A new multidrug-resistant enterotoxigenic Escherichia coli pulsed-field gel electrophoresis cluster associated with enrofloxacin non-susceptibility in diseased pigs.

AIMS: To describe the temporal trends in Escherichia coli pathotypes and antimicrobial resistance detected in isolates from diseased-pig cases submitted to the EcL from 2008 to 2016, in Quebec, Canada, and to investigate the presence of spatiotemporal and phylogenetic clusters. METHODS AND RESULTS: Detection of 12 genes coding for virulence factors in pathogenic E. coli in pigs by PCR and antimicrobial resistance standard disc diffusion assay were performed. Demographic and clinical data were entered in the Animal Pathogenic and Zoonotic E. coli (APZEC) database. ETEC:F4 was the most prevalent pathovirotype among the 3773 cases submitted. The LT:STb:F4 virotype was predominant until 2014, then was overtaken by the LT:STb:STa:F4 virotype. More than 90% of the ETEC:F4 isolates were multidrug resistant. A spatiotemporal cluster of LT:STb:STa:F4 isolates non-susceptible to enrofloxacin was detected between 4/2015 and 9/2016. Pulsed-field gel electrophoresis analysis of 137 ETEC:F4 isolates revealed the presence of a cluster composed mainly of LT:STb:STa:F4 isolates non-susceptible to enrofloxacin. CONCLUSIONS: The APZEC database was useful to highlight temporal trends in E. coli pathotypes. A high-risk ETEC:F4 clone might disseminate in the pig population in Quebec since 2015. SIGNIFICANCE AND IMPACT OF THE STUDY: Surveillance is crucial to identify new clones and develop control strategies.

Isolation of enterotoxigenic Staphylococcus aureus harboring seb gene and enteropathogenic Escherichia coli (serogroups O18, O114, and O125) from soft and hard artisanal cheeses in Egypt.

Background: Soft and hard artisanal cheeses are regularly consumed in Egypt. These products are usually processed from raw milk which may harbor many pathogenic and spoilage microorganisms. Aim: To evaluate the safety of some artisanal cheeses in Egypt, such as Ras, Domiati, and Mish, through chemical and microbiological examination. Methods: One hundred and fifty random samples of traditional Ras, Domiati, and Mish cheeses (50 each) were microbiologically and chemically analyzed. Counts of total bacteria, presumptive coliform, staphylococci, yeast, and mold were estimated. Furthermore, isolation of Escherichia coli and Staphylococcus aureus was performed, followed by PCR confirmation; isolates of E. coli were examined for the presence of virulence genes; on the other hand, the detection of the five classical enterotoxin genes of S. aureus was performed using multiplex PCR. Regarding chemical analysis, moisture, salt, and acidity content were measured. Correlations between chemical and microbial findings were investigated. Results: Mean counts of total bacteria, presumptive coliform, staphylococci, yeast, and mold were (2 × 108, 3 × 106 and 1 × 107 ), (3 × 105, 5 × 10 and 5 × 102), (1 × 106, 4 × 105and 1 × 105), (3 × 105, 1 × 105 and 5 × 105), and (7 × 103, 4 × 103 and 3 × 104) for Ras, Domiati and Mish cheeses, respectively. Serological identification of suspected E. coli revealed that E. coli O125 was isolated from Ras and Domiati samples, E. coli O18 was recovered from Ras samples, while E. coli O114 was isolated from Mish samples. PCR results revealed that all detected isolates of E. coli were positive for both iss (increased serum survival) and fimH (type 1 fimbriae) genes. Concerning isolated S. aureus, all examined products were harboring S. aureus enterotoxigenic strains, with seb and sed genes being the most common. The mean values of moisture, salt, and acidity were (30.03, 56.44, and 58.70), (3.30, 6.63, and 7.56) and (0.65, 0.68, and 0.50) for Ras, Domiati, and Mish cheeses, respectively. Conclusion: Enterotoxigenic S. aureus harboring seb gene and enteropathogenic E. coli (serogroups O18, O114, and O125) were frequently isolated from soft and hard artisanal cheeses in Egypt. Therefore, strict hygienic measures should be applied during their manufacture, handing, and distribution.

A new plasmid carrying mphA causes prevalence of azithromycin resistance in enterotoxigenic Escherichia coli serogroup O6.

BACKGROUND: At present, azithromycin has become an effective treatment for severe diarrhea caused by Enterotoxigenic Escherichia coli (ETEC) infection. However, enterobacteria have begun to develop resistance to azithromycin and have attracted attention in recent years. This study conducted to described the emergence of a high proportion of azithromycin-resistant ETEC serogroup O6 strains in Shanghai and to analyzed the mechanisms of azithromycin resistance. RESULTS: Strains from adult diarrhea patients with ETEC serogroup O6 infections were collected by Shanghai Diarrhea Surveillance Network and the Foodborne Surveillance Network from 2016 to 2018. We tested 30 isolates of ETEC O6 serogroup, 26 of which were resistant to azithromycin. Phylogenetic analysis revealed that these ETEC serogroup O6 strains have formed an independent dominant clone. S1-PFGE and southern blotting revealed the presence of the mphA gene on the 103 kb plasmid. Illumina and Nanopore sequencing and plasmid coverage analysis further confirmed that azithromycin-resistant strains carried a novel IncFII plasmid harboring mphA and blaTEM-1 resistance genes. CONCLUSIONS: This is the first study to report a high proportion of azithromycin resistance in a particular ETEC serogroup due to a specific plasmid carrying mphA. Our findings indicate the rapid spread of azithromycin resistance, highlighting the urgency of stringent surveillance and control measure.

Polymorphism of the alpha-1-fucosyltransferase (FUT1) gene in several wild boar (Sus scrofa) populations in France and link to edema disease.

BACKGROUND: In 2013, an outbreak of edema disease in a population of wild boars (Sus scrofa) took place. This was the first described case as reported worldwide. An enterotoxigenic Escherichia coli (presenting the Stx2e and F18 virulence factors) is the main pathogen for this disease in wild boar. The alpha-1-fucosyltransferase gene (FUT1) has been identified as the gene regulating the expression of the receptor for E. coli stx2e F18 bacteria in domestic pigs affected by the disease. The genotypic frequencies of the FUT1 gene in European wild boars have not yet been investigated. The genotypes of wild boars for this gene were determined in four French departments with or without edema diseases cases. RESULTS: All of the wild boars analyzed had a genotype susceptible to the disease (GG or AG). The recessive, resistant A allele was found for the first time in wild boars, but in a very small proportion of individuals (7/222). No statistical differences were found between healthy hunted wild boars versus wild boars found dead by edema disease or among the four French departments. CONCLUSIONS: These results suggest that further mortality due to edema disease remains possible in wild boars in France.

[The first identification of epidemic clone of enterotoxic Escherichia coli O∶6 serogroup highly associated with azithromycin resistance in Shanghai].

Objective: To investigate the molecular characterization of adult diarrhea cases caused by enterotoxic Escherichia coli (ETEC) and explore the practical model of epidemiology for laboratory technique and data needs based on the surveillance network. Methods: Epidemiological design and sampling targeted adult cases ETEC caused diarrhea in epidemic season. The enterotoxin type, serogroup, resistance, colonization factor and molecular type of ETEC were identified. Multiple dynamic phenotypic characteristics of ETEC were indicated by multidimensional and multivariable data. Results: From 2016 to 2018, 84 eligible ETEC strains were detected. The dominant serums/toxins were O∶6 (STh), O∶25 (LT), O∶159 (STh), O∶153 (STh). O∶6 (STh+CS21), which replaced O∶25 and O∶159 as the popular clones in 2018. Six cases of O∶153 (STh+CFA/I+CS8+PT34) in outbreak in 2017 were imported ones. The resistance rates of ETEC strains detected in adults to sulfasoxazole, naproxinic acid, ampicillin and azithromycin were more than 30%, multidrug resistance (MDR) reached 58.3%. Serum/toxin types suggested that attenuated strains were more likely to become MDR. Molecular typing confirmed that the genetic similarity of the dominant clone of O∶6 serogroup (PT20-24) was higher than O∶25 and O∶159. There was a high correlation between the minimal inhibitory concentration (MIC) of azithromycin and the resistant gene mphA (87.5%, 28/32). O∶6 (STh+CS21+mphA) resistant clone was first detected in 2016. Conclusion: A new epidemic clone in adult ETEC diarrhea cases in Shanghai was O∶6 (STh+CS21+mphA). For the first time the association between azithromycin resistance gene mphA and a serum group of ETEC was observed. Multidimensional and multivariate analysis techniques based on epidemiology can help reveal the potential transmission pattern of ETEC for the accurate surveillance and early warning of outbreaks.

Conservation and global distribution of non-canonical antigens in Enterotoxigenic Escherichia coli.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) cause significant diarrheal morbidity and mortality in children of resource-limited regions, warranting development of effective vaccine strategies. Genetic diversity of the ETEC pathovar has impeded development of broadly protective vaccines centered on the classical canonical antigens, the colonization factors and heat-labile toxin. Two non-canonical ETEC antigens, the EtpA adhesin, and the EatA mucinase are immunogenic in humans and protective in animal models. To foster rational vaccine design that complements existing strategies, we examined the distribution and molecular conservation of these antigens in a diverse population of ETEC isolates. METHODS: Geographically diverse ETEC isolates (n = 1159) were interrogated by PCR, immunoblotting, and/or whole genome sequencing (n = 46) to examine antigen conservation. The most divergent proteins were purified and their core functions assessed in vitro. RESULTS: EatA and EtpA or their coding sequences were present in 57.0% and 51.5% of the ETEC isolates overall, respectively; and were globally dispersed without significant regional differences in antigen distribution. These antigens also exhibited >93% amino acid sequence identity with even the most divergent proteins retaining the core adhesin and mucinase activity assigned to the prototype molecules. CONCLUSIONS: EtpA and EatA are well-conserved molecules in the ETEC pathovar, suggesting that they serve important roles in virulence and that they could be exploited for rational vaccine design.

Comparative genomic analysis and molecular examination of the diversity of enterotoxigenic Escherichia coli isolates from Chile.

Enterotoxigenic Escherichia coli (ETEC) is one of the most common diarrheal pathogens in the low- and middle-income regions of the world, however a systematic examination of the genomic content of isolates from Chile has not yet been undertaken. Whole genome sequencing and comparative analysis of a collection of 125 ETEC isolates from three geographic locations in Chile, allowed the interrogation of phylogenomic groups, sequence types and genes specific to isolates from the different geographic locations. A total of 80.8% (101/125) of the ETEC isolates were identified in E. coli phylogroup A, 15.2% (19/125) in phylogroup B, and 4.0% (5/125) in phylogroup E. The over-representation of genomes in phylogroup A was significantly different from other global ETEC genomic studies. The Chilean ETEC isolates could be further subdivided into sub-clades similar to previously defined global ETEC reference lineages that had conserved multi-locus sequence types and toxin profiles. Comparison of the gene content of the Chilean ETEC identified genes that were unique based on geographic location within Chile, phylogenomic classifications or sequence type. Completion of a limited number of genomes provided insight into the ETEC plasmid content, which is conserved in some phylogenomic groups and not conserved in others. These findings suggest that the Chilean ETEC isolates contain unique virulence factor combinations and genomic content compared to global reference ETEC isolates.

Visualization-assisted binning of metagenome assemblies reveals potential new pathogenic profiles in idiopathic travelers' diarrhea.

BACKGROUND: Travelers' diarrhea (TD) is often caused by enterotoxigenic Escherichia coli, enteroaggregative E. coli, other bacterial pathogens, Norovirus, and occasionally parasites. Nevertheless, standard diagnostic methods fail to identify pathogens in more than 40% of TD patients. It is predicted that new pathogens may be causative agents of the disease. RESULTS: We performed a comprehensive amplicon and whole genome shotgun (WGS) metagenomic study of the fecal microbiomes from 23 TD patients and seven healthy travelers, all of which were negative for the known etiologic agents of TD based on standard microbiological and immunological assays. Abnormal and diverse taxonomic profiles in TD samples were revealed. WGS reads were assembled and the resulting contigs were visualized using multiple query types. A semi-manual workflow was applied to isolate independent genomes from metagenomic pools. A total of 565 genome bins were extracted, 320 of which were complete enough to be characterized as cellular genomes; 160 were viral genomes. We made predictions of the etiology of disease for many of the individual subjects based on the properties and features of the recovered genomes. Multiple patients with low-diversity metagenomes were predominated by one to several E. coli strains. Functional annotation allowed prediction of pathogenic type in many cases. Five patients were co-infected with E. coli and other members of Enterobacteriaceae, including Enterobacter, Klebsiella, and Citrobacter; these may represent blooms of organisms that appear following secretory diarrhea. New "dark matter" microbes were observed in multiple samples. In one, we identified a novel TM7 genome that phylogenetically clustered with a sludge isolate; it carries genes encoding potential virulence factors. In multiple samples, we observed high proportions of putative novel viral genomes, some of which form clusters with the ubiquitous gut virus, crAssphage. The total relative abundance of viruses was significantly higher in healthy travelers versus TD patients. CONCLUSION: Our study highlights the strength of assembly-based metagenomics, especially the manually curated, visualization-assisted binning of contigs, in resolving unusual and under-characterized pathogenic profiles of human-associated microbiomes. Results show that TD may be polymicrobial, with multiple novel cellular and viral strains as potential players in the diarrheal disease.

[Major Vehicles and O-Serogroups in Foodborne Enterotoxigenic Escherichia coli Outbreaks in Japan, and Effective Detection Methods of the Pathogen in Food Associated with An Outbreak].

Enterotoxigenic Escherichia coli (ETEC) is a common pathogen in developing countries, and causes foodborne infections through contaminated vegetables and water. ETEC also caused some foodborne infections in developed countries, though the vehicles are often unclear. We analyzed ETEC foodborne outbreaks in Japan based on the National Food Poisoning Statistics. Vegetables and private well water accounted for 50% and 22.2% of vehicles, respectively. The main vehicles were similar to those in developing countries. Serogroups of ETEC were also analyzed, and O6, O25, O27, O148, O153, O159, and O169 were the seven major O-serogroups. We investigated suitable detection methods for the pathogen (O148) in food samples associated with an outbreak of ETEC in Japan in 2011. We show that ETEC O148 could be effectively detected in cut leeks by means of a two-step enrichment and real-time PCR assay targeting heat-stable enterotoxin gene. Our survey of the vehicles and the major O-serogroups of ETEC outbreaks in Japan indicates that ETEC survives in the environment in Japan.

Molecular networks affected by neonatal microbial colonization in porcine jejunum, luminally perfused with enterotoxigenic Escherichia coli, F4ac fimbria or Lactobacillus amylovorus.

The development of an early complex gut microbiota may play an important role in the protection against intestinal dysbiosis later in life. The significance of the developed microbiota for gut barrier functionality upon interaction with pathogenic or beneficial bacteria is largely unknown. The transcriptome of differently perfused jejunal loops of 12 caesarian-derived pigs, neonatally associated with microbiota of different complexity, was studied. Piglets received pasteurized sow colostrum at birth (d0), a starter microbiota (Lactobacillus amylovorus (LAM), Clostridium glycolicum, and Parabacteroides) on d1-d3, and a placebo inoculant (simple association, SA) or an inoculant consisting of sow's diluted feces (complex association, CA) on d3-d4. On d 26-37, jejunal loops were perfused for 8 h with either enterotoxigenic Escherichia coli F4 (ETEC), purified F4 fimbriae, LAM or saline control (CTRL). Gene expression of each intestinal loop was analyzed by Affymetrix Porcine Gene 1.1_ST array strips. Gene Set Enrichment Analysis was performed on expression values. Compared to CTRL, 184 and 74; 2 and 139; 2 and 48 gene sets, were up- and down-regulated by ETEC, F4 and LAM, respectively. ETEC up-regulated networks related to inflammatory and immune responses, RNA processing, and mitosis. There was a limited overlap in up-regulated gene sets between ETEC and F4 fimbriae. LAM down-regulated genes related to inflammatory and immune responses, as well as to cellular compound metabolism. In CA pigs, 57 gene sets were up-regulated by CA, while 73 were down-regulated compared to SA. CA up-regulated gene sets related to lymphocyte modulation and to cellular defense in all loop perfusions. In CA pigs, compared to SA pigs, genes for chemokine and cytokine activity and for response to external stimuli were down-regulated in ETEC-perfused loops and up-regulated in CTRL. The results highlight the importance of the nature of neonatal microbial colonization in the response to microbial stimuli later in life.

Extended-spectrum ß-lactamase prevalence and virulence factor characterization of enterotoxigenic Escherichia coli responsible for acute diarrhea in Nepal from 2001 to 2016.

Background: Multidrug-resistant (MDR) Gram-negative bacterial species are an increasingly dangerous public health threat, and are now endemic in many areas of South Asia. However, there are a lack of comprehensive data from many countries in this region determining historic and current MDR prevalence. Enterotoxigenic Escherichia coli (ETEC) is a leading cause of both acute infant diarrhea and traveler's diarrhea in Nepal. The MDR prevalence and associated resistance mechanisms of ETEC isolates responsible for enteric infections in Nepal are largely unknown. Methods: A total of 265 ETEC isolates were obtained from acute diarrheal samples (263/265) or patient control samples (2/265) at traveler's clinics or regional hospitals in Nepal from 2001 to 2016. Isolates were screened for antibiotic resistance, to include extended spectrum beta-lactamase (ESBL) production, via the Microscan Automated Microbiology System. ETEC virulence factors, specifically enterotoxins and colonization factors (CFs), were detected using multiplex PCR, and prevalence in the total isolate population was compared to ESBL-positive isolates. ESBL-positive isolates were assessed using multiplex PCR for genetic markers potentially responsible for observed resistance. Results: A total of 118/265 (44.5%) ETEC isolates demonstrated resistance to ≥2 antibiotics. ESBL-positive phenotypes were detected in 40/265 isolates, with isolates from 2008, 2013, 2014, and 2016 demonstrating ESBL prevalence rates of 1.5, 34.5, 31.2, and 35.0% respectively. No difference was observed in overall enterotoxin characterization between the total ETEC and ESBL-positive populations. The CFs CS2 (13.6%), CS3 (25.3%), CS6 (30.2%), and CS21 (62.6%) were the most prevalent in the total ETEC population. The ESBL-positive ETEC isolates exhibited a higher association trend with the CFs CS2 (37.5%), CS3 (35%), CS6 (42.5%), and CS21 (67.5%). The primary ESBL gene identified was blaCTX-M-15 (80%), followed by blaSHV-12 (20%) and blaCTX-M-14 (2.5%). The beta-lactamase genes blaTEM-1 (40%) and blaCMY-2 (2.5%) were also identified. It was determined that 42.5% of the ESBL-positive isolates carried multiple resistance genes. Conclusion: Over 30% of ETEC isolates collected post-2013 and evaluated in this study demonstrated ESBL resistance. Persistent surveillance and characterization of enteric ETEC isolates are vital for tracking the community presence of MDR bacterial species in order to recommend effective treatment strategies and help mitigate the spread of resistant pathogens.

Evaluation of a Culture-Dependent Algorithm and a Molecular Algorithm for Identification of Shigella spp., Escherichia coli, and Enteroinvasive E. coli.

Identification of Shigella spp., Escherichia coli, and enteroinvasive E. coli (EIEC) is challenging because of their close relatedness. Distinction is vital, as infections with Shigella spp. are under surveillance of health authorities, in contrast to EIEC infections. In this study, a culture-dependent identification algorithm and a molecular identification algorithm were evaluated. Discrepancies between the two algorithms and original identification were assessed using whole-genome sequencing (WGS). After discrepancy analysis with the molecular algorithm, 100% of the evaluated isolates were identified in concordance with the original identification. However, the resolution for certain serotypes was lower than that of previously described methods and lower than that of the culture-dependent algorithm. Although the resolution of the culture-dependent algorithm is high, 100% of noninvasive E. coli, Shigella sonnei, and Shigella dysenteriae, 93% of Shigella boydii and EIEC, and 85% of Shigella flexneri isolates were identified in concordance with the original identification. Discrepancy analysis using WGS was able to confirm one of the used algorithms in four discrepant results. However, it failed to clarify three other discrepant results, as it added yet another identification. Both proposed algorithms performed well for the identification of Shigella spp. and EIEC isolates and are applicable in low-resource settings, in contrast to previously described methods that require WGS for daily diagnostics. Evaluation of the algorithms showed that both algorithms are capable of identifying Shigella species and EIEC isolates. The molecular algorithm is more applicable in clinical diagnostics for fast and accurate screening, while the culture-dependent algorithm is more suitable for reference laboratories to identify Shigella spp. and EIEC up to the serotype level.

Enteroaggregative Escherichia coli is the predominant diarrheagenic E. coli pathotype among irrigation water and food sources in South Africa.

Diarrheagenic E. coli (DEC) has been implicated in foodborne outbreaks worldwide and have been associated with childhood stunting in the absence of diarrhoea. Infection is extraordinarily common, but the routes of transmission have not been determined. Therefore, determining the most prevalent pathotypes in food and environmental sources may help provide better guidance to various stakeholders in ensuring food safety and public health and advancing understanding of the epidemiology of enteric disease. We characterized 205 E. coli strains previously isolated from producer distributor bulk milk (PDBM)(118), irrigation water (48), irrigated lettuce (29) and street vendor coleslaw (10) in South Africa. Enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC) and diffusely adherent E. coli (DAEC) were sought. We used PCR and partial gene sequencing for all 205 strains while 46 out of 205 that showed poor resolution were subsequently characterized using cell adherence (HeLa cells). PCR and partial gene sequencing of aatA and/or aaiC genes confirmed EAEC (2%, 5 out of 205) as the only pathotype. Phylogenetic analysis of sequenced EAEC strains with E. coli strains in GenBank showing ≥80% nucleotide sequence similarity based on possession of aaiC and aatA generated distinct clusters of strains separated predominantly based on their source of isolation (food source or human stool) suggesting a potential role of virulence genes in source tracking. EAEC 24%, 11 out of 46 strains (PDBM = 15%, irrigation water = 7%, irrigated lettuce = 2%) was similarly the predominant pathotype followed by strains showing invasiveness to HeLa cells, 4%, 2 out of 46 (PDBM = 2%, irrigated lettuce = 2%), among stains characterized using cell adherence. Therefore, EAEC may be the leading cause of DEC associated food and water-borne enteric infection in South Africa. Additionally, solely using molecular based methods targeting virulence gene determinants may underestimate prevalence, especially among heterogeneous pathogens such as EAEC.

Pathotyping of Escherichia coli isolated from community toilet wastewater and stored drinking water in a slum in Bangladesh.

This study investigated the occurrence of Escherichia coli pathotypes in sanitary wastewater and drinking water in a Bangladeshi urban slum and the potential associations between these sources. We examined 621 E. coli isolates from sanitary wastewater and stored drinking water by multiplex PCR and dual-index sequencing, classifying them into eight pathotypes based on 14 virulence genes and additionally evaluating the possession of the human-specific E. coli genetic biomarker H8. The proportions of pathogenic E. coli were significantly different (P < 0·001) between wastewater (18·6%) and drinking water (1·7%). StIb-positive enterotoxigenic E. coli (ETEC) were predominant in wastewater, indicating that people in the site carried ETEC. In contrast, no ETEC was present in drinking water and the proportion of H8-positive isolates was significantly smaller (7·8%) than that in wastewater (16·3%) (P = 0·001). Our findings indicate that sanitary wastewater from the slum was heavily contaminated with pathogenic E. coli, posing a great health risk. Furthermore, E. coli contamination of drinking water could be derived from not only human but also other sources. SIGNIFICANCE AND IMPACT OF THE STUDY: Sanitary wastewater from an urban slum was heavily contaminated with pathogenic Escherichia coli. It is worth noting a great health risk of accidental exposure to pathogenically contaminated wastewater improperly discharged in and around urban slums. The distinct difference in pathotypes between wastewater and drinking water and the significantly smaller positive proportion of the human-specific E. coli genetic biomarker (H8) in drinking water indicate that drinking water contamination could be derived from not only human but also other sources. This highlights that pathotyping in association with the H8 marker provides an indication of pathogen contamination sources of environmental transmission media.

An untypeable enterotoxigenic Escherichia coli represents one of the dominant types causing human disease.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhoea in children below 5 years of age in endemic areas, and is a primary cause of diarrhoea in travellers visiting developing countries. Epidemiological analysis of E. coli pathovars is traditionally carried out based on the results of serotyping. However, genomic analysis of a global ETEC collection of 362 isolates taken from patients revealed nine novel O-antigen biosynthesis gene clusters that were previously unrecognized, and have collectively been called unclassified. When put in the context of all isolates sequenced, one of the novel O-genotypes, OgN5, was found to be the second most common ETEC O-genotype causing disease, after O6, in a globally representative ETEC collection. It's also clear that ETEC OgN5 isolates have spread globally. These novel O-genotypes have now been included in our comprehensive O-genotyping scheme, and can be detected using a PCR-based and an in silico typing method. This will assist in epidemiological studies, as well as in ETEC vaccine development.

Characterization of enterotoxigenic Escherichia coli strains isolated from the massive multi-pathogen gastroenteritis outbreak in the Antofagasta region following the Chilean earthquake, 2010.

In March 2010, a massive outbreak of gastroenteritis started in the region of Antofagasta (northern Chile). The outbreak was mainly attributed to Norovirus genogroup II although ETEC strains were also isolated with high frequency from clinical samples. We review this outbreak and determined that ETEC was an underestimated etiologic agent.

Characterization of virulence factors and phylogenetic group determination of Escherichia coli isolated from diarrheic and non-diarrheic calves from Brazil.

This study aimed to detect virulence factors, pathovars, and phylogenetic groups of Escherichia coli strains obtained from feces of calves with and without diarrhea up to 70 days old and to determine the association between occurrence of diarrhea, phylogenetic groups, and pathovars. Phylo-typing analysis of the 336 E. coli strains isolated from calves with Clermont method showed that 21 (6.25 %) belong to phylogroup A, 228 (67.85 %) to phylogroup B1, 2 (0.6 %) to phylogroup B2, 5 (1.49 %) to phylogroup C, 57 (16.96 %) to phylogroup E, and 3 (0.9 %) to phylogroup F. Phylogroup D was not identified and 20 strains (5.95 %) were assigned as "unknown." The distribution of phylogenetic groups among pathovars showed that NTEC belong to phylogroups B1 (17) and C (4); EPEC to phylogroups B1 (6) and E (8); STEC to phylogroups A (5), B1 (56), B2 (2), C (1), and E (15); EHEC to phylogroups B1 (95) and E (5); and ETEC to phylogroups A (3), B1 (7), and E (10). The EAST-1 strains were phylogroups A (13), B1 (47), E (19), and F (3); E. coli strains of "unknown" phylogroups belonged to pathovars EPEC (1), EHEC (2), STEC (7), and EAST-1 strains (6). ETEC was associated with diarrhea (P = 0.002). Our study did not find association between the phylogenetic background and occurrence of diarrhea (P = 0.164) but did find some relationship in phylogenetic group and pathovar. The study showed that EHEC and STEC are classified as phylogroup B1, EAST-1 phylogroup A, ETEC, and EPEC phylogroup E.

Immunogenicity of recombinant Lactobacillus casei-expressing F4 (K88) fimbrial adhesin FaeG in conjunction with a heat-labile enterotoxin A (LTAK63) and heat-labile enterotoxin B (LTB) of enterotoxigenic Escherichia coli as an oral adjuvant in mice.

AIMS: The aims of this study were to develop an effective oral vaccine against enterotoxigenic Escherichia coli (ETEC) infection and to design new and more versatile mucosal adjuvants. METHODS AND RESULTS: Genetically engineered Lactobacillus casei strains expressing F4 (K88) fimbrial adhesin FaeG (rLpPG-2-FaeG) and either co-expressing heat-labile enterotoxin A (LTA) subunit with an amino acid mutation associated with reduced virulence (LTAK63) and a heat-labile enterotoxin B (LTB) subunit of E. coli (rLpPG-2-LTAK63-co-LTB) or fused-expressing LTAK63 and LTB (rLpPG-2-LTAK63-fu-LTB) were constructed. The immunogenicity of rLpPG-2-FaeG in conjunction with rLpPG-2-LTAK63-co-LTB or rLpPG-2-LTAK63-fu-LTB as an orally administered mucosal adjuvant in mice was evaluated. Results showed that the levels of FaeG-specific serum IgG and mucosal sIgA, as well as the proliferation of lymphocytes, were significantly higher in mice orally co-administered rLpPG-2-FaeG and rLpPG-2-LTAK63-fu-LTB compared with those administered rLpPG-2-FaeG alone, and were lower than those co-administered rLpPG-2-FaeG and rLpPG-2-LTAK63-co-LTB. Moreover, effective protection was observed after challenge with F4+ ETEC strain CVCC 230 in mice co-administered rLpPG-2-FaeG and rLpPG-2-LTAK63-co-LTB or rLpPG-2-FaeG and rLpPG-2-LTAK63-fu-LTB group compared with those that received rLpPG-2-FaeG alone. CONCLUSIONS: rLpPG-2-FaeG showed greater immunogenicity in combination with LTAK63 and LTB as molecular adjuvants. SIGNIFICANCE AND IMPACT OF THE STUDY: Recombinant Lactobacillus provides a promising platform for the development of vaccines against F4+ ETEC infection.

Flooding adds pathogenic Escherichia coli strains to the water sources in southern Khyber Pakhtunkhwa, Pakistan.

PURPOSE: Seasonal rains in Pakistan result in heavy floods across the country, whereby faecal contaminants will be added to the water bodies and cause numerous food-borne outbreaks. The present study was aimed to determine the prevalence of diarrheagenic Escherichia coli (DEC) strains in the water sources. MATERIALS AND METHODS: Two hundred water samples collected during (2011-2012) were processed for the isolation of E. coli (EC) strains. EC strains were further analysed for antibiotic susceptibility patterns, and pathogroups-specific virulence factors stx1, stx2, stx2c, eae, tir, hlyA, bfpA, estA and eltA were detected using multiplex polymerase chain reaction. RESULTS: Thirty-three percent of the water samples were contaminated with EC pathotypes. Fifty percent (33/66) of the DEC pathotypes were identified as enterotoxigenic EC (ETEC). Seventy-two percent (13/18) of the enteropathogenic EC (EPEC) strains were identified as typical EPEC and 28% (5/18) as atypical EPEC. Eleven percent (7/66) of the Shiga toxin EC (STEC) isolates carried a combination of stx1 and stx2 genes. Summer was found as a peak season with 47% (31/66) for EC pathogroups' activities. Eighty-nine percent of the strains showed resistance against tetracycline. CONCLUSION: ETEC and EPEC are the primary causes of water contamination in southern regions of Khyber Pakhtunkhwa province, Pakistan. Firm adherence to the prescribed drugs can decrease trends in antibiotic resistance.