| Aminoglycosides are clinically effective agents for treatinga broad range of life-threatening infections caused byboth Gram-negative and Gram-positive pathogens,usually in combination with β-lactam agents.However, resulting from the overuse of aminoglycosides, resistance has become increasingly prevalent, thus compromising theirtherapeutic efficacy.Resistance to aminoglycosides is frequently due to theacquisition of modifying enzymes that vary in their substrate ranges, such as acetyltransferases, phosphorylasesand adenylyltransferases,unlike modifying enzymes, post-transcriptional methylation of the 16S rRNA, has been reported since 2003,and results in high-level resistance to most aminoglycoside antibiotics such as arbekacin, gentamicin, tobramycin, andamikacin.The horizontal transfer of 16S rRNA methylase genes through plasmids, integrons andtransposons has been found to play an important role in thedissemination of aminoglycosides resistance genes.The changing status of pets in society has allowed more and closer physical contactbetween humans and their pets(eg, through shared living spaces) and has created anopportunity for humans and pets to exchange microorganisms.Thus, we decide to identify the prevalence of 16S rRNA methylase genes as well as its molecular characterization among Enterobacteriaceaeisolates from hospitalized animals in several veterinary hospitals in Guangdong, China. 1. Prevalence of 16S rRNAmethylases genesAmong 381 Enterobacteriaceae isolates from pets in China from 2008 to 2012, 77 isolates which exhibiting animoglycosides resistancewere selected and screened for 16S rRNA methylase genes(arm A, rmt A, rmt B, rmt C, rmt D, rmt E, rmt F, rmt G, and npm A) by PCR and sequencing. Among them, 61(16.01%,61/381)isolates were positive for rmt B, 4(1.05%,4/381) isolates harboring arm A,and 2(0.52%,2/381) isolates were positive for both rmt B and arm A.Thermt A, rmt C, rmt D, rmt E, rmt F, rmt G and npm A genes were not detected in any of the tested isolates.All isolates resistant to both gentamicin and amikacin were tested for susceptibility to 14 antimicrobial agentsby agar dilution method.The results of MICs of antimicrobial revealed that there was a very high frequency(>90.0%) of tetracycline, gentamicin, sulfamethoxazole/trimethoprim, ampicillin, kanamycinresistance among these strains harboring16S rRNA methylase genes; the resistance rates to cefotaxime, norfloxacin, florfenicol, ciprofloxacin, chloramphenicol, streptomycin, amikacin, neomycin were varied from 50.0% to 90.0%; the resistance rate below 50% was apramycin. The top five were tetracycline(98.70%), ampicillin(97.40%), sulfamethoxazole/trimethoprim(96.10%),gentamicin(94.81%) and kanamycin(94.81%); the last three were apramycin(41.56%), florfenicol(53.25%) and cefotaxime(55.84%).These data illustrate that multiple drug resistance problemin 16S rRNA methylase-producing Enterobacteriaceae isolates from pets is very serious. 2. Transmission mechanisms of 16S rRNA methylase genesIn order to analyse whether clonal spread was responsible for the dissemination of thermt B and arm A genes, PFGEwas carried out.Most E.coli strains wereclonally unrelated, while theexisting of strains with the same PFGE profiles indicated the clonal transmission ofrmt B-positive Klebsiella pneumoniaeisolates in the same veterinary hospital. Conjugation, RFLP and Southern blotwere also used to study the horizontal transmission of rmt B and/or arm A. Conjugation experiments showed that rmt B transferred to the recipients, bla CTX-Malso co-transfered with rmt B in some cases. Rmt B were located on Inc F33 and Inc F2 conjugative plasmids in size ranging from ~60kb to ~196kb in E.coli, whereas,in K. pneumoniae, rmt B were located on ~97kb Inc F33 plasmids.Nearly the same Eco RI digestion patterns among the same subtypes of Inc Findicated that Inc F conjugative plasmids were responsible for the dissemination andtransmission of both rmt B and bla CTX-M-55 in Klebsiella pneumoniae.Some rmt B-bearing plasmids of E.coli and K. pneumoniaesharing similar RFLP profile indicating that this plasmid may transferbetween different bacteria.Thermt Bgene was distributed widely in companion animals in China and spread rapidly all around the country.Both colonal transfer and horizontal transfer of Inc F plasmids contributed to the dissemination of rmt B and blaCTX-Min Klebsiella pneumoniae, horizontal transmission of the same plasmid existed in some E.coli strains. The rmt Bin E.coliwere located on Inc Fconjugative plasmids in different sizes,while rmt B and blaCTX-M-55 were on the same size Inc F33 conjugative plasmids in Klebsiella pneumoniae. Spread ofcompanion animal origin Enterobacteriaceae harboring both rmt BblaCTX-M genes have grown up to be a serious problem in public health. Dissemination of resistance in companion animals will undoubtedly continue to be a challenge in veterinary medicine, from both patient health and public health standpoints, organized surveillance is required to better understand the scope of the prevalence, and to identify measures that may be used to decrease the impact of this problem. |
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