Enterotoxigenic Escherichia Coli Strains Research Articles

307 Awardee Talk: Effects of dietary valerate glycerides on weanling piglet diarrhea and immunity

Abstract The use of various feed additives for their non-nutrient, immunomodulatory qualities is under investigation for application in swine production, especially during the post-weaning period. Butyrate (C4) is a known modulator of intestinal physiology, produced endogenously by commensal microbes and found in other organic sources. However, the understanding of the role of valerate (C5) as a feed additive for pigs has not been well-evaluated. Two experiments were conducted utilizing glycerides of butyrate or valerate as feed additives for weanling piglets under enterotoxigenic Escherichia coli (ETEC) infection conditions. In both experiments, daily diarrhea scores were recorded to monitor the frequency of diarrhea. The first pilot experiment utilized 20 weanling piglets (21 to 24 d of age) housed individually and assigned to either control diet, or control diet supplemented with 0.10% butyrate glycerides or 0.10% valerate glycerides. This experiment also aimed to establish a coinfection disease model, administering two strains of ETEC at a dose of 0.5 × 109 CFU/1.5 mL for each strain after a 7-d adaptation period (F4 and F18; two highly prevalent strains in swine production). Overall, piglets had significantly (P < 0.05) decreased frequency of diarrhea in either treatment group compared with control, suggesting similar modulatory effects by dietary valerate glycerides and butyrate glycerides. In addition, pigs fed valerate glycerides tended (P = 0.061) to have less neutrophils and had significantly (P < 0.05) less serum TNF-α on d 4 post-inoculation (PI). Moving forward, a full-scale experiment was performed using 60 weanling piglets (21 to 24 d of age) to further investigate the effect of dietary valerate glycerides on piglet health under ETEC F18 infection conditions. Treatments included nursery basal diet (control), 0.075% or 0.10% monovalerin, or 0.1% trivalerin added to control. Pigs fed 0.1% trivalerin exhibited significantly (P < 0.05) reduced frequency of diarrhea and fecal shedding of ETEC F18. Serum samples were collected throughout the experimental period and six replicate pigs per treatment group were sacrificed on d 7 PI to collect intestinal samples for morphological analysis and intestinal mucosa RT-qPCR analysis. Pigs fed trivalerin had reduced (P < 0.05) serum TNF-α on d 0 before inoculation and d 6 PI compared with pigs in control. The mRNA expression of TNFA in ileal mucosa from pigs fed 0.1% monovalerin tended (P < 0.10) to be lower compared with control. In addition, ZO1 mRNA expression tended (P < 0.10) to be increased in jejunal mucosa from pigs fed 0.1% trivalerin. Overall, the second experiment indicated that dietary supplementation of valerate glycerides reduced diarrhea and fecal shedding of ETEC F18 in piglets infected with ETEC. In conclusion, both experiments provide evidence in support of supplementing 0.075 or 0.10% valerate glycerides could enhance disease resistance of weanling piglets experiencing ETEC-associated diarrhea.

A Novel Multiepitope Fusion Antigen as a Vaccine Candidate for the Prevention of Enterotoxigenic E. coli-Induced Calf Diarrhea.

Calf diarrhea caused by enterotoxigenic E. coli (ETEC) poses an enormous economic challenge in the cattle industry. Fimbriae and enterotoxin are crucial virulence factors and vaccine targets of ETEC. Since these proteins have complicated components with large molecular masses, the development of vaccines by directly expressing these potential targets is cumbersome Therefore, this study aimed to develop a multiepitope fusion antigen designated as MEFA by integrating major epitopes of FanC and Fim41a subunits and a toxoid epitope of STa into the F17G framework. The 3D modeling predicted that the MEFA protein displayed the epitopes from these four antigens on its surface, demonstrating the desired structural characteristics. Then, the MEFA protein was subsequently expressed and purified for mouse immunization. Following that, our homemade ELISA showed that the mouse antiserum had a consistent increase in polyclonal antibody levels with the highest titer of 1:217 to MEFA. Furthermore, the western blot assay demonstrated that this anti-MEFA serum could react with all four antigens. Further, this antiserum exhibited inhibition on ETEC adhesion to HCT-8 cells with inhibitory rates of 92.8%, 84.3%, and 87.9% against F17+, F5+, and F41+ ETEC strains, respectively. Additionally, the stimulatory effect of STa toxin on HCT-8 cells was decreased by approximately 75.3% by anti-MEFA serum. This study demonstrates that the MEFA protein would be an antigen candidate for novel subunit vaccines for preventing ETEC-induced diarrhea in cattle.

Validation of a Human Challenge Model Using an LT-Expressing Enterotoxigenic E. coli Strain (LSN03-016011) and Characterization of Potential Amelioration of Disease by an Investigational Oral Vaccine Candidate (VLA1701).

Controlled human infection models are important tools for the evaluation of vaccines against diseases where an appropriate correlate of protection has not been identified. Enterotoxigenic Escherichia coli (ETEC) strain LSN03-016011/A (LSN03) is an LT enterotoxin and CS17-expressing ETEC strain useful for evaluating vaccine candidates targeting LT-expressing strains. We sought to confirm the ability of the LSN03 strain to induce moderate-to-severe diarrhea in a healthy American adult population, as well as the impact of immunization with an investigational cholera/ETEC vaccine (VLA-1701) on disease outcomes. A randomized, double-blinded pilot study was conducted in which participants received two doses of VLA1701 or placebo orally, one week apart; eight days after the second vaccination, 30 participants (15 vaccinees and 15 placebo recipients) were challenged with approximately 5 × 109 colony-forming units of LSN03. The vaccine was well tolerated, with no significant adverse events. The vaccine also induced serum IgA and IgG responses to LT. After challenge, 11 of the placebo recipients (73.3%; 95%CI: 48.0-89.1) and 7 of the VLA1701 recipients (46.7%; 95%CI: 24.8-68.8) had moderate-to-severe diarrhea (p = 0.26), while 14 placebo recipients (93%) and 8 vaccine recipients (53.3%) experienced diarrhea of any severity, resulting in a protective efficacy of 42.9% (p = 0.035). In addition, the vaccine also appeared to provide protection against more severe diarrhea (p = 0.054). Vaccinees also tended to shed lower levels of the LSN03 challenge strain compared to placebo recipients (p = 0.056). In addition, the disease severity score was lower for the vaccinees than for the placebo recipients (p = 0.046). In summary, the LSN03 ETEC challenge strain induced moderate-to-severe diarrhea in 73.3% of placebo recipients. VLA1701 vaccination ameliorated disease severity, as observed by several parameters, including the percentage of participants experiencing diarrhea, as well as stool frequency and ETEC severity scores. These data highlight the potential value of LSN03 as a suitable ETEC challenge strain to evaluate LT-based vaccine targets (NCT03576183).

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.

Preparation of Bispecific IgY-scFvs Inhibition Adherences of Enterotoxigenic Escherichia coli (K88 and F18) to Porcine IPEC-J2 Cell.

Enterotoxigenic Escherichia coli (ETEC) strains are significant contributors to postweaning diarrhea in piglets. Of the ETEC causing diarrhea, K88 and F18 accounted for 92.7%. Despite the prevalence of ETEC K88 and F18, there is currently no effective vaccine available due to the diversity of these strains. This study presents an innovative approach by isolating chicken-derived single-chain variable fragment antibodies (scFvs) specific to K88 and F18 fimbrial antigens from chickens immunized against these ETEC virulence factors. These scFvs effectively inhibited adhesion of K88 and F18 to porcine intestinal epithelial cells (IPEC-J2), with the inhibitory effect demonstrating a dose-dependent increase. Furthermore, a bispecific scFv was designed and expressed in Pichia pastoris. This engineered construct displayed remarkable potency; at a concentration of 25.08 μg, it significantly reduced the adhesion rate of ETEC strains to IPEC-J2 cells by 72.10% and 69.11% when challenged with either K88 or F18 alone. Even in the presence of both antigens, the adhesion rate was notably decreased by 57.92%. By targeting and impeding the initial adhesion step of ETEC pathogenesis, this antibody-based intervention holds promise as a potential alternative to antibiotics, thereby mitigating the risks associated with antibiotic resistance and residual drug contamination in livestock production. Overall, this study lays the groundwork for the development of innovative treatments against ETEC infections in piglets.

MecVax supplemented with CFA MEFA-II induces functional antibodies against 12 adhesins (CFA/I, CS1-CS7, CS12, CS14, CS17, and CS21) and 2 toxins (STa, LT) of enterotoxigenic Escherichia coli (ETEC).

Enterotoxigenic Escherichia coli (ETEC) strains that produce various adhesins and one or two enterotoxins are the leading causes of children's diarrhea and travelers' diarrhea. MecVax, a multivalent ETEC vaccine candidate, consists of two proteins, an adhesin multiepitope fusion antigen (MEFA) that stimulates antibodies to the seven most important ETEC adhesins (CFA/I and CS1-CS6) and a toxoid fusion antigen which stimulates antibodies against ETEC enterotoxins (heat-labile toxin and heat-stable toxin). CFA MEFA-II, another polyvalent MEFA protein, has been demonstrated to stimulate antibodies to another five important ETEC adhesins (CS7, CS12, CS14, CS17, and CS21). We hypothesize that MecVax coverage and efficacy can be expanded if MecVax could stimulate antibodies to all 12 adhesins. In this study, we supplemented MecVax with CFA MEFA-II, examined broad immunity to the 12 targeted ETEC adhesins and 2 ETEC toxins (STa, LT) in mice, and assessed mouse antibody functions for inhibiting the adherence of the 12 adhesins and neutralizing the enterotoxicity of 2 toxins, thus assessing the potential application of a broadly protective pan-ETEC vaccine. Mice intramuscularly immunized with MecVax and CFA MEFA-II developed robust antibody responses to the 12 ETEC adhesins and 2 toxins; furthermore, mouse serum antibodies showed functional activities against the adherence from each of the targeted adhesins and the enterotoxicity of either toxin. Data also indicated that CFA MEFA-II was antigenically compatible with MecVax. These results demonstrated that the inclusion of CFA MEFA-II further expands MecVax broad immunogenicity and protection coverage, suggesting the feasibility of developing a vaccine against all important diarrheal ETEC strains.IMPORTANCEThere are no vaccines licensed for Enterotoxigenic Escherichia coli (ETEC), a leading cause of children's diarrhea and the most common cause of travelers' diarrhea. Since ETEC strains produce over 25 adhesins and 2 distinctive enterotoxins, heterogeneity is a key obstacle to vaccine development. MecVax, a multivalent ETEC vaccine candidate, induces protective antibodies against the seven most important adhesins (CFA/I and CS1-CS6) associated with two-thirds of ETEC clinical cases. However, ETEC prevalence shifts chronically and geographically, and other adhesins are also associated with clinical cases. MecVax would become a pan-ETEC vaccine if it also protects against the remaining important adhesins. This study demonstrated that MecVax supplemented with adhesin protein CFA MEFA-II induces functional antibodies against 12 important ETEC adhesins (CFA/I, CS1-CS7, CS12, CS14, CS17, and CS21), enabling the development of a more broadly protective ETEC vaccine and further validating the application of the MEFA vaccinology platform for multivalent vaccine development.

The Antimicrobial Resistance of Enterotoxigenic Escherichia coli from Diarrheal Patients and the Environment in Two Geographically Distinct Rural Areas in Bangladesh over the Years.

Antimicrobial resistance (AMR) is an unprecedented global health challenge, involving the transfer of bacteria and genes between humans and the environment. We simultaneously and longitudinally determined the AMR of enterotoxigenic Escherichia coli (ETEC) strains isolated from diarrheal patients and an aquatic environment over two years from two geographically distinct locations, Coastal Mathbaria and Northern Chhatak in Bangladesh. A total of 60% and 72% of ETEC strains from the patients in Mathbaria and Chhatak, respectively, were multi-drug resistant (MDR) with a high proportion of ETEC resistant to nalidixic acid (80.7%), macrolides (49.1-89.7%), ampicillin (57.9-69%), and trimethoprim/sulfamethoxazole (55.2%). From the surface water, 68.8% and 30% of ETEC were MDR in Mathbaria and Chhatak, respectively, with a high proportion of ETEC strains resistant to macrolides (87.5-100%), ampicillin (50-75%), ceftriaxone (62.5%), and nalidixic acid (40%). Notably, 80-100% of the ETEC strains were susceptible to tetracycline and quinolones (ciprofloxacin and norfloxacin), both in clinical and aquatic ETEC. The AMR varied by the ETEC toxin types. The patterns of excessive or limited consumption of drugs to treat diarrhea over time in Bangladesh were reflected in the ETEC AMR from the patients and the environment. The high prevalence of MDR-ETEC strains in humans and the environment is of concern, which calls for vaccines and other preventative measures against ETEC.

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.

A Review on Ecoli 0157: H7 (EHEC)

Escherichia coli O157:H7 is an important food borne and water borne pathogen is first isolated in 1982, shiga toxin. producing Ecoli O157:H7 which causes diarrhea, hemorrhagic colitis and haemolytic uremic Syndrome in humans. Haemolytic uremic. Syndrome (Hus) is a Serious condition and upto 50% of Hus patients can develop renal acute dysfunction or blood pressure. Ecoli 0157:H7 infects the alimentary tract and induces abdominal cramps with hemorrhagic diarrhea and it can be transmitted by person to person through fecal shedding and 11%. of infection are estimated. Enterotoxigenic Escherichia coli(ETEC) strain are the main cause of the diarrheal disease problems. ETEC strains are the bacteria which cause the acute watery diarrhea in chinldren. Diarrheagenic Ecoli infection are more common in Africa countries. The occurance of antimicrobial resistance depends upon the type of antimicrobialagent,bacterial strain,dose,time and mode of ministration. some of Ecoli 0517:H7 occurs after taking the undercooked meat or deficiency pasteurized dairy products. And some of Ecoli 0157:H7 occurs due ro exposal to contaminated water from potable drinking sources,pools and lakes and foods such as inappropriate washed leafy green vegetables and fruits,apple juice and direct contact with contaminated animals. Ecoli 0157:H7 shiga toxin causes leukocyte aggregation,platelet aggregation,microthrombin formation, apoptosis of affected cells ,renal dysfunction. shiga toxin not only effect kidney but it can also effect in diffuse vasculitic injuries which cause the effect on multiple organ system and organ failure.

Protein-based vaccine candidate MecVax broadly protects against enterotoxigenic Escherichia coli intestinal colonization in a rabbit model.

There are no vaccines licensed against enterotoxigenic Escherichia coli (ETEC), a leading cause of children's diarrhea and the most common cause of travelers' diarrhea. Multivalent vaccine candidate MecVax unprecedentedly targets two ETEC enterotoxins (heat-stable toxin, STa; heat-labile toxin, LT) and the seven most prevalent ETEC adhesins (colonization factor antigen, CFA/I, coli surface antigens, CS1-CS6) and has been demonstrated preclinically to protect against STa- and LT-mediated ETEC clinical diarrhea and prevent intestinal colonization from ETEC strain H10407 (CFA/I, STa, LT). However, it is unattested whether MecVax broadly protects against intestinal colonization from ETEC strains producing the other six adhesins (CS1-CS6) also targeted by this product. In this study, we immunized rabbits with MecVax and challenged them with heterogeneous ETEC strains that express CS1-CS6 adhesins to evaluate MecVax's efficacy against bacterial intestinal colonization, thus providing broad vaccine protection against ETEC infection. Data revealed that rabbits intramuscularly immunized with MecVax developed robust responses to both ETEC enterotoxins (STa, LT) and seven adhesins (CFA/I, CS1-CS6), and when challenged with ETEC isolates expressing CS1/CS3, CS2/CS3, CS4/CS6, CS5/CS6, or CS6 adhesin, the immunized rabbits prevented over two logs (>99%) of bacteria from colonization in small intestines. Additionally, compared to a CFA-toxoid fusion protein, which is another potential ETEC vaccine antigen to target two ETEC enterotoxins and the seven adhesins, MecVax exhibited better protection against ETEC intestinal colonization. These results, in conjunction with the protection data from early studies, evidenced that MecVax is broadly protective, validating MecVax's candidacy as an effective vaccine against ETEC-associated diarrhea and accelerating ETEC vaccine development.

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-1β, IL-12p35; in moDCs, decreased mRNA levels for IL-1β). 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-1β, 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.

Structure, function, and potential application of F18 in vaccine development

Post-weaning diarrhea is one of the most common diseases in pigs, affecting pigs in the first two weeks after weaning, causing significant economic losses. The main cause of this disease is Enterotoxigenic Escherichia coli (ETEC), which is characterized by two pathogenic factors: fimbriae and enterotoxins. These fimbriae are considered to mediate bacterial adhesion through binding to the receptor present in the pig intestine. Several studies indicate that F4 and F18 fimbriae account for a high percentage of the fimbriae in ETEC and are detected in most of the isolated ETEC strains. Currently, oral vaccines are a potential preventive measure used with the ability to generate IgA, stimulate the mucosal immune system, and be widely noticed by researchers. However, the vaccines are only effective against ETEC-F4 and less effective against ETEC-F18, so more research is needed to improve the effectiveness of ETEC-F18 vaccines in the future. In this review, we presented the structure, function, and potential application of the F18 in vaccine development.

Hyperimmune bovine colostrum containing lipopolysaccharide antibodies (IMM124-E) has a nondetrimental effect on gut microbial communities in unchallenged mice.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of bacterial diarrhea with the potential to cause long-term gastrointestinal (GI) dysfunction. Preventative treatments for ETEC-induced diarrhea exist, yet the effects of these treatments on GI commensals in healthy individuals are unclear. Whether administration of a prophylactic preventative treatment for ETEC-induced diarrhea causes specific shifts in gut microbial populations in controlled environments is also unknown. Here, we studied the effects of a hyperimmune bovine colostrum (IMM-124E) used in the manufacture of Travelan (AUST L 106709) on GI bacteria in healthy C57BL/6 mice. Using next-generation sequencing, we aimed to test the onset and magnitude of potential changes to the mouse gut microbiome in response to the antidiarrheagenic hyperimmune bovine colostrum product, rich in immunoglobulins against select ETEC strains (Travelan, Immuron Ltd). We show that in mice administered colostrum containing lipopolysaccharide (LPS) antibodies, there was an increased abundance of potentially gut-beneficial bacteria, such as Akkermansia and Desulfovibrio, without disrupting the underlying ecology of the GI tract. Compared to controls, there was no difference in overall weight gain, body or cecal weights, or small intestine length following LPS antibody colostrum supplementation. Overall, dietary supplementation with colostrum containing LPS antibodies produced subtle alterations in the gut bacterial composition of mice. Primarily, Travelan LPS antibody treatment decreased the ratio of Firmicutes/Bacteroidetes in gut microbial populations in unchallenged healthy mice. Further studies are required to examine the effect of Travelan LPS antibody treatment to engineer the microbiome in a diseased state and during recovery.

Anti-Toxin Responses to Natural Enterotoxigenic Escherichia coli (ETEC) Infection in Adults and Children in Bangladesh.

A sero-epidemiology study was conducted in Dhaka, Bangladesh between January 2020 and February 2021 to assess the immune responses to ETEC infection in adults and children. (1) Background: Enterotoxigenic Escherichia coli infection is a main cause of diarrheal disease in endemic countries. The characterization of the immune responses evoked by natural infection can guide vaccine development efforts. (2) Methods: A total of 617 adult and 480 pediatric diarrheal patients were screened, and 43 adults and 46 children (below 5 years of age) with an acute ETEC infection completed the study. The plasma samples were analyzed for antibody responses against the ETEC toxins. (3) Results: Heat-stable toxin (ST)-positive ETEC is the main cause of ETEC infection in adults, unlike in children in an endemic setting. We detected very low levels of anti-ST antibodies, and no ST-neutralizing activity. However, infection with ETEC strains expressing the heat-labile toxin (LT) induced systemic antibody responses in less than 25% of subjects. The antibody levels against LTA and LTB, as well as cholera toxin (CT), correlated well. The anti-LT antibodies were shown to have LT- and CT- neutralizing activity. The antibody reactivity against linear LT epitopes did not correlate with toxin-neutralizing activity. (4) Conclusions: Unlike LT, ST is a poor antigen and even adults have low anti-ST antibody levels that do not allow for the detection of toxin-neutralizing activity.

Feasibility of crude F4 fimbriae extract as a vaccine candidate for preventing Escherichia coli-induced diarrhea in piglets.

Enterotoxigenic Escherichia coli (ETEC) poses a substantial risk of neonatal diarrhea and post-weaning diarrhea among piglets, with F4+ ETEC strains emerging as a particularly challenging issue within the pig farming industry. This study aimed to introduce a straightforward approach for generating a crude extract of F4 fimbriae that shows promise as an antigenic determinant for potential vaccination strategies. A crude F4 fimbriae extract was obtained from F4+ ETEC using a combination of heat shock and homogenization techniques. Subsequently, three 4-week-old piglets were immunized with a primary dose of 150 μg and a booster dose 2 weeks later. Blood samples were collected to evaluate the level of serum F4-specific antibodies using an enzyme-linked immunosorbent assay. Analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography tandem-mass spectrometry techniques unveiled crucial insights into the composition of the crude F4 fimbriae extract. Notably, a distinct prominent band (~24 kDa) was identified, corresponding to the size of FaeG, the major subunit of F4 fimbriae. Regarding antibody response, there was a remarkable disparity between the levels of serum immunoglobulin (Ig)G and IgA antibodies targeting F4 compared with other E. coli strains (F18+ ETEC, F41+ ETEC, and F4-F18-F41- EC), as well as with the unvaccinated control group (p < 0.01). Specifically, the levels of IgG antibodies against other E. coli strains were also significantly higher than those observed in the unvaccinated control group (p < 0.05). Our findings suggest that the crude F4 fimbriae extracts obtained using our simple extraction method induce specific immune responses against F4+ E. coli and stimulate cross-immunity against other E. coli strains. Therefore, our method shows potential for use in future vaccine development against diarrhea in pigs caused by E. coli.

A Polyvalent Adhesin-Toxoid Multiepitope-Fusion-Antigen-Induced Functional Antibodies against Five Enterotoxigenic Escherichia coli Adhesins (CS7, CS12, CS14, CS17, and CS21) but Not Enterotoxins (LT and STa).

The increasing prevalence and association with moderate-to-severe diarrhea make enterotoxigenic Escherichia coli (ETEC) adhesins CS7, CS12, CS14, CS17, and CS21 potential targets of ETEC vaccines. Currently, there are no vaccines licensed to protect against ETEC, a top cause of children's diarrhea and travelers' diarrhea. Recently, a polyvalent adhesin protein (adhesin MEFA-II) was demonstrated to induce antibodies that inhibited adherence from these five ETEC adhesins and reduced the enterotoxicity of ETEC heat-stable toxin (STa), which plays a key role in causing ETEC-associated diarrhea. To improve adhesin MEFA-II for functional antibodies against STa toxin and the other ETEC toxin, heat-labile toxin (LT), we modified adhesin MEFA-II by adding another STa toxoid and an LT epitope; we examined the new antigen immunogenicity (to five adhesins and two toxins) and more importantly antibody functions against ETEC adherence and STa and LT enterotoxicity. Data show that mice intramuscularly immunized with the new antigen (adhesin MEFA-IIb) developed robust IgG responses to the targeted adhesins (CS7, CS12, CS14, CS17, and CS21) and toxins (STa and LT). Mouse antibodies inhibited the adherence of ETEC strains expressing any of these five adhesins but failed to neutralize STa or LT enterotoxicity. In further studies, rabbits intramuscularly immunized with adhesin MEFA-IIb developed robust antigen-specific antibodies; when challenged with an ETEC isolate expressing CS21 adhesin (JF2101, CS21, and STa), the immunized rabbits showed a significant reduction in intestinal colonization by ETEC bacteria. These data indicate that adhesin MEFA-IIb is broadly immunogenic and induces functional antibodies against the targeted ETEC adhesins but not the toxins.

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

Enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of infant diarrhea in low- and middle-income countries (LMICs). Diarrheal pathogens are transmitted through environmental reservoirs; however, the bacterial clones that spread across the human-environment interface remain unexplored. We aimed to determine the relationship and clonal dissemination of ETEC between children with diarrhea and polluted water samples from a local river in La Paz, Bolivia. By using WGS and the PhenePlates phenotypic system to analyze ETEC strains, we showed that ST218 and ST410 LT+STh ETEC expressing the colonization factor (CF) CS23 were found with high frequency in both samples. The CS23 ETEC isolates were found within several STs, E. coli phylogroups, and across ETEC lineages. Comparative genomic evaluation and PhenePlate screening of globally distributed clinical ETEC strains suggest that the CS23 gene is likely carried on plasmids acquired independently of the bacterial chromosomal background. Clinical strains were more often multidrug-resistant (MDR) than environmental isolates and harbored the class 1 integron-integrase gene intI1 next to the MDR cassettes. Retrospective analysis of antibiotic resistance in ETEC revealed a high frequency of MDR in clinical isolates. The LT+STh CS23 environmental ETEC isolates, showed an increased biofilm ability at environmental temperature, equal cytotoxicity, and significantly lower adherence to human epithelial cells compared to ETEC expressing other CFs. Together, we suggest that CS23 is more prevalent in ETEC than previously estimated, and the Choqueyapu River is a reservoir for LT+STh CS23 ETEC containing strains capable of causing diarrheal cases in children. 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.

Developing and Evaluating a Weaned Piglet Model of Enterotoxigenic Escherichia Coli Infection

Enterotoxigenic Escherichia Coli (ETEC) remains the main pathogen associated with post-weaning diarrhea in piglets. Here, we introduce a reliable method to develop an ETEC-infected weaned piglet model. For the first 24 h post-birth, neonatal piglets were separated from dams so that they did not receive colostrum. Afterwards, piglets were returned to the care of their mothers, until weaning (21-day-old). On days 25, 26 and 27 post-birth, piglets were orally challenged with 1011 Colony-Forming Units (CFU) of virulent ETEC strain incubated in colony-forming antigen medium and delivered in chitosan-coated capsules. In all infected piglets, severe diarrhea and typical body weight loss were observed post-infection. To simplify the work, for the newly developed method, the number of challenging ETEC cells was lowered to 1010 CFU/head and the incubation medium was changed to tryptic soy. These modifications influenced neither the diarrheal defecation ratio nor the body weight loss in piglets. To induce experimentally ETEC infection in future work, we recommend that 1010 CFU/head of ETEC cell grown in tryptic soy broth be delivered in chitosan-coated capsules to colostrum restricted piglets, because this methodology stably caused 100% diarrheal defecation and growth reduction.

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.

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.