Prevalence of 16S rRNA methylase genes in Enterobacteriaceae isolates from a Greek University Hospital

Methylation of 16S ribosomal RNA (rRNA) has recently emerged as a new mechanism of resistance against aminoglycosides among gram-negative pathogens [1]. It confers high-level resistance to all parenterally administered aminoglycosides that are currently in clinical use, as well as ACHN-490, a next-generation aminoglycoside, currently in early clinical development [2]. Six distinct genes, rmtA, rmtB, rmtC, rmtD, armA and npmA encoding their respective enzymes (16S rRNA methylases) have been identified in clinical and veterinary strains from various geographic areas, including East Asia, Europe and the Americas, since 2003 [1,3,4]. Those genes are mostly located on transposons within transferable plasmids, which provides them with the potential to spread horizontally and may in part explain the already worldwide distribution of this novel resistance mechanism [5–8]. Some of these organisms have been found to co-produce extended-spectrum β-lactamases or carbapenemases, contributing to their multidrug-resistant phenotypes.

The aim of this study was to investigate the occurrence of 16S rRNA methylase genes in Enterobacteriaceae isolates from a university hospital in Athens.

The computerized database of Infectious Diseases Research Laboratory was retrospectively searched for Enterobacteriaceae strains isolated during a 2-year period (November 2007–October 2009) from inpatients and outpatients of the Attikon University General Hospital, Athens, Greece. Only one isolate per patient was included in the study. Species identification of isolated bacteria and MIC determinations were performed using an automated system (BD Phoenix automated microbiology system; BD Diagnostic Systems, Sparks, MD, USA). Isolates resistant to amikacin and gentamicin were tested further for MIC determination to amikacin, gentamicin, tobramycin, netilmicin, apramycin and neomycin (Sigma-Aldrich, St Louis, MO, USA) with the agar dilution technique following the Clinical and Laboratory Standards Institute (CLSI) guidelines [9]. Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as control strains. Results were interpreted in accordance with European Committee on Antimicrobial Susceptibility Testing criteria [10].

Isolates with MICs ≥256 mg/L to amikacin, gentamicin, tobramycin and netilmicin, were examined for the presence of 16S rRNA methylase genes by multiplex PCR [1]. Genomic fingerprinting was carried out by repetitive-element PCR (rep-PCR) analysis using primers 5′ III GCG CCG ICA TCA GGC 3′, and 5′ ACG TCT TAT CAG GCC TAC 3′. β-Lactamases were determined by isoelectric focusing and the presence of the responsible genes was confirmed by PCR [11,12]. Sequencing of the PCR products was performed by MWG-THE Genomic Company (Ebersberg, Germany). Conjugation experiments were performed by the broth mating technique, with E. coli K12 strain RC85 R– (rifampicin-resistant; MIC >128 mg/L) as the recipient. Transconjugants were selected on agar supplemented with rifampicin (128 mg/L) and gentamicin (20 mg/L).

Gram-negative isolates belonging to species: Klebsiella pneumoniae (n = 1534), E. coli (n = 1398), Proteus mirabilis (n = 734) and Enterobacter spp. (n = 414) were included in the study. In K. pneumoniae the percentage of resistance to amikacin and gentamicin was 48.2% and 8.1%, respectively, whereas resistance to both aminoglycosides was 0.9%. In E. coli amikacin was more active with 3.6% of the isolates being resistant, whereas 15.1% were resistant to gentamicin and only 0.4% of the isolates were resistant to both aminoglycosides. In P. mirabilis 8.0%, 40.5% and 1.1% were resistant to amikacin, gentamicin and both aminoglycosides, respectively. Finally Enterobacter spp. isolates exhibited resistance rates of 12.5% to amikacin, 8.5% to gentamicin and 1.0% to both aminoglycosides, respectively.

A high percentage of resistance to amikacin among K. pneumoniae isolates and to gentamicin among P. mirabilis isolates was observed. However, resistance to both amikacin and gentamicin was less common (0.4–1.1%). Fourteen K. pneumoniae, five E. coli, eight P. mirabilis and four Enterobacter spp. isolates were further evaluated by agar dilution for high-level resistance to amikacin, gentamicin, tobramycin and netilmicin. Among them, three K. pneumoniae (0.2%) and three P. mirabilis isolates (0.4%) were highly resistant to all four aminoglycosides (MICs ≥256 mg/L) whereas MICs to apramycin ranged from 4 to 16 mg/L (Table 1). All isolates were positive for rmtB.

All rmtB-bearing isolates were clonally unrelated. Two of the K. pneumoniae isolates were KPC-2 and OXA-10 producers whereas the third one was a DHA-1 producer (Table 1). One P. mirabilis isolate was a VIM-1 and OXA-10 producer, one was an OXA-10 producer and one was not an ESBL producer. None of the E. coli or Enterobacter spp. isolates was positive for armA, rmtA, rmtB, rmtC or rmtD genes. Conjugation experiments were successful for one K. pneumoniae (m2573A) and two P. mirabilis (m642C, m2651A) isolates. RmtB was co-transferred with bla_OXA-10 and bla_TEM-1 in all three cases. Additionally bla_VIM-1 was co-transferred from P. mirabilis m642C to the E. coli transconjugant.

This is the first report on the occurrence of 16S rRNA methylases in Enterobacteriaceae in Greece and the first report on the occurrence of rmtB in P. mirabilis in Europe. We have found a low prevalence of rmtB (0.2% among K. pneumoniae and 0.4% among P. mirabilis), lower than that found in Turkey (0.7%) and France (1.3%) among ESBL-producing Enterobacteriaceae and higher than that reported in Belgium (0.12%) among Enterobacteriaceae consecutively collected between 2000 and 2005 [13,14]. Currently, the 16S rRNA methylase seems to be more prevalent in Asia than in Europe and the Americas [1]. RmtB was associated with carbapenemase or acquired AmpC-type production and OXA-10 in K. pneumoniae and P. mirabilis isolates. The spread of extensively drug-resistant isolates co-producing a carbapenemase or an acquired AmpC, an ESBL and 16S rRNA methylases raises clinical concern and may become a major therapeutic threat in the future. Furthermore, these ribosomal methylases may compromise the usefulness of ACHN-490, a much anticipated novel aminoglycoside with potent activity against a variety of gram-negative pathogens.

Funding

This work was supported by funds from Athens University to H. Giamarellou.

Transparency Declaration

Nothing to declare.