Journal of Associated Medical Sciences (Nov 2018)
High-resolution melting-curve analysis for serotyping of Salmonella spp. group B isolated from minced pork in the Northern part of Thailand
Abstract
Background: Nontyphoidal Salmonella spp. is the major bacterial cause of food poisoning. Conventional serotyping is complicated and time consuming. Objectives: To establish a rapid molecular - based screening for Salmonella serotypes Materials and methods: Several aspects of Salmonella isolates were characterized by both rapid multiplex real-time PCR and high-resolution melting-curve (HRM) analysis. Group B Salmonella isolates (n=29) were isolated from 165 of minced pork samples randomly collected from local markets in six provinces of the Northern Thailand. Several genetic determinants responsible to specific phenotypes were selected, including Salmonella spp. (InvA), Salmonella Serotypes (fljB, gyrB and ycfQ), Beta lactam resistance including serious ESBL determinants (blaTEM, blaCTX-M). Results: HRM serotyping successfully revealed the epidemiological prevalence of three Salmonella serotypes from all group B Salmonella isolates, including 38% S. Stanley, 24% S. Typhimurium, and 17% S. Monophasic. Further conventional serotyping showed five unknown HRM patterns as S. Agona, S. Schwarzengrund, S. Saintpaul, S. Brandenburg and one unknown serotype. Fifty-five percent of the isolates showed multidrug-resistant phenotype. The high prevalence of blaTEM gene totally corresponded to the observed ampicillin-resistant phenotype. However, the presence of blaCTX-M group 1 was widely observed but not corresponded to its expected ESBL phenotype. Melt curve analysis of the observed blaCTX-M group 1 amplicons compared with the positive ESBL gene (blaCTX-M -55) showed the high difference in the melting temperature (Tm) of those amplicons which indicated that the observed blaCTX-M group 1 amplicons were less likely to be ESBL gene. Only one ESBL Salmonella isolate from Nan province showed the presence of blaCTX -M group 9 with ESBL phenotype. The highly virulent ESBL Salmonella serovar Typhimurium encoding blaCTX-M group 9 in contaminated minced pork from the Nan province suggested the high alert for the rapid screening of ESBL producing Salmonella spp. in meat and animals to prevent a potential future outbreak. Conclusion: By performing the molecular analysis, this study successfully revealed the importantly epidemiological aspects of the Salmonella isolates group B collected from the Northern Thailand. This approach should simplify the screening for Salmonella serotypes in minced pork.
