The vast MIC differences between MRSA strains, the population het

The vast MIC differences between MRSA strains, the population heterogeneity within single strains and the dependence of resistance levels on external factors are reflected in these many structural genes and global regulators, which can influence resistance levels. While typically considered nosocomial pathogens, new faster growing and apparently more virulent MRSA have begun spreading in the community. Interestingly, these emerging strains often express very low methicillin resistance, e.g. the MRSA clone spreading amongst intravenous drug users in the Zurich area, which has an in vitro check details doubling time of 25 min, but oxacillin MICs of only 0.5

to 4 μg/ml [23]. This particular clone’s low-level resistance is partially due to a promoter mutation, leading to tight repression of mecA, but resistance levels appear to be mainly restricted by unknown factors within its genomic background [12]. To identify potential factors involved in mecA regulation

or methicillin resistance levels in such an extremely low level resistant MRSA, we performed DNA-binding protein purification assays, using the mecA operator region as bait. A novel, uncharacterized protein, SA1665, was found to bind to this DNA fragment, and shown to increase methicillin resistance levels when deleted. Results Identification of SA1665 MRSA strain CHE482 is the type strain for GSK 3 inhibitor the so-called “”drug clone”" spreading amongst intravenous drug users in the Zurich area [12, 23]. This strain carries mecA and expresses PBP2a, but appears phenotypically methicillin susceptible by conventional phenotypic tests. However, like most other low-level resistant MRSA, it can segregate a small proportion of higher resistant subclones in the presence of β-lactams. We hypothesized that regulation of methicillin resistance in such low-level resistant clonal lineages may differ qualitatively from classical heterogeneously- or highly-resistant MRSA. A DNA-binding protein purification assay was performed to identify new potential factors involved in the regulation of mecA/PBP2a. The mecA/mecR1 intergenic DNA region, including the 5′

9 bp of mecR1 and the first 52 bp of mecA, was used as bait against crude protein extract from strain CHE482. Proteins Ureohydrolase binding to this DNA fragment were analysed by SDS-PAGE. Even though CHE482 contained BlaI, which is known to bind to the mec operator, this band could not be identified on gels due to co-migrating, non-specific bands the same size as BlaI (14.9 KDa) that bound to both the DNA-coated and uncoated control beads. The most prominent protein band of ~16–20 kDa, isolated from DNA-labelled but not from control beads, was identified as the hypothetical protein SA1665 (N315 genome annotation [BA000018]) (Figure 1A). SA1665 encodes a predicted 17-kDa protein with an n-terminal helix-turn-helix (HTH) motif characteristic of DNA-binding transcriptional regulators.

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