Characterization of Leuconostoc carnosum and Latilactobacillus sakei during Cooked Pork Ham Processing.

Cooked ham is a popular, ready-to-eat product made of pork meat that is susceptible to microbial growth throughout its shelf life. In this study, we aimed to monitor the microbial growth and composition of nine vacuum-packed cooked ham lots using plate counting until the microbial limit of 7.4 log10 AMC/LAB CFU/g was exceeded. Eight out of nine lots exceeded the microbial limit after 20 days of storage. Lactic acid bacteria strains, particularly Leuconostoc carnosum and Latilactobacillus sakei, prevailed in vacuum-packed cooked ham. Leuconostoc carnosum 2 (Leuc 2) and Latilactobacillus sakei 4 (Sakei 4) were isolated from raw meat and the post-cooking area of the food processing facility. Carbohydrate utilization patterns of Leuc. carnosum PFGE types isolated from raw meat and the food processing environment differed from those isolated from cooked ham. These findings demonstrate how raw meat and its processing environment impact the quality and shelf life of cooked ham.

Biofilms in Water Hoses of a Meat Processing Environment Harbor Complex Microbial Communities.

Safe and hygienic water distribution is essential for maintaining product quality and safety. It is known that biofilms alter the appearance and microbial quality of water along the distribution chain. Yet, biofilms in water hoses throughout the food processing environment have not been investigated in detail. Here, microbial communities from water hoses and other environmental sites in contact with water, in addition to the source water itself, were studied in the meat processing environment. Biofilms were present in all water hoses as determined by the presence of bacterial DNA and biofilm matrix components (carbohydrates, extracellular DNA, and proteins). The microbial community of the biofilms was dominated by Proteobacteria, represented mainly by Comamonadaceae and Pseudoxanthomonas. Moreover, genera that are associated with an intracellular lifestyle (e.g., Neochlamydia and Legionella) were present. Overall, the microbial community of biofilms was less diverse than the water microbial community, while those from the different sample sites were distinct from each other. Indeed, only a few phyla were shared between the water hose biofilm and the source water or associated environmental samples. This study provides first insights towards understanding the microbiota of water hose biofilms in the food processing environment.

Autochthonous fungi are central components in microbial community structure in raw fermented sausages.

Raw meat sausage represents a unique ecological niche rich in nutrients for microbial consumption, making it particularly vulnerable to microbial spoilage. Starter cultures are applied to improve product stability and safety as well as flavour characteristics. However, the influence of starter cultures on microbial community assembly and succession throughout the fermentation process is largely unknown. In particular the effect on the fungal community has not yet been explored. We evaluate the microbiological status of four different raw meat sausages using high-throughput 16S rRNA gene and ITS2 gene sequencing. The objective was to study temporal changes of microbial composition during the fermentation process and to identify potential keystone species that play an important role within the microbial community. Our results suggest that fungi assigned to the species Debaryomyces hansenii and Alternaria alternata play a key role in microbial community dynamics during fermentation. In addition, bacteria related to the starter culture Lactobacillus sakei and the spoilage-associated genera Acinetobacter, Pseudomonas and Psychrobacter are central components of the microbial ecosystem in raw fermented sausages. Elucidating the exact role and interactions of these microorganisms has the potential to have direct impacts on the quality and safety of fermented foods.

Deciphering the virulence potential of Listeria monocytogenes in the Norwegian meat and salmon processing industry by combining whole genome sequencing and in vitro data.

Whole genome sequencing (WGS) of foodborne pathogens such as Listeria monocytogenes is globally on the rise in the food industry. It provides an improvement for proactive surveillance and source-tracking and allows in-depth genetic characterization of the pathogen. In the present study, the virulence gene profile including 99 virulence genes of 767 L. monocytogenes isolates from the Norwegian meat and salmon processing industry was characterized. The isolate collection comprised 28 clonal complexes (CCs) that occur globally. We additionally determined the in vitro virulence potential for 13 major CCs in human intestinal epithelial Caco2 cells using cocktails of three to six representative isolates. Our aim was to test whether the virulence potential could be predicted from the virulence gene profiles to estimate the application potential of WGS in risk assessment in the food industry. The virulence gene profiles were highly conserved within the individual CCs and similar among phylogenetically closely related CCs. We observed a CC-associated distribution of accessory virulence genes in addition to different length polymorphisms. Furthermore, we detected different premature stop codons (PMSC) in the inlA gene, which were mainly present in CC9, CC121 and CC5 isolates. Accordingly, CC9 and CC5 were unable to invade Caco2 cells, whereas CC121 showed moderate virulence potential due to the presence of an isolate harboring full-length inlA. The highest invasion was observed for CC403 and CC415, potentially due to the presence of accessory virulence genes. We demonstrated that CC14, which harbored full-length inlA, was unable to invade Caco2 cells due to a low inlA gene expression. Reconstruction of inlA in CC9 and CC121 isolates showed that without the presence of InlA on the cell wall (as detected in the CC9 isolates), invasion into host cells failed. Our study showed that predicting the virulence potential based on genetic virulence profiles provides valuable information for risk assessment in the food industry but also has its limitations. The mere presence of a full-length inlA gene is not sufficient for virulence, but gene expression and the presence of the protein on the cell wall is required for the successful invasion of L. monocytogenes into host cells. Moreover, hypovirulent CCs like CC121 were among the most abundant human clinical isolates in Norway despite harboring a PMSC mutation in the inlA gene. In conclusion, our study highlights that combining genotypic and phenotypic data is of great importance to improve the informative value of applying WGS in the food industry.

Presence of Microbial Contamination and Biofilms at a Beer Can Filling Production Line.

ABSTRACT: Contamination of beer arises in 50% of all events at the late stages of production, in the filling area. This is where biofilms, a consortia of microorganisms embedded in a matrix composed of extracellular polymeric substances, play a critical role. To date, most studies have focused on the presence of (biofilm-forming) microorganisms in the filling environment. Our aim was to characterize the microbial status as well as the presence of possible biofilms at a can filling line for beer by determining the presence of microorganisms and their associated matrix components (carbohydrates, proteins and extracellular DNA [eDNA]). For 23 sampling sites, targeted quantitative PCR confirmed the presence of microorganisms at 10 sites during operation and at 3 sites after cleaning. The evaluation of carbohydrates, eDNA, and proteins showed that 16 sites were positive for at least one component during operation and 4 after cleaning. We identified one potential biofilm hotspot, namely the struts below the filler, harboring high loads of bacteria and yeast, eDNA, carbohydrates, and proteins. The protein pattern was different from that of beer. This work deepens our understanding of biofilms and microorganisms found at the filling line of beer beverages at sites critical for production.

Co-Occurrence of Listeria spp. and Spoilage Associated Microbiota During Meat Processing Due to Cross-Contamination Events.

A large part of foodborne outbreaks related to Listeria monocytogenes are linked to meat and meat products. Especially, recontamination of meat products and deli-meat during slicing, packaging, and repackaging is in the focus of food authorities. In that regard, L. monocytogenes persistence in multi-species biofilms is one major issue, since they survive elaborate cleaning and disinfection measures. Here, we analyzed the microbial community structure throughout a meat processing facility using a combination of high-throughput full-length 16S ribosomal RNA (rRNA) gene sequencing and traditional microbiological methods. Samples were taken at different stages during meat cutting as well as from multiple sites throughout the facility environment to capture the product and the environmental associated microbiota co-occurring with Listeria spp. and L. monocytogenes. The listeria testing revealed a widely disseminated contamination (50%; 88 of 176 samples were positive for Listeria spp. and 13.6%; 24 of 176 samples were positive for L. monocytogenes). The pulsed-field gel electrophoresis (PFGE) typing evidenced 14 heterogeneous L. monocytogenes profiles with PCR-serogroup 1/2a, 3a as most dominant. PFGE type MA3-17 contributed to the resilient microbiota of the facility environment and was related to environmental persistence. The core in-house microbiota consisted mainly of the genera Acinetobacter, Pseudomonas, Psychrobacter (Proteobacteria), Anaerobacillus, Bacillus (Firmicutes), and Chryseobacterium (Bacteroidota). While the overall microbial community structure clearly differed between product and environmental samples, we were able to discern correlation patterns regarding the presence/absence of Listeria spp. in both sample groups. Specifically, our longitudinal analysis revealed association of Listeria spp. with known biofilm-producing Pseudomonas, Acinetobacter, and Janthinobacterium species on the meat samples. Similar patterns were also observed on the surface, indicating dispersal of microorganisms from this multispecies biofilm. Our data provided a better understanding of the built environment microbiome in the meat processing context and promoted more effective options for targeted disinfection in the analyzed facility.

Identification of biofilm hotspots in a meat processing environment: Detection of spoilage bacteria in multi-species biofilms.

Biofilms are comprised of microorganisms embedded in a self-produced matrix that normally adhere to a surface. In the food processing environment they are suggested to be a source of contamination leading to food spoilage or the transmission of food-borne pathogens. To date, research has mainly focused on the presence of (biofilm-forming) bacteria within food processing environments, without measuring the associated biofilm matrix components. Here, we assessed the presence of biofilms within a meat processing environment, processing pork, poultry and beef, by the detection of microorganisms and at least two biofilm matrix components. Sampling included 47 food contact surfaces and 61 non-food contact surfaces from eleven rooms within an Austrian meat processing plant, either during operation or after cleaning and disinfection. The 108 samples were analysed for the presence of microorganisms by cultivation and targeted quantitative real-time PCR based on 16S rRNA. Furthermore, the presence of the major matrix components carbohydrates, extracellular DNA and proteins was evaluated. Overall, we identified ten biofilm hotspots, among them seven of which were sampled during operation and three after cleaning and disinfection. Five biofilms were detected on food contact surfaces (cutters and associated equipment and a screw conveyor) and five on non-food contact surfaces (drains and water hoses) resulting in 9.3 % of the sites being classified as biofilm positive. From these biofilm positive samples, we cultivated bacteria of 29 different genera. The most prevalent bacteria belonged to the genera Brochothrix (present in 80 % of biofilms), Pseudomonas and Psychrobacter (isolated from 70 % biofilms). From each biofilm we isolated bacteria from four to twelve different genera, indicating the presence of multi-species biofilms. This work ultimately determined the presence of multi-species biofilms within the meat processing environment, thereby identifying various sources of potential contamination. Especially the identification of biofilms in water hoses and associated parts highlights the need of a frequent monitoring at these sites. The knowledge gained about the presence and composition of biofilms (i.e. chemical and microbiological) will help to prevent and reduce biofilm formation within food processing environments.

The sources and transmission routes of microbial populations throughout a meat processing facility.

Microbial food spoilage is responsible for a considerable amount of waste and can cause food-borne diseases in humans, particularly in immunocompromised individuals and children. Therefore, preventing microbial food spoilage is a major concern for health authorities, regulators, consumers, and the food industry. However, the contamination of food products is difficult to control because there are several potential sources during production, processing, storage, distribution, and consumption, where microorganisms come in contact with the product. Here, we use high-throughput full-length 16S rRNA gene sequencing to provide insights into bacterial community structure throughout a pork-processing plant. Specifically, we investigated what proportion of bacteria on meat are presumptively not animal-associated and are therefore transferred during cutting via personnel, equipment, machines, or the slaughter environment. We then created a facility-specific transmission map of bacterial flow, which predicted previously unknown sources of bacterial contamination. This allowed us to pinpoint specific taxa to particular environmental sources and provide the facility with essential information for targeted disinfection. For example, Moraxella spp., a prominent meat spoilage organism, which was one of the most abundant amplicon sequence variants (ASVs) detected on the meat, was most likely transferred from the gloves of employees, a railing at the classification step, and the polishing tunnel whips. Our results suggest that high-throughput full-length 16S rRNA gene sequencing has great potential in food monitoring applications.

Culture-Independent Evaluation of Bacterial Contamination Patterns on Pig Carcasses at a Commercial Slaughter Facility.

Traditionally, the microbiological status of meat is determined by culture-based techniques, although many bacteria are not able to grow on conventional media. The aim of this study was to obtain quantitative data on total bacterial cell equivalents, as well as taxa-specific abundances, on carcass surfaces during pig slaughter using quantitative real-time PCR. We evaluated microbial contamination patterns of total bacteria, Campylobacter, Escherichia coli, Lactobacillus group, Listeria monocytogenes, Salmonella, and Pseudomonas species throughout slaughtering and on different carcass areas. In addition, we compared contamination levels of breeding sow carcasses with fattening pig carcasses, and we assessed the efficacy of carcass polishing machines under two water amount conditions. Our results demonstrate that relevant meat-spoilage organisms show similar contamination patterns to total bacteria. The highest bacterial load was detected in the stunning chute (4.08 × 105 bacterial cell equivalents per cm2) but was reduced by 3 log levels after singeing and polishing (P < 0.001). It increased again significantly by a 4.73-fold change until the classification step. Levels of Campylobacter, Lactobacillus, and Pseudomonas species and of E. coli followed a similar trend but varied between 0 and 2.49 × 104 bacterial cell equivalents per cm2. Microbial levels did not vary significantly between sampled carcass areas for any analyzed taxa. Running the polishing machine with a low water amount proved to be less prone to microbial recontamination compared with a high water amount (17.07-fold change, P = 0.024). In the studied slaughterhouse, slaughter of breeding sows did not produce microbiologically safe meat products (>104 cells per cm2) and the implementation of specific hazard analysis critical control point systems for the slaughter of breeding sows should be considered. A larger cohort from different abattoirs is needed to confirm our results and determine whether this is universally valid.