Effect of SA1665 deletion on β-lactam resistance To analyse the effect of SA1665 inactivation on methicillin resistance, nonpolar markerless deletions of SA1665 (Figure 1B) were constructed in a selection of clinical MRSA isolates, which varied in their genetic background, SCCmec type, and mecA regulation . Strain CHE482, belongs to clonal this website complex CC45 and sequence type ST45, and contains a novel SCCmec (SCCmec N1 ); while strains ZH37 (CC45/ST45) and ZH73 (CC22/ST22) contain type IV SCCmecs. All three of these strains have truncated mecI/mecR1 regulatory
loci but intact BlaI/BlaR1 loci controlling mecA expression. Strain ZH44 (CCT8/ST8) contained a type A mec complex (mecI-mecR1-mecA) within a type II SCCmec, and had no β-lactamase locus; so mecA was only under the control of its cognate regulators MecI/MecR1. Deletion of SA1665 increased oxacillin resistance in all mutants compared to their corresponding parent strains, as demonstrated on oxacillin SAR302503 gradient plates (Figure 3A); with mutants ΔCHE482 and ΔZH37 approximately doubling in resistance
and ΔZH44 and ΔZH73 expressing considerably higher resistance. Population Natural Product Library analysis resistance profiles of the mutants showed a distinct shift at the top of the curve, indicating that the higher resistance was due to increased basal oxacillin resistance levels (Figure 3B). Strains CHE482/ΔCHE482 and ZH37/ΔZH37 had very similar resistance profiles, despite having different SCCmec elements, suggesting that it was their common clonal background (CC45) that determined their resistance levels and the extent of second resistance increase upon SA1665 deletion. Figure 3 Effect of SA1665 deletion on oxacillin resistance. A, Growth of MRSA strains and their SA1665 deletion mutants, containing empty plasmid vector pAW17 or pBUS1, and trans complemented mutants, containing pME26 or pME27, was compared on plates containing appropriate oxacillin
gradients, as indicated. Plates were supplemented with either kanamycin (25 μg/ml) or tetracycline (5 μg/ml) to ensure plasmid maintainence. B, Representative population analysis profiles of MRSA strains CHE482, ZH37, ZH44, and ZH73 and their corresponding mutants. Wildtype strains are indicated by squares and mutants by triangles. x- and y-axis show the oxacillin concentrations (μg/ml) and the cfu/ml, respectively. Oxacillin concentrations used were two-fold dilutions ranging from 0.1–256 μg/ml for strains CHE482 and ZH37 and 1–1024 μg/ml for strains ZH44 and ZH73. C, Growth curves of wildtype strains (solid lines, closed symbols) and their corresponding SA1665 mutants (dashed lines, open symbols); CHE482 (diamonds), ZH37 (triangles), ZH44 (circles), ZH73 (squares).