PBP 2a mutations producing very-high-level resistance to beta-lactams

Abstract

Resistance to the beta-lactam class of antibiotics in methicillin-resistant Staphylococcus aureus (MRSA) is mediated by PBP 2a, a synthetic bacterial cell wall penicillin-binding protein with a low affinity of binding to beta-lactams that is encoded by mecA. Beta-lactams that bind to PBP 2a with a high affinity and that are highly active against MRSA are under development. The potential for the emergence of resistance to such compounds was investigated by passage of homogeneous MRSA strain COL in L-695,256, an investigational carbapenem. A highly resistant mutant, COL52, expressed PBP 2a in which a two-amino-acid deletion mutation and three single-amino-acid substitution mutations were present. To examine the effects of these mutations on the resistance phenotype and PBP 2a production, plasmids carrying (i) PBP 2a with two or three of the four mutations, (ii) wild-type PBP 2a, or (iii) COL52 PBP 2a were introduced into methicillin-susceptible COL variants COLnex and COL52ex, from which the staphylococcus cassette chromosome mec (SCCmec) has been excised, as indicated by the "ex" suffix. Two amino acids substitutions, E-->K(237) within the non-penicillin-binding domain and V-->E(470) near the SDN(464) conserved penicillin-binding motif in the penicillin-binding domain in COL52, were important for high-level resistance. The highest level of resistance was observed when all four mutations were present. The emergence of PBP 2a-mediated resistance to beta-lactams that bind to PBP 2a with a high affinity is likely to require multiple mutations in mecA; chromosomal mutations appear to have a minor role.

FIG. 1.

Structural map of PBP 2a in strains COLn and COL52 and positions of the substituted amino acid sequences on the six plasmids. The arrows in the map of PBP 2a indicate the direction of mecA transcription. The striped, white, and gray regions indicate the transmembrane anchor, the nPBD, and the transpeptidase domain, respectively. The locations of three penicillin-binding motifs in the penicillin-binding (transpeptidase) domain are indicated by black lines. The amino acid mutations in PBP 2a of strain COL52 are indicated by dotted lines. The positions of the primers for PCR amplification are indicated by arrowheads. The table at the bottom shows the mutations, indicated by a plus sign, present in the plasmid constructs used to express wild-type or mutant PBP 2a.

FIG. 2.

Population analysis showing heterogeneous or homogeneous resistance phenotypes for five MRSA strains and one MSSA strain, RN4220, transformed with pYK20_COLn (open triangles) expressing wild-type PBP 2a or with pYK21_COL52 (open squares) expressing mutant PBP 2a. COLn and COL52 naturally express homogeneous methicillin resistance; BB270 and N315 express heterogeneous resistance; COL8a, a spontaneous mutant of COL that does not express PBP 2a, and RN4220 are methicillin susceptible. The phenotypes of the recipient parent and the mutant of the parent from which mecA was excised and that was transformed with plasmid vector pAW8 are indicated by solid and open circles, respectively. The y axis indicates the number of cells (in log₁₀ CFU per milliliter) growing on nafcillin-containing agar; the concentration of nafcillin is shown on the x axis.

FIG. 3.

Population analysis of COL52 and COL52ex transformants (a) or COLn and COLnex transformants (b), as follows: parent recipient containing pAW8 (closed circles), recipient from which mecA was excised containing pYK20_COLn (open squares), recipient from which mecA was excised containing pYK21_COL52 (open circles), recipient from which mecA was excised containing pYK26 (multiplication signs), recipient from which mecA was excised containing pYK27 (open triangles), recipient from which mecA was excised containing pYK28 (open diamonds), and recipient from which mecA was excised containing pYK29 (plus signs).

FIG. 4.

Western blot analysis for expression of mecA in transformants with plasmid vector pAW8 or a plasmid encoding wild-type mecA (pYK20_COLn) or mutant mecA (pYK21_COL52, pYK26, pYK27, pYK28, pYK29).