Incidence And Characterization Of Integrons In Escherichia Coli And Salmonella Spp. Strains Isolated From Food

Obejective: To invesitigate the level of antibiotic resistance in Escherichia coli and Salmonella spp. strains isolated from food collected in Shenzhen, the frequency of the multidrug resistance strains carrying integrons, the relationship between the gene cassettes of the integron carried by the strain with its antibiotic resistance profile, and find out whether and to what extent the horizontal transfer of integrons contributed to the development of miltidrug resistance in foodborne strains.Methods: Escherichia coli and Salmonella spp. strains isolated from chicken, beef and pork samples were characterized. Testing of susceptibility to nineteen different antimicrobial agents was done using the agar diffusion method. The break points were those recommended by the National Committee for Clinical Laboratory Standards. To determine whether the high prevelance of multidrug resistance strains were the results of an epidemic spread of a few strains or merely an increase in unrelated strains, all multidrug resistant isolates were typed by plasmid profile analysis. To identify the presence of an integron and to determine the size of any inserted gene cassette, PCRs amplifing intl gene and the variable region between the conserved segments (CS-PCR) were performed for each integron class. And the integron content of every isolate was characterized. That is, each CS-PCR amplicon of the variable region that had a unique size (number of base pairs) was sequenced. The size of the CS-PCR product of each newstrain was compared with the size of the already sequenced products. If the new product had the same size and RFLP pattern as the already sequenced product, the 2 integrons were considered to be identical; Otherwise, the new one was sequenced. To obtain further evidence for in vivo horizontal transfer of resistance genes, conjugation experiments were done between E.coli or Salmonella spp. strains with integron and those without integron or with integrons of different size. Film mating was performed on LB agar, and transconjugants were selected on LB, EMB or HE agar containing the antibiotics.Results: The isolation rate of Esherichia coli from the raw meat samples was 96.2%, and Salmonella was 14.05%. The serotype of Salmonella isolates, S.typhi, S.agona, S. derby, S.entertidis and S.typhimuim were the top five serotypes. Also, a S.wetevreden strain was detected in this study. Susceptibility testing of the isolates gave the following results: more than 30% of all the strains were resistant or intermediately susceptibility to Ampicillin, Streptomycin, Tetracycline, Sulfonamides, and Nalidixic acid, and the resistance to some antibiotics used in clinical was considerably high. Some of the tested agents were found to be highly effective against all strains, such as the third generation of cephalosporin antibiotics. The antibiotic resistance of E.coli strains was more severe than Salmonella strains, not only the resistance to several antibiotics, but also the number of antibiotics they resistant to. The number and size of plasmids the strain carried had no significant relationship with its resistant profile.All the strains tested for antibiotic susceptibility were screened for the presence of class 1 to 3 integrons. In the 31 multidrug resistant E.coli strains, 19 isolates were identified as being positive for integrase1 gene in the total DNA, 13 had detectable amplification products, and the positive rates of intM and integron 1 in 27 Salmonella strains resistant to more than 3 agents were 74% and 70.4%, respectively. Two E.coli isolates had the class 2 integron. Most of the gene cassettes integrated in the integrons conferred resistant against trimethoprim and streptomycin-spectinomycin, and the integrons with the dfr1-aadA1 or dfr17-aadA5 were predominant. Strains of the same antibiotic resistance profile or plasmid profile did not mean having the same integron, and the strains with different phenotype or genotype may have the same integron. These indicated that the integrons had transferred horizontally between strains. By conjugation, we obtained further evidence for in vivo horizontal transfer of resistance genes. Integrons were founded among transconjugants of the recipient strains using integron specific PCR assays. And in this process, gain or loss of gene cassettes might happened in some integrons, for we found some transconjugants had integrons different from the donor or recipient strains in size.Conclusion:The features of the bacteria contaminated in the food indicate that the food safty of Shenzhen is potentially at risk, and the government devision responsed to should reinforce their survey to prevent the outbreak of foodborne desease.Commensal bacteria constitute a reservior of resistance genes for (potentially) pathogenic bacteria. It is important to invesitigate the antoibiotic resistance level of indicator strain of intestinal, Escherichia coli, for undersatand the situation of selection pressure posed by antibiotic use and resistance problems to be expected in pathogens,and for better control of foodborn deseases.Integrons, which occur widely and are significantly associated with resistance to multiple classes of antimicrobial compounds, have an important role in the development and dissemination of multidrug resistance in Escherichia coli and Salmonella spp. isolates. PCR screen for the presence of integrons may be a useful method in antibiotic susceptibility testing, especially in fast identification of multidrug resistance strains and comprehension of the mechanisms for development and dissemination of resistance.The gene cassettes gain or loss of integrons in the film mating conjugation indicates that the structure of integron could not as stable as had been suggested previously. The horizontal transfer mechanisms of integrons are complicate, which need more information to interprete.Study of the antibiaotic resistance in bacteria isolated from food facilitates the understanding of mechanisms by which the clinical strains build their resistance.It is an urgent need to restrict the use of antimicrobial agents in animals bred as a food source for humans, both the veterinary use and the use as feed additives for growth promotion. This would not only diminish the public health risk of dissemination of resistant bacteria or resistant genes from animals to humans, but would also be of major importance in maintaining the efficacy of antibiotics in veterinary medicine.