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Insertional inactivation of staphylococcal methicillin resistance by Tn551
Abstract:
Transposon Tn551 was translocated into the chromosome of a methicillin-resistant (mec) strain of Staphylococcus aureus by heat inactivation of a thermo-sensitive plasmid carrying Tn551 and selection for erythromycin-resistant (Emr) survivors. Two independent chromosomal insertions of Tn551 were obtained which reduced the level of the methicillin resistance by a factor of 50 to 100, making the strains phenotypically methicillin sensitive (Mecs). Each of the Tn551 insertions was on the largest fragment produced by EcoRI digestion of the chromosomal DNA of these strains. The integration sites lie about 1 kilobase apart. These Mecs strains reverted to Mecr at frequencies of 2.4 X 10(-8) and 3.6 X 10(-5), respectively. The majority of Mecr revertants still were Emr; only a few lost the Emr phenotype concomitantly with reversion to the Mecr phenotype. Hybridization data with labeled Tn551 showed complex rearrangements and deletions in the region of the insertion. These two Tn551 insertions do not lie on the same linkage group, II, as the mec determinant. The phenotypic expression of methicillin resistance, therefore, is also dependent upon a chromosomal genetic marker not physically linked to the mec determinant.
Insights
Transposon Tn551 insertions in Staphylococcus aureus reduced methicillin resistance, creating methicillin-sensitive strains. These insertions indicate a new genetic marker, unlinked to the mec determinant, influences resistance 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 the mec determinant, is well-studied.
- However, other factors influencing the phenotypic expression of methicillin resistance are less understood.
Purpose of the Study:
- To investigate the genetic factors influencing methicillin resistance in Staphylococcus aureus.
- To identify potential chromosomal genetic markers that modulate the expression of methicillin resistance.
- To understand the relationship between transposon insertion, methicillin resistance, and other antibiotic resistance phenotypes.
Main Methods:
- Transposon mutagenesis using Tn551 on a methicillin-resistant Staphylococcus aureus strain.
- Selection for erythromycin resistance (Emr) to identify transposon insertion mutants.
- Phenotypic characterization of methicillin resistance levels (Mecs) in mutant strains.
- Chromosomal DNA analysis using EcoRI digestion and Southern hybridization with labeled Tn551.
- Analysis of reversion frequencies from methicillin-sensitive (Mecs) to methicillin-resistant (Mecr) phenotypes.
Main Results:
- Two independent Tn551 insertions into the S. aureus chromosome resulted in a 50- to 100-fold decrease in methicillin resistance, yielding phenotypically methicillin-sensitive (Mecs) strains.
- These Tn551 insertion sites were located on large EcoRI fragments and were approximately 1 kilobase apart.
- Reversion to methicillin resistance (Mecr) occurred at low frequencies, with most revertants retaining erythromycin resistance (Emr).
- Hybridization data revealed complex genomic rearrangements and deletions around the Tn551 insertion sites.
- The Tn551 insertion sites were found not to be linked to the established mec determinant (linkage group II).
Conclusions:
- The phenotypic expression of methicillin resistance in Staphylococcus aureus is influenced by chromosomal genetic markers not physically linked to the mec determinant.
- Transposon Tn551 can identify and disrupt genes or regulatory regions that modulate antibiotic resistance.
- Further research is needed to identify the specific genetic marker(s) responsible for modulating methicillin resistance expression.