Novel polymorphisms in mec genes and a new mec complex type in methicillin-resistant Staphylococcus aureus isolates obtained in rural Wisconsin

Abstract

We determined allelic polymorphisms in the mec complexes of 524 methicillin-resistant Staphylococcus aureus isolates by partial or complete sequencing of three mec genes, mecA, mecI, and mecR1. The isolates had been collected over a 10-year period from patients living in rural Wisconsin, where the use of antibiotics was expected to be lower than in the bigger cities. Of the 18 mutation types identified, 16 had not been described previously. The five most common mutations were a mutation 7 nucleotides (nt) upstream from the start site (G-->T) in the mecA promoter (34.7%), an E246G change encoded by mecA (2.2%), a cytosine insertion at codon 257 in mecA (13.2%), an N121K change encoded by mecI (7.8%), and a major mecI-mecR1 deletion designated as a class B1 mec complex deletion type (25.4%). There was a high degree of conservation of the amino acid sequence of MecR1. Strains with the same mutations had variable resistance to oxacillin, and the median MIC for isolates that harbored the 7-nt-upstream mutation was lower than that for strains harboring other mutations. Our data suggest that the mecA promoter mutation plays a more important role in determining methicillin resistance than mutations in mecI and mecA do. Eighty-five percent of the tested isolates (n = 148) with the mecA promoter mutation and the class B1 mec complex deletion belonged to the same major clonal group, identified as MCG-2, and harbored the type IV staphylococcal cassette chromosome mec DNA. There was also a wide range of oxacillin MICs for strains with wild-type mecA, mecI, and mecR1 sequences, suggesting that the genetic backgrounds of clinical strains are significant in determining susceptibility to methicillin.

FIG. 1.

An ethidium bromide-stained agarose gel showing amplicons from the primers used in the present study. Primer pairs for the respective amplicons are listed at the top of each lane. DNA markers are loaded in the first and last lanes.

FIG. 2.

(Top) Schematic diagram of the class A mec complex showing the mecA promoter-operator region and the MecA, MecR1, and MecI proteins with relevant nucleotide positions and amino acids where significant mutations were found. Transcriptional directions and the size of each protein are shown below the line diagram. (Bottom) Schematic diagram of the class B1 mec complex along with the mutation identified in this group of isolates.

FIG. 3.

Chromatogram showing the nucleotide position of the deletion junction in the mecR1 gene of the class B1 mec complex compared with the wild-type gene. The arrow at nt position 782 indicates the deletion junction.

FIG. 4.

Histogram showing the frequency distribution (left y axis) of MRSA isolates with five nonsynonymous mutations in the mec complex. Each bar represents the frequency (number at the top of the bar) of isolates with either a single mutation or a combination of mutations. Also shown are the mean (filled triangles) and median (open circles) oxacillin MICs (right y axis) for isolates with each category of mutation. The differently shaded sections of each bar represent the frequencies of isolates for a given range of MICs, as shown in the key.