Molecular detection of Listeria monocytogenes from different dairy and street food sources in North Karnataka, India.

BACKGROUND: Food-borne pathogen Listeria monocytogenes is abundantly present in nature and accountable for sporadic and epidemic cases of listeriosis in humans. The objective of this study was to screen common food sources for L. monocytogenes using biochemical and molecular methods to detect and characterise its toxin genes as well as for biofilm formation. METHODS: A total of 92 samples, comprising dairy and street food products, were randomly collected from various sources for this investigation. The collected samples were processed for biochemical and molecular methods to detect L. monocytogenes. Additionally, virulence factors associated genes, antibiogram profiles and biofilm formation related assays were determined. RESULTS: L. monocytogenes presence was confirmed using molecular detection methods targeting prs and lmo1030 genes, along with MALDI-TOF MS. Following 16 S rRNA sequencing, the identified Listeria species were further categorised into two groups. L. monocytogenes was detected in two (2.17%) food samples tested (L-23 and L-74). Multiplex PCR indicated the presence of seven virulence-related genes in L. monocytogenes isolates, i.e., inlA, inlB, prfA, iap, actA, plcB, and hlyA. In addition, 17 antibiotics were tested, whereby two isolates showed resistance to clindamycin and azithromycin, while one isolate (L-74) was also resistant to nalidixic acid, co-trimoxazole, ampicillin, norfloxacin, and cefotaxime. L-23 and L-74 isolates showed biofilm formation, especially at pH 8.6 and 37°C. CONCLUSIONS: Besides the demonstration of the presence of L. monocytogenes in some dairy and street food products, this study underscores the need to increase the standards of hygiene on the one hand and the importance of the surveillance of food-borne pathogens on the other.

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.

Antioxidant and Anti-Inflammatory Activity on LPS-Stimulated RAW 264.7 Macrophage Cells of White Mulberry (Morus alba L.) Leaf Extracts.

The white mulberry (Morus alba L.) is widely used as a medicinal plant in Asia. In this study, the bioactive compounds of ethanolic extracts of white mulberry leaves from the Sakon Nakhon and Buriram cultivars were evaluated. The ethanolic extracts of mulberry leaves from the Sakon Nakhon cultivar showed the highest total phenolic content of 49.68 mg GAE/g extract and antioxidant activities of 4.38 mg GAE/g extract, 4.53 mg TEAC/g extract, and 92.78 mg FeSO4/g extract using 2,2 diphenyl-1-picrylhydrazyl (DPPH), 2,20-azinobis-(3-ethylbenzothiazolin-6-sulfonic acid) (ABTS), and ferric reducing antioxidant power (FRAP) assays, respectively. The resveratrol and oxyresveratrol compounds in mulberry leaves were also investigated by high-performance liquid chromatography (HPLC). The mulberry leaf extracts from the Sakon Nakhon and Buriram cultivars showed oxyresveratrol contents of 1.20 ± 0.04 mg/g extract and 0.39 ± 0.02 mg/g extract, respectively, whereas resveratrol was not detected. It was also found that the potent anti-inflammatory properties of mulberry leaf extracts and its compounds, resveratrol and oxyresveratrol, suppressed the LPS-stimulated inflammatory responses in RAW 264.7 macrophage cells by significantly reducing nitric oxide production in a concentration-dependent manner. These compounds further inhibited interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) production and suppressed the mRNA and protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-stimulated RAW 264.7 macrophage cells. Therefore, it is established that mulberry leaf extract and its bioactive compounds contribute to its anti-inflammatory activity.

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.

Crystal structure of a subtilisin-like autotransporter passenger domain reveals insights into its cytotoxic function.

Autotransporters (ATs) are a large family of bacterial secreted and outer membrane proteins that encompass a wide range of enzymatic activities frequently associated with pathogenic phenotypes. We present the structural and functional characterisation of a subtilase autotransporter, Ssp, from the opportunistic pathogen Serratia marcescens. Although the structures of subtilases have been well documented, this subtilisin-like protein is associated with a 248 residue ß-helix and itself includes three finger-like protrusions around its active site involved in substrate interactions. We further reveal that the activity of the subtilase AT is required for entry into epithelial cells as well as causing cellular toxicity. The Ssp structure not only provides details about the subtilase ATs, but also reveals a common framework and function to more distantly related ATs. As such these findings also represent a significant step forward toward understanding the molecular mechanisms underlying the functional divergence in the large AT superfamily.

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.

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.

Variation of Antigen 43 self-association modulates bacterial compacting within aggregates and biofilms.

The formation of aggregates and biofilms enhances bacterial colonisation and infection progression by affording protection from antibiotics and host immune factors. Despite these advantages there is a trade-off, whereby bacterial dissemination is reduced. As such, biofilm development needs to be controlled to suit adaptation to different environments. Here we investigate members from one of largest groups of bacterial adhesins, the autotransporters, for their critical role in the assembly of bacterial aggregates and biofilms. We describe the structural and functional characterisation of autotransporter Ag43 variants from different Escherichia coli pathotypes. We show that specific interactions between amino acids on the contacting interfaces of adjacent Ag43 proteins drives a common mode of trans-association that leads to cell clumping. Furthermore, subtle variation of these interactions alters aggregation kinetics and the degree of compacting within cell clusters. Together, our structure-function investigation reveals an underlying molecular basis for variations in the density of bacterial communities.

Comparative Study on Electrical Conductivity of CeO2-Doped AlN Ceramics Sintered by Hot-Pressing and Spark Plasma Sintering.

Aluminum nitride (AlN) ceramics were prepared by both Hot-pressing (HP) and Spark-Plasma-Sintering (SPS) using cerium oxide as the sintering aid. The characterization of AlN raw powder denoted the presence of an amorphous layer that led to the formation of aluminum oxide. During the sintering process, CeO2 introduced as a sintering aid was reduced into Ce2O3. The latter reacted with aluminum oxide to form a transient liquid phase that promotes sintering by both HP and SPS. A reactional path leading to the formation of secondary phases, such as CeAlO3 and CeAl11O18, has been proposed according to the pseudo-binary Al2O3 - Ce2O3. Ceramics obtained from HP and SPS are presented as similar, except for the secondary-phase distribution. The influences of secondary phase composition and distribution on electrical conductivity were evaluated by leakage current measurements. The mechanism of DC conduction and the global conductivity of ceramics were discussed according to the sintering process and the number of secondary phases.

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.

Deep impact of the inactivation of the SecA2-only protein export pathway on the proteosurfaceome of Listeria monocytogenes.

Listeria monocytogenes presents a dimorphism associated to the SecA2 activity with cells having a normal rod shape or a dysmorphic elongated filamentous form. Besides variation of the cell and colony morphotype, this cell differentiation has profound ecophysiological and physiopathological implications with collateral effects on virulence and pathogenicity, biotope colonisation, bacterial adhesion and biofilm formation. This suggests the SecA2-only protein export could influence the listerial cell surface, which was investigated first by characterising its properties in L. monocytogenes wt and ΔsecA2. The degree of hydrophilicity and Lewis acid-base properties appeared significantly affected upon SecA2 inactivation. As modification of electrostatic properties would owe to modification in the composition of cell-surface proteins, the proteosurfaceome was further investigated by shotgun label-free proteomic analysis with a comparative relative quantitative approach. Following secretomic analysis, the protein secretion routes of the identified proteins were mapped considering the cognate transport and post-translocational maturation systems, as well as protein categories and subcellular localisation. Differential protein abundance profiles coupled to network analysis revealed the SecA2 dependence of 48 proteins, including some related to cell envelope biogenesis, translation and protein export, which could account for modifications of adhesion and surface properties of L. monocytogenes upon SecA2 inactivation. This investigation unravelled the profound influence of SecA2 activity on the cell surface properties and proteosurfaceome of L. monocytogenes, which provides advanced insights about its ecophysiopathology. SIGNIFICANCE: L. monocytogenes is a foodborne zoonotic pathogen and etiological agent of human listeriosis. This species presents a cellular dimorphism associated to the SecA2 activity that has profound physiopathological and ecophysiological implications with collateral effects on bacterial virulence and colonisation. To explore the influence of the SecA2-only protein export on the listerial cell, the surface properties of L. monocytogenes expressing or depleted of SecA2 was characterised by microelectrophoresis, microbial affinity to solvents and contact angles analyses. As modifications of hydrophilicity and Lewis acid-base electrostatic properties would owe to modification in the composition of cell-surface proteins, the proteinaceous subset of the surfaceome, i.e. the proteosurfaceome, was investigated further by shotgun label-free proteomic analysis. This subproteome appeared quite impacted upon SecA2 inactivation with the identification of proteins accounting for modifications in the cell surface properties. The profound influence of SecA2 activity on the cell surface of L. monocytogenes was unravelled, which provides advanced insights about its ecophysiopathology.

Evaluation of Antioxidant and Antibacterial Activities of White Mulberry (Morus alba L.) Fruit Extracts.

The fruit of mulberry trees (Morus sp.), mulberries, are traditionally utilised as a nutritional food and provide health benefits as well as skin nourishment in Thailand. White mulberries (Morus alba L.) from Chiang Mai and Mae Hong Son provinces were evaluated for their antioxidant and antibacterial activities. The antioxidant activities as well as the total phenolic, flavonoid and anthocyanin content of the aqueous and ethanolic extracts were determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azinobis-(3-ethylbenzothiazolin-6-sulfonic acid) (ABTS) and ferric reducing antioxidant power (FRAP) assays. The aqueous extracts of mulberries exhibited the highest antioxidant activity, which was associated with a higher phenolic and anthocyanin content. In testing the potent antibacterial activity against Escherichia coli, Salmonella Typhi, Shigella dysenteriae, Staphylococcus aureus and Vibrio cholerae, the mulberry extracts proved to be quite efficient, especially following water extraction. Time-kill and antibacterial adhesion assays further indicated that aqueous mulberry extracts could inhibit bacterial growth and prevent adhesions of pathogenic enteric bacteria on intestinal epithelial cells. It thus appears that mulberries can potentially be consumed as a good source of antioxidants, containing antimicrobial properties against some pathogenic bacteria which cause gastrointestinal tract infections.

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.

Pathogenicity Factors of Genomic Islands in Intestinal and Extraintestinal Escherichia coli.

Escherichia coli is a versatile bacterial species that includes both harmless commensal strains and pathogenic strains found in the gastrointestinal tract in humans and warm-blooded animals. The growing amount of DNA sequence information generated in the era of "genomics" has helped to increase our understanding of the factors and mechanisms involved in the diversification of this bacterial species. The pathogenic side of E. coli that is afforded through horizontal transfers of genes encoding virulence factors enables this bacterium to become a highly diverse and adapted pathogen that is responsible for intestinal or extraintestinal diseases in humans and animals. Many of the accessory genes acquired by horizontal transfers form syntenic blocks and are recognized as genomic islands (GIs). These genomic regions contribute to the rapid evolution, diversification and adaptation of E. coli variants because they are frequently subject to rearrangements, excision and transfer, as well as to further acquisition of additional DNA. Here, we review a subgroup of GIs from E. coli termed pathogenicity islands (PAIs), a concept defined in the late 1980s by Jörg Hacker and colleagues in Werner Goebel's group at the University of Würzburg, Würzburg, Germany. As with other GIs, the PAIs comprise large genomic regions that differ from the rest of the genome by their G + C content, by their typical insertion within transfer RNA genes, and by their harboring of direct repeats (at their ends), integrase determinants, or other mobility loci. The hallmark of PAIs is their contribution to the emergence of virulent bacteria and to the development of intestinal and extraintestinal diseases. This review summarizes the current knowledge on the structure and functional features of PAIs, on PAI-encoded E. coli pathogenicity factors and on the role of PAIs in host-pathogen interactions.

An Overview of the Elusive Passenger in the Gastrointestinal Tract of Cattle: The Shiga Toxin Producing Escherichia coli.

For approximately 10,000 years, cattle have been our major source of meat and dairy. However, cattle are also a major reservoir for dangerous foodborne pathogens that belong to the Shiga toxin-producing Escherichia coli (STEC) group. Even though STEC infections in humans are rare, they are often lethal, as treatment options are limited. In cattle, STEC infections are typically asymptomatic and STEC is able to survive and persist in the cattle GIT by escaping the immune defenses of the host. Interactions with members of the native gut microbiota can favor or inhibit its persistence in cattle, but research in this direction is still in its infancy. Diet, temperature and season but also industrialized animal husbandry practices have a profound effect on STEC prevalence and the native gut microbiota composition. Thus, exploring the native cattle gut microbiota in depth, its interactions with STEC and the factors that affect them could offer viable solutions against STEC carriage in cattle.

Interplay between enterohaemorrhagic Escherichia coli and nitric oxide during the infectious process.

Enterohaemorrhagic Escherichia coli (EHEC) are bacterial pathogens responsible for life-threatening diseases in humans such as bloody diarrhoea and the hemolytic and uremic syndrome. To date, no specific therapy is available and treatments remain essentially symptomatic. In recent years, we demonstrated in vitro that nitric oxide (NO), a major mediator of the intestinal immune response, strongly represses the synthesis of the two cardinal virulence factors in EHEC, namely Shiga toxins (Stx) and the type III secretion system, suggesting NO has a great potential to protect against EHEC infection. In this study, we investigated the interplay between NO and EHEC in vivo using mouse models of infection. Using a NO-sensing reporter strain, we determined that EHEC sense NO in the gut of infected mice. Treatment of infected mice with a specific NOS inhibitor increased EHEC adhesion to the colonic mucosa but unexpectedly decreased Stx activity in the gastrointestinal tract, protecting mice from renal failure. Taken together, our data indicate that NO can have both beneficial and detrimental consequences on the outcome of an EHEC infection, and underline the importance of in vivo studies to increase our knowledge in host-pathogen interactions.

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.

Identification of lipid A deacylase as a novel, highly conserved and protective antigen against enterohemorrhagic Escherichia coli.

Enterohemorrhagic E. coli (EHEC) is a major cause of large outbreaks worldwide associated with hemorrhagic colitis and hemolytic uremic syndrome. While vaccine development is warranted, a licensed vaccine, specific for human use, against EHEC is not yet available. In this study, the reverse vaccinology approach combined with genomic, transcriptional and molecular epidemiology data was applied on the EHEC O157:H7 genome to select new potential vaccine candidates. Twenty-four potential protein antigens were identified and one of them (MC001) was successfully expressed onto Generalized Modules for Membrane Antigens (GMMA) delivery system. GMMA expressing this vaccine candidate was immunogenic, raising a specific antibody response. Immunization with the MC001 candidate was able to reduce the bacterial load of EHEC O157:H7 strain in feces, colon and caecum tissues after murine infection. MC001 is homologue to lipid A deacylase enzyme (LpxR), and to our knowledge, this is the first study describing it as a potential vaccine candidate. Gene distribution and sequence variability analysis showed that MC001 is present and conserved in EHEC and in enteropathogenic E. coli (EPEC) strains. Given the high genetic variability among and within E. coli pathotypes, the identification of such conserved antigen suggests that its inclusion in a vaccine might represent a solution against major intestinal pathogenic strains.

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.