Evaluation of microbial contamination of different pork carcass areas through culture-dependent and independent methods in small-scale slaughterhouses

Highlights

• Contamination levels on four pork carcass areas were not significant different.

• Bacterial communities on the four carcasses areas were dominated by the same genera.

• Sampling only one area is appropriate for the evaluation of the slaughter's hygiene.

• Microbial communities on the carcasses were distinct between the two slaughterhouses.

• Bacterial community on the carcass is influenced by the slaughterhouse community.

Abstract

Routine evaluation of the slaughter process is performed by the enumeration of the aerobic colony count, Enterobacteriaceae and Salmonella spp. on the carcass through destructive or non-destructive methods. With non-destructive methods, bacteria are counted from a minimum area of 100 cm² in different sampling sites on the pork carcasses, and the results of these investigated areas are pooled to one value for the complete carcass evaluation (a total of 400 cm²). However, the composition of the bacterial community present on the different sampling areas remains unknown. The aim of the study was to characterize the microbial population present on four areas (ham, back, jowl and belly) of eight pork carcasses belonging to two different slaughterhouses through culture-dependent (Matrix-assisted laser desorption/ionization time-of-flight Mass Spectrometry MALDI-TOF MS, combined with 16S rRNA gene sequencing) and complementary culture-independent (16S rRNA amplicon sequencing) methods. The presence of Salmonella spp. and Y. enterocolitica was additionally assessed. Using MALDI-TOF MS, Staphylococcus, Pseudomonas, and Escherichia coli were found to dominate the bacterial cultures isolated from the 8 carcasses. Based on the 16S rRNA amplicon sequencing analyses however, no specific genus clearly dominated the bacterial community composition. By using this culture-independent method, the most abundant genera in microbial populations of the ham, back, jowl and belly were found to be similar, but important differences between the two slaughterhouses were observed. Thus, present data suggests that the indigenous bacterial population of individual animals is overruled by the microbial population of the slaughterhouse in which the carcass is handled. Also, our data suggests that sampling of only one carcass area by official authorities may be appropriate for the evaluation of the hygienic status of the carcasses and therefore of the slaughter process.

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.