Shang Wei Wu

Tracking the in vivo evolution of multidrug resistance in Staphylococcus aureus by whole-genome sequencing

The spread of multidrug-resistant Staphylococcus aureus (MRSA) strains in the clinical environment has begun to pose serious limits to treatment options. Yet virtually nothing is known about how resistance traits are acquired in vivo. Here, we apply the power of whole-genome sequencing to identify steps in the evolution of multidrug resistance in isogenic S. aureus isolates recovered periodically from the bloodstream of a patient undergoing chemotherapy with vancomycin and other antibiotics. After extensive therapy, the bacterium developed resistance, and treatment failed. Sequencing the first vancomycin susceptible isolate and the last vancomycin nonsusceptible isolate identified genome wide only 35 point mutations in 31 loci. These mutations appeared in a sequential order in isolates that were recovered at intermittent times during chemotherapy in parallel with increasing levels of resistance. The vancomycin nonsusceptible isolates also showed a 100-fold decrease in susceptibility to daptomycin, although this antibiotic was not used in the therapy. One of the mutated loci associated with decreasing vancomycin susceptibility (the vraR operon) was found to also carry mutations in six additional vancomycin nonsusceptible S. aureus isolates belonging to different genetic backgrounds and recovered from different geographic sites. As costs drop, whole-genome sequencing will become a useful tool in elucidating complex pathways of in vivo evolution in bacterial pathogens.

Overexpression of Genes of the Cell Wall Stimulon in Clinical Isolates of Staphylococcus aureus Exhibiting Vancomycin-Intermediate- S. aureus-Type Resistance to Vancomycin

Custom-designed gene chips (Affymetrix) were used to determine genetic relatedness and gene expression profiles in Staphylococcus aureus isolates with increasing MICs of vancomycin that were recovered over a period of several weeks from the blood and heart valve of a patient undergoing extensive vancomycin therapy. The isolates were found to be isogenic as determined by the GeneChip based genotyping approach and thus represented a unique opportunity to study changes in gene expression that may contribute to the vancomycin resistance phenotype. No differences in gene expression were detected between the parent strain, JH1, and JH15, isolated from the nares of a patient contact. Few expression changes were observed between blood and heart valve isolates with identical vancomycin MICs. A large number of genes had altered expression in the late stage JH9 isolate (MIC = 8 microg/ml) compared to JH1 (MIC = 1 microg/ml). Most genes with altered expression were involved in housekeeping functions or cell wall biosynthesis and regulation. The sortase-encoding genes, srtA and srtB, as well as several surface protein-encoding genes were downregulated in JH9. Two hypothetical protein-encoding genes, SAS016 and SA2343, were dramatically overexpressed in JH9. Interestingly, 27 of the genes with altered expression in JH9 grown in drug-free medium were found to be also overexpressed when the parental strain JH1 was briefly exposed to inhibitory concentrations of vancomycin, and more than half (17 of 27) of the genes with altered expression belonged to determinants that were proposed to form part of a general cell wall stress stimulon (S. Utaida et al., Microbiology 149:2719-2732, 2003).

Development of Methicillin Resistance in Clinical Isolates of Staphylococcus sciuri by Transcriptional Activation of the mecA Homologue Native to the Species

The β-lactam resistance gene mecA was acquired by Staphylococcus aureus from an extraspecies source. The search for the possible origin of this gene has led to the identification of a close structural homologue of mecA as a native gene in the animal species Staphylococcus sciuri. Surprisingly, the overwhelming majority of S. sciuri isolates were fully susceptible to β-lactam antibiotics in spite of the ubiquitous presence of the mecA homologue in the bacteria. We now describe two unusual S. sciuri strains isolated from humans—SS-37 and SS-41—that showed resistance to methicillin associated with high rates of transcription of the mecA homologue and production of a protein resembling penicillin binding protein 2a, the gene product of S. aureus mecA. In strain SS-37 increased transcription of the mecA homologue was related to insertion of an IS256 element upstream of the structural gene, and strain SS-41 had single nucleotide alterations in the promoter region of the mecA homologue which appear to be related to up-regulation of the rate of transcription. A third methicillin-resistant human isolate of S. sciuri that carries both the native mecA homologue and a methicillin-resistant S. aureus (MRSA) type mecA, strain K3, was now shown to be unstable in the absence of drug selection, causing the segregation of antibiotic-susceptible cells accompanied by the loss of the MRSA type mecA. These observations illustrate the remarkable variety of strategies available to bacteria for acquiring mechanisms of drug resistance in the in vivo environment.

Penicillin-binding protein 2 is essential for expression of high-level vancomycin resistance and cell wall synthesis in vancomycin-resistant Staphylococcus aureus carrying the enterococcal vanA gene complex.

ABSTRACT A combination of biochemical and genetic experiments were performed in order to better understand the mechanism of expression of high-level vancomycin resistance in Staphylococcus aureus. The transcription of pbp2 of the highly vancomycin- and oxacillin-resistant strain COLVA200 and its mutant derivative with inactivated mecA were put under the control of an inducible promoter, and the dependence of oxacillin and vancomycin resistance and cell wall composition on the concentration of the isopropyl-β-d-thiogalactopyranoside inducer was determined. The results indicate that mecA—the genetic determinant of oxacillin resistance—while essential for oxacillin resistance, is not involved with the expression of vancomycin resistance. Penicillin binding protein 2A, the protein product of mecA, appears to be unable to utilize the depsipeptide cell wall precursor produced in the vancomycin-resistant cells for transpeptidation. The key penicillin binding protein essential for vancomycin resistance and for the synthesis of the abnormally structured cell walls characteristic of vancomycin-resistant S. aureus (A. Severin, K. Tabei, F. Tenover, M. Chung, N. Clarke, and A. Tomasz, J. Biol. Chem. 279:3398-3407, 2004) is penicillin binding protein 2.

High-level β-lactam resistance and cell wall synthesis catalyzed by the mecA homologue of Staphylococcus sciuri introduced into Staphylococcus aureus

A close homologue of mecA, the determinant of broad-spectrum beta-lactam resistance in Staphylococcus aureus was recently identified as a native gene in the animal commensal species Staphylococcus sciuri. Introduction of the mecA homologue from a methicillin-resistant strain of S. sciuri into a susceptible strain of S. aureus caused an increase in drug resistance and allowed continued growth and cell wall synthesis of the bacteria in the presence of high concentrations of antibiotic. We determined the muropeptide composition of the S. sciuri cell wall by using a combination of high-performance liquid chromatography, mass spectrometric analysis, and Edman degradation. Several major differences between the cell walls of S. aureus and S. sciuri were noted. The pentapeptide branches in S. sciuri were composed of one alanine and four glycine residues in contrast to the pentaglycine units in S. aureus. The S. sciuri wall but not the wall of S. aureus contained tri- and tetrapeptide units, suggesting the presence of dd- and ld-carboxypeptidase activity. Most interestingly, S. aureus carrying the S. sciuri mecA and growing in methicillin-containing medium produced a cell wall typical of S. aureus and not S. sciuri, in spite of the fact that wall synthesis under these conditions had an absolute dependence on the heterologous S. sciuri gene product. The protein product of the S. sciuri mecA can efficiently participate in cell wall biosynthesis and build a cell wall using the cell wall precursors characteristic of the S. aureus host.