NDM-1 Metallo-β-Lactamase and ArmA 16S rRNA methylase producing Providencia rettgeri clinical isolates in Nepal

Background Drug-resistant Providencia rettgeri producing metallo-β-lactamase and 16S rRNA methylase has been reported in several countries. We analyzed P. rettgeri clinical isolates with resistance to carbapenems and aminoglycosides in a hospital in Nepal. Methods Five clinical isolates of multidrug-resistant P. rettgeri were obtained in a hospital in Nepal. Antimicrobial susceptibilities were determined using the microdilution method and entire genomes were sequenced to determine drug-resistant genes. Epidemiological analysis was performed by pulsed-field gel electrophoresis. Results Four of the 5 isolates were resistant to carbapenems (imipenem and meropenem), with MICs ≥16 mg/L, with the remaining isolate showing intermediate resistance to imipenem, with an MIC of 2 mg/L and susceptibility to meropenem with an MIC ≤1 mg/L. All 5 isolates had blaVEB-1. Of the 4 carbapenem-resistant strains, 3 had blaNDM-1 and 1 had blaOXA-72. All isolates were highly resistant to aminoglycosides (MICs ≥1,024 mg/L) and harbored armA. As the result of pulsed-field gel electrophoresis pattern analysis in the 5 P. rettgeri isolates, 4 had identical PFGE patterns and the fifth showed 95.7% similarity. Conclusions This is the first report describing multidrug-resistant P. rettgeri strains harboring blaNDM-1 or blaOXA-72 and armA isolated from patients in Nepal.

Since then, NDM-1-producing Enterobacteriaceae have been isolated in various parts of the world [9,10].
Exogenously acquired 16S rRNA methylase genes responsible for very high levels of resistance to various aminoglycosides are widely distributed among Enterobacteriaceae and glucose-nonfermentative microbes [11]. Gram-negative pathogens producing 16S rRNA methylase ArmA have been isolated in various countries [11].

Bacterial strains
Five P. rettgeri clinical isolates were obtained from May to July 2012 from 5 patients at Tribhuvan University Teaching Hospital in Kathmandu, Nepal. Three isolates were from sputum and 2 from pus at surgical sites. Samples were obtained as part of standard patient care. Phenotypical identification [17] was confirmed by API 32GN (BioMérieux, Mercy l'Etoile, France) and 16S rRNA sequencing (1,497 bp) [18,19].

Antimicrobial susceptibilities
MICs were determined using the microdilution method, according to the guidelines of the Clinical Laboratory Standards Institute (CLSI) [20]. Breakpoints to antibiotics were determined. The modified Hodge test, the meropenem-sodium mercaptoacetic acid double-disk synergy test (Eiken Chemical, Tokyo, Japan) and E-test (imipenem/ EDTA) (AB Biodisk, Solna, Sweden) were performed.

Entire genome sequencing
The entire genomes of these isolates were extracted and sequenced by MiSeq (Illumina, San Diego, CA). CLC genomics workbench version 5.5 (CLC bio, Tokyo, Japan) was used for de novo assembly of reads and to search for 923 drug-resistance genes, including genes encoding β-lactamases, 16S rRNA methylases and aminoglycoside-acethyl/adenylyltransferases; point mutations in the gyrA, parC and pmrCAB operons; and point mutations in the fos genes, including fosA, fosA2, fosA3, fosC and fosC2.

Nucleotide sequence accession numbers
The nucleotide sequences surrounding bla NDM-1 and bla OXA-72 have been deposited in GenBank with the accession number AB828598 and AB857844, respectively.

Ethical approval
The study protocol was reviewed and approved by the Institutional Review Board of the Institute of Medicine,

Antimicrobial susceptibilities
Four of the 5 isolates were resistant to carbapenems (doripenem, imipenem and meropenem) and piperacillin/tazobactam, whereas the fifth was susceptible to piperacillin/tazobactam, doripenem and meropenem and showed intermediate resistance to imipenem ( Table 1). All 5 isolates were highly resistant to cephalosporins (ceftazidime and cefepime), aztreonam, aminoglycosides (arbekacin, amikacin and gentamicin), ciprofloxacin, colistin and fosfomycin, and all 5 showed intermediate resistance to tigecycline. The four isolates resistant to carbapenems were negative with the modified Hodge test, but three of the four isolates were positive with the meropenem-sodium mercaptoacetic acid double-disk synergy test and E-test/EDTA.

Drug-resistant genes
All 5 isolates tested had several genes associated with β-lactam and aminoglycoside-resistance (Table 1). These isolates had bla VEB-1 , bla OXA-10 , bla TEM-1 , bla ADC-67 (ampC), armA and aadA1; 3 had bla NDM-1 ; and 1 had bla OXA-72 . None of these isolates had any other βlactamase encoding genes, including the class A genes bla SHVs and bla CTX-Ms ; the class B genes bla AIM , bla DIM , bla FIM , bla GIM , bla IMPs , bla INDs , bla KHM , bla SIM , bla SMB , bla SPM , bla TMBs , and bla VIMs ; or the class D gene bla OXAs except for bla OXA-10 and bla OXA-72 . None had other genes encoding 16S rRNA methylases or aminoglycoside acetyl/adenylyltransferases. All 5 isolates had point mutations in the quinolone-resistance-determining regions of gyrA and parC, with amino acid substitutions of S83I and D87E in GyrA and S80I in ParC, but none had any mutations in the pmrCAB operon and fos genes. All sequences of the drug-resistant genes tested were identical to those registered in GenBank.

PFGE and southern hybridyzation
Of the 5 P. rettgeri isolates, 4 had identical PFGE patterns and the fifth showed 95.7% similarity (Figure 1). Three of these isolates had a plasmid harboring bla NDM-1 and one had a plasmid harboring bla OXA-72 , with plasmid sizes ranging from 9.42 to 23.1 kbp (data not shown).

Conclusions
To our knowledge, this is the first report describing P. rettgeri strains harboring bla NDM-1 or bla OXA-72 and armA isolated from patients in Nepal. These 5 strains were highly resistant to both β-lactams and aminoglycosides and expanded in a clonal manner in the hospital.

Competing interests
The authors declare that they have no competing interest.
Authors' contributions TT: Performed PCR and sequencing, analyzed data and drafted the manuscript. TMA: Performed entire genome sequencing. RKD and MKS: Performed drug susceptibility tests. HO: Supervised this study. KS: Performed pulsed-field gel electrophoresis and its pattern analysis. TK and BMP: Designed protocols and supervised this study. All authors read and approved the final manuscript.