High Level Oxacillin and Vancomycin Resistance and Altered Cell Wall Composition in Staphylococcus aureus Carrying the Staphylococcal mecA and the Enterococcal vanA Gene Complex

Abstract

Recently, for the first time in the history of this bacterial species, methicillin-resistant Staphylococcus aureus (MRSA) carrying the enterococcal vanA gene complex and expressing high level resistance to vancomycin was identified in clinical specimens (CDC (2002) MMWR 51, 565-567). The purpose of our studies was to understand how vanA is expressed in the heterologous background of S. aureus and how it interacts with the mecAbased resistance mechanism, which is also present in these strains and is targeted on cell wall biosynthesis. The vanA-containing staphylococcal plasmid was transferred from the clinical vancomycin-resistant S. aureus (VRSA) strain HIP11714 (CDC (2002) MMWR 51, 565-567) to the methicillin-resistant S. aureus (MRSA) strain COL for which extensive genetic and biochemical information is available on staphylococcal cell wall biochemistry and drug resistance mechanisms. The transconjugant named COLVA showed high and homogeneous resistance to both oxacillin and vancomycin. COLVA grown in vancomycin-containing medium produced an abnormal peptidoglycan: all pentapeptides were replaced by tetrapeptides, and the peptidoglycan contained at least 22 novel muropeptide species that frequently showed a deficit or complete absence of pentaglycine branches. The UDP-MurNAc-pentapeptide, the major component of the cell wall precursor pool in vancomycin-sensitive cells was replaced by UDPMurNAc-depsipeptide and UDP-MurNAc-tetrapeptide. Transposon inactivation of the ␤-lactam resistance gene mecA caused complete loss of ␤-lactam resistance but had no effect on the expression of vancomycin resistance. The two major antibiotic resistance mechanisms encoded by mecA and vanA residing in the same S. aureus appear to use different sets of enzymes for the assembly of cell walls.Until the late 1990s, clinical isolates of Saccharomyces aureus have retained a uniform high sensitivity to vancomycin with minimal inhibitory concentration (MIC) 1 values in the vicinity of 1 g/ml. Beginning with the late 1990s, isolates with reduced susceptibility to vancomycin began to be reported from several countries (1), but the MIC value of these vancomycin intermediate-resistant S. aureus isolates was limited to the range of 8 -16 g/ml of the antibiotic. The first highly vancomycin-resistant S. aureus (VRSA) isolates (MIC over 32 g/ml) were only detected during the last year in two hospitals in the United States (2, 3). The appearance of such VRSA strains in clinical specimens is of obvious and grave concern, because the spread of VRSA isolates may seriously jeopardize the chemotherapy of multidrug-resistant S. aureus disease and raise the specter of untreatable staphylococcal infections (4, 5). The VRSA strain HIP11714 recovered from a dialysis patient in Detroit, Michigan has acquired the vanA gene complex most likely from a vancomycin-resistant Enterococcus faecalis strain coinfecting the diabetic wound and catheter insertion site of the patient (1). HIP11714 was also methicillin-resistant: it carried the heterologous mecA gene, t...

Functional and Morphological Adaptation to Peptidoglycan Precursor Alteration in Lactococcus lactis

“…These results show that modified stem peptides can efficiently be used as a donor but also as an acceptor for transpeptidation in L. lactis. By comparison, D-Ala-D-Lac termini were not detected in the peptidoglycan extracted from vancomycinresistant strains of enterococci and S. aureus expressing the vanA/B gene cluster, although the level of cross-linking was unchanged (33)(34)(35)(36)(37). It was proposed that removing D-Lac from the peptide chains is necessary in these bacterial species to supply transpeptidases with an appropriate acceptor substrate, which seems not to be the case in L. lactis (33,37).…”

Genomic and expression analysis of the vanG-like gene cluster of Clostridium difficile

“…Peptidoglycan precursors were extracted and analysed according to established procedures (Billot-Klein et al, 1994) with some modifications. Cultures of C. difficile were grown in the absence or presence of vancomycin (0.375 mg ml 21 ) to an optical density at 600 nm of 0.5, bacitracin was added (768 mg ml 21 when vancomycin is absent and 100 mg ml 21 when vancomycin is added) and incubation continued for 2 h to amplify the amount of cell-wall precursors (Severin et al, 2004). The cells were rapidly cooled in an ice bath and harvested by centrifugation.…”

“…To investigate further the possible expression of the vanG-like operon, we analysed in the present work the cell-wall precursors in C. difficile 630 grown in the presence (1/4 MIC) or absence of vancomycin, by blocking the cell-wall assembly with the addition of bacitracin (Severin et al, 2004). In the absence of vancomycin, bacitracin at 770 mg ml 21 was added to completely stop the bacterial growth, but the addition of the same bacitracin concentration in culture grown in the presence of vancomycin led to a rapid lysis of the cells, due to a synergistic effect of the two antibiotics.…”

Specificity of L,D-Transpeptidases from Gram-positive Bacteria Producing Different Peptidoglycan Chemotypes

“…Successful modifications that have spread in Gram-positive pathogens under the selective pressure of glycopeptides include incorporation of D-lactate or D-Ser instead of D-Ala at the fifth position of pentapeptide stems (7). Strikingly, production of D-lactate-ending precursors can also have an impact on the activity of ␤-lactams in the enterococci and staphylococci, presumably because low affinity PBPs responsible for ␤-lactam resistance cannot function with modified precursors (6,8). Total elimination of D-Ala 5 by hydrolysis of the C-terminal residue of pentapeptide stems is an alternative mechanism of glycopeptide resistance in mutants of Enterococcus faecium selected in laboratory conditions (9).…”