Analysis of the virulence genes, phenotypes of Salmonella Typhimurium and the development of immunomagnetic separation -PCR system for the simultancous detection of Listeria monocytogenes and Salmonella

• Main Authors: Hsien-Yee Hsih, 石心怡
• Other Authors: Hau-Yang Tsen
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/43589059491463070599

博士 === 國立中興大學 === 食品科學系 === 90 === 45 Salmonella Typhimurium strains isolated between 1991-1994 were collected from the National Institute of Preventive Medicine, Department of Health, Executive Yuan. These S. Typhimurium strains were divided into two groups，ie, high and low virulence groups, according to the virulence study to the mice. Five strains randomly selected from the major PFGE subtypes were subjected to the virulence study to mice. It was found that they were all high virulent strains. Virulence plasmids could not be isolated from all of these 45 strains. However, 13 S. Typhimurium strains (28.9 %) of the major PFGE subtype carry the virulence plasmid and 30 strains (66.7 %) of the clinical isolates carry the virulence plasmid. Virulence plasmid were not isolated from 33.3 % (15/45) of S. Typhimurium strains studied. On the other hand, the inv and stn genes were found in all of the 45 strains. The restriction fragment length polymorphism (RFLP) patterns of stn gene show no difference for these 45 Salmonella Typhimurium strains. The relationships between invasive ability to human and the enterotoxin titers of S. Typhimurium strains were not found. Also, the relationship between invasion ability and replication capability to the virulence of S. Typhimurium cells in Raw 264.7 macrophage cell line was not found. Thus, the abilities of invasion and replication of S. Typhimurium to Raw 264.7 were not useful for the analysis of Salmonella virulence The relationship between cytotoxic to macrophage and virulence was not found. Strains in PFGE subtypes X6A7S6 and X17 were belonging to low virulence strains and were more susceptible to H2O2. Strains of high virulence subtypes were more acid tolerant. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of the secretion proteins for those Salmonella Typhimurium strains were not relative to their virulence. Strains from PFGE major subtype were more acid tolerant, more resistant to H2O2, and possess higher enterotoxin titer, which might be the reasons of being virulent and epidemic. Tumor necrosis factor alpha (TNF-a) formations in macrophage and biofilm formation ability were also not related to the virulence of these S. Typhimurium strains. The method mentioned above could not replace the method of animal models for the study of the virulence of S. Typhimurium. In recently years, the number of multidrug resistant Salmonella Typhimurium strains was increasing. In this study, the antibiograms of 45 human isolates and 87 animal isolates of Salmonella Typhimurium in Taiwan was investigated. Analysis of the antibiotic resistant genes in molecular level may help us to realize the origins of antibiotic resistance strains. It was found that the antibiograms for human and animal isolates are quite similar. Both the human and animal isolates are highly resistant to first-line antibiotics, such as tetracycline, sulfisoxazole, ampicillin, streptomycin and chloramphenicol; but are sensitive to gentamicin, fluoroquinolone antibiotics, such as norfloxacin and the third generation antibiotic of cephalosporin, such as cefoperazone. Between 93 % and 100 % of the local strains is inhibited by these antibiotics. Also, a significant fraction of these S. Typhimurium isolates are multidrug resistant strains. For example, 58.6 % of the animal isolates and 68.9 % of the human isolates are multidrug-resistant. Strains from PFGE major subtype were resistant to ampicillin, chloramphenicol, streptomycin, sulfonamide, tetracycline (ACSSuT). When sizes of the integrons were analyzed using PCR primers 5’CS/3’CS, the integron sizes were the same for human and animal isolates, which show identical PFGE and antibiogram patterns. Thus, antibiotic resistant human isolates may be circulated through foods from the infectious animals. The increasing of multidrug resistant Salmonella Typhimurium strains in recent years and the predominance of these multidrug resistant strains as major infectious strains of Salmonellosis all over the world become a serious problem. Such fact has induced the interest of probiotic use. Probiotic are capable of protecting the gastrointestinal tract from invasion by pathogens. The effectiveness of probiotic could be assessed by their prevention of pathological conditions and by their form as biotherapeutic agents. Due to the probiotic properties of lactic acid bacteria (LAB), we have used some LAB strains for the study of their prevention of Salmonella invasion. The basic probiotic properties evaluated for LAB strains are their capability to tolerate to gastric acid and bile salts, the ability to inhibit pathogenic bacteria and the adherence capability to human intestinal epithelium cells. In this study, four LAB strains LA5, LF33, MP012 and CCRC 10069 have been shown to be tolerant to pH 2.0 buffer, and resistant to 0.3% bile salts. However, for the adhesive ability study, only LA5 and LF33 strains were found adhesive to the human Int 407 or Caco-2 cell lines. The protective effectiveness for the orally administered LAB strains to the invasion of mice spleen and liver organs by S. Typhimurium was investigated. Results showed that the cell numbers of S. Typhimurium in liver and spleen organs of mice measured at the 6th day after Salmonella infection was lower for LAB administered mice as compared to the control mice. Of them, strain LA5 strain was most effective; followed by strains CCRC 10069 and LF33. CCRC 10069 was not able to adhere to Int 407 or Caco-2 cell lines but it provides the protective effect too. These four LAB strains were also studied for their adhesion ability to the mice intestinal cells. CCRC 10069 show the adherence to the mice intestinal cells, and this may be the reason why CCRC 10069 provides the protective effect for S. Typhimurium infection too. Owing to Salmonella and Listeria monocytogenes to human health, in this study, we also tried to develop a method for the simultaneous and rapid detection of these two kinds of organisms. The method we developed was the immunomagnetic separation followed by PCR method. A method that combined the immunomagnetic separation (IMS) technique and the multiplex polymerase chain reaction (PCR) method (i. e., the IMS-mPCR method) were developed for simultaneous detection of Listeria monocytogenes and Salmonella spp. in food samples. When only the multiplex PCR method was used, it was found that if cell numbers of each of the two target organisms (L. monocytogenes and Salmonella spp.) were above the detection limit, but differed by more than 2 logs-e. g., n x 107 to n x 104 or n x 106 to n x 103 — the organism presenting the lower numbers might go undetected. Listeria and anti-Salmonella immunomagnetic beads was performed prior to PCR, both pathogens could be detected unambiguously. Such results could be obtained for target organisms in food samples, such as milk, dairy, and meat products, if similar enrichment and IMS steps were performed prior to PCR.