Enterotoxigenic Escherichia Coli Isolates Research Articles

Predominance of enterotoxigenic Escherichia coli among ESBL/plasmid-mediated AmpC-producing strains isolated from diarrheic foals: a public health concern

BackgroundThe upsurge of diarrheagenic E. coli pathotypes carrying extended-spectrum beta-lactamases (ESBLs)/plasmid-mediated AmpC β-lactamase (pAmpC) among animals constitutes an emerging threat for humans and animals. This study investigated the burden of ESBL-/pAmpC-producing diarrheagenic E. coli among diarrheic foals and its potential public health implications. Rectal swabs were collected from 80 diarrheic foals. These swabs were processed to isolate and identify ESBL/pAmpC-producing E. coli using a selective culture medium, biochemical tests, phenotypic identification, and molecular identification of ESBL- and pAmpC-encoding genes. Moreover, all ESBL-/pAmpC-producing E. coli isolates were examined for different virulence genes related to diarrheagenic E. coli pathotypes.ResultsOut of 80 examined foals, 26 (32.5%) were confirmed as ESBL-/pAmpC-producing E. coli, of which 14 (17.5%) animals carried only ESBL-producing E. coli, whereas 12 (15%) animals possessed ESBL-pAmpC-producing E. coli. The only detected diarrheagenic pathotype was enterotoxigenic, encoded by the heat-stable enterotoxin gene (ST) with a prevalence rate of 80.8% (21/26). The ST gene was further characterized where STa, STb, and STa + STb were found in one, four, and 16 strains, respectively. Moreover, all enterotoxigenic E. coli (ETEC) isolates exhibited a multidrug-resistance pattern. The phylogenetic analysis of 3 obtained partial STb sequences revealed high genetic relatedness to ETEC isolates retrieved from humans, conferring such sequences' public health significance.ConclusionsThese findings highlight that diarrheic foals could serve as a potential reservoir for multidrug-resistant ESBL-/pAmpC-producing enterotoxigenic E. coli.

Development of an antibody against EtpA from enterotoxigenic Escherichia coli and evaluation of its use for bacterial isolation using magnetic beads.

The enterotoxigenic Escherichia coli (ETEC) strain is one of the most frequent causative agents of childhood diarrhea and travelers' diarrhea in low-and middle-income countries. Among the virulence factors secreted by ETEC, the exoprotein EtpA has been described as an important. In the present study, a new detection tool for enterotoxigenic E. coli bacteria using the EtpA protein was developed. Initially, antigenic sequences of the EtpA protein were selected via in silico prediction. A chimeric recombinant protein, corresponding to the selected regions, was expressed in an E. coli host, purified and used for the immunization of mice. The specific recognition of anti-EtpA IgG antibodies generated was evaluated using flow cytometry. The tests demonstrated that the antibodiesdeveloped were able to recognize the native EtpA protein. By coupling these antibodies to magnetic beads for the capture and detection of ETEC isolates, cytometric analyses showed an increase in sensitivity, specificity and the effectiveness of the method of separation and detection of these pathogens. This is the first report of the use of this methodology for ETEC separation. Future trials may indicate their potential use for isolating these and other pathogens in clinical samples, thus accelerating the diagnosis and treatment of diseases.

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.

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.

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.

Heat-Labile Enterotoxin Decreases Macrophage Phagocytosis of Enterotoxigenic Escherichia coli.

Enterotoxigenic E. coli (ETEC) are endemic in low-resource settings and cause robust secretory diarrheal disease in children less than five years of age. ETEC cause secretory diarrhea by producing the heat-stable (ST) and/or heat-labile (LT) enterotoxins. Recent studies have shown that ETEC can be carried asymptomatically in children and adults, but how ETEC subvert mucosal immunity to establish intestinal residency remains unclear. Macrophages are innate immune cells that can be exploited by enteric pathogens to evade mucosal immunity, so we interrogated the ability of ETEC and other E. coli pathovars to survive within macrophages. Using gentamicin protection assays, we show that ETEC H10407 is phagocytosed more readily than other ETEC and non-ETEC isolates. Furthermore, we demonstrate that ETEC H10407, at high bacterial burdens, causes nitrite accumulation in macrophages, which is indicative of a proinflammatory macrophage nitric oxide killing response. However, at low bacterial burdens, ETEC H10407 remains viable within macrophages for an extended period without nitrite accumulation. We demonstrate that LT, but not ST, intoxication decreases the number of ETEC phagocytosed by macrophages. Furthermore, we now show that macrophages exposed simultaneously to LPS and LT produce IL-33, which is a cytokine implicated in promoting macrophage alternative activation, iron recycling, and intestinal repair. Lastly, iron restriction using deferoxamine induces IL-33 receptor (IL-33R) expression and allows ETEC to escape macrophages. Altogether, these data demonstrate that LT provides ETEC with the ability to decrease the perceived ETEC burden and suppresses the initiation of inflammation. Furthermore, these data suggest that host IL-33/IL-33R signaling may augment pathways that promote iron restriction to facilitate ETEC escape from macrophages. These data could help explain novel mechanisms of immune subversion that may contribute to asymptomatic ETEC carriage.

Serotypes, Pathotypes, Shiga Toxin Variants and Antimicrobial Resistance in Diarrheagenic Escherichia coli Isolated from Rectal Swabs and Sheep Carcasses in an Abattoir in Mexico

Sheep represent one of the main reservoirs of diarrheagenic Escherichia coli; this microorganism is an etiological agent of food-borne diseases; therefore, this work aimed to identify and characterize the principal pathotypes of diarrheagenic E. coli (DEC) obtained through rectal swabs and carcasses samples from sheep slaughtered in an abattoir at the central region of Mexico. The isolates were subjected to bacteriological identification, serotyping; phylogenetic classification; detection for virulence factors, and antimicrobial sensibility. A total of 90 E. coli isolates were obtained. It was observed through 49 E. coli isolates (54%), 8 of them from carcasses, and 43 from feces was DEC. DEC serotypes with health public relevance were found: O76:H19 (n = 5), O146:H21 (n = 3), O91:H10 (n = 1), O6:NM (n = 1), and O8:NM (n = 1). Regarding the presence of Shiga toxin-producing E.coli (STEC), 43/90 (47.7%) isolates have the stx1 w/o stx2 genes, and therefore were assigned as STEC non-O157; only one isolate expressed stx1 and eae genes and was classified as t-STEC (typical STEC). Additionally, 3/90 (3.3%) harbored only the eae gene and were classified as enteropathogenic E. coli (EPEC), the stp gene was found in 2/90 isolates (2.2%) and were classified as enterotoxigenic E. coli (ETEC); 1/90 (1.1%) isolates harboring the ipaH were classified as enteroinvasive E. coli EIEC. Regarding stx1 genes subtypes, stx1c only was found in 60.5% (26/43), followed by stx1a-stx1c 20.9% (9/43) and stx1a-stx1d 2.3% (1/43). The presence of both, stx1 and stx2 genes was found in 7/43 isolates (16.3%) from rectal swabs; the combination stx1c-stx2g was detected in 3/43 isolates (6.9%), while 4 (9.4%) isolates showed different patterns (stx1a-stx1c-stx2g; stx1c-stx2b-stx2g; stx1c-stx2b and stx1a-stx1c-stx2b-stx2g). STEC isolates showed the major diversity of phylogenetic groups, although phylogroup B1 was predominant in 90.6% (39/43) while there was only one isolate (2.3%) in each remaining phylogroup (A, B2, C, and F). All EPEC, ETEC, and EIEC isolates were clustered in phylogroup B1. We observed that 27.9% (12/43) of STEC isolates carried at least one antibiotic resistance: nine isolates expressed the tetB gene, one isolate the tetA gene, two isolates the sul2 gene, one isolate the sul1 and one isolate the sul1-tetB genes. These results highlight the importance of diarrheagenic E. coli as a potential risk for public health during the slaughtering process.

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.

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.

Colonizing and Virulence Factors in Enterotoxigenic Escherichia coli from Peru.

Enterotoxigenic Escherichia coli (ETEC) ranks among the most relevant diarrheagenic pathogens. Efforts to design vaccines to fight ETEC have been focused on colonizing factors (CFs) and atypical virulence factors (AVF). An effective vaccine must account for differences in the regional prevalence of these CFs and AVFs to be truly effective in a given area. In the present study, the presence of 16 CFs and 9 AVFs, as well as the heat-stable (ST) variants (STh or STp), was established by polymerase chain reaction in 205 Peruvian ETEC isolates (120 from diarrhea cases and 85 from healthy controls). Ninety-nine (48.3%) isolates were heat-labile, 63 (30.7%) ST, and 43 (21.0%) presented both toxins. Of ST isolates, 59 (28.8%) possessed STh, 30 (14.6%) STp, five (2.4%) both STh and STp, and 12 (5.8%) were not amplified for any variant tested. The presence of CFs was associated with diarrhea (P < 0.0001). The presence of eatA as well as concomitant presence of CSI, CS3, and CS21 and of C5 and C6 was statistically related to diarrhea cases. The present results suggests that, if effective, a vaccine considering CS6, CS20, and CS21, together with EtpA, would provide protection against 64.4% of the isolates analyzed, whereas the addition of CS12 and EAST1 would lead to 83.9% coverage. Large studies are needed to establish both the ideal candidates to be considered to develop a vaccine effective in the area, and continuous surveillance is needed to detect displacement of circulating isolates that may compromise future vaccines.

Antimicrobial Resistance and Virulence Factors Assessment in Escherichia coli Isolated from Swine in Italy from 2017 to 2021

Prudent antibiotic use in pigs is critical to ensuring animal health and preventing the development of critical resistance. We evaluated the antimicrobial resistance (AMR) pattern in commensal and enterotoxigenic Escherichia coli (ETEC) isolates obtained in 2017−2021 from pigs suffering from enteric disorders. Overall, the selected 826 E. coli isolates showed the highest level of resistance to ampicillin (95.9%), tetracycline (89.7%), cefazolin (79.3%), and trimethoprim/sulfamethoxazole (74.8%). The resistance rates of the isolates to ampicillin increased (p < 0.05), reaching 99.2% of resistant strains in 2021. Regarding isolates harboring virulence genes, ETEC F18+ were significantly more resistant to florfenicol, gentamicin, kanamycin, and trimethoprim/sulfamethoxazole than ETEC F4+ strains. E. coli lacking virulence factor genes were more resistant to amoxicillin with clavulanic acid and cefazolin, but less resistant to gentamicin (p < 0.01) than isolates harboring virulence factors. Throughout the study period, a significant number of ETEC F18+ isolates developed resistance to florfenicol, gentamicin, and kanamycin. Finally, ETEC 18+ significantly (p < 0.05) increased resistance to all the tested antibiotics. In conclusion, AMR varied for E. coli over time and showed high levels for molecules widely administered in the swine industry, emphasizing the need for continuous surveillance. The observed differences in AMR between commensal and ETEC isolates may lead to the hypothesis that plasmids carrying virulence genes are also responsible for AMR in E. coli, suggesting more research on genetic variation between pathogenic and nonpathogenic E. coli.

Molecular detection of Enterotoxigenic Escherichia coli Toxins and Colonization Factors from Diarrheic Children in Pediatric Teaching Hospital, Sulaymaniyah, Iraq

Enterotoxigenic Escherichia coli (ETEC) is one well-established causative agent of diarrhea in the developing countries among young children. This prospective study was performed at Laboratories of University of Sulaimani (in Sulaymaniyah City/Iraq) from September to October 2021which aimed to determine the prevalence of ETEC among children and the most prevalence colonization factor (CFA/I) among ETEC. One hundred and twenty-five fresh stool samples were collected from hospitalized – children with diarrhea at Dr. Jamal Ahmed Rashid’s Pediatric Teaching Hospital. The collected samples were cultured on MacConkey and eosin methylene blue agar as selective and differential media for Gram- negative bacteria. Colonies were identified through Gram staining and biochemical tests including: Indole, methyl red, and catalase reaction test. Vitek-2 machine was depended to test some obtained isolates. Most of isolates (60%) showed positive results for E. coli – out of this percentage, 14 (18.66%) were positive for ETEC using polymerase chain reaction assay identifying stable and labile toxins (LTs). It was noticed that all of the ETEC isolates were stable toxin producer isolates whereas LT producer isolates were not identified. Colonization factor 5 (CS5) has been detected among three ETEC isolates (21.42%), meanwhile, 11 isolates (78.57%) have not expressed colonization factors at all.

The role of CFA/I in adherence and toxin delivery by ETEC expressing multiple colonization factors in the human enteroid model.

Enterotoxigenic Escherichia coli (ETEC) is a primary causative agent of diarrhea in travelers and young children in low-to-middle-income countries (LMICs). ETEC adhere to intestinal epithelia via colonization factors (CFs) and secrete heat-stable toxin (ST) and/or heat-labile toxin (LT), causing dysregulated cellular ion transport and water secretion. ETEC isolates often harbor genes encoding more than one CF that are targets as vaccine antigens. CFA/I is a major CF that is associated with ETEC that causes moderate-to-severe diarrhea and plays an important role in pathogenesis. The Global Enteric Multicenter Study finding that 78% of CFA/I-expressing ETEC also encode the minor CF CS21 prompted investigation of the combined role of these two CFs. Western blots and electron microscopy demonstrated growth media-dependent and strain-dependent differences in CFA/I and CS21 expression. The critical role of CFA/I in adherence by ETEC strains expressing CFA/I and CS21 was demonstrated using the human enteroid model and a series of CFA/I- and CS21-specific mutants. Furthermore, only anti-CFA/I antibodies inhibited adherence by global ETEC isolates expressing CFA/I and CS21. Delivery of ST and resulting cGMP secretion was measured in supernatants from infected enteroid monolayers, and strain-specific ST delivery and time-dependent cGMP production was observed. Interestingly, cGMP levels were similar across wildtype and CF-deficient strains, reflecting a limitation of this static aerobic infection model. Despite adherence by ETEC and delivery of ST, the enteroid monolayer integrity was not disrupted, as shown by the lack of decrease in transepithelial electrical resistance and the lack of IL-8 cytokines produced during infection. Taken together, these data demonstrate that targeting CFA/I in global clinical CFA/I-CS21 strains is sufficient for adherence inhibition, supporting a vaccine strategy that focuses on blocking major CFs. In addition, the human enteroid model has significant utility for the study of ETEC pathogenesis and evaluation of vaccine-induced functional antibody responses.

Relationship between virulence factors and antimicrobial resistance genes of pathogenic Escherichia coli from diarrheic weaned piglets

A total of 690 pathogenic Escherichia (E.) coli isolates from weaned piglets were examined for antimicrobial resistance phenotypes, resistance genes, and virulence gene profiles. Also, 29 enterotoxigenic E. coli (ETEC) and 35 Shiga-toxin producing E. coli (STEC) isolates were analyzed using multi-locus sequence typing (MLST). Comparisons of the associations between antimicrobial resistance phenotypes, resistance genes, and virulence genes were performed separately by assessing odds ratio (OR). Although majorities of associations were not confirmed however, we found that associations between specific virulence factors-antimicrobial resistance. F18 encoding isolates showed association with resistance to cefazolin (OR = 3.08) and cefoxitin (OR = 3.65), and also with antimicrobial resistance gene mcr-3 (OR = 4.58). There was a high correlation between F4-STb (OR = 13.56), F4-LT (OR = 8.77), F4-EAST-I (OR = 4.97), and F18-Stx2e (OR = 3.83). Most of ETEC (21 of 29, 72.4%) isolates were assigned to ST100, and 20 of 35 STEC isolates (57.1%) were ST1. There were 5 clusters, and each cluster showed specific antimicrobial resistance patterns. Cluster I showed resistance to gentamicin, streptomycin, neomycin, nalidixic acid, ciprofloxacin, norfloxacin, trimethoprim / sulfamethoxazole, and tetracyclines whereas, cluster V showed resistance to ampicillin, amoxicillin / clavulanic acid, cephalothin, cefoxitin, cefazolin, norfloxacin, and colistin. Although there is need to do more experiments to clarify why certain virulence factors showed relationship with antimicrobial resistance, it is clear that there is a significant association between specific virulence genes and antimicrobial resistance in E. coli from weaned piglets with enteric colibacillosis in Korea.

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

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

Genomic Epidemiology and Antimicrobial Susceptibility Profile of Enterotoxigenic Escherichia coli From Outpatients With Diarrhea in Shenzhen, China, 2015–2020

Enterotoxigenic Escherichia coli (ETEC) is the leading cause of severe diarrhea in children and the most common cause of diarrhea in travelers. However, most ETEC infections in Shenzhen, China were from indigenous adults. In this study, we characterized 106 ETEC isolates from indigenous outpatients with diarrhea (77% were adults aged >20 years) in Shenzhen between 2015 and 2020 by whole-genome sequencing and antimicrobial susceptibility testing. Shenzhen ETEC isolates showed a remarkable high diversity, which belonged to four E. coli phylogroups (A: 71%, B1: 13%, E: 10%, and D: 6%) and 15 ETEC lineages, with L11 (25%, O159:H34/O159:H43, ST218/ST3153), novel L2/4 (21%, O6:H16, ST48), and L4 (15%, O25:H16, ST1491) being major lineages. Heat-stable toxin (ST) was most prevalent (76%, STh: 60% STp: 16%), followed by heat-labile toxin (LT, 17%) and ST + LT (7%). One or multiple colonization factors (CFs) were identified in 68 (64%) isolates, with the common CFs being CS21 (48%) and CS6 (34%). Antimicrobial resistance mutation/gene profiles of genomes were concordant with the phenotype testing results of 52 representative isolates, which revealed high resistance rate to nalidixic acid (71%), ampicillin (69%), and ampicillin/sulbactam (46%), and demonstrated that the novel L2/4 was a multidrug-resistant lineage. This study provides novel insight into the genomic epidemiology and antimicrobial susceptibility profile of ETEC infections in indigenous adults for the first time, which further improves our understanding on ETEC epidemiology and has implications for the development of vaccine and future surveillance and prevention of ETEC infections.

Sequence variations in the ETEC CS6 operon affect transcript and protein expression

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) is a leading cause of diarrheal disease in developing nations where it accounts for a significant disease burden in children between the ages of 0 to 59 months. It is also the number one bacterial causative agent of traveler’s diarrhea. ETEC infects hosts through the fecal-oral route and utilizes colonization factors (CF) to adhere within the small intestine. Over 25 CFs have been identified; 7 are considered major CFs and a vaccine targeting these is predicted to provide protection against up to 66% of ETEC associated disease. Coli Surface Antigen 6 (CS6) is a major CF and is associated with disease-causing ETEC isolates. Analysis of the CS6 operon sequence led to the identification of two regions of variability among clinical isolates which we predicted exert effects on CS6 transcript and protein expression. A total of 7 recombinant E. coli strains were engineered to encode the CS6 operon in wild-type, hybrid, and mutant configurations. Western blot analysis and RT-qPCR provided evidence to support the importance of an intergenic hairpin structure on CS6 expression. Our results reveal the significance of CS6 sequence selection regarding ETEC vaccine development and present novel information regarding CS6 sequence variation in WT ETEC strains.

Distribution of virulence factors and its relatedness towards the antimicrobial response of enterotoxigenic Escherichia coli strains isolated from patients in Kolkata, India.

Enterotoxigenic Escherichia coli (ETEC) is one of the most widely recognized diarrhoeal pathogens in developing countries. The advancement of ETEC vaccine development depends on the antigenic determinants of the ETEC isolates from a particular geographical region. So, the aim here was to comprehend the distribution of virulence determinants of the clinical ETEC strains of this region. Additionally, an attempt was made to find any correlation with the antimicrobial response pattern. Multiplex PCR was employed to identify virulence determinants followed by confirmatory singleplex PCR. For observation of antibiotic response, the Kirby-Bauer method was used. Out of 379strains, 46% of strains harboured both the enterotoxins ST and LT, whereas 15% were LT only. Among the major colonization factors (CFs), CS6 (41%) was the most prevalent followed by CFA/I (35%) and CFA/III was the lowest (3%). Among the minor CFs, CS21 (25%) was most prevalent, while CS15showed the lowest (3%) presence. Among the non-classical virulence factors, EatA (69%) was predominant. ETEC strains harbouring CS6showed resistance towards the commonly used drug Ciprofloxacin (70%). CS6 and elt+est toxin genes co-occurred covering 51% of the isolates. CS21 was found in most strains with est genes (43%). EatA was found to occur frequently when ST was present alone or with LT. CS6-harbouring strains showed an independent correlation to antimicrobial resistance. This study would aid in identifying the commonly circulating ETEC isolates of Kolkata, India, and their prevalent virulence determinants. Knowledge of antibiotic resistance patterns would also help in the appropriate use of antibiotics. Furthermore, the study would aid in identifying the multivalent antigens suitable for region-specific ETEC vaccines with maximum coverage.

Articles of Significant Interest in This Issue

Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen that encodes two enterotoxins, the heat-labile toxin (LT) and the heat-stable toxin (ST). Each toxin alone is sufficient to cause diarrheal disease, but many wild ETEC isolates carry both enterotoxins. Motyka et al. (e00707-20) show that ST modulates host epithelial physiology and the epithelial-immune axis. ST intoxication causes extracellular and luminal cGMP accumulation and alteration of epithelial type 1 cytokine expression, including nuclear alarmin IL-33. ETEC immunization studies also show that ST suppresses LT-based adjuvanted mucosal IgA, suggesting that two toxins act synergistically to help ETEC to evade host immune responses.