Extensive Homoplasy but No Evidence of Convergent Evolution of Repeat Numbers at MIRU Loci in Modern Mycobacterium tuberculosis Lineages.

More human deaths have been attributable to Mycobacterium tuberculosis than any other pathogen, and the epidemic is sustained by ongoing transmission. Various typing schemes have been developed to identify strain-specific differences and track transmission dynamics in affected communities, with recent introduction of whole genome sequencing providing the most accurate assessment. Mycobacterial interspersed repetitive unit (MIRU) typing is a family of variable number tandem repeat schemes that have been widely used to study the molecular epidemiology of M. tuberculosis. MIRU typing was used in most well-resourced settings to perform routine molecular epidemiology. Instances of MIRU homoplasy have been observed in comparison with sequence-based phylogenies, limiting its discriminatory value. A fundamental question is whether the observed homoplasy arises purely through stochastic processes, or whether there is evidence of natural selection. We compared repeat numbers at 24 MIRU loci with a whole genome sequence-based phylogeny of 245 isolates representing three modern M. tuberculosis lineages. This analysis demonstrated extensive homoplasy of repeat numbers, but did not detect any evidence of natural selection of repeat numbers, at least since the ancestral branching of the three modern lineages of M. tuberculosis. In addition, we observed good sensitivity but poor specificity and positive predictive values of MIRU-24 to detect clusters of recent transmission, as defined by whole-genome single nucleotide polymorphism analysis. These findings provide mechanistic insight, and support a transition away from VNTR-based typing toward sequence-based typing schemes for both research and public health purposes.

Whole Genome Sequencing Demonstrates Limited Transmission within Identified Mycobacterium tuberculosis Clusters in New South Wales, Australia.

Australia has a low tuberculosis incidence rate with most cases occurring among recent immigrants. Given suboptimal cluster resolution achieved with 24-locus mycobacterium interspersed repetitive unit (MIRU-24) genotyping, the added value of whole genome sequencing was explored. MIRU-24 profiles of all Mycobacterium tuberculosis culture-confirmed tuberculosis cases diagnosed between 2009 and 2013 in New South Wales (NSW), Australia, were examined and clusters identified. The relatedness of cases within the largest MIRU-24 clusters was assessed using whole genome sequencing and phylogenetic analyses. Of 1841 culture-confirmed TB cases, 91.9% (1692/1841) had complete demographic and genotyping data. East-African Indian (474; 28.0%) and Beijing (470; 27.8%) lineage strains predominated. The overall rate of MIRU-24 clustering was 20.1% (340/1692) and was highest among Beijing lineage strains (35.7%; 168/470). One Beijing and three East-African Indian (EAI) clonal complexes were responsible for the majority of observed clusters. Whole genome sequencing of the 4 largest clusters (30 isolates) demonstrated diverse single nucleotide polymorphisms (SNPs) within identified clusters. All sequenced EAI strains and 70% of Beijing lineage strains clustered by MIRU-24 typing demonstrated distinct SNP profiles. The superior resolution provided by whole genome sequencing demonstrated limited M. tuberculosis transmission within NSW, even within identified MIRU-24 clusters. Routine whole genome sequencing could provide valuable public health guidance in low burden settings.

Luciferase Reporter Gene System to Detect Cell Wall Stress Stimulon Induction in Staphylococcus aureus.

Luciferase reporter gene fusions provide an extremely rapid and sensitive tool for measuring the induction or repression of stress responses in bacteria. Staphylococcus aureus activates the expression of a cell wall stress stimulon (CWSS) in response to the inhibition or disruption of cell wall synthesis. The highly sensitive promoter-reporter gene fusion construct psas016 p-luc+ can be used to quantify and compare any changes in CWSS expression levels and induction kinetics. Potential uses of this system include identifying and characterizing novel cell wall-targeting antibacterial agents, identifying genomic loci influencing cell envelope synthesis and detecting changes in CWSS expression that could be linked to decreased antibiotic susceptibility profiles in clinical isolates.

Genomic Epidemiology of Clostridium botulinum Isolates from Temporally Related Cases of Infant Botulism in New South Wales, Australia.

Infant botulism is a potentially life-threatening paralytic disease that can be associated with prolonged morbidity if not rapidly diagnosed and treated. Four infants were diagnosed and treated for infant botulism in NSW, Australia, between May 2011 and August 2013. Despite the temporal relationship between the cases, there was no close geographical clustering or other epidemiological links. Clostridium botulinum isolates, three of which produced botulism neurotoxin serotype A (BoNT/A) and one BoNT serotype B (BoNT/B), were characterized using whole-genome sequencing (WGS). In silico multilocus sequence typing (MLST) found that two of the BoNT/A-producing isolates shared an identical novel sequence type, ST84. The other two isolates were single-locus variants of this sequence type (ST85 and ST86). All BoNT/A-producing isolates contained the same chromosomally integrated BoNT/A2 neurotoxin gene cluster. The BoNT/B-producing isolate carried a single plasmid-borne bont/B gene cluster, encoding BoNT subtype B6. Single nucleotide polymorphism (SNP)-based typing results corresponded well with MLST; however, the extra resolution provided by the whole-genome SNP comparisons showed that the isolates differed from each other by >3,500 SNPs. WGS analyses indicated that the four infant botulism cases were caused by genomically distinct strains of C. botulinum that were unlikely to have originated from a common environmental source. The isolates did, however, cluster together, compared with international isolates, suggesting that C. botulinum from environmental reservoirs throughout NSW have descended from a common ancestor. Analyses showed that the high resolution of WGS provided important phylogenetic information that would not be captured by standard seven-loci MLST.

Temporal dynamics of Mycobacterium tuberculosis genotypes in New South Wales, Australia.

BACKGROUND: Molecular epidemiology of Mycobacterium tuberculosis, its transmission dynamics and population structure have become important determinants of targeted tuberculosis control programs. Here we describe recent changes in the distribution of M. tuberculosis genotypes in New South Wales (NSW), Australia and compared strain types with drug resistance, site of disease and demographic data. METHODS: We evaluated all culture-confirmed newly identified tuberculosis cases in NSW, Australia, from 2010-2012. M. tuberculosis population structure and clustering rates were assessed using 24-loci Mycobacterial interspersed repetitive unit (MIRU) analysis and compared to MIRU data from 2006-2008. RESULTS: Of 1177 tuberculosis cases, 1128 (95.8%) were successfully typed. Beijing and East African Indian (EAI) lineage strains were most common (27.6% and 28.5%, respectively) with EAI strains increasing in relative abundance from 11.8% in 2006-2008 to 28.5% in 2010-2012. Few cases of multi-drug resistant tuberculosis were identified (18; 1.7%). Compared to 12-loci, 24-loci MIRU provided improved cluster resolution with 695 (61.6%) and 227 (20.1%) clustered cases identified, respectively. Detailed analysis of the largest cluster identified (an 11 member Beijing cluster) revealed wide geographic diversity in the absence of documented social contact. CONCLUSIONS: EAI strains of M. tuberculosis recently overtook Beijing family as a prevalent cause of tuberculosis in NSW, Australia. This lineage appeared to be less commonly related to multi-drug resistant tuberculosis as compared to Beijing strain lineage. The resolution provided by 24-loci MIRU typing was insufficient for reliable assessment of transmissions, especially of Beijing family strains.

Mutation in the C-di-AMP cyclase dacA affects fitness and resistance of methicillin resistant Staphylococcus aureus.

Faster growing and more virulent strains of methicillin resistant Staphylococcus aureus (MRSA) are increasingly displacing highly resistant MRSA. Elevated fitness in these MRSA is often accompanied by decreased and heterogeneous levels of methicillin resistance; however, the mechanisms for this phenomenon are not yet fully understood. Whole genome sequencing was used to investigate the genetic basis of this apparent correlation, in an isogenic MRSA strain pair that differed in methicillin resistance levels and fitness, with respect to growth rate. Sequencing revealed only one single nucleotide polymorphism (SNP) in the diadenylate cyclase gene dacA in the faster growing but less resistant strain. Diadenylate cyclases were recently discovered to synthesize the new second messenger cyclic diadenosine monophosphate (c-di-AMP). Introduction of this mutation into the highly resistant but slower growing strain reduced resistance and increased its growth rate, suggesting a direct connection between the dacA mutation and the phenotypic differences of these strains. Quantification of cellular c-di-AMP revealed that the dacA mutation decreased c-di-AMP levels resulting in reduced autolysis, increased salt tolerance and a reduction in the basal expression of the cell wall stress stimulon. These results indicate that c-di-AMP affects cell envelope-related signalling in S. aureus. The influence of c-di-AMP on growth rate and methicillin resistance in MRSA indicate that altering c-di-AMP levels could be a mechanism by which MRSA strains can increase their fitness levels by reducing their methicillin resistance levels.

Deletion of hypothetical wall teichoic acid ligases in Staphylococcus aureus activates the cell wall stress response.

The Staphylococcus aureus cell wall stress stimulon (CWSS) is activated by cell envelope-targeting antibiotics or depletion of essential cell wall biosynthesis enzymes. The functionally uncharacterized S. aureus LytR-CpsA-Psr (LCP) proteins, MsrR, SA0908 and SA2103, all belong to the CWSS. Although not essential, deletion of all three LCP proteins severely impairs cell division. We show here that VraSR-dependent CWSS expression was up to 250-fold higher in single, double and triple LCP mutants than in wild type S. aureus in the absence of external stress. The LCP triple mutant was virtually depleted of wall teichoic acids (WTA), which could be restored to different degrees by any of the single LCP proteins. Subinhibitory concentrations of tunicamycin, which inhibits the first WTA synthesis enzyme TarO (TagO), could partially complement the severe growth defect of the LCP triple mutant. Both of the latter findings support a role for S. aureus LCP proteins in late WTA synthesis, as in Bacillus subtilis where LCP proteins were recently proposed to transfer WTA from lipid carriers to the cell wall peptidoglycan. Intrinsic activation of the CWSS upon LCP deletion and the fact that LCP proteins were essential for WTA-loading of the cell wall, highlight their important role(s) in S. aureus cell envelope biogenesis.

LytR-CpsA-Psr proteins in Staphylococcus aureus display partial functional redundancy and the deletion of all three severely impairs septum placement and cell separation.

Staphylococcus aureus contains three members of the LytR-CpsA-Psr (LCP) family of membrane proteins: MsrR, SA0908 and SA2103. The characterization of single-, double- and triple-deletion mutants revealed distinct phenotypes for each of the three proteins. MsrR was involved in cell separation and septum formation and influenced ß-lactam resistance; SA0908 protected cells from autolysis; and SA2103, although displaying no apparent phenotype by itself, enhanced the properties of msrR and sa0908 mutants when deleted. The deletion of sa0908 and sa2103 also further attenuated the virulence of msrR mutants in a nematode-killing assay. The severely defective growth phenotype of the triple mutant revealed that LytR-CpsA-Psr proteins are essential for optimal cell division in S. aureus. Growth could be rescued to varying degrees by any one of the three proteins, indicating some functional redundancy within members of this protein family. However, differing phenotypic characteristics of all single and double mutants and complemented triple mutants indicated that each protein played a distinct role(s) and contributed differently to phenotypes influencing cell separation, autolysis, cell surface properties and virulence.

Induction kinetics of the Staphylococcus aureus cell wall stress stimulon in response to different cell wall active antibiotics.

BACKGROUND: Staphylococcus aureus activates a protective cell wall stress stimulon (CWSS) in response to the inhibition of cell wall synthesis or cell envelope damage caused by several structurally and functionally different antibiotics. CWSS induction is coordinated by the VraSR two-component system, which senses an unknown signal triggered by diverse cell wall active agents. RESULTS: We have constructed a highly sensitive luciferase reporter gene system, using the promoter of sas016 (S. aureus N315), which detects very subtle differences in expression as well as measuring > 4 log-fold changes in CWSS activity, to compare the concentration dependence of CWSS induction kinetics of antibiotics with different cell envelope targets. We compared the effects of subinhibitory up to suprainhibitory concentrations of fosfomycin, D-cycloserine, tunicamycin, bacitracin, flavomycin, vancomycin, teicoplanin, oxacillin, lysostaphin and daptomycin. Induction kinetics were both strongly antibiotic- and concentration-dependent. Most antibiotics triggered an immediate response with induction beginning within 10 min, except for tunicamycin, D-cycloserine and fosfomycin which showed lags of up to one generation before induction commenced. Induction characteristics, such as the rate of CWSS induction once initiated and maximal induction reached, were strongly antibiotic dependent. We observed a clear correlation between the inhibitory effects of specific antibiotic concentrations on growth and corresponding increases in CWSS induction kinetics. Inactivation of VraR increased susceptibility to the antibiotics tested from 2- to 16-fold, with the exceptions of oxacillin and D-cycloserine, where no differences were detected in the methicillin susceptible S. aureus strain background analysed. There was no apparent correlation between the induction capacity of the various antibiotics and the relative importance of the CWSS for the corresponding resistance phenotypes. CONCLUSION: CWSS induction profiles were unique for each antibiotic. Differences observed in optimal induction conditions for specific antibiotics should be determined and taken into account when designing and interpreting CWSS induction studies.

GeneXpert captures unstable methicillin-resistant Staphylococcus aureus prone to rapidly losing the mecA gene.

A cefoxitin-susceptible Staphylococcus aureus strain was identified by the Cepheid GeneXpert as methicillin-resistant S. aureus (MRSA). This strain was highly unstable and rapidly lost SCCmec upon subculturing in vitro, indicating that unstable MRSA is best detected by gene amplification-based methods.

Regulation of antibiotic resistance in Staphylococcus aureus.

Staphylococcus aureus has a formidable ability to adapt to varying environmental conditions and an extraordinary capacity to rapidly become resistant to virtually all antibiotics. Resistance develops either through mutations and rearrangements within the staphylococcal genome, or by the acquisition of resistance determinants. Antibiotic resistances often impose a fitness burden on the host. Such biological costs can be reduced by tight regulation and antibiotic-inducible expression of resistance genes, or by compensatory mutations. Resistance induction by antibiotics can be mediated by dedicated, antibiotic-recognizing signal transducers or by mechanisms relieving translational attenuation. Antibiotic tolerance and the expression of resistance phenotypes can also be strongly influenced by the genetic backgrounds of strains and several other factors. Modification and indirect regulation of resistance levels can occur by mutations that alter gene expression or substrate specificity of genes contributing to resistance. Insertion elements can alter resistance profiles by turning relevant genes on or off. Environmental conditions and stress response mechanisms triggered by perturbation of the cell envelope, DNA damage, or faulty intermediary metabolism can also have an impact on resistance development and expression. Clinically relevant resistance is often built up through multiple steps, each of which contributes to an increase in resistance. The driving force behind resistance formation is antibiotic stress, and under clinical conditions selection for resistance is continuously competing with selection for bacterial fitness.

MsrR contributes to cell surface characteristics and virulence in Staphylococcus aureus.

MsrR, a factor contributing to methicillin resistance in Staphylococcus aureus, belongs to the LytR-CpsA-Psr family of cell envelope-associated proteins. Deletion of msrR increased cell size and aggregation, and altered envelope properties, leading to a temporary reduction in cell surface hydrophobicity, diminished colony-spreading ability, and an increased susceptibility to Congo red. The reduced phosphorus content of purified cell walls of the msrR mutant suggested a reduction in wall teichoic acids, which may explain some of the observed phenotypes. Microarray analysis of the msrR deletion mutant revealed only minor changes in the global transcriptome, suggesting that MsrR has structural rather than regulatory functions. Importantly, virulence of the msrR mutant was decreased in a nematode-killing assay as well as in rat experimental endocarditis. MsrR is therefore likely to play a role in cell envelope maintenance, cell separation, and pathogenicity of S. aureus.

A novel DNA-binding protein modulating methicillin resistance in Staphylococcus aureus.

BACKGROUND: Methicillin resistance in Staphylococcus aureus is conferred by the mecA-encoded penicillin-binding protein PBP2a. Additional genomic factors are also known to influence resistance levels in strain specific ways, although little is known about their contribution to resistance phenotypes in clinical isolates. Here we searched for novel proteins binding to the mec operator, in an attempt to identify new factor(s) controlling methicillin resistance phenotypes. RESULTS: Analysis of proteins binding to a DNA fragment containing the mec operator region identified a novel, putative helix-turn-helix DNA-binding protein, SA1665. Nonpolar deletion of SA1665, in heterogeneously methicillin resistant S. aureus (MRSA) of different genetic backgrounds, increased methicillin resistance levels in a strain dependent manner. This phenotype could be fully complemented by reintroducing SA1665 in trans. Northern and Western blot analyses, however, revealed that SA1665 had no visible influence on mecA transcription or amounts of PBP2a produced. CONCLUSION: SA1665 is a new chromosomal factor which influences methicillin resistance in MRSA. Although SA1665 bound to the mecA promoter region, it had no apparent influence on mecA transcription or translation, suggesting that this predicted DNA-binding protein modulates resistance indirectly, most likely through the control of other genomic factors which contribute to resistance.

Impact of mecA promoter mutations on mecA expression and beta-lactam resistance levels.

The reason for the extremely low-level oxacillin resistance in a so-called 'drug clone', a methicillin-resistant Staphylococcus aureus circulating among injection drug users in Zurich, Switzerland, could be traced back to the mecA promoter sequence and particularly to the strain's genetic background. Sequencing of its mec complex identified a point mutation (TATACT to TATATT), creating a perfect palindrome in the -10 region of the mecA promoter/operator region containing the binding sites for the mecA repressors MecI and BlaI. Two strains with vastly different beta-lactam resistance phenotypes, the low-level resistant drug clone type strain CHE482 and the highly homogeneously resistant strain COLn, were cured of their SCCmec elements and subsequently transformed with plasmids containing mecA under the control of either the wild-type or mutant promoter. Expression studies showed that this mutation had significant effects on both mecA transcription and corresponding PBP2a production, but only small effects on beta-lactam resistance levels within a given genetic background. A further mutation in the mecA ribosomal binding site (GGAGG to GGAGT), common to SCCmec type IV strains, was found to have no discernable effect on mecA transcription and PBP2a content, and only minimal effects on beta-lactam resistance. Factors associated with the genetic backgrounds into which these differently controlled mecA genes were introduced had a much higher impact on beta-lactam resistance levels than the rates of mecA transcription. The tight repression of mecA expression in this drug clone in the absence of beta-lactams could contribute to the apparent fitness of this fast growing strain.

Functional characterization of TcaA: minimal requirement for teicoplanin susceptibility and role in Caenorhabditis elegans virulence.

The inactivation of TcaA contributes to intrinsic teicoplanin resistance in experimental and clinical isolates of glycopeptide-intermediate resistant Staphylococcus aureus. PhoA fusions confirmed that TcaA is a transmembrane protein with a short intracellular N-terminal domain containing a C-4 zinc finger binding motif, a single membrane-spanning domain, and a large extracellular C-terminal domain. The region conferring teicoplanin susceptibility was narrowed down to the transmembrane part and the first third of the extracellular domain of TcaA, suggesting that neither the C-4 zinc finger binding motif nor the C terminus contributed to teicoplanin susceptibility. TcaA belongs to the cell wall stress stimulon, which comprises a set of genes universally upregulated by cell wall damage. Induction of tcaA was shown to be fully dependent on the two-component regulatory system VraSR. A 66-bp region upstream of the transcriptional start site, which contained an inverted repeat partially covering the promoter box, was shown to be essential for VraSR-mediated induction by cell wall stress. Interestingly, the induction or overexpression of tcaA did not contribute further to teicoplanin susceptibility, suggesting that small amounts of TcaA, such as those present under normal uninduced conditions, were sufficient for TcaA-mediated teicoplanin susceptibility. The strong attenuation of tcaA deletion mutants in a Caenorhabditis elegans survival assay suggested that TcaA may, in addition to affecting glycopeptide susceptibility, also play a role in virulence.

Variability in SCCmecN1 spreading among injection drug users in Zurich, Switzerland.

BACKGROUND: An extremely low level methicillin resistant Staphylococcus aureus (MRSA) belonging to ST45, circulates among intravenous drug users in the Zurich area. This clone can be misinterpreted as an MSSA by phenotypic oxacillin resistance tests, although it carries a staphylococcal cassette chromosome mec (SCCmec) element encoding a functional mecA gene and it produces PBP2a. RESULTS: This clone carried a new 45.7-kb element, termed SCCmecN1, containing a class B mec complex (mecA-DeltamecR1::IS1272), a truncated Tn4003 harbouring the dfrA gene, and a fusB1 gene, conferring methicillin, trimethoprim and low level fusidic acid resistance, respectively. In addition to the two insertion site sequences (ISS) framing the SCCmec, a third ISS (ISS*) was identified within the element. SCCmecN1 also harboured two distinct ccrAB complexes belonging to the class 4 subtype, both of which were shown to be active and to be able to excise the SCCmecN1 or parts thereof. Slight variations in the SmaI-PFGE pattern of the clinical MRSA isolates belonging to this clone were traced back to differences in the sizes of the SCCmec J2 regions and/or to a 6.4-kb deletion extending from ISS* to the right end ISS. This latter deletion led to a variant right SCCmec-chromosomal junction site. MRSA clones carrying the shorter SCCmec with the 6.4-kb deletion were usually ciprofloxacin resistant, while strains with the complete SCCmecN1 were co-trimoxazole resistant or had no additional resistances. This suggested that the genetic backbone of the host S. aureus, although identical by PFGE pattern, had at some stage diverged with one branch acquiring a sulfonomide resistance mutation and the other ciprofloxacin resistance. CONCLUSION: This description of the structure and variations of SCCmecN1 will allow for quicker and easier molecular detection of this clone and monitoring of its spread.

State of the knowledge of bacterial resistance.

Bacteria have adapted a variety of different ways to acquire antibiotic resistance, fostering the rapid development of resistance within a short evolutionary time. The general genetic basis of events leading to and promoting antibiotic resistance formation in bacteria are presented and exemplified by showing the evolution of methicillin, glycopeptide, linezolid, and ketolide resistance in Staphylococcus aureus.

Molecular epidemiology of methicillin-resistant Staphylococcus aureus in Zurich, Switzerland (2003): prevalence of type IV SCCmec and a new SCCmec element associated with isolates from intravenous drug users.

The majority of methicillin-resistant Staphylococcus aureus (MRSA) isolates, recovered in 2003 at the Department of Medical Microbiology in Zürich, Switzerland, belonged to major clones that are circulating worldwide. Staphylococcal cassette chromosome mec type IV (SCCmec-IV), harbored by half of the isolates, was found in sequence type 217 (ST 217), which is an allelic variant of epidemic MRSA-15 (designated EMRSA-15), in a new local ST 617 descending from clonal complex CC 8 and in low-level oxacillin-resistant strains of multiple genetic lineages characteristic of community-onset MRSA. SCCmec-I, SCCmec-II, and SCCmec-III were in the minority, and four MRSA isolates had complex, rearranged SCCmec elements. A novel SCCmec-N1 of approximately 30 kb, associated with a dfrA gene and a ccr 4-related recombinase complex, was identified in a large number of low-level oxacillin-resistant isolates, which descended from the successful clonal complex CC 45 and are spreading among intraveneous drug users. In contrast, the SCCmec types of oxacillin-resistant coagulase-negative staphylococci (MRCNS) were of completely different composition. SCCmec type I (SCCmec-I) and SCCmec-II were more frequent than in the MRSA, while fewer contained SCCmec-IV. The other MRCNS displayed 11 different, complex patterns, suggesting frequent recombination between different SCCmec elements. With one ccr-negative exception, these strains amplified between one and three different ccr products, indicating either new varied complexes or multiple ccr loci. This suggests the presence of novel SCCmec types in MRCNS and no extensive interspecies SCCmec transfer between MRSA and MRCNS.

Reduced content of lysyl-phosphatidylglycerol in the cytoplasmic membrane affects susceptibility to moenomycin, as well as vancomycin, gentamicin, and antimicrobial peptides, in Staphylococcus aureus.

An association between moenomycin resistance and vancomycin intermediate resistance in Staphylococcus aureus was demonstrated previously. Thus, to elucidate the mechanism of vancomycin intermediate resistance, we searched for factors contributing to moenomycin resistance. Random Tn551 insertional mutagenesis of methicillin-resistant S. aureus strain COL yielded three mutants with decreased susceptibilities to moenomycin. Correspondingly, these mutants also exhibited slightly decreased susceptibilities to vancomycin. Genetic analysis revealed that two of the mutants had Tn551 insertions in the fmtC (mprF) gene, which is associated with the synthesis of lysyl-phosphatidylglycerol. The third Tn551 insertion was located in the lysC gene, which is involved in the biosynthesis of lysine from aspartic acid. Consequently, mutations in both of these loci reduced the lysyl-phosphatidylglycerol content in the cell membrane, giving it a more negative net charge. The positively charged antibiotic gentamicin and cationic antimicrobial peptides such as beta-defensins and CAP18 were more effective against the mutants. The levels of moenomycin and vancomycin binding to intact cells was also greater in the mutants than in the wild type, while the binding affinity was not altered when cells boiled in sodium dodecyl sulfate were used, indicating that both agents had higher affinities for the negatively charged membranes of the mutants. Therefore, the membrane charge of S. aureus appears to influence the efficacies of moenomycin, vancomycin, and other cationic antimicrobial agents.

The gate controlling cell wall synthesis in Staphylococcus aureus.

Glucosamine-6-P occupies a central position between cell wall synthesis and glycolysis. In the initial steps leading to peptidoglycan precursor formation glucosamine-6-P is processed sequentially to UDP-N-acetylglucosamine, while to enter the glycolysis pathway, glucosamine-6-P is isomerized by NagB to fructose-6-P. Although we could not demonstrate NagB activity, nagB inactivation significantly reduced growth. Mutational analysis showed that NagA was involved in glucosamine-6-P formation from N-acetylglucosamine-6-P, and GlmS in that from fructose-6-P. Inactivation of glmS prevented growth on glucose as sole carbon source, which resumed after complementation with N-acetylglucosamine. Transcription of glmS as well as the amount of GlmS was reduced in the presence of N-acetylglucosamine. This and the preferential incorporation of N-acetylglucosamine over glucose into cell wall material showed that N-acetylglucosamine was used exclusively for cell wall synthesis, while glucose served both cell wall synthesis and glycolysis. These observations suggest furthermore GlmS to be the key and only enzyme leading from glucose to cell wall synthesis in Staphylococcus aureus, and show that there exists a tight regulation and hierarchy in sugar utilization. Inactivation of nagA, nagB or glmS affected the susceptibility of S. aureus to cell wall synthesis inhibitors, suggesting an interdependence between efficiency of cell wall precursor formation and resistance levels.