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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.

Enterotoxigenic Escherichia coli (ETEC) is the principal pathogen responsible for post-weaning diarrhea in newly weaned piglets. Expansion of ETEC at weaning is thought to be the consequence of various stress factors such as transient anorexia, dietary change or increase in intestinal inflammation and permeability, but the exact mechanisms remain to be elucidated. As the use of animal experiments raise more and more ethical concerns, we used a recently developed in vitro model of piglet colonic microbiome and mucobiome, the MPigut-IVM, to evaluate the effects of a simulated weaning transition and pathogen challenge at weaning. Our data suggested that the tested factors impacted the composition and functionality of the MPigut-IVM microbiota. The simulation of weaning transition led to an increase in relative abundance of the Prevotellaceae family which was further promoted by the presence of the ETEC strain. In contrast, several beneficial families such as Bacteroidiaceae or Ruminococcaceae and gut health related short chain fatty acids like butyrate or acetate were reduced upon simulated weaning. Moreover, the incubation of MPigut-IVM filtrated effluents with porcine intestinal cell cultures showed that ETEC challenge in the in vitro model led to an increased expression of pro-inflammatory genes by the porcine cells. This study provides insights about the etiology of a dysbiotic microbiota in post-weaning piglets.

Enterotoxigenic Escherichia coli (ETEC) is the primary aetiologic agent of traveller’s diarrhoea and a significant cause of diarrhoeal disease and death in developing countries. ETEC O169:H41 strains are known to cause both traveller’s diarrhoea and foodborne outbreaks in developed countries and are cause for concern. Here, whole-genome sequencing (WGS) was used to assemble 46 O169:H41 (ST182) E. coli draft genomes derived from two airplane waste samples sourced from a German international airport. The ST182 genomes were compared with all 84 publicly available, geographically diverse ST182 genomes to construct a core genome-based phylogenetic tree. ST182 isolates were all phylogroup E, the majority serotype O169:H41 (n = 121, 93%) and formed five major clades. The airplane waste isolates differed by an average of 15 core SNPs (range 0–45) but their accessory genome content was diverse. While uncommon in other ST182 genomes, all airplane-derived ST182 isolates carried: (i) extended-spectrum β-lactamase gene blaCTX–M–15 notably lacking the typical adjacent ISEcp1; (ii) qnrS1 and the S83L mutation in gyrA, both conferring resistance to fluoroquinolones; and (iii) a class 1 integron structure (IS26-intI1Δ648-dfrA17-aadA5-qacEΔ1-sul1-ORF-srpC-padR-IS6100-mphR-mrx-mphA-IS26) identified previously in major extraintestinal pathogenic E. coli STs but not in ETEC. ST182 isolates carried ETEC-specific virulence factors STp + CS6. Adhesin/invasin tia was identified in 89% of aircraft ST182 isolates (vs 23%) and was located on a putative genomic island within a hotspot region for various insertions including PAI I536 and plasmid-associated transposons. The most common plasmid replicons in this collection were IncFII (100%; F2:A-:B-) and IncB/O/K/Z (89%). Our data suggest that potentially through travel, E. coli ST182 are evolving a multidrug-resistant profile through the acquisition of class 1 integrons and different plasmids.

Lactobacillus casei (L. casei), a normal resident of the gastrointestinal tract of mammals, has been extensively studied over the past few decades for its probiotic properties in clinical and animal models. Some studies have shown that some bacterium of Lactobacillus stimulate the production of antimicrobial peptides in intestinal cells to clear enteric pathogens, however, which antimicrobial peptides are produced by L. casei stimulation and its function are still not completely understood. In this study, we investigated the changes of antimicrobial peptides’ expression after intragastric administration of L. casei to mice. The bioinformatics analysis revealed there were nine genes strongly associated with up-regulated DEGs. But, of these, only the antimicrobial peptide mReg3a gene was continuously up-regulated, which was also confirmed by qRT-PCR. We found out the mReg3a expressed in engineering E.coli promoted cell proliferation and wound healing proved by CCK-8 assay and wound healing assay. Moreover, the tight junction proteins ZO-1 and E-cadherin in mReg3a treatment group were significantly higher than that in the control group under the final concentration of 0.2 mg/ml both in Porcine intestinal epithelial cells (IPEC-J2) and Mouse intestinal epithelial cells (IEC-6) (p < 0.05). Surprisingly, the recombinant mReg3a not only inhibited Enterotoxigenic Escherichia coli (ETEC), but also reduced the copy number of the piglet diarrheal viruses, porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and porcine rotavirus (PoRV), indicating the antimicrobial peptides mReg3a may be feed additives to resist the potential of the intestinal bacterial and viral diarrhea disease.

The European epidemic monophasic variant of Salmonella enterica serovar Typhimurium (S. 1,4,[5],12:i:-) characterized by the multi locus sequence type ST34 and the antimicrobial resistance ASSuT profile has become one of the most common serovars in Europe (EU) and the United States (US). In this study, we reconstructed the time-scaled phylogeny and evolution of this Salmonella in Europe. The epidemic S. 1,4,[5],12:i:- ST34 emerged in the 1980s by an acquisition of the Salmonella Genomic Island (SGI)-4 at the 3′ end of the phenylalanine phe tRNA locus conferring resistance to copper and arsenic toxicity. Subsequent integration of the Tn21 transposon into the fljAB locus gave resistance to mercury toxicity and several classes of antibiotics used in food-producing animals (ASSuT profile). The second step of the evolution occurred in the 1990s, with the integration of mTmV and mTmV-like prophages carrying the perC and/or sopE genes involved in the ability to reduce nitrates in intestinal contents and facilitate the disruption of the junctions of the host intestinal epithelial cells. Heavy metals are largely used as food supplements or pesticide for cultivation of seeds intended for animal feed so the expansion of the epidemic S. 1,4,[5],12:i:- ST34 was strongly related to the multiple-heavy metal resistance acquired by transposons, integrative and conjugative elements and facilitated by the escape until 2011 from the regulatory actions applied in the control of S. Typhimurium in Europe. The genomic plasticity of the epidemic S. 1,4,[5],12:i:- was demonstrated in our study by the analysis of the plasmidome. We were able to identify plasmids harboring genes mediating resistance to phenicols, colistin, and fluoroquinolone and also describe for the first time in six of the analyzed genomes the presence of two plasmids (pERR1744967-1 and pERR2174855-2) previously described only in strains of enterotoxigenic Escherichia coli and E. fergusonii.

This study aimed to investigate the effects of dietary anethole supplementation on the growth performance, intestinal barrier function, inflammatory response, and intestinal microbiota of piglets challenged with enterotoxigenic Escherichia coli K88. Thirty-six weaned piglets (24 ± 1 days old) were randomly allocated into four treatment groups: (1) sham challenge (CON); (2) Escherichia coli K88 challenge (ETEC); (3) Escherichia coli K88 challenge + antibiotics (ATB); and (4) Escherichia coli K88 challenge + anethole (AN). On day 12, the piglets in the ETEC, ATB, and AN group were challenged with 10 mL E. coli K88 (5 × 109 CFU/mL), whereas the piglets in the CON group were orally injected with 10 mL nutrient broth. On day 19, all the piglets were euthanized for sample collection. The results showed that the feed conversion ratio (FCR) was increased in the Escherichia coli K88-challenged piglets, which was reversed by the administration of antibiotics or anethole (P < 0.05). The duodenum and jejunum of the piglets in ETEC group exhibited greater villous atrophy and intestinal morphology disruption than those of the piglets in CON, ATB, and AN groups (P < 0.05). Administration of anethole protected intestinal barrier function and upregulated mucosal layer (mRNA expression of mucin-1 in the jejunum) and tight junction proteins (protein abundance of ZO-1 and Claudin-1 in the ileum) of the piglets challenged with Escherichia coli K88 (P < 0.05). In addition, administration of antibiotics or anethole numerically reduced the plasma concentrations of IL-1β and TNF-α (P < 0.1) and decreased the mRNA expression of TLR5, TLR9, MyD88, IL-1β, TNF-α, IL-6, and IL-10 in the jejunum of the piglets after challenge with Escherichia coli K88 (P < 0.05). Dietary anethole supplementation enriched the abundance of beneficial flora in the intestines of the piglets. In summary, anethole can improve the growth performance of weaned piglets infected by ETEC through attenuating intestinal barrier disruption and intestinal inflammation.

Enterotoxigenic E. coli (ETEC) is a common cause of diarrhea in children in low- and middle-income countries, and in travelers to these countries. ETEC is also an important cause of morbidity and premature mortality in piglets, calves, goat kids and lambs. The major virulence determinants of ETEC are enterotoxins and colonization factors, which enable the pathogen to colonize the small intestine and deliver enterotoxins, such as the heat-stable enterotoxins, STp and STh, to epithelial cells. Because most ETEC strains are host-specific, there are few convenient animal models to investigate the pathogenesis of ETEC infections or to evaluate specific anti-ETEC interventions, such as drugs and vaccines. An exception is ETEC strains bearing F41 pili, which mediate intestinal colonization of various young animals, including neonatal mice, to cause disease and in some cases death. In this study, we used the archetypal F41-producing bovine ETEC strain, B41 (O101:NM; K99, F41, STp) to validate and further explore the contribution of F41 and STp to bacterial virulence. By using targeted gene deletion and trans-complementation studies, augmented by whole genome sequencing, and in vitro and animal studies of virulence, we established that F41 mediates colonization of the mouse intestine and is essential for bacterial virulence. In addition, we showed for the first time that STp is as important as F41 for virulence. Together, these findings validate the use of neonatal mice to study the pathogenesis of F41-bearing ETEC and to investigate possible specific anti-ETEC interventions including vaccines that target heat-stable enterotoxins.

Applying probiotics to improve gut health and growth performance of pigs is considered an effective approach to reduce use of antimicrobial growth promoters in swine production. Understanding the properties of these probiotics is a prerequisite for the selection of probiotic strains for pigs. Host-adapted probiotic strains were suggested to exert probiotic effects by different mechanisms when compared to free-living or nomadic probiotic strains. This study assessed the effect of probiotic intervention with Limosilactobacillus reuteri TMW1.656, a host-adapted species producing the antimicrobial compound reutericyclin, its isogenic and reutericyclin-negative L. reuteri TMW1.656ΔrtcN, and with Limosilactobacillus fermentum and Lacticaseibacillus casei, two species with a nomadic lifestyle. Probiotic strains were supplemented to the post weaning diet in piglets by fermented feed or as freeze-dried cultures. The composition of fecal microbiota was determined by high throughput sequencing of 16S rRNA gene sequence tags; Enterotoxigenic Escherichia coli and Clostridium perfringens were quantified by qPCR targeting specific virulence factors. Inclusion of host-adapted L. reuteri effectively reduced ETEC abundance in swine intestine. In contrast, nomadic L. fermentum and L. casei did not show inhibitory effects on ETEC but reduced the abundance of Clostridium spp. In addition, the increasing abundance of Bacteriodetes after weaning was correlated to a reduction of ETEC abundance. Remarkably, the early colonization of piglets with ETEC was impacted by maternal-neonatal transmission; the pattern of virulence factors changed significantly over time after weaning. Probiotic intervention or the production of reutericyclin showed limited effect on the overall composition of commensal gut microbiota. In conclusion, the present study provided evidence that the lifestyle of lactobacilli is a relevant criterion for selection of probiotic cultures while the production of antimicrobial compounds has only minor effects.

CexE is a 12 kDa protein that was originally reported to be present in just three strains of enterotoxigenic Escherichia coli (ETEC); a frequent cause of diarrheal illnesses worldwide. However, an examination of sequenced genomes has revealed that CexE is actually present in a majority of ETEC strains. In addition, homologs of CexE are present in enteroaggregative E. coli (EAEC), Yersinia enterocolitica, Providencia alcalifaciens, and Citrobacter rodentium. Although it has been hypothesized that CexE and its homologs are virulence factors, this has yet to be tested. Thus the primary aim of this study was to determine if these proteins contribute to pathogenicity. Our secondary aim was determine if they are secreted coat proteins. Here we report that all neonatal mice infected with a wild-type strain of C. rodentium perished. In contrast a cexE mutant was significantly attenuated with 45% neonate survival. In adult mice the wild-type strain reached significantly higher loads in the large intestines and were shed in greater numbers than cexE mutants. Secretion of the CexE homolog in EAEC is dependent upon an atypical Type I secretion system that accepts its client from the periplasm rather than the cytoplasm. Insertion mutants of cexC, the putative ATPase of the CexE secretion system, were attenuated in our murine model. In vitro we found that CexC is required for the secretion of CexE to the outer membranes of both ETEC and C. rodentium. Secretion is not constitutive because CexE accumulates in the periplasm when the two pathogens are cultured under noninducing conditions. Although secretion conditions differ between ETEC and C. rodentium, secreted CexE remains predominantly associated with the outer membranes of both species. In aggregate these findings demonstrate that CexE is a secreted coat protein and virulence factor that promotes colonization of host intestinal tissues by enteric pathogens.

Besides the major nutrients, phosphorus (P) is an essential mineral for both the host animal and the porcine gut microbiota. Different strategies including phytase supplementation and more recently lactic acid (LA) are used to enhance the P availability from cereals in pig diets; however, their impact on the gut microbiota has been rarely related to fecal shedding of opportunistic pathogens. The present study investigated the effect of phytase supplementation and the treatment of dietary cereals with 2.5% LA on the fecal microbiome composition of metabolic active bacteria and expression of virulence factor genes of enterotoxigenic Escherichia coli and Clostridium perfringens in growing pigs. Phytase supplementation reduced the fecal abundance of the most abundant Lactobacillaceae family, whereas the LA-treatment of cereals had a stronger impact on the bacterial community, reducing amylolytic, pullulanolytic and hemicellulolytic Lactobacillaceae, Lachnospiraceae and Ruminococcaceae as well as the fecal bacterial species richness (Chao1) and diversity (Shannon index). Mainly the family Clostridiaceae benefited from the decline in the aforementioned families, being enriched by both dietary treatments. Multigroup data integration using sparse partial least squares-discriminant analysis showed that among the most discriminative operational taxonomic units (OTU) especially two unclassified Clostridiaceae-OTUs, one Prevotella copri-like OTU and one OTU within the vadinCA11 group were associated with calcium and P levels but were negatively linked with complex carbohydrates in feces. Heat-stable toxin A (Sta) of enterotoxigenic E. coli and Stx2e of Shiga-toxin producing E. coli were expressed in feces but were similar among feeding groups. Without modifying the total bacterial gene copies and virulence factor expression of E. coli, both dietary phytase supplementation and LA-treatment of cereals drastically altered the bacterial community composition in pig feces. Results thereby allowed for the characterization of bacterial nutrient dependencies, indicating a link between fecal P availability, complex carbohydrate composition and alterations in the predominant genera.

Enterotoxigenic Escherichia coli produces a myriad of adhesive structures collectively named colonization factors (CFs). CS3 is a CF, which is assembled into fine wiry fibrillae encoded by the cstA-H gene cluster. In this work we evaluated the influence of environmental cues such as temperature, osmolarity, pH, and carbon source on the expression of CS3 genes. The transcription of cstH major pilin gene was stimulated by growth of the bacteria in colonization factor broth at 37°C; the presence of glycerol enhanced cstH transcription, while glucose at high concentration, high osmolarity, and the depletion of divalent cations such as calcium and magnesium repressed cstH expression. In addition, we studied the role of H-NS, CpxRA, and CRP global regulators in CS3 gene expression. H-NS and CpxRA acted as repressors and CRP as an activator of cstH expression. Under high osmolarity, H-NS, and CpxRA were required for cstH repression. CS3 was required for both, bacterial adherence to epithelial cells and biofilm formation. Our data strengthens the existence of a multi-factorial regulatory network that controls transcription of CS3 genes in which global regulators, under the influence of environmental signals, control the production of this important intestinal colonization factor.

This study aimed to investigate the jejunal metabolic variations in enterotoxigenic Escherichia coli (ETEC)-infected piglets. Piglets were infected with 1 × 1010 CFUs (colony-forming units) of ETEC W25K and assigned into diarrheal, recovered, control, and resistant groups. Jejunal samples were harvested at day 6 and metabolic profiles were analyzed via gas chromatography coupled to time-of-flight mass spectrometry (GC/TOFMS). The results showed that 33 metabolites in the jejunum were identified in ETEC-induced diarrhea, including amino acids, fatty acids, sugars, and organic acids. Compared with the control, resistant, and recovered piglets, diarrheal piglets showed higher concentrations of 4-aminobutyric acid (GABA) and glycine in the jejunum. Compared with the control and resistant piglets, six metabolites were markedly decreased in diarrheal piglets, including ornithine, asparagine, glutamine, citric acid, citrulline, and lysine. Collectively, this study provides insights into jejunal metabolic response to ETEC infection and ETEC induced diarrhea in piglets.

Weaning disturbs the intestinal barrier function and increases the risk of infection in piglets. Probiotics exert beneficial health effects, mainly by reinforcing the intestinal epithelium and modulating the gut microbiota. However, the mechanisms of action, and especially, the specific regulatory effects of modulated microbiota by probiotics on the intestinal epithelium have not yet been elucidated. The present study aimed to decipher the protective effects of the probiotic Lactobacillus plantarum strain ZLP001 on the intestinal epithelium and microbiota as well as the effects of modulated microbiota on epithelial function. Paracellular permeability was measured with fluorescein isothiocyanate-dextran (FD-4). Gene and protein expression levels of tight junction (TJ) proteins, proinflammatory cytokines, and host defense peptides were determined by RT-qPCR, ELISA, and western blot analysis. Short-chain fatty acid (SCFA) concentrations were measured by ion chromatography. Fecal microbiota composition was assessed by high-throughput sequencing. The results showed that pretreatment with 108 colony forming units (CFU) mL-1 of L. plantarum ZLP001 significantly counteracted the increase in gut permeability to FD-4 induced by 106 CFU mL-1 enterotoxigenic Escherichia coli (ETEC). In addition, L. plantarum ZLP001 pretreatment alleviated the reduction in TJ proteins (claudin-1, occludin, and ZO-1) and downregulated proinflammatory cytokines IL-6 and IL-8, and TNFα expression and secretion caused by ETEC. L. plantarum ZLP001 also significantly increased the expression of the host defense peptides pBD2 and PG1-5 and pBD2 secretion relative to the control. Furthermore, L. plantarum ZLP001 treatment affected piglet fecal microbiota. The abundance of butyrate-producing bacteria Anaerotruncus and Faecalibacterium was significantly increased in L. plantarum ZLP001-treated piglets, and showed a positive correlation with fecal butyric and acetic acid concentrations. In addition, the cell density of Clostridium sensu stricto 1, which may cause epithelial inflammation, was decreased after L. plantarum ZLP001 administration, while the beneficial Lactobacillus was significantly increased. Our findings suggest that L. plantarum ZLP001 fortifies the intestinal barrier by strengthening epithelial defense functions and modulating gut microbiota.

Enterotoxigenic Escherichia coli (ETEC) bacteria remain a leading cause of children's diarrhea and travelers' diarrhea. Vaccines that induce antibodies to block ETEC bacterial adherence and to neutralize toxin enterotoxicity can be effective against ETEC-associated diarrhea. Recent studies showed that 6xHis-tagged CFA/I/II/IV multiepitope fusion antigen (MEFA) induced broad-spectrum antibodies to inhibit adherence of the seven most important ETEC adhesins (CFA/I, CS1 to CS6) (Ruan et al., 2014a) and 6xHis-tagged toxoid fusion antigen 3xSTaN12S-mnLTR192G/L211A (previously named as 3xSTaN12S-dmLT) elicited antibodies to neutralize both heat-labile toxin (LT) and heat-stable toxin (STa) produced by ETEC strains (Ruan et al., 2014b). In this study, we constructed two new genes to express tag-less toxoid fusion 3xSTaN12S-mnLTR192G/L211A and tag-less CFA/I/II/IV MEFA and then examined immunogenicity of each tag-less protein in mouse immunization. We further combined two tag-less proteins and investigated antigen co-administration in mice. Data showed that mice immunized with tag-less 3xSTaN12S-mnLTR192G/L211A or tag-less CFA/I/II/IV MEFA developed antigen-specific IgG antibody responses, and mice co-administered with two tag-less proteins induced neutralizing antibodies against seven adhesins and both toxins. These results indicated tag-less toxoid fusion 3xSTaN12S-mnLTR192G/L211A and tag-less CFA/I/II/IV MEFA administered individually or combined induced neutralizing antitoxin and/or anti-adhesin antibodies, and suggested the potential application of two tag-less proteins for ETEC vaccine development.

Weaning stress renders piglets susceptible to pathogen infection, which leads to post-weaning diarrhea, a severe condition characterized by heavy diarrhea and mortality in piglets. Enterotoxigenic Escherichia coli (ETEC) is one of typical strains associated with post-weaning diarrhea. Thus, prevention and inhibition of ETEC infection are of great concern. Probiotics possess anti-pathogenic activity and can counteract ETEC infection; however, their underlying mechanisms and modes of action have not yet been clarified. In the present study, the direct and indirect protective effects of Lactobacillus plantarum ZLP001 against ETEC infection were investigated by different methods. We found that bacterial culture and culture supernatant of L. plantarum ZLP001 prevented ETEC growth by the Oxford cup method, and ETEC growth inhibition was observed in a co-culture assay as well. This effect was suggested to be caused mainly by antimicrobial metabolites produced by L. plantarum ZLP001. In addition, adhesion capacity of L. plantarum ZLP001 to IPEC-J2 cells were observed using microscopy and counting. L. plantarum ZLP001 also exhibited a concentration-dependent ability to inhibit ETEC adhesion to IPEC-J2 cells, which mainly occurred via exclusion and competition mode. Furthermore, quantitative real time polymerase chain reaction (qPCR) analysis showed that L. plantarum ZLP001 upregulated the expression of host defense peptides (HDPs) but did not trigger an inflammatory response. In addition, L. plantarum ZLP001 induced HDP secretion, which enhanced the potential antimicrobial activity of IPEC-J2 cell-culture supernatant after incubation with L. plantarum ZLP001. Our findings demonstrate that L. plantarum ZLP001, an intestinal Lactobacillus species associated with piglet health, possesses anti-ETEC activity. L. plantarum ZLP001 might prevent ETEC growth, inhibit ETEC adhesion to the intestinal mucosa, and activate the innate immune response to secret antimicrobial peptides. L. plantarum ZLP001 is worth investigation as a potential probiotics.

In this study, we discovered a novel mobilized colistin resistance (mcr-1) gene variant, named mcr-1.9, which was identified in a colistin-resistant enterotoxigenic Escherichia coli (ETEC) strain from a clinical diarrhea case. The mcr-1.9 gene differs from mcr-1 at position 1036 due to a single nucleotide polymorphism (G→A), which results in an aspartic acid residue being replaced by an asparagine residue (Asp346→Asn) in the MCR-1 protein sequence. Antimicrobial susceptibility testing showed that the mcr-1.9-harboring ETEC strain is resistant to colistin at a minimum inhibitory concentration of 4 μg/ml. Plasmid profiling and conjugation experiments also suggest that the mcr-1.9 variant can be successfully transferred into the E. coli strain J53, indicating that the gene is located on a transferable plasmid. Bioinformatics analysis of data obtained from genome sequencing indicates that the mcr-1.9 gene is located on a 64,005 bp plasmid which has been named pEC26. This plasmid was found to have high similarity to the mcr-1-bearing IncI2-type plasmids pWF-5-19C (99% identity and 99% coverage) and pmcr1-IncI2 (99% identity and 98% coverage). The mcr-1.9-harboring ETEC also shows multidrug resistance to nine classes of antibiotics, and contains several virulence and antimicrobial-resistance genes suggested by the genome sequence analysis. Our report is the first to identify a new mcr-1 variant in an ETEC isolated from a human fecal sample, raising concerns about the existence of more such variants in human intestinal flora. Therefore, we believe that an undertaking to identify new mcr-1 variants in the bacterial communities of human intestines is of utmost importance, and that measures need to be taken to control the spread of mcr-1 and its variants in human intestinal microflora.

Table olives are increasingly recognized as a vehicle as well as a source of probiotic bacteria, especially those fermented with traditional procedures based on the activity of indigenous microbial consortia, originating from local environments. In the present study, we report characterization at the species level of 49 Lactic Acid Bacteria (LAB) strains deriving from Nocellara del Belice table olives fermented with the Spanish or Castelvetrano methods, recently isolated in our previous work. Ribosomal 16S DNA analysis allowed identification of 4 Enterococcus gallinarum, 3 E. casseliflavus, 14 Leuconostoc mesenteroides, 19 Lactobacillus pentosus, 7 L. coryniformis, and 2 L. oligofermentans. The L. pentosus and L. coryniformis strains were subjected to further screening to evaluate their probiotic potential, using a combination of in vitro and in vivo approaches. The majority of them showed high survival rates under in vitro simulated gastro-intestinal conditions, and positive antimicrobial activity against Salmonella enterica serovar Typhimurium, Listeria monocytogenes and enterotoxigenic Escherichia coli (ETEC) pathogens. Evaluation of antibiotic resistance to ampicillin, tetracycline, chloramphenicol, or erythromycin was also performed for all selected strains. Three L. coryniformis strains were selected as very good performers in the initial in vitro testing screens, they were antibiotic susceptible, as well as capable of inhibiting pathogen growth in vitro. Parallel screening employing the simplified model organism Caenorhabditis elegans, fed the Lactobacillus strains as a food source, revealed that one L. pentosus and one L. coryniformis strains significantly induced prolongevity effects and protection from pathogen-mediated infection. Moreover, both strains displayed adhesion to human intestinal epithelial Caco-2 cells and were able to outcompete foodborne pathogens for cell adhesion. Overall, these results are suggestive of beneficial features for novel LAB strains, which renders them promising candidates as starters for the manufacturing of fermented table olives with probiotic added value.

Enterotoxigenic Escherichia coli (ETEC) is the most common bacterial cause of diarrhea in children in developing countries, as well as in travelers to these countries. To cause disease, ETEC needs to produce a series of virulence proteins including enterotoxins, colonization factors and secretion pathways, which enable this pathogen to colonize the human small intestine and deliver enterotoxins to epithelial cells. Previously, a number of studies have demonstrated that CfaD, an AraC-like transcriptional regulator, plays a key role in virulence gene expression by ETEC. In this study, we carried out a transcriptomic analysis of ETEC strain, H10407, grown under different conditions, and determined the complete set of genes that are regulated by CfaD. In this way, we identified a number of new target genes, including rnr-1, rnr-2, etpBAC, agn43, flu, traM and ETEC_3214, whose expression is strongly activated by CfaD. Using promoter-lacZ reporters, primer extension and electrophoretic mobility shift assays, we characterized the CfaD-mediated activation of several selected target promoters. We also showed that the gut-associated environmental signal, sodium bicarbonate, stimulates CfaD-mediated upregulation of its virulence target operons. Finally, we screened a commercial small molecule library and identified a compound (CH-1) that specifically inhibited the regulatory function of CfaD, and by 2-D analoging, we identified a second inhibitor (CH-2) with greater potency.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of morbidity in children under 5 years of age in low- and middle-income countries and a leading cause of traveler's diarrhea worldwide. The ability of ETEC to colonize the intestinal epithelium is mediated by fimbrial adhesins, such as CS21 (Longus). This adhesin is a type IVb pilus involved in adherence to intestinal cells in vitro and bacterial self-aggregation. Fourteen open reading frames have been proposed to be involved in CS21 assembly, hitherto only the lngA and lngB genes, coding for the major (LngA) and minor (LngB) structural subunit, have been characterized. In this study, we investigated the role of the LngA, LngB, LngC, LngD, LngH, and LngP proteins in the assembly of CS21 in ETEC strain E9034A. The deletion of the lngA, lngB, lngC, lngD, lngH, or lngP genes, abolished CS21 assembly in ETEC strain E9034A and adherence to HT-29 cells was reduced 90%, compared to wild-type strain. Subcellular localization prediction of CS21 proteins was similar to other well-known type IV pili homologues. We showed that LngP is the prepilin peptidase of LngA, and that ETEC strain E9034A has another peptidase capable of processing LngA, although with less efficiency. Additionally, we present immuno-electron microscopy imagens to show that the LngB protein could be localized at the tip of CS21, and probably helps to control CS21 length. In conclusion, our results demonstrate that the LngA, LngB, LngC, LngD, LngH, and LngP proteins are essential for CS21 assembly, as well as for bacterial aggregation and adherence to HT-29 cells.

Background: Enterotoxigenic Escherichia coli (ETEC) colonize the human intestinal mucosa using pili and non-pili colonization factors (CFs). CS21 (also designated Longus) is one of the most prevalent CFs encoded by a 14 kb lng DNA cluster located in a virulence plasmid of ETEC; yet limited information is available on the prevalence of CS21 positive ETEC isolates in different countries. The aim of this study was to evaluate the prevalence of CS21 among ETEC clinical isolates from Mexican and Bangladeshi children under 5 years old with diarrhea and to determine the phenotypic and genotypic features of these isolates. Methods: ETEC clinical isolates positive to lngA gene were characterized by genotype, multidrug-resistance, self-aggregation, biofilm formation, and adherence to HT-29 cell line. Results: A collection of 303 E. coli clinical isolates were analyzed, the 81.51% (247/303) were identified as ETEC, 30.76% (76/247) were st+/lt+, and 25.10% (62/247) were positive for the lngA gene. Among the lngA+ ETECs identified, 50% of isolates (31/62) were positive for LngA protein. The most frequent serotype was O128ac:H12 found in 19.35% (12/62) of lngA+ ETEC studied. Multidrug-resistance (MDR) lngA+ ETEC isolates was identified in 65% (39/60), self-aggregation in 48.38% (30/62), and biofilm formation in 83.87% (52/62). ETEC lngA+ isolates were able to adhere to HT-29 cells at different levels. Two lngA isogenic mutants were constructed in the ETEC E9034A and ETEC73332 clinical isolate, showing a 77% and 98% reduction in adherence, respectively with respect to the wild type. Conclusion: ETEC isolates that have the lngA gene showed features associated with self-aggregation, and adherence to HT-29 cells, important characteristics in the human gut colonization process and pathogenesis.

Enteric infections induced by pathogens like Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) remain a massive burden in developing countries with increasing morbidity and mortality rates. Previously, we showed that the immunization with genetically detoxified outer membrane vesicles (OMVs) derived from V. cholerae elicits a protective immune response based on the generation of O antigen antibodies, which effectively block the motility by binding to the sheathed flagellum. In this study, we investigated the potential of lipopolysaccharide (LPS)-modified and toxin negative OMVs isolated from V. cholerae and ETEC as a combined OMV vaccine candidate. Our results indicate that the immunization with V. cholerae or ETEC OMVs induced a species-specific immune response, whereas the combination of both OMV species resulted in a high-titer, protective immune response against both pathogens. Interestingly, the immunization with V. cholerae OMVs alone resulted in a so far uncharacterized and cholera toxin B-subunit (CTB) independent protection mechanism against an ETEC colonization. Furthermore, we investigated the potential use of V. cholerae OMVs as delivery vehicles for the heterologously expression of the ETEC surface antigens, CFA/I, and FliC. Although we induced a detectable immune response against both heterologously expressed antigens, none of these approaches resulted in an improved protection compared to a simple combination of V. cholerae and ETEC OMVs. Finally, we expanded the current protection model from V. cholerae to ETEC by demonstrating that the inhibition of motility via anti-FliC antibodies represents a relevant protection mechanism of an OMV-based ETEC vaccine candidate in vivo.

The coli surface antigen 26 (CS26) of enterotoxigenic Escherichia coli (ETEC) had been described as a putative adhesive pilus based on the partial sequence of the crsH gene, detected in isolates from children with diarrhea in Egypt. However, its production and activity as adherence determinant has not been experimentally addressed. The crsH was identified as a homolog of genes encoding structural subunits of ETEC colonization factors (CFs) CS12, CS18, and CS20. These CFs, along with the recently discovered CS30, belong to the γ2 family of pili assembled by the chaperone-usher pathway (CU pili). Further, the complete CS26 locus, crsHBCDEFG, was described in an O141 ETEC strain (ETEC 100664) obtained from a diarrhea case in The Gambia, during the Global Enterics Multicenter Study. Here, we report that CS26 is a pilus of ∼10 nm in diameter, with the capacity to increase the cell adherence of the non-pathogenic strain E. coli DH10B. As for other related pili, production of CS26 seems to be regulated by phase variation. Deletion of crsHBCDEFG in ETEC 100664 significantly decreased its adherence capacity, which was recovered by in trans complementation. Furthermore, CrsH was cross-recognized by polyclonal antibodies directed against the major structural subunit of CS20, CsnA, as determined by Western blotting and immunogold labeling. ETEC CS26+ strains were found to harbor the heat-labile enterotoxin only, within three different sequence types of phylogroups A and B1, the latter suggesting acquisition through independent events of horizontal transfer. Overall, our results demonstrate that CS26 is an adhesive pilus of human ETEC. In addition, cross-reactivity with anti-CsnA antibodies indicate presence of common epitopes in γ2-CFs.