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.

Investigating Extracellular DNA Release in Staphylococcus xylosus Biofilm In Vitro.

Staphylococcus xylosus forms biofilm embedded in an extracellular polymeric matrix. As extracellular DNA (eDNA) resulting from cell lysis has been found in several staphylococcal biofilms, we investigated S. xylosus biofilm in vitro by a microscopic approach and identified the mechanisms involved in cell lysis by a transcriptomic approach. Confocal laser scanning microscopy (CLSM) analyses of the biofilms, together with DNA staining and DNase treatment, revealed that eDNA constituted an important component of the matrix. This eDNA resulted from cell lysis by two mechanisms, overexpression of phage-related genes and of cidABC encoding a holin protein that is an effector of murein hydrolase activity. This lysis might furnish nutrients for the remaining cells as highlighted by genes overexpressed in nucleotide salvage, in amino sugar catabolism and in inorganic ion transports. Several genes involved in DNA/RNA repair and genes encoding proteases and chaperones involved in protein turnover were up-regulated. Furthermore, S. xylosus perceived osmotic and oxidative stresses and responded by up-regulating genes involved in osmoprotectant synthesis and in detoxification. This study provides new insight into the physiology of S. xylosus in biofilm.

Investigation of Escherichia coli O157:H7 Survival and Interaction with Meal Components during Gastrointestinal Digestion.

Escherichia coli O157:H7 is responsible for foodborne poisoning, incriminating contaminated animal food and especially beef meat. This species can survive in the digestive tract, but, up to now, very few studies have considered its survival during the gastrointestinal digestion of meat. The present study aimed to investigate the survival of the pathogenic strain E. coli O157:H7 CM454 during the gastrointestinal digestion of ground beef meat and its interactions with meal components using a semidynamic digestive model. The CM454 strain in meat survived throughout digestion despite acidic pH (pH 2) and the presence of bile salts. The addition of nitrite and ascorbate in the digestion medium led to a decrease in strain survival. During digestion, a release of free iron was observed, which was accentuated in the presence of the CM454 strain. In addition, the strain modified the Fe2+/Fe3+ ratio, in favor of Fe2+ compared to the noninoculated meat sample. In the presence of nitrite, nitroso compounds such as nitrosamines, nitrosothiols, and nitrosylheme were formed. E. coli O157:H7 CM454 had no impact on N-nitrosation but seemed to decrease S-nitrosation and nitrosylation.

Physicochemical and microbiological characteristics of El-Guedid from meat of different animal species.

El-Guedid is an Algerian traditional meat-based product that is prepared from red meats. It belongs to the wide diversity of salted/dried meat products. This study described the physicochemical and microbiological properties of different products from four animal origins and during all the conservation. Results indicated that these products were mainly characterized by a low moisture with an average decrease of water content between 15.6% and 16.3% for all the samples, and a decrease in water activity ranging from 0.66 to 0.68, while the salt content ranged from 8.8 to 19.3%. A decrease in pH values oscillated from (6.3-6.4) to reach (5.2-5.5) at T0 and T365 consecutively, in all the samples. Microbial analyses revealed the absence of pathogenic bacteria such as Listeria and Salmonella but the sporadic contamination by Staphylococcus aureus up to one month of ripening. Lactic acid bacteria and coagulase negative staphylococci were the dominant populations in El-Guedid with Leuconostoc mesenteroides, Lactobacillus sakei, and Staphylococcus saprophyticus as the main species identified. All these populations decreased along the process and reached low levels (2 log CFU/g) at the end of storage (365 days). The drastic drying of El-Guedid led to safe traditional meat product that could promote its production.

Transcriptomic Analysis of Staphylococcus xylosus in Solid Dairy Matrix Reveals an Aerobic Lifestyle Adapted to Rind.

Staphylococcus xylosus is found in the microbiota of traditional cheeses, particularly in the rind of soft smeared cheeses. Despite its frequency, the molecular mechanisms allowing the growth and adaptation of S. xylosus in dairy products are still poorly understood. A transcriptomic approach was used to determine how the gene expression profile is modified during the fermentation step in a solid dairy matrix. S. xylosus developed an aerobic metabolism perfectly suited to the cheese rind. It overexpressed genes involved in the aerobic catabolism of two carbon sources in the dairy matrix, lactose and citrate. Interestingly, S. xylosus must cope with nutritional shortage such as amino acids, peptides, and nucleotides, consequently, an extensive up-regulation of genes involved in their biosynthesis was observed. As expected, the gene sigB was overexpressed in relation with general stress and entry into the stationary phase and several genes under its regulation, such as those involved in transport of anions, cations and in pigmentation were up-regulated. Up-regulation of genes encoding antioxidant enzymes and glycine betaine transport and synthesis systems showed that S. xylosus has to cope with oxidative and osmotic stresses. S. xylosus expressed an original system potentially involved in iron acquisition from lactoferrin.

Interaction in dual species biofilms between Staphylococcus xylosus and Staphylococcus aureus.

Staphylococcus xylosus, a coagulase-negative Staphylococcus, is frequently isolated from food products of animal origin and used as a starter culture in these products in which it contributes to their flavour, while Staphylococcus aureus, a coagulase-positive bacterium, causes foodborne intoxication and is implicated in a broad diversity of infections in medical sector, notably in nosocomial infections. S. xylosus and S. aureus are both capable of forming a biofilm and share the same ecological niches, thus we explored their interaction in biofilms with a view to limiting the risks associated with S. aureus. Cell-free supernatants of different strains of S. xylosus were able to inhibit the biofilm formation of S. aureus. The S. xylosus C2a strain released into the supernatant a molecule of molecular weight above 30 kDa that is resistant to proteolytic enzymes and inhibits the formation of S. aureus MW2 biofilm, though the mechanism involved has yet to be elucidated. Furthermore, S. xylosus C2a modified the architecture of S. aureus MW2 in co-culture biofilm. Confocal laser scanning microscopy revealed that S. aureus formed a biofilm with a flat and compact structure while in co-culture with S. xylosus the two species formed large juxtaposed aggregates throughout the period of incubation. This architecture made the S. aureus biofilm more susceptible to detachment.

Cell-Wall Hydrolases as Antimicrobials against Staphylococcus Species: Focus on Sle1.

Some staphylococcal species are opportunistic pathogens of humans and/or animals with Staphylococcus epidermidis as one of the most important. It causes a broad spectrum of diseases in humans and animals. This species is able to form biofilms and has developed antibiotic resistance, which has motivated research on new antibacterial agents. Cell-wall hydrolases (CWHs) can constitute a potential alternative. Following a hijacking strategy, we inventoried the CWHs of S. epidermidis. The lytic potential of representative CWHs that could be turned against staphylococci was explored by turbidity assays which revealed that cell wall glycosidases were not efficient, while cell wall amidases and cell wall peptidases were able to lyse S. epidermidis. Sle1, which is encoded by chromosomal gene and composed of three anchoring LysM domains and a C-terminal CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain, was one of the most active CWHs. The phylogeny of Sle1 revealed seven clusters mostly identified among staphylococci. Sle1 was able to lyse several staphylococcal species, including Staphylococcus aureus, both in planktonic and sessile forms, but not Micrococcus.

Physicochemical and microbiological characteristics of kitoza, a traditional salted/dried/smoked meat product of Madagascar.

Kitoza samples collected from producers in Madagascar were analyzed for their physicochemical and microbial properties. Lactic acid bacteria and coagulase-negative staphylococci were the two codominant populations with average counts of 6-7 log cfu/g. Good hygienic practices were sometimes lacking but samples were not contaminated with Salmonella, Clostridium perfringens, and Bacillus cereus and only once with Listeria monocytogenes. Staphylococcus aureus was found occasionally with higher counts in salted/dried products than in salted/smoked products. Moisture, protein, fat, and salt contents varied considerably and were on average 41.5, 43.5, 14.3, and 3.3 g/100 g, respectively, and water activity was 0.893 on average. Smoked kitoza showed higher moisture content compared to dried kitoza. Most of the smoked kitoza had a water activity higher than 0.9 which is not in accordance with their storage at ambient temperatures. Benzo(a)pyrene content was above 2 µg/kg in 11 out of 30 smoked samples (17 ± 16.5 µg/kg on average).

Cell Wall Hydrolases in Bacteria: Insight on the Diversity of Cell Wall Amidases, Glycosidases and Peptidases Toward Peptidoglycan.

The cell wall (CW) of bacteria is an intricate arrangement of macromolecules, at least constituted of peptidoglycan (PG) but also of (lipo)teichoic acids, various polysaccharides, polyglutamate and/or proteins. During bacterial growth and division, there is a constant balance between CW degradation and biosynthesis. The CW is remodeled by bacterial hydrolases, whose activities are carefully regulated to maintain cell integrity or lead to bacterial death. Each cell wall hydrolase (CWH) has a specific role regarding the PG: (i) cell wall amidase (CWA) cleaves the amide bond between N-acetylmuramic acid and L-alanine residue at the N-terminal of the stem peptide, (ii) cell wall glycosidase (CWG) catalyses the hydrolysis of the glycosidic linkages, whereas (iii) cell wall peptidase (CWP) cleaves amide bonds between amino acids within the PG chain. After an exhaustive overview of all known conserved catalytic domains responsible for CWA, CWG, and CWP activities, this review stresses that the CWHs frequently display a modular architecture combining multiple and/or different catalytic domains, including some lytic transglycosylases as well as CW binding domains. From there, direct physiological and collateral roles of CWHs in bacterial cells are further discussed.

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.

Nitric oxide synthase: What is its potential role in the physiology of staphylococci in meat products?

Coagulase-negative staphylococci are frequently isolated from meat products and two species are used as starter cultures in dry fermented sausages. In these products, they face various environmental conditions such as variation of redox potential and oxygen levels that can lead to oxidative stress. Furthermore, when nitrate and nitrite are added as curing salts, staphylococci also experience nitrosative stress. A nos gene encoding a nitric oxide synthase (NOS) is present in the genome of all staphylococci. NOS produces nitric oxide (NO) and citrulline from arginine, but its activity is still poorly characterized, particularly in coagulase-negative staphylococci. NO is highly reactive with a broad spectrum of activity resulting from targeting metal centres (heme and non-heme) and protein thiols. At low concentration, NO acts as a signalling molecule, while at higher concentration it generates stress. Thus, it was initially suggested that staphylococcal NOS counteract oxidative stress in relation to PerR and Fur regulators. In the physiology of staphylococci, it has recently been highlighted that NO controls the rate of aerobic respiration and regulates the transition from aerobic to nitrate respiration and also helps maintain the membrane potential in relation to the two-component systems SrrAB and AirRS. As NO interacts with heme centres, it binds the heme iron atom of myoglobin to form nitrosomyglobin, which is the typical red pigment of cured meat. However, the contribution of NOS to this reaction in meat products has yet to be evaluated.

Contribution of nitric oxide synthase from coagulase-negative staphylococci to the development of red myoglobin derivatives.

As part of the microbial community of meat or as starter cultures, coagulase-negative staphylococci (CNS) serve several essential technological purposes in meat products, such as color development through the reduction of nitrate to nitrite. As the safety of nitrite as an additive has been questioned, we explored the potential of CNS to develop red myoglobin derivatives such as oxymyoglobin and nitrosomyoglobin. Nitrosoheme was extracted to evaluate NO production. This production could be due to a nitric oxide synthase (NOS) activity. In all CNS strains, a nos gene was identified. The NOS sequences deduced were highly conserved within CNS. A phylogenetic tree based on the NOS sequences revealed that the strains within species were clustered. Ninety-one percent of the strains, whatever the species, were able to form red myoglobin derivatives in aerobic conditions, but a high variability was observed between strains within species. However, NO production was low as nitrosomyoglobin represented 8% to 16% of the red pigments according to the species. Formation of oxymyoglobin, especially under aerobic conditions, was substantial, but varied greatly within species. The mechanism involved in the formation of oxymyoglobin could rely on staphylococcal reductases and remains to be explored.

Special Issue: Beneficial Microorganisms for Food Manufacturing-Fermented and Biopreserved Foods and Beverages.

Food fermentation is an ancient technology, disseminated worldwide, which harness microorganisms and their enzymes to improve and diversify the human diet [...].

Insight into the Genome of Staphylococcus xylosus, a Ubiquitous Species Well Adapted to Meat Products.

Staphylococcus xylosus belongs to the vast group of coagulase-negative staphylococci. It is frequently isolated from meat products, either fermented or salted and dried, and is commonly used as starter cultures in sausage manufacturing. Analysis of the S. xylosus genome together with expression in situ in a meat model revealed that this bacterium is well adapted to meat substrates, being able to use diverse substrates as sources of carbon and energy and different sources of nitrogen. It is well-equipped with genes involved in osmotic, oxidative/nitrosative, and acidic stress responses. It is responsible for the development of the typical colour of cured meat products via its nitrate reductase activity. It contributes to sensorial properties, mainly by the the catabolism of pyruvate and amino acids resulting in odorous compounds and by the limiting of the oxidation of fatty acids, thereby avoiding rancidity.

Colonisation of Meat by Escherichia coli O157:H7: Investigating Bacterial Tropism with Respect to the Different Types of Skeletal Muscles, Subtypes of Myofibres, and Postmortem Time.

Escherichia coli O157:H7 is an enterohaemorrhagic E. coli (EHEC) responsible for serious diseases, especially pediatric, and of great concern for the meat industry. Meat contamination by EHEC occurs at slaughtering, especially at dehiding stage, where bacteria can be transferred from hides to carcasses. The skeletal muscle tissues comprise four major types of myofibres, which differ in their contraction velocity and metabolism. Myofibres are surrounded by the extracellular matrix (ECM). Adhesion of E. coli O157:H7 to meat was investigated considering well-defined types of skeletal muscle and their constituent myofibres as well as postmortem changes in muscle, using fluorescence microscopy and immunohistochemical analyses. By analysing the adhesion of E. coli O157:H7 to model oxidative (soleus) and glycolytic [extensor digitorum longus (EDL)] skeletal muscles, it first appeared that differential adhesion occurred at the surface of these extreme skeletal muscle types. At a cellular level, bacterial adhesion appeared to occur essentially at the ECM. Considering the different constituent myofibres of types I, IIA, IIX and IIB, no significant differences were observed for adhering bacteria. However, bacterial adhesion to the ECM was significantly influenced by postmortem structural modifications of muscle tissues. By providing information on spatial localisation of E. coli O157:H7 on meat, this investigation clearly demonstrated their ability to adhere to skeletal muscle, especially at the ECM, which consequently resulted in their heterogeneous distribution in meat. As discussed, these new findings should help in reassessing and mitigating the risk of contamination of meat, the food chain and ultimately human infection by EHEC.

Evidence for Nitric Oxide Synthase Activity in Staphylococcus xylosus Mediating Nitrosoheme Formation.

Staphylococcus xylosus is used as a starter culture in fermented meat products and contributes to color formation by the reduction of nitrate to nitrite. Nitrite is a food additive that is chemically turned to nitric oxide (NO) in meat but its safety has been questioned. The objective of this study was to determine the ability of NO synthase (NOS) of S. xylosus C2a to produce NO. For this purpose, a nos deletion mutant (Δnos) in S. xylosus was constructed and NO production was evaluated in a test based on its ability to form nitrosomyoglobin and nitrosoheme. Production of NO was abrogated in the Δnos mutant under aerobic conditions and reduced about 35-40% comparing to the wild type C2a under limited oxygenation. This mutant was sensitive to oxidative stress. The expression of genes encoding catalase was modulated in the mutant with an up-regulation of katA and a down-regulation of katB and katC. The Δnos mutant displayed high colony pigmentation after prolonged growth on agar medium. Finally, the Δnos mutant showed no growth in minimal medium. Growth was not restored in the minimal medium by complementation with nos, but was restored by either addition of phenylalanine or complementation with pdt, a gene that encodes a prephenate dehydratase involved in phenylalanine biosynthesis and co-transcribed with nos. Our findings clearly demonstrate NOS-mediated NO production in S. xylosus, a meat-associated coagulase-negative Staphylococcus.

Staphylococcal ecosystem of kitoza, a traditional malagasy meat product.

Kitoza is a traditional meat product from Madagascar manufactured with strips of pork or beef. The process includes a first step of salting and mixing with spices followed by sun-drying or smoking step. As salting and drying select coagulase-negative staphylococci (CNS), our aim was to identify the CNS species in kitoza with the objective in the future of developing indigenous starters. Microbial analyses revealed that the only pathogenic bacterium enumerated was Staphylococcus aureus, which was found in 54% of the samples. The level of Enterobacteriaceae revealed a rather good hygienic quality of these products. CNS were confirmed in all the samples at high levels ranging from 5 to 7logcfu/g. Identification of CNS species in a large collection of 829 isolates revealed 9 identified species, 7 for beef and 8 for pork kitoza. There were significant difference in the distribution of CNS species according to the type of meat and the process. Staphylococcus saprophyticus was the dominant species for sun-dried or smoked beef and sun-dried pork kitoza (73-75%), while for smoked pork kitoza Staphylococcus equorum (26%), S. saprophyticus (23%), Staphylococcus succinus (23%) and Staphylococcus epidermidis (17%) co-dominated. Some CNS could be used as indigenous starters in particular to compete against S. aureus.

Ferritin, an iron source in meat for Staphylococcus xylosus?

Staphylococcus xylosus is frequently isolated from food of animal origin. Moreover, this species is one of the major starter cultures used for meat fermentation. Iron is a key element for growth and survival of bacteria. Meat is particularly rich in haemic (myoglobin and haemoglobin) and non-haemic (ferritin and transferrin) iron sources. Ferritin is a storage protein able to capture large quantities of iron. It is highly resistant to microbial attack and few microorganisms can use it as an iron source. Surprisingly, we found that the S. xylosus C2a strain grows in the presence of ferritin as a sole iron source. A three-cistron operon was highly overexpressed under ferritin iron growth conditions. We generated a deletion-insertion in the first gene of the operon and evaluated the phenotype of the mutant. The mutant showed decreased growth because it was less able to acquire iron from ferritin. Transcriptional analysis of the mutant revealed downregulation of several genes involved in the response to oxidative stress. This study characterized for the first time the capacity of a Staphylococcus to use iron from ferritin and revealed that a potential reductive pathway was involved in this acquisition. We hypothesize that this ability could give an advantage to S. xylosus in meat products.