Aminoglycoside-modifying enzyme and 16S ribosomal RNA methyltransferase genes among a global collection of Gram-negative isolates

Highlights

• Surveillance studies of aminoglycoside modifying enzymes (AMEs) are scarce.

• 200 Gram-negative clinical isolates were screened for AMEs and 16S rRNA methyltransferase genes Isolates were collected worldwide and resistant to amikacin and/or gentamicin and/or tobramycin AMEs were very prevalent among Enterobacterales, Acinetobacter spp. and P. aeruginosa.

• Cloning of a new rmtB variant and AMEs not previously characterized was also performed.

Abstract

Objectives

The prevalence of genes encoding aminoglycoside-modifying enzymes (AMEs) and 16S rRNA methyltransferases among 200 Gram-negative clinical isolates resistant to different aminoglycosides and collected worldwide during 2013 was evaluated.

Methods

Selected AMEs and 16S rRNA methyltransferase genes were screened by PCR/sequencing among 49 Acinetobacter spp., 52 Pseudomonas aeruginosa and 99 Enterobacterales.

Results

In total 72 isolates carried aac(6′)-lb variants (36.0% overall; 55.6% Enterobacterales): 30 aac(6′)-Ib-cr, 21 aac(6′)-Ib and 21 aac(6′)-Ib-like displaying substitutions L119S (alone or in combination with V71A or R173K) or S100G. Ten aph(3′)-VI variants were detected among 35 isolates (46.9% of Acinetobacter spp.). Nineteen isolates carried variants of aac(3)-I, with aac(3)-Ia (n = 13, mostly Acinetobacter spp.) being the most prevalent. Other AME genes detected were ant(3″)-Ia (n = 41), ant(2″)-Ia (n = 24), aac(3)-IIe (n = 23), aac(3)-IId (n = 21), aac(6′)-Im (n = 13, mostly P. aeruginosa), aacA8 (n = 3), aac(3)-IIf (n = 1) and aac(3)-IVa (n = 1). Among 42 isolates resistant to amikacin, gentamicin and tobramycin tested for 16S rRNA methyltransferase genes, 21 (50.0%) tested positive; armA was most common (n = 14), but 4 isolates carried rmtB1, 2 rmtF1 and 1 new variant rmtB4. Over 60 gene combinations, consisting of one to four AMEs and 16S rRNA methyltransferases, were observed. Cloning genes not previously characterised revealed diverse aminoglycoside resistance patterns for some AMEs, but expected results for rmtB4.

Conclusions

Studies broadly evaluating these aminoglycoside resistance genes are needed. Using agents stable in the presence of these resistance genes might help overcome resistance.

Introduction

Aminoglycosides play an important role in the treatment of aerobic, Gram-negative infections, usually in combination with β-lactam agents. In the USA, the most commonly prescribed aminoglycosides for the treatment of serious infections include gentamicin, tobramycin and amikacin [1]. These and other aminoglycoside agents act by binding at the A-site of the 30S ribosomal subunit where codon–anticodon accuracy is assessed; the binding interferes with the function of the 16S rRNA ribosomal subunit and inhibits protein synthesis [2]. Many bacterial species have developed resistance mechanisms to aminoglycosides, including antimicrobial modification, ribosomal alteration and decreased permeability.

In the clinical setting, resistance to aminoglycosides is primarily mediated by aminoglycoside-modifying enzymes (AMEs) [3], [4]. AMEs have significant clinical importance because the genes encoding these enzymes can be disseminated by plasmids or transposons and are often detected as part of gene cassettes carried by integrons that usually harbour other resistance markers, including metallo-β-lactamases, facilitating their selection [4]. AMEs inactivate aminoglycosides by catalysing the modified amino or hydroxyl groups through the process of acetylation (AAC), phosphorylation (APH) and/or adenylation (ANT) [4]. Each enzyme has a unique resistance phenotype owing to its varying effects on particular aminoglycosides [2].

Previous studies have noted that aminoglycoside resistance in Acinetobacter baumannii is usually encoded by APH and ANT and in Pseudomonas aeruginosa by AAC [5]. Because the AMEs in A. baumannii and P. aeruginosa are often associated with transferable plasmids and integrons, these genes can be shared with other Gram-negative species through horizontal gene transfer [5], [6], [7]. It has also been observed that the prevalence of aminoglycoside resistance mechanisms in Enterobacterales (previously Enterobacteriaceae) isolates correlates with aminoglycoside usage and changes with time and location [5].

Although less common, methylation of the 16S ribosomal subunit through the action of plasmid-mediated methyltransferases is a serious threat to the aminoglycoside class of antimicrobials. These enzymes methylate specific nucleotides of the ribosomal target sites that restrict binding of the aminoglycoside and result in high-level resistance to almost all aminoglycosides [8]. Currently, ten 16S rRNA methyltransferase enzymes have been identified (RmtA–H, ArmA and NpmA). The genes encoding these enzymes have all been shown to be plasmid-mediated [8] and are often associated with β-lactamases, including carbapenemases, which can facilitate their selection and dissemination [8], [9], [10].

The aim of this study was to evaluate the prevalence of common aminoglycoside resistance genes, including genes encoding AMEs and 16S rRNA methyltransferases, among 200 Gram-negative clinical isolates composed of Acinetobacter spp., P. aeruginosa and Enterobacterales species collected worldwide in 2013 displaying distinct resistance patterns for amikacin, tobramycin and gentamicin.