Evaluation of microbial contamination of different pork carcass areas through culture-dependent and independent methods in small-scale slaughterhouses
Introduction
Pork carcasses and pork cuts may support the growth and serve as a source of different microorganisms which may have important consequences for the quality and safety of the product (Koutsoumanis and Sofos, 2004). The microbial load on pork carcasses strongly depends on the spread of microorganisms during the slaughtering process (stunning, bleeding, scalding, dehairing, singeing, evisceration, splitting and cooling) (Mann et al., 2016). In particular, microbial surface contamination may occur from the animals' hide and gastrointestinal tract, or from equipment, contact surfaces and slaughterhouse workers (Mrdovic et al., 2017). To reduce or inhibit the bacterial growth, the EU regulation 853/2004 establish that carcasses must be cooled down to a temperature of no more than 7 °C after the dressing stage, as 7 °C is recognized as the limit temperature below which most pathogens do not grow (Koutsoumanis and Sofos, 2004). However, the growth of some pathogens, as well as a range of food spoilage organisms, is not completely inhibited. Thus, the microbial population on the carcasses after the dressing stage is still composed of a mixture of mesophilic and psychrotrophic bacteria that may affect meat quality and cause spoilage (such as Aeromonas, Brochothrix, Serratia, and Pseudomonas spp.) and/or may be responsible of human illness (Mann et al., 2016). Among pathogens, a good deal have an enteric origin (such as Salmonella and Yersinia), and the presence of them on the carcass surface is mainly the result of an improper evisceration (Choi et al., 2013; Mrdovic et al., 2017; Sánchez-Rodríguez et al., 2018). To indirectly evaluate the hygiene of the slaughter process, the EU regulation 1441/07 require the obligatory control through the excision method or the swabbing method of Total Aerobic Bacterial count (TAB) at 30 °C, of the Enterobacteriaceae and of Salmonella spp. on different sampling sites on the pork carcasses, sites should be chosen to target the areas with the highest level of contamination (ISO 176604/2015). In particular, the excision method (destructive) is performed incising and removing a specific sampling area of skin or tissue from the carcasses while swabbing is a non-destructive method that includes the use of absorbent material (e.g. sponges, swabs, tampons, cloths).However, while Enterobacteriaceae and Salmonella spp. indicate fecal contamination and, thus, improper evisceration, the determination of TAB at 30 °C provides only an indication about the level of culturable bacteria present on the carcasses, without giving any additional information at taxonomic level and on the bacterial diversity between the different sampling sites. Thus, as no or only some colonies are picked for further identification, knowledge on the microbial diversity of the counted microorganisms is still lacking.
Culture-independent techniques are commonly used to study complex microbial communities and therefore, to obtain information at taxonomic level on bacteria present in different ecosystems (Ercolini, 2013). Among them 16S amplicon sequencing is the most commonly employed because it is quick, simple and cost-effective (Knight et al., 2018) even if it still subject to some biases and a large number of unknown taxa are produced (Knight et al., 2018). However, culturing in combination with sequencing has been shown to enable the identification of organisms belonging to unknown taxa generated with the culture-independent methods (Lagier et al., 2016).
For the culture-dependent techniques, the 16S gene is also used for the identification of bacterial isolates. Upon isolation and DNA extraction, sequencing of the whole 16S gene of isolates is considered the “gold standard” method for identification, however, it is still expensive and, moreover, time-consuming (Singhal et al., 2015). Identification of bacterial isolates by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid and accurate method for routine identification of clinical isolates (Cherkaoui et al., 2010), however, the database for identification is still limited to these clinical relevant microorganism.
Combined identification of bacterial strains using MALDI TOF MS and 16S gene sequencing with overall community profiling using a culture-independent method is complementary and yields important insights into the complex relationship between microorganisms in a food (Peruzy et al., 2019a; Yu et al., 2019). The aim of the present study was to evaluate the microbial diversity occurring on four sampling sites of pork carcasses slaughtered in two different slaughterhouses through the culture-dependent and independent approaches.
Section snippets
Sampling
A total of 8 pork carcasses (C1 to C8) were examined. They originated from different Italian farms and were slaughtered in two different abattoirs named SA (C1-C4) and SB (C5-C8) in the Campania region of southern Italy. The slaughterhouses were regularly inspected by the competent authority and the daily production capacity of SA and SB was around 150 and 48 carcasses, respectively. The layout of the slaughter processes was similar, only differing in the singeing step. In SA, singeing was
Bacterial isolation
Bacterial counts for the 8 carcasses (C1-C8), per sampling point and per slaughterhouse are shown in Table 2. The mean (±SD) of the total aerobic counts on PCA ranged from 3 ± 0.45 (C4) to 5.36 ± 0.05 (C2) log CFU/cm2, from 1.44 ± 1.68 (C2) to 3.38 ± 0.52 (C3) log CFU/cm2 and from 2.54 ± 0.63 (C7) to 3.84 ± 0.80 (C8) log CFU/cm2 for mesophilic, psychrotrophic and anaerobic bacteria, respectively. The count of typical purple/pink Enterobacteriaceae colonies on VRBG and the blue E. coli colonies
Discussion
According to the EC Regulation No.1441/07, TAB 30 °C is supposed to be, along with the Enterobacteriaceae, an indicator of the slaughter hygiene process. In the present study, although the counts were slightly higher in slaughterhouse A, no significant differences were observed between the two slaughterhouses (p > 0.05). In order to enable comparison with other studies, when pooling the results of the investigated areas to one value for complete carcass evaluation, the mean of TAB 30 °C was
Conclusions
In conclusion, in the small slaughterhouses studied, the bacterial community of each carcass may depend mainly on the microbial population of the slaughterhouse to which it belongs rather than on the indigenous microbiota of the slaughtered animals. However, to confirm this hypothesis further studies on the environmental population of the slaughterhouse's facilities should be performed. Moreover, the results of the comparison of different sampling areas show the absence of clear and
Declaration of competing interest
The authors declare no potential conflict of interests.
Acknowledgements
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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2022, Meat ScienceCitation Excerpt :The growth of the microbial flora, as assessed by overall and specific counts, agrees with maximum counts for MAP pork chops observed by Sørheim, Nissen, and Nesbakken (1999). Moreover, the initial microbial flora showed a diversity mostly typical for pork carcasses including the presence of Acinetobacter spp., Brevundimonas spp., Carnobacterium spp., Chryseobacterium spp., Leuconostoc spp., Pseudomonas spp. but excluding e.g. Staphylococcus spp. (Peruzy et al., 2021). L. carnosum, predominant during most of the storage time, is commonly associated with raw and processed meat products (Audenaert et al., 2010; Candeliere et al., 2020; Laursen, Byrne, Kirkegaard, & Leisner, 2009; Raimondi et al., 2019; Shaw & Harding, 1989; Vasilopoulos et al., 2008).