Holistic integration of omics data reveals the drivers that shape the ecology of microbial meat spoilage scenarios.

Background: The use of omics data for monitoring the microbial flow of fresh meat products along a production line and the development of spoilage prediction tools from these data is a promising but challenging task. In this context, we produced a large multivariate dataset (over 600 samples) obtained on the production lines of two similar types of fresh meat products (poultry and raw pork sausages). We describe a full analysis of this dataset in order to decipher how the spoilage microbial ecology of these two similar products may be shaped differently depending on production parameter characteristics. Methods: Our strategy involved a holistic approach to integrate unsupervised and supervised statistical methods on multivariate data (OTU-based microbial diversity; metabolomic data of volatile organic compounds; sensory measurements; growth parameters), and a specific selection of potential uncontrolled (initial microbiota composition) or controlled (packaging type; lactate concentration) drivers. Results: Our results demonstrate that the initial microbiota, which is shown to be very different between poultry and pork sausages, has a major impact on the spoilage scenarios and on the effect that a downstream parameter such as packaging type has on the overall evolution of the microbial community. Depending on the process, we also show that specific actions on the pork meat (such as deboning and defatting) elicit specific food spoilers such as Dellaglioa algida, which becomes dominant during storage. Finally, ecological network reconstruction allowed us to map six different metabolic pathways involved in the production of volatile organic compounds involved in spoilage. We were able connect them to the different bacterial actors and to the influence of packaging type in an overall view. For instance, our results demonstrate a new role of Vibrionaceae in isopropanol production, and of Latilactobacillus fuchuensis and Lactococcus piscium in methanethiol/disylphide production. We also highlight a possible commensal behavior between Leuconostoc carnosum and Latilactobacillus curvatus around 2,3-butanediol metabolism. Conclusion: We conclude that our holistic approach combined with large-scale multi-omic data was a powerful strategy to prioritize the role of production parameters, already known in the literature, that shape the evolution and/or the implementation of different meat spoilage scenarios.

Effects of sodium nitrite reduction, removal or replacement on cured and cooked meat for microbiological growth, food safety, colon ecosystem, and colorectal carcinogenesis in Fischer 344 rats.

Epidemiological and experimental evidence indicated that processed meat consumption is associated with colorectal cancer risks. Several studies suggest the involvement of nitrite or nitrate additives via N-nitroso-compound formation (NOCs). Compared to the reference level (120 mg/kg of ham), sodium nitrite removal and reduction (90 mg/kg) similarly decreased preneoplastic lesions in F344 rats, but only reduction had an inhibitory effect on Listeria monocytogenes growth comparable to that obtained using the reference nitrite level and an effective lipid peroxidation control. Among the three nitrite salt alternatives tested, none of them led to a significant gain when compared to the reference level: vegetable stock, due to nitrate presence, was very similar to this reference nitrite level, yeast extract induced a strong luminal peroxidation and no decrease in preneoplastic lesions in rats despite the absence of NOCs, and polyphenol rich extract induced the clearest downward trend on preneoplastic lesions in rats but the concomitant presence of nitrosyl iron in feces. Except the vegetable stock, other alternatives were less efficient than sodium nitrite in reducing L. monocytogenes growth.

Inactivation of Toxoplasma gondii in dry sausage and processed pork, and quantification of the pathogen in pig tissues prior to production.

Toxoplasma gondii is an important zoonotic foodborne parasite. Meat of infected animals appears to be a major source of infection in Europe. Pork is the most consumed meat in France, with dry sausages well represented. The risk of transmission via consumption of processed pork products is largely unknown, mainly since processing will affect viability but may not entirely inactivate all T. gondii parasites. We investigated the presence and concentration of T. gondii DNA in the shoulder, breast, ham, and heart of pigs orally inoculated with 1000 oocysts (n = 3) or tissue cysts (n = 3) and naturally infected pigs (n = 2), by means of magnetic capture qPCR (MC-qPCR). Muscle tissues of experimentally infected pigs were further used to evaluate the impact of manufacturing processes of dry sausages, including different concentrations of nitrates (0, 60, 120, 200 ppm), nitrites (0, 60, 120 ppm), and NaCl (0, 20, 26 g/kg), ripening (2 days at 16-24 °C) and drying (up to 30 days at 13 °C), by a combination of mouse bioassay, qPCR and MC-qPCR. DNA of T. gondii was detected in all eight pigs, including in 41.7% (10/24) of muscle samples (shoulder, breast and ham) and 87.5% (7/8) of hearts by MC-qPCR. The number of parasites per gram of tissue was estimated to be the lowest in the hams (arithmetic mean (M) = 1, standard deviation (SD) = 2) and the highest in the hearts (M = 147, SD = 233). However, the T. gondii burden estimates varied on the individual animal level, the tissue tested and the parasitic stage used for the experimental infection (oocysts or tissue cysts). Of dry sausages and processed pork, 94.4% (51/54) were positive for T. gondii by MC-qPCR or qPCR, with the mean T. gondii burden estimate equivalent to 31 parasites per gram (SD = 93). Only the untreated processed pork sample collected on the day of production was positive by mouse bioassay. The results suggest an uneven distribution of T. gondii in the tissues examined, and possibly an absence or a concentration below the detection limit in some of them. Moreover, the processing of dry sausages and processed pork with NaCl, nitrates, and nitrites has an impact on the viability of T. gondii from the first day of production. Results are valuable input for future risk assessments aiming to estimate the relative contribution of different sources of T. gondii human infections.

Microbial Ecology of French Dry Fermented Sausages and Mycotoxin Risk Evaluation During Storage.

Dry fermented sausages are produced worldwide by well-controlled fermentation processes involving complex microbiota including many bacterial and fungal species with key technological roles. However, to date, fungal diversity on sausage casings during storage has not been fully described. In this context, we studied the microbial communities from dry fermented sausages naturally colonized or voluntarily surface inoculated with molds during storage using both culture-dependent and metabarcoding methods. Staphylococci and lactic acid bacteria largely dominated in samples, although some halotolerant genera (e.g., Halomonas, Tetragenococcus, and Celerinatantimonas spp.) were also frequently observed. Fungal populations varied from 7.2 to 9.8 log TFU/cm2 sausage casing during storage, suggesting relatively low count variability among products. Fungal diversity identified on voluntarily inoculated casings was lower (dominated by Penicillium nalgiovense and Debaryomyces hansenii) than naturally environment-inoculated fermented sausages (colonized by P. nalgiovense, Penicillium nordicum, and other Penicillium spp. and sporadically by Scopulariopsis sp., D. hansenii, and Candida zeylanoïdes). P. nalgiovense and D. hansenii were systematically identified, highlighting their key technological role. The mycotoxin risk was then evaluated, and in situ mycotoxin production of selected mold isolates was determined during pilot-scale sausage productions. Among the identified fungal species, P. nalgiovense was confirmed not to produce mycotoxins. However, some P. nordicum, Penicillium chrysogenum, Penicillium bialowienzense, Penicillium brevicompactum, and Penicillium citreonigrum isolates produced one or more mycotoxins in vitro. P. nordicum also produced ochratoxin A during pilot-scale sausage productions using "worst-case" conditions in the absence of biotic competition. These data provide new knowledge on fermented sausage microbiota and the potential mycotoxin risk during storage.

Spoilage of fresh turkey and pork sausages: Influence of potassium lactate and modified atmosphere packaging.

Fresh poultry and pork meat products represent highly perishable products which are susceptible to spoil within a few days after production. Lactate addition and modified atmosphere packaging are common preservation strategies used to overcome spoilage. This study aimed to identify the effects of these strategies and their possible interactions on spoilage indicators simultaneously on fresh pork and turkey sausages. Ten batches of raw meat (turkey or pork) sausages were industrially produced with different lactate concentrations (0, 1 or 2% w/w in turkey and 0, 0.57 and 1.13% w/w in pork), packed under different gas mixtures (air, MAP1: 70% O2 - 30% CO2 and MAP2: 50% CO2 - 50% N2) and chill stored during 22 days. Spoilage responses including enumeration of total aerobic mesophilic and lactic acid bacteria, measurement of pH and colour, evaluation of visual defects and off-odour, were monitored. Effects of lactate and modified atmosphere packaging (MAP) as well as random effect of the batch variability were studied using a mixed effect model. Despite initial batch variability, significant effects of lactate and gas packaging were observed but in a different way in turkey and pork. Our results suggest that for fresh turkey sausages, the gas mixture enriched in oxygen enhanced off-odour perception and sausage discolouration from red to dark grey / brown colour. Unlike turkey sausages, in pork sausages, lactate did not significantly influence the monitored spoilage responses, whereas MAP (70% O2-30% CO2) reduced the off-odour perception. The developed model could be useful to estimate the effect of preservation strategies on spoilage occurrence while considering industrial batch variability.

Large-scale multivariate dataset on the characterization of microbiota diversity, microbial growth dynamics, metabolic spoilage volatilome and sensorial profiles of two industrially produced meat products subjected to changes in lactate concentration and packaging atmosphere.

Data in this article provide detailed information on the diversity of bacterial communities present on 576 samples of raw pork or poultry sausages produced industrially in 2017. Bacterial growth dynamics and diversity were monitored throughout the refrigerated storage period to estimate the impact of packaging atmosphere and the use of potassium lactate as chemical preservative. The data include several types of analysis aiming at providing a comprehensive microbial ecology of spoilage during storage and how the process parameters do influence this phenomenon. The analysis includes: the gas content in packaging, pH, chromametric measurements, plate counts (total mesophilic aerobic flora and lactic acid bacteria), sensorial properties of the products, meta-metabolomic quantification of volatile organic compounds and bacterial community metagenetic analysis. Bacterial diversity was monitored using two types of amplicon sequencing (16S rRNA and GyrB encoding genes) at different time points for the different conditions (576 samples for gyrB and 436 samples for 16S rDNA). Sequencing data were generated by using Illumina MiSeq. The sequencing data have been deposited in the bioproject PRJNA522361. Samples accession numbers vary from SAMN10964863 to SAMN10965438 for gyrB amplicon and from SAMN10970131 to SAMN10970566 for 16S.

European survey and evaluation of sampling methods recommended by the standard EN ISO 18593 for the detection of Listeria monocytogenes and Pseudomonas fluorescens on industrial surfaces.

The ready-to-eat products can be contaminated during processing by pathogen or spoilage bacteria, which persist in the industrial environment. Some bacterial species are able to form biofilms which protect them from environmental conditions. To check the bacterial contamination of the surfaces in the food industries, the professionals must regularly use surface sampling methods to detect the pathogen such as Listeria monocytogenes or the spoilage such as Pseudomonas fluorescens. In 2010, we designed and carried out a European survey to collect surface sampling information to detect or enumerate L. monocytogenes in food processing plants. A total of 137 questionnaires from 14 European Union Member States were returned. The outcome of this survey showed that the professionals preferred friction sampling methods with gauze pad, swab and sponges versus contact sampling methods. After this survey, we compared the effectiveness of these three friction sampling methods and the contact plates, as recommended in the standard EN ISO 18593 that was revised in 2018, on the recovery of L. monocytogenes and of P. fluorescens in mono-specie biofilms. This study showed no significant difference between the effectiveness of the four sampling methods to detach the viable and culturable bacterial population of theses mono-specie biofilms.

Prevalence of methicillin-resistant staphylococci species isolated from computer keyboards located in secondary and postsecondary schools.

Methicillin-resistant Staphylococcus aureus (MRSA) is a public health threat within the general community, thereby warranting identification of MRSA reservoirs within the community. Computer terminals in schools were sampled for S. aureus and methicillin-resistant staphylococci. The overall prevalence of MRSA on computer keyboards was low: 0.68% for a postsecondary institution and 2% and 0% for two secondary institutes. The MRSA isolate from the postsecondary institution did not correspond to the Canadian epidemic clusters, but is related to the USA 700 cluster, which contains strains implicated in outbreaks within the U.S. The isolate from the secondary institute's keyboard was typed as CMRSA7 (USA 400), a strain that has been implicated in both Canadian and U.S. epidemics. Methicillin-resistant S. haemolyticus and S. epidermidis were also isolated from keyboards, indicating that a mixed community of methicillin-resistant staphylococci can be present on keyboards. Although the prevalence was low, the presence of MRSA combined with the high volume of traffic on these student computer terminals demonstrates the potential for public-access computer terminals and computer rooms at educational institutes to act as reservoirs.

Persistence of host-associated Bacteroidales gene markers and their quantitative detection in an urban and agricultural mixed prairie watershed.

Microbial source tracking is an emerging tool developed to protect water sources from faecal pollution. In this study, we evaluated the suitability of real time-quantitative PCR (qPCR) Taqman assays developed for detection of host-associated Bacteroidales markers in a prairie watershed. The qPCR primers and probes used in this study exhibited high accuracy (88-96% sensitivity and ≥ 99% host specificity) in detecting Bacteroidales spp. that are associated with faeces from humans, ruminants, bovines, and horses. The ruminant- and human-associated markers were also found in high concentrations within individual faecal samples, ranging from 3.4 to 7.3 log(10) marker copy numberg(-1) of individual host faeces. Following validation of host sensitivity and specificity, the host-associated Bacteroidales markers were detected in the Qu'Appelle Valley watershed of Saskatchewan, Canada which experiences a diversity of anthropogenic inputs. Concentrations of the ruminant marker were well-correlated with proximity to cattle operations and there was a correlation between the marker and Escherichia coli concentrations at these sites. Low concentrations of the human faecal marker were measured throughout the sampling sites, and may indicate a consistent influx of human faecal pollution into the watershed area. Persistence of each of the Bacteroidales host-associated marker was also studied in situ. The results indicated that the markers persist for shorter periods of time (99% decay in <8 days) compared with the conventional E. coli marker (99% decay in >15 days), suggesting they are effective at detecting recent faecal contamination events. The levels of Bacteroidales markers and E. coli counts did not correlate with the presence of the pathogenic bacteria, Salmonella spp. or Campylobacter spp. detected in the Qu'Appelle Valley. Collectively, the results obtained in this study demonstrated that the qPCR approach for detecting host-associated Bacteroidales spp. markers can be a useful tool in helping to determine host-specific impacts of faecal pollution into a prairie watershed.