Stable classes of phenotypic expression in methicillin-resistant clinical isolates of staphylococci

Abstract

A collection of coagulase-positive and -negative clinical strains of staphylococci, all of which gave a positive reaction with a mec-specific DNA probe, was analyzed for the mode of phenotypic expression of methicillin resistance by using population analysis on agar plates containing different concentrations of the antibiotic. Strains could be divided into four arbitrary expression classes. Cultures of class 4 strains were composed of uniformly and highly resistant bacteria (MIC 2 800 pg/ml). In contrast, cultures of strains belonging to classes 1, 2, and 3 were heterogeneous: they were composed of two or more subpopulations of cells that differed from one another in MICs and frequencies. In cultures of strains belonging to expression class 1, most of the cells had methicillin MICs of 1.5 to 3 ,ug/ml, i.e., only two to three times higher than those for truly susceptible strains. In cultures of strains belonging to expression classes 2 and 3, the methicillin MICs for the majority of bacteria ranged from 6 to 12 and up to 50 to 200 ,Lg/ml, respectively. While the definition of the expression classes was arbitrary, the modes of phenotypic expression were specific and reproducible: randomly picked colonies of a given strain produced identical population profiles. The strain-specific mode of expression was also retained after numerous single-colony picks and sequential passages in antibiotic-free medium. We suggest that these classes represent stages in an evolutionary sequence leading to progressively improved phenotypic expression of methicillin resistance in staphylococci.The phenotypic expression of methicillin resistance in staphylococci presents several intriguing features. Methicillin-resistant strains appear to be uniform in that they all contain the unique, foreign-born mec gene and its product, the 78-kDa penicillin-binding protein 2A or 2' (for a recent review, see reference 3). On the other hand, the degree of antibiotic resistance (i.e., MIC, as determined by the conventional techniques of susceptibility testing) varies tremendously from one strain to another, spanning the range of a few micrograms per milliliter (values not far above the MIC for susceptible Staphylococcus aureus) to several milligrams per milliliter. By using a technique of higher resolution (population analysis), one can also demonstrate an even more peculiar feature of these bacteria: broth cultures of most methicillin-resistant S. aureus (MRSA) strains are not composed of cells ofuniform MICs, but rather they are made up of several subpopulations of bacteria that differ widely in their degrees of antibiotic resistance (4). Thus, there exists a surprising degree of nonuniformity in the phenotypic expression of antibiotic resistance, both from one strain to another and also within the progeny of a single MRSA isolate. While both genetic and environmental factors are known to influence the phenotypic expression of methicillin resistance, the mechanistic basis of this diversity is not understood. To see if some pattern in this complexity...

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