Reconstruction of the phenotypes of methicillin-resistant Staphylococcus aureus by replacement of the staphylococcal cassette chromosome mec with a plasmid-borne copy of Staphylococcus sciuri pbpD gene

Abstract

The mecA gene, the central determinant of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA), is not native to this bacterial species but may have originated in the animal commensal species Staphylococcus sciuri. All S. sciuri strains carry a close homologue of mecA in the form of pbpD, the genetic determinant of penicillin binding protein 4 (PBP 4) of S. sciuri. Here we describe an experimental system that could be used for additional tests for this proposition. The S. sciuri pbpD gene was cloned into a shuttle plasmid and introduced into methicillin-susceptible S. aureus strain COL-S derived from parental MRSA strain COL from which the resistance cassette staphylococcal cassette chromosome mec was excised. The S. sciuri pbpD determinant was transcribed and translated in the S. aureus transductants producing large amounts of the 84-kDa S. sciuri PBP 4 and was then deposited in the plasma membrane of the host bacterium. Transductants carrying the heterologous S. sciuri pbpD gene exhibited properties typical of those of parental MRSA strain COL, including broad-spectrum, high-level, and homogeneous resistance to structurally different beta-lactams. Antibiotic resistance was dependent on the functioning of S. aureus PBP 2 and was suppressed by the specific regulatory genes mecI and mecR and by inhibitors of an early step in cell wall biosynthesis. S. sciuri PBP 4 was also able to replace the essential physiological function(s) of the native PBP 2 of S. aureus and produce peptidoglycan typical of that of parental MRSA strain COL. Our results provide further support for the proposition that the resistance determinant mecA of MRSA strains has evolved from S. sciuri pbpD.

FIG. 1.

Transcription of S. aureus mecA and pbpB genes and S. sciuri pbpD gene. RNA was purified from cultures grown at 30°C to an OD₆₂₀ of 0.7. RNA (5 μg) was resolved by electrophoresis on agarose-formaldehyde gels. After transfer, the membranes were hybridized with ³²P-labeled S. aureus mecA and pbpB and S. sciuri pbpD DNA probes.

FIG. 2.

PBP patterns and detection of protein products of S. aureus mecA (PBP 2A) and S. sciuri pbpD (PBP 4). (A) Membrane preparations (150 μg of proteins) were incubated with a single saturating concentration of [¹⁴C]benzylpenicillin. After SDS-PAGE, the gel was exposed to a tritium storage phosphor screen for 2 weeks. (B) Membrane preparations (80 μg of proteins) were tested by Western blot analysis for the production of proteins that react with a monoclonal antibody raised against S. aureus PBP 2A. The results for S. aureus COL and S. sciuri SS37 are provided as positive controls and size markers for PBP 2A and PBP 4, respectively. (C) Protein patterns on an SDS-polyacrylamide gel stained with Coomassie blue.

FIG. 3.

Effect of suppression of pbpB transcription and introduction of the plasmid-borne S. aureus mecA or S. sciuri pbpD gene on growth in S. aureus. Overnight cultures supplemented with IPTG were centrifuged, washed twice, and suspended in fresh medium to an initial OD₆₂₀ of 0.01 in the presence of 500 μM IPTG (closed symbols and solid lines) or in the absence of IPTG (open symbols and dashed lines). The turbidities of the cultures were monitored at 30°C for 12 h. (A) COL-S_spac::pbpB (▪ and □) and COL_spac::pbpB (▴ and ▵); (B) COL-S_{spac::pbpB/SAmecA} (• and ○) and COL-S_{spac::pbpB/SSpbpD} (♦ and ⋄).

FIG. 4.

HPLC elution profiles of purified peptidoglycans. Peptidoglycans were purified from 1-liter cultures grown at 30°C to an OD₆₂₀ of 0.4, digested with mutanolysin, and separated by HPLC. The peptidoglycan compositions of S. aureus COL (A), transductants COL-S_{spac::pbpB/SAmecA} (B) and COL-S_{spac::pbpB/SSpbpD} (C) grown in the absence of IPTG, and S. sciuri SS37 (D) were determined. Muropeptides were detected by measurement of the absorbance at 206 nm.

FIG. 5.

Effect of introduction of the plasmid-borne S. aureus mecA or S. sciuri pbpD genes and suppression of pbpB transcription on the expression of oxacillin resistance in S. aureus. Aliquots of overnight cultures were plated on tryptic soy agar containing increasing concentrations of oxacillin. The numbers of CFU were counted after incubation for 72 h at 30°C. The oxacillin susceptibility profiles were determined for COL (♦), COL_spac::pbpB grown in the presence (▴) and absence (▵) of IPTG, COL-S_spac::pbpB grown in the presence of IPTG (▾), COL-S_{spac::pbpB/SAmecA} grown in the presence (•) and absence (○) of IPTG, and COL-S_{spac::pbpB/SSpbpD} grown in the presence (▪) and absence (□) of IPTG.