Virtual Screening Assisted Search for Inhibitors of the Translocated Intimin Receptor of Enteropathogenic Escherichia Coli.

This study aimed to identify inhibitors of the translocated intimin receptor (Tir) of enteropathogenic Escherichia coli (EPEC). EPEC is an intestinal pathogen that causes diarrhea and is a major health concern worldwide. Because Tir is a key virulence factor involved in EPEC pathogenesis, inhibiting its function is a potential strategy for controlling EPEC infections. Virtual screening was applied to chemical libraries to search for compounds that inhibit Tir-mediated bacterial adherence to host cells. Three sites were targeted using the cocrystal structure published earlier. A selection of compounds was then assessed in a cell-based infection model and fluorescence microscopy assay. The results of this study provide a basis for further optimization and testing of Tir inhibitors as potential therapeutic agents for EPEC infections.

Genetic Characterization of Intimin Gene (eae) in Clinical Shiga Toxin-Producing Escherichia coli Strains from Pediatric Patients in Finland.

Shiga toxin (Stx)-producing Escherichia coli (STEC) infections cause outbreaks of severe disease in children ranging from bloody diarrhea to hemolytic uremic syndrome (HUS). The adherent factor intimin, encoded by eae, can facilitate the colonization process of strains and is frequently associated with severe disease. The purpose of this study was to examine and analyze the prevalence and polymorphisms of eae in clinical STEC strains from pediatric patients under 17 years old with and without HUS, and to assess the pathogenic risk of different eae subtypes. We studied 240 STEC strains isolated from pediatric patients in Finland with whole genome sequencing. The gene eae was present in 209 (87.1%) strains, among which 49 (23.4%) were from patients with HUS, and 160 (76.6%) were from patients without HUS. O157:H7 (126, 60.3%) was the most predominant serotype among eae-positive STEC strains. Twenty-three different eae genotypes were identified, which were categorized into five eae subtypes, i.e., γ1, ß3, ε1, θ and ζ3. The subtype eae-γ1 was significantly overrepresented in strains from patients aged 5-17 years, while ß3 and ε1 were more commonly found in strains from patients under 5 years. All O157:H7 strains carried eae-γ1; among non-O157 strains, strains of each serotype harbored one eae subtype. No association was observed between the presence of eae/its subtypes and HUS. However, the combination of eae-γ1+stx2a was significantly associated with HUS. In conclusion, this study demonstrated a high occurrence and genetic variety of eae in clinical STEC from pediatric patients under 17 years old in Finland, and that eae is not essential for STEC-associated HUS. However, the combination of certain eae subtypes with stx subtypes, i.e., eae-γ1+stx2a, may be used as risk predictors for the development of severe disease in children.

Genetic Diversity and Expression of Intimin in Escherichia albertii Isolated from Humans, Animals, and Food.

Escherichia albertii (E. albertii) is an emerging diarrheagenic pathogen associated with sporadic infections and human gastroenteric outbreaks. The eae gene, which encodes intimin in the locus of enterocyte effacement (LEE) operon, contributes to the establishment of the attaching and effacing (A/E) lesion. Increasing collection of E. albertii strains from various sources has resulted in a rapid increase in the number of eae subtypes. This study systematically investigated the prevalence and genetic diversity of eae among E. albertii strains isolated from humans, animals, and food. The eae gene was present in 452/459 (98.5%) strains and 23 subtypes were identified including two novel subtypes, named eae-α11 and η3. The eae-σ subtype was the most predominant among humans, animals, and food-derived strains, while eae-γ3, τ, and α11 were unique in human-derived strains. Additionally, the LEE island was also analyzed at genomic, transcriptional, and functional levels through genomic analysis, quantitative reverse transcription PCR, and HEp-2 cell adherence assays, respectively. The eae transcript levels were variable and associated with eae subtypes. Three different adherence patterns, including localized adherence-like (LAL), diffuse adherence (DA), and detachment (DE), were observed among E. albertii strains. This study demonstrated a high diversity of functional intimin in E. albertii strains isolated from humans, animals, and food. Further in vivo and in vitro studies are warranted to better elucidate the role of intimin or LEE in different genetic backgrounds.

Genomic and Phenotypic Characterization of Shiga Toxin-Producing Escherichia albertii Strains Isolated from Wild Birds in a Major Agricultural Region in California.

Escherichia albertii is an emerging foodborne pathogen. To better understand the pathogenesis and health risk of this pathogen, comparative genomics and phenotypic characterization were applied to assess the pathogenicity potential of E. albertii strains isolated from wild birds in a major agricultural region in California. Shiga toxin genes stx2f were present in all avian strains. Pangenome analyses of 20 complete genomes revealed a total of 11,249 genes, of which nearly 80% were accessory genes. Both core gene-based phylogenetic and accessory gene-based relatedness analyses consistently grouped the three stx2f-positive clinical strains with the five avian strains carrying ST7971. Among the three Stx2f-converting prophage integration sites identified, ssrA was the most common one. Besides the locus of enterocyte effacement and type three secretion system, the high pathogenicity island, OI-122, and type six secretion systems were identified. Substantial strain variation in virulence gene repertoire, Shiga toxin production, and cytotoxicity were revealed. Six avian strains exhibited significantly higher cytotoxicity than that of stx2f-positive E. coli, and three of them exhibited a comparable level of cytotoxicity with that of enterohemorrhagic E. coli outbreak strains, suggesting that wild birds could serve as a reservoir of E. albertii strains with great potential to cause severe diseases in humans.

Characterisation of typical enteropathogenic Escherichia coli (tEPEC) lineages and novel bfpA variants detected in Australian fruit bats (Pteropus poliocephalus).

Enteropathogenic Escherichia coli (EPEC) is an important cause of diarrhoeal disease in human infants. EPEC strains are defined by the presence of specific virulence factors including intimin (encoded by the eae gene) and bundle forming pili (Bfp). Bfp is encoded by the bfp operon and includes the bfpA gene for the major pilus subunit. By definition, Bfp are only present in typical EPEC (tEPEC), for which, humans are considered to be the only known natural host. This study detected tEPEC in faecal samples from a wild Australian fruit bat species, the grey-headed flying-fox (Pteropus poliocephalus). Whole genome sequencing of 61 E. coli isolates from flying-foxes revealed that 21.3 % (95%CI: 13 %-33 %) were tEPEC. Phylogenetic analyses showed flying-fox tEPEC shared evolutionary lineages with human EPEC, but were predominantly novel sequence types (9 of 13) and typically harboured novel bfpA variants (11 of 13). HEp-2 cell adhesion assays showed adherence to human-derived epithelial cells by all 13 flying-fox tEPEC, indicating that they all carried functional Bfp. Using an EPEC-specific duplex PCR, it was determined that tEPEC comprised 17.4 % (95%CI: 13 %-22 %) of 270 flying-fox E. coli isolates. Furthermore, a tEPEC-specific multiplex PCR detected the eae and bfpA virulence genes in 18.0 % (95%CI: 8.0 %-33.7 %) of 506 flying-fox faecal DNA samples, with occurrences ranging from 1.3 % to 87.0 % across five geographic areas sampled over a four-year period. The identification of six novel tEPEC sequence types and five novel bfpA variants suggests flying-foxes carry bat-specific tEPEC lineages. However, their close relationship with human EPEC and functional Bfp, indicates that flying-fox tEPEC have zoonotic potential and that dissemination of flying-fox tEPEC into urban environments may pose a public health risk. The consistent detection of tEPEC in flying-foxes over extensive geographical and temporal scales indicates that both wild grey-headed flying-foxes and humans should be regarded as natural tEPEC hosts.

Escherichia albertii as a Potential Enteropathogen in the Light of Epidemiological and Genomic Studies.

Escherichia albertii is a new enteropathogen of humans and animals. The aim of the study was to assess the prevalence and pathogenicity of E. albertii strains isolated in northeastern Poland using epidemiological and genomic studies. In 2015-2018, a total of 1154 fecal samples from children and adults, 497 bird droppings, 212 food samples, 92 water samples, and 500 lactose-negative E. coli strains were tested. A total of 42 E. albertii strains were isolated. The PCR method was suitable for their rapid identification. In total, 33.3% of E. albertii isolates were resistant to one antibiotic, and 16.7% to two. Isolates were sensitive to cefepime, imipenem, levofloxacin, gentamicin, trimethoprim/sulfamethoxazole, and did not produce ESBL ß-lactamases. High genetic variability of E. albertii has been demonstrated. In the PFGE method, 90.5% of the strains had distinct pulsotypes. In MLST typing, 85.7% of strains were assigned distinct sequence types (STs), of which 64% were novel ST types. Cytolethal distending toxin (CDT) and Paa toxin genes were found in 100% of E. albertii isolates. Genes encoding toxins, IbeA, CdtB type 2, Tsh and Shiga (Stx2f), were found in 26.2%, 9.7%, 1.7%, and 0.4% of E. albertii isolates, respectively. The chromosome size of the tested strains ranged from 4,573,338 to 5,141,010 bp (average 4,784,003 bp), and at least one plasmid was present in all strains. The study contributes to a more accurate assessment of the genetic diversity of E. albertii and the potential threat it poses to public health.

PLGA nanoparticles containing Intimin-Flagellin fusion protein for E. coli O157:H7 nano-vaccine.

Escherichia coli O157:H7 is a foodborne pathogen that can lead to severe gastrointestinal diseases in humans. Vaccination is a promising strategy for preventing E. coli O157:H7 infections, which offers socio-economic benefits and provides the possibility of stimulating both humoral and cellular immune responses at systemic and mucosal sites. In this study, we developed a needle-free vaccine candidate against E. coli O157:H7 using poly(lactic-co-glycolic acid) (PLGA) nanoparticles entrapping a chimeric Intimin-Flagellin (IF) protein. The IF protein was expressed and verified using SDS-PAGE and western blot analysis, with a yield of 1/7 mg/L and a molecular weight of approximately 70 kDa. The prepared nanoparticles showed uniformly shaped spherical particles in the 200-nm range, as confirmed by SEM and DLS analysis. Three different routes of vaccine administration were used, including intranasal, oral, and subcutaneous, and the groups vaccinated with NPs protein had a higher antibody response compared to those receiving free protein. Subcutaneous administration of IF-NPs resulted in the highest level of IgG antibody titer, while oral administration of IF-NPs produced the highest amount of IgA antibody titer. Finally, all mice in the nanoparticle- intranasal and oral administered groups challenged with 100LD50 survived, while all control mice died before day 5. Based on these findings, we conclude that the PLGA-encapsulated IF protein has the potential to serve as a promising needle-free vaccine candidate against E. coli O157:H7.

In silico design, production and immunization evaluation of a recombinant bivalent fusion protein candidate vaccine against E. coli O157:H7.

In silico techniques are highly suited for both the discovery of new and development of available vaccines. Escherichia coli O157: H7, a main cause of food poisoning can infect humans through the consumption of contaminated water or food. Vaccination is a choice strategy to combat the bacterium. In the present study, we designed, expressed and purified a chimeric protein comprising two antigens of Escherichia coli O157: H7, including intimin and flagellin proteins, as a vaccine candidate and evaluated its immunization ability in mice. Thein silicoresults showed that the proposed antigen has a high antigenicity and conformation to be used as a potent vaccine candidate. The protein was successfully expressed in E. coli expression system with a proper level of expression (0/8g/L). Immunization evaluation showed that the protein is able to evoke the mice's humoral immunity and can confer a protective immunity against E. coli O157:H7, so that 80 % of the immunized animals were survived following the intraperitoneal injection of 100 LD50 of the live bacteria. Shedding analysis also showed the protectivity power of the protein. Bacterial excretion in control animals remained stable at about 108 CFU after 15 days, while the excreted bacteria in the feces of immunized mice's decreased to about 102 after the same time. According to the results, the proposed protein is able to stimulate the immune responses of mice and protect them against E. coli O157:H7.

Escherichia coli O157:H7 tir 255 T > A allele strains differ in chromosomal and plasmid composition.

Shiga toxin-producing Escherichia coli (STEC) O157:H7 strains with the T allele in the translocated intimin receptor polymorphism (tir) 255 A > T gene associate with human disease more than strains with an A allele; however, the allele is not thought to be the direct cause of this difference. We sequenced a diverse set of STEC O157:H7 strains (26% A allele, 74% T allele) to identify linked differences that might underlie disease association. The average chromosome and pO157 plasmid size and gene content were significantly greater within the tir 255 A allele strains. Eighteen coding sequences were unique to tir 255 A allele chromosomes, and three were unique to tir 255 T allele chromosomes. There also were non-pO157 plasmids that were unique to each tir 255 allele variant. The overall average number of prophages did not differ between tir 255 allele strains; however, there were different types between the strains. Genomic and mobile element variation linked to the tir 255 polymorphism may account for the increased frequency of the T allele isolates in human disease.

Nanobody-Based Bispecific Neutralizer for Shiga Toxin-Producing E. coli.

Currently, no specific therapeutics are available for foodborne Shiga toxin-producing Escherichia coli (STEC) infections that cause severe gastroenteritis and life-threatening complications of hemolytic uremic syndrome (HUS). As STEC attachment to intestinal epithelium might increase the host absorption of Shiga toxins and severity of the disease, we were inspired to develop a bispecific neutralizer capable of blocking its Shiga toxin and adhesin intimin simultaneously. Two nanobodies against the B subunit of Shiga toxin 2 (Stx2B) and the C terminus of Intimin (IntC280) were genetically fused together as the bispecific neutralizer, and it can be efficiently produced in a conventional E. coli expression system. We demonstrated that each of the nanobody modules in the bispecific format showed increased antigen binding capability and was able to functionally neutralize the binding of Stx2B or IntC280 to the respective host receptors even in the presence of the two virulence factors together. Moreover, the bispecific neutralizer was relatively stable to harsh storage conditions and gastrointestinal pH extremes. Taking into account its easy and economical production and superior pharmaceutical properties, we believe that a nanobody-based bispecific neutralizer would be more favorable and practical to be developed as a therapeutic to fight STEC in the developing world.

Loss of an Intimin-Like Protein Encoded on a Uropathogenic E. coli Pathogenicity Island Reduces Inflammation and Affects Interactions with the Urothelium.

Uropathogenic Escherichia coli (UPEC) causes the majority of uncomplicated urinary tract infections (UTI), which affect nearly half of women worldwide. Many UPEC strains carry an annotated intimin-like adhesin (ila) locus in their genome related to a well-characterized virulence factor in diarrheagenic E. coli pathotypes. Its role in UPEC uropathogenesis, however, remains unknown. In prototype UPEC strain CFT073, there is an ila locus that contains three predicted intimin-like genes, sinH, sinI, and ratA. We used in silico approaches to determine the phylogeny and genomic distribution of this locus among uropathogens. We found that the currently annotated intimin locus-encoded proteins in CFT073 are more closely related to invasin proteins found in Salmonella. Deletion of the individual sinH, sinI, and ratA genes did not result in measurable effects on growth, biofilm formation, or motility in vitro. On average, sinH was more highly expressed in clinical strains during active human UTI than in human urine ex vivo. Unexpectedly, we found that strains lacking this ila locus had increased adherence to bladder cells in vitro, coupled with a decrease in bladder cell invasion and death. The sinH mutant displayed a significant fitness defect in the murine model of ascending UTI, including reduced inflammation in the bladder. These data confirmed an inhibitory role in bladder cell adherence to facilitate invasion and inflammation; therefore, the ila locus should be termed invasin-like rather than intimin-like. Collectively, our data suggest that loss of this locus mediates measurable interactions with bladder cells in vitro and contributes to fitness during UTI.

Engineering Tropism of Pseudomonas putida toward Target Surfaces through Ectopic Display of Recombinant Nanobodies.

Gram-negative bacteria are endowed with complex outer membrane (OM) structures that allow them to both interact with other organisms and attach to different physical structures. However, the design of reliable bacterial coatings of solid surfaces is still a considerable challenge. In this work, we report that ectopic expression of a fibrinogen-specific nanobody on the envelope of Pseudomonas putida cells enables controllable formation of a bacterial monolayer strongly bound to an antigen-coated support. To this end, either the wild type or a surface-naked derivative of P. putida was engineered to express a hybrid between the ß-barrel of an intimin-type autotransporter inserted in the outer membrane and a nanobody (VHH) moiety that targets fibrinogen as its cognate interaction partner. The functionality of the thereby presented VHH and the strength of the resulting cell attachment to a solid surface covered with the cognate antigen were tested and parametrized with Quartz Crystal Microbalance technology. The results not only demonstrated the value of using bacteria with reduced OM complexity for efficient display of artificial adhesins, but also the potential of this approach to engineer specific bacterial coverings of predetermined target surfaces.

Folding Control in the Path of Type 5 Secretion.

The type 5 secretion system (T5SS) is one of the more widespread secretion systems in Gram-negative bacteria. Proteins secreted by the T5SS are functionally diverse (toxins, adhesins, enzymes) and include numerous virulence factors. Mechanistically, the T5SS has long been considered the simplest of secretion systems, due to the paucity of proteins required for its functioning. Still, despite more than two decades of study, the exact process by which T5SS substrates attain their final destination and correct conformation is not totally deciphered. Moreover, the recent addition of new sub-families to the T5SS raises additional questions about this secretion mechanism. Central to the understanding of type 5 secretion is the question of protein folding, which needs to be carefully controlled in each of the bacterial cell compartments these proteins cross. Here, the biogenesis of proteins secreted by the Type 5 secretion system is discussed, with a focus on the various factors preventing or promoting protein folding during biogenesis.

Identification of Nanobodies Blocking Intimate Adherence of Shiga Toxin-Producing Escherichia coli to Epithelial Cells.

Therapeutic antibodies (Abs) inhibiting bacterial adhesion to host epithelia are an attractive option to reduce the load of Shiga toxin-producing E. coli (STEC) in the intestine of the patient and also in the bovine reservoir, thereby minimizing the risk of STEC contamination in the food chain. Of particular interest are recombinant single-domain Ab fragments called nanobodies (Nbs) derived from the variable domain of camelid heavy chain-only antibodies (VHH). The outer membrane adhesin intimin and the translocated intimin receptor (Tir) are essential for the attachment of STEC to host epithelia. In addition, EspA filaments of the bacterial type III protein secretion system are needed for Tir translocation into the host cell. Given their importance for bacterial adhesion and colonization, we developed Nbs against intimin, Tir and EspA proteins of STEC serotype O157:H7. Here, we report the screening methods used to isolate inhibitory Nbs blocking intimin-Tir protein-protein interaction, actin-pedestal formation, and intimate adhesion of STEC to epithelial cells in vitro. First, we describe how VHH gene repertoires can be produced as Nbs secreted by E. coli using the α-hemolysin (HlyA) protein secretion system. Next, we report the methods for identification of inhibitors of intimin-Tir protein-protein interaction and of STEC intimate adhesion to HeLa cells in culture. These methods can be adapted for the screening of Nbs against different adhesin-receptor complexes to block the adhesion of other pathogens to host cells.

Molecular characteristics of eae-positive clinical Shiga toxin-producing Escherichia coli in Sweden.

Shiga toxin (Stx)-producing Escherichia coli (STEC) can cause a wide range of symptoms from asymptomatic carriage, mild diarrhea to bloody diarrhea (BD) and hemolytic uremic syndrome (HUS). Intimin, encoded by the eae gene, also plays a critical role in STEC pathogenesis. Herein, we investigated the prevalence and genetic diversity of eae among clinical STEC isolates from patients with diarrhea, BD, HUS as well as from asymptomatic STEC-positive individuals in Sweden with whole-genome sequencing. We found that 173 out of 239 (72.4%) of clinical STEC strains were eae positive. Six eae subtypes (ϵ1, γ1, ß3, θ, ζ and ρ) were identified eae and its subtype γ1 were significantly overrepresented in O157:H7 strains isolated from BD and HUS patients. ϵ1 was associated with O121:H19 and O103:H2 strains, and ß3 to O26:H11 strains. The combination of eae subtype γ1 and stx subtype (stx 2 or stx 1+stx 2) is more likely to cause severe disease, suggesting the possibility of using eae genotypes in risk assessment of STEC infection. In summary, this study demonstrated a high prevalence of eae in clinical STEC strains and considerable genetic diversity of eae in STEC strains in Sweden from 1994 through 2018, and revealed association between eae subtypes and disease severity.

Heterologous expression of Intimin and IpaB fusion protein in Lactococcus lactis and its mucosal delivery elicit protection against pathogenicity of Escherichia coli O157 and Shigella flexneri in a murine model.

Escherichia coli O157:H7 and Shigella flexneri are the predominant diarrhoeal pathogens and those strains producing Shiga toxins cause life-threatening sequelae including hemolytic uremic syndrome (HUS) upon their entry into the host. Intimate adherence of E. coli O157 and invasion of S. flexneri in the host intestinal epithelial cells is mainly mediated by Intimin and IpaB proteins, respectively. In this study, we have synthesized chimera of immunodominant regions of Intimin (eae) and IpaB (ipaB) designated as EI and expressed it in Lactococcus lactis (LL-EI) to develop a combinatorial oral vaccine candidate. Immune parameters and protective efficacy of orally administered LL-EI were assessed in the murine model. Significant EI-specific serum IgG, IgA, and fecal IgA antibody titer were observed in the LL-EI group. Considerable increase in EI-specific splenocyte proliferation and a concurrent upregulation of both Th1 and Th2 cytokines was observed in LL-EI immunized mice. Flow cytometry analysis also revealed a significant increase in CD4 and CD8 cell counts in LL-EI immunized group compared to PBS, LL control group.In vitro studies using LL-EI immunized mice sera showed substantial protection against bacterial adhesion and invasion caused by E. coli O157 and Shigella flexneri¸ respectively. LL-EI immunized group challenged with E. coli O157 ceased fecal shedding within 6 days, and mice challenged with S. flexneri showed 93% survival with minimal bacterial load in the lungs. Our results indicate that LL-EI immunization elicits systemic, mucosal and cell-mediated immune responses, and can be a promising candidate for oral vaccine development against these pathogens.

Detection of Shiga toxin-producing Escherichia coli (STEC) in beef products using droplet digital PCR.

Many of the current accredited methods for the molecular detection of Shiga toxin-producing Escherichia coli (STEC) in foods rely on a PCR-based screen for the pathotype-specific genetic markers stx and eae. Unfortunately, these methods can inaccurately conclude the presence of E.coli containing both stx and eae because of the inability of the methods to determine if the two genes originated from a single organism as opposed to a mixture of organisms. This study was undertaken to evaluate if a droplet digital PCR (ddPCR)-based method that does not require DNA isolation could reliably identify the presence of an STEC containing eae in beef samples by confirming that both genes reside within the same cell, even when present in a mixed culture. The ddPCR system used in this study, dd-Check STEC Solution (Bio-Rad), works without the need for DNA isolation by partitioning intact cells into emulsion droplets, where they are lysed, and subsequently undergo multiplexed endpoint PCR. This enables the assay to differentiate between samples where a single organism contains both stx and eae from samples in which stx and eae reside in different organisms. Comparisons were made between the dd-Check STEC Solution, the BAX System Real-Time PCR STEC assay suite (Hygiena), and the iQ-Check STEC PCR detection kit (Bio-Rad) using 37 unique simulations of E. coli contamination in ground beef. While no single platform was consistently superior at detecting eae and stx across all pathogens tested, the results indicated that the dd-Check STEC Solution has the potential to reduce the number of inaccurately identified samples when screening for E. coli with a stx+, eae+ genotype because it can identify the co-existence of multiple virulence genes within a cell even when in the presence of a mixed microbial population containing identical genes. Ultimately, incorporation of this system could result in substantial cost savings by reducing the expenses incurred when product samples are incorrectly classified as containing E. coli with a stx+, eae+ genotype.

Macrophage inflammatory response mediated by intimin and bundle-Forming pilus from Enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) are important agents of acute diarrhea in children living in developing countries. A severe dysfunction of the intestinal epithelial barrier occurs during EPEC infection, leading to diarrhea and inflammation as consequences. EPEC main virulence factors include the adhesins intimin and bundle-forming pilus (BFP), as well as several effector proteins translocated to the enterocyte by the type-three secretion system. The initial interaction of EPEC with the host cell and the role of effector proteins in this process are well known. However, the role of the EPEC virulence factors in macrophage activation is not fully understood. Hence, we analyzed the ability of intimin and bundle-forming pilus (BfpA) to activate the innate response mediated by macrophages, where the production of the proinflammatory cytokines TNF-α, IL-1, IL-6 and IL-12, as well as the anti-inflammatory cytokine IL-10 and chemokine MCP-1, were evaluated. Our results showed that recombinant intimin and BfpA activate macrophages in a dose-dependent manner, and the stimulated cells produced TNF-α, IL-12, IL-6, IL-10 and MCP-1, but not IL-1β. No synergistic effect was observed in the production of pro-inflammatory cytokines by combining BfpA and intimin, although production of IL-10, an anti-inflammatory mediator, was potentiated at a higher dose. The effect observed was largely attributed to these proteins, as the treatment of proteins with polymyxin B did not alter the production of TNF-α. Thus, herein we showed that intimin and BfpA can activate the innate immune response, inducing the production of pro- and anti-inflammatory cytokines, as well as chemokines, playing additional role as inflammatory molecules in the early steps of EPEC infection.

Antimicrobial resistance genes and virulence gene encoding intimin in Escherichia coli and Enterococcus isolated from wild rabbits (Oryctolagus cuniculus) in Tunisia.

The spread of antimicrobial-resistant bacteria in wildlife must be viewed as a major concern with serious implications for human and animal health. Escherichia coli and enterococcal isolates were recovered from faecal samples of 49 wild rabbits (Oryctolagus cuniculus) on specific media and were characterised using biochemical and molecular tests. For all isolates, antimicrobial susceptibility testing was performed, and resistance genes were detected by PCR. Molecular typing of isolates was carried out by pulsed-field gel-electrophoresis, and E. coli strains were also tested for the presence of intimin (eae) gene characteristic of rabbit enteropathogenic E. coli. A total of 34 E. coli and 36 enterococci [E. hirae (52.8%) and E. faecalis (47.2%)] were obtained. For E. coli, resistance to tetracycline (94%), streptomycin (62%), ciprofloxacin (47%), trimethoprim-sulphamethoxazole (35%) and chloramphenicol (6%) was observed. Resistance to third-generation cephalosporins was detected in one E. coli strain that carried the blaCMY-2 and blaTEM-1 genes. Class 1 integrons were detected in eight isolates. For enterococci, resistance to tetracycline (63.9%), erythromycin (30.5%), streptomycin (18.2%), and chloramphenicol (5.5%) was detected. The tet(M)+tet(L), erm(B) and ant (6)-Ia genes were identified in thirteen, seven and three resistant Enterococcus strains, respectively. Molecular typing showed a high diversity among our strains. Wild rabbits could represent a reservoir of E. coli, and enterococci carrying antimicrobial resistance genes and E. coli additionally carrying the eae gene of enteropathogenic pathotypes could both contaminate the environment. our finding seems to represent the first report of eae-positive E. coli in wild rabbits.

Prevention of EHEC infection by chitosan nano-structure coupled with synthetic recombinant antigen.

One of highly effective methods for prevention and control of Entrohemorragic Esherichia coli (EHEC) infections is to use vaccination against extremely immunogenic part of attachment factors. In this study rEIT (EspA, Intimin, Tir) was produced in bacteria and then encapsulated with chitosan nanoparticle as a candidate nanovaccine. A chimeric trivalent recombinant protein which was previously found to provide reasonable immunogenicity against E.coli O157:H7 was used as a base. Mice immunized orally with chitosan based nanoparticle containing rEIT antigen. The rEIT-specific immune responses (IgG and IgA) were measured by indirect ELISA. In challenging tests different groups of immunized mice were infected orally with E.coli O157:H7. The results showed that the recombinant nanovaccine candidate could induce the strong humoral and mucosal immune responses and protect the mice from live EHEC O157:H7 challenge. Higher titers of serum anti rEIT IgG were achieved after the last immunization in all of the groups. Comparison of the amount of IgA titers in serum and feces showed higher values for the latter. In vitro study of binding inhibition assay on Caco-2 cell monolayers by pre-incubated antisera with EHEC bacteria, showed that immunized mice antibody could reduce adhesion properties of E. coli O157:H7. In a challenging study with EHEC bacteria, reduction in number of colonies was observed in all of the immunized groups for over two weeks. Results from the present study prove that nanovaccine candidate with rEIT can reduce signs and symptoms of EHEC infections. This novel approach can be a new strategy for inducing immunity against E. coli O157:H7. This study suggests the use of oral -injection combined vaccination routes comparing to other methods available in order to achieve higher humoral and mucosal immunogenicity levels.