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Antimicrobial Agents and Chemotherapy, June 2007, p. 2247-2248, Vol. 51, No. 6
0066-4804/07/$08.00+0     doi:10.1128/AAC.00241-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Transient Loss of High-Level Mupirocin Resistance in Staphylococcus aureus Due to MupA Polymorphism{triangledown}

Daniel G. Driscoll, Casey L. Young, and Urs A. Ochsner*

Replidyne, Inc., Louisville, Colorado 80027

Received 16 February 2007/ Returned for modification 18 March 2007/ Accepted 5 April 2007


   ABSTRACT
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Spontaneous loss of MupA-mediated high-level mupirocin resistance was observed in Staphylococcus aureus, although the isolate gave a PCR-positive test result for mupA. Sequencing of the mupA gene identified a single base-pair deletion that resulted in a frameshift mutation and loss of functional protein. Reversion to the wild-type allele and restoration of high-level resistance occurred with high frequency (>10–6), indicating the transient nature of MupA polymorphism.


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Mupirocin is an antibiotic produced by Pseudomonas fluorescens that inhibits protein synthesis via inhibition of isoleucyl tRNA synthetase, thus effectively preventing amino acid activation (6, 7). Topical formulations of mupirocin have been used for many years to treat skin infections such as impetigo and secondarily infected traumatic skin lesions due to staphylococci and streptococci and for the prevention of methicillin-resistant Staphylococcus aureus infections in methicillin-resistant Staphylococcus aureus carriers (4, 9). Increasing resistance to mupirocin has compromised its effectiveness and can lead to clinical failures (11, 15). Recent surveillance data showed that 1.9 to 5.6% of S. aureus isolates and 12.8 to 39.9% of coagulase-negative staphylococci were mupirocin resistant (3). Low-level mupirocin resistance (MIC = 8 to 256 µg/ml) in S. aureus is caused by point mutations in the Rossman fold of the chromosomal ileS gene (1) High-level mupirocin resistance (MIC ≥ 512 µg/ml) is conferred by the acquisition of a second and phylogenetically distinct isoleucyl tRNA synthetase gene, mupA, which encodes an alternate isoleucyl tRNA synthetase whose function is not inhibited by mupirocin (5). Transfer of a conjugative plasmid expressing mupA between staphylococcal strains has been demonstrated in vitro and in vivo and is believed to contribute to the spread of mupirocin resistance (8, 12).

A recent collection of >100 S. aureus isolates showing high levels of mupirocin resistance (Mt. Sinai Hospital, Toronto, Canada) was profiled to determine the MIC of mupirocin and by PCR to detect mupA. DNA of all strains was purified using a QIAGEN DNeasy tissue kit (QIAGEN Inc., Valencia, CA), and PCR was performed using High Fidelity PCR SuperMix (Invitrogen Corporation, Carlsbad, CA), Mup1 and Mup2 primers (Table 1), and published amplification conditions (10). A good correlation between the high-level mupirocin resistance phenotype and a mupA-positive genotype (PCR) was observed. However, one particular isolate, S. aureus MB1348, which had originally been shown to be mupirocin resistant at a high level by Etest (MIC > 256 µg/ml), was reproducibly shown to be mupirocin susceptible (MIC = 0.25 µg/ml) when retested using broth microdilution. The strain had been properly stored as a frozen stock and had undergone only a limited number of passages on swabs and agar plates during shipping and routine strain maintenance. Interestingly, this strain tested positive for the mupA gene by PCR.


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  		TABLE 1. Primer sequences used to amplify portions of the S. aureus mupA gene

 
In order to determine why this mupA-positive isolate was mupirocin susceptible, the entire mupA gene of strain MB1348, including the upstream promoter region, was sequenced. A series of six overlapping primer pairs (Table 1) were designed to amplify the 3.3 kb mupA region, corresponding to bases 2041 to 5341 of the known pUSA03 plasmid sequence of S. aureus strain USA300 (GenBank accession number CP000258). Using these six primer pairs, each fragment was amplified in duplicate by PCR (2 min at 94°C followed by 30 cycles of 1 min each at 94°C, 55°C, and 72°C, with a final extension for 10 min at 72°C). PCR products were purified using a QIAGEN QIAquick 8 PCR purification kit (QIAGEN Inc., Valencia, CA) and directly sequenced in both directions using the same forward and reverse primers (Molecular Biology Core Facility, Barbara Davis Center, University of Colorado Health Sciences Center, Aurora, CO). The sequences were assembled using Sequencher software (Gene Codes Corporation, Ann Arbor, MI), resulting in the 3.3 kb mupA gene of S. aureus strain MB1348. DNA sequence alignments of mupA from MB1348 using plasmid pUSA03 and ClustalW (13) revealed a single base-pair deletion corresponding to position 2441 of the pUSA03 GenBank sequence. The wild-type mupA sequence contains a string of nine adenine residues in this region, the last of which is at position 2441. In the MB1348 mupA sequence, there are only eight adenine residues, resulting in a frameshift mutation that causes out-of-frame translation of 13 codons followed by a premature stop. The wild-type MupA protein contains 1,024 amino acid residues, but the mutant MupA protein from strain MB1348 possesses only the first 93 residues. The truncated MupA protein lacks the conserved Rossman-fold motif KMSKS that is located at amino acid residues 585 to 589 in the wild-type protein and is essential for enzymatic function. As a consequence, the mutated mupA allele does not confer mupirocin resistance.

To investigate whether the mupA mutation was reversible, a single colony of the mupirocin-susceptible strain MB1348 was inoculated into Mueller-Hinton broth, the culture was grown overnight, and 10-fold serial dilutions were plated on agar with or without 512 µg/ml mupirocin. Colonies showing high levels of mupirocin resistance emerged with a calculated frequency of 6.8 x 10–5 after 48 h at 37°C. In an attempt to identify the compensatory mutations in the mupirocin-resistant revertants, DNA was extracted from eight colonies grown on agar containing 512 µg/ml mupirocin and was subjected to PCR amplification and sequencing of the mupA gene as described above. In each of these mupirocin-resistant MB1348 derivatives, the adenine residue at position 2441 was reinserted, thus effectively restoring the wild-type mupA gene and the mupirocin resistance phenotype. Other compensatory mutations, such as single base insertions in the vicinity of the poly(A) string that would restore the proper reading frame, were not detected. It appears that the poly(A) site in the mupA coding sequence constitutes a hot spot for small deletions and insertions. Replication slippage resulting in mononucleotide repeat instability is a particular type of error caused by DNA polymerases that has been described as occurring in both bacterial and eukaryotic cells (2, 14).

In conclusion, our data demonstrate the existence of strains showing potentially high levels of mupirocin resistance that exhibit a mupirocin susceptibility phenotype. Specifically, antimicrobial surveillance that relies entirely on susceptibility testing would not account for such strains unless PCR-based methods to detect resistance markers were also used. Such "wolves in sheep's clothing" are particularly worrisome in the case of nasal carriage or a skin infection treated with mupirocin, since such strains, given the high reversion frequency, would readily become resistant at high levels upon initial exposure to the drug and would likely result in treatment failures.


   ACKNOWLEDGMENTS
 
We thank Allison McGeer at Mount Sinai Hospital in Toronto for providing a collection of mupirocin-resistant S. aureus strains.

The mupA DNA sequence of S. aureus strain MB1348 was deposited in GenBank (accession EF433950).


   FOOTNOTES
 
* Corresponding author. Mailing address: Replidyne, Inc., Dept. Microbiology, 1450 Infinite Dr., Louisville, CO 80027. Phone: (303) 996-5532. Fax: (303) 996-5599. E-mail: uochsner{at}replidyne.com Back

{triangledown} Published ahead of print on 16 April 2007. Back


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Antimicrobial Agents and Chemotherapy, June 2007, p. 2247-2248, Vol. 51, No. 6
0066-4804/07/$08.00+0     doi:10.1128/AAC.00241-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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