SECurité des ALIments et Microbiologie

Biofilms are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions. They can be beneficial or have a negative impact particularly when formed in industrial settings or on medical devices. As such, research into the formation and elimination of biofilms is important for many disciplines. Several new methodologies have been recently developed for, or adapted to, biofilm studies that have contributed to deeper knowledge on biofilm physiology, structure and composition. In this review, traditional and cutting-edge methods to study biofilm biomass, viability, structure, composition and physiology are addressed. Moreover, as there is a lack of consensus among the diversity of techniques used to grow and study biofilms. This review intends to remedy this, by giving a critical perspective, highlighting the advantages and limitations of several methods. Accordingly, this review aims at helping scientists in finding the most appropriate and up-to-date methods to study their biofilms.

In combination with other strategies, hyperosmolarity and desiccation are frequently used by the food processing industry as a means to prevent bacterial proliferation, and particularly that of foodborne pathogens, in food products. However, it is increasingly observed that bacteria, including human pathogens, encode mechanisms to survive and withstand these stresses. This review provides an overview of the mechanisms employed by Salmonella spp., Shiga toxin producing E. coli, Cronobacter spp., Listeria monocytogenes and Campylobacter spp. to tolerate osmotic and desiccation stresses and identifies gaps in knowledge which need to be addressed to ensure the safety of low water activity and desiccated food products.

Foodborne zoonoses have a major health impact in industrialised countries. New European food safety regulations were issued to apply risk analysis to the food chain. The severity of foodborne zoonoses and the exposure of humans to biological hazards transmitted by food must be assessed. For meat, inspection at the slaughterhouse is historically the main means of control to protect consumers. However, the levels of detection of biological hazards during meat inspection have not been established in quantitative terms yet. Pork is the most frequently consumed meat in Europe. The aim of this study was to provide elements for quantifying levels of risk for pork consumers and lack of detection by meat inspection. Information concerning hazard identification and characterisation was obtained by the compilation and statistical analysis of data from 440 literature references. The incidence and severity of human cases due to pork consumption in Europe were assessed in order to calculate risk scores. A ratio of non-control was calculated for each biological hazard identified as currently established in Europe, i.e. the incidence of human cases divided by the prevalence of hazards on pork. Salmonella enterica, Yersinia enterocolitica and Campylobacter spp. were characterised by high incidence rates. Listeria monocytogenes, Clostridium botulinum and Mycobacterium spp. showed the highest severity scores. The three main high risk hazards involved in foodborne infections, Y. enterocolitica, S. enterica and Campylobacter spp. are characterised by high non-control ratios and cannot be detected by macroscopic examination of carcasses. New means of hazard control are needed to complement the classical macroscopic examination.

Various types of surfaces are used today in the food industry, such as plastic, stainless steel, glass, and wood. These surfaces are subject to contamination by microorganisms responsible for the cross-contamination of food by contact with working surfaces. The HACCP-based processes are now widely used for the control of microbial hazards to prevent food safety issues. This preventive approach has resulted in the use of microbiological analyses of surfaces as one of the tools to control the hygiene of products. A method of recovering microorganisms from different solid surfaces is necessary as a means of health prevention. No regulation exists for surface microbial contamination, but food companies tend to establish technical specifications to add value to their products and limit contamination risks. The aim of this review is to present the most frequently used methods: swabbing, friction or scrubbing, printing, rinsing or immersion, sonication and scraping or grinding and describe their advantages and drawbacks. The choice of the recovery method has to be suitable for the type and size of the surface tested for microbiological analysis. Today, quick and cheap methods have to be standardized and especially easy to perform in the field.

Ionization radiations are used to ensure food safety and quality. This irradiation process uses ions of beta or gamma rays to inactivate or destroy the food spoilage pests, microorganisms and their toxins without significantly increasing the temperature of the treated product. Meanwhile, various intrinsic and extrinsic factors are involved in determining the efficacy of ionization irradiation against these organisms. Therefore, the dose of radiations is recommended according to the type of irradiation, substrate and microorganisms. However, controversies are surrounding the use of irradiations in the food industry due to a negative perception of irradiations. This manuscript described the use of ionization radiations to control the foodborne biological hazards and increase shelf life. Firstly, the characteristics and mode of action of irradiations were discussed. Secondly, the role of extrinsic and intrinsic factors influencing the radioresistance of biological hazards were elaborated. This literature review also detailed the differential effects of irradiations on different microorganisms and pests having a role in food safety and deterioration. Finally, the regulatory status and the consumer values along with the controversies surrounding the use of ionization irradiations in the food sector were explained.

The development of a multi-omics approach has provided a new approach to the investigation of microbial communities allowing an integration of data, which can be used to better understand the behaviour of and interactions between community members. Metagenomics, metatranscriptomics, metaproteomics and metabolomics have the potential of producing a large amount of data in a very short time, however an important challenge is how to exploit and interpret these data to assist risk managers in food safety and quality decisions. This can be achieved by integrating multi-omics data in microbiological risk assessment. In this paper we identify limitations and challenges of the multi-omics approach, underlining promising potentials, but also identifying gaps, which should be addressed for its full exploitation. A view on how this new way of investigation will impact the traditional microbiology schemes in the food industry is also presented.

Whole genome sequencing (WGS) of important foodborne pathogens is a technology under development, but is already employed in routine surveillance by public health agencies and is being increasingly exploited in tracing transmission routes and identifying contamination events (source tracking) that take place in the farm-to-fork continuum. Furthermore, data generated from WGS, complemented by other -omics data, have the potential to be integrated into and strengthen microbiological risk assessment. In this paper, we discuss the contribution of WGS in diverse areas important to food safety and public health. Additionally, an outlook of future WGS applications, which should contribute to our understanding of the ecology and physiology of foodborne microorganisms, is presented.

Food-borne human infection with Campylobacter jejuni is a medical concern in both industrialized and developing countries. Efficient eradication of C. jejuni reservoirs within live animals and processed foods is limited by the development of antimicrobial resistances and by practical problems related to the use of conventional antibiotics in food processes. We have investigated the bacteriostatic and bactericidal activities of two phytochemicals, allyl-isothiocyanate (AITC), and benzyl isothiocyanate (BITC), against 24 C. jejuni isolates from chicken feces, human infections, and contaminated foods, as well as two reference strains NCTC11168 and 81-176. AITC and BITC displayed a potent antibacterial activity against C. jejuni. BITC showed a higher overall antibacterial effect (MIC of 1.25-5 μg mL(-1)) compared to AITC (MIC of 50-200 μg mL(-1)). Both compounds are bactericidal rather than bacteriostatic. The sensitivity levels of C. jejuni isolates against isothiocyanates were neither correlated with the presence of a GGT (γ-Glutamyl Transpeptidase) encoding gene in the genome, with antibiotic resistance nor with the origin of the biological sample. However the ggt mutant of C. jejuni 81-176 displayed a decreased survival rate compared to wild-type when exposed to ITC. This work determined the MIC of two ITC against a panel of C. jejuni isolates, showed that both compounds are bactericidal rather than bacteriostatic, and highlighted the role of GGT enzyme in the survival rate of C. jejuni exposed to ITC.

Food packaging is multifunctional: it protects from harvest to table. Four main groups of materials for direct food contact are mentioned in the literature: wood, glass, plastic, and metal. In this review, the focus is on wooden packaging for direct contact with food. In Europe, wood as a food contact material is subject to European Regulation (EC) No 1935/2004 states that materials must not transfer their constituents to food. Today, wooden packaging, like other packaging materials, does not have a Europe-wide harmonized specific regulation, so member countries legislate at different levels. Wood has been safely used for centuries in contact with food but is usually questioned because of its microbiological behavior compared with smooth surfaces. Based on a review of published conclusions from scientific studies over the last 20 y and after a description of the general properties of wooden packaging, we focus on the microbiological status of natural wood. Then, we discuss the parameters influencing the survival of microorganisms on wood. Finally, we report on the transfer of microorganisms from wood to food and the factors influencing this phenomenon. This review demonstrates that the porous nature of wood, especially when compared with smooth surfaces, is not responsible for the limited hygiene of the material used in the food industry and that it may even be an advantage for its microbiological status. In fact, its rough or porous surface often generates unfavorable conditions for microorganisms. In addition, wood has the particular characteristic of producing antimicrobial components able to inhibit or limit the growth of pathogenic microorganisms.

A literature search was performed on spoilage of fresh meat products by combining keyword query, text mining and expert elicitation. From the 258 collected studies, a quantitative analysis was first performed to identify the methods which are the most used to evaluate spoilage beside the preservation strategies suggested. In a second step focusing on a subset of 24 publications providing quantitative data on spoilage occurrence time, associations between spoilage occurrence time of meat products and specific spoilage indicators were investigated. The analysis especially focused on factors well represented in the 24 publications, i.e., gas packaging (O2 and CO2) and storage temperature. Relationships between spoilage occurrence and several microbiological indicators were also sought. The results point out possible advantages of removing dioxygen in packaging to delay spoilage occurrence, whereas, in the presence of dioxygen, the carbon dioxide proportion in the gas mixtures was shown to influence spoilage occurrence. The collected data clearly reveal a potentially protective role of lactic acid bacteria. Besides, while a spoilage role could be attributed to Pseudomonas spp., the growth of mesophilic aerobic microbes, Brochothrix spp. and Enterobacteriaceae seemed independent of spoilage occurrence time.

In food safety and public health risk evaluations, microbiological exposure assessment plays a central role as it provides an estimation of both the likelihood and the level of the microbial hazard in a specified consumer portion of food and takes microbial behaviour into account. While until now mostly phenotypic data have been used in exposure assessment, mechanistic cellular information, obtained using omics techniques, will enable the fine tuning of exposure assessments to move towards the next generation of microbiological risk assessment. In particular, metagenomics can help in characterizing the food and factory environment microbiota (endogenous microbiota and potentially pathogens) and the changes over time under the environmental conditions associated with processing, preservation and storage. The difficulty lies in moving up to a quantitative exposure assessment, because the development of models that enable the prediction of dynamics of pathogens in a complex food ecosystem is still in its infancy in the food safety domain. In addition, collecting and storing the environmental data (metadata) required to inform the models has not yet been organised at a large scale. In contrast, progress in biomarker identification and characterization has already opened the possibility of making qualitative or even quantitative connection between process and formulation conditions and microbial responses at the strain level. In term of modelling approaches, without changing radically the usual model structure, changes in model inputs are expected: instead of (or as well as) building models upon phenotypic characteristics such as for example minimal temperature where growth is expected, exposure assessment models could use biomarker response intensity as inputs. These new generations of strain-level models will bring an added value in predicting the variability in pathogen behaviour. Altogether, these insights based upon omics techniques will increase our (quantitative) knowledge on pathogenic strains and consequently will reduce our uncertainty; the exposure assessment of a specific combination of pathogen and food will be then more accurate. This progress will benefit the whole community of safety assessors and research scientists from academia, regulatory agencies and industry.

Bedding material is an important requirement of floor-based poultry production systems to meet the health and welfare requirements. This substrate can be an organic material like wood or plant-based material or inorganic material like clay and sand and should generally be a good absorbant, easily available, comfortable and nontoxic to birds. Wood is an organic renewable resource having good humidity regulation and antimicrobial properties and its by-products are commonly used as poultry litter in many countries. For example, in France, wood-based bedding is used in 80% of turkey production. This litter has approximately 50% lower NH3 production compared to other materials like sand and hulls, and has multiple environment-friendly disposal options. Moreover, the birds grown on such litter show better health and welfare and they prefer to practice their natural behaviours on this material which is helpful for the amelioration of physiological status and reduction of locomotion injuries. Moreover, according to some studies, the birds show improvement in different performance parameters, for example, up to 5-7% improvement in daily weight gain, live body weight, feed conversion ratio (FCR) and survivability. These improvements may differ depending upon the production system and type of wood material used in experiments, still it can be concluded that wood-based bedding materials do not negatively affect the production potential of poultry. In addition, the antimicrobial potential of wood may help to counter the load of certain pathogens in environment of birds. This review describes the physiochemical properties of wood-based bedding materials and their role in production and welfare of poultry.

During the last years, Campylobacter has emerged as the leading cause of bacterial foodborne infections in developed countries. Described as an obligate microaerophile, Campylobacter has puzzled scientists by surviving a wide range of environmental oxidative stresses on foods farm to retail, and thereafter intestinal transit and oxidative damage from macrophages to cause human infection. In this study, confocal laser scanning microscopy (CLSM) was used to explore the biofilm development of two well-described Campylobacter jejuni strains (NCTC 11168 and 81-176) prior to or during cultivation under oxygen-enriched conditions. Quantitative and qualitative appraisal indicated that C. jejuni formed finger-like biofilm structures with an open ultrastructure for 81-176 and a multilayer-like structure for NCTC 11168 under microaerobic conditions (MAC). The presence of motile cells within the biofilm confirmed the maturation of the C. jejuni 81-176 biofilm. Acclimation of cells to oxygen-enriched conditions led to significant enhancement of biofilm formation during the early stages of the process. Exposure to these conditions during biofilm cultivation induced an even greater biofilm development for both strains, indicating that oxygen demand for biofilm formation is higher than for planktonic growth counterparts. Overexpression of cosR in the poorer biofilm-forming strain, NCTC 11168, enhanced biofilm development dramatically by promoting an open ultrastructure similar to that observed for 81-176. Consequently, the regulator CosR is likely to be a key protein in the maturation of C. jejuni biofilm, although it is not linked to oxygen stimulation. These unexpected data advocate challenging studies by reconsidering the paradigm of fastidious requirements for C. jejuni growth when various subpopulations (from quiescent to motile cells) coexist in biofilms. These findings constitute a clear example of a survival strategy used by this emerging human pathogen.

As fragile food commodities, microbial and organoleptic qualities of fishery and seafood can quickly deteriorate. In this context, microbial quality and security improvement during the whole food processing chain (from catch to plate), using hurdle technology, a combination of mild preserving technologies such as biopreservation, modified atmosphere packaging and superchilling, are of great interest. Thirty-five lactic acid bacteria (LAB) belonging to the species Carnobacterium maltaromaticum, Carnobacterium inhibens, Lactococcus piscium, Leuconostoc gelidum, Vagococcus fluvialis, Vagococcus penaei and Aerococcus viridans, known to possess interesting antimicrobial activity, were chosen for their potential application as bioprotective agents as a part of hurdle technology applied to fishery products. The selection approach was based on 7 criteria including antimicrobial activity, alteration potential, resistance to chitosan coating and superchilling process, cross inhibition, biogenic amines production (histamine, tyramine) and antibiotics resistance. Antimicrobial activity was assessed against 6 common spoiling bacteria in fishery products (Shewanella baltica, Photobacterium phosphoreum, Brochothrix thermosphacta, Lactobacillus sakei, Hafnia alvei, Serratia proteamaculans) and 1 pathogenic bacteria (Listeria monocytogenes) in co-culture inhibitory assays miniaturized in 96-well microtiter plates. Antimicrobial activity and spoilage evaluation, both performed in cod and salmon juice, highlighted the existence of sensory signatures and inhibition profiles, which seem to be species related. Finally 6 LAB with no unusual antibiotics resistance profile nor histamine production ability were selected as bioprotective agents for further in situ inhibitory assays in cod and salmon based products, alone or in combination with other hurdles (chitosan, modified atmosphere packing and superchilling).

Some wood species have antimicrobial properties, making them a better choice over inert surfaces in certain circumstances. However, the organic and porous nature of wood raises questions regarding the use of this material in hygienically important places. Therefore, it is reasonable to investigate the microbial survival and the antimicrobial potential of wood via a variety of methods. Based on the available literature, this review classifies previously used methods into two broad categories: one category tests wood material by direct bacterial contact, and the other tests the action of molecules previously extracted from wood on bacteria and fungi. This article discusses the suitability of these methods to wood materials and exposes knowledge gaps that can be used to guide future research. This information is intended to help the researchers and field experts to select suitable methods for testing the hygienic safety and antimicrobial properties of wood materials.

The world population’s constant increase and the continuous need to ensure food safety and security are among the major challenges to be faced in the next 30 years. In addition, human diet is evolving with a decreased inclusion of proteins from animal sources. In this context, consumption of insects by humans (entomophagy) could be an alternative solution to the intake of protein derived from conventional livestock, due to the lower environmental impact of insect rearing compared to traditional farming. Furthermore, various insect species have promising nutritional profiles regarding both macro and micronutrients. Nowadays, it is recognized that about 2 billion people consume insects at a worldwide scale, with more than 2000 different species to have been reported. Since the beginning of the 2000s, mass rearing of insects for human consumption has been developing all over the world. Nevertheless edible insects are foodstuffs of animal origin and are usually consumed in their entirety, including the digestive tract, meaning that they may contain biological agents with hazardous potential (e.g. bacteria, parasites, viruses, prions, yeasts, molds, mycotoxins, histamine, and antibiotic resistance genes) and they must undergo a thorough analysis. Therefore, establishing the synthesis of the current knowledge on entomophagy and the related biological hazards is the main purpose of this review.

Seafood and fishery products are very fragile commodities with short shelf-lives owing to rapid deterioration of their organoleptic and microbiological quality. Microbial growth and activity are responsible for up to 25% of food losses in the fishery industry. In this context and to meet consumer demand for minimally processed food, developing mild preservation technologies such as biopreservation represents a major challenge. In this work, we studied the use of six lactic acid bacteria (LAB), previously selected for their properties as bioprotective agents, for salmon dill gravlax biopreservation. Naturally contaminated salmon dill gravlax slices, with a commercial shelf-life of 21 days, were purchased from a French industrial company and inoculated by spraying with the protective cultures (PCs) to reach an initial concentration of 106 log CFU/g. PC impact on gravlax microbial ecosystem (cultural and acultural methods), sensory properties (sensory profiling test), biochemical parameters (pH, TMA, TVBN, biogenic amines) and volatilome was followed for 25 days of storage at 8° C in vacuum packaging. PC antimicrobial activity was also assessed in situ against Listeria monocytogenes. This polyphasic approach underlined two scenarios depending on the protective strain. Carnobacterium maltaromaticum SF1944, Lactococcus piscium EU2229 and Leuconostoc gelidum EU2249, were very competitive in the product, dominated the microbial ecosystem, and displayed antimicrobial activity against the spoilage microbiota and L. monocytogenes. The strains also expressed their own sensory and volatilome signatures. However, of these three strains, C. maltaromaticum SF1944 did not induce strong spoilage and was the most efficient for L. monocytogenes growth control. By contrast, Vagococcus fluvialis CD264, Carnobacterium inhibens MIP2551 and Aerococcus viridans SF1044 were not competitive, did not express strong antimicrobial activity and produced only few organic volatile compounds (VOCs). However, V. fluvialis CD264 was the only strain to extend the sensory quality, even beyond 25 days. This study shows that C. maltaromaticum SF1944 and V. fluvialis CD264 both have a promising potential as bioprotective cultures to ensure salmon gravlax microbial safety and sensorial quality, respectively.

The genus Lactococcus comprises 12 species, some known for decades and others more recently described. Lactococcus piscium, isolated in 1990 from rainbow trout, is a psychrotrophic lactic acid bacterium, probably disregarded because most of the strains are unable to grow at 30°C. During the last 10 years, this species has been isolated from a large variety of food: meat, seafood and vegetables, mostly packed under vacuum (VP) or modified atmosphere (MAP) and stored at chilled temperature. Recently, culture-independent techniques used for characterization of microbial ecosystems have highlighted the importance of Lc. piscium in food. Its role in food spoilage varies according to the strain and the food matrix. However, most studies have indicated that Lc. piscium spoils meat, whereas it does not degrade the sensory properties of seafood. Lactococcus piscium strains have a large antimicrobial spectrum, including Gram-positive and negative bacteria. In various seafoods, some strains have a protective effect against spoilage and can extend the sensory shelf-life of the products. They can also inhibit the growth of Listeria monocytogenes, by a cell-to-cell contact-dependent. This article reviews the physiological and genomic characteristics of Lc. piscium and discusses its spoilage or protective activities in food.

The virulence of Viable But Non-Culturable (VBNC) cells of 4 strains of Listeria monocytogenes was investigated in both a human adenocarcinoma cell line (HT-29) and a mouse model. LO 28, ATCC 19115 and CNL 895807 strains of Listeria monocytogenes became VBNC when incubated in microcosm water at 20 degrees C and Scott A strain at 4 degrees C. No culturable bacteria were detected in the VBNC state, although 104 active cells/mL were found by the Direct Viable Count (DVC) and CTC-DAPI double staining methods. A comparison of virulence in both human adenocarcinoma cell line HT-29 and the mouse model showed that culturable controls were more virulent than VBNC cells, which appeared to be avirulent regardless of the virulence methods applied. Pathogenicity was tested in each model and was lost concomitantly with culturability, whereas some cells were still metabolically active (determined by CTC and DVC). Moreover, amplification of a 388 bp fragment with Immunocapture-PCR revealed the presence of Listeria monocytogenes DNA in all mixed spleen samples after intravenous injection of VBNC cells. These results demonstrate that VBNC cells were present in the mouse spleens. The results of the study suggest that Listeria monocytogenes strains might remain in the aquatic environment for prolonged periods in the VBNC state but these cells were not pathogenic in the conditions tested. These findings demonstrate the value of VBNC studies and show the need to investigate the role of VBNC cells in environmental transmission of Listeria monocytogenes. Further studies are needed in order to investigate the virulence of VBNC cells of Listeria monocytogenes after recovery of a culturable state.

Each year in Europe, meat is associated with 2.3 million foodborne illnesses, with a high contribution from beef meat. Many of these illnesses are attributed to pathogenic bacterial contamination and inadequate operations leading to growth and/or insufficient inactivation occurring along the whole farm-to-fork chain. To ensure consumer health, decision-making processes in food safety rely on Quantitative Microbiological Risk Assessment (QMRA) with many applications in recent decades. The present study aims to conduct a critical analysis of beef QMRAs and to identify future challenges. A systematic approach, the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, was used to collate beef QMRA models, identify steps of the farm-to-fork chain considered, and analyze inputs and outputs included as well as modelling methods. A total of 2343 articles were collected and 67 were selected. These studies focused mainly on western countries and considered Escherichia coli (EHEC) and Salmonella spp. pathogens. Future challenges were identified and included the need of whole-chain assessments, centralization of data collection processes, and improvement of model interoperability through harmonization. The present analysis can serve as a source of data and information to inform QMRA framework for beef meat and will help the scientific community and food safety authorities to identify specific monitoring and research needs.