Antimicrobial Susceptibility Testing And Drug-resistant Mechanism Of Vibrio Cholerae O139 Isolated In China

ObjectivesThe extensive use of antibiotics,even abusive,makes bacteria face strong selective pressure,resulting these bacteria into resistant strains through a variety of mechanisms,such as gene mutations and transfer of drug-resistant genes.Until now,there are more and more reports of multi-drug resistant bacteria.The objectives of this study are to determine the antimicrobial resistance profiles among a representative sample of 300 Vibrio cholerae O139 isolated in China,and make a further study on the mechanisms of drug resistance,such as the mobile multi-resistant integrons and gene mutations related to the quinolone resistance.MethodsAccording to the CLSI standard recommended by the Clinical Laboratory Standards Institute about antibiotics sensitivity test of Vibrio cholerae and Enterobacteriaceae in clinical microbiology laboratory,we select 17 kinds of representative antibiotics,and use the dilution method to test antibiotics sensitivity of 300 Vibrio cholerae O139 strains from Chinese Center for Disease Control and Prevention of infectious diseases,which is recommended by WHO.Quality control is regularly performed by running specific control strains as E.coli ATCC25922 and ATCC29213.Further interpretation of the MIC and analysis of Vibrio cholerae O139 spectrum resistance are done according to the NCCLS recommendation.We searched the sequences of the class1-4 integron and the SXT conjugative transfer gene elements in GenBank,and designed primers in the conserved region of their specific integrase.We detected the distribution of these gene elements through PCR amplification,and analyzed their roles of gene-mediated bacterial resistance.Further,we also designed primers in the topoisomerase gene such as gyrA,gyrB,parC and parE,which are related to the quinolone resistance in Vibrio cholerae.The PCR amplification products are sequenced,and the roles of mutation in genes encoding topoisomerase are analyzed in gene-mediated quinolone resistance. ResultsThe results of drug susceptibility tests about 300 Vibrio cholerae O139 strains showed that all strains are susceptible to the third generations of cephalosporin such as ceftriaxone and cefixime.The majority of strains are susceptible to cefuroxime,Cephalothin,Doxycycline and Ciprofloxacin,with the sensitivity of 99.3%,83.3%,91.3%and 95.0%,respectively.More than 50%of the strains are resistant to chloramphenicol,kanamycin,nalidixic acid, tetracycline,ampicillin and Sulfamethoxazole/Rimethoprim with low sensitivity of 39.0%,31.0%,27.3%,27.0%,25.3%and 17.0%,respectively.Most strains show little sensitivity to streptomycin,erythromycin and polymyxin B,with the sensitivity of 1.0%,6.0%and 3.3%.99.0%of the strains show resistance to more than two kinds of antibiotics.47.0%(140) of the strains show the same multi-drug resistant profiles,which are ampicillin,streptomycin,tetracycline, chloramphenicol,Sulfamethoxazole/Rimethoprim,nalidixic acid,erythromycin and kanamycin.Distribution of integrons shows that 64.3%of the Vibrio cholerae O139 strains contain the class 1 integron,and the class 2 and 3 integron was negative in Vibrio cholerae,and the class 4 of integron,also known as super-integron, are found in all strains.90.1%of the variable regions of the class 1 integron showed that there was only one gene cassette,such as aadA gene,but a small number of strains found the dfrA and arr-3 gene cassette.The gene sequencing of the topoisomerase resistance-determining region of these nalidixic acid resistant strains showed that:95.3%of the strains have two gene mutations,such as the 83 amino acid of GyrA and the 85 amino acids of ParC,In addition to the 83 amino acid mutation in GyrA and 85 amino acid mutation in ParC,7 Ciprofloxacin-resistant strains have additional gene mutation,such as the 87 amino acids in GyrA from Asp into Asn and the 87 amino acids in ParE from Asp into Asn.ConclusionsAccording to the drug sensitivity testing of 300 Vibrio cholerae O139,these strains show little sensitivity to chloramphenicol,tetracycline,ampicillin, cotrimoxazole,streptomycin and erythromycin,and high sensitivity to ciprofloxacin,although some Ciprofloxacin-resistant strains have appeared. The class 1,4 integron and SXT are greatly distributed in 300 Vibrio cholerae O139,which transfer the resistance genes.The gene mutations encoding topoisomerase in Vibrio cholerae O139 are more common,most of which are single point mutations,and the majority of strains with nalidixic acid resistance show that the 83 amino acid in GyrA and 85 amino acid in ParC are replaced,so does the 87 amino acid in the GyrA.The gene mutation in the 420 amino acid of ParE associated with ciprofloxacin-resistance in Vibrio cholerae is firstly described in this study.To conclude,the study show us the importance of the regular drug-resistance testing of Vibrio cholerae,and we should strengthen the surveillance of drug-resistant profile of Vibrio cholerae, and give the clinicians good suggestion of rational use of drugs.