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Novel determinants of antibiotic resistance: identification of mutated loci in highly methicillin-resistant subpopulations of methicillin-resistant Staphylococcus aureus.

ABSTRACT: We identified mutated genes in highly resistant subpopulations of methicillin-resistant Staphylococcus aureus (MRSA) that are most likely responsible for the historic failure of the ?-lactam family of antibiotics as therapeutic agents against these important pathogens. Such subpopulations are produced during growth of most clinical MRSA strains, including the four historically early MRSA isolates studied here. Chromosomal DNA was prepared from the highly resistant cells along with DNA from the majority of cells (poorly resistant cells) followed by full genome sequencing. In the highly resistant cells, mutations were identified in 3 intergenic sequences and 27 genes representing a wide range of functional categories. A common feature of these mutations appears to be their capacity to induce high-level ?-lactam resistance and increased amounts of the resistance protein PBP2A in the bacteria. The observations fit a recently described model in which the ultimate controlling factor of the phenotypic expression of ?-lactam resistance in MRSA is a RelA-mediated stringent response. IMPORTANCE It has been well established that the level of antibiotic resistance (i.e., minimum concentration of a ?-lactam antibiotic needed to inhibit growth) of a methicillin-resistant Staphylococcus aureus (MRSA) strain depends on the transcription and translation of the resistance protein PBP2A. Here we describe mutated loci in an additional novel set of genetic determinants that appear to be essential for the unusually high resistance levels typical of subpopulations of staphylococci that are produced with unique low frequency in most MRSA clinical isolates. We propose that mutations in these determinants can trigger induction of the stringent stress response which was recently shown to cause increased transcription/translation of the resistance protein PBP2A in parallel with the increased level of resistance.

SUBMITTER: Dordel J

PROVIDER: S-EPMC3993859 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Novel determinants of antibiotic resistance: identification of mutated loci in highly methicillin-resistant subpopulations of methicillin-resistant Staphylococcus aureus.

Dordel Janina J Kim Choonkeun C Chung Marilyn M Pardos de la Gándara María M Holden Matthew T J MT Parkhill Julian J de Lencastre Hermínia H Bentley Stephen D SD Tomasz Alexander A

mBio 20140408 2

We identified mutated genes in highly resistant subpopulations of methicillin-resistant Staphylococcus aureus (MRSA) that are most likely responsible for the historic failure of the β-lactam family of antibiotics as therapeutic agents against these important pathogens. Such subpopulations are produced during growth of most clinical MRSA strains, including the four historically early MRSA isolates studied here. Chromosomal DNA was prepared from the highly resistant cells along with DNA from the m ...[more]

PMID: 24713324

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Project description:Antimicrobial susceptibility testing standards driving clinical decision-making have centered around the use of cation-adjusted Mueller-Hinton broth (CA-MHB) as the medium with the notion of supporting bacterial growth, without consideration of recapitulating the in vivo environment. However, it is increasingly recognized that various medium conditions have tremendous influence on antimicrobial activity, which in turn may have major implications on the ability of in vitro susceptibility assays to predict antibiotic activity in vivo. To elucidate differential growth optimization and antibiotic resistance mechanisms, adaptive laboratory evolution was performed in the presence or absence of the antibiotic nafcillin with methicillin-resistant Staphylococcus aureus (MRSA) TCH1516 in either (i) CA-MHB, a traditional bacteriological nutritionally rich medium, or (ii) Roswell Park Memorial Institute (RPMI), a medium more reflective of the in vivo host environment. Medium adaptation analysis showed an increase in growth rate in RPMI, but not CA-MHB, with mutations in apt, adenine phosphoribosyltransferase, and the manganese transporter subunit, mntA, occurring reproducibly in parallel replicate evolutions. The medium-adapted strains showed no virulence attenuation. Continuous exposure of medium-adapted strains to increasing concentrations of nafcillin led to medium-specific evolutionary strategies. Key reproducibly occurring mutations were specific for nafcillin adaptation in each medium type and did not confer resistance in the other medium environment. Only the vraRST operon, a regulator of membrane- and cell wall-related genes, showed mutations in both CA-MHB- and RPMI-evolved strains. Collectively, these results demonstrate the medium-specific genetic adaptive responses of MRSA and establish adaptive laboratory evolution as a platform to study clinically relevant resistance mechanisms.IMPORTANCE The ability of pathogens such as Staphylococcus aureus to evolve resistance to antibiotics used in the treatment of infections has been an important concern in the last decades. Resistant acquisition usually translates into treatment failure and puts patients at risk of unfavorable outcomes. Furthermore, the laboratory testing of antibiotic resistance does not account for the different environment the bacteria experiences within the human body, leading to results that do not translate into the clinic. In this study, we forced methicillin-resistant S. aureus to develop nafcillin resistance in two different environments, a laboratory environment and a physiologically more relevant environment. This allowed us to identify genetic changes that led to nafcillin resistance under both conditions. We concluded that not only does the environment dictate the evolutionary strategy of S. aureus to nafcillin but also that the evolutionary strategy is specific to that given environment.

Dataset Information

Novel determinants of antibiotic resistance: identification of mutated loci in highly methicillin-resistant subpopulations of methicillin-resistant Staphylococcus aureus.

Publications

Novel determinants of antibiotic resistance: identification of mutated loci in highly methicillin-resistant subpopulations of methicillin-resistant Staphylococcus aureus.

Similar Datasets

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