Abstract
Staphylococcus aureus resistance to mupirocin is often caused by acquisition of a novel isoleucyl-tRNA synthetase encoded on the plasmid gene mupA. We tested S. aureus isolates from children at Texas Children's Hospital with recurrent skin and soft tissue infections for mupirocin resistance and mupA. Of 136 isolates, 20 were resistant to mupirocin (14.7%). Fifteen isolates (11%) carried mupA, and the gene was more common in methicillin-susceptible S. aureus (21.4%) than methicillin-resistant S. aureus (8.3%; P = 0.03). Seven of 20 mupirocin-resistant isolates displayed clindamycin resistance.
INTRODUCTION
Mupirocin inhibits isoleucyl-tRNA synthetase, an enzyme necessary for bacterial protein synthesis. Mupirocin resistance exists as high-level resistance (HMR), with a MIC that is ≥512 μg/ml, or low-level resistance (LMR), with a MIC between 8 and 256 μg/ml (7). HMR is mediated by mupA, a plasmid-associated gene, which encodes a novel isoleucyl-tRNA synthetase (8, 9). LMR is facilitated by a mutation in the native gene ileS-1 (1).
Mupirocin resistance in adults ranges from 2 to 28% (14, 16). Few data are available on mupirocin resistance in children. We determined the prevalence of mupirocin resistance among S. aureus isolates causing recurrent skin and soft tissue infections (SSTI) at Texas Children's Hospital (TCH).
Isolates were chosen from a Staphylococcus aureus database approved by the Baylor College of Medicine Institutional Review Board (12). We reviewed medical records for patients who had at least three SSTI separated by at least 6 months between 2001 and 2009; patients with immunocompromising or chronic skin diseases were excluded. The initial and last isolates were analyzed. Isolates were classified as community acquired (CA), community-onset health care associated (CO-HCA), or nosocomial (11).
Oxacillin susceptibility was determined following Clinical and Laboratory Standards Institute guidelines (3). Mupirocin MICs were determined by Etest (Biomériux, Sweden). Mupirocin agar dilution was used for a mupA+ strain with an LMR phenotype to identify resistant subpopulations.
A mupA probe was obtained using S. aureus control strain FPR3757, published mupA primers (4), and the DIG-High Prime labeling kit (Roche, Mannheim, Germany) according to the manufacturer's instructions and then used for a dot blot hybridization assay. Lysed bacterial suspensions were transferred to a Hybond-N+ membrane (GE Healthcare, Piscataway, NJ) by using a dot blot apparatus (Schleicher & Schuell, Keene, NH).
Pulsed-field gel electrophoresis (PFGE) was performed on SmaI digestions of mupA-positive and/or resistant isolates and their pairs (11). For two isolates with a discrepancy between their MICs and the presence or absence of mupA, the mupA and ileS-1 genes were sequenced (Seqwright, Inc.).
A total of 136 isolates representing 68 patients were available for molecular testing and were included in the final analysis. A total of 108 (79.5%) isolates were methicillin-resistant S. aureus (MRSA) and 28 (20.5%) were methicillin-susceptible S. aureus (MSSA). Twenty (14.7%) mupirocin-resistant isolates were identified from 16 patients; 16 of the isolates were HMR (Table 1).
Table 1.
Prevalence of mupirocin resistance and mupA
| Isolate group | Total no. of isolates with indicated level of mupirocin resistance/total no. of isolates (%) |
Total no. of mupA-positive isolates/total no. analyzed (%) | ||
|---|---|---|---|---|
| Resistant | HMR | LMR | ||
| Overall | 20/136 (14.7) | 16/136 (11.7) | 4/136 (2.9) | 15/136 (11) |
| Initial isolates | 7/68 (10.3) | 5/68 (7.4) | 2/68 (2.9) | 5/68 (7.3) |
| Recurrent isolates | 13/68 (19.1) | 11/68 (16.1) | 2/68 (2.9)a | 10/68 (14.7)b |
| MRSA | 14/108 (13) | 11/108 (10.1) | 3/108 (2.7) | 9/108 (8.3) |
| MSSA | 6/28 (21.4) | 5/28 (17.8) | 1/28 (3.5)c | 6/28 (21.4)d |
P = 0.06 for comparison of initial versus recurrent isolates for mupirocin resistance.
P = 0.09 for comparison of initial versus recurrent isolates for mupA positivity.
P = 0.1 for comparison of MRSA versus MSSA for mupirocin resistance.
P = 0.03 for comparison of MRSA versus MSSA for mupA positivity.
The mupirocin MIC50 was 0.064 μg/ml (range, 0.064 to >1,024 μg/ml), which did not change over time. Resistance occurred more commonly among recurrent (19.1%) than initial (10.3%) isolates (P = 0.06). The rate of mupirocin resistance was higher among MSSA (21.4%) than MRSA (13%) isolates.
Eleven of 16 (68.8%) patients with mupirocin-resistant isolates were nonwhite, which was similar to the frequency for susceptible isolates (35/52, or 67.3%; P = 0.46). The median age of patients with resistant isolates was 61.5 months versus 29.5 months among susceptible isolates (P = 0.07).
Fifteen isolates carried mupA, occurring more commonly among recurrent (14.7%) than initial (7.3%) isolates (Table 1). mupA carriage was greater among MSSA (21.4%) than MRSA (8.3%) isolates (P = 0.03). One mupA-positive isolate displayed an LMR phenotype; sequencing revealed a single adenine residue deletion that caused a premature stop codon. Mupirocin agar dilution from 33 colonies from this isolate revealed that 31/33 clones had a mupirocin MIC of <2 μg/ml; one isolate had a MIC of 4 μg/ml, and another had a MIC of 16 μg/ml. The subclones were found to have identical PFGE patterns. Two mupA-negative isolates were HMR. Sequencing of both isolates identified no mutations in the ileS-1 gene.
From review of medical records, mupirocin was used in 16/68 patients prior to acquisition of the isolate. Five of these 16 patients with resistant isolates had previous mupirocin exposure.
A total of 28/136 organisms exhibited clindamycin resistance (20.4%). Seven of 20 mupirocin-resistant organisms were clindamycin resistant (35%; P = 0.05); 5/15 mupA-positive isolates were also clindamycin resistant.
Isolates were derived from CA (n = 78), CO-HCA (n = 56), and nosocomial (n = 2) infections; 13/20 mupirocin-resistant isolates and 12/15 mupA-positive isolates were CA. By PFGE, 18 mupirocin-resistant organisms were USA300 (Table 2).
Table 2.
Comparison of paired patient isolates
| Patient no.a | Initial or recurrence isolateb | Diagnosis | Methicillin resistance phenotype | Mupirocin resistance phenotype | Mupirocin MIC (μg/ml) | mupA | PFGE type |
|---|---|---|---|---|---|---|---|
| 1 | Initial | Pustule | MRSA | LMR | 16 | Negative | USA300.0114 |
| Recurrence | Abscess | MRSA | S | 0.064 | Negative | USA300.0114 | |
| 2 | Initial | Paronychia | MRSA | S | 0.064 | Negative | USA300.0114 |
| Recurrence | Pustule | MRSA | HMR | >1,024 | Positive | Non-USA300 | |
| 3 | Initial | Pustule | MRSA | LMR | 128 | Negative | USA300.0114 |
| Recurrence# | Abscess | MRSA | S | 0.5 | Negative | USA300.0114 | |
| 4 | Initial | Abscess | MRSA | S | 0.094 | Negative | USA300.0114 |
| Recurrence | Abscess | MRSA | LMR | 128 | Negative | USA300.0114 | |
| 5 | Initial | Abscess | MRSA | HMR | 512 | Negative | USA300.0114 |
| Recurrence | Abscess | MRSA | S | 0.094 | Negative | USA300.0114 | |
| 6 | Initial | Abscess | MRSA | HMR | >1,024 | Positive | USA300.0114 |
| Recurrence# | Abscess | MSSA | S | 0.25 | Negative | USA300 var 1c | |
| 7 | Initial# | Abscess | MRSA | HMR | >1,024 | Positive | USA300.0114 |
| Recurrence# | Abscess | MRSA | HMR | >1,024 | Positive | USA300 var 2 | |
| 8 | Initial | Abscess | MSSA | HMR | >1,024 | Positive | USA300 var 3 |
| Recurrence | Abscess | MSSA | HMR | >1,024 | Positive | Non-USA300 | |
| 9 | Initial | Abscess | MSSA | S | 0.064 | Negative | USA300 var 4 |
| Recurrence | Abscess | MRSA | HMR | >1,024 | Positive | USA300.0114 | |
| 10 | Initial | Pustule | MRSA | S | 0.19 | Negative | USA300.0114 |
| Recurrence# | Abscess | MSSA | HMR | >1,024 | Positive | USA300 var 1 | |
| 11 | Initial# | Abscess | MRSA | S | 0.125 | Negative | USA300.0114 |
| Recurrence | Abscess | MRSA | HMR | >1,024 | Positive | USA300.0114 | |
| 12 | Initial | Abscess | MRSA | S | 0.094 | Negative | USA300.0114 |
| Recurrence | Abscess | MRSA | HMR | >1,024 | Positive | USA300.0114 | |
| 13 | Initial | Lymphadenitis, cellulitis | MSSA | LMR | 192 | Positive | USA300 var 1 |
| Recurrence | Abscess | MSSA | HMR | >1,024 | Positive | USA300 var 1 | |
| 14 | Initial | Abscess | MRSA | HMR | 512 | Positive | USA300.0114 |
| Recurrence | Abscess | MRSA | HMR | 512 | Negative | USA300.0114 | |
| 15 | Initial | Abscess | MSSA | S | 0.064 | Negative | USA300 var 1 |
| Recurrence# | Abscess | MSSA | HMR | >1,024 | Positive | USA300 var 1 | |
| 16 | Initial# | Abscess | MRSA | S | 0.125 | Negative | USA300 var 5 |
| Recurrence | Abscess | MRSA | HMR | >1,024 | Positive | USA300 var 5 |
Patients include all those with at least one mupirocin-resistant and/or mupA-positive isolate along with its pair.
#, the isolate was obtained after mupirocin exposure.
var denotes a USA300 variant.
This is one of two studies examining mupirocin resistance in children in a community setting. We found mupirocin resistance in 14.7% of isolates, which is higher than the rate of 1.8% determined in the other community-based study of children (10).
mupA was carried significantly more frequently by MSSA than MRSA, in contrast to previous studies (2). USA300 has been associated with carriage of multidrug resistance plasmids, such as pUSA03, which confers resistance to mupirocin and clindamycin (5). Studies of S. aureus USA300 isolates from HIV-positive men found that all isolates harboring pUSA03 were mupirocin resistant (5).
Eighteen of 20 mupirocin-resistant isolates were USA300 by PFGE. Of the mupA-positive isolates, one-third were clindamycin resistant, consistent with acquisition of multidrug resistance determinants by USA300. Studies at our institution have shown higher rates of clindamycin resistance among MSSA than among MRSA (12). This may explain the higher prevalence of mupA among MSSA. Furthermore, both clindamycin- and mupirocin-resistant S. aureus isolates have disseminated in our community; four of five clindamycin-resistant, mupA-positive isolates were isolated from community-acquired infections.
One isolate with an LMR phenotype possessed mupA. Such a discrepancy has been described when mutations occur within mupA or when the plasmid-mediated gene becomes incorporated into the chromosome (6, 13). Our sequencing data revealed a single nucleotide deletion that caused a frameshift and a nonfunctional peptide. Population analysis further revealed that this isolate contained an LMR subpopulation. The LMR clone may have obtained further mutations within ileS-1, partially restoring the resistance phenotype, or it may have lacked the single nucleotide deletion in mupA. As noted by others (15), two isolates were found to have an HMR phenotype but lacked mupA. No mutations were noted within the native ileS-1 gene, and it is possible that these isolates acquired HMR by another unknown mechanism.
Our retrospective design limits a complete determination of mupirocin use in our community. Furthermore, the true incidence of mupirocin resistance may be underrepresented. We only evaluated SSTI, and from these data it is difficult to state the impact that mupirocin resistance may have on invasive disease.
The rate of mupirocin resistance among pediatric S. aureus isolates in the Houston area is approximately 15% and is particularly a problem for infections by community-acquired MSSA and clindamycin-resistant isolates. Continued surveillance of mupirocin resistance is warranted.
Acknowledgments
This study was supported in part by Pfizer.
Footnotes
Published ahead of print on 31 January 2011.
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