Jumping the barrier to beta-lactam resistance in Staphylococcus aureus

Abstract

Although the staphylococcal methicillin resistance determinant, mecA, resides on a mobile genetic element, staphylococcus cassette chromosome mec (SCCmec), its distribution in nature is limited to as few as five clusters of related methicillin-resistant Staphylococcus aureus (MRSA) clones. To investigate the potential role of the host chromosome in clonal restriction of the methicillin resistance determinant, we constructed plasmid pYK20, carrying intact mecA, and introduced it into several methicillin-susceptible Staphylococcus aureus strains, five of which were naive hosts (i.e., mecA not previously resident on the host chromosome) and five of which were experienced hosts (i.e., methicillin-susceptible variants of MRSA strains from which SCCmec was excised). We next assessed the effect of the recipient background on the methicillin resistance phenotype by population analysis, by assaying the mecA expression of PBP2a by Western blot analysis, and by screening for mutations affecting mecA. Each experienced host transformed with pYK20 had a resistance phenotype and expressed PBP2a similar to that of the parent with chromosomal SCCmec, but naive hosts transformed with pYK20 selected against its expression, indicative of a host barrier. Either inducible beta-lactamase regulatory genes blaR1-blaI or homologous regulatory genes mecR1-mecI, which control mecA expression, acted as compensatory elements, permitting the maintenance and expression of plasmid-carried mecA.

FIG. 1.

Population analysis with nafcillin for five MRSA strains and experienced host MRSA (MRSAex) transformants. Symbols: •, wild-type MRSA with SCCmec present plus cloning vector pAW8 only; ○, MRSAex with SCCmec excised plus cloning vector only; ▵, MRSAex transformed with pYK20, carrying unregulated mecA; ×, MRSAex transformed with pYK644, carrying the intact mecI-mecR1-mecA complex. Shown on the y axis is the number of cells (per milliliter) growing on nafcillin-containing agar.

FIG. 2.

Population analysis with nafcillin for four naive host methicillin-susceptible S. aureus transformants. Symbols: •, wild-type naive host with cloning vector pAW8 only; ▵, naive host transformed with pYK20, carrying unregulated mecA; ×, naive host transformed with pYK644, carrying the intact mecI-mecR1-mecA complex; ▴, naive host cotransformed with pYK20 and pZRI, carrying the intact blaI-blaR1-blaZ complex.

FIG. 3.

Detection by Western blot analysis of PBP2a (78 kDa) encoded by mecA. COLn(pAW8) is the positive control; COLnex(pAW8), RN4220(pAW8), and RN4220(pZRI)(pAW8) are the negative controls. (a) Comparison of experienced host and naive host transformants carrying pYK20. (b) Representative transformants with cloning vector pAW8 only, pYK644 (carrying intact mecR1-mecI-mecA), and pYK20 (carrying unregulated mecA) and cotransformants with pZRI (carrying blaR1-blaI-blaZ). All were grown under noninducing conditions.