Sampling and Survival of Salmonella Typhimurium on Surfaces

Abstract

It is important to determine both presence and number of viable microorganisms on the surface of utensils, work surfaces and other equipment in contact with food. Cross-contamination of foodborne pathogens on food-contact surfaces occurs readily during food-preparation, and the pathogens might remain viable in the kitchen for prolonged time. The Salmonella-genus is listed as a Category B food safety threat agent by CDC. This is the category for second highest priority agents. Salmonella is responsible for food infections in both the industrialized and developing parts of the world, and has the ability to survive under unfavorable environmental conditions. From a microbiological safety point of view, it is necessary to evaluate survival-ability of such pathogens. The traditional methods for detection and identification of foodborne pathogens are both time consuming and labor demanding. The protocols for determination of species and serotype in Salmonella-detection are comprehensive and include culturing and serologic tests, which take 4-6 days to complete. PCR-based techniques have proved to be very promising methods for rapid and accurate detection of Salmonella, and have been frequently studied for the past years. PCR is faster compared to culture-based detection. Absolute quantification with external standard is easy to implement in the assay, and detection limits can be very low. In this thesis, survival of Salmonella enterica serovar Typhimurium on surfaces was investigated, with surrounding temperature and matrix as effect factors. Plastic (polystyrene) surfaces were contaminated with S. Typhimurium of a known concentration and stored at room-temperature (22oC), in cold surroundings (4oC) or warm surroundings (37oC). S. Typhimurium was either diluted in PBS, or mixed with thaw water from chicken or drain water as matrix. Samples were collected with swab, according to a protocol developed for this study. The samples were investigated with plate count for survival and real-time quantitative PCR for total cell count. EMA treatment in combination with real-time PCR was used to decide total viable count. Samples of non-viable cells were subject to resuscitation to determine if the cells were dead or had entered a VBNC state. In PBS, survival was observed for 3-4 days at 22oC, up till one week at 4oC and 9 hours at 37oC. With thaw water from chicken as matrix, viable cells were observed for 6 weeks at 22oC, and four weeks at 4oC. Mixed with drain water, the survival was observed for up till 10 days and four days at 22oC and 4oC, respectably. Total cell count and total-viability count did always give higher yield compared to plate count. Total viability count did always provide lower yield compared to total count. Resuscitation of non-viable cells was possible by enrichment in buffered peptone water. Surface sampling with swab does not provide full recovery or quantification of a sample. EMA-treatment before real-time PCR can distinguish live and dead cells, but is probably not sufficient to discriminate between viable cells and VBNC-cells. It was not possible to determine if the observed resuscitation was resulted by true resuscitation from VBNC back to viable form. In conclusion, surrounding temperature and matrix have an effect on survival of S. Typhimurium on surfaces. There is highlighted that S. Typhimurium remain viable and detectable on a surface up till six weeks and may present a possible hazard for a considerable period of time.