Field evaluation of a simple and rapid diagnostic test, RLDT to detect Shigella and enterotoxigenic E. coli in Indian children.

The diagnostic assays currently used to detect Shigella spp. (Shigella) and enterotoxigenic Escherichia coli (ETEC) are complex or elaborate which make them difficult to apply in resource poor settings where these diseases are endemic. The simple and rapid nucleic acid amplification-based assay "Rapid LAMP-based Diagnostic Test (RLDT)" was evaluated to detect Shigella spp (Shigella) and enterotoxigenic Escherichia coli (ETEC) and determine the epidemiology of these pathogens in Kolkata, India. Stool samples (n = 405) from children under five years old with diarrhea seeking care at the hospitals were tested, and 85(21%) and 68(17%) by RLDT, 91(23%) and 58(14%) by quantitative PCR (qPCR) and 35(9%) and 15(4%) by culture, were positive for Shigella and ETEC, respectively. The RLDT showed almost perfect agreement with qPCR, Kappa 0.96 and 0.89; sensitivity 93% and 98%; specificity 100% and 97% for Shigella and ETEC, respectively. While RLDT detected additional 12% Shigella and 13% ETEC than culture, all culture positives for Shigella and ETEC except one each were also positive by the RLDT, sensitivity 97% and 93% respectively. RLDT is a simple, sensitive, and rapid assay that could be implemented with minimum training in the endemic regions to strengthen the disease surveillance system and rapid outbreak detection.

Epidemiology of enterotoxigenic Escherichia coli and impact on the growth of children in the first two years of life in Lima, Peru.

Background: Enterotoxigenic E. coli (ETEC) is a leading cause of diarrheal morbidity and mortality in children, although the data on disease burden, epidemiology, and impact on health at the community level are limited. Methods: In a longitudinal birth cohort study of 345 children followed until 24 months of age in Lima, Peru, we measured ETEC burden in diarrheal and non-diarrheal samples using quantitative PCR (LT, STh, and STp toxin genes), studied epidemiology and measured anthropometry in children. Results: About 70% of children suffered from one or more ETEC diarrhea episodes. Overall, the ETEC incidence rate (IR) was 73 per 100 child-years. ETEC infections began early after birth causing 10% (8.9-11.1) ETEC-attributable diarrheal burden at the population level (PAF) in neonates and most of the infections (58%) were attributed to ST-ETEC [PAF 7.9% (1.9-13.5)] and LT + ST-ETEC (29%) of which all the episodes were associated with diarrhea. ETEC infections increased with age, peaking at 17% PAF (4.6-27.7%; p = 0.026) at 21 to 24 months. ST-ETEC was the most prevalent type (IR 32.1) with frequent serial infections in a child. The common colonization factors in ETEC diarrhea cases were CFA/I, CS12, CS21, CS3, and CS6, while in asymptomatic ETEC cases were CS12, CS6 and CS21. Only few (5.7%) children had repeated infections with the same combination of ETEC toxin(s) and CFs, suggested genotype-specific immunity from each infection. For an average ETEC diarrhea episode of 5 days, reductions of 0.060 weight-for-length z-score (0.007 to 0.114; p = 0.027) and 0.061 weight-for-age z-score (0.015 to 0.108; p = 0.009) were noted in the following 30 days. Conclusion: This study showed that ETEC is a significant pathogen in Peruvian children who experience serial infections with multiple age-specific pathotypes, resulting in transitory growth impairment.

The colonization factor CS6 of enterotoxigenic Escherichia coli contributes to host cell invasion.

Enterotoxigenic Escherichia coli (ETEC) is one of the main causes of diarrhea in children and travelers in low-income regions. The virulence of ETEC is attributed to its heat-labile and heat-stable enterotoxins, as well as its colonization factors (CFs). CFs are essential for ETEC adherence to the intestinal epithelium. However, its invasive capability remains unelucidated. In this study, we demonstrated that the CS6-positive ETEC strain 4266 can invade mammalian epithelial cells. The invasive capability was reduced in the 4266 ΔCS6 mutant but reintroduction of CS6 into this mutant restored the invasiveness. Additionally, the laboratory E. coli strain Top 10, which lacks the invasive capability, was able to invade Caco-2 cells after gaining the CS6-expressing plasmid pCS6. Cytochalasin D inhibited cell invasion in both 4266 and Top10 pCS6 cells, and F-actin accumulation was observed near the bacteria on the cell membrane, indicating that CS6-positive bacteria were internalized via actin polymerization. Other cell signal transduction inhibitors, such as genistein, wortmannin, LY294002, PP1, and Ro 32-0432, inhibited the CS6-mediated invasion of Caco-2 cells. The internalized bacteria of both 4266 and Top10 pCS6 strains were able to survive for up to 48 h, and 4266 cells were able to replicate within Caco-2 cells. Immunofluorescence microscopy revealed that the internalized 4266 cells were present in bacteria-containing vacuoles, which underwent a maturation process indicated by the recruitment of the early endosomal marker EEA-1 and late endosomal marker LAMP-1 throughout the infection process. The autophagy marker LC3 was also observed near these vacuoles, indicating the initiation of LC-3-associated phagocytosis (LAP). However, intracellular bacteria continued to replicate, even after the initiation of LAP. Moreover, intracellular filamentation was observed in 4266 cells at 24 h after infection. Overall, this study shows that CS6, in addition to being a major CF, mediates cell invasion. This demonstrates that once internalized, CS6-positive ETEC is capable of surviving and replicating within host cells. This capability may be a key factor in the extended and recurrent nature of ETEC infections in humans, thus highlighting the critical role of CS6.

Survey on resistance occurrence for F4+ and F18+ enterotoxigenic Escherichia coli (ETEC) among pigs reared in Central Italy regions.

Porcine Post Weaning Diarrhoea (PWD) is one of the most important swine disease worldwide, caused by Enterotoxigenic Escherichia coli (ETEC) strains able to provoke management, welfare and sanitary issues. ETEC is determined by proteinaceous surface appendages. Numerous studies conducted by now in pigs have demonstrated, at the enterocytes level, that, the genes mucin 4 (MUC4) and fucosyltransferase (FUT1), coding for ETEC F4 and F18 receptors respectively, can be carriers of single nucleotide polymorphisms (SNPs) associated with natural resistance/susceptibility to PWD. The latter aspect was investigated in this study, evaluating the SNPs of the MUC4 and FUT1 genes in slaughtered pigs reared for the most in Central Italy. Genomic DNA was extracted from 362 swine diaphragmatic samples and then was subjected to the detection of known polymorphisms on MUC4 and FUT1candidate target genes by PCR-RFLP. Some of the identified SNPs were confirmed by sequencing analysis. Animals carrying the SNPs associated with resistance were 11% and 86% for the FUT1 and MUC4 genes respectively. Therefore, it can be assumed that the investigated animals may be an important resource and reservoir of favorable genetic traits for the breeding of pigs resistant to enterotoxigenic E.coli F4 variant.

Immunomodulatory effect of probiotic exopolysaccharides in a porcine in vitro co-culture model mimicking the intestinal environment on ETEC infection.

The aim of this study was to evaluate the immunomodulatory effect of EPS-L26 isolated from the probiotic strain Lactobacillus (Limosilactobacillus) reuteri L26 Biocenol™, in a model of infection with an enterotoxigenic E. coli (ETEC) by establishing monocultures consisting of the IPEC-J2 cell line or monocyte-derived dendritic cells (moDCs) and creating a 3D model of cell co-cultures established with IPEC-J2 cells and moDCs. The immunomodulatory and immunoprotective potential of used EPS-L26 was confirmed in monocultures in an experimental group of pretreated cells, where our study showed that pretreatment of cells with EPS-L26 and subsequent exposure to infection resulted in significantly down-regulated mRNA levels of genes encoding inflammatory cytokines compared to ETEC challenge in single cell cultures (in IPEC-J2, decreased mRNA levels for TNF-α, IL-6, IL-1ß, IL-12p35; in moDCs, decreased mRNA levels for IL-1ß). Similar to monocultures, we also demonstrated the immunostimulatory potential of the ETEC strain in the co-culture model on directly treated IPEC-J2 cells cultivated on insert chambers (apical compartment) and also on indirectly treated moDCs cultivated in the lower chamber (basolateral compartment), however in the co-culture model the expression of inflammatory cytokines was attenuated at the mRNA level compared to monocultures. Pretreatment of the cells on the insert chambers pointed to the immunoprotective properties of EPS-L26, manifested by decreased mRNA levels in both cell lines compared to ETEC challenge (in IPEC-J2 decreased mRNA levels for IL-12p35; in moDCs decreased mRNA levels for IL-1ß, IL-6). Our results suggest intercellular communication via humoral signals derived from IPEC-J2 cells by influencing the gene expression of indirectly treated moDC cells located in the basolateral compartment.

Pathogenicity of enterotoxigenic Escherichia coli in Caenorhabditis elegans as an alternative model host.

Enterotoxigenic Escherichia coli (ETEC), one of the diarrheagenic E. coli, is the most common cause of diarrhea in developing country and in travelers to those areas. In this study, Caenorhabditis elegans was used as an alternative model host to evaluate ETEC infections. The ETEC strain ETEC1, which was isolated from a patient with diarrhea, possessed enterotoxins STh, LT1, and EAST1 and colonization factors CS2 and CS3. Live ETEC1 shortened the life span and body size of C. elegans in association with increased expression of enterotoxin genes and intestinal colonization. In contrast, heat-killed ETEC1 did not affect the life span of C. elegans. Caenorhabditis elegans infected with ETEC1 showed upregulated expression of genes related to insulin-like peptides and host defense responses. These results suggest that ETEC1 exhibits pathogenicity through intestinal colonization and enterotoxin production in C. elegans. This system is useful as an ETEC infection model.

Colicins and T6SS-based competition systems enhance enterotoxigenic E. coli (ETEC) competitiveness.

Diarrheal diseases are still a significant problem for humankind, causing approximately half a million deaths annually. To cause diarrhea, enteric bacterial pathogens must first colonize the gut, which is a niche occupied by the normal bacterial microbiota. Therefore, the ability of pathogenic bacteria to inhibit the growth of other bacteria can facilitate the colonization process. Although enterotoxigenic Escherichia coli (ETEC) is one of the major causative agents of diarrheal diseases, little is known about the competition systems found in and used by ETEC and how they contribute to the ability of ETEC to colonize a host. Here, we collected a set of 94 fully assembled ETEC genomes by performing whole-genome sequencing and mining the NCBI RefSeq database. Using this set, we performed a comprehensive search for delivered bacterial toxins and investigated how these toxins contribute to ETEC competitiveness in vitro. We found that type VI secretion systems (T6SS) were widespread among ETEC (n = 47). In addition, several closely related ETEC strains were found to encode Colicin Ia and T6SS (n = 8). These toxins provide ETEC competitive advantages during in vitro competition against other E. coli, suggesting that the role of T6SS as well as colicins in ETEC biology has until now been underappreciated.

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.

ArcA positively regulates the expression of virulence genes and contributes to virulence of porcine Shiga toxin-producing enterotoxigenic Escherichia coli.

IMPORTANCE: Enterotoxigenic Escherichia coli (ETEC) cause severe diarrhea in humans and animals, leading to death and huge economic loss worldwide. Thus, elucidation of ETEC's pathogenic mechanisms will provide powerful data for the discovery of drugs serving as prevention or therapeutics against ETEC-caused diarrheal diseases. Here, we report that ArcA plays an essential role in the pathogenicity and virulence regulation in ETEC by positively regulating the expression of several key virulence factors including F18 fimbriae, heat-labile and heat-stable toxins, Shiga toxin 2e, and hemolysin, under microaerobic conditions and in vivo. Moreover, we found that positive regulation of several virulence genes by ArcA requires a global repressor H-NS (histone-like nucleoid structuring), implying that ArcA may exert positive effects by antagonizing H-NS. Collectively, our data established a key role for ArcA in the pathogenicity of porcine ETEC and ETEC strains isolated from human infections. Moreover, our work reveals another layer of regulation in relation to oxygen control of virulence factors in ETEC.

The role of the minor colonization factor CS14 in adherence to intestinal cell models by geographically diverse ETEC isolates.

Enterotoxigenic Escherichia coli (ETEC) is a primary causative agent of diarrhea in travelers and young children in low- to middle-income countries. ETEC adheres to small intestinal epithelia via colonization factors (CFs) and secretes heat-stable toxin and/or heat-labile toxin, causing dysregulated ion transport and water secretion. There are over 30 CFs identified, including major CFs associated with moderate-to-severe diarrhea (MSD) and minor CFs for which a role in pathogenesis is less clear. The Global Enteric Multicenter Study identified CS14, a class 5a fimbriae, as the only minor CF significantly associated with MSD and was recommended for inclusion in ETEC vaccines. Despite detection of CS14 in ETEC isolates, the sequence conservation of the CS14 operon, its role in adherence, and functional cross-reactivity to other class 5a fimbriae like CFA/I and CS4 are not understood. Sequence analysis determined that the CS14 operon is >99.9% identical among seven geographically diverse isolates with expanded sequence analysis demonstrating SNPs exclusively in the gene encoding the tip adhesin CsuD. Western blots and electron microscopy demonstrated that CS14 expression required the growth of isolates on CFA agar with the iron chelator deferoxamine mesylate. CS14 expression resulted in significantly increased adherence to cultured intestinal cells and human enteroids. Anti-CS14 antibodies and anti-CS4 antibodies, but not anti-CFA/I antibodies, inhibited the adherence of a subset of ETEC isolates, demonstrating CS14-specific inhibition with partial cross-reactivity within the class 5a fimbrial family. These data provide support for CS14 as an important fimbrial CF and its consideration as a vaccine antigen in future strategies. IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) infection causes profuse watery diarrhea in adults and children in low- to middle-income countries and is a leading cause of traveler's diarrhea. Despite increased use of rehydration therapies, young children especially can suffer long-term effects including gastrointestinal dysfunction as well as stunting and malnutrition. As there is no licensed vaccine for ETEC, there remains a need to identify and understand specific antigens for inclusion in vaccine strategies. This study investigated one adhesin named CS14. This adhesin is expressed on the bacterial surface of ETEC isolates and was recently recognized for its significant association with diarrheal disease. We demonstrated that CS14 plays a role in bacterial adhesion to human target cells, a critical first step in the disease process, and that adherence could be blocked by CS14-specific antibodies. This work will significantly impact the ETEC field by supporting inclusion of CS14 as an antigen for ETEC vaccines.

Genetic background of neomycin resistance in clinical Escherichia coli isolated from Danish pig farms.

Neomycin is the first-choice antibiotic for the treatment of porcine enteritis caused by enterotoxigenic Escherichia coli. Resistance to this aminoglycoside is on the rise after the increased use of neomycin due to the ban on zinc oxide. We identified the neomycin resistance determinants and plasmid contents in a historical collection of 128 neomycin-resistant clinical E. coli isolates from Danish pig farms. All isolates were characterized by whole-genome sequencing and antimicrobial susceptibility testing, followed by conjugation experiments and long-read sequencing of eight selected representative strains. We detected 35 sequence types (STs) with ST100 being the most prevalent lineage (38.3%). Neomycin resistance was associated with two resistance genes, namely aph(3')-Ia and aph(3')-Ib, which were identified in 93% and 7% of the isolates, respectively. The aph(3')-Ia was found on different large conjugative plasmids belonging to IncI1α, which was present in 67.2% of the strains, on IncHI1, IncHI2, and IncN, as well as on a multicopy ColRNAI plasmid. All these plasmids except ColRNAI carried genes encoding resistance to other antimicrobials or heavy metals, highlighting the risk of co-selection. The aph(3')-Ib gene occurred on a 19 kb chimeric, mobilizable plasmid that contained elements tracing back its origin to distantly related genera. While aph(3')-Ia was flanked by either Tn903 or Tn4352 derivatives, no clear association was observed between aph(3')-Ib and mobile genetic elements. In conclusion, the spread of neomycin resistance in porcine clinical E. coli is driven by two resistance determinants located on distinct plasmid scaffolds circulating within a highly diverse population dominated by ST100. IMPORTANCE Neomycin is the first-choice antibiotic for the management of Escherichia coli enteritis in pigs. This work shows that aph(3')-Ia and to a lesser extent aph(3')-Ib are responsible for the spread of neomycin resistance that has been recently observed among pig clinical isolates and elucidates the mechanisms of dissemination of these two resistance determinants. The aph(3')-Ia gene is located on different conjugative plasmid scaffolds and is associated with two distinct transposable elements (Tn903 and Tn4352) that contributed to its spread. The diffusion of aph(3')-Ib is mediated by a small non-conjugative, mobilizable chimeric plasmid that likely derived from distantly related members of the Pseudomonadota phylum and was not associated with any detectable mobile genetic element. Although the spread of neomycin resistance is largely attributable to horizontal transfer, both resistance determinants have been acquired by a predominant lineage (ST100) associated with enterotoxigenic E. coli, which accounted for approximately one-third of the strains.

Circulation of enterotoxigenic Escherichia coli (ETEC) isolates expressing CS23 from the environment to clinical settings.

IMPORTANCE: The importance of clean water cannot be overstated. It is a vital resource for maintaining health and well-being. Unfortunately, water sources contaminated with fecal discharges from animal and human origin due to a lack of wastewater management pose a significant risk to communities, as they can become a means of transmission of pathogenic bacteria like enterotoxigenic E. coli (ETEC). ETEC is frequently found in polluted water in countries with a high prevalence of diarrheal diseases, such as Bolivia. This study provides novel insights into the circulation of ETEC between diarrheal cases and polluted water sources in areas with high rates of diarrheal disease. These findings highlight the Choqueyapu River as a potential reservoir for emerging pathogens carrying antibiotic-resistance genes, making it a crucial area for monitoring and intervention. Furthermore, the results demonstrate the feasibility of a low-cost, high-throughput method for tracking bacterial pathogens in low- and middle-income countries, making it a valuable tool for One Health monitoring efforts.

Molecular and Genomic Analysis of the Virulence Factors and Potential Transmission of Hybrid Enteropathogenic and Enterotoxigenic Escherichia coli (EPEC/ETEC) Strains Isolated in South Korea.

Hybrid strains Escherichia coli acquires genetic characteristics from multiple pathotypes and is speculated to be more virulent; however, understanding their pathogenicity is elusive. Here, we performed genome-based characterization of the hybrid of enteropathogenic (EPEC) and enterotoxigenic E. coli (ETEC), the strains that cause diarrhea and mortality in children. The virulence genes in the strains isolated from different sources in the South Korea were identified, and their phylogenetic positions were analyzed. The EPEC/ETEC hybrid strains harbored eae and est encoding E. coli attaching and effacing lesions and heat-stable enterotoxins of EPEC and ETEC, respectively. Genome-wide phylogeny revealed that all hybrids (n = 6) were closely related to EPEC strains, implying the potential acquisition of ETEC virulence genes during ETEC/EPEC hybrid emergence. The hybrids represented diverse serotypes (O153:H19 (n = 3), O49:H10 (n = 2), and O71:H19 (n = 1)) and sequence types (ST546, n = 4; ST785, n = 2). Furthermore, heat-stable toxin-encoding plasmids possessing estA and various other virulence genes and transporters, including nleH2, hlyA, hlyB, hlyC, hlyD, espC, espP, phage endopeptidase Rz, and phage holin, were identified. These findings provide insights into understanding the pathogenicity of EPEC/ETEC hybrid strains and may aid in comparative studies, virulence characterization, and understanding evolutionary biology.

Distribution of Escherichia coli Pathotypes along an Urban-Rural Gradient in Ecuador.

Diarrheal diseases are a leading cause of mortality and morbidity in low- and middle-income countries. Diarrhea is associated with a wide array of etiological agents including bacterial, viral, and parasitic enteropathogens. Previous studies have captured between- but not within-country heterogeneities in enteropathogen prevalence and severity. We conducted a case-control study of diarrhea to understand how rates and outcomes of infection with diarrheagenic pathotypes of Escherichia coli vary across an urban-rural gradient in four sites in Ecuador. We found variability by site in enteropathogen prevalence and infection outcomes. Any pathogenic E. coli infection, coinfections, diffuse adherent E. coli (DAEC), enteroinvasive E. coli (EIEC), and rotavirus were significantly associated with acute diarrhea. DAEC was the most common pathotype overall and was more frequently associated with disease in urban areas. Enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) were more common in rural areas. ETEC was only associated with diarrhea in one site. Phylogenetic analysis revealed that associations with disease were not driven by any single clonal complex. Higher levels of antibiotic resistance were detected in rural areas. Enteropathogen prevalence, virulence, and antibiotic resistance patterns vary substantially by site within Ecuador. The variations in E. coli pathotype prevalence and virulence in this study have important implications for control strategies by context and demonstrate the importance of capturing within-country differences in enteropathogen disease dynamics.

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.

Complete association between CHCF1 genotype and enterotoxigenic Escherichia coli F4ab-associated post-weaning diarrhea in a pig challenge trial.

Enterotoxigenic E. coli (ETEC) susceptibility in pigs is highly influenced by their genotype. The aim of this study was to determine the association between CHCF1 genotype and ETEC F4ab susceptibility in experimentally infected pigs. We investigated ETEC diarrhea development in CHCF1 heterozygous susceptible (RS) (n = 12 pigs) compared to CHCF1 homozygous resistant (RR) (n = 12 pigs) for six days after ETEC F4ab challenge. Afterwards, we genotyped with MUC4 and MUC13 markers to relate performance in identifying ETEC F4ab diarrhea susceptible pigs. In the CHCF1 RS group, 12/12 pigs developed ETEC diarrhea compared with 0/12 pigs in the CHCF1 RR group. Weight gain was lower in CHCF1 RS pigs compared with RR pigs (mean ± SD: 208 ± 323 g and 987 ± 615 g, p = 0.0007). Further, the shedding of hemolytic E. coli was significantly higher in CHCF1 RS pigs from 2 to 6 days post inoculation and they shed the challenge strain for more days (mean ± SD: 3.5 ± 1.6 days versus 0.5 ± 0.5 days, p < 0.0001). Twelve pigs with ETEC diarrhea were misclassified as resistant with the MUC4 marker and four pigs without ETEC diarrhea were misclassified as susceptible with the MUC13 marker. We found complete association between CHCF1 genotype and ETEC diarrhea development in pigs from a herd with Danbred genetics. The CHCF1 marker was more likely to determine the true host susceptibility to ETEC F4ab than the other markers. The marker shows potential for improving reliability of PWD challenge models and potentially for use in breeding for ETEC F4ab/ac resistance.

In Vitro Effect on Piglet Gut Microbiota and In Vivo Assessment of Newly Isolated Bacteriophages against F18 Enterotoxigenic Escherichia coli (ETEC).

Enterotoxigenic Escherichia coli (ETEC) causing post-weaning diarrhea (PWD) in piglets have a detrimental impact on animal health and economy in pig production. ETEC strains can adhere to the host's small intestinal epithelial cells using fimbriae such as F4 and F18. Phage therapy could represent an interesting alternative to antimicrobial resistance against ETEC infections. In this study, four bacteriophages, named vB_EcoS_ULIM2, vB_EcoM_ULIM3, vB_EcoM_ULIM8 and vB_EcoM_ULIM9, were isolated against an O8:F18 E. coli strain (A-I-210) and selected based on their host range. These phages were characterized in vitro, showing a lytic activity over a pH (4-10) and temperature (25-45 °C) range. According to genomic analysis, these bacteriophages belong to the Caudoviricetes class. No gene related to lysogeny was identified. The in vivo Galleria mellonella larvae model suggested the therapeutic potential of one selected phage, vB_EcoS_ULIM2, with a statistically significant increase in survival compared to non-treated larvae. To assess the effect of this phage on the piglet gut microbiota, vB_EcoS_ULIM2 was inoculated in a static model simulating the piglet intestinal microbial ecosystem for 72 h. This study shows that this phage replicates efficiently both in vitro and in vivo in a Galleria mellonella model and reveals the safety of the phage-based treatment on the piglet microbiota.

Isolation, characterization and whole genome analysis of the novel genus Lederbergvirus, phage vB_EcoP_E212 infecting enterotoxigenic Escherichia coli K88.

The newly discovered phage vB_EcoP_E212 (also known as E212) was characterized and its genome was annotated in this study, which was conducted in Jilin, China. Transmission electron microscopy indicates that phage E212 belongs to the class Caudoviricetes. This phage exclusively infects enterotoxigenic E. coli K88. E212 was found to have a short latent period of 20 min, and a burst size of 125 PFU/cell. Additionally, E212 remained stable at all pH levels (3.0-12.0) and temperatures between -20 and 60 ºC. The genome of the phage E212 consists of 38,252 bp dsDNA molecule with a G + C content of 46.98%. The genome is projected to include 53 ORFs but no tRNAs. This phage lacks homologs of virulence factors or antimicrobial resistance genes, but it has lysogeny-related genes. Phage E212 was placed in the genus Lederbergvirus as a result of nucleotide sequence alignment and phylogenetic analysis.

The CpxRA two-component system represses gene expression of the heat-labile toxin of enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) strains produce at least one of two types of enterotoxins: the heat-labile (LT) and heat-stable (ST) toxins, which are responsible for the watery secretory diarrhoea that is a hallmark of the human ETEC infection. One regulatory system that controls the transcription of virulence genes in pathogenic bacteria is the CpxRA two-component system (TCS). We reported that the eltAB bicistronic operon, which encodes for the A and B subunits of LT, was repressed for the CpxRA TCS by direct binding of CpxR-P from -12 to +6 bp with respect to the transcription start site of eltAB. Moreover, the Cpx-response activation down-regulated the transcription of eltAB genes, and this negative effect was CpxRA-dependent. Our data show that CpxRA TCS is a negative regulator of the LT, one of the main virulence determinants of ETEC.

Genomic characterization of endemic diarrheagenic Escherichia coli and Escherichia albertii from infants with diarrhea in Vietnam.

BACKGROUND: Diarrheagenic Escherichia coli (DEC) is a group of bacterial pathogens that causes life-threatening diarrhea in children in developing countries. However, there is limited information on the characteristics of DEC isolated from patients in these countries. A detailed genomic analysis of 61 DEC-like isolates from infants with diarrhea was performed to clarify and share the characteristics of DEC prevalent in Vietnam. PRINCIPAL FINDINGS: DEC was classified into 57 strains, including 33 enteroaggregative E. coli (EAEC) (54.1%), 20 enteropathogenic E. coli (EPEC) (32.8%), two enteroinvasive E. coli (EIEC) (3.3%), one enterotoxigenic E. coli (ETEC), and one ETEC/EIEC hybrid (1.6% each), and surprisingly into four Escherichia albertii strains (6.6%). Furthermore, several epidemic DEC clones showed an uncommon combination of pathotypes and serotypes, such as EAEC Og130:Hg27, EAEC OgGp9:Hg18, EAEC OgX13:H27, EPEC OgGp7:Hg16, and E. albertii EAOg1:HgUT. Genomic analysis also revealed the presence of various genes and mutations associated with antibiotic resistance in many isolates. Strains that demonstrate potential resistance to ciprofloxacin and ceftriaxone, drugs recommended for treating childhood diarrhea, accounted for 65.6% and 41%, respectively. SIGNIFICANCE: Our finding indicate that the routine use of these antibiotics has selected resistant DECs, resulting in a situation where these drugs do not provide in therapeutic effects for some patients. Bridging this gap requires continuous investigations and information sharing regarding the type and distribution of endemic DEC and E. albertii and their antibiotic resistance in different countries.