Spatially localised expression of the glutamate decarboxylase gadB in Escherichia coli O157:H7 microcolonies in hydrogel matrices.

Functional diversity within isogenic spatially organised bacterial populations has been shown to trigger emergent community properties such as stress tolerance. Considering gadB gene encoding a key glutamate decarboxylase involved in E. coli tolerance to acidic conditions, we investigated its expression in hydrogels mimicking the texture of some structured food matrices (such as minced meat or soft cheese). Taking advantage of confocal laser scanning microscopy combined with a genetically-engineered dual fluorescent reporter system, it was possible to visualise the spatial patterns of bacterial gene expression from in-gel microcolonies. In E. coli O157:H7 microcolonies, gadB showed radically different expression patterns between neutral (pH 7) or acidic (pH 5) hydrogels. Differential spatial expression was determined in acidic hydrogels with a strong expression of gadB at the microcolony periphery. Strikingly, very similar spatial patterns of gadB expression were further observed for E. coli O157:H7 grown in the presence of L. lactis. Considering the ingestion of contaminated foodstuff, survival of E. coli O157:H7 to acidic stomachal stress (pH 2) was significantly increased for bacterial cells grown in microcolonies in acidic hydrogels compared to planktonic cells. These findings have significant implications for risk assessment and public health as they highlight inherent differences in bacterial physiology and virulence between liquid and structured food products. The contrasting characteristics observed underscore the need to consider the distinct challenges posed by these food types, thereby emphasising the importance of tailored risk mitigation strategies.

Specific Proteomic Identification of Collagen-Binding Proteins in Escherichia coli O157:H7: Characterisation of OmpA as a Potent Vaccine Antigen.

Escherichia coli is a versatile commensal species of the animal gut that can also be a pathogen able to cause intestinal and extraintestinal infections. The plasticity of its genome has led to the evolution of pathogenic strains, which represent a threat to global health. Additionally, E. coli strains are major drivers of antibiotic resistance, highlighting the urgent need for new treatment and prevention measures. The antigenic and structural heterogeneity of enterohaemorrhagic E. coli colonisation factors has limited their use for the development of effective and cross-protective vaccines. However, the emergence of new strains that express virulence factors deriving from different E. coli diarrhoeagenic pathotypes suggests that a vaccine targeting conserved proteins could be a more effective approach. In this study, we conducted proteomics analysis and functional protein characterisation to identify a group of proteins potentially involved in the adhesion of E. coli O157:H7 to the extracellular matrix and intestinal epithelial cells. Among them, OmpA has been identified as a highly conserved and immunogenic antigen, playing a significant role in the adhesion phenotype of E. coli O157:H7 and in bacterial aggregation. Furthermore, antibodies raised against recombinant OmpA effectively reduced the adhesion of E. coli O157:H7 to intestinal epithelial cells. The present work highlights the role of OmpA as a potent antigen for the development of a vaccine against intestinal pathogenic E. coli.

Genome-Wide Analysis of Antigen 43 (Ag43) Variants: New Insights in Their Diversity, Distribution and Prevalence in Bacteria.

Antigen 43 (Ag43) expression induces aggregation and biofilm formation that has consequences for bacterial colonisation and infection. Ag43 is secreted through the Type 5 subtype "a" secretion system (T5aSS) and is a prototypical member of the family of self-associating autotransporters (SAATs). As a T5aSS protein, Ag43 has a modular architecture comprised of (i) a signal peptide, (ii) a passenger domain that can be subdivided into three subdomains (SL, EJ, and BL), (iii) an autochaperone (AC) domain, and (iv) an outer membrane translocator. The cell-surface SL subdomain is directly involved in the "Velcro-handshake" mechanism resulting in bacterial autoaggregation. Ag43 is considered to have a ubiquitous distribution in E. coli genomes and many strains harbour multiple agn43 genes. However, recent phylogenetic analyses indicated the existence of four distinct Ag43 classes exhibiting different propensities for autoaggregation and interactions. Given the knowledge of the diversity and distribution of Ag43 in E. coli genomes is incomplete, we have performed a thorough in silico investigation across bacterial genomes. Our comprehensive analyses indicate that Ag43 passenger domains cluster in six phylogenetic classes associated with different SL subdomains. The diversity of Ag43 passenger domains is a result of the association of the SL subtypes with two different EJ-BL-AC modules. We reveal that agn43 is almost exclusively present among bacterial species of the Enterobacteriaceae family and essentially in the Escherichia genus (99.6%) but that it is not ubiquitous in E. coli. The gene is typically present as a single copy but up to five copies of agn43 with different combinations of classes can be observed. The presence of agn43 as well as its different classes appeared to differ between Escherichia phylogroups. Strikingly, agn43 is present in 90% of E. coli from E phylogroup. Our results shed light on Ag43 diversity and provide a rational framework for investigating its role in E. coli ecophysiology and physiopathology.

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.

Genetic, physiological, and cellular heterogeneities of bacterial pathogens in food matrices: Consequences for food safety.

In complex food systems, bacteria live in heterogeneous microstructures, and the population displays phenotypic heterogeneities at the single-cell level. This review provides an overview of spatiotemporal drivers of phenotypic heterogeneity of bacterial pathogens in food matrices at three levels. The first level is the genotypic heterogeneity due to the possibility for various strains of a given species to contaminate food, each of them having specific genetic features. Then, physiological heterogeneities are induced within the same strain, due to specific microenvironments and heterogeneous adaptative responses to the food microstructure. The third level of phenotypic heterogeneity is related to cellular heterogeneity of the same strain in a specific microenvironment. Finally, we consider how these phenotypic heterogeneities at the single-cell level could be implemented in mathematical models to predict bacterial behavior and help ensure microbiological food safety.

Antioxidants of Fruit Extracts as Antimicrobial Agents against Pathogenic Bacteria.

Fruit is an essential part of the human diet and is of great interest because of its richness in phytochemicals. Various fruit extracts from citrus, berries and pomegranates have been shown to possess a broad spectrum of medicinal properties. Fruit phytochemicals are of considerable interest because of their antioxidant properties involving different mechanisms of action, which can act against different pathogenic bacteria. The antioxidant capacity of fruit phytochemicals involves different kinds of reactions, such as radical scavenging and chelation or complexation of metal ions. The interaction between fruit phytochemicals and bacteria has different repercussions: it disrupts the cell envelope, disturbs cell-cell communication and gene regulation, and suppresses metabolic and enzymatic activities. Consequently, fruit phytochemicals can directly inhibit bacterial growth or act indirectly by modulating the expression of virulence factors, both of which reduce microbial pathogenicity. The aim of this review was to report our current knowledge on various fruit extracts and their major bioactive compounds, and determine the effectiveness of organic acids, terpenes, polyphenols, and other types of phenolic compounds with antioxidant properties as a source of antimicrobial agents.

Spatial organisation of Listeria monocytogenes and Escherichia coli O157:H7 cultivated in gel matrices.

The spatial organisation of bacterial pathogens in food matrices remains poorly understood, but is important in improving risk assessment and preventing infection of consumers by contaminated foodstuff. By combining confocal laser scanning microscopy with genetic fluorescent labelling of Listeria monocytogenes and Escherichia coli O157:H7, it was possible to investigate the spatial patterns of colonisation of both foodborne pathogens in gel matrices, alone or in combination, in various environmental conditions. Increasing low melting point agarose (LMPA) concentrations triggers the transition between a motile single-cell lifestyle to a sessile population spatially organised as microcolonies. The size, number and morphology of microcolonies were highly affected by supplementations in NaCl or lactic acid, two compounds frequently used in food products. Strikingly, single-cell motility was partially restored at higher LMPA concentration in the presence of lactic acid for Escherichia coli O157:H7 and in the presence of NaCl for Listeria monocytogenes. Co-culture of both species in the hydrogel affected pathogen colonisation features; Listeria monocytogenes was better able to colonise gel matrices containing lactic acid in the presence of Escherichia coli O157:H7. Altogether, this investigation provides insights into the spatial distribution and structural dynamics of bacterial pathogens in gel matrices. Potential impacts on food safety are discussed.

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.

The Secretome landscape of Escherichia coli O157:H7: Deciphering the cell-surface, outer membrane vesicle and extracellular subproteomes.

Among diarrheagenic E. coli (DEC), enterohaemorrhagic E. coli (EHEC) are the most virulent anthropozoonotic agents. The ability of bacterial cells to functionally interact with their surrounding essentially relies on the secretion of different protein effectors. To experimentally determine the repertoire of extracytoproteins in E. coli O157:H7, a subproteomic analysis was performed not only considering the extracellular milieu but the cell surface and outer membrane vesicles. Following a secretome-based approach, the proteins trafficking from the interior to the exterior of the cell were depicted considering cognate protein transport systems and subcellular localisation. Label-free quantitative analysis of the proteosurfaceome, proteovesiculome and exoproteome from E. coli O157:H7 grown in three different nutrient media revealed differential protein expression profiles and allowed defining the core and variant subproteomes. Network analysis further revealed the higher abundance of some protein clusters in chemically defined medium over rich complex medium, especially related to some outer membrane proteins, ABC transport and Type III secretion systems. This first comprehensive study of the EHEC secretome unravels the profound influence of environmental conditions on the extracytoplasmic proteome, provides new insight in the physiology of E. coli O157:H7 and identifies potentially important molecular targets for the development of preventive strategies against EHEC/STEC. SIGNIFICANCE: Escherichia coli O157:H7 is responsible for severe diarrhoea especially in young children. Despite years of investigations, the global view of the extracytoplasmic proteins expressed in this microorganism was eluded. To provide the first comprehensive view of the secretome landscape of E. coli O157:H7, the exoproteome, proteosurfaceome and proteovesiculome were profiled using growth conditions most likely to induce changes in bacterial protein secretion. The profound influence of growth conditions on the extracytoplasmic proteome was unravelled and allowed identifying the core and variant subproteomes. Besides new insight in the physiology of enterohaemorrhagic E. coli, these proteins potentially constitute important molecular targets for the development of preventive strategies.

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.

Molecular determinants of surface colonisation in diarrhoeagenic Escherichia coli (DEC): from bacterial adhesion to biofilm formation.

Escherichia coli is primarily known as a commensal colonising the gastrointestinal tract of infants very early in life but some strains being responsible for diarrhoea, which can be especially severe in young children. Intestinal pathogenic E. coli include six pathotypes of diarrhoeagenic E. coli (DEC), namely, the (i) enterotoxigenic E. coli, (ii) enteroaggregative E. coli, (iii) enteropathogenic E. coli, (iv) enterohemorragic E. coli, (v) enteroinvasive E. coli and (vi) diffusely adherent E. coli. Prior to human infection, DEC can be found in natural environments, animal reservoirs, food processing environments and contaminated food matrices. From an ecophysiological point of view, DEC thus deal with very different biotopes and biocoenoses all along the food chain. In this context, this review focuses on the wide range of surface molecular determinants acting as surface colonisation factors (SCFs) in DEC. In the first instance, SCFs can be broadly discriminated into (i) extracellular polysaccharides, (ii) extracellular DNA and (iii) surface proteins. Surface proteins constitute the most diverse group of SCFs broadly discriminated into (i) monomeric SCFs, such as autotransporter (AT) adhesins, inverted ATs, heat-resistant agglutinins or some moonlighting proteins, (ii) oligomeric SCFs, namely, the trimeric ATs and (iii) supramolecular SCFs, including flagella and numerous pili, e.g. the injectisome, type 4 pili, curli chaperone-usher pili or conjugative pili. This review also details the gene regulatory network of these numerous SCFs at the various stages as it occurs from pre-transcriptional to post-translocational levels, which remains to be fully elucidated in many cases.

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.

Differential homotypic and heterotypic interactions of antigen 43 (Ag43) variants in autotransporter-mediated bacterial autoaggregation.

Antigen 43 (Ag43) is a cell-surface exposed protein of Escherichia coli secreted by the Type V, subtype a, secretion system (T5aSS) and belonging to the family of self-associating autotransporters (SAATs). These modular proteins, comprising a cleavable N-terminal signal peptide, a surface-exposed central passenger and an outer membrane C-terminal translocator, self-recognise in a Velcro-like handshake mechanism. A phylogenetic network analysis focusing on the passenger revealed for the first time that they actually distribute into four distinct classes, namely C1, C2, C3 and C4. Structural alignment and modelling analyses demonstrated these classes arose from shuffling of two different subdomains within the Ag43 passengers. Functional analyses revealed that homotypic interactions occur for all Ag43 classes but significant differences in the sedimentation kinetics and aggregation state were present when Ag43C3 was expressed. In contrast, heterotypic interaction occurred in a very limited number of cases. Single cell-force spectroscopy demonstrated the importance of specific as well as nonspecific interactions in mediating Ag43-Ag43 recognition. We propose that structural differences in the subdomains of the Ag43 classes account for different autoaggregation dynamics and propensities to co-interact.

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.