agr-deficiency and expression changes in regulatory and cell-wall genes responsible for hVISA and VISA phenotypes
Abstract number: R2145
Objectives: Glycopeptides are still the gold standard to treat serious MRSA infections, but their widespread use has led to the emergence of glycopeptide-low-level resistant isolates (hVISA and VISA). The molecular basis of this reduced susceptibility is not clear, but different genetic loci encoding regulatory systems or proteins involved in autolysis and in cell-wall turnover have been implicated. We investigated the molecular mechanisms of glycopeptide intermediate resistance on prototype microorganisms, i.e. NRS149 (VSSA), Mu3 (h-VISA), Mu50 (VISA) of agr-II and on our Quasi-VISA clinical isolate of agr-II.
Methods: We analyzed delta-hemolysin production on 5% sheep-blood-agar. We investigated, with or without sub-inhibitory vancomycin concentrations, both the autolytic activity by TRITON X-100 induction and, by real-time RT-PCR, the expression levels of hld, graR/S (regulatory genes), atl, SAV2095 (sceD-like gene), mprF (cell-wall genes), all involved in h-VISA and VISA phenotypes.
Results: We observed the lack of delta-hemolysin (in sheep-blood-agar) in the VISA and its decreased production at 48 h in the hVISA. In both culture conditions, the VISA showed the lowest autolytic activity, the Q-VISA an intermediate level with respect to hVISA and VISA, whereas in hVISA an autolysis similar to VSSA was observed. These data correlate with the gene expression showing a gradual but substantial hld down-regulation as follows: VISA < Q-VISA < hVISA < VSSA. An atl down-regulation was found in VISA, whereas a low level of sceD transcripts was found in hVISA only with vancomycin. The regulator graR/S down-regulation, related to the atl down-regulation, was found in VISA. mprF up-regulation was found in all phenotypes towards VSSA.
Conclusions: Among agr-II, hVISA had a dysfunctional agr-locus and a regular autolytic activity, but, with vancomycin, this strain reduced its cell-wall turnover. In VISA, the lack of agr functionality and reduced autolysis were independent from the presence of vancomycin. The increased expression of mprF in hVISA and VISA could be related to a lower vancomycin binding than in VSSA.
|Session name:||Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Vienna, Austria, 10 - 13 April 2010|
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