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Involvement of multiple genetic determinants in high-level methicillin resistance in Staphylococcus aureus
1Rockefeller University, New York, New York 10021-6399.
Abstract:
A methicillin-susceptible, novobiocin-resistant strain of Staphylococcus aureus (RN2677; methicillin MIC, 0.8 micrograms/ml) was transformed with DNA prepared from highly and homogeneously methicillin-resistant S. aureus strains (methicillin MIC, greater than or equal to 400 micrograms/ml) or from heterogeneous strains in which the majority of cells had a low level of resistance (methicillin MIC, 6.3 micrograms/ml). All methicillin-resistant transformants showed low and heterogeneous resistance (methicillin MIC, 3.1 micrograms/ml) irrespective of the resistance level of DNA donors. All transformants examined produced normal amounts of the low-affinity penicillin-binding protein (PBP) 2a, and methicillin resistance and the capacity to produce PBP 2a showed the same degree of genetic linkage to the novobiocin resistance marker with both homogeneous and heterogeneous DNA donors. Next, we isolated a methicillin-susceptible mutant from a highly and homogeneously resistant strain which had a Tn551 insertion near or within the PBP 2a gene and thus did not produce PBP 2a. With this mutant used as the recipient, genetic transformation of the methicillin resistance gene was repeated with DNA isolated either from highly and homogeneously resistant strains or from heterogeneous (low-resistance) strains. All transformants obtained expressed high and homogeneous resistance and produced PBP 2a irrespective of the resistance level of the DNA donors. Our findings suggest that (i) the methicillin resistance locus is identical to the structural gene for PBP 2a, (ii) although the ability to produce PBP 2a is essential for resistance, the MICs for the majority of cells are not related to the cellular concentration of PBP 2a, and (iii) high MICs and homogeneous expression of resistance require the products of other distinct genetic elements as well.
Insights
Methicillin resistance in Staphylococcus aureus is linked to the PBP 2a gene. High-level resistance requires PBP 2a production and additional genetic factors for homogeneous expression.
Area of Science:
- Microbiology
- Genetics
- Molecular Biology
Background:
- Methicillin resistance in Staphylococcus aureus (MRSA) is a significant public health concern.
- The genetic basis of MRSA, particularly the role of penicillin-binding protein 2a (PBP 2a), is complex and not fully understood.
- Understanding the genetic determinants of methicillin resistance is crucial for developing effective treatment strategies.
Purpose of the Study:
- To investigate the genetic locus responsible for methicillin resistance in Staphylococcus aureus.
- To determine the relationship between the production of PBP 2a and the level of methicillin resistance.
- To elucidate the genetic mechanisms underlying homogeneous and heterogeneous expression of methicillin resistance.
Main Methods:
- Genetic transformation experiments using methicillin-susceptible and resistant strains of Staphylococcus aureus.
- Analysis of penicillin-binding protein 2a (PBP 2a) production in transformants.
- Tn551 mutagenesis to isolate mutants deficient in PBP 2a production.
- Determination of methicillin minimum inhibitory concentrations (MICs) and genetic linkage analysis.
Main Results:
- Genetic transformation with DNA from resistant strains resulted in transformants with low and heterogeneous resistance, all producing PBP 2a.
- A mutant lacking PBP 2a production, when transformed, yielded high and homogeneous resistance, with PBP 2a production restored.
- Methicillin resistance showed consistent genetic linkage to the novobiocin resistance marker, regardless of donor strain resistance levels.
- High-level, homogeneous methicillin resistance required PBP 2a production and additional genetic elements.
Conclusions:
- The methicillin resistance locus in Staphylococcus aureus is identical to the structural gene for PBP 2a.
- While PBP 2a production is essential for methicillin resistance, cellular concentrations do not directly correlate with MICs.
- High-level and homogeneous methicillin resistance necessitate the involvement of other genetic factors beyond the PBP 2a gene.