Pcr-Based Detection Of Food Isolates Of Yersinia Species And Its Behavioural Pattern In Selected Foods.

Divya, K. H. (2011) Pcr-Based Detection Of Food Isolates Of Yersinia Species And Its Behavioural Pattern In Selected Foods. PhD thesis, University of Mysore.

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Abstract

In the spectrum of changing foodborne diseases, Yersinia enterocolitica, an important food and waterborne gastrointestinal causative organism is regarded as a pathogen of public health significance. Presence of Y. enterocolitica in food products is of special concern, since these organisms are tolerant to low temperature and can continue to grow at refrigeration temperature and also survive in frozen food subjected to freezing and thawing. Several PCR-based markers including primers for species specificity, rRNA sequences and different toxin genes have been evolved to detect for the presence of virulent strains of this bacterial species. It becomes imperative, that the focus of research investigation should aim at assessing the prevalence and identification of toxigenic traits in food isolates of Y. enterocolitica, their heat and cold treatments profile in line with those existing in food processing, molecular basis for the diversity of native isolates and predictive models for the behaviour in broth and food systems under defined intrinsic and extrinsic factors. These aspects would give a comprehensive approach from the point of food safety and public health perspective leading to the betterment of quality of human life. In a total of 120 food samples of diverse varieties using the alkaline post-enrichment and plating onto Cefsulodin-Irgasan-Novobiocin (CIN) agar plates resulted in the characterization of two isolates of Y. enterocolitica and three isolates of Y. intermedia. Both the isolates of Y. enterocolitica belonged to the highly pathogenic biotype 1B. The isolates of Y. enterocolitica were further tested for pathogenicity markers through different culture-based methods and a similar antibiogram pattern was observed. The toxigenic/virulence traits in native isolates of Yersinia spp. were determined by polymerase chain reaction (PCR) by designing oligonucleotide primers for selected target genes. The two isolates of Y. enterocolitica showed positive amplification in PCR for the selected virulence target genes of (i) attachment invasion locus (ail), (ii) heat stable enterotoxin (yst), (iii) regulator of virulence (rovA), (iv) regulator of myfA, which codes for the virulence factor- mucoid Yersinia factor A (myfF), (v) Yersinia phospholipase (yplA), (vi) Yersinia type 2 secretion system (yts1). The isolates of Y. intermedia were positive only for the gene of yplA. Force type neighbor-joining phylograms generated for Y. enterocolitica showed 100% homology with two to three strains of Y. enterocolitica and about 75% homology with several strains of Y. pestis. a Abstract of Ph.D. thesis The factors affecting the secretion of phospholipase in Y. enterocolitica was investigated, wherein the production of phospholipase was found to be affected by temperature, sodium chloride and also glucose levels in the medium. The lag phase duration (LPD) and growth rate (GR) values of a native food isolate of Y. enterocolitica CFR 2301 as influenced by selected factors and its variables were studied. A central composite design based on the factors and variables of temperature, pH level, concentrations of sodium chloride and sodium nitrite was used to generate growth curves. The observed LPD values based on Baranyi and Gompertz functions were similar ranging from 2.02 to 19.96 h and these values showed were almost close to the estimated values. Similarly, the observed GR values based on the two functions ranged from 0.05 to 0.50/h and revealed a good agreement with the estimated values. The R2 value obtained for LPD and GR were close to unity making the model acceptable. The response surface plots generated for LPD and GR values based on the functional attributes of varying pH and NaNO2 levels under defined concentrations of NaCl (0.5, 2.5 and 5.5%) and incubation temperatures (12, 26 and 40°C) did indicate a definite behavioural pattern under the influence of a combination of factors and its variables. The effect of heat and cold treatments on isolates of Y. enterocoltica CFR 2301 and Y. intermedia CFR 2303 was studied. The heat sensitivity of selected isolates were in the order of water > normal saline > skimmed milk > beef gravy. The D-values ranged from 0.08 min at 65oC to 18.52 min at 50oC for Y. enterocolitica. The isolate of Y. intermedia had the lowest and highest heat sensitivity in beef gravy with D-values of 8.134 min at 50°C and 0.08 min at 65°C. As a means of ascertaining the relationship between growth incubation temperatures and heat sensitivity in the native isolate of Y. enterocoltica CFR 2301, the culture was pre-grown at 5, 10, 15, 37 and 43oC and then D-value determined at 55°C. The D-value was higher (4.02 min) in those cells obtained after growth at 43oC and lower (1.35 min) for cells grown at 5oC. In the case of cold treatment, the survival of Yersinia spp. was higher in skim milk, followed by normal saline. However, the susceptibility was quite significant in water at all the 3 treatment temperatures. In all the 3 menstra, isolate of Y. enterocolitica survived efficiently at -20°C, while the isolate of Y. intermedia CFR 2303 was more tolerant at 0°C. b Abstract of Ph.D. thesis c A few of the commonly used spice essential oil active principles such as trans-cinnamaldehyde, eugenol, cuminaldehyde, piperine and curcumin were used to assess the antibacterial activity against Y. enterocolitica CFR 2301. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of trans-cinnamaldehyde was observed to be 0.1 μl/ml and 0.3 μl/ml, respectively and that of eugenol was 0.4 μl/ml and 0.6 μl/ml, respectively. Scanning electron microscopy of cells of Y. enterocolitica CFR 2301 treated with trans-cinnamaldehyde and eugenol at their MIC concentration revealed that the cells appeared to have lost the contents and exhibited a shrunken morphology. As a means to develop the predictive model for the antibacterial activity of trans-cinnamaldehyde against Y. enterocolitica in chocolate milk, the experimental design was a complete 3x3x3 factorial of initial inoculums, storage temperature and trans-cinnamaldehyde concentration. In the presence of trans-cinnamaldehyde, an inactivation model was generated for the behaviour of Y. enterocolitica in chocolate milk, which revealed that irrespective of initial inoculum levels, the death time (DT) was primarily influenced by trans-cinnamaldehyde concentration and incubation temperature. The death times were found to decrease with higher cinnamaldehyde concentrations and incubation temperatures. The present study established a low prevalence of pathogenic biotype 1B Y. enterocolitica in the tested food samples. The isolates of Y. enterocolitica did harbour potent virulent traits. The target genes selected in this study can be used as markers of toxigenicity. The behaviour of Y. enterocolitica and Y. intermedia under different heat and cold temperature treatments in different media showed that these species were considerably sensitive to high temperatures and resistant to low temperatures. However, isolation of this organism from heat processed product did indicate the doubts about the hygiene and sanitation practices in food chain establishment. At the same time, excessive use of cold chain needs to be re-looked in view of the psychrotrophic character of Y. enterocolitica.

Item Type:	Thesis (PhD)
Uncontrolled Keywords:	Yersinia enterocolitica, foodborne diseases, gastrointestinal causative organism
Subjects:	600 Technology > 08 Food technology > 10 Food Microorganisms
Divisions:	Human Resource Development
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	13 Nov 2013 06:34
Last Modified:	13 Nov 2013 06:34
URI:	http://ir.cftri.res.in/id/eprint/11285

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