Molecular epidemiology of 16S rRNA methylase genes in Escherichia colifrom humans and animals

Abstract

Background

Aminoglycosides are one of the clinically relevant antibiotics. Plasmid-encoded 16S rRNA methylase enzymes have emerged in clinical isolates of Gram-negative bacteria worldwide. The spread of these resistance determinants has become a great concern.

Objectives

The objectives of this study were to investigate the prevalence of 16S rRNA methylases and aminoglycoside modifying enzyme, AAC(3)-II in Escherichia coli isolated from human blood cultures and faecal samples of animals. E. coli isolates with unexplained aminoglycoside resistance phenotypes were investigated by detection of four aminoglycoside modifying enzymes, AAC(6’)-I, ANT(2”)-I, ANT(4’)-II and APH(3’)-VI.

Methodology

This study included 188 E. coli clinical isolates obtained from blood cultures of patients in one regional hospital between January 2004 and September 2010 and 81 E. coli isolates obtained from faecal samples of chickens, pigs, cattle, cats, dogs and rats between September 2008 and August 2011. All 269 E. coli isolates in this study were screened for the aac(3)-II gene and six 16S rRNA methylase genes(armA, rmtA, rmtB, rmtC, rmtD and rmtE)by two individual sets of multiplex PCR assays. A subset of 88E. coli isolates with aminoglycoside resistance phenotypes, which could not be explained by the genes detected, were subjected to detection of the aac(6’)-Ib, ant(2”)-Ia, ant(4’)-IIaand aph(3’)-Via genes by four individual PCR assays. The transfer of resistance of the rmtB gene was studied by conjugation experiments. The clonal relationship between rmtB-producing strains was investigated by pulsed-field gel electrophoresis.

Results

67.6% (25/37) and 63.4% (26/41) of the Gen-R/Amk-NS group isolates from human and animal sources, respectively, were found to possess the aac(3)-IIgene. The aac(3)-IIgene was also found in 96.7% (146/151) Gen-R/Amk-S group human isolates. 21.6% (8/37) and 61%(25/41) of the Gen-R/Amk-NS isolates from human and animal sources, respectively, were found to possess the rmtB gene. The armA gene was found in one human and one animal isolates, which were both resistant to gentamicin and amikacin. No rmtA, rmtC, rmtD orrmtE genes were found in this study. Among 88E. coli isolates with unexplained aminoglycoside resistance phenotypes, the aac(6’)-Ib gene was found in51.2%(22/43) and 10% (4/40) of the Gen-R/Amk-NS group and the Gen-S/Amk-NS group, respectively. The ant(2”)-Ia gene was found in 11.6% (5/43) of the Gen-R/Amk-NS group E. coli isolates. No ant(4’)-IIa or aph(3’)-Via genes were found. No major PFGE cluster was observed among 32 rmtB-positive isolates by pulsed-field gel electrophoresis.In addition, amikacin resistance could be transferred by conjugation from 12rmtB-positive donors.

Conclusion

The present study showed that the rmtB gene was the most prevalent 16S rRNA methylase gene in both human and animal E. coli isolates. A high incidence of the aac(3)-IIgene was found among gentamicin-resistant strains. The spread of 16S rRNA methylases has aroused clinical concern and become a major therapeutic threat in the future.