The aim of the study was to evaluate and adapt the PCR-based protocol that utilizes the developed serotype-specific primers to identify Salmonella enterica species and its serotypes that are most frequently isolated from poultry samples in Vojvodina. Using the slide agglutination test, 64 and 33 out of 107 Salmonella isolates were identified as S. Infantis and S. Enteritidis, respectively, while ten isolates were identified as eight different Salmonella serovars. Using the same isolates, presence of 993-bp (bcfC gene), 636-bp (steB gene) and 293-bp (sdf locus) amplicons in multiplex PCR unambiguously identified 31 isolates as S. Enteritidis. Two isolates identified as Enteritidis in slide agglutination test were not identified as such in PCR-based approach since they both were missing 293-bp long PCR product. Thirty-nine isolates produced a 727-bp amplicon in the specific simplex PCR, and thus were identified as S. Infantis. The greatest discrepancy in comparison to the results of conventional serotyping has been observed in the case of S. Infantis, since 25 more isolates were noted as S. Infantis by conventional serotyping. Seven isolates, with unexpected PCR profiles stayed unidentified by molecular typing, although they were serotyped as S. Typhimurium (1) and S. Infantis (6). S. Gallinarum serovar has to be additionally confirmed, since it shares the same PCR profile with S. Livingstone. Clearly, PCR-based identification has to be thoroughly checked, verified and adapted if it is to be applied as the routine identification protocol.
If the inline PDF is not rendering correctly, you can download the PDF file here.
1. McDermott PF: Antimicrobial resistance in nontyphoidal Salmonellae. In: Antimicrobial resistance in bacteria of animal origin. Washington DC USA: ASM Press; 2006 293-315.
2. CDC (2014) Foodborne Diseases Active Surveillance Network (FoodNet): FoodNet Surveillance Report for 2012 (Final Report). Atlanta Georgia: U.S. Department of Health and Human Services CDC.
3. Commission regulation (EC) No 2073/2005 on microbiological criteria for foodstuffs Official Journal of European Union L388 Volume 48 2005 1-25.
4. Brands DA: Salmonella and Food-borne illness. In: Deadly diseases and epidemics-Salmonella. Langhorne PA USA: Chelsea House Publishers; 2006 19-27.
5. World Health Organisation Microbiological Risk Assessment Series: Salmonella and Campylobacter in chicken meat Meeting Report 2009.
6. European Food Safety Authority Scientific Report of EFSA and ECDC: The European Union summary report on trends and sources of zoonoses zoonotic agents and foodborne outbreaks in 2013 EFSA Journal 2015 13 (1):3991.
7. European Food Safety Authority Scientific Report of EFSA: Analysis of the baseline survey on the prevalence of Campylobacter in broiler batches and of Campylobacter and Salmonella on broiler carcasses in the EU 2008. Part B: Analysis of factors associated with Salmonella contamination of broiler carcasses EFSA Journal 2011 9 (2):2017.
8. Andino A Hanning I: Salmonella enterica: survival colonization and virulence differences among serovars. Sci World J 2015 DOI 10.1155/2015/520179.
9. Carlton LG Prescott JF Songer G Thoen CO: Pathogenesis of bacterial infection. Hoboken NY USA: Wiley and Blackwell 4th Edition; 2010.
10. Winn W Allen S Janda W Koneman E Procop G Schreckenberger P Woods G: Koneman’s color atlas and textbook of diagnostic microbiology. Philadelphia PA USA: Lippincott Williams & Wilkins 6th Edition; 2006.
11. McQuiston JR Parrenas R Ortiz-Rivera M Gheesling L Brenner F Fields PI: Sequencing and comparative analysis of flagellin genes fliCfljB and flpA from Salmonella. J Clin Microbiol 2004 42:1923-32.
12. Kim S Frye JG Hu J Fedorka-Cray PJ Gautom R Boyle DS: Multiplex PCR-based method for identification of common clinical serotypes of Salmonella enterica subsp. enterica. J Clin Microbiol 2006 44:3608-3615.
13. Tennant SM Diallo S Levy H Livio S Sow SO Tapia M Fields PI Mikoleit M Tamboura B Kotloff KL Nataro JP Galen JE Levine MM: Identification by PCR of non-typhoidal Salmonella enterica serovars associated with invasive infections among febrile patients in Mali. PLoS Negl Trop Dis 2010 4:e621.
14. Salazar JK Wang Y Yu S Wang H Zhang W: Polymerase chain reaction-based serotyping of pathogenic bacteria in food. J Microbiol Methods 2015 110:18-26.
15. Krawiec M Kuczkowski M Kruszewicz AG Wieliczko A: Prevalence and genetic characteristics of Salmonella in free-living birds in Poland. BMC Vet Res 2015 11:15.
16. Pugliese N Circella E Pazzani C Pupillo A Camarda A. Validation of a seminested PCR approach for rapid detection of Salmonella enteric subsp. enterica serovar Gallinarum. J Microbiol Methods 2011 85:22-27.
17. Zhu C Yue M Rankin S Weill F-X Frey J Schifferli DM: One-step identification of five prominent chicken Salmonella serovars and biotypes. J Clin Microbiol 2015 53: 3881-3883.
18. Kardos G Farkas T Antal M Nogrady N Kiss I: Novel PCR assay for identification of Salmonella enterica serovars Infantis. Lett Appl Microbiol 2007 45:421-425.
19. ISO 6579:2002 (E). Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Salmonella spp. (2002) 4th Ed. International Standard Geneva Switzerland.
20. Altschul S Gish W Miller W Myers E Lipman D: Basic local alignment search tool. J Mol Biol 1990 215:403-410.
21. Velhner M Kozoderović G Grego E Galić N Stojanov I Jelesić Z Kehrenberg C: Clonal spread of Salmonella enterica serovar Infantis in Serbia: Acquisition of mutations in the topoisomerase genes gyrA and parC leads to increased resistance to fluoroquinolones. Zoonoses Public Hlth 2014 61:364-70.
22. Agron PG Walker RL Kinde H Sawyer SJ Hayes DC Wollard J Andersen GL: Identification by subtractive hybridization of sequences specific for Salmonella enteric serovars Enteritidis. Appl Environ Microbiol 2001 67:4984–4991.
23. Shivaprasad HL Methner U Barrow PA: Salmonella infections in the domestic fowl. In: Salmonella in domestic animals. Wallingford United Kingdom: CABI; 2013 162-192.
24. Nogrady N Kardos G Bistyak A Turcsanyi I Meszaros J GalantaiZs Juhasz A Samu P Kaszanyitzky JE Paszti J Kiss I: Prevalence and characterization of Salmonella Infantis isolates originating from different points of the broiler chicken-human food in Hungary. Int J Food Microbiol 2008 127:162-167.
25. Chironna M Tafuri S Gallone MS Sallustio A Martinelli D Prato R Germinario C: Outbreak of Salmonella Infantis gastroenteritis among people who had eaten at a hash house in southern Italy. Public Health 2014 128:438-443.
26. Shimizu R Osawa K Shigemura K Yoshida H Fujiwara M Iijima Y Fujisawa M Shirakawa T: Development of multiplex PCR for rapid identification of four Salmonella serovars most commonly isolated in Japan. Southeast Asian J Trop Med Public Health 2014 45:654-661.
27. Ghoddusi A Nayeri Fasaei B Karimi V AshrafiTamai I Moulana Z Zahraei Salehi T: Molecular identification of Salmonella Infantis isolated from backyard chickens and detection of their resistance genes by PCR. Iran J Vet Res 2015 16:293-297.
28. Guedda I Taminiau B Ferjani A Boukadida J Bertrand S Daube G: Antimicrobial and molecular analysis of Salmonella serovar Livingstone strains isolated from humans in Tunisia and Belgium. J Infect Dev Ctries 2014 8:973-980.