Conversion of oxacillin-resistant staphylococci from heterotypic to homotypic resistance expression

Abstract

Staphylococci that acquire the mecA gene are usually resistant to beta-lactam antibiotics (methicillin or oxacillin resistance). mecA encodes a penicillin-binding protein (PBP 2a) that has a reduced affinity for beta-lactams. In some isolates with methicillin or oxacillin resistance, only a small proportion (< or =0.1%) of the population expresses resistance to > or =10 microg of oxacillin per ml (heterotypic resistance [HeR]), while in other isolates most of the population expresses resistance (homotypic resistance [HoR]). In the present study, growth of Staphylococcus aureus or Staphylococcus epidermidis strains with HeR in concentrations of oxacillin (0.3 to 0.7 microg/ml) that produced a fall or a lag in optical density converted the strains from the HeR to the HoR phenotype. The conversion from the HeR to the HoR phenotype appeared to be due to the selection of a highly resistant mutant population, as determined by fluctuation analysis and the failure of populations with HoR to revert to HeR after 60 generations of growth in antibiotic-free media. The frequencies of conversion were as high as 10(-3) to 10(-2). Conversion to HoR required an intact mecA gene and an increase in the level of mecA transcription since no highly resistant subpopulation could be selected after growth in oxacillin when mecA transcription was constitutively repressed or when mecA had been inactivated. In addition, in both S. epidermidis and S. aureus the level of resistance to vancomycin, which also acts directly on the staphylococcal cell wall, was greater among convertants with HoR than their isogenic parents. The conversion of a population from HeR to HoR involves the selection of a mutation(s) that occurs at a high frequency and most likely requires abundant PBP 2a.

FIG. 1.

Time course and phenotypic expression of conversion to homotypic oxacillin resistance of S. aureus strain RN450M. The linear OD₆₀₀ is on the y axis, and time is on the x axis. (A) Growth curve of oxacillin-resistant strain RN450M and its isogenic oxacillin-susceptible parent, RN450, in the presence of oxacillin (ox.; 0.5 μg/ml). A growth curve for RN450M in the absence of oxacillin is included for comparison. (B) Oxacillin gradient plates streaked with cells of RN450M grown in the presence and absence of oxacillin. The concentrations in the gradient are indicated to the left. The time points correspond to those in the graph in panel A.

FIG. 2.

EOP curves before conversion to a homotypic resistance phenotype (▪), immediately after conversion (•), and following conversion to homotypy and after passage for 60 generations in antibiotic-free BHI broth (▴). Shown on the y axis are the numbers of S. aureus or S. epidermidis cells (in log₁₀ CFU per milliliter on oxacillin/CFU per milliliter on MHA) remaining on the plates containing increasing concentrations of oxacillin (shown on the x axis). Strains RN450M, N315, and 67-0 are S. aureus; strain SE42 is S. epidermidis.

FIG. 3.

EOP curves generated by testing cells obtained directly from plates containing RN450M colonies growing on various concentrations of oxacillin. Colonies to be tested were resuspended in 1 ml of BHI broth. Shown on the y axis is the number of S. aureus cells (in log₁₀ CFU per milliliter on oxacillin/CFU per milliliter on MHA) remaining on the plates containing various concentrations of oxacillin (shown on the x axis). Not shown on the x axis are numbers for 0.5, 1.0, 2.0, and 5.0 μg. Cells were obtained from agar containing 1 μg (solid squares), 5 μg (solid crosses), 10 μg (solid circles), and 50 μg (solid triangles) of oxacillin per ml.

FIG. 4.

Kinetics of transcription of a chromosomal mecA::lacZ fusion during conversion to homotypic oxacillin resistance. β-Galactosidase activity, indicated by the bars, is expressed as relative light units (RLU)/OD₆₀₀ of the sample. The mean and standard error are provided for each sample, and a growth curve (circles or diamonds) is also shown. No measurements were performed between 4 and 44 h due to small numbers of cells. (A) Transcription of a mecA::lacZ fusion (RN450M::pGO630) over time in the presence of 0.3 μg of oxacillin per ml with both the mecA repressor (mecI) and the mecA inducer (mecR1) intact. (B) Transcription of a mecA::lacZ fusion (RN450M::pGO630ΔR1) over time in the presence of 0.3 μg of oxacillin per ml with mecI intact but mecR1 inactivated.