Microarray Transcription Analysis of Clinical Staphylococcus aureus Isolates Resistant to Vancomycin

Abstract

The transcriptomes of vancomycin intermediate-resistance Staphylococcus aureus (VISA) clinical isolates HIP5827 and Mu50 (MIC ‫؍‬ 8 g/ml) were compared to those of highly vancomycin-resistant S. aureus (VRSA; MIC ‫؍‬ 32 g/ml) passage derivatives by microarray. There were 35 genes with increased transcription and 16 genes with decreased transcription in common between the two VRSAs compared to those of their VISA parents. Of the 35 genes with increased transcription, 15 involved purine biosynthesis or transport, and the regulator (purR) of the major purine biosynthetic operon (purE-purD) was mutant. We hypothesize that increased energy (ATP) is required to generate the thicker cell walls that characterize resistant mutants.Vancomycin is the treatment of choice for serious infections caused by oxacillin-resistant Staphylococcus aureus, and some isolates are reported that are susceptible only to this antibiotic (8, 12). Oxacillin-resistant S. aureus isolates with reduced susceptibility to vancomycin (vancomycin intermediate-resistance S. aureus [VISA] isolates; the vancomycin MIC increases from 1 to 8 g/ml) have been recovered from patients receiving prolonged courses of vancomycin (1-3). A number of abnormalities have been noted among VISA isolates, including reduced rate of growth, decreased cell wall cross-linking (6, 16), increased cell wall thickness (4, 10), decreased autolysis (17), changes in penicillin binding proteins (6, 9, 15), and alterations in glutamate amidation of the peptidoglycan stem peptide (5). No unifying molecular hypothesis has been proposed that explains the VISA phenotype, but the current hypothesis is that the thickened, poorly cross-linked cell wall provides more peripheral targets for vancomycin, trapping the antibiotic before it can reach its site of lethal action at the cell membrane (17). It is also assumed that because of the long clinical vancomycin exposure times required to generate resistance, multiple genes and, probably, multiple metabolic pathways have been altered.We sought to assess genomic changes in gene transcription (the transcriptome) by DNA microarray and took a number of approaches different from those taken by others in trying to understand genomic changes associated with the VISA phenotype. First, we used clinical VISA isolates as parents rather than vancomycin-susceptible laboratory strains. There is evidence that there is something unique about VISA isolates that allows them to become resistant to vancomycin more easily than other S. aureus isolates (13). Second, we sought to amplify the phenotype by continued exposure of VISA isolates to vancomycin in vitro, further increasing the vancomycin MIC. We hypothesized that in this way, we would create isogenic strain sets that exaggerate the changes present in clinical VISA isolates. Third, we compared stable mutants of VISA strains for which vancomycin MICs were increased and we did not grow strains in the presence of the antibiotic, removing any direct effect of the antibiotic on transcription.Generation of mutant...

Spontaneous Deletion of the Methicillin Resistance Determinant, mecA , Partially Compensates for the Fitness Cost Associated with High-Level Vancomycin Resistance in Staphylococcus aureus

“…However, high-level vancomycin resistance (MICs Ն 32 g/ml) due to this resistance mechanism is not seen among clinical isolates. Here we show that high-level, passage-induced vancomycin resistance is associated with a decrease in the growth rate and the loss of SCCmec, including mecA, observations that have also been made by others (1,31,36,41). These SCCmec deletions resulted in strains with a faster growth rate and an ability to outcompete their mecA-containing vancomycin-resistant counterparts in mixed culture competition experiments (Fig.…”

“…The loss of mecA during the passage of S. aureus to high-level vancomycin resistance has been observed previously (1,31,36,41). In order to further characterize the loss of mecA during vancomycin passage, we examined three sets of strains.…”

Vancomycin-Intermediate Staphylococcus aureus Strains Have Impaired Acetate Catabolism: Implications for Polysaccharide Intercellular Adhesin Synthesis and Autolysis

“…Increased resistance to vancomycin is associated with changes in bacterial morphology, physiology, and growth characteristics (8,19,27). In particular, increased resistance to vancomycin decreased the growth rate and cell size relative to isogenic strains of lower resistance (19).…”

“…Most studies that have attempted to ascertain the adaptive changes occurring during the transition to growth in the presence of vancomycin have focused on alterations in cell wall synthesis and morphology (8,11,21,27), autolysis (11,28,35), and global transcriptional changes (7,18,19,34). These studies have led to a model of vancomycin resistance in which decreased cell wall turnover and autolysis result in increased cell wall thickness and resistance to vancomycin.…”

“…To circumvent this barrier, Mongodin et al grew two VISA strains (Mu50 and HIP5827) in medium containing increasing concentrations of vancomycin until the derivative strains had vancomycin MICs of 32 g/ml (strains Mu50-32 and VP-32), thus providing an alternative to isogenic VSSA and VISA pairs. Using these isogenic strain pairs of low and high vancomycin resistance provided insight into the transcriptional changes that occur during adaptation to growth in the presence of vancomycin (19). In the present study, we used these isogenic pairs as a means to identify common physiological differences and then examined if these differences were present in naturally occurring VISA strains.…”