| The history of aminoglycosides development is also a history of aminoglycosides resistance In 2003,plasmid-mediated resistance mechanisms of 16S rRNA methylation enzymes which can confer high level resistance to aminoglycosides was reported,and continuous reports were observed in the next 10 more years.To date,10 kinds of 16S rRNA methylase genes such as rmtA have been reported.Although studies of this class of antibiotics in human have become even more clear,it is still difficult to change the status of high level resistance of bacteria.As an important class of antibiotics,aminoglycosides are still widely used in the breeding industry today.In this study,157 fish samples collected from 9 markets in Guangzhou were used as research objects.59 isolates were isolated from 50 samples using MacConkey media containing amikacin and gentamicin,which included 54 E.coli,3 Aeromonas hydrophila,and 2 Citrobacter.The 16S rRNA methylase genes and other resistant genes were detected by PCR and gel electrophoresis.The rmtB detection rate was 98.3%（58/59）,and the armA detection rate was 1.7%（1/59）.The MIC test was performed on the 59 resistant isolates by agar dilution method and found that they were highly resistant against nine drugs such as ampicillin,gentamycin,amikacin,kanamycin,neomycin,florfenicol,tetracycline,and ciprofloxacin,with drug resistance rates ranging from 90%to 100%;the resistance rate to cefotaxime,chloramphenicol,enrofloxacin,and olaquindox were between 80%and 90%;some isolates were resistant to colistin,fosfomycin,tigecycline,and cefoxitin,and the drug resistance rates were all below 15%.At the same time,PCR results showed that,apart from aminoglycoside resistance genes,β-lactams,amides,and fluoroquinolones resistance genes also had a high detection rate.These data indicated that multidrug resistance can be observed even in normal bacterial communities in healthy aquatic animals.Pulsed-field gel electrophoresis,conjugation experiment,S1-PFGE and Southern blotting of rmtB genes were performed on resistant strains to further investigate the prevalence characteristics of the 16S rRNA methylase genes.54 resistant E.coli isolates can be divided into 10 PFGE clusters.One of the clusters containing 34 resistant strains included samples from 8 markets,and the similarity reached more than 91.2%.A total of 10transconjugants were obtained,with a conjugation rate of 16.9%.During the conjugation experiment,the rmtB gene of the original bacterium was successfully transferred.In this process,blaCTX-M-1G,oqxAB,floR and bla NDM genes can co-transfer with the rmtB gene.These co-transfer genes were also successfully expressed after they were transferred to the recipient strain E.coli C600.Among them,the rmtB gene of 7 strains was located on the plasmid of66kb to77kb in length,and the rmtB gene of the remaining 3 strains were located on the plasmids of104kb,138kb,and170kb in length,respectively.All the ten plasmids were IncF plasmids.The data from this study indicated that the 16S rRNA methylase gene was detected in Enterobacteriaceae of freshwater fish in Guangzhou,and the rmtB gene is predominant.In these isolates carrying the 16S rRNA methylase genes,one or more kinds of drug resistance genes are often co-existed,which confered the multidrug resistance;the plasmid carrying the resistance genes can also transfer to other bacteria.These isolates have high levels of resistance to fishery drugs thet are not approved by the Ministry of Agriculture,suggesting that there might be irrational drug use in the breeding process.In such a high-pressure antibiotic environment,bacterial resistance might become more severe. |
