VIRULENCE CHARACTERIZATION AND GENOTYPIC ANALYSIS OF SALMONELLA TYPHIMURIUM ISOLATED FROM FOOD AND PROCESSING ENVIRONMENTS

Introduction:

Nontyphoid salmonellosis is a worldwide disease of humans and animals. Infections due to Salmonella spps. range from gastroenteritis to enteric fever. Salmonella Typhimurium may cause nontyphoid salmonellosis and is a very important food-borne pathogen (World Health Organization, 2005). The causative organisms can pass through the food chain from primary production to households or food-service establishments and institutions. Large number of Salmonella outbreaks from contaminated food such as sprouts, poultry, fish, meat, and eggs has been reported (National Advisory Committee on Microbiological Criteria for Foods, 1999; van Duynhoven et al., 2002; Dominguez et al., 2007). The recent-most reported outbreak was in 2006 and was connected with the consumption of tomatoes contaminated with S. Typhimurium (Center for Disease Control and Prevention, 2006). Diarrheal diseases are common in Pakistan due to the consumption of contaminated water and food. No systematic guideline exists for the surveillance of Salmonella infections in human and animal in Pakistan. Therefore, the magnitude of nontyphoidal Salmonella infection is not well documented.

Salmonellae cause disease by invading the intestinal epithelium after ingestion. Researchers have studied the internalization of salmonellae, such as S. typhi, S. typhimurium, and S. choleraesuis, in epithelial cells and found that they multiply within formed vacuoles (Gahring et al., 1990). In vitro systems using different cell lines have been used to study the interaction between salmonellae and eukaryotic cells. Galan and Curtiss (1989) first characterized the Salmonella invasion gene invA, the first gene in an operon which is thought to trigger the internalization of S. typhimurium in cultured epithelial cells. Mutations within this operon render salmonellae incapable of invading Madin Darby canine kidney (MDCK) cells in culture. Virulence plasmids are one of several Salmonella virulence determinants involved in survival and growth in host cells. Virulence plasmids are thought to not be involved in the initial interaction between salmonellae and the intestinal mucosa or required for invasion into deeper tissue. However, they enable the organism to persist in the reticuloendothelial cells of liver and spleen (Gulig, 1990).

Present study is designed to determine the relationship between the presence of both the invA and spvC genes and the degree of invasiveness among salmonella isolates recovered from diverse food sources. A comparison of the strains recovered of different origins will clarify the nature of human salmonellosis. The data generated by genotypic analysis, antibiotic profile, plasmid profile and presence of virulence genes in S. Typhimurium isolates from foods can be useful in epidemiological investigation of Salmonellosis.

Objectives:

1. To compare the isolates for the presence of virulence genes invA and spvC.

2. To compare Salmonella isolates obtained from various sources for invasiveness in epithelial tissues.
3. To characterize isolates in terms of antibiotic resistance and genetic relatedness

Methodology:

Bacterial strains:

Salmonella will be isolated from commercially processed spent hens, broilers, and eggs and the egg production environment. Poultry associated samples including ovaries and oviduct tissues, cecum tissue sections, whole egg contents and the samples from egg production environment will be taken. Other food products such as fish, meat, beef, fruits, vegetables, sprouts will also be sampled. Salmonella samples will be cultured and identified by API system. Stereotyping of recovered isolates will be performed.

InvA and spvC Primers and probes:

Primers for Salmonella spvC and invA will be designed using published sequences (GenBank accession number M64295 and M90846).Probes will be prepared using PCR and labeled PCR products will be used as probes in DNA-DNA hybridization with colonies or plasmid DNA.

Plasmid DNA isolation:

Plasmid DNA will be isolated and purified using Mini-Prep kit.Plasmid DNA will be characterized by gel electrophoresis on a 1% agarose gel according to standard methods (Sambrook et al., 1989).

Hybridization:

Colonies will be grown on Brain heart infusion agar and then patched onto nitrocellulose membrane for the colony blots. The colonies will be lysed and the DNA will be denatured and neutralized (Sambrook et al., 1989). The DNA-DNA hybridization will be performed by prehybridization for 1 h at 68C in 5XSSC. Hybridization will be conducted at 68C overnight in buffer containing boiled digoxigenin-labelled probe. Enzyme immunoassay for the detection of the presence of digoxigenin labeled probe will be performed.

Invasin assay:

Invasin assay of Finlay and Falkow will be used with modifications. Monolayers will be disrupted by pipetting and titres of internalized bacteria will be determined on MacConkey’s agar. Inoculum level will be determined and percent invasion will be calculated by the formula: (CFU recovered after cell lysis/CFU of innoculum) x 100. Each invasion assay will be run in triplicate along with positive and negative controls.

Genome typing by PFGE:

Intact genomic DNA isolation and PFGE analysis will be carried out using protocol described by Gautum (1997). Salmonella Typhimurium isolates will be incubated at 37C in 25ml Luria broth. 1ml of bacterial cells will be harvested and washed three times using TE buffer and cell density will be adjusted. 100ml of the cell suspension will be incubated with proteinase K and lysozyme at 37C for 15 minutes. Restriction digestion will be carried out with 50U of Xbal at 37C. PFGE will be performed in 1% agarose gel in Tris Borate EDTA buffer. Strains differentiating in one band will be considered as different pulsed field profiles (PFP’S).

Antimicrobial Susceptibility Test:

Antimicrobial susceptibility test, constituting commonly used antimicrobial agents, was performed on Muller-Hinton Agar by disc diffusion method as described by National Committee for Clinical Laboratory Standards (National Committee for Clinical Laboratory Standards, 2002).

References:

Centers for Disease Control and Prevention (2006). Salmonellosis. Available online at http://www.cdc.gov/ncidod/dbmd/diseaseinfo/ salmonellosis_2006/outbreak_notice.htm. Accessed 10 April2008.

Dominguez A., Torner N., Ruiz L., Martinez A., Bartolome R., Sulleiro E., Teixido A. and Plasencia A. (2007). Foodborne Salmonella-caused outbreaks in Catalonia (Spain), 1990 to 2003. Journal of Food Protection 70: 209–213.

Gautom R.K. (1997). Rapid pulsed-field gel electrophoresis protocol for typing of Escherichia coli O157:H7 and other gram negative organisms in 1 day. Journal of ClinicalMicrobiology 35: 2977–2980.

Gahring, L. C., F. Heffron, B. B. Finlay, and S. Falkow. 1990. Invasion and replication of Salmonella typhimurium in animal cells. Infect. Immun. 58: 443–448.

Galan, J. E., and R. Curtiss III. 1989. Cloning and molecular characterization of genes whose products allow S. typhimurium to penetrate tissue culture cells. Proc. Natl. Acad. Sci. USA 86:6383–6387.

Gulig, P. A. 1990. Virulence plasmids of S. typhimurium and other salmonellae. Microb. Pathog. 8:3–11.

National Advisory Committee on Microbiological Criteria for Foods. (1999). Microbiological safety evaluation and recommendation of sprouted seeds. International Journal of Food Microbiology 52: 123–153.

National Committee for Clinical Laboratory Standards (NCCLS) (2002). Performance standards for Antimicrobial Susceptibility Testing, NCCLS Document no. M100-S12. Villanova, PA: National Committee for Clinical Laboratory Standards.

Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed., vol. 1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

van Duynhoven Y.T., Widdowson M.A., de Jager C.M., Fernandes T., Neppelenbroek S., van den B.W., Wannet W.J., van Kooij J.A., Rietveld H.J. and van Pelt W. (2002). Salmonella enterica serotype Enteritidis phage type 4b outbreak associated with bean sprouts. Emerging Infectious Disease8: 440–443.

World Health Organization. (2005). Drug resistant Salmonella. Available online at http://www.who.int/mediacentre/factsheets/ fs139/en. Accessed 10 April 2008.