Rapid detection and specific differentiation of Salmonella enterica subsp. enterica Enteritidis, Typhimurium and its monophasic variant 4,[5],12:i:- by real-time multiplex PCR.

Salmonella enterica is one of the most common zoonotic pathogens worldwide causing clinical diseases in human and animal hosts. Targeting a reduction of Salmonella prevalence in poultry, the EU set up a microbiological criterion that demands the absence of S. enterica subsp. enterica serovars Enteritidis and Typhimurium including its monophasic variant with seroformula 4,[5],12:i:- in 25 g of poultry neck skin samples and fresh meat according to regulation (EU) no 1086/2011. We developed and in-house validated a method that detects and differentiates these Salmonella serovars based on a 5-plex real-time PCR assay within 24 h after sampling. The inclusivity and exclusivity were between 98 and 99% analysing 456 bacterial strains. Validation according to ISO 16140:2003 against the traditional cultural reference method ISO 6579:2002 was performed using 60 artificially contaminated and 31 presumably naturally contaminated chicken neck skin samples resulting in a relative accuracy of 100%. The detection probability reached 100% between 3 and 5 CFU/25 g sample. We were also able to assign rough and non-motile strains to S. enterica subsp. enterica serovars Enteritidis and Typhimurium. In conclusion, we provide diagnostic laboratories a fast and accurate method to monitor these Salmonella serovars in chicken neck skin samples. Other matrices could be easily adapted.

[1]  Sherilyn J. Sawyer,et al.  Identification by Subtractive Hybridization of Sequences Specific for Salmonella enterica Serovar Enteritidis , 2001, Applied and Environmental Microbiology.

[2]  D. Maskell,et al.  Genomic Comparison of the Closely Related Salmonella enterica Serovars Enteritidis and Dublin , 2012, The open microbiology journal.

[3]  Maido Remm,et al.  Enhancements and modifications of primer design program Primer3 , 2007, Bioinform..

[4]  B. Faircloth,et al.  Primer3—new capabilities and interfaces , 2012, Nucleic acids research.

[5]  V. Beneš,et al.  The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. , 2009, Clinical chemistry.

[6]  Efsa Journal,et al.  European Food Safety Authority, European Centre for Disease Prevention and Control; The European Union Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Food-borne Outbreaks in 2009 , 2011 .

[7]  S. Herrera-León,et al.  Genetic Evolution of the Spanish Multidrug-Resistant Salmonella enterica 4,5,12:i:- Monophasic Variant , 2010, Journal of Clinical Microbiology.

[8]  BarcoLisa,et al.  Molecular characterization of "inconsistent" variants of Salmonella Typhimurium isolated in Italy. , 2014 .

[9]  W. Rabsch,et al.  Pork Contaminated with Salmonellaenterica Serovar 4,[5],12:i:−, an Emerging Health Risk for Humans , 2010, Applied and Environmental Microbiology.

[10]  K. Ohnishi,et al.  Gene fliA encodes an alternative sigma factor specific for flagellar operons in Salmonella typhimurium , 1990, Molecular and General Genetics MGG.

[11]  D. Maskell,et al.  Role in virulence and protective efficacy in pigs of Salmonella enterica serovar Typhimurium secreted components identified by signature-tagged mutagenesis. , 2007, Microbiology.

[12]  T. Barry,et al.  Development of a real-time multiplex PCR assay for the detection of multiple Salmonella serotypes in chicken samples , 2008, BMC Microbiology.

[13]  M. Wiedmann,et al.  Salmonella enterica Serotype 4,5,12:i:−, an Emerging Salmonella Serotype That Represents Multiple Distinct Clones , 2009, Journal of Clinical Microbiology.

[14]  R. Sack,et al.  The flagellin N-methylase gene fliB and an adjacent serovar-specific IS200 element in Salmonella typhimurium. , 1997, Microbiology.

[15]  M. Levine,et al.  Identification by PCR of Non-typhoidal Salmonella enterica Serovars Associated with Invasive Infections among Febrile Patients in Mali , 2010, PLoS neglected tropical diseases.

[16]  M. Hartung,et al.  Erreger von Zoonosen in Deutschland im Jahr 2008 , 2010 .

[17]  M. Wiedmann,et al.  Emergence, distribution, and molecular and phenotypic characteristics of Salmonella enterica serotype 4,5,12:i:-. , 2009, Foodborne pathogens and disease.

[18]  Burkhard Malorny,et al.  Making Internal Amplification Control Mandatory for Diagnostic PCR , 2003, Journal of Clinical Microbiology.

[19]  Valentina Rizzi,et al.  The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2010. , 2012, Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin.

[20]  J. Hoorfar,et al.  A PCR-based strategy for simple and rapid identification of rough presumptive Salmonella isolates. , 1999, Journal of microbiological methods.

[21]  Zhemin Zhou,et al.  Multilocus Sequence Typing as a Replacement for Serotyping in Salmonella enterica , 2012, PLoS pathogens.

[22]  S. Fanning,et al.  A multiplex real-time PCR assay for the identification and differentiation of Salmonella enterica serovar Typhimurium and monophasic serovar 4,[5],12:i:-. , 2013, International journal of food microbiology.

[23]  Reiner Helmuth,et al.  A real-time PCR for the detection of Salmonella Enteritidis in poultry meat and consumption eggs. , 2007, Journal of microbiological methods.

[24]  G. Olsen,et al.  Genomic Comparison of the Closely-Related Salmonella enterica Serovars Enteritidis, Dublin and Gallinarum , 2015, PloS one.

[25]  E. Orlova,et al.  Structural analysis of the Saf pilus by electron microscopy and image processing. , 2008, Journal of Molecular Biology.