| Abstract | Salmonella is a Gram-negative, motile, facultative anaerobic bacterium which typically causes an intestinal infection that is accompanied by fever, abdominal cramps and diarrhea. Contaminated egg, meat, sea foods and poultry products are the main sources of Salmonella infection and a major threat to food safety. At present, the inspection of food due to the presence of Salmonella has become routine all over the world and is done by conventional methods. However, a number of rapid methods for the detection of Salmonella in foods are being developed. Molecular techniques, such as Polymerase chain reaction (PCR) based techniques and Fluorescent In Situ Hybridization (FISH) are the most popular among them. However, there are problems associated with the mentioned methods which include low detection sensitivity and inability to differentiate between viable and non-viable cells. Reasons for low sensitivity in molecular detection techniques are difficulties in isolating microorganisms from food matrixes, inhibition of the detection by chemical inhibitors in food samples, and the requirement of minimum number of cells to give positive results. The isolation of Salmonella enterica cells from food matrixes using metal hydroxide immobilization was tested in this work as a means to concentrate bacterial cells. Five different DNA extraction methods were tested for their efficiency for high purity DNA extraction and detection sensitivity improvement. Nested-PCR method, a modified-PCR method, was tested for its ability to increase the sensitivity of Salmonella enterica detection compared with the general PCR method. Metal hydroxide immobilization prior to DNA extraction was successful in isolation and concentration of Salmonella enterica cells from meat food matrix. Immobilization with metal hydroxide increased the DNA yield of all five extraction methods tested. The increase of the DNA yield of Zr(OH)4 immobilization was higher than Ti(OH)4 immobilization. Spectrophotometic readings revealed that the DNA extracted by all of the five methods were within acceptable purity levels. Out of the five tested DNA extraction methods, Fontana et al., (2005) method gave the highest DNA yield. The application of Nested PCR was able to increase the detection sensitivity by 100 fold compared to the conventionl PCR. The combination of the application of metal hydroxide immobilization, DNA extraction by Fontana et al., (2005) method and Nested PCR amplification was able to increase the detection sensitivity up to 100-101cfu/mL after 4 hours of enrichment. The inability of molecular detection techniques like the PCR to differentiate between viable and non-viable cells is because of the persistence of nucleic acid (DNA and rRNA) in cells for a long time even after cell death. We expected this to be overcome this problem by treating DNA in dead cells (more porous cell walls) by DNAase enzyme before the DNA extraction (the DTD-PCR method) and by using the Fluorescent in situ Hybridization (FISH) method. The persistence of DNA and RNA in Salmonella enterica cells heat-killed using different thermal treatments (800C, 1000C and 1210C for 15min) were examined using the DTD-PCR and FISH technique respectively after different time intervals (5min - 48h) after thermal treatments. Moreover, the FISH technique was modified by introducing a cell vivification step using antibiotic treatment to increase the reliability of viable cell detection. In order to find out the best method for viable and non-viable cell differentiation, general and modified FISH techniques, DTD-PCR technique and three salmonella specific culture media namely, Rambach agar, Rainbow Agar Salmonella, and XLT4 agar were compared with each other.
DNA persisted in all heat-killed bacterial samples through out the time periods studied in our experiments. rRNA persisted for 12h, 3h and 1h after thermal treatments in the samples
iv
heated to 800C, 1000C and 1210C respectively. This was the case even though there were no growth (colonies) in plate culture. In all FISH samples, bacterial cells were stained to different levels and visualized to different intensities. Number of well-stained cells were less and reduced with time. The reduction of staining intensity of the cells with time increased with the increase of treatment temperature. Compared to culture treated at 800C, staining decreased considerably when treated at 1000C temperature. In autoclaved culture, staining of cells was very less and cells were hardly detectable. The modified FISH technique using antibiotic treatment step gave higher fluorescent signals compared to general FISH technique. When five different methods used for differentiating live and dead cells were tested, significantly lower count compared to actual live cell count (Hemocytometer count) were given by all three Salmonella specific culture media tested. The conventional FISH technique gave significantly higher viable cell count compared to actual count. The viable cell count of modified FISH method was not significantly different from actual count. In conclusion it can be said that, the combined application of metal hydroxide immobilization and Nested-PCR was found to be a powerful and sensitive method for the detection of Salmonella enterica in meat food samples. Out of five tested DNA extraction methods, Modified Fontana et al., (2005) method was identified as the best method for Salmonella enterica DNA extraction from contaminated meat samples. Study on the persistence of DNA and rRNA in heat-killed cells revealed the inapplicability of DTD-PCR technique in differentiation of dead and live cells. The effect of DNAase seems to be determined by many factors as the results contradict previous works. Compared to the PCR method, the FISH technique was found to be more promising in differentiation of viable Salmonella enterica cells from heat-killed cells. Moreover, the introduction of antibiotic treatment step in FISH increased the accuracy of viable microbial counts. The results of the comparison of different viable cell counting methods using Salmonella specific culture media confirmed the low reliability of cultural technique and general FISH technique for viable cell counts as compared to the actual counts made on a hemocytometer. FISH technique with vivification step by antibiotic treatment was identified as a reliable technique in enumeration of viable-food-borne Salmonella enterica. |