Construction of the ETECFinder database for the characterization of enterotoxigenic Escherichia coli (ETEC) and revision of the VirulenceFinder web tool at the CGE website.

The identification of pathogens is essential for effective surveillance and outbreak detection, which lately has been facilitated by the decreasing cost of whole-genome sequencing (WGS). However, extracting relevant virulence genes from WGS data remains a challenge. In this study, we developed a web-based tool to predict virulence-associated genes in enterotoxigenic Escherichia coli (ETEC), which is a major concern for human and animal health. The database includes genes encoding the heat-labile toxin (LT) (eltA and eltB), heat-stable toxin (ST) (est), colonization factors CS1 through 30, F4, F5, F6, F17, F18, and F41, as well as toxigenic invasion and adherence loci (tia, tibAC, etpBAC, eatA, yghJ, and tleA). To construct the database, we revised the existing ETEC nomenclature and used the VirulenceFinder webtool at the CGE website [VirulenceFinder 2.0 (dtu.dk)]. The database was tested on 1,083 preassembled ETEC genomes, two BioProjects (PRJNA421191 with 305 and PRJNA416134 with 134 sequences), and the ETEC reference genome H10407. In total, 455 new virulence gene alleles were added, 50 alleles were replaced or renamed, and two were removed. Overall, our tool has the potential to greatly facilitate ETEC identification and improve the accuracy of WGS analysis. It can also help identify potential new virulence genes in ETEC. The revised nomenclature and expanded gene repertoire provide a better understanding of the genetic diversity of ETEC. Additionally, the user-friendly interface makes it accessible to users with limited bioinformatics experience. IMPORTANCE: Detecting colonization factors in enterotoxigenic Escherichia coli (ETEC) is challenging due to their large number, heterogeneity, and lack of standardized tests. Therefore, it is important to include these ETEC-related genes in a more comprehensive VirulenceFinder database in order to obtain a more complete coverage of the virulence gene repertoire of pathogenic types of E. coli. ETEC vaccines are of great importance due to the severity of the infections, primarily in children. A tool such as this could assist in the surveillance of ETEC in order to determine the prevalence of relevant types in different parts of the world, allowing vaccine developers to target the most prevalent types and, thus, a more effective vaccine.

Carrier state of enterotoxigenic Escherichia coli virulence markers in pigs: Effects on gut microbiota modulation and immune markers transcription.

Enterotoxigenic Escherichia coli (ETEC) causes diarrhea in pigs at early age, leading to high mortality rates and significant economic losses in the swine industry. ETEC effect on gut microbiota and immune system is mostly studied in diarrheic model under controlled laboratory conditions, however its impact on asymptomatic carriers remains unknown. Thus, we investigated whether ETEC can modulate gut microbiota or regulate the transcription of immune markers in asymptomatic pigs in farm environment. Stool samples from newborn piglets, nursery and growing pigs, and sows were screened for ETEC markers, then submitted to 16S-rDNA sequencing to explore gut microbiota composition in carriers (ETEC+) and non-carriers (ETEC-) animals. We observed a reduced α-diversity in ETEC+ animals (p < 0.05), while bacterial compositions were mostly driven by ageing (p > 0.05). Prevotella marked ETEC-carrier group, while Rikenellaceae RC9 gut group was a marker for a healthy gut microbiota, suggesting that they might be biomarker candidates for surveillance and supplementation purposes. Furthermore, we observed transcription regulation of il6 and tff2 genes in ETEC+ in newborn and nursery stages, respectively. Our findings indicate that ETEC presence modulate gut microbiota and the immune response in asymptomatic pigs; nevertheless, further studies using a probabilistic design must be performed to assess the effect of ETEC presence on gut imbalance in pigs despite the age bias.

Impact of an alpha helix and a cysteine-cysteine disulfide bond on the resistance of bacterial adhesion pili to stress.

Escherichia coli express adhesion pili that mediate attachment to host cell surfaces and are exposed to body fluids in the urinary and gastrointestinal tracts. Pilin subunits are organized into helical polymers, with a tip adhesin for specific host binding. Pili can elastically unwind when exposed to fluid flow forces, reducing the adhesin load, thereby facilitating sustained attachment. Here we investigate biophysical and structural differences of pili commonly expressed on bacteria that inhabit the urinary and intestinal tracts. Optical tweezers measurements reveal that class 1a pili of uropathogenic E. coli (UPEC), as well as class 1b of enterotoxigenic E. coli (ETEC), undergo an additional conformational change beyond pilus unwinding, providing significantly more elasticity to their structure than ETEC class 5 pili. Examining structural and steered molecular dynamics simulation data, we find that this difference in class 1 pili subunit behavior originates from an α-helical motif that can unfold when exposed to force. A disulfide bond cross-linking ß-strands in class 1 pili stabilizes subunits, allowing them to tolerate higher forces than class 5 pili that lack this covalent bond. We suggest that these extra contributions to pilus resiliency are relevant for the UPEC niche, since resident bacteria are exposed to stronger, more transient drag forces compared to those experienced by ETEC bacteria in the mucosa of the intestinal tract. Interestingly, class 1b ETEC pili include the same structural features seen in UPEC pili, while requiring lower unwinding forces that are more similar to those of class 5 ETEC pili.

Tannic acid alleviates ETEC K88-induced intestinal damage through regulating the p62-keap1-Nrf2 and TLR4-NF-κB-NLRP3 pathway in IPEC-J2 cells.

BACKGROUND: Tannic acid (TA), a naturally occurring polyphenol, has shown diverse potential in preventing intestinal damage in piglet diarrhea induced by Enterotoxigenic Escherichia coli (ETEC) K88. However, the protective effect of TA on ETEC k88 infection-induced post-weaning diarrhea and its potential mechanism has not been well elucidated. Therefore, an animal trial was carried out to investigate the effects of dietary supplementation with TA on the intestinal diarrhea of weaned piglets challenged with ETEC K88. In addition, porcine intestinal epithelial cells were used as an in vitro model to explore the mechanism through which TA alleviates intestinal oxidative damage and inflammation. RESULTS: The results indicated that TA supplementation (2 and 4 g kg-1) reduced diarrhea rate, enzyme activity (diamine oxidase [DAO] and Malondialdehyde [MAD]) and serum inflammatory cytokines concentration (TNF-α and IL-1ß) (P < 0.05) compared to the Infection group (IG), group in vivo. In vitro, TA treatment effectively alleviated ETEC-induced cytotoxicity, increased the expression of ZO-1, occludin and claudin-1 at both mRNA and protein levels. Moreover, TA pre-treatment increased the activity of antioxidant enzymes (such as T-SOD) and decreased serum cytokine levels (TNF-α and IL-1ß). Furthermore, TA increased cellular antioxidant capacity by activating the Nrf2 signaling pathway and decreased inflammatory response by down-regulating the expression of TLR4, MyD88, NF-kB and NLRP3. CONCLUSION: The present study showed that TA reduced the diarrhea rate of weaned piglets by restoring the intestinal mucosal mechanical barrier function, alleviating oxidative stress and inflammation. The underlying mechanism was achieved by modulating the p62-keap1-Nrf2 and TLR4-NF-κB-NLRP3 pathway. © 2024 Society of Chemical Industry.

[Molecular characterization of diarrheagenic Escherichia coli from an outpatient pediatric population with diarrhea attended in two hospitals from Buenos Aires, Argentina]. / Caracterización molecular de Escherichia coli diarreogénica proveniente de población pediátrica ambulatoria con diarrea, atendida en dos hospitales de Buenos Aires, Argentina.

Diarrheagenic Escherichia coli comprises a heterogeneous group of pathotypes or pathogenic variants that share phenotypic characteristics with marked differences in virulence genes, colonization sites, pathogenesis, clinical presentation, and epidemiology of infection. The most studied pathotypes are Shiga toxin-producing E.coli (STEC), enterotoxigenic E.coli (ETEC), enteropathogenic E.coli (EPEC), enteroaggregative E.coli (EAEC), and enteroinvasive E.coli (EIEC). The objective of the study was to characterize the isolates of diarrheagenic E.coli from an outpatient pediatric population with diarrhea attended in two public hospitals from Buenos Aires, Argentina. Diarrheagenic E.coli pathotypes were investigated by amplifying characteristic virulence gene fragments: intimin (eae), heat-labile toxin (lt), heat-stable toxins (stp, sth), invasion plasmid antigen H (ipaH), transcriptional activator R (aggR) and Shiga toxins (stx1, stx2). Molecular subtyping of isolates was performed using PFGE (XbaI). Diarrheagenic E.coli was detected in 14% (84/601) of cases. The EAEC pathotype was prevalent, while ETEC, STEC, EPEC and EIEC were found in a lower proportion. EAEC isolates exhibited a high degree of genetic diversity. All pathotypes were found in children under 5years of age, while only EAEC, EIEC and ETEC were detected in the older population. Future studies that include the characterization of isolates from a greater number of genes and populations from other geographical areas will be necessary to determine the relevance of diarrheagenic E.coli in Argentina.

Comprehensive analysis of circular RNAs in porcine small intestine epithelial cells associated with susceptibility to Escherichia coli F4ac diarrhea.

BACKGROUND: Diarrhea is one of the most common diseases in pig industry, which seriously threatens the health of piglets and causes huge economic losses. Enterotoxigenic Escherichia coli (ETEC) F4 is regarded as the most important cause of diarrhea in piglets. Some pigs are naturally resistant to those diarrheas caused by ETEC-F4, because they have no F4 receptors (F4R) on their small intestine epithelial cells that allow F4 fimbriae adhesion. Circular RNA (circRNA) has been shown to play an important regulatory role in the pathogenesis of disease. We hypothesized that circRNAs may also regulate the adhesion of piglet small intestinal epithelial cells to ETEC F4 fimbriae. However, the circRNA expression profiles of piglets with different Enterotoxigenic Escherichia coli F4 fimbriae (ETEC-F4ac) adhesion phenotypes are still unclear, and the intermediate regulatory mechanisms need to be explored. Hence, the present study assessed the circRNA expression profiling in small intestine epithelial cells of eight male piglets with different ETEC-F4 adhesion phenotypes and ITGB5 genotypes to unravel their regulatory function in susceptibility to ETEC-F4ac diarrhea. Piglets were divided into two groups: non-adhesive group (n = 4) with CC genotype and adhesive group (n = 4) with TT genotype. RESULTS: The RNA-seq data analysis identified 13,199 circRNAs from eight samples, most of which were exon-derived. In the small intestine epithelial cells, 305 were differentially expressed (DE) circRNAs between the adhesive and non-adhesive groups; of which 46 circRNAs were upregulated, and 259 were downregulated. Gene ontology and KEGG enrichment analysis revealed that most significantly enriched DE circRNAs' host genes were linked to cytoskeletal components, protein phosphorylation, cell adhesion, ion transport and pathways (such as adherens junction, gap junction) associated with ETEC diarrhea. The circRNA-miRNA-mRNA interaction network was also constructed to elucidate their underlying regulatory relationships. Our results identified several candidate circRNAs that affects susceptibility to ETEC diarrhea. Among them, circ-SORBS1 can adsorb ssc-miR-345-3p to regulate the expression of its host gene SORBS1, thus improving cell adhesion. CONCLUSION: Our results provided insights into the regulation function of circRNAs in susceptibility to ETEC diarrhea of piglets, and enhanced our understanding of the role of circRNAs in regulating ETEC diarrhea, and reveal the great potential of circRNA as a diagnostic marker for susceptibility of ETEC diarrhea in piglets.

Polyvalent Protein Adhesin MEFA-II Induces Functional Antibodies against Enterotoxigenic Escherichia coli (ETEC) Adhesins CS7, CS12, CS14, CS17, and CS21 and Heat-Stable Toxin (STa).

There are no licensed vaccines for enterotoxigenic Escherichia coli (ETEC), a common cause of children's diarrhea and travelers' diarrhea. ETEC strains producing enterotoxins (heat-labile toxin, LT; heat-stable toxin, STa) and adhesins CFA/I, CFA/II (CS1-CS3) or CFA/IV (CS4-CS6) attributed to a majority of ETEC-associated diarrheal cases, thus the two toxins (STa, LT) and the seven adhesins (CFA/I, CS1 to CS6) are historically the primary targets in ETEC vaccine development. Recent studies, however, revealed that ETEC strains with adhesins CS14, CS21, CS7, CS17, and CS12 are also prevalent and cause moderate-to-severe diarrhea; these adhesins are now considered antigen targets as well for ETEC vaccines. In this study, we applied the epitope- and structure-based multiepitope-fusion-antigen (MEFA) vaccinology platform and constructed a polyvalent protein to present immuno-dominant continuous B-cell epitopes of these five adhesins (also an STa toxoid); we then characterized this protein antigen's (termed as adhesin MEFA-II) broad immunogenicity and evaluated antibody functions against each targeted adhesin and STa toxin. Data showed that mice intramuscularly immunized with adhesin MEFA-II protein developed robust IgG to the targeted adhesins and toxin STa. Importantly, the antigen-derived antibodies significantly inhibited adherence of ETEC bacteria expressing adhesin CS7, CS12, CS14, CS17, or CS21 and reduced STa enterotoxicity. These results indicated that adhesin MEFA-II protein is broadly immunogenic and induces cross-functional antibodies, suggesting adhesin MEFA-II can be an effective ETEC vaccine antigen; if included in an ETEC vaccine candidate, adhesin MEFA-II can expand vaccine coverage and increase efficacy against ETEC-associated children's diarrhea and travelers' diarrhea. IMPORTANCE An effective vaccine is lacking against ETEC, a primary cause of children's diarrhea and traveler's diarrhea and a threat to global health. The key challenge in ETEC vaccine development is that ETEC bacteria express heterogeneous virulence determinants (>25 adhesins and two toxins). While the current strategy to target the seven most prevalent ETEC adhesins (CFA/I, CS1 to CS6) potentially lead to a vaccine against many clinical cases, the prevalence of ETEC strains shifts chronically and geographically, and ETEC expressing other adhesins, mainly CS7, CS12, CS14, CS17, and CS21, also cause moderate-to-severe diarrhea. However, it is impossible to develop an ETEC vaccine to target as many as 12 adhesins under conventional approaches. This study used a unique vaccinology platform to create a polyvalent antigen and demonstrated the antigen's broad immunogenicity and functions against the targeted ETEC adhesins, enabling the development of a broadly protective vaccine essentially against all of the important ETEC strains.

Structural and functional characterization of colonization factors AIBI-CS6 and AIIBII-CS6 of enterotoxigenic Escherichia coli.

Over time, the structure and function of the broadly dispersed colonization factor (CF) CS6 of enterotoxigenic Escherichia coli (ETEC) have become more significant. CS6 is composed of tightly-associated subunits, CssA and CssB which due to presence of natural point mutation gave rise to CS6 subtypes. In contrast to the other ETEC CFs, CS6 is an afimbrial, spherical-shaped oligomers of (CssA-CssB)n complex where 'n' is concentration dependent. In this study, we have compared AIBI-CS6 and AIIBII-CS6 structurally and functionally. The Mw of CssAI was 18.5 kDa but Mw of CssAII was 15.1 kDa. Both CssBI and CssBII had Mw of 15.9 kDa. The substitution of Gly39 with Ala39 in CssAI leads to reduction in Mw from 18.5 to 15.1 kDa. Due to higher Mw of CssAI, the size of AIBI concentration-dependent oligomers should be higher. However, the Mw of AIIBII oligomers were higher and AIIBII also showed higher oligomeric forms compared to AIBI both in native PAGE and electron microscopy. The oligomers of both subtypes could withstand greater temperatures and denaturant concentrations. In terms of cellular response, the levels of inflammatory cytokines were significantly higher in case of AIBI-CS6 expressing ETEC as compared to AIIBII-CS6 expressing ETEC both in vitro and in vivo. When inflammatory cytokines were evaluated after infecting suckling mice with these ETEC strains, the results were consistent. In conclusion, even though there was subtle structural difference between AIBI-CS6 and AIIBII-CS6 due to natural point mutations but ETEC strains expressing these subtypes displayed great variability in pathogenicity.

Effect of phage vB_EcoM_FJ1 on the reduction of ETEC O9:H9 infection in a neonatal pig cell line.

Enterotoxigenic Escherichia coli (ETEC) colonizes the intestine of young pigs causing severe diarrhoea and consequently bringing high production costs. The rise of antibiotic selective pressure together with ongoing limitations on their use, demands new strategies to tackle this pathology. The pertinence of using bacteriophages as an alternative is being explored, and in this work, the efficacy of phage vB_EcoM_FJ1 (FJ1) in reducing the load of ETEC EC43-Ph (serotype O9:H9 expressing the enterotoxin STa and two adhesins F5 and F41) was assessed. Foreseeing the oral application on piglets, FJ1 was encapsulated on calcium carbonate and alginate microparticles, thus preventing phage release under adverse conditions of the simulated gastric fluid (pH 3.0) and allowing phage availability in simulated intestinal fluid (pH 6.5). A single dose of encapsulated FJ1, provided to IPEC-1 cultured cells (from intestinal epithelium of piglets) previously infected by EC43, provided bacterial reductions of about 99.9% after 6 h. Although bacteriophage-insensitive mutants (BIMs) have emerged from treatment, the consequent fitness costs associated with this new phenotype were demonstrated, comparatively to the originating strain. The higher competence of the pig complement system to decrease BIMs' viability, the lower level of colonization of IPEC-1 cells observed with these mutants, and the increased survival rates and health index recorded in infected Galleria mellonella larvae supported this observation. Most of all, FJ1 established a proof-of-concept of the efficiency of phages to fight against ETEC in piglet intestinal cells.

Protective effects of anti-CfaB-EtpA-LTB IgY antibody against adherence and toxicity of enterotoxigenic Escherichia coli (ETEC).

AIM: Production of IgY antibodies against CfaB-EtpA-LTB (CEL) chimeric protein and evaluation of its protective effects against enterotoxigenic Escherichia coli (ETEC) by in vivo and in vitro investigation. METHODS AND RESULTS: Indirect ELISA and immunoblotting methods were applied to assess the immunogenicity and specificity of IgYs and also to evaluate the efficacy of IgYs in binding prevention and neutralizing the heat-labile (LT) toxin of ETEC bacteria. The results indicated that the anti-CEL IgY at a concentration of 2 mg ml-1 could decrease the bacterial adhesion to HT-29 cells by 74% compared to the control group.At a concentration of 750 µg ml-1, the IgY antibody managed to neutralize the disruptive LT toxin effect on the Y1 cell line. At a concentration of 2 mg ml-1, 81% reduction was observed in the fluid accumulation in the ileal loop assay. CONCLUSION: According to our findings, passive immunotherapy with anti-CEL IgY can prevent bacterial colonization and toxicity, thus facilitating in controlling the enteric diseases caused by ETEC infection.

Effects of Catecholamine Stress Hormones Norepinephrine and Epinephrine on Growth, Antimicrobial Susceptibility, Biofilm Formation, and Gene Expressions of Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is a significant contributor to diarrhea. To determine whether ETEC-catecholamine hormone interactions contribute to the development of diarrhea, we tested the effects of catecholamine hormones acting on ETEC in vitro. The results showed that in the presence of norepinephrine (NE) and epinephrine (Epi), the growth of 9 out of 10 ETEC isolates was promoted, the MICs of more than 60% of the isolates to 6 antibiotics significantly increased, and the biofilm formation ability of 10 ETEC isolates was also promoted. In addition, NE and Epi also significantly upregulated the expression of the virulence genes feaG, estA, estB, and elt. Transcriptome analysis revealed that the expression of 290 genes was affected by NE. These data demonstrated that catecholamine hormones may augment the diarrhea caused by ETEC.

Effects of mixed fibres and essential oils blend on growth performance and intestinal barrier function of piglets challenged with enterotoxigenic Escherichia coli K88.

This study was to evaluate the effects of supplementing mixed dietary fibres (MDF) and essential oils blend (EOB) either alone or in combination on growth performance and intestinal barrier function in weaned piglets challenged with enterotoxigenic Escherichia coli K88 (ETEC). Forty-two piglets (28 days old) were randomly allocated into six treatments in a 25-day experiment, and fed the basal diet (CON or ETEC) either with antibiotics (AT), MDF, EOB or MDF + EOB. On Day 22 of the experiment, pigs in CON and challenged groups (ETEC, AT, MDF, EOB and MDF + EOB) were orally administered sterile saline and ETEC containing 6 × 1010 CFU/kg body weight respectively. On Day 26, all pigs were euthanized to collect samples. Before ETEC challenge, piglets in MDF and EOB had lower diarrhoea incidence (p < 0.01) than others. After ETEC challenge, piglets in ETEC had lower average daily gain and higher diarrhoea incidence (p < 0.05) than those of CON. Furthermore, compared to CON, ETEC group increased the serum lipopolysaccharide concentration and diamine oxidase activity, and decreased mRNA levels of genes relating to barrier function (aquaporin 3, AQP3; mucin1, MUC1; zonula occludens-1, ZO-1; Occludin), and increased the concentration of cytokines (interleukin-1ß/4/6/10, IL-1ß/4/6/10) and secretory immunoglobulin A (sIgA) in jejunal mucosa (p < 0.05). However, these deleterious effects induced by ETEC were partly alleviated by MDF, EOB, MDF + EOB and AT. Additionally, compared to ETEC group, MDF increased Bifidobacterium abundance in cecal digesta and butyrate concentration in colonic digesta (p < 0.05). Also, EOB improved propionate concentration in cecal digesta, and MDF + EOB decreased IL-10 concentration in jejunal mucosa (p < 0.05) compared with ETEC. Conclusively, MDF and EOB either alone or in combination can improve growth performance and alleviate diarrhoea via improving intestinal barrier function of piglets after ETEC challenge, and all may serve as potential alternatives to AT for piglets.

Enterotoxigenic Escherichia coli Enterotoxins Regulate Epithelial to Immune Relay of IL-33 and IL-1Ra Cytokines.

Enterotoxigenic Escherichia coli (ETEC) remain a major cause of diarrheal mortality and morbidity in children in low-resource settings. Few studies have explored the consequences of simultaneous intoxication with heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT) despite the increased prevalence of wild ETEC isolates expressing both toxins. We therefore used a combination of tissue culture and murine models to explore the impact of simultaneous ST + LT intoxication on epithelial and myeloid cells. We report that LT induces sustained production of interleukin 33 (IL-33) and interleukin 1 receptor antagonist (IL-1Ra) in T84 intestinal epithelial cells via cAMP production and protein kinase A activation. We demonstrate that combined ST + LT intoxication hastens epithelial transcriptional responses induced more slowly by LT alone. ST- and LT-mediated luminal fluid accumulation in vivo correlates with significant increases in IL-33 and IL-1Ra in small intestinal mucosal scrapings. Additionally, IL-33 receptor (IL-33R)-deficient mice are significantly less susceptible to ST-mediated secretion than wildtype mice. In the immune compartment, IL-33 is sensed by myeloid cells, and LT suppresses IL-33-induced tumor necrosis factor α (TNF-α) secretion from macrophages and bone marrow-derived dendritic cells (BMDCs) but amplifies IL-33-mediated induction of IL-6 from BMDCs. In conclusion, our studies suggest that enterotoxin-induced IL-33 and IL-1Ra modulate intestinal inflammation and IL-1 receptor signaling in the intestinal mucosa in response to ETEC enterotoxins.

Wild Boars as Reservoir of Highly Virulent Clone of Hybrid Shiga Toxigenic and Enterotoxigenic Escherichia coli Responsible for Edema Disease, France.

Edema disease is an often fatal enterotoxemia caused by specific strains of Shiga toxin-producing Escherichia coli (STEC) that affect primarily healthy, rapidly growing nursery pigs. Recently, outbreaks of edema disease have also emerged in France in wild boars. Analysis of STEC strains isolated from wild boars during 2013-2019 showed that they belonged to the serotype O139:H1 and were positive for both Stx2e and F18 fimbriae. However, in contrast to classical STEC O139:H1 strains circulating in pigs, they also possessed enterotoxin genes sta1 and stb, typical of enterotoxigenic E. coli. In addition, the strains contained a unique accessory genome composition and did not harbor antimicrobial-resistance genes, in contrast to domestic pig isolates. These data thus reveal that the emergence of edema disease in wild boars was caused by atypical hybrid of STEC and enterotoxigenic E. coli O139:H1, which so far has been restricted to the wildlife environment.

Genetic Characteristics of the Transmissible Locus of Stress Tolerance (tLST) and tLST Harboring Escherichia coli as Revealed by Large-Scale Genomic Analysis.

The transmissible locus of stress tolerance (tLST) confers resistance to multiple stresses in E. coli. Utilizing 18,959 E. coli genomes available in the NCBI database, we investigated the prevalence, phylogenetic distribution, and configuration patterns of tLST, and correlations between tLST, and virulence and antimicrobial resistance (AMR) genes in E. coli. Four tLST variants were found in 2.7% of E. coli, with the most prevalent (77.1%) variant being tLST1 followed by tLST2 (8.3%), tLST3b (8.3%) and tLST3a (6.3%). The majority (93%) of those tLST were in E. coli belonging to phylogroup A in which the prevalence was 10.4%. tLST was also found in phylogroup B1 (0.5%) and C (0.5%) but not found in B2 or D-G. An additional 1% of the 18,959 E. coli genomes harbored tLST fragments to various extent. Phylogenetic analysis revealed both intra- and interspecies transmission of both chromosomal and plasmid-borne tLST, with E. coli showing a preference of chromosomal over plasmid-borne tLST. The presence of tLST and virulence genes in E. coli was overall negatively correlated, but tLST was found in all genomes of a subgroup of enterotoxigenic E. coli (ST2332). Of note, no Shiga toxin-producing E. coli (n = 3,492) harbored tLST. The prevalence of tLST and AMR genes showed different temporal trends over the period 1985 to 2019. However, a substantial fraction of tLST positive E. coli harbor AMR genes, posing a threat to public health. In conclusion, this study improves our understanding of the genetic characteristics of tLST and E. coli harboring tLST. IMPORTANCE This study, through a large-scale genomic analysis, demonstrated that the genomic island tLST related to multiple stress resistance (such as extreme heat resistance and oxidative stress tolerance) in E. coli is differentially present in subgroups of E. coli and is strongly associated with certain phylogenetic background of the host strain. The study also shows the transmission mechanisms of tLST in E. coli and other bacterial species. The overall negative association of tLST, and virulence genes and antimicrobial (AMR) genes suggest the selective pressures for the acquisition and transmission of these traits likely differ. Even so, the high prevalence of tLST in the enterotoxigenic E. coli clone ST2332 and co-occurrence of tLST and AMR genes in E. coli are concerning. Thus, the findings better our understanding of tLST evolution and provide information for risk assessment of tLST harboring bacteria.

Intradermally Administered Enterotoxigenic Escherichia coli Vaccine Candidate MecVax Induces Functional Serum Immunoglobulin G Antibodies against Seven Adhesins (CFA/I and CS1 through CS6) and Both Toxins (STa and LT).

There are no vaccines licensed for enterotoxigenic Escherichia coli (ETEC), a leading bacterial cause of children's diarrhea and travelers' diarrhea. MecVax, a multivalent E. coli vaccine candidate composed of two epitope- and structure-based polyvalent proteins (toxoid fusion 3xSTaN12S-mnLTR192G/L211A and colonization factor antigen [CFA]/I/II/IV multiepitope fusion antigen [MEFA]), is designed to induce broad antiadhesin and antitoxin antibodies against heterogeneous ETEC pathovars. When administered intraperitoneally or intramuscularly, MecVax was shown to induce antibodies against seven ETEC adhesins (CFA/I and CS1 to CS6) produced by ETEC pathovars that cause over 60% of ETEC-associated diarrheal cases and moderate-to-severe cases and both toxins (heat-labile toxin [LT] and heat-stable toxin [STa]) expressed by all ETEC strains. To further characterize the immunogenicity of this protein-based injectable subunit vaccine candidate and to explore other parenteral administration routes for the product, in this study we immunized mice intradermally (i.d.) with MecVax and measured antigen-specific antibody responses and further antibody functional activities against the adhesins and toxins targeted by the vaccine. Data showed that mice immunized i.d. with MecVax developed robust anti-CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, LT and anti-STa IgG responses. Furthermore, antibodies derived from MecVax administered via the i.d. route inhibited the adherence of ETEC or E. coli strains expressing any of the seven target adhesins (CFA/I and CS1 to CS6) and neutralized the enterotoxicity of LT and STa. These results confirmed broad immunogenicity of MecVax and suggested that this multivalent ETEC subunit vaccine candidate can be effectively delivered via the i.d. route. IMPORTANCE ETEC is a leading bacterial cause of diarrhea in children living in developing countries and international travelers. Developing an effective vaccine for ETEC diarrhea has been hampered because of the challenges of virulence heterogeneity and the difficulties of inducing neutralizing antibodies against the key toxin STa. MecVax, a subunit vaccine candidate carrying two polyvalent protein antigens, for the first time induces functional antibodies against the most important ETEC adhesins, which are associated with a majority of diarrheal cases and moderate-to-severe cases, and also against the enterotoxicity of LT and more importantly STa, which plays a key role in children's diarrhea and travelers' diarrhea, potentially leading to the development of a truly effective ETEC vaccine. Data from this study may also indicate that this ETEC subunit vaccine can be administered effectively via the i.d. route, expanding clinical administration options for this vaccine product.

Evaluation of Multivalent Enterotoxigenic Escherichia coli Vaccine Candidate MecVax Antigen Dose-Dependent Effect in a Murine Model.

There are no licensed vaccines against enterotoxigenic Escherichia coli (ETEC), a leading cause of children's diarrhea and travelers' diarrhea. Recently, protein-based vaccine candidate MecVax was demonstrated to induce functional antibodies against both ETEC toxins (heat-stable toxin [STa] and heat-labile toxin [LT]) and seven ETEC adhesins (CFA/I and CS1 to CS6) and to protect against ETEC clinical diarrhea or intestinal colonization preclinically. Those studies used intraperitoneal, intramuscular, and intradermal routes, and a dose range for MecVax protein antigens, toxoid fusion 3xSTaN12S-mnLTR192G/L211A, and adhesin CFA/I/II/IV MEFA has not been investigated. Here, we further characterized MecVax broad immunogenicity, utilizing a subcutaneous route, and examined vaccine dose-dependent antibody response effects and also antibody functional activities against ETEC enterotoxicity and bacterial adherence. Data showed that mice immunized subcutaneously with MecVax developed robust IgG responses to seven ETEC adhesins (CFA/I, as well as CS1 to CS6) and two toxins (STa and LT). At a subcutaneous dose of 25, 20, or 10 µg or at an intramuscular dose of 12, 6, or 3 µg, MecVax induced similar levels IgG responses to the targeted toxins and adhesins, and these antibodies exhibited equivalent functional activities against ETEC toxin enterotoxicity and bacterial adherence. Once the intramuscular dose was decreased to 1 µg, vaccine-induced antibodies were significantly reduced and no longer neutralized STa enterotoxicity. The results indicated that MecVax administered subcutaneously is broadly immunogenic and, at an intramuscular dose of 3 µg, can induce functional antitoxin and anti-adhesin antibodies in mice, providing instructive information for future vaccine dose studies in humans and accelerating MecVax vaccine development. IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) is a leading cause of children's diarrhea and the most common cause of travelers' diarrhea. ETEC infections are responsible for >200 million diarrhea clinical cases and near 100,000 deaths annually. Currently, there are no licensed vaccines for ETEC diarrhea. The protein-based vaccine candidate MecVax unprecedentedly targets two ETEC toxins (STa and LT, produced by all ETEC strains) and seven ETEC adhesins (CFA/I, as well as CS1 to CS6, associated with >60% of ETEC clinical diarrhea cases) and has been demonstrated to be broadly immunogenic and cross protective; as such, it represents a potentially effective multivalent vaccine against ETEC-associated children's and travelers' diarrhea. This study further confirmed MecVax broad immunogenicity and evaluated the vaccine antigen dose effect on the induction of antigen-specific antibody responses in mice and on antibody functional activities against ETEC toxin enterotoxicity and bacterial adherence, yielding useful information for future human volunteer studies and the development of MecVax as an effective ETEC vaccine.

Assessment the role of some Bacillus strains in improvement rex rabbits resistance against ETEC challenge.

Increasing reports have indicated that specific strains of probiotic Bacillus have the potential to prevent diseases. The purpose of this study was to explore the effects of three Bacillus strains (Bacillus subtilis BSWJ2017001, Bacillus pumilus BSWJ2017002, and B. subtilis BSWJ2017003) mixture dietary supplementation on rex rabbits infected with enterotoxigenic Escherichia coli (ETEC). In this study, 60 35-day-old weaning rex rabbits were separated into two groups randomly: control group (fed basal diet with no antibiotics) and Bacillus strains group (fed basal diet containing 1.0 × 106 CFU/g Bacillus strains mixture). After 8 weeks of feeding, the rex rabbits were inoculated orally with 5.0 mL of ETEC (1.0 × 109 CFU/mL) and assessed at 0, 12, and 24 h. The Bacillus strains mixture attenuated the oxidative damage, diarrhea severity, and intestinal damage of ETEC infected rabbits. It also significantly increased the population of Lactobacillus spp., and Bifidobacterium spp., and decreased the population of Enterococcus spp.. Moreover, Bacillus strains group exhibited higher levels of toll-like receptor (TLR) 2, anti-inflammatory cytokines, secretory immunoglobulin A, and intestinal barrier-related genes than control group, as well as lower levels of TLR-4 and pro-inflammatory cytokines. These results demonstrated that Bacillus strains mixture could attenuate injury caused by ETEC and enhance disease resistance by improving specific intestinal microbiota members and immunity in weaning rex rabbits.

The Controlled Human Infection Model for Enterotoxigenic Escherichia coli.

The controlled human infection model (CHIM) for enterotoxigenic Escherichia coli (ETEC) has been instrumental in defining ETEC as a causative agent of acute watery diarrhea, providing insights into disease pathogenesis and resistance to illness, and enabling preliminary efficacy evaluations for numerous products including vaccines, immunoprophylactics, and drugs. Over a dozen strains have been evaluated to date, with a spectrum of clinical signs and symptoms that appear to replicate the clinical illness seen with naturally occurring ETEC. Recent advancements in the ETEC CHIM have enhanced the characterization of clinical, immunological, and microbiological outcomes. It is anticipated that omics-based technologies applied to ETEC CHIMs will continue to broaden our understanding of host-pathogen interactions and facilitate the development of primary and secondary prevention strategies.

Burden of Diarrhoeagenic Escherichia coli in Santa Rosa, Guatemala in active health-services surveillance during 2008-2009 and 2014-2015.

OBJECTIVE: To describe the epidemiology of laboratory-confirmed Diarrhoeagenic Escherichia coli (DEC) cases from active facility-based surveillance in Guatemala. METHODS: We collected clinical and risk factor data on enrolled patients (aged 0-52 years) with acute diarrhoea at government healthcare facilities (1 hospital and 6 clinics) in Santa Rosa, Guatemala, during 2008-2009 and 2014-2015. Stool samples were analysed, E. coli identified through culture and biochemical tests, PCR amplification of genes encoding pathotype-specific virulence factors identified specific DEC pathotypes. Healthcare-seeking adjusted incidence rates were calculated. RESULTS: A total of 3041 diarrhoea cases were captured by surveillance (647 hospitalisations (H), 2394 clinic visits (CV)); general E. coli prevalence was 17.9%. DEC pathotypes were identified in 19% (n = 95/497) and 21% (n = 450/2113) in diarrhoea H and CV, respectively. Enteropathogenic E. coli (EPEC) was most frequently isolated (8.2% (n = 41) in diarrhoea H, 12.0% (n = 255) in diarrhoea CV), followed by ETEC (6.8% (n = 34) in H, 6% (n = 128) in CV) and STEC (0.6% (n = 3) in H, 0.6% (n = 13) in CV). We did not find evidence of a difference in severity between DEC and non-DEC diarrhoea. Incidence of DEC clinic visits and hospitalisations was 648.0 and 29.3, respectively, per 10,000 persons aged ≤5 years and 36.8 and 0.4, respectively, per 10,000 persons aged >5 years. CONCLUSIONS: DEC pathotypes, especially EPEC and ETEC, were detected frequently from patients presenting with diarrhoeal illness in Santa Rosa, Guatemala. Our findings suggest that preventive interventions should be prioritised for young children.