Contribution of omics to biopreservation: Toward food microbiome engineering.

Biopreservation is a sustainable approach to improve food safety and maintain or extend food shelf life by using beneficial microorganisms or their metabolites. Over the past 20 years, omics techniques have revolutionised food microbiology including biopreservation. A range of methods including genomics, transcriptomics, proteomics, metabolomics and meta-omics derivatives have highlighted the potential of biopreservation to improve the microbial safety of various foods. This review shows how these approaches have contributed to the selection of biopreservation agents, to a better understanding of the mechanisms of action and of their efficiency and impact within the food ecosystem. It also presents the potential of combining omics with complementary approaches to take into account better the complexity of food microbiomes at multiple scales, from the cell to the community levels, and their spatial, physicochemical and microbiological heterogeneity. The latest advances in biopreservation through omics have emphasised the importance of considering food as a complex and dynamic microbiome that requires integrated engineering strategies to increase the rate of innovation production in order to meet the safety, environmental and economic challenges of the agri-food sector.

Tetracycline Gene Transfer in Staphylococcus xylosus in situ During Sausage Fermentation.

The presence of determinants of resistance to antibiotics can constitute a possible safety hazard in coagulase-negative staphylococci (CNS), which are widely present in food of animal origin. Among CNS, S. xylosus is a species frequently isolated from fermented meat products. Resistance to tetracycline was found to be one of the most distributed resistances occurring in S. xylosus strains isolated from fermented sausages. We evaluated the transfer of tetracycline resistance in vitro and in situ between S. xylosus strains. We selected three strains isolated from dry fermented sausages, resistant to tetracycline but not to minocycline, their resistance occurring by a mechanism of active efflux encoded by the tetK gene. Only one strain was able to transfer its tetracycline resistance to a recipient strain initially susceptible and plasmid-free using a filter mating procedure. Transfer of tetracycline resistance was observed at very low frequencies of 3.4 × 10-9 per recipient. To further investigate the transferability of this tetracycline resistance, the donor and recipient strains were tested in pilot-scale fermented sausage production. This transfer was possible but at a low rate, 1.4 × 10-7, and only under conditions of a high inoculation level of 108 CFU/g of meat. The tetK gene is located on a small mobilizable plasmid close to Staphylococcus aureus pT181 plasmid. In conclusion, the transfer of tetracycline resistance between strains of S. xylosus is possible, but at a really low frequency in vitro and in situ in fermented sausages. Even if this represents a very moderate risk, it should be taken into account as required by the European approach of Qualified Presumption of Safety (QPS) and AFSSA safety recommendations, advising that strains used as starter cultures should not carry any transferable antibiotic resistance.

Mechanisms of the bactericidal effects of nitrate and nitrite in cured meats.

For cured meat products, nitrite is recognized for its antimicrobial effects against pathogenic bacteria, even though the specific inhibitory mechanisms are not well known. Nitrite contributes to oxidative stress by being the precursor of peroxynitrite (ONOO-), which is the major strong oxidant. Thus, bacterial stress (highly pH-very low partial pressure of oxygen-dependent) is enhanced by the nitrate-nitrite-peroxynitrite system which is also highly pH- and low partial pressure of oxygen-dependent. Nitrite is a hurdle technology which effectiveness depends on several other hurdle technologies including sodium chloride (accelerating the autoxidation of oxymyoglobin and promote peroxynitrite formation), ascorbate (increasing ONOO- synthesis), and Aw. In this environment, certain species are more resistant than others to acidic, oxidative, and nitrative stresses. The most resistant are gram-negative aerobic/facultative anaerobic bacteria (Escherichia coli, Salmonella), and the most fragile are gram-positive anaerobic bacteria (Clostridium botulinum). This position review highlights the major chemical mechanisms involved, the active molecules and their actions on bacterial metabolisms in the meat ecosystem.

Impact of reducing nitrate/nitrite levels on the behavior of Salmonella Typhimurium and Listeria monocytogenes in French dry fermented sausages.

Salmonella and Listeria monocytogenes are two pathogenic bacteria that most frequently contaminate pork meat. In dry fermented sausages, several hurdles are used for controlling bacterial growth such as nitrite and salt addition. In Europe, practices consist of adding potassium nitrate (250ppm expressed as NaNO3) or a combination of nitrate/nitrite (150/150ppm expressed as NaNO3/NaNO2 respectively). However, involvement of these additives in nitrosamine formation is a matter of concern. Consequently, a decrease in nitrite/nitrate amounts is proposed. The aim of this study was to evaluate the impact of reducing levels of these additives on Listeria and Salmonella behavior. Using challenge-tests, five trials were carried out by varying the concentration of nitrate and nitrate/nitrite. Results shown that nitrite is a relevant hurdle for control Salmonella and Listeria. At the end of drying, the most significant reductions of pathogens are obtained in sausages with nitrite added at the both tested concentrations (120 or 80ppm NaNO2).

Origin and ecological selection of core and food-specific bacterial communities associated with meat and seafood spoilage.

The microbial spoilage of meat and seafood products with short shelf lives is responsible for a significant amount of food waste. Food spoilage is a very heterogeneous process, involving the growth of various, poorly characterized bacterial communities. In this study, we conducted 16S ribosomal RNA gene pyrosequencing on 160 samples of fresh and spoiled foods to comparatively explore the bacterial communities associated with four meat products and four seafood products that are among the most consumed food items in Europe. We show that fresh products are contaminated in part by a microbiota similar to that found on the skin and in the gut of animals. However, this animal-derived microbiota was less prevalent and less abundant than a core microbiota, psychrotrophic in nature, mainly originated from the environment (water reservoirs). We clearly show that this core community found on meat and seafood products is the main reservoir of spoilage bacteria. We also show that storage conditions exert strong selective pressure on the initial microbiota: alpha diversity in fresh samples was 189±58 operational taxonomic units (OTUs) but dropped to 27±12 OTUs in spoiled samples. The OTU assemblage associated with spoilage was shaped by low storage temperatures, packaging and the nutritional value of the food matrix itself. These factors presumably act in tandem without any hierarchical pattern. Most notably, we were also able to identify putative new clades of dominant, previously undescribed bacteria occurring on spoiled seafood, a finding that emphasizes the importance of using culture-independent methods when studying food microbiota.

Extending the gamma concept to non-thermal inactivation: a dynamic model to predict the fate of Salmonella during the dried sausages process.

The process of dried fermented sausages is recognized to be favourable to the reduction of the Salmonella population. The objective of this study was to develop a model describing the evolution of Salmonella during the fabrication process of dried sausages and to optimize the food formulation to prevent pathogen presence at the end of the process. An experimental design was set to investigate the effects of the fermentation and drying process for several formulations, taking into account the type of starter culture, the sodium chloride concentration, the dextrose and lactose concentration on the Salmonella Typhimurium strain behaviour. A growth-inactivation model based on the gamma concept was then developed to quantify Salmonella behaviour in dynamic process conditions of temperature, pH, lactic acid and water activity. This behaviour was characterized by a first growth step, followed by an inactivation step. The Salmonella fate was well described by the model in terms of population size variation and transition from growth to inactivation. The Salmonella behaviour was influenced by the initial sugar concentration and the starter type but not by sodium chloride content. This model can be a valuable tool to design the food process and formulation to control Salmonella.

Impact of cleaning and disinfection on the non-culturable and culturable bacterial loads of food-contact surfaces at a beef processing plant.

We assessed the impact of industrial cleaning and disinfection (C&D) on colony-forming units (CFUs), viable (culturable and viable but non-culturable) cells and on total cells (viable and dead cells). Bacterial loads on polyvinyl chloride (PVC) and stainless steel surfaces in a cutting room at a beef processing plant were determined before and after C&D by real-time PCR to quantify cells from successive swabs from surfaces with or without an ethidium monoazide pre-treatment and by CFU counts on tryptone soy agar. Agar contact plates were also applied after C&D for comparison. Before C&D, total cells reached 5.4 and 4.7 log cells/cm(2), viable cells 4.0 and 4.4 log cells/cm(2) and CFUs 3.1 and 2.9 log CFU/cm(2) on PVC and stainless steel surfaces, respectively. Although C&D left surfaces visually clean, it did not lead to a significant reduction in total cells. Significant reductions were only observed on PVC for CFUs: 0.8 log and on stainless steel surfaces for viable cells and CFUs: 0.8 and 1.5 log, respectively. Our results show that CFUs were both more easily detached and killed on stainless steel surfaces than on PVC surfaces. Other important results include the following observations: 1) a single swabbing detached only between 2 and 27% of the actual bacterial load; 2) after C&D, the difference between the actual culturable population and the one assessed by one agar contact plate was 1.9 and 2.7 log CFU/cm(2) on PVC and stainless steel surfaces, respectively.

Biodiversity of coagulase-negative Staphylococci in French cheeses, dry fermented sausages, processing environments and clinical samples.

In this study, the biodiversity of Coagulase-Negative Staphylococci (CNS) strains isolated in France from cheese related samples (227 isolates) and dry sausage related samples (204 isolates) was compared to the biodiversity of 297 clinical isolates. Species identification was performed using different molecular methods (specific PCR, "Staph array" hybridization and sodA gene sequencing). Infraspecific biodiversity of strains belonging to the main CNS species found in both food and clinical samples was then assessed by pulse-field gel electrophoresis (PFGE). For food-related samples, the main species encountered corresponded to Staphylococcus equorum (28.5%), S. xylosus (28.3%), S. saprophyticus (12.5%) and S. succinus (7.7%); while, for clinical isolates, the main species encountered corresponded to S. epidermidis (69.4%), S. capitis (9.8%), S. hominis (4.5%), S. warneri (4.5%) and S. haemolyticus (3.8%). The two main species common to both food and clinical samples corresponded to S. epidermidis and S. saprophyticus. Concerning infraspecific biodiversity, PFGE profiles of S. equorum, S. saprophyticus and S. epidermidis showed a large genomic biodiversity. Comparatively, S. xylosus exhibited a lower biodiversity. No correlation could be observed between PFGE patterns and either the geographical origin or the sample type. This study highlighted that no food strains had similar PFGE profiles to clinical ones and that the two main food-related species, S. equorum and S. xylosus, were not found in clinical samples. The identification of CNS species and the characterisation of the genetic diversity of the strains constitute a first step towards CNS safety assessment.