin in the methicillin resistant S. aureus (MRSA) strains N315 and JH1, and/or by other cell wall active antibiotics in RN450; while other ORFs appeared to be induced strain specifically in Newman. Northern analyses showed that the induction pathway for several of the ORFs appeared to be altered in a number of clinical NARSA isolates. Induction was found to be dependent on inhibitory concentrations of antibiotics.

In vivo survival of teicoplanin-resistant Staphylococcus aureus and fitness cost of teicoplanin resistance.

Glycopeptide resistance, in a set of in vitro step-selected teicoplanin-resistant mutants derived from susceptible Staphylococcus aureus SA113, was associated with slower growth, thickening of the bacterial cell wall, increased N-acetylglucosamine incorporation, and decreased hemolysis. Differential transcriptome analysis showed that as resistance increased, some virulence-associated genes became downregulated. In a mouse tissue cage infection model, an inoculum of 10(4) CFU of strain SA113 rapidly produced a high-bacterial-load infection, which triggered MIP-2 release, leukocyte infiltration, and reduced leukocyte viability. In contrast, with the same inoculum of the isogenic glycopeptide-resistant derivative NM67, CFU initially decreased, resulting in the elimination of the mutant in three out of seven cages. In the four cages in which NM67 survived, it partially regained wild-type characteristics, including thinning of the cell wall, reduced N-acetylglucosamine uptake, and increased hemolysis; however, the survivors also became teicoplanin hypersusceptible. The elimination of the teicoplanin-resistant mutants and selection of teicoplanin-hypersusceptible survivors in the tissue cages indicated that glycopeptide resistance imposes a fitness burden on S. aureus and is selected against in vivo, with restoration of fitness incurring the price of resistance loss.

Rapidly destructive Staphylococcus epidermidis endocarditis.

A 29-year-old man with rapidly destructive Staphylococcus epidermidis endocarditis after mitral valve reconstruction is presented. Resistance to rifampin and teicoplanin occurred during antibiotic treatment resulting in clinical failure and valve destruction. Subsequently, the patient was successfully treated, by combining valve replacement with antibiotic therapy including quinupristin/dalfopristin, levofloxacin, and vancomycin. In conclusion, S. epidermidis can cause rapid valve destruction with large vegetations, and combination of surgery and antibiotic therapy may be necessary.

Improved methods for detection of methicillin-resistant Staphylococcus aureus.

In order to assess the performance of two detection methods, a set of 93 recent clinical isolates of Staphylococcus aureus, including a large number of strains that demonstrated low-level methicillin-resistance were evaluated using the MRSA-Screen (Denka Seiken, Japan), a commercial latex agglutination test to detect penicillin-binding protein 2' (PBP2'), and a polymerase chain reaction assay using the LightCycler Instrument (Roche Diagnostics, Switzerland). The results show that the latex agglutination test is highly sensitive if performed after induction by cefoxitin. Inconclusive results can be rapidly confirmed on the same day by real-time polymerase chain reaction used to detect mecA and femA genes.

Teicoplanin stress-selected mutations increasing sigma(B) activity in Staphylococcus aureus.

A natural rsbU mutant of Staphylococcus aureus, unable to activate the alternative transcription factor sigma(B) via the RsbU pathway and therefore forming unpigmented colonies, produced first-step teicoplanin-resistant mutants upon selection for growth in the presence of teicoplanin, of which the majority were of an intense orange color. By using an asp23 promoter-luciferase fusion as an indicator, the pigmented mutants were shown to express increased sigma(B) activity. Increased sigma(B) activity was associated with point mutations in rsbW, releasing sigma(B) from sequestration by the anti-sigma factor RsbW, or to promoter mutations increasing the sigma(B)/RsbW ratio. Genetic manipulations involving the sigB operon suggested that the mutations within the operon were associated with the increase in teicoplanin resistance. The upregulation of sigma(B) suggests that a sigma(B)-controlled gene(s) is directly or indirectly involved in the development of teicoplanin resistance in S. aureus. Carotenoids do not contribute to teicoplanin resistance, since inactivation of the dehydrosqualene synthase gene crtM abolished pigment formation without affecting teicoplanin resistance. The relevant sigma(B)-controlled target genes involved in teicoplanin resistance remain to be identified.

In vivo emergence of subpopulations expressing teicoplanin or vancomycin resistance phenotypes in a glycopeptide-susceptible, methicillin-resistant strain of Staphylococcus aureus.

Several reports indicate the emergence of subpopulations resistant to glycopeptides in some clinical isolates of Staphylococcus aureus. While the development of glycopeptide resistance in S. aureus is easily observed in vitro, the in vivo conditions promoting emergence of glycopeptide-resistant subpopulations are unknown. Using a rat model, subcutaneous implants were chronically infected with a methicillin-resistant strain of S. aureus, MRGR3, devoid of a significant (>10(-7)) glycopeptide-resistant subpopulation at 2 mg/L of either teicoplanin or vancomycin. After 3 weeks of infection in antibiotic-untreated animals, subpopulations emerged, growing on agar containing 10 mg/L of either glycopeptide. These subpopulations were detected in all tissue cage fluids containing >7 log cfu/mL at average frequencies of 4 x 10(-5) and 2 x 10(-5) on teicoplanin- and vancomycin-containing agar, respectively. While teicoplanin MICs increased two- to 16-fold, vancomycin MICs increased by less than two-fold. Population analysis and survival kinetic studies of three teicoplanin-selected subclones indicated that transfer from solid to liquid medium conditions decreased expression of teicoplanin resistance in the bacterial population. In Mueller-Hinton broth, >90% of cells remained fully resistant to antibiotic, but did not grow in the presence of teicoplanin for an initial period of at least 6 h. All three teicoplanin-resistant subclones expressed stable teicoplanin resistance with slight cross-resistance to vancomycin after a few transfers on teicoplanin-supplemented agar. These data suggest that some in vivo conditions may lead to selection of S. aureus subpopulations exhibiting decreased glycopeptide susceptibility and growing in the presence of otherwise inhibitory concentrations of these antimicrobial agents.

The AbcA transporter of Staphylococcus aureus affects cell autolysis.

Increased production of penicillin-binding protein PBP 4 is known to increase peptidoglycan cross-linking and contributes to methicillin resistance in Staphylococcus aureus. The pbp4 gene shares a 400-nucleotide intercistronic region with the divergently transcribed abcA gene, encoding an ATP-binding cassette transporter of unknown function. Our study revealed that methicillin stimulated abcA transcription but had no effects on pbp4 transcription. Analysis of abcA expression in mutants defective for global regulators showed that abcA is under the control of agr. Insertional inactivation of abcA by an erythromycin resistance determinant did not influence pbp4 transcription, nor did it alter resistance to methicillin and other cell wall-directed antibiotics. However, abcA mutants showed spontaneous partial lysis on plates containing subinhibitory concentrations of methicillin due to increased spontaneous autolysis. Since the autolytic zymograms of cell extracts were identical in mutants and parental strains, we postulate an indirect role of AbcA in control of autolytic activities and in protection of the cells against methicillin.

Involvement of multiple genetic loci in Staphylococcus aureus teicoplanin resistance.

Teicoplanin resistance was transformed from a teicoplanin-resistant Staphylococcus aureus into the susceptible strain BB255 to give strain BB938. The cell wall composition, amidation of the iD-glutamate, and peptide crosslinking were identical in BB938 as in BB255 except for a 60% increased length of the glycan chain. Transductional crosses revealed that at least two distinct loci contributed in a cumulative fashion to teicoplanin resistance. One of these loci correlated with a mutation inactivating the anti-sigma factor RsbW. This mutation must have occurred during transformation and selection for teicoplanin resistance in BB938. Genetic manipulations involving the sigB operon showed that transcription factor SigB contributed to decreased teicoplanin susceptibility.

Inactivation of a novel three-cistronic operon tcaR-tcaA-tcaB increases teicoplanin resistance in Staphylococcus aureus.

A novel teicoplanin-associated operon termed tcaR-tcaA-tcaB was identified by Tn917-mediated insertional mutagenesis. Resistance to teicoplanin rose 4-fold by insertional inactivation of tcaA or by deletion of the entire operon. tcaA encodes a hypothetical transmembrane protein with a metal-binding motif, possibly a sensor-transducer. tcaB codes for a membrane-associated protein, which has sequence homologies to a bicyclomycin resistance protein. The two genes are preceded by tcaR encoding a putative regulator with sequence homologies to the transcriptional regulator MarR. The fact that tcaA inactivation as well as deletion of tcaRAB produced the same increase in teicoplanin resistance confirmed the association of tcaRAB with teicoplanin susceptibility. Cotransductional crosses showed that the level of teicoplanin resistance produced by these insertions was strain-dependent and that in the methicillin-resistant strain COL, it was paired with a remarkable decrease in methicillin resistance. This allowed to postulate that tcaRAB may be involved in some way in cell wall biosynthesis, and that teicoplanin may interact with TcaA and/or TcaB either directly or indirectly.

Site-specific serine incorporation by Lif and Epr into positions 3 and 5 of the Staphylococcal peptidoglycan interpeptide bridge.

The FemAB-like factors Lif and Epr confer resistance to glycylglycine endopeptidases lysostaphin and Ale-1, respectively, by incorporating serine residues into the staphylococcal peptidoglycan interpeptide bridges specifically at positions 3 and 5. This required the presence of FemA and/or FemB, in contrast to earlier postulations.

False-positive beta-lactamase results with Staphylococcus lugdunensis in the Vitek AutoMicrobic system.

The Vitek AutoMicrobic system in combination with the Gram-positive susceptibility card detects beta-lactamase in staphylococci by utilizing penicillin as the substrate coupled with oxacillin as an inducer. The beta-lactamase activity of 21 clinical isolates and two reference strains of Staphylococcus lugdunensis was determined with this automated system and compared with a liquid nitrocefin assay after induction with oxacillin. Eight (38%) clinical isolates and the reference strain ATCC 49576 of S. lugdunensis showed production of beta-lactamase in both tests. Thirteen (62%) clinical isolates and the type strain ATCC 43809 were nitrocefin-negative. The Vitek AutoMicrobic system reported false-positive beta-lactamase results for 9 of those 13 isolates and for the type strain of S. lugdunensis. Results for disk diffusion (ampicillin) were concordant with the nitrocefin assay. With one exception, the MICs for penicillin of the nitrocefin-negative strains were in the equivocal range of 0.06-0.12 mg/l according to NCCLS. However, none of the nitrocefin-negative and Vitek-positive strains revealed any of the known staphylococcal genes for beta-lactamase as investigated by Southern hybridization, supporting the fact that false-positive beta-lactamase results may occur in the Vitek AutoMicrobic system. We conclude from our data that it may be justified to include S. lugdunensis in the quality control of Vitek cards containing beta-lactamase tests.

The essential Staphylococcus aureus gene fmhB is involved in the first step of peptidoglycan pentaglycine interpeptide formation.

The factor catalyzing the first step in the synthesis of the characteristic pentaglycine interpeptide in Staphylococcus aureus peptidoglycan was found to be encoded by the essential gene fmhB. We have analyzed murein composition and structure synthesized when fmhB expression is reduced. The endogenous fmhB promoter was substituted with the xylose regulon from Staphylococcus xylosus, which allowed glucose-controlled repression of fmhB transcription. Repression of fmhB reduced growth and triggered a drastic accumulation of uncrosslinked, unmodified muropeptide monomer precursors at the expense of the oligomeric fraction, leading to a substantial decrease in overall peptidoglycan crosslinking. The composition of the predominant muropeptide was confirmed by MS to be N-acetylglucosamine-(beta-1,4)-N-acetylmuramic acid(-L-Ala-D-iGln-L-Lys-D-Ala-D-Ala), proving that FmhB is involved in the attachment of the first glycine to the pentaglycine interpeptide. This interpeptide plays an important role in crosslinking and stability of the S. aureus cell wall, acts as an anchor for cell wall-associated proteins, determinants of pathogenicity, and is essential for the expression of methicillin resistance. Any shortening of the pentaglycine side chain reduces or even abolishes methicillin resistance, as occurred with fmhB repression. Because of its key role FmhB is a potential target for novel antibacterial agents that could control the threat of emerging multiresistant S. aureus.

Identification of three additional femAB-like open reading frames in Staphylococcus aureus.

Three new proteins, FmhA, FmhB and FmhC, with significant identities to FemA and FemB were identified in the Staphylococcus aureus (ATCC 55748) genome database. They were mapped to the SmaI-C, SmaI-H and SmaI-A fragments of the S. aureus 8325 chromosome, respectively. Whereas insertional inactivation of fmhA and fmhC had no effects on growth, antibiotic susceptibility, lysostaphin resistance, or peptidoglycan composition of the strains, fmhB could not be inactivated, strongly suggesting that fmhB may be an essential gene. As deduced from the functions of FemA and FemB which are involved in the synthesis of the peptidoglycan pentaglycine interpeptide, FmhB may be a candidate for the postulated FemX thought to add the first glycine to the nascent interpeptide.