Enterotoxigenic Escherichia Coli Research Articles

PCR diagnostics are insufficient for the detection of Diarrhoeagenic Escherichia coli in Ibadan, Nigeria.

Understanding the contribution of different diarrhoeagenic Escherichia coli pathotypes to disease burden is critical to mapping risk and informing vaccine development. Targeting select virulence genes by PCR is the diagnostic approach of choice in high-burden, least-resourced African settings. We compared the performance of a commonly-used multiplex protocol to whole genome sequencing (WGS). PCR was applied to 3,815 E. coli isolates from 120 children with diarrhoea and 357 healthy controls. Three or more isolates per specimen were also Illumina-sequenced. Following quality assurance, ARIBA and Virulencefinder database were used to identify virulence targets. Root cause analysis of deviant PCR results was performed by examining target sensitivity using BLAST, Sanger sequencing false-positive amplicons, and identifying lineages prone to false-positivity using in-silico multilocus sequence typing and a Single Nucleotide Polymorphism phylogeny constructed using IQTree. The sensitivity and positive predictive value of PCR compared to WGS ranged from 0-77.8% while specificity ranged from 74.5-94.7% for different pathotypes. WGS identified more enteroaggregative E. coli (EAEC), fewer enterotoxigenic E. coli (ETEC) and none of the Shiga toxin-producing E. coli detected by PCR, painting a considerably different epidemiological picture. Use of the CVD432 target resulted in EAEC under-detection, and enteropathogenic E. coli eae primers mismatched more recently described intimin alleles common in our setting. False positive ETEC were over-represented among West Africa-predominant ST8746 complex strains. PCR precision varies with pathogen genome so primers optimized for use in one part of the world may have noticeably lower sensitivity and specificity in settings where different pathogen lineages predominate.

Drug resistance and molecular typing of diarrheagenic escherichia coli from the diarrhea clinics in Qingpu District, Shanghai City from 2015 to 2019

Objective: To analyze the drug resistance and multilocus sequence typing of five types of diarrheagenic Escherichia coli (DEC) isolated from diarrhea outpatients of diarrhea comprehensive monitoring designated hospital in Qingpu District, Shanghai City from 2015 to 2019. Methods: From January 2015 to December 2019, five types of DEC, isolated and identified from diarrhea outpatient cases' anal swabs of the Qingpu branch of Zhongshan Hospital were collected to determine the minimal inhibitory concentration by using the micro broth dilution susceptibility test. The strains, resistant to the third-generation cephalosporins or carbapenems, or producing ESBLs, were selected based on the results of sensitivity tests and determined by WGS. The MLST typing of DEC was analyzed based on the WGS technology and the minimum spanning tree was constructed by BioNumerics 7.6 software to analyze the local dominant flora. Results: A total of 513 strains of DEC were detected and isolated from 4 494 anal swabs, with a detection rate of 11.42%. About 500 strains were tested for drug sensitivity to nine antibiotics in four classes, including 330 strains of enterotoxigenic E.coli (ETEC), 72 strains of enteroaggregative E.coli (EAEC), 95 strains of enteropathogenic E.coli (EPEC), 1 strain of enterohemorrhagic E.coli (EHEC), and 2 strains of enteroinvasive E.coli (EIEC). From 2015 to 2019, the resistance rate of cefotaxime-clavulanic acid was significantly different (P<0.05). The resistance rate of virulence types of DEC to nalixic acid was significantly different (P<0.05). About 71 strains of DEC were determined by WGS, and 77 drug-resistant genes were detected. Strains were classified into 32 ST subtypes, with the dominant genotypes being ST-1491 (29.6%, 21/71) and ST-10 Complex (23.9%, 17/71). All ST-1491 produced ESBLs, which were blaCTX-M gene mutant strains. The dominant type of ST-10 complex was ST-218 (35.3%, 6/17). In addition, 8 strains of EAEC, 14 strains of EPEC and 49 strains of ETEC were classified into 7, 14 and 18 ST subtypes, respectively. Conclusion: The drug resistance of DEC strains from the diarrhea outpatient case of Qingpu District is serious. The ST types of EAEC and EPEC are highly polymorphic. The dominant ST types of DEC are basically consistent with the common genotypes in southeast China.

Reduced Plasma Guanylin Levels Following Enterotoxigenic Escherichia coli-Induced Diarrhea.

The intestinal peptide hormones guanylin (GN) and uroguanylin (UGN) interact with the epithelial cell receptor guanylate cyclase C to regulate fluid homeostasis. Some enterotoxigenic Escherichia coli (ETEC) produce heat-stable enterotoxin (ST), which induces diarrhea by mimicking GN and UGN. Plasma concentrations of prohormones of GN (proGN) and UGN (proUGN) are reportedly decreased during chronic diarrheal diseases. Here we investigate whether prohormone concentrations also drop during acute diarrhea caused by ST-producing ETEC strains TW10722 and TW11681. Twenty-one volunteers were experimentally infected with ETEC. Blood (n = 21) and urine (n = 9) specimens were obtained immediately before and 1, 2, 3, and 7 days after ETEC ingestion. Concentrations of proGN and proUGN were measured by ELISA. Urine electrolyte concentrations were measured by photometry and mass spectrometry. Ten volunteers developed diarrhea (D group), and eleven did not (ND group). In the D group, plasma proGN, but not proUGN, concentrations were substantially reduced on days 2 and 3, coinciding with one day after diarrhea onset. No changes were seen in the ND group. ETEC diarrhea also seemed to affect diuresis, the zinc/creatinine ratio, and sodium and chloride secretion levels in urine. ETEC-induced diarrhea causes a reduction in plasma proGN and could potentially be a useful marker for intestinal isotonic fluid loss.

Economic costs related to foodborne disease in Burkina Faso and Ethiopia in 2017

IntroductionFoodborne diseases (FBD) are a significant problem in low- and middle-income countries, especially in Africa. Country-specific estimates of the economic costs related to FBD caused by different hazards in different food products can inform decision makers about this problem’s magnitude. This aids policy makers in prioritising actions to mitigate risks and prevent illness. Although estimates exist for many high-income countries, they are lacking for African countries.MethodsThis study estimates economic cost in Burkina Faso and Ethiopia related to FBD caused by non-typhoidal Salmonella enterica (NTS), Campylobacter spp. (CAMP) and enterotoxigenic Escherichia coli (ETEC) in all foods, chicken meat and tomatoes. The World Health Organization’s estimates of FBD burden, updated from 2010 to 2017, were used as a basis. Economic cost estimates were the sum of estimates of willingness-to-pay (WTP) to reduce risk of death and of pain and suffering, and lost productivity. WTP was based on value of statistical life (VSL) and value of statistical life year (VSLY), extrapolated from USA data. We used sensitivity options to account for uncertainty in these values.ResultsMean economic costs related to FBD caused by NTS, CAMP and ETEC in 2017 were estimated at 391 million constant 2017 international dollars in Burkina Faso and 723 million in Ethiopia. These represent 3.0% of gross national income in Burkina Faso and 0.9% in Ethiopia. Lost productivity contributed 70%, WTP to reduce risk of death 30%, and WTP to reduce risk of pain and suffering 1-2%. NTS caused the highest costs, followed by ETEC and CAMP. Chicken meat caused 9-12 times higher costs than tomatoes. Children under five years (14.6-17.1% of populations) bore 20-75% of the costs. Due to uncertainty in disease burden and economic data, costs could be four times higher than mean estimates.DiscussionPolicies to control FBD likely result in substantial benefits, especially efforts aiming at this study’s hazards in chicken meat and children under 5 years. Efforts to reduce uncertainty in VSL, VSLY and disease burden estimates, estimate costs for other countries, attribute costs to other food products and hazards, further standardise estimation methodologies, and estimate treatment costs and illness prevention expenditures are recommended.

Genomic traits of multidrug resistant enterotoxigenic Escherichia coli isolates from diarrheic pigs.

Diarrhea caused by enterotoxigenic Escherichia coli (ETEC) infections poses a significant challenge in global pig farming. To address this issue, the study was conducted to identify and characterize 19 ETEC isolates from fecal samples of diarrheic pigs sourced from large-scale farms in Sichuan Province, China. Whole-genome sequencing and bioinformatic analysis were utilized for identification and characterization. The isolates exhibited substantial resistance to cefotaxime, ceftriaxone, chloramphenicol, ciprofloxacin, gentamicin, ampicillin, tetracycline, florfenicol, and sulfadiazine, but were highly susceptible to amikacin, imipenem, and cefoxitin. Genetic diversity among the isolates was observed, with serotypes O22:H10, O163orOX21:H4, and O105:H8 being dominant. Further analysis revealed 53 resistance genes and 13 categories of 195 virulence factors. Of concern was the presence of tet(X4) in some isolates, indicating potential public health risks. The ETEC isolates demonstrated the ability to produce either heat-stable enterotoxin (ST) alone or both heat-labile enterotoxin (LT) and ST simultaneously, involving various virulence genes. Notably, STa were linked to human disease. Additionally, the presence of 4 hybrid ETEC/STEC isolates harboring Shiga-like toxin-related virulence factors, namely stx2a, stx2b, and stx2e-ONT-2771, was identified. IncF plasmids carrying multiple antimicrobial resistance genes were prevalent, and a hybrid ETEC/STEC plasmid was detected, highlighting the role of plasmids in hybrid pathotype emergence. These findings emphasized the multidrug resistance and pathogenicity of porcine-origin ETEC strains and the potential risk of epidemics through horizontal transmission of drug resistance, which is crucial for effective control strategies and interventions to mitigate the impact on animal and human health.

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.

Protective effect of Limosilactobacillus reuteri-fermented yogurt on mouse intestinal barrier injury induced by enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is a pathogen that causes traveler's diarrhea, for which an effective vaccine is lacking. Previous studies showed that Limosilactobacillus reuteri could inhibit E. coli, effectively increase the expression of its tight junction protein, and reduce the adhesion of ETEC to the intestinal epithelial Caco-2 cell line. In this study, three kinds of yogurt with different starter cultures were first prepared: Lm. reuteri yogurt (fermented by Lm. reuteri alone), traditional yogurt (fermented by Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus at a ratio of 1:1) and mixed yogurt (fermented by Lm. reuteri, S. thermophilus and L. delbrueckii subsp. bulgaricus at a ratio of 1:1:1). The physiological properties, oxidative stress, intestinal barrier function, tight junction protein, pathological conditions and intestinal microbiota composition were investigated. The data showed that Lm. reuteri-fermented yogurt pregavage could effectively alleviate the intestinal barrier impairment caused by ETEC in mice. It alleviated intestinal villus shortening and inflammatory cell infiltration, decreased plasma diamine oxidase concentration and increased claudin-1 and occludin expression in the jejunum of ETEC-infected mice. In addition, Lm. reuteri-fermented yogurt significantly reduced the ETEC load in fecal samples, reversed the increase in Pseudomonadota abundance and decreased Bacteroidota abundance caused by ETEC infection. Furthermore, the composition of the intestinal microbiota could maintain a stable state similar to that in healthy mice. These findings indicate that Lm. reuteri-fermented yogurt could alleviate intestinal barrier damage, inhibit ETEC growth and maintain the stability of the intestinal microbiota during ETEC infection. © 2023 Society of Chemical Industry.

Molecular Classification and Antimicrobial Profiles of Chlorination-Resistant Escherichia Coli at Wastewater Treatment Plant in the North West Province of South Africa

The resistance of different pathogenic variants of E. coli to antibiotics, is a health concern globally. The study assessed the resistance of 90 E. coli isolates that survived chlorination at a Wastewater Treatment Plant (WWTP) in North West, South Africa (NW-SA), to 12 different antibiotics using the Kirby-Bauer disk diffusion method. The study further assessed the diarrheagenic pathotypes origin of the isolates. The molecular characterization revealed diarrheagenic E. coli pathotypes ranged as follows: Enteroaggregative E. coli (EAEC) 16 (17.78%), Enteroinvasive E. coli (EIEC) 6 (6.67%), Enterotoxigenic E. coli (ETEC) 5 (5.56%) and Enteropathogenic E. coli (EPEC) 3 (3.33%). A high degree of resistance was observed against sulphamethoxazol (92.22%), while lower resistance was observed against Kanamycin (3.33%), chloramphenicol (5.56%) and ciprofloxacin (6.67%). Multiple drug resistance of three and more antibiotics was observed in 81.11% of the E. coli isolates. The detected diarrheagenic E. coli pathotypes showed multiple resistance to different studied antibiotics with Multiple Antibiotic Resistance Indexing (MARI) equal to 0.9 for EIEC and EAEC respectively, followed by ETEC at 0.8 and EPEC at 0.2. The study reveals that the wastewater effluent from the studied plant serves as an important reservoir for the distribution of antibiotic resistant diarrheagenic E. coli pathotypes and other potential pathogens to the aquatic milieu, thus confirming potential risk to public health.

L-Alanine promotes anti-infectious properties of Bacillus subtilis S-2 spores via the germination receptor gerAA.

Bacillus species, which have two cell-type forms (vegetative cells and spores), demonstrate a variety of probiotic functions in animal feed additives and human nutrition. We previously found that the probiotic effect of Bacillus subtilis S-2 spores with high germination response to L-alanine was specifically enhanced by the L-alanine pretreatment. The germination response of Bacillus is highly associated with the germination receptors of spores. However, how L-alanine-induced germination of spores exerts anti-infectious effect in epithelial cells remains unclear. In this study, we constructed the mutant strain of B. subtilis S-2 with germination receptor gerAA knockout to further explore the role of spore germination in resisting pathogen infection to cells. The differential probiotic effects of B. subtilis S-2 and S-2ΔgerAA spores pretreated with L-alanine were evaluated in intestinal porcine epithelial cells (IPEC-J2) or Caco2 cells infected with enterotoxigenic Escherichia coli (ETEC) or following IL-1β stimulation. The results showed that the germination response of the S-2ΔgerAA spores to L-alanine was significantly reduced. Compared with the S-2ΔgerAA spores, the L-alanine-induced germination of B. subtilis S-2 spores significantly increased the activity of anti-adhesion of ETEC to IPEC-J2 cells and reduced the expression of inflammatory factors and cell receptors. L-alanine induction also significantly promoted the expression of autophagy-related proteins in the B. subtilis S-2 spores. These findings demonstrate that the gerAA germination receptor is essential for the probiotic function of Bacillus spores and that L-alanine treatment promotes the anti-infectious properties of the germinated spores in porcine intestinal epithelial IPEC-J2 cells. The result suggests the importance of germination receptor gerAA in helping spore germination and enhancing anti-infectious activity. The findings in the study benefit to screening of potential Bacillus probiotics and increasing probiotic efficacy induced by L-alanine as an adjuvant.

High variation across E. coli hybrid isolates identified in metabolism-related biological pathways co-expressed with virulent genes

ABSTRACT Virulent genes present in Escherichia coli (E. coli) can cause significant human diseases. These enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) isolates with virulent genes show different expression levels when grown under diverse laboratory conditions. In this research, we have performed differential gene expression analysis using publicly available RNA-seq data on three pathogenic E. coli hybrid isolates in an attempt to characterize the variation in gene interactions that are altered by the presence or absence of virulent factors within the genome. Almost 26.7% of the common genes across these strains were found to be differentially expressed. Out of the 88 differentially expressed genes with virulent factors identified from PATRIC, nine were common in all these strains. A combination of Weighted Gene Co-Expression Network Analysis and Gene Ontology Enrichment Analysis reveals significant differences in gene co-expression involving virulent genes common among the three investigated strains. The co-expression pattern is observed to be especially variable among biological pathways involving metabolism-related genes. This suggests a potential difference in resource allocation or energy generation across the three isolates based on genomic variation.

Protective Effects of Bacillus subtilis HH2 against Oral Enterotoxigenic Escherichia coli in Beagles.

This study evaluated the protective effect of Bacillus subtilis HH2 on beagles orally challenged with enterotoxigenic Escherichia coli (ETEC). We assessed the physiological parameters and the severity of diarrhea, as well as the changes in three serum immunoglobulins (IgG, IgA, and IgM), plasma diamine oxidase (DAO), D-lactate (D-LA), and the fecal microbiome. Feeding B. subtilis HH2 significantly reduced the severity of diarrhea after the ETEC challenge (p < 0.05) and increased serum levels of IgG, IgA, and IgM (p < 0.01). B. subtilis HH2 administration also reduced serum levels of DAO at 48 h after the ETEC challenge (p < 0.05), but no significant changes were observed in D-LA (p > 0.05). Oral ETEC challenge significantly reduced the richness and diversity of gut microbiota in beagles not pre-fed with B. subtilis HH2 (p < 0.05), while B. subtilis HH2 feeding and oral ETEC challenge significantly altered the gut microbiota structure of beagles (p < 0.01). Moreover, 14 days of B. subtilis HH2 feeding reduced the relative abundance of Deinococcus-Thermus in feces. This study reveals that B. subtilis HH2 alleviates diarrhea caused by ETEC, enhances non-specific immunity, reduces ETEC-induced damage to the intestinal mucosa, and regulates gut microbiota composition.

Transferable mechanisms of quinolone resistance are more frequent among enterotoxigenic Escherichia coli isolates displaying low-level quinolone resistance.

This study analysed the mechanisms of quinolone resistance among enterotoxigenic Escherichia coli (ETEC) in a periurban area of Lima, Peru. The susceptibility to nalidixic acid and ciprofloxacin, the role of Phe-Arg-b-Naphtylamyde inhibitable-(PAbN) efflux pumps, the presence of mutations in gyrA and parC as well as the presence of aac(6')Ib-cr, qepA, qnrA, qnrB, qnrC, qnrD, qnrVC and oqxAB were determined in 31 ETEC from previous case/control studies of children's diarrhoea. Discordances between disk diffusion, with all isolates showing intermediate or fully resistance to nalidixic acid, and minimal inhibitory concentration (MIC), with 7 isolates being below considered resistance breakpoint, were observed. Twenty-one isolates possessed gyrA mutations (19 S83L, 2 S83A). AAC(6') Ib-cr, QnrS, QnrB and QepA were found in 7, 6, 2 and 1 isolates respectively, with 3 isolates presenting 2 transferable mechanisms of quinolone resistance (TMQR) concomitantly. TMQR were more frequent among isolates with MIC to nalidixic acid ranging from 2 to 16 mg/L (p=0.03), while gyrA mutations were more frequent among isolates with nalidixic acid MIC >= 128 mg/L (p=0.0002). In summary, the mechanisms of quinolone resistance present in ETEC isolates in Peru have been described. Differences in the prevalence of underlying mechanisms associated with final MIC levels were observed. The results suggest two different evolutive strategies to survive in the presence of quinolones related to specific bacterial genetic background.

Prevalence and Antibiotic Resistance Pattern of Diarrheagenic Escherichia coli Pathotypes Among Children under 5 Years in Khuzestan, Iran

Background: Diarrhea is a life-threatening cause of high mortality, especially among children living in areas with poor sanitation. Enterobacteriaceae is one of the serious causes of bacterial diarrhea in children and adults. In this family, infection with diarrheagenic Escherichia coli (DEC) pathotypes in children is associated with extensive health risks and is of particular importance. In this study, we compared the distribution of pathotypes, epidemiological patterns, and antibiotic resistance of DEC in two diarrheal and non-diarrheal groups among children less than 5 years. Methods: In this study, 303 stool samples were collected from patients admitted to Golestan hospitals in Ahvaz and Dr. Ganjavian in Dezful, Khuzestan. To this end, 201 samples from children with diarrhea (case group) and 102 samples from healthy children (control group) were examined. DEC was characterized by polymerase chain reaction (PCR) for each stool sample, and DEC isolates were tested with antibiotic resistance tests against different antibiotic agents to identify the prevalence of multidrug-resistant (MDR) strains in both groups. Results: DEC was found in 24% (48 out of 200) of the children with diarrhea and 3.8% (4 out of 103) of the healthy children. Enteroaggregative E. coli (EAEC) was the DEC most frequently associated with diarrhea (32 out of 48, 66.6%), which was followed by enteropathogenic E. coli (EPEC) 22.9% (11 out of 48, 22.9%), and enterotoxigenic E. coli (ETEC) (5 out of 48, 10.4%) from children with diarrhea. Four DEC isolates were identified in healthy children: EAEC (2 out of 4, 50%) and EPEC (2 out of 4, 50%) in the healthy group, but no enteroinvasive E. coli (EIEC) or enterohemorrhagic E. coli (EHEC) strains were found in both groups in this study group. In general, DEC isolates exhibited high resistance to ceftriaxone and cefotaxime, and 33 (63.4%) isolates of DEC were MDR. Conclusion: A high prevalence of DEC strains was observed in the group of children with diarrhea and healthy children. Accordingly, further attention should be paid to continuous monitoring of the prevalence and pattern of antibiotic resistance of diarrheal bacterial isolates among children and the whole community.

Dynamics of circulating lymphocytes responding to human experimental enterotoxigenic Escherichia coli infection.

Enterotoxigenic Escherichia coli (ETEC) is an important cause of children's and travelers' diarrhea, with no licensed vaccine. This study aimed to explore the role of cellular immunity in protection against human ETEC infection. Nine volunteers were experimentally infected with ETEC, of which six developed diarrhea. Lymphocytes were collected from peripheral blood buffy coats, before and 3, 5, 6, 7, 10, and 28 days after dose ingestion, and 34 phenotypic and functional markers were examined by mass cytometry. Thirty-three cell populations, derived by manually merging 139 cell clusters from the X-shift unsupervised clustering algorithm, were analyzed. Initially, the diarrhea group responded with increased CD56dim CD16+ natural killer cells, dendritic cells tended to rise, and mucosal-associated invariant T cells decreased. On day 5-7, an increase in plasmablasts was paralleled by a consistent rise in CD4+ Th17-like effector memory and regulatory cell subsets. CD4+ Th17-like central memory cells peaked on day 10. All Th17-like cell populations showed increased expression of activation, gut-homing, and proliferation markers. Interestingly, in the nondiarrhea group, these same CD4+ Th17-like cell populations expanded earlier, normalizing around day 7. Earlier development of these CD4+ Th17-like cell populations in the nondiarrhea group may suggest a recall response and a potential role in controlling ETEC infections.

Structural and biophysical characterization of the secreted, β-helical adhesin EtpA of Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is a diarrhoeal pathogen associated with high morbidity and mortality especially among young children in developing countries. At present, there is no vaccine for ETEC. One candidate vaccine antigen, EtpA, is a conserved secreted adhesin that binds to the tips of flagellae to bridge ETEC to host intestinal glycans. EtpA is exported through a Gram-negative, two-partner secretion system (TPSS, type Vb) comprised of the secreted EtpA passenger (TpsA) protein and EtpB (TpsB) transporter that is integrated into the outer bacterial membrane. TpsA proteins share a conserved, N-terminal TPS domain followed by an extensive C-terminal domain with divergent sequence repeats. Two soluble, N-terminal constructs of EtpA were prepared and analysed respectively including residues 67 to 447 (EtpA67-447) and 1 to 606 (EtpA1-606). The crystal structure of EtpA67-447 solved at 1.76 Å resolution revealed a right-handed parallel β-helix with two extra-helical hairpins and an N-terminal β-strand cap. Analyses by circular dichroism spectroscopy confirmed the β-helical fold and indicated high resistance to chemical and thermal denaturation as well as rapid refolding. A theoretical AlphaFold model of full-length EtpA largely concurs with the crystal structure adding an extended β-helical C-terminal domain after an interdomain kink. We propose that robust folding of the TPS domain upon secretion provides a template to extend the N-terminal β-helix into the C-terminal domains of TpsA proteins.

Characterization of the Inflammatory Response Evoked by Bacterial Membrane Vesicles in Intestinal Cells Reveals an RIPK2-Dependent Activation by Enterotoxigenic Escherichia coli Vesicles.

Although the immunomodulatory potency of bacterial membrane vesicles (MVs) is widely acknowledged, their interactions with host cells and the underlying signaling pathways have not been well studied. Herein, we provide a comparative analysis of the proinflammatory cytokine profile secreted by human intestinal epithelial cells exposed to MVs derived from 32 gut bacteria. In general, outer membrane vesicles (OMVs) from Gram-negative bacteria induced a stronger proinflammatory response than MVs from Gram-positive bacteria. However, the quality and quantity of cytokine induction varied between MVs from different species, highlighting their unique immunomodulatory properties. OMVs from enterotoxigenic Escherichia coli (ETEC) were among those showing the strongest proinflammatory potency. In depth analyses revealed that the immunomodulatory activity of ETEC OMVs relies on a so far unprecedented two-step mechanism, including their internalization into host cells followed by intracellular recognition. First, OMVs are efficiently taken up by intestinal epithelial cells, which mainly depends on caveolin-mediated endocytosis as well as the presence of the outer membrane porins OmpA and OmpF on the MVs. Second, lipopolysaccharide (LPS) delivered by OMVs is intracellularly recognized by novel caspase- and RIPK2-dependent pathways. This recognition likely occurs via detection of the lipid A moiety as ETEC OMVs with underacylated LPS exhibited reduced proinflammatory potency but similar uptake dynamics compared to OMVs derived from wild-type (WT) ETEC. Intracellular recognition of ETEC OMVs in intestinal epithelial cells is pivotal for the proinflammatory response as inhibition of OMV uptake also abolished cytokine induction. The study signifies the importance of OMV internalization by host cells to exercise their immunomodulatory activities. IMPORTANCE The release of membrane vesicles from the bacterial cell surface is highly conserved among most bacterial species, including outer membrane vesicles (OMVs) from Gram-negative bacteria as well as vesicles liberated from the cytoplasmic membrane of Gram-positive bacteria. It is becoming increasingly evident that these multifactorial spheres, carrying membranous, periplasmic, and even cytosolic content, contribute to intra- and interspecies communication. In particular, gut microbiota and the host engage in a myriad of immunogenic and metabolic interactions. This study highlights the individual immunomodulatory activities of bacterial membrane vesicles from different enteric species and provides new mechanistic insights into the recognition of ETEC OMVs by human intestinal epithelial cells.

Dietary Supplementation with Puerarin Improves Intestinal Function in Piglets Challenged with Escherichia coli K88

The objective of this study was to investigate the effect of puerarin supplementation on the growth performance and intestinal function of piglets challenged with enterotoxigenic Escherichia coli (ETEC) K88. Twenty-four ternary crossbred piglets were randomly assigned to three treatment groups: control group, ETEC group (challenged with ETEC K88 on day 8), and ETEC + Puerarin group (supplemented with 5 mg/kg puerarin and challenged with ETEC K88 on day 8). All piglets were orally administered D-xylose (0.1 g/kg body weight) on day 10, and blood samples were collected after 1 h. Subsequently, piglets were killed and intestinal samples were collected for further analysis. The results showed that puerarin supplementation significantly decreased the adverse effects of ETEC K88-challenged piglets; significantly improved growth performance; increased the number of Bifidobacterium in the colon and Lactobacillus in the jejunum, cecum and colon; decreased the number of Escherichia coli in the jejunum and cecum; reduced the hydrogen peroxide content in the jejunum and myeloperoxidase activity in the jejunum and ileum; and increased the activities of catalase and superoxide dismutase in the jejunum and ileum. In addition, puerarin supplementation alleviated ETEC K88-induced intestinal injury in piglets, significantly downregulated the mRNA level of Interleukin-1β and upregulated the mRNA levels of intercellular cell adhesion molecule-1, myxovirus resistance protein 1, myxovirus resistance protein 2, and guanylate-binding protein-1 in the small intestine of piglets. In conclusion, dietary supplementation with puerarin could attenuate ETEC K88-induced intestinal injury by increasing the antioxidant and anti-inflammatory capacity and the number of beneficial intestinal bacteria in piglets.

Site-Specific Analysis of the Incidence Rate of Enterotoxigenic Escherichia coli Infection Elucidates an Association with Childhood Stunting, Wasting, and Being Underweight: A Secondary Analysis of the MAL-ED Birth Cohort.

Asymptomatic infection by fecal enteropathogens is a major contributor to childhood malnutrition. Here, we investigated the incidence rate of asymptomatic infection by enterotoxigenic Escherichia coli (ETEC) and assessed its association with childhood stunting, wasting, and being underweight among children under 2 years of age. The Malnutrition and Enteric Disease birth cohort study included 1,715 children who were followed from birth to 24 months of age from eight distinct geographic locations including Bangladesh, Brazil, India, Peru, Tanzania, Pakistan, Nepal, and South Africa. The TaqMan array card assay was used to determine the presence of ETEC in the nondiarrheal stool samples collected from these children. Poisson regression was used to estimate the incidence rate, and multiple generalized estimating equations with binomial family, logit link function, and exchangeable correlation were used to analyze the association between asymptomatic ETEC infection and anthropometric indicators such as stunting, wasting, and being underweight. The site-specific incidence rates of asymptomatic ETEC infections per 100 child-months were also higher at the study locations in Tanzania (54.81 [95% CI: 52.64, 57.07]) and Bangladesh (46.75 [95% CI: 44.75, 48.83]). In the Bangladesh, India, and Tanzania sites, the composite indicator of anthropometric failure was significantly associated with asymptomatic ETEC infection. Furthermore, a significant association between asymptomatic heat-stable toxin ETEC infections and childhood stunting, wasting, and being underweight was found in only the Bangladesh and Tanzania sites.

Isolation and characterization of bacteriophages from soil against food spoilage and foodborne pathogenic bacteria

Microbial food spoilage and foodborne disease are the main challenges in the food industry regarding food shelf life. Current preservation methods are frequently associated with changes in organoleptic characteristics and loss of nutrients. For this reason, bacteriophage offers an alternative natural method as a biocontrol agent that can reduce bacterial contamination in food without altering the organoleptic properties. This study was conducted to isolate and characterize bacteriophage from soil to control food spoilage bacteria, such as Bacillus cereus and Bacillus subtilis, and foodborne pathogenic bacteria, such as enterotoxigenic Escherichia coli (ETEC) and enterohemorrhagic E. coli (EHEC). Isolation was done by agar overlay assay method, and phages BC-S1, BS-S2, ETEC-S3, and EHEC-S4 were recovered. The host range of all isolated phages tended to be narrow and had high specificity towards the specific bacteria. The phage efficiency were measured where ETEC-S3 showed no effectivity against B. cereus and EHEC-S4 showed low efficiency against Enteropathogenic E. coli (EPEC). Morphology analysis was conducted for phage BC-S1 and ETEC-S3 with Transmission Electron Microscopy (TEM), and it is shown to belong to the Caudovirales order. Phages BC-S1 and BS-S2 significantly reduced the host bacteria when applied to the cooked rice and pasteurized milk samples with miMOI of 0.1. While phage ETEC-S3 at miMOI of 0.001 and phage EHEC-S4 at miMOI of 1 also showed a significant reduction when applied to chicken meat and lettuce samples at storage temperatures of 4 °C and 28 °C. The highest bacterial reduction of 100% was shown by phage BC-S1 on pasteurized milk samples and reduction up to 96.06% by phage ETEC-S3 on chicken meat samples at 28 °C incubation.

E. coli infection disrupts the epithelial barrier and activates intrinsic neurosecretory reflexes in the pig colon.

This study aims to assess the barrier integrity and possible activation of enteric neural pathways associated with secretion and motility in the pig colon induced by an enterotoxigenic Escherichia coli (ETEC) challenge. 50 Danbred male piglets were used for this study. 16 were challenged with an oral dose of the ETEC strain F4+ 1.5 × 109 colony-forming unit. Colonic samples were studied 4- and 9-days post-challenge using both a muscle bath and Ussing chamber. Colonic mast cells were stained with methylene blue. In control animals, electrical field stimulation induced neurosecretory responses that were abolished by tetrodotoxin (10-6M) and reduced by the combination of atropine (10-4M) and α-chymotrypsin (10U/mL). Exogenous addition of carbachol, vasoactive intestinal peptide, forskolin, 5-HT, nicotine, and histamine produced epithelial Cl- secretion. At day 4 post-challenge, ETEC increased the colonic permeability. The basal electrogenic ion transport remained increased until day 9 post-challenge and was decreased by tetrodotoxin (10-6M), atropine (10-4M), hexamethonium (10-5M), and ondansetron (10-5M). In the muscle, electrical field stimulation produced frequency-dependent contractile responses that were abolished with tetrodotoxin (10-6M) and atropine (10-6M). Electrical field stimulation and carbachol responses were not altered in ETEC animals in comparison with control animals at day 9 post-challenge. An increase in mast cells, stained with methylene blue, was observed in the mucosa and submucosa but not in the muscle layer of ETEC-infected animals on day 9 post-challenge. ETEC increased the response of intrinsic secretory reflexes and produced an impairment of the colonic barrier that was restored on day 9 post-challenge but did not modify neuromuscular function.