Peruzy, Maria Francesca (2019) Assessment of the microbial contamination on pork and wild boar meat by a culture-dependent and independent approach. [Tesi di dottorato]

Preview

Text ThesisPeruzy.pdf Download (3MB) | Preview

Collection description

Domestic pigs and wild boars are members of the same species (Sus scrofa) and they are classified in the subspecies domestica and scrofa respectively. Pork meat, in particular, minced pork meat, is widely consumed in the world and the consumption of the wild boar meat, although is still low compared to pork, is increasing. The carcasses and the cuts of these animals may support the growth and serve as a source of various spoilage and pathogen microorganisms (e.g. Salmonella, Yersinia enterocolitica and Listeria monocytogenes) which may have important consequences for the quality and safety of the product. In pork chain the food hygiene monitoring (1441/07) is based on the count of indicator microorganisms, however, knowledge of the microbial diversity on the agar plates of the counted bacteria is lacking. Moreover, the hygiene criteria for pork are commonly applied to assess the microbiological quality of wild boar meat since no microbial limits are settled for this animal. For the past few years culture independent methods allowed to study different ecosystems overcoming the limits of the classic microbial cultivation and bacteria identification. Recently, Matrix-assisted laser desorption/ionization time-of-flight Mass Spectrometry has been introduced in clinical microbiology for routine identification of clinical isolates. Moreover, MALDI TOF technology combined with 16S amplicon sequencing have resulted a promising approach to study the microbiota. The general aim of this thesis was to gain more insight in the microbiological contamination of pork and wild boar meat with culture-dependent based on MALDI TOF MS technique combined with 16S amplicon sequencing and an independent method. Moreover, with the purpose of evaluating the contamination of the samples with Y. enterocolitica and with the necessity to select gene target for the real-time PCR, an initial study on the distribution of virulence genes in Y. enterocolitica strains has been conducted. Therefore, in Chapter 1 a total of 161 Y. enterocolitica strains grouped into four biotypes (1A, 2 (serotypes O3, O5 and O9); 3 (serotypes O3 and O9), and 4 (serotype O3)) and isolated in eight countries were analyzed in order to evaluate the distribution of yadA, virF, inv, ystA, ystB, myfA, hreP and ymoA virulence genes. The results showed that not all the strains analyzed were positive to all the virulence genes tested. However, the most common virulence-associated gene in pathogenic Y. enterocolitica proved to be ystA, which can, therefore, be considered the best target gene to be amplified in order to evaluate the presence of pathogenic biotypes. While to identify Y. enterocolitica 1A strains, ystB can be proposed. In Chapter 2, the microbial community of 14 unrelated pork minced meat samples was evaluated through a culture-independent method (16S amplicon sequencing) compared to classical isolation methods combined with identification by Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI TOF MS) and 16S rRNA amplicon sequencing. Moreover, the impact of three sample preparation (direct colony identification, bacterial suspension and extraction method) for MALDI TOF analysis and different DNA extraction methods for the culture-independent method (16S amplicon sequencing) was evaluated. The results of the present study illustrate that the suspension method resulted in more low-level identifications, instead, the direct colony identification method can be recommended as a as first screening tool when large amounts of isolates have to be examined. Bacteria identified by MALDI-TOF MS and 16S amplicon sequencing were assigned to 16 families and 20 genera. Members of the genus Pseudomonas, along with the genera Brochothrix and Carnobacterium were commonly identified among the mesophilic and psychrotrophic population. Moreover, statistical analysis showed that the temperature of incubation of the PCA plates (30° or 7°C) did not have a significant impact on the bacterial colony counts (p>0.05) and the analysis of bacterial communities among the 14 samples between PCA at 7°C and 30°C showed that the microbial diversity was higher in mesophilic conditions. Comparing the results of the culture dependent methods to 16S rRNA amplicon sequencing, some contrasting data were obtained. Except for Brochothrix spp. and Pseudomonas spp., that were abundant and always detected, genera obtained with the two methods in the same sample were not always the same. Moreover, different results were obtained among the DNA extraction methods used. In Chapter 3, the microbial community on four areas (ham, back, jowl and belly) of 8 different pork carcasses belonging to two different slaughterhouses (Slaughterhouse A= C1, C2, C3 and C4; Slaughterhouse B= C5, C6, C7, C8) was evaluated through 16S rRNA amplicon sequencing compared to classical isolation methods combined with identification by Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI TOF MS) and 16S rRNA amplicon sequencing. Moreover, the presence of Salmonella spp. and Y. enterocolitica was also evaluated using qPCR from the enrichment broths. Salmonella was isolated in 7 carcasses. The serotypes identified in the present work (monophasic S. Typhimurium, S. Brandenburg, S. derbyand S. Rissen) are commonly associated with pigs and pork meat. Concerning Y. enterocolitica, the gene ystA was never detected in the samples, though ystB was present on the ham of one carcass. The dominant bacterial communities isolated from the 8 carcasses belonged to Staphylococcus, Pseudomonas, and E. coli, while with 16S amplicon sequencing, the diversity indices showed that no genus clearly dominated the 8 carcasses. However, Brochothrix was the most abundant genus detected with 16S amplicon sequencing, immediately followed by Pseudomonas. Important differences have been observed between the two slaughterhouses. However, regardless of the method used (culture dependent and independent) the present data illustrate, that the microbial population of the ham, back, jowl and belly were dominated by the same genera. In Chapter 4, the microbial community of 22 unrelated wild boar meat samples by a culture-dependent approach using ISO-methods combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification, and 16S rRNA amplicon sequencing was evaluated. Moreover, the presence of Salmonella, Y. enterocolitica and L. monocytogenes was examined by qPCR and confirmed by classical isolation. The samples resulted highly contaminated by mesophilic bacteria and Enterobacteriaceae. Moreover, 31.82% were positive to Salmonella spp.; three serovars were identified (monophasic S. Typhimurium, S. Stanleyville and S. Kasenyi) of which Salmonella Kasenyi has not been reported from wild boar meat yet. Concerning Y. enterocolitica, the gene ystB was present in three samples. Instead, L. monocytogenes was never detected. A great variability of the microbial contamination between were not always detected with 16S amplicon sequencing, and vice versa. As described in the general conclusion, this thesis demonstrated that the study of a microbial community in an ecosystem can be best achieved by using a combination of culturomics based on the application of MALDI TOF technology and 16S amplicon sequencing and culture independent methods. In fact, every time that in this thesis culture dependent and independent methods were compared, contrasting data were obtained. Concerning the three samples preparation of the MALDI TOF analysis the use first of “direct colony identification” and then “extraction method” on a selection is efficient in term of time and cost when a lot of isolates must be examined. Moreover, when applying culture independent techniques, it is important to adapt the DNA extraction method to the research questions asked. The data of the present thesis illustrate, furthermore, that the bacterial community of each carcass mainly depends on the microbial population of the slaughterhouse and the sampling of only one area for the evaluation of the hygienic status of the carcasses by the official authority may be sufficient. Moreover, for the first time, the bacterial population of wild boar meat has been in depth studied and the data showed that it can be a great public health risk. Thus, particular attention has to be paid to the non-inspected meat supplied directly to consumers.

Downloads

Actions (login required)

View Item