LETTER
Pseudomonas aeruginosa is the most prevalent microorganism isolated from the respiratory tract of cystic fibrosis (CF) patients (1). Aminoglycosides are frequently used in the therapy of airway infection for CF patients, especially in the context of nebulized inhaled therapy. In the last decade, acquired 16S rRNA methyltransferases (16S-RMTases) have been described as a novel and high-level resistance mechanism against all aminoglycosides (2–4). RmtD was first described in a P. aeruginosa clinical isolate in Brazil and has subsequently been detected in multiple species of Enterobacteriaceae in Brazil, Argentina, and Chile (3, 5–9). We recently described the second 16S-RMTase, RmtG, which was coproduced with KPC and CTX-M β-lactamases among K. pneumoniae isolates identified in São Paulo State in Brazil between 2010 and 2011 (5).
In this study, we sought to identify 16S-RMTases in P. aeruginosa clinical isolates from CF patients. Clinical samples recovered from the respiratory tract of patients attending the outpatient clinic of the Instituto da Criança (University of São Paulo Medical School), collected during three periods from 2003 to 2004, 2006 to 2007, and in 2009, were sent to the Instituto Adolfo Lutz for culture and biochemical identification. A total of 580 P. aeruginosa isolates from 98 patients were identified. Of these, isolates from patients who had at least 5 isolates in at least two of the periods of time cited above were studied further; these included 129 P. aeruginosa isolates from 11 patients. Disk diffusion tests showed that nine P. aeruginosa isolates were completely resistant to all aminoglycosides (gentamicin, amikacin, and tobramycin). The MICs were determined by Etest, and the isolates were screened for the known 16S-RMTase genes by PCR as previously described (2, 5). As a result, seven serial isolates from patient 3 were found to be positive for rmtG, which was confirmed by DNA sequencing (Table 1). These isolates also presented resistance or diminished susceptibility to ciprofloxacin and levofloxacin and susceptibility to imipenem, meropenem, ceftazidime, cefepime, and polymyxin B (10). Two isolates presented intermediate susceptibility to ticarcillin-clavulanate (Table 1) (10). The P. aeruginosa isolates were subjected to pulsed-field gel electrophoresis (PFGE) using the restriction enzyme SpeI (New England Biolabs) (11) and to multilocus sequence typing (MLST) (12). The database http://pubmlst.org/paeruginosa was used to determine the sequence types (STs). Patient 3 carried P. aeruginosa isolates with the same PFGE profile A, with 3 subtypes, A1, A2, and A3, isolated between 2004 and 2007, and isolates obtained from patient 9 showed PFGE profile B. MLST revealed that all strains belonged to ST235, an international clone that has successfully spread worldwide. Several attempts to obtain transconjugants by mating or transformants by electroporation were unsuccessful. PFGE of S1 nuclease-treated genomic DNA (13), followed by hybridization using an rmtG probe, was also unsuccessful. The gene rmtG is therefore likely located in the chromosome. This is also the first description of 16S-RMTase production in a CF patient to our knowledge. Our case shows that acquired resistance can play a significant role in multiresistance of CF isolates.
TABLE 1.
Antimicrobial susceptibility, PCR results for rmtD and rmtG, and PFGE and MLST profiles of nine P. aeruginosa isolates resistant to aminoglycosides obtained from cystic fibrosis patients
| Isolate | Patient | Age | Mo/yr of isolation | MIC (μg/ml) ofa: |
PCR result for: |
PFGE profile | MLST | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| AK | GM | TM | TZ | PM | TLc | IP | MP | CI | LE | PO | rmtD | rmtG | ||||||
| 302-2 | 3 | 13 | 2/2004 | >256 | >256 | >256 | 3 | 4 | 64/2 | 0.75 | 1.5 | >32 | >32 | 1 | − | + | A | 235 |
| 538 | 3 | 15 | 1/2007 | >256 | >256 | >256 | 0.38 | 1.5 | 0.25/2 | 0.125 | 0.023 | >32 | >32 | 0.19 | − | + | A | 235 |
| 653-1 | 3 | 16 | 5/2007 | >256 | >256 | >256 | 0.75 | 2 | 0.25/2 | 0.25 | 0.047 | >32 | >32 | 0.5 | − | + | A | 235 |
| 653-2 | 3 | 16 | 5/2007 | >256 | >256 | >256 | 0.75 | 1.5 | 0.25/2 | 0.5 | 0.047 | 4 | 2 | 1 | − | + | A1 | 235 |
| 783-1 | 3 | 16 | 8/2007 | >256 | >256 | 192 | 0.75 | 3 | 0.38/2 | 0.38 | 0.094 | 4 | 4 | 0.75 | − | + | A2 | 235 |
| 783-2 | 3 | 16 | 8/2007 | >256 | >256 | >256 | 2 | 4 | 64/2 | 0.75 | 1 | >32 | >32 | 0.75 | − | + | A3 | 235 |
| 783-3 | 3 | 16 | 8/2007 | >256 | >256 | >256 | 0.5 | 1 | 0.25/2 | 0.5 | 0.047 | 4 | 2 | 0.75 | − | + | A2 | 235 |
| 19 | 9 | 8 | 6/2003 | >256 | >256 | 128 | NDb | ND | ND | ND | ND | ND | ND | ND | − | − | B | ND |
| 487 | 9 | 11 | 11/2006 | >256 | 24 | 8 | ND | ND | ND | ND | ND | ND | ND | ND | − | − | B | ND |
AK, amikacin; GM, gentamicin; TM, tobramycin; TZ, ceftazidime; PM, cefepime; TLc, ticarcillin-clavulanate; IP, imipenem; MP, meropenem; CI, ciprofloxacin; LE, levofloxacin; PO, polymyxin B.
ND, not determined.
ACKNOWLEDGMENTS
Part of this work was supported by a research grant from FAPESP (2012/21709-5) to D.D.O.G.
We thank Vera Simonsen, from Instituto Adolfo Lutz, São Paulo, Brazil, for helping us with hybridization experiments.
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