Presence of Salmonella in retail grade a eggs determined by the International Organization for Standardization 6579 method and a LightCycler polymerase chain reaction system

Abstract

This study aims to determine the presence of Salmonella in naturally contaminated grade A eggs by the standard culture method International Organization for Standardization Method 6579 (ISO) and a specific real-time PCR system (LightCycler PCR-LCPCR) to complement ISO. A total of 1635 eggs pooled into 101 samples were randomly collected within one year period from 20 different retail markets in Bursa, Turkey, carrying eggs of 16 large egg producers/suppliers of 5 cities with intensive layer production. Preparation of the egg and shell for analyses, Salmonella isolations and identifications, and detections were performed according to ISO 6887-4:2003, ISO 6579 and LCPCR, respectively. Overall Salmonella detection rate by ISO and LCPCR were 15.8 % (16/101) and 46.5 % (47/101), respectively. Out of 101 inner parts, Salmonella was detected in 11 (10.9 %) samples by ISO, and in 31 (30.7 %) samples by LCPCR. Six of 101 shell samples (5.9 %) were found to harbor Salmonella by ISO, while 18 (17.8 %) shells were positive by LCPCR. All isolates were determined as Salmonella enterica subsp. enterica serovar Enteritidis. These findings indicate considerably high Salmonella contamination in retail grade A eggs. This should be under routine monitoring by rapid methods such as PCR, complemented by standard culture to evaluate and assess the significance of risk for public health

Keywords

• ISO 6579
• real-time polymerase chain reaction
• retail egg
• Salmonella

Perakende A sınıfı yumurtalarda Salmonella varlığının ISO 6579 metodu ve LightCycler polimerazzincir reaksiyonu ile belirlenmesi

Abstract

Bu çalışmada doğal kontamine A sınıfı yumurtalarda Salmonella varlığının ISO 6579 standart kültür metodu ve bu metodun spesifik gerçek zamanlı bir PCR sistemi (LightCycler PCR-LCPCR) ile desteklenmesini belirlemek amaçlanmıştır. Bursa’da 20 farklı perakende marketten 1 yıl süresince, yoğun yumurta üretimi olan 3 bölge (Ege, Marmara, Orta Anadolu)’nin büyük üretici/tedarikçisi olan 16 farklı firmaya ait 101 birleştirilmiş, toplam 1635 adet yumurta rastgele olarak örneklendi. Yumurtaların analize hazırlanması, Salmonella izolasyonu ile identifikasyonu ve deteksiyonu sırasıyla ISO 6887-4:2003, ISO 6579’a göre ve LCPCR ile yapıldı. Yumurtalarda genel Salmonella deteksiyon oranları ISO ve LCPCR ile sırası ile %15.8 (16/101) ve % 46.5 (47/101) olarak belirlendi. Yüzbir iç örneğinin ISO ile 11’inde (% 10.9), LCPCR ile 31’inde (% 30.7) Salmonella saptandı. Kabuk örneklerinden 6’sında (% 5.9) ISO ile Salmonella bulunurken, 18’i (17.8 %) LCPCR ile pozitif olarak tespit edildi. Tüm izolatların Salmonella enterica subsp. enterica serovar Enteritidis olduğu belirlendi. Bu bulgular perakende A sınıfı yumurtalarda Salmonella kontaminasyonunun oldukça yüksek olduğunu ve bu durumun halk sağlığı açısından önemini belirlemek ve değerlendirebilmek için standart kültür metodu ile desteklenen PCR gibi hızlı bir metotla rutin olarak izlenmesi gerektiğini göstermektedir

Keywords

• ISO 6579
• gerçek zamanlı polimeraz zincir reaksiyonu
• perakende yumurta
• Salmonella

References

1. Adesiyun A, Offiah N, Seepersadsingh N, Rodrigo S, Lashley V, Musai L (2007): Antimicrobial resistance of Salmonella spp. and Escherichia coli isolated from table eggs. Food Control, 18, 306-311.
2. Betancor L, Pereira M, Martinez A, Giossa G, M. Fookes, K. Flores, P. Barrios, V. Repiso, R. Vignoli, N. Cordeiro, G. Algorta, N. Thomson, D. Maskell, F. Schelotto, JA Chabalgoity (2010): Prevalence of Salmonella enterica in poultry and eggs in Uruguay during an epidemic due to Salmonella enterica serovar Enteritidis. J Clin Microbiol 48, 2413-2423.
3. Bohaychuk VM, Gensler GE, McFall ME, King RK, Renter DG (2007): A real time PCR assay for the detection of Salmonella in a wide variety of food and food- animal matrices. J Food Prot, 70, 1080-1087.
4. Cakiroglu HS, Gumussoy KS (2005): Ankara garnizonunda tüketime sunulan tavuk yumurtalarının Salmonella spp. yönünden analizi. Sag Bil Derg, 14, 158-162.
5. Chen J, Zhang L, Paoli GC, Shi C, Tu S, Shi X: (2010) A real-time PCR method for the detection of Salmonella enterica from food using a target sequence identiŞed by comparative genomic analysis. Int J Food Microbiol, 137, 16-174.
6. Cogan TA, Humphrey TJ (2003): The rise and fall of Salmonella Enteritidis in the UK. J Appl Microbiol, 94 (Suppl), 114-119.
7. EFSA (2007): Report of the task force on zoonoses data collection on the analysis of the baseline survey on the prevalence of Salmonella in broiler flocks of Gallus gallus, in the EU, 2005–2006. Part A: Salmonella prevalence estimates. EFSA J, 1-85.
8. Erkan ME, Vural A, Güran HŞ (2008): Diyarbakır ilinde satışa sunulan köy ve market yumurtalarının hijyenik kalitesi üzerine bir araştırma. J Dicle Univ Vet Fac, 1, 11-16.

9. Eyigor A, Carli KT, Unal CB (2002): Implementation of real-time PCR to tetrathionate broth enrichment step of Salmonella detection in poultry. Lett Appl Microbiol, 34, 37-41.
10. Eyigor A, Temelli S, Carli KT (2010): Evaluation of ISO 6579 and FDA-BAM methods to complement real-time polymerase chain reaction for the detection of Salmonella in naturally contaminated poultry meat and red meat. Foodborne Pathog Dis, 7, 921-927. 11. Food and Drug Administration Bacteriological
11. Analytical Manual (2011): FDA Chapter 5: Salmonella.
12. Ed. Andrews WH, Jacobson A, Hammack T. Available at
13. http://www.fda.gov/Food/FoodScienceResearch/Laborator
14. yMethods/ucm070149.htm (accessed September 2013).
15. Gole VC, Chousalkar KK, Roberts JR (2013): Survey of Enterobacteriaceae contamination of table eggs collected from layer flocks in Australia. Int J Food Microbiol, 164, 161-165.
16. Grimont PAD, Weill F (2007): Antigenic formulae of the Salmonella serovars: White-Kauffmann-Le Minor Scheme, 9th edition. WHO Collaborating Centre for Reference and Research http://www.pasteur.fr/ip/portal/action/WebdriveActionEve nt/oid/01s-000036-089 (accessed September 2013).
17. Guibourdenche M, Roggent P, Mikoleit M, Fields PI, Bockemühl J, Grimont PA, Weill FX (2010): Supplement 2003-2007 (No. 47) to the White-Kauffmann- Le Minor scheme. Res Microbiol, 161, 26-29.
18. International Organization for Standardization (2002): Microbiology of food and animal feeding stuffs. Horizontal method for the detection of Salmonella spp. ISO 6579:2002 (E).
19. Jones DR, Anderson KE, Musgrove MT (2011): Comparison of environmental and egg microbiology associated with conventional and free-range laying hen management. Poultry Sci, 90, 2063-2068.
20. Lee SK, Chon JW, Song KY, Hyeon JY, Moon JS, Seo KH antimicrobial susceptibility of Salmonella Gallinarum isolated from eggs produced in conventional or organic farms in South Korea. Poultry Sci, 92, 2789-2797. and
21. Malorny B, Löfström C, Wagner M, Kramer N, Hoorfar J (2008): Enumeration of Salmonella bacteria in food and feed samples by real-time PCR for quantitative microbial risk assessment. Appl Environ Microbiol, 74, 1299-1304.
22. Musgrove MT, Jones DR, Northcutt JK, Harrison MA, Cox NA, Ingram KD, Hinton AJ Jr (2005): Recovery of Salmonella from commercial shell eggs by shell rinse and shell crush methodologies. Poultry Sci, 84, 1955-1958.
23. Pangloli P, Dje Y, Ahmed O, Doane CA, Oliver SP, Draughon FA (2008): Seasonal incidence and molecular characterization of Salmonella from dairy cows, calves, and farm environment. Foodborne Pathog Dis, 5, 87–96.
24. Rebolledo J, Garvey P, Ryan A, O'Donnell J, Cormican M, Jackson S, Cloak F, Cullen L, Swaan CM, Schimmer B, Appels RW, Nygard K, Finley R, Sreenivasan N, Lenglet A, Gossner C, McKeown P (2013): International outbreak investigation of Salmonella Heidelberg associated with in-flight catering. Epidemiol Infect, doi:10.1017/S0950268813001714.
25. Sasaki Y, Tsujiyama Y, Asai T, Noda Y, Katayama S, Yamada Y (2011): Salmonella prevalence in commercial raw shell eggs in Japan: A survey. Epidemiol Infect, 139, 1060-1064.
26. Soria MA, Soria MC, Bueno DJ (2012a): A comparative study of culture methods and polymerase chain reaction for Salmonella detection in egg content. Poultry Sci, 91, 2668-2676.
27. Soria MC, Soria MA, Bueno DJ, Colazo JL (2011): A comparative study of culture methods and polymerase chain reaction assay for Salmonella detection in poultry feed. Poultry Sci, 90, 2606-2618.
28. Soria, MC, Soria MA, Bueno DJ (2012b): Comparison of 2 culture methods and PCR assays for Salmonella detection in poultry feces. Poultry Sci, 91, 616-626.
29. Suresh T, Hatha AAM, Sreenivasan D, Sangeetha N, Lashmanaperumalsamy P (2006): Prevalence and antimicrobial resistance of Salmonella enteritidis and other salmonellas in the eggs and egg-storing trays from retails markets of Coimbatore, South India. Food Microbiol, 23, 294-299.
30. Wilson IG (1997): Inhibition and facilitation of nucleic acid amplification. Appl Environ Microb, 63, 3741-3751. Geliş tarihi: 17.02.2014/ Kabul tarihi: 14.07.2014 Address for correspondence: Prof. Dr. Ayşegül Eyigör Uludağ University, Faculty of Veterinary Medicine, Department of Food Hygiene and Technology, 16059, Görükle Campus, Bursa, Turkey e-mail: [e-mail]