| In recent years, accompanied with the mass production and usage of antibiotics, bacterial resistance(AR) has became a global public health problem. The antibiotic genes were detected in various bacteria from soil, water and sewage. Procambarus clarkii live in freshwater where bacteria exist, these bacteria not only carry various AR genes, genes of different species can also exchange by mobile elements such as plasmids, insertion sequences and transposons,and they can transfer resistance genes to other organisms or humans. Living in these environment, Procambarus clarkii can carry bacteria with AR genes. At present, reports on monitoring and researching of antibiotic resistance from aquatic bacteria are less available in China.In order to explore the prevalence and mechanism of antibiotic resistance in ESBLs-producing E.coli from Procambarus clarkii, we investigated AR gene profiles,susceptibility to common antibiotics, mobile genetic elements and plasmids transferability of these bacteria from Procambarus clarkii. 107 ESBLs-producing E. coli from 198 procambarus clarkii were collected from five provinces in China(Shandong, Jiangsu, Zhejiang, Dalian,Hubei). The antimicrobial susceptibility and ESBLs phenotypes of these stains were determined by cefotaxime-containing medium, PCR and BD Phoenix TM-100 system. Following analysis, the resistance genes of ESBLs, PMQR and 16 s r RNA methyltransferase were detected by PCR.Then conjugation experiment was conducted to confirm mechanisms of horizontal transferability of the plasmids and to confirm the resistance genes in transconjugants. The results showed:The ratio of ESBLs-producing E. coli was 54%. All ESBLs-producing E. coli stains were resistant to penicillins, the first generation cephalosporins and part of the second generation cephalosporins. The average drug-resistance rate to tetracyclines, fluoroquinolones,chloramphenicols and sulfonamides was above 60%(>65/107). The resistance rate to aminoglycosides was lower. All strains were sensitive to the carbapenems. Among these ESBLs-producing E.coli strains, the percentage of multi-drug resistant(R≥3classes) isolates was86.7%(96/107), isolates with resistance to five(30%, 31/107) or six(28%, 30/107) classes of drugs were predominant.All of the 107 ESBLs-producing E.coli isolates harbored bla CTX-M genes, in which the bla CTX-M-1group was the most popular(74%, 78/107) and followed by bal CTX-M-9group(20%,21/107) and bla CTX-M-2group(7.48%, 8/107). 54 bla TEM strains(50.5%) were detected in the 107ESBLs-producing E.coli strains and they are all bla TEM-1 type. Nine isolates carried bla Amp C genes(8.4%) were detected, among them, 7 isolates carried bla CMY-2 genes(87.5%, 7/8) and one strains carried bla DHA gene(0.9%). Another isolate carried bla CMY-42 type ESBLs, this is the first time that bla CMY-42 type ESBLs was detected in ESBLs-producing E.coli stains from Animal Derived Food, other genes including bla MOX, bla ACC, bla EBC, bla FOX and bla SHV were not detected.Bla CTX-M-1 group genes was the most common gene in the five provinces. As for non-ESBLs PMQR genes, there were 38 isolates harboring qnr S genes(35.5%, 38/107), including qnr S1 type and qnr S2 type, followed by oqx A genes(33.6%,36/107), oqx B genes(32.7%, 35/107),aac(6’)-Ib-cr genes(18.7%, 20/107) and qep A genes(1.87%, 2/107). Among the 107ESBLs-positive E.coli isolates, there were 37 isolates harboring over 2 kinds of PMQR genes and the combination of oqx A-oqx B were the most common. Twelve 16 s r RNA methyltransferase-positive isolates were detected and the rates of arm A gene and rmt B genes were respectively 0.93%(1/107) and 11.2%(12/107). The rmt B genes were mainly distributed in the Shandong province and Jiangsu province, only one isolates harboring arm A and rmt B genes was detected in Liaoning province.Eleven types of plasmids were detected from 107 ESBLs-producing E.coli isolates,Inc FIIA type(52.3%, 56/107) was the most popular, and most of them carried 2 types(31.8%,34/107). Meanwhile, there were 77 isolates carried class I integrase genes(72.0%, 77/107), only1 isolate carried class II integrase gene(0.9%). Among the 77 class I integrase-positive isolates,there were 43 isolates(55.8%, 43/77) harbored gene cassettes, dominated by dfr A17-aad A5 gene cassettes(32.6%, 14/43), the class II integrase-positive isolates carried drf A12-sat2-aad A1 gene cassette. Conjugation experiments was conducted in which fifty-seven isolates that carrying the bla CTX-M genes were randomly selected and used as donors, E.coli J53 bacteria was used as the recipients, The results showed that 43 isolates were conjugated successfully(75.4%, 43/57) and all of the transconjugants carried bla CTX-M genes. Meanwhile, PMQR genes were co-transfered with most of the bla CTX-M genes just few 16 s r RNA methyltransferase genes and the integrons genes were co-transfered. This results showed that the 107 isolates carried self-transferable plasmids harbored bla CTX-M genes, even some plasmids harbored not only one AR genes, and they could transfer the AR genes with plasmids to other bacteria.This assay obtained epidemic data about AR genes in ESBLs-producing E. coli of Procambarus clarkii from five provinces in China. All the results were beneficial for monitoring public hygiene of aquatic product and provided support for clinical medicine usage. |
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