Phenotypic and genotypic characterization of Zoonotic Salmonella Typhimurium and Salmonella Enteritidis

Douadi, Benacer (2009) Phenotypic and genotypic characterization of Zoonotic Salmonella Typhimurium and Salmonella Enteritidis. Masters thesis, University of Malaya.

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Salmonella is a leading cause of food-borne illness in humans, as well as in domestic and wild animals, worldwide. The two most common etiologic agents of nontyphoidal salmonellosis are Salmonella Typhimurium and Salmonella Enteritidis. The infection caused by these two serotypes is mainly contracted by ingestion of contaminated food or water, or through contact with an infected host. Salmonella Typhimurium and Salmonella Enteritidis are widely dispersed in nature and are common inhabitants of the intestinal tract of domestic and wild mammals, reptiles, birds, and even the insects. The increase in antimicrobial resistance in zoonotic Salmonella species have been a serious problem for public health worldwide. To better control the spread of multidrugresistant Salmonella, it is important to understand the mechanisms responsible for drug resistance and the factors responsible for dissemination. An effective subtyping scheme is needed to determine the epidemiology and relatedness of strains association. A total of 47 Salmonella Typhimurium (33 zoonotic, 14 clinical), and 59 zoonotic Salmonella Enteritidis strains isolated between 1969 and 2006 were tested for their antimicrobial resistance using standard disk diffusion method. The presence of relevant resistance genes and class 1 integrons were carried out by using PCR. The genomic diversity of Salmonella Typhimurium and Salmonella Enteritidis strains was analysed by pulsed gel electrophoresis (PFGE), ERIC-PCR, REP-PCR and plasmid profiling. Seventy per cent of Salmonella Typhimurium and 22.0% of Salmonella Enteritidis strains were resistant to one or more antimicrobial agents tested. High rates of resistance were found among the strains to tetracycline: 70.2% of Salmonella Typhimurium and 19.0% of Salmonella Enteritidis. For Salmonella Typhimurium strains showed high rates of resistance were seen to sulphonamides (57.4%), streptomycin (53.2%), ampicillin (30.0%), nalidixic acid (27.7%), kanamycin (23.4%), chloramphenicol (21.3%), and trimethoprim (19.1%). Additional resistance towards cephalosporins was detected: cephalothin (27.7%), cephradine (21.1%), amoxicillin clavulanate acid (17.0%) and cephalexin (17.0%). Compared to Salmonella Typhimurium, the rate of resistance of Salmonella Enteritidis strains was lower (3.4%) to sulphonamides, trimethoprim, trimethoprimsulfamethoxazole and amoxicillin-clavulanate acid. Only 1.7% of the strains were resistant to ampicillin, gentamicin, streptomycin, cephalexin and cephalothin respectively. PCR detection of antimicrobial resistance genes and class 1 integrons in Salmonella Typhimurium strains showed that all the 14 ampicillin-resistant and 25 streptomycin-resistant strains had the blaTEM and strA genes, respectively. However, 54% of the streptomycin resistant strains had a sequence similar to the aadA gene, 37% of the 27 sulphonamidesresistant strains harboured sul1 and 89% harboured sul2. Among the 33 tetracycline resistant strains, 70%, 15%, and 12 % had tet(A), tet(B) and tet(C) genes, respectively. 33(87%) of the strains were carried class 1 integrons. Based on the numbers and size of integrons obtained, the strains were grouped into 9 different integron profiles (IPs). The sequencing analysis identified sat, aadA, pse-1 and dhfr genes in variable regions on class 1 integrons. Thirty-five (75%) of Salmonella Typhimurium strains (clinical = 12, zoonotic = 23) harboured 1-6 plasmids each, while 12 strains lacked any type of visible plasmids bands. A total of 22 different plasmids ranging from 2.0 to 95 kb were extracted. The major plasmid was 90 kb which was present in 17 (49%) plasmid-positive strains. Among the Salmonella Enteritidis, one ampicillin-resistant, 2 sulphonamides-resistant strains and the 11 tetracycline-resistant strains harboured blaTEM, sul2 and tet(A) gene, respectively. The genetic diversity of Salmonella Typhimurium and Salmonella Enteritidis strains was determined by PFGE, REP-PCR, ERIC1R-PCR and ERIC2-PCR. Among the 47 Salmonella Typhimurium PFGE, REP-PCR, ERIC1R-PCR and ERIC2-PCR subtyped the strains into 39, 41, 43, 44 profiles, respectively. The three genotyping approaches (PFGE, REP- PCR and ERIC-PCR) had equally high discriminative power (D = 0.99), which suggests that PCR fingerprinting is as useful as PFGE in typing of Salmonella Typhimurium. Compared to PCR fingerprinting, PFGE was more reproducible. The major difficulties associated with PFGE relate to the technical demands of expensive equipment and time-consuming process, varying from 1 – 3 days dependent upon individual protocols. PFGE, REP-PCR, ERIC1R-PCR and ERIC2-PCR subtyped the 59 Salmonella Enteritidis strains into 11, 13, 14, 15 profiles, respectively. Among the 11 distinct XbaIpulsotypes found in Salmonella Enteritidis, PFP X2 was the predominant PFGE profile and was represented by 44 strains. Based on the discriminatory power (D), ERIC1R and REP-PCR fingerprinting (D = 0.90) were found to be the most discriminative methods for Salmonella Enteritidis strains, followed by ERIC2-PCR (D = 0.80) and PFGE (D = 0.44). These results suggest that the PCR fingerprinting may be an alternative tool to PFGE for subtyping Salmonella Enteritidis strains. In conclusion, a high rate of multidrug-resistance was found among the Salmonella Typhimurium strains as indicated by the prevalence of resistance genes. More than 87% and 75% of the Salmonella Typhimurium strains carried class 1 integrons and plasmids.Salmonella Typhimurium resistant to cephalosporins (amoxicillin clavulanic acid, cephalothin, cephradine, cephalexin, ceftriaxone, cefotaxime, ceftiofur, ceftaxidime, cefepime aztreonam and cefuroxime) was also detected among 9 zoonotic and 7 clinical isolates. Based on PFGE and PCR fingerprinting, the strains were very diverse and no particular genotype seems to predominate and persist over the years (1969- 2006). On the other hand, low rate of resistance in Salmonella Enteritidis strains was found, and the strains had high genetic homogeneity. A combination of one or two molecular typing methods, such as PCR and PFGE are needed to enhance the ability to discriminate Salmonella Enteritidis strains.

Item Type: Thesis (Masters)
Subjects: Q Science > Q Science (General)
Date Deposited: 31 Jul 2013 03:10
Last Modified: 31 Jul 2013 03:10

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