Identification of chromosomal location of mupA gene, encoding low-level mupirocin resistance in staphylococcal isolates.

Low- and high-level mupirocin resistance have been reported in Staphylococcus aureus. The expression of plasmid-encoded mupA is responsible for high-level mupirocin resistance. Low-level mupirocin-resistant strains do not contain plasmid-encoded mupA, and a chromosomal location for this gene has not previously been reported. We examined high- and low-level mupirocin-resistant S. aureus strains to determine if mupA was present on the chromosome of low-level-resistant isolates. Southern blot analysis of DNA from four mupirocin-resistant strains identified mupA in both high- and low-level mupirocin-resistant strains. Low-level mupirocin-resistant strains contained a copy of mupA on the chromosome, while the high-level mupirocin-resistant isolate contained a copy of the gene on the plasmid. PCR amplification of genomic DNA from each mupirocin-resistant strain resulted in a 1.65-kb fragment, the predicted product from the intragenic mupA primers. This is the first report of a chromosomal location for the mupA gene conferring low-level mupirocin resistance.

Characterization of a conjugative staphylococcal mupirocin resistance plasmid.

We studied conjugative plasmids encoding high-level mupirocin resistance. These plasmids were found in Staphylococcus aureus isolates from two geographic locations in the United States. Transfer genes on three mupirocin resistance plasmids with different restriction endonuclease profiles were indistinguishable by DNA hybridization from those on pG01, a conjugative aminoglycoside resistance plasmid representative of similar plasmids that are prevalent in the United States. One mupirocin resistance plasmid, pG0400 (34 kb), was smaller than pG01 (52 kb) because of the absence from pG0400 of DNA, found on pG01, that contained genes encoding resistance to aminoglycosides, trimethoprim, and quaternary ammonium compounds flanked by directly repeated copies of the insertion sequence (IS)-like element IS431-IS257. The plasmids pG0400 and pG01 were otherwise indistinguishable except for the presence in pG0400 of a 4.5-kb HinDIII fragment encoding mupirocin resistance. The added mupirocin resistance gene was flanked by two directly repeated copies of IS431/257. The nucleotide sequence of DNA contiguous to the outside of the IS elements, as well as those of the elements themselves, was identical in both pG01 and pG0400, and there were no target site duplications flanking either copy of the element. We conclude that the mupirocin resistance gene was added to an existing conjugative plasmid in conjunction with the deletion of other resistance genes by recombination at IS elements. The construction of conjugative plasmids carrying a mupirocin resistance gene may be a model for the mobility of other resistance genes newly acquired by staphylococci.

Mupirocin resistance: clinical and molecular epidemiology.

The antistaphylococcal activity of topical mupirocin has made it an attractive agent for the treatment of asymptomatic colonization with Staphylococcus aureus. Increasing use has been associated with the emergence of mupirocin resistance in staphyloccoci, and failure of therapy has been associated with the isolation of strains exhibiting high-level resistance (MIC > 500 micrograms/mL). Fortunately, low-level mupirocin resistance (MIC < 100 micrograms/mL) occurs most commonly. Because a novel gene encoding for mupirocin resistance resides in both low-level and high-level resistant strains, the emergence of low-level mupirocin resistance may not be as epidemiologically insignificant as previously thought.

Transcriptional regulation by TrsN of conjugative transfer genes on staphylococcal plasmid pGO1.

The major conjugative transfer gene cluster of staphylococcal plasmid pGO1 (trs) consists of 13 open reading frames (trsA to trsM) transcribed from one DNA strand and a single 189-bp open reading frame (trsN) within the first 348 bp of trs that is transcribed divergently. Promoter regions for trsN and trsA partially overlap. TrsN, a 7,181-Da protein, was purified as a fusion to glutathione S-transferase and found to have DNA-binding activity. Increasing concentrations of the fusion protein progressively retarded the gel migration of PCR-generated DNA fragments containing predicted promoters 5' to trsL, trsA, and trsN. The target sequences contained areas of identity, including regions of dyad symmetry, that were protected in DNase I footprinting studies. The binding of TrsN to its trsL target was required for this target DNA to be stably introduced into Staphylococcus aureus on a high-copy-number vector. Provision of excess TrsN from this high-copy-number vector in S. aureus decreased beta-galactosidase activity from a trsL-lacZ transcriptional fusion and decreased pGO1 conjugation frequency. Conversely, both transcription and conjugation increased in the presence of excess trsL target. We propose that TrsN negatively regulates the transcription of genes essential for conjugative transfer by binding to regions 5' to their translational start sites.

DNA sequence and units of transcription of the conjugative transfer gene complex (trs) of Staphylococcus aureus plasmid pGO1.

The conjugative transfer genes of 52-kb staphylococcal R plasmid pGO1 were localized to a single BglII restriction fragment and cloned in Escherichia coli. Sequence analysis of the 13,612-base transfer region, designated trs, identified 14 intact open reading frames (ORFs), 13 of which were transcribed in the same direction. Each ORF identified was preceded by a typical staphylococcal ribosomal binding sequence, and 10 of the 14 proteins predicted to be encoded by these ORFs were seen when an E. coli in vitro transcription-translation system was used. Functional transcription units were identified in a Staphylococcus aureus host by complementation of Tn917 inserts that abolished transfer and by Northern (RNA) blot analysis of pGO1 mRNA transcripts. These studies identified three complementation groups (trsA through trsC, trsD through trsK, and trsL-trsM) and four mRNA transcripts (trsA through trsC [1.8 kb], trsA-trsB [1.3 kb], trsL-trsM [1.5 kb], and trsN [400 bases]). No definite mRNA transcript was seen for the largest complementation group, trsD through trsK (10 kb). Comparison of predicted trs-encoded amino acid sequences to those in the data base showed 20% identity of trsK to three related genes necessary for conjugative transfer of plasmids in gram-negative species and 32% identity of trsC to a gene required for conjugative mobilization of plasmid pC221 from staphylococci.