Pulsed-field Gel Electrophoresis Genotyping And Characterization Of Multi-drug-resistant Salmonella Enterica Isolates

The bacterial genus Salmonella are important pathogens in humans and animals. Some of these pathogens are host specific, e.g., Salmonella enterica serovar pullorum, which infect only poultry and aquatic birds. Pullorum disease is still a disease of significance in China. It has largely been eradicated from Latin America, South America, the Middle East, the Indian subcontinent, and parts of Africa.Samonella enterica isolates from clinical specimens may be resistant to multiple antimicrobial agents and a substantial proportion of multi-resistant Salmonella isolates carry class I integrons. Resistance to ampicillin, chloramphenicol, streptomycin, sulphonamides and tetracycline (abbreviated ACSSuT) is common, although resistance to other antibiotics is prevalent in other countries. These strains are a potential reservoir for antimicrobial resistance genes and play an important role in the ecology of antimicrobial resistance of bacterial populations. Since many gene cassettes of integrons contain antimicrobial resistance genes in Gram-negative bacteria, the horizontal transfer of integrons through plasmids and transposons has been found to play an important role in the dissemination of antimicrobial resistance genes and the development of multi-resistance. The aim of the current study was to characterize antimicrobial susceptibility and the mechanisms of antimicrobial resistance found in S. pullorum from poultry and Salmonella isolates from meats and human. The study was also to determine if there was any possible transfer of antimicrobial resistance between these isolates and to evaluate genetic relationships of Salmonella isolates by PFGE from different regions during the 10-year period.1. Characterization of multi-drug-resistant Salmonella serovars of different originsAntimicrobial resistance was investigated in 358 Salmonella isolates in 10 provinces in China, of which 241 Salmonella pullorum isolates were collected from chickens with clinical signs of pullorum disease from January 1962 to July 2006 in China, and the other isolates (n=117) were isolated from meats and fecal samples from human. The antimicrobial susceptibility of these isolates to 18 antibiotics was determined by broth microdilution method. A total of 117 Salmonella strains isolated from retail meats and human in some regions of Jiangsu were assayed for their antimicrobial susceptibility. The majority of Salmonella isolates were resistant to the antimicrobials tested. 111 (94.9%) were resistant to at least two antimicrobial agents. Resistance against streptomycin(92.3%,108/117)was the most common. Resistance was also observed to trimethoprim (88%), cefamandole (57%), ampicillin (44%), kanamycin (40%) and nalidixic acid (36.8%). Whereas 59 (50.4%) of these strains were resistant to at least five antimicrobials. Twelve isolates showed resistance to six antimicrobials: ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, tetracycline, and kanamycin (R type ACSSuTK). For Salmonella pullorum isolates derived between 1962 and 1979, the rate of resistance to 17 antibiotics was low in general, with the highest rate to sulfonamides (32.1%). Multi-drug-resistant Salmonella pullorum isolates emerged in 1997, and the most common phenotype was ASSuT (resistance to ampicillin, streptomycin, sulfonamide, and tetracycline). The resistance to nalidixic acid (82.0%) was detected in isolates from 2000 onward.2. Antibiotic resistance genes, class I integrons and multiple antibiotic resistance in Salmonella enterica isolates in China39 of 117 Salmonella strains isolated from retail meats and human and 60 of 237 Salmonella pullorum isolates were assayed for the presence of integrons and antimicrobial resistance genes, and the ability of horizontal transfer of the characterized antimicrobial resistance determinants via conjugation. A total of 15 different antimicrobial resistance genes were identified in 99 multi-drug-resistant Salmonella isolates. Genes such as blatem-1, tetA, sul2, strA-B and dfrA12 were found in Salmonella Pullorum; whereas genes such as blatem-1, aadA1, blapse1, sul1, sul2, tetA, tetB, tetG, strA-B, floR, cmlA and dfrA12 were present in Salmonella strains isolated from retail meats and human. Among the 39 Salmonella strains isolated from retail meats and human, while 32 (82%) of these isolates tested carried integrons ranging in size from 0.3 to 1.6 kb. The most common integron was 1.6 kb which carried aadA5 and dfr17 gene cassettes. 27 of 60 Salmonella pullorum isolates were positive for class 1 integrons. All of integron-positive isolates were isolated between 1998 and 2006. The gene cassette arrangements were determined in the positive isolates, which harbored one (aadA2, sul1) or two [aadA5-dfrA17, dfrA1-orf1 (unknown function)] cassettes in their variable regions. The aadA5-dfrA17 gene cassette was found most frequently in all these isolates. Conjugation studies demonstrated that there was plasmid-mediated transfer of antimicrobial resistance genes. These data showed that the Salmonella isolates recovered from meats and human in some regions of Jiangsu were commonly resistant to multiple antimicrobials, and genes conferring antimicrobial resistance in Salmonella were often carried on integrons and plasmids and could be transmitted through conjugation. The mobile DNA elements might play a very important role in transmission and dissemination of antimicrobial resistance determinants among Salmonella strains. They contributed to the multi-resistance phenotype observed in these isolates, but most resistance genes were located outside the integron structure, as independent genes.3. Contribution of mutations of gyrA and acrB, ompF, and marA to decreased susceptibility of Salmonella enterica serovar isolates to quinolones and other antimicrobialsThe mechanisms involved in fluoroquinolone resistance in Salmonella enterica include target alterations, decreased expression of the porin Omp F and overexpression of efflux pumps. MarA mediates drug resistance by causing decreased expression of the porin Omp F and overexpression of the multi-drug effux pump ArcB. The present study evaluated the role of known and putative multi-drug resistance efflux pumps and mutations in topoisomerase genes among quinolone-resistant Salmonella enterica strains. We determined by broth microdilution method for the susceptibility to nalidixic acid and three fluoroquinolones of Salmonella pullorum and Salmonella strains isolated from human and food between 2000 and 2006. These isolates were ciprofloxacin susceptible except one isolate, but exhibited reduced susceptibility to three fluoro- quinolones. A mutation of the gyrA in codon 83 or 87 could be demonstrated in all the nalidixic acid-resistant isolates but in none of the nalidixic acid-susceptible isolates analyzed. All isolates tested with a mutation in codon 83 were highly resistant to nalidixic acid (MIC,≥1024μg/ml in Salmonella strains isolated from human and food; MIC, 512~1024μg/ml in Salmonella pullorum). Of the 23 isolates with the base substitutions in a sp 87, all had a lower level of resistance to nalidixic acid (MIC, 32~ 512μg/ml). This study illustrated the simultaneous presence of nalidixic acid-resistance and decreased ciprofloxacin susceptibility in Salmonella isolates, and that the resistance was mainly associated with mutations in gyrA. Strains with nalidixic acid MICs of 2, 256, 512, and 1024μg/mL were selected to analyze the role of acrB, marA and ompF on antimicrobial susceptibility to fluoroquinolones and other tested antimicrobials. In these strains, fluoroquinolone MIC for the strains with deletion of acrB increased by from 8 to16-fold, whereas MIC for the strains with mutations of marA and ompF increased by from 2 to 8 fold. These results indicate that a combination of topoisomerase gene mutations, as well as enhanced antimicrobial efflux, played a critical role in the development of fluoroquinolone resistance in the quinolone-resistant strains.4. Molecular analysis of Salmonella isolates of different origins by pulsed- field gel electrophoresisThe incidence of food-borne salmonellosis and pullorum is reported to be on the increase in China. The pulsed-field gel electrophoresis (PFGE) subtyping method was used to assess the extent of genetic diversity and clonality of Salmonella strains from humans and the environment. PFGE of Xba I-digested chromosomal DNA from 237 strains of Salmonella pullorum gave distinct profiles with a range of Dice coefficients (0.6 to 1.00), indicating that PFGE was very discriminative and that multiple clones of Salmonella pullorum existed among clinical isolates. Salmonella isolates (n=85) of meats from Jiangsu province were analyzed via PFGE with Xba I. PFGE subtypes were analyzed by cluster analysis and compared to conventional serotyping results. The analysis showed a correlation of serotype to PFGE subtype. In addition, conserved fragments were identified within the restriction patterns that were unique to each serotype. PFGE using Xba I restriction provided a possible alternative method for screening and identifying food-borne Salmonella serotypes. In summary, the following conclusions were drawn: 1)111 of 117 Salmonella strains isolated from retail meats and human in some regions of Jiangsu were resistant to at least two antimicrobial agents. The most common phenotype was ACSSuTK (resistance to ampicillin, chloramphenicol, streptomycin, sulfonamide, kanamycin, and tetracycline) among MDR isolates.2)The rate of resistance to 17 antibiotics was increasing from 1962 to 1979. Multi-drug-resistant Salmonella enterica serovar Pullorum isolates emerged in 1997, and the most common phenotype was ASSuT (resistance to ampicillin, streptomycin, sulfonamide, and tetracycline).3) A total of 15 different antimicrobial resistance genes were identified in multi-drug-resistant Salmonella isolates. Genes such as blatem-1, tetA, sul2, strA-B and dfrA12 were found in Salmonella pullorum; whereas genes such as blatem-1, aadA1, blapse1, sul1, sul2, tetA, tetB, tetG, strA-B, floR, cmlA and dfrA12 were present in Salmonella strains isolated from retail meats and human.4) Salmonella strains tested carried integrons ranging in size from 0.3 to 1.6 kb. The most common integron was 1.6 kb which carried aadA5 and dfr17 gene cassettes.5) The mobile DNA elements might play a very important role in transmission and dissemination of antimicrobial resistance determinants among Salmonella strains. But most resistance genes were located outside the integron structure, as independent genes.6) These results indicated that a combination of topoisomerase gene mutations, as well as enhanced antimicrobial efflux, plays a critical role in the development of fluoroquinolone resistance in naturally occurring quinolone-resistant Salmonella strains.7) The mechanisms involved in fluoroquinolone resistance in Salmonella enterica include target alterations, decreased expression of the porin OmpF and overexpression of efflux pumps.8) PFGE using Xba I restriction enzyme provided a possible alternative method for screening and identifying food-borne Salmonella serotypes.