Detection and Molecular Identification of Plasmid Virulence Genes in Salmonella enterica serovar typhimurium Isolated from Human and Animals by Multiplex PCR Method
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Abstract
Background: Salmonella enterica is a zoonotic species that can acquire its resistance in livestock. In humans, Salmonella typhimurium is a major etiological agent of food-borne salmonellosis. The identification of Salmonella spp. by traditional cultural techniques requires 4 to 5 days. The polymerase chain reaction (PCR) offers a simple tool for the rapid detection of Salmonella.
Methods: Fifty-five S. typhimurium isolates from bovine, poultry and human sources were isolated and analyzed with biochemical and serological tests. Firstly, multiplex PCR assay with four sets of primers was selected for invA, rfbj, fliC and fljB genes. In the second stage, a simple PCR method with one set primer was applied to detect spvA and spvB genes. Also, multiplex PCR assay with two set primers was carried out to simultaneously detect and identify invA and spvC genes in S. typhimurium.
Results: Analysis of the samples showed that while the presence of spvA, spvB and spvC genes in S. typhimurium from the bovine source was 100% (15/15), these same genes were present in 65% (13/20) of the poultry sources. The study also showed that spvA, spvB and spvC genes were present in 85% of human source.
Conclusion: This study showed that M- PCR of invA, rfbJ, fljB, and fliC genes were fast, simple, less expensive, accurate and specific in identification S. typhimurium. The advantage of multiplex PCR was that it could simultaneously identify the Salmonella strains which had a virulence plasmid thus facilitating the search for specific etiologic Salmonella serovars. The higher prevalence of spv genes among bovine sources can be injurious for public health.
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2. Majtan V, Szaboova M. The virulence markers of Salmonella serovar typhimurium different phage type strains isolated in Slovakia. Folia microbiol 2005; 50(6):516-23.
3. Gay J. Bovine herd Salmonellosis. Am J Infect Control 1995; 23:56-62.
4. Lim YH, Hirose K, Izumiya H, et al. Multiplex polymerase chain reaction assay for selective detection of Salmonella entrica serovar typhimurium. Jpn J Infect Dis 2003; 56(4):151-5.
5. Pan TM, liu YJ. Identification of Salmonella enteritidis isolate by polymerase chain reaction and multiplex polymerase chain reaction. J Microbiol Immunol Infect 2002; 35(3):147-51.
6. Soumet C, Ermel G, Rose N, et al. Evaluation of a multiplex PCR assay for simultaneous identification of Salmonella serovars, Salmonella enteritidis and Salmonella typhimurium from environmental swabs of poultry houses. Lett Appl Microbiol 1999; 28(2):113-7.
7. Malkawi HI, Gharaibe R. Rapid and simultaneous Identification of two Salmonella entrica serotypes enteritidis and typhimurium from chicken and meat product by multiplex PCR. Biotech 2004; 3(1):44-8.
8. Jamshidi A, Bassami MR, Afshari-Nic S. Identification of Salmonella serovars typhimurium by a multiplex PCR-based assay from poultry carcasses in Mashhad-Iran. Int J Vet Res 2008; 3(1):43-8.
9. Aabo S, Rasmussen F, Rossen L, et al. Salmonella identification by the polymerase chain reaction. Mol Cell Probes 1993; 7(3):171-8.
10. Jangjou A, Mokhtari A, Panahi P, et al. Salmonella enteritidis in captive siberian Tiger: A case report from Iran. Glob Vet 2011; 6(5):502-5.
11. Chu C, Chiu CH. Evolutions of the virulence plasmid of non-typhoid Salmonella and its association with antimicrobial resistance. Microbes Infect 2006; 8(7):1931-6.
12. Gothoh H, Okada N, kim YG, et al. Extracellular secretion of the virulence plasmid- encoded ADP-ribosyl transferase SPVB in Salmonella. Microb pathog 2003; 34(5):227-38.
13. Hong Sf, chiu CH, Chu C, et al. Complete nucleotide sequence of a virulence of a virulence plasmid of Salmonella entrica serovar dublin and its phylogenetic relationship to the virulence plasmids of serovars choleraesuis, enteritidis and typhmurium. FEMS microbial lett 2008; 282(1):39-43.
14. Rotger R, Casadesus J. The virulence plasmids of Salmonella. Int Microbiol 1999; 2(3):177-84.
15. Ahmer MMB, Tran M, Heffron f. The virulence plasmid of Salmonella typhimurium is self-transmissible. J Bacteriol 1998; 181(4):1364-8.
16. Boyed EF, Hartl DL. Salmonella virulence plasmid modular acquisition of the spv virulence Region by an F-Plasmid in Salmonella entrica subspecies I and Insertion into the chromosome of subspecies II, IIIa, IV and VII Isolate. Genetics 1998; 149(3):1183-90.
17. Gebreyes WA, Thakur S, Dorr P, et al. Occurrence of spvA virulence gene and clinical significance for multidrug-resistant Salmonella strains. J clin Microbiol 2008; 47(3):777-80.
18. Chu C, Hong SF, Tsai C, et al. Comparative physical and genetic maps of the virulence plasmids of Salmonella entrica serovar typhimurium, enteritidis, choleraesuis and dublin. Infect Immun 1999; 67(5):2611-5.
19. Brian M, Ahmer M, Tran M, et al. The virulence plasmid of Salmonella typhimurium is self-transmissible. J Bacteriol 1998; 181(4):1364-8.
20. Del cerro A, Soto SM, Mendoza MC. Virulence and antimicrobial-resistance gene Profiles determined by PCR-Based Procedures for Salmonella isolated from samples of animal origin. Food microbiol 2002; 20(4):431-6.
21. Ziemer CJ, Steadham SR. Evaluation of the specifity of Salmonella PCR Primers using various intestinal bacterial species. Lett Appl Microbiol 2003; 37(6):463-9.
22. Rahn K, De Grandees SA, Clarke RC, et al. Amplification of an inv-A sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella. Mol Cell Probes 1992; 6(4);271-9.
23. Saroj SD, Shashidhar R, Bandekar JR. Note Genetic diversity of food isolates of Salmonella entrica serovar typhimurium in India. Food Sci Tec Int 2007; 14(2):151-6.
24. Madadgar O, Zahraei Salehi T, Tadjbaksh H, et al. Genomic and Phenotypic evaluation of Salmonella typhimurium and Salmonella entritidis in Iran. Com clin Pathol 2008; 17(4):229-35.
25. Malorny B, Hoofer j, Bunge C, et al. Multicenter validation of the analytical accuracy of Salmonella PCR Towards an international standard. Appl Environ Microbiol 2003; 69(3):290-6.
26. Scholz HC, Arnold T, Marg H, et al. Improvement of an invA- based PCR for the specific detection of Salmonella typhimurium in organs of pigs. Berl Munch Tierarztl Wochenschr 2001; 114(9-10):401-3.
27. Hughes AL, Shopland S, Wigley P, et al. Characterisation of Salmonella enterica serovar typhimurium isolates from wild bird in northern England from 2005-2006. BMC Vet Res 2008; 4:4-16.
28. El-Feky MA, Hassan MA, Mohamed WA, et al. Detection of InvA gene in non typhoidal Salmonellae isolated from food products and clinical cases Egypt. J Med Microbiol 2014; 23(1):33-41.
29. Chiu CH, Su LH, Chu CH, et al. Detection of multidrug-resistant Salmonella entrica serovar typhimurium phage types DT102, DT 104 and U302 by multiplex PCR. J Clin Microbiol 2006; 44(7):2354-8.
30. Derakhshandeh A, Firouzi R, Khoshbakht R. Association of three plasmid-encoded spv genes among different Salmonella serotypes isolated from different origins. Indian J Microbiol 2013; 53(1):106-10.
31. Zahraei Salehi T, Amini K, Nikbakht G, et al. Molecular detection of invA and spv virulence genes in Salmonella enteritidis isolated from human and animals in Iran. Afr J Microbiol Res 2008; 4(21):2202-10.
32. Moussa IM, Aleslamboly YS, Al-Arfaj AA, et al. Molecular characterization of Salmonella virulence genes isolated from different sources relevant to human health. J Food Agric Environ 2013; 11(2):197-201.
33. Namimatsu T, Asai T, Osumi T, et al. Prevalence of the virulence plasmid in Salmonella typhimurium isolates from pigs. J Vet Med Sci 2006; 68 (2): 187-188.
34. Ling JML. Rapid detection of food-borne pathogens in clinical specimens, food and environmental samples. Hong Kong Med J 2009; 15(1):26-9.
35. Nikbakht Gh, Tadjbakhsh H. Study of plasmid virulence genes (spv) in Salmonella enterica serovars isolated in Iran. J Fac Vet Med Univ Tehran 2004; 59(2):137-40.
36. Matsui H, Bacot MC, Garlington AW, et al. virulence plasmid- Borne spvB and spvC genes can replace the 90-kilobase plasmid in conferring virulence to Salmonella entrica serovar typhimurium in subcutaneously inculcated mice. J Bacteriol 2001; 183(15): 2652-8.
37. Heithoff DM, Shimp WR, Iau PW, et al. Human salmonella clinical isolates distinct from those of animal origin. Appl Environ Microbiol 2008; 74(6):1757-66.
38. Bakshi CS, Singh VP, Malik M, et al. 55kb plasmid and virulence associated genes are positively correlated with Salmonella enteritidis pathogenicity in mice and chickens. Vet Res Comm 2003; 27(6):425-32.
2. Majtan V, Szaboova M. The virulence markers of Salmonella serovar typhimurium different phage type strains isolated in Slovakia. Folia microbiol 2005; 50(6):516-23.
3. Gay J. Bovine herd Salmonellosis. Am J Infect Control 1995; 23:56-62.
4. Lim YH, Hirose K, Izumiya H, et al. Multiplex polymerase chain reaction assay for selective detection of Salmonella entrica serovar typhimurium. Jpn J Infect Dis 2003; 56(4):151-5.
5. Pan TM, liu YJ. Identification of Salmonella enteritidis isolate by polymerase chain reaction and multiplex polymerase chain reaction. J Microbiol Immunol Infect 2002; 35(3):147-51.
6. Soumet C, Ermel G, Rose N, et al. Evaluation of a multiplex PCR assay for simultaneous identification of Salmonella serovars, Salmonella enteritidis and Salmonella typhimurium from environmental swabs of poultry houses. Lett Appl Microbiol 1999; 28(2):113-7.
7. Malkawi HI, Gharaibe R. Rapid and simultaneous Identification of two Salmonella entrica serotypes enteritidis and typhimurium from chicken and meat product by multiplex PCR. Biotech 2004; 3(1):44-8.
8. Jamshidi A, Bassami MR, Afshari-Nic S. Identification of Salmonella serovars typhimurium by a multiplex PCR-based assay from poultry carcasses in Mashhad-Iran. Int J Vet Res 2008; 3(1):43-8.
9. Aabo S, Rasmussen F, Rossen L, et al. Salmonella identification by the polymerase chain reaction. Mol Cell Probes 1993; 7(3):171-8.
10. Jangjou A, Mokhtari A, Panahi P, et al. Salmonella enteritidis in captive siberian Tiger: A case report from Iran. Glob Vet 2011; 6(5):502-5.
11. Chu C, Chiu CH. Evolutions of the virulence plasmid of non-typhoid Salmonella and its association with antimicrobial resistance. Microbes Infect 2006; 8(7):1931-6.
12. Gothoh H, Okada N, kim YG, et al. Extracellular secretion of the virulence plasmid- encoded ADP-ribosyl transferase SPVB in Salmonella. Microb pathog 2003; 34(5):227-38.
13. Hong Sf, chiu CH, Chu C, et al. Complete nucleotide sequence of a virulence of a virulence plasmid of Salmonella entrica serovar dublin and its phylogenetic relationship to the virulence plasmids of serovars choleraesuis, enteritidis and typhmurium. FEMS microbial lett 2008; 282(1):39-43.
14. Rotger R, Casadesus J. The virulence plasmids of Salmonella. Int Microbiol 1999; 2(3):177-84.
15. Ahmer MMB, Tran M, Heffron f. The virulence plasmid of Salmonella typhimurium is self-transmissible. J Bacteriol 1998; 181(4):1364-8.
16. Boyed EF, Hartl DL. Salmonella virulence plasmid modular acquisition of the spv virulence Region by an F-Plasmid in Salmonella entrica subspecies I and Insertion into the chromosome of subspecies II, IIIa, IV and VII Isolate. Genetics 1998; 149(3):1183-90.
17. Gebreyes WA, Thakur S, Dorr P, et al. Occurrence of spvA virulence gene and clinical significance for multidrug-resistant Salmonella strains. J clin Microbiol 2008; 47(3):777-80.
18. Chu C, Hong SF, Tsai C, et al. Comparative physical and genetic maps of the virulence plasmids of Salmonella entrica serovar typhimurium, enteritidis, choleraesuis and dublin. Infect Immun 1999; 67(5):2611-5.
19. Brian M, Ahmer M, Tran M, et al. The virulence plasmid of Salmonella typhimurium is self-transmissible. J Bacteriol 1998; 181(4):1364-8.
20. Del cerro A, Soto SM, Mendoza MC. Virulence and antimicrobial-resistance gene Profiles determined by PCR-Based Procedures for Salmonella isolated from samples of animal origin. Food microbiol 2002; 20(4):431-6.
21. Ziemer CJ, Steadham SR. Evaluation of the specifity of Salmonella PCR Primers using various intestinal bacterial species. Lett Appl Microbiol 2003; 37(6):463-9.
22. Rahn K, De Grandees SA, Clarke RC, et al. Amplification of an inv-A sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella. Mol Cell Probes 1992; 6(4);271-9.
23. Saroj SD, Shashidhar R, Bandekar JR. Note Genetic diversity of food isolates of Salmonella entrica serovar typhimurium in India. Food Sci Tec Int 2007; 14(2):151-6.
24. Madadgar O, Zahraei Salehi T, Tadjbaksh H, et al. Genomic and Phenotypic evaluation of Salmonella typhimurium and Salmonella entritidis in Iran. Com clin Pathol 2008; 17(4):229-35.
25. Malorny B, Hoofer j, Bunge C, et al. Multicenter validation of the analytical accuracy of Salmonella PCR Towards an international standard. Appl Environ Microbiol 2003; 69(3):290-6.
26. Scholz HC, Arnold T, Marg H, et al. Improvement of an invA- based PCR for the specific detection of Salmonella typhimurium in organs of pigs. Berl Munch Tierarztl Wochenschr 2001; 114(9-10):401-3.
27. Hughes AL, Shopland S, Wigley P, et al. Characterisation of Salmonella enterica serovar typhimurium isolates from wild bird in northern England from 2005-2006. BMC Vet Res 2008; 4:4-16.
28. El-Feky MA, Hassan MA, Mohamed WA, et al. Detection of InvA gene in non typhoidal Salmonellae isolated from food products and clinical cases Egypt. J Med Microbiol 2014; 23(1):33-41.
29. Chiu CH, Su LH, Chu CH, et al. Detection of multidrug-resistant Salmonella entrica serovar typhimurium phage types DT102, DT 104 and U302 by multiplex PCR. J Clin Microbiol 2006; 44(7):2354-8.
30. Derakhshandeh A, Firouzi R, Khoshbakht R. Association of three plasmid-encoded spv genes among different Salmonella serotypes isolated from different origins. Indian J Microbiol 2013; 53(1):106-10.
31. Zahraei Salehi T, Amini K, Nikbakht G, et al. Molecular detection of invA and spv virulence genes in Salmonella enteritidis isolated from human and animals in Iran. Afr J Microbiol Res 2008; 4(21):2202-10.
32. Moussa IM, Aleslamboly YS, Al-Arfaj AA, et al. Molecular characterization of Salmonella virulence genes isolated from different sources relevant to human health. J Food Agric Environ 2013; 11(2):197-201.
33. Namimatsu T, Asai T, Osumi T, et al. Prevalence of the virulence plasmid in Salmonella typhimurium isolates from pigs. J Vet Med Sci 2006; 68 (2): 187-188.
34. Ling JML. Rapid detection of food-borne pathogens in clinical specimens, food and environmental samples. Hong Kong Med J 2009; 15(1):26-9.
35. Nikbakht Gh, Tadjbakhsh H. Study of plasmid virulence genes (spv) in Salmonella enterica serovars isolated in Iran. J Fac Vet Med Univ Tehran 2004; 59(2):137-40.
36. Matsui H, Bacot MC, Garlington AW, et al. virulence plasmid- Borne spvB and spvC genes can replace the 90-kilobase plasmid in conferring virulence to Salmonella entrica serovar typhimurium in subcutaneously inculcated mice. J Bacteriol 2001; 183(15): 2652-8.
37. Heithoff DM, Shimp WR, Iau PW, et al. Human salmonella clinical isolates distinct from those of animal origin. Appl Environ Microbiol 2008; 74(6):1757-66.
38. Bakshi CS, Singh VP, Malik M, et al. 55kb plasmid and virulence associated genes are positively correlated with Salmonella enteritidis pathogenicity in mice and chickens. Vet Res Comm 2003; 27(6):425-32.
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| Issue | Vol 8 No 3-4 (2019) | |
| Section | Original Articles | |
| Keywords | ||
| Multiplex PCR Salmonella typhimurium Virulence genes. | ||
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How to Cite
1.
Amini K, Mobasseri P. Detection and Molecular Identification of Plasmid Virulence Genes in Salmonella enterica serovar typhimurium Isolated from Human and Animals by Multiplex PCR Method. J Med Bacteriol. 2019;8(3-4):31-39.
