Prevalence of Slow-Growth Vancomycin Nonsusceptibility in Methicillin-Resistant Staphylococcus aureus.

Yuki Katayama,Miwa Sekine,Motoyasu Miyazaki,Tomonori Ochiai,Hideaki Hanaki,Tohru Takata,Norio Komatsu,Henry F Chambers,Kota Asakura,Takuya Azechi,Hiroshi Sasano,Koji Yahara,Tomoiku Takaku,Hidehito Matsui

doi:10.1128/aac.00452-17

Abstract

ABSTRACTWe previously reported a novel phenotype of vancomycin-intermediate Staphylococcus aureus (VISA), i.e., “slow VISA,” whose colonies appear only after 72 h of incubation. Slow-VISA strains can be difficult to detect because prolonged incubation is required and the phenotype is unstable. To develop a method for detection of slow-VISA isolates, we studied 23 slow-VISA isolates derived from the heterogeneous VISA (hVISA) clinical strain Mu3. We identified single nucleotide polymorphisms (SNPs) in genes involved in various pathways which have been implicated in the stringent response, such as purine/pyrimidine synthesis, cell metabolism, and cell wall peptidoglycan synthesis. We found that mupirocin, which also induces the stringent response, caused stable expression of vancomycin resistance. On the basis of these results, we developed a method for detection of slow-VISA strains by use of 0.032 μg/ml mupirocin (Yuki Katayama, 7 March 2017, patent application PCT/JP2017/008975). Using this method, we detected 53 (15.6%) slow-VISA isolates among clinical methicillin-resistant S. aureus (MRSA) isolates. In contrast, the VISA phenotype was detected in fewer than 1% of isolates. Deep-sequencing analysis showed that slow-VISA clones are present in small numbers among hVISA isolates and proliferate in the presence of vancomycin. This slow-VISA subpopulation may account in part for the recurrence and persistence of MRSA infection.

Highlights

We previously reported a novel phenotype of vancomycin-intermediate Staphylococcus aureus (VISA), i.e., “slow VISA,” whose colonies appear only after 72 h of incubation
HVISA is distinguishable from VSSA by the presence of cells resistant to 4 ␮g/ml vancomycin within the cell population, at a frequency of 1 ϫ 10Ϫ6 or greater. heterogeneous VISA (hVISA) is thought to be an intermediate step in the development of resistance from VSSA to VISA. hVISA and slow-VISA cells may lose their resistance in the process of repeated isolation and incubation or may not be detected at all under the standard incubation period of 48 h recommended by the Clinical and Laboratory Standards Institute (CLSI) guidelines
We found numerous mutations in hVISA strain Mu3-derived slow-VISA strains, in genes involved in various metabolic pathways, including purine/pyrimidine synthesis and 5P-D-ribosyl-1-pyrophosphate (PRPP) synthesis, as well as in rpoB and rpoC

Summary

Introduction

We previously reported a novel phenotype of vancomycin-intermediate Staphylococcus aureus (VISA), i.e., “slow VISA,” whose colonies appear only after 72 h of incubation. We found numerous mutations in hVISA strain Mu3-derived slow-VISA strains, in genes involved in various metabolic pathways, including purine/pyrimidine synthesis and 5P-D-ribosyl-1-pyrophosphate (PRPP) synthesis, as well as in rpoB and rpoC. These genes are known to be involved in the stringent response, a phenomenon originally identified in cells subjected to the stress of amino acid starvation, during which cells undergo massive physiological changes to save energy and conserve cellular resources [7]. The stringent stress response induces high-level, homogeneous resistance to beta-lactams and depends on the transglycosylase activity of PBP2 [13, 14]

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