Quantification of Zoonotic Bacterial Pathogens within Commercial Poultry Processing Water Samples Using Droplet Digital PCR

A robust 5' nuclease (TaqMan) real-time PCR was developed and validated in-house for the specific detection of Salmonella in food. The assay used specifically designed primers and a probe target within the ttrRSBCA locus, which is located near the Salmonella pathogenicity island 2 at centisome 30.5. It is required for tetrathionate respiration in Salmonella. The assay correctly identified all 110 Salmonella strains and 87 non-Salmonella strains tested. An internal amplification control, which is coamplified with the same primers as the Salmonella DNA, was also included in the assay. The detection probabilities were 70% when a Salmonella cell suspension containing 10(3) CFU/ml was used as a template in the PCR (5 CFU per reaction) and 100% when a suspension of 10(4) CFU/ml was used. A pre-PCR sample preparation protocol including a preenrichment step in buffered peptone water followed by DNA extraction-purification was applied when 110 various food samples (chicken rinses, minced meat, fish, and raw milk) were investigated for Salmonella. The diagnostic accuracy was shown to be 100% compared to the traditional culture method. The overall analysis time of the PCR method was approximately 24 h, in contrast to 4 to 5 days of analysis time for the traditional culture method. This methodology can contribute to meeting the increasing demand of quality assurance laboratories for standard diagnostic methods. Studies are planned to assess the interlaboratory performance of this diagnostic PCR method.

Campylobacters are a primary cause of human bacterial enteritis worldwide. They are usually considered susceptible to the disinfectant molecules used in the food industry. The purpose of this study was to see if campylobacters could survive cleaning and disinfection in poultry slaughterhouses and whether the strains recovered could contaminate carcasses during processing. Samples obtained from the environment before and after cleaning and disinfection (transport crates, processing equipment surfaces, scald tank water) and from birds (fresh droppings, neck skins) were collected during 7 investigations in 4 different slaughterhouses. Out of 41 samples collected, 30 Campylobacter jejuni strains were recovered from the surfaces of processing equipment before cleaning and disinfection procedures in three slaughterhouses and 9 C. jejuni out of 51 samples collected were found after cleaning. The study was then focused on one slaughterhouse to trace passage of the pathogen on poultry carcasses. The antimicrobial resistance phenotypes (P) (minimum inhibitory concentration, MIC) of the C. jejuni isolates collected in this slaughterhouse were determined. Nine phenotypes could be distinguished. Three of these were of interest as they were found in isolates recovered after cleaning and disinfection procedures. The genotypes (G) were determined by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) of isolates with one of the three phenotypes of interest. Clusters constructed by combining the phenotype and genotyping observations (PG type) were compared between isolates obtained after cleaning and disinfection, and isolates from droppings, neck skin and transport crate samples of slaughtered poultry flocks. Only one PG type of strain was recovered from surfaces after cleaning and disinfection and from neck skin samples but was also recovered from transport crates. Our findings indicate that C. jejuni is able to survive overnight on food processing equipment surfaces, after cleaning and disinfection procedures, and that these strains may contaminate carcasses during the slaughter process. These results add to our understanding of poultry carcass contamination and highlight the need to develop ways of reducing the risk of human infection with Campylobacter through the consumption of poultry products.