Evaluation of a simple, rapid and field-adapted diagnostic assay for enterotoxigenic E. coli and Shigella.

Understanding the global burden of enterotoxigenic E. coli (ETEC) and Shigella diarrhea as well as estimating the cost effectiveness of vaccines to control these two significant pathogens have been hindered by the lack of a diagnostic test that is rapid, simple, sensitive, and can be applied to the endemic countries. We previously developed a simple and rapid assay, Rapid Loop mediated isothermal amplification based Diagnostic Test (RLDT) for the detection of ETEC and Shigella spp. (Shigella). In this study, the RLDT assay was evaluated in comparison with quantitative PCR (qPCR), culture and conventional PCR for the detection of ETEC and Shigella. This validation was performed using previously collected stool samples from endemic countries, from the travelers to the endemic countries, as well as samples from a controlled human infection model study of ETEC. The performance of RLDT from dried stool spots was also validated. RLDT resulted in excellent sensitivity and specificity compared to qPCR (99% and 99.2% respectively) ranging from 92.3 to 100% for the individual toxin genes of ETEC and 100% for Shigella. Culture was less sensitive compared to RLDT. No significant differences were noted in the performance of RLDT using samples from various sources or stool samples from moderate to severe diarrhea or asymptomatic infections. RLDT performed equally well in detection of ETEC and Shigella from the dried stool samples on filter papers. This study established that RLDT is sufficiently sensitive and specific to be used as a simple and rapid diagnostic assay to detect ETEC and Shigella in endemic countries to determine disease burden of these pathogens in the national and subnational levels. This information will be important to guide public health and policy makers to prioritize resources for accelerating the development and introduction of effective preventative and/or treatment interventions against these enteric infections.

Development of a simple, rapid, and sensitive diagnostic assay for enterotoxigenic E. coli and Shigella spp applicable to endemic countries.

Enterotoxigenic E. coli (ETEC) and Shigella spp (Shigella) are complex pathogens. The diagnostic assays currently used to detect these pathogens are elaborate or complicated, which make them difficult to apply in resource poor settings where these diseases are endemic. The culture methods used to detect Shigella are not sensitive, and the methods used to detect ETEC are only available in a few research labs. To address this gap, we developed a rapid and simple diagnostic assay-"Rapid LAMP based Diagnostic Test (RLDT)." The six minutes sample preparation method directly from the fecal samples with lyophilized reaction strips and using established Loop-mediated Isothermal Amplification (LAMP) platform, ETEC [heat labile toxin (LT) and heat stable toxins (STh, and STp) genes] and Shigella (ipaH gene) detection was made simple, rapid (<50 minutes), and inexpensive. This assay is cold chain and electricity free. Moreover, RLDT requires minimal equipment. To avoid any end user's bias, a battery-operated, handheld reader was used to read the RLDT results. The results can be read as positive/negative or as real time amplification depending on the end user's need. The performance specifications of the RLDT assay, including analytical sensitivity and specificity, were evaluated using fecal samples spiked with ETEC and Shigella strains. The limit of detection was ~105 CFU/gm of stool for LT, STh, and STp and ~104 CFU/gm of stool for the ipaH gene, which corresponds to about 23 CFU and 1 CFU respectively for ETEC and Shigella per 25uL reaction within 40 minutes. The RLDT assay from stool collection to result is simple, and rapid and at the same time sufficiently sensitive. RLDT has the potential to be applied in resource poor endemic settings for the rapid diagnosis of ETEC and Shigella.

Detection of Enterotoxigenic Escherichia coli in Rotavirus-Infected Ghanaian Children Diagnosed with Acute Gastroenteritis.

Diarrhea is a notable global health problem in several developing countries, especially in children. Prior to the introduction of the rotavirus vaccination program in Ghana, a surveillance study was conducted to investigate the prevalence of the disease caused by rotavirus in children. In this report, we re-used archival stool samples from the pre-vaccine surveillance study to provide information on prevalence of enterotoxigenic Escherichia coli in Ghanaian children. Re-analysis of the stool samples revealed co-infection of enterotoxigenic E. coli and rotavirus in 2% of the children whose samples were selected for this study. As Ghana is approaching 10 years post-implementation of the rotavirus vaccination program, the preliminary data presented in this report are a vital reference for subsequent studies aimed at ascertaining the effect of the vaccine on both rotavirus and enterotoxigenic E. coli.

Functional analysis of colonization factor antigen I positive enterotoxigenic Escherichia coli identifies genes implicated in survival in water and host colonization.

Enterotoxigenic Escherichia coli (ETEC) expressing the colonization pili CFA/I are common causes of diarrhoeal infections in humans. Here, we use a combination of transposon mutagenesis and transcriptomic analysis to identify genes and pathways that contribute to ETEC persistence in water environments and colonization of a mammalian host. ETEC persisting in water exhibit a distinct RNA expression profile from those growing in richer media. Multiple pathways were identified that contribute to water survival, including lipopolysaccharide biosynthesis and stress response regulons. The analysis also indicated that ETEC growing in vivo in mice encounter a bottleneck driving down the diversity of colonizing ETEC populations.

Long-read-sequenced reference genomes of the seven major lineages of enterotoxigenic Escherichia coli (ETEC) circulating in modern time.

Enterotoxigenic Escherichia coli (ETEC) is an enteric pathogen responsible for the majority of diarrheal cases worldwide. ETEC infections are estimated to cause 80,000 deaths annually, with the highest rates of burden, ca 75 million cases per year, amongst children under 5 years of age in resource-poor countries. It is also the leading cause of diarrhoea in travellers. Previous large-scale sequencing studies have found seven major ETEC lineages currently in circulation worldwide. We used PacBio long-read sequencing combined with Illumina sequencing to create high-quality complete reference genomes for each of the major lineages with manually curated chromosomes and plasmids. We confirm that the major ETEC lineages all harbour conserved plasmids that have been associated with their respective background genomes for decades, suggesting that the plasmids and chromosomes of ETEC are both crucial for ETEC virulence and success as pathogens. The in-depth analysis of gene content, synteny and correct annotations of plasmids will elucidate other plasmids with and without virulence factors in related bacterial species. These reference genomes allow for fast and accurate comparison between different ETEC strains, and these data will form the foundation of ETEC genomics research for years to come.

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.

Clones of enterotoxigenic and Shiga toxin-producing Escherichia coli implicated in swine enteric colibacillosis in Spain and rates of antibiotic resistance.

Shiga toxin-producing E. coli (STEC) and enterotoxigenic E. coli (ETEC) are the main agents of swine colibacillosis, an infectious disease which implies important economic losses. We characterized here 186 diarrheagenic E. coli from Spanish industrial pig farms (2005-2017) to know which clones were involved in this syndrome, and the rates of antibiotic resistance. The PCR based on pathotype-associated virulence genes determined that 161 of 186 isolates (86.5 %) exhibited the ETEC pathotype, 10 (5.4 %) the STEC pathotype, and 15 (8.1 %) the hybrid ETEC/STEC pathotype. The majority of the isolates showed phylogroup A (85.5 %), clonotype CH11-24 (72 %) and belonged to the clonal complex (CC) 10, including two ETEC clones accounting for around 50 % of the 186 isolates: O157:HNM-A-ST10 (CH11-24), which exhibited mostly the fimbrial antigen F4ac; and O108:HNM-A-ST10 (CH11-24), which exhibited mainly F18. Other associations were O139:H1-E-ST1 (CH2-54) with the STEC pathotype, and both O141:H4-A-CC10 (CH11-24) and O138:HNM-E-ST42 (CH28-41) with ETEC/STEC. We found that 87.1 % of the isolates were multidrug-resistant, including 9% ESBL-producers, with the highest rates to nalidixic acid (82 %), colistin (77 %), ticarcillin (76 %) and ampicillin (76 %). Besides, more than 50 % of isolates showed non-susceptibility to gentamicin, tobramycin, doxycycline, ciprofloxacin, trimethoprim-sufamethoxazole and chloramphenicol. Additionally, 11 out of 17 ESBL-producing isolates were mcr-carriers. Results suggest that O108:HNM-A-ST10 (CH11-24) F18 is an emerging clone taking space left by other classical serogroups. Further follow-up studies on predominant clones in pig colibacillosis are essential for the update of vaccines, as alternative to the use of antibiotics.

Refinement of the CS6-expressing enterotoxigenic Escherichia coli strain B7A human challenge model: A randomized trial.

BACKGROUND: Human challenge models for enterotoxigenic Escherichia coli (ETEC) facilitate vaccine down-selection. The B7A (O148:H28 CS6+LT+ST+) strain is important for vaccine development. We sought to refine the B7A model by identifying a dose and fasting regimen consistently inducing moderate-severe diarrhea. METHODS: An initial cohort of 28 subjects was randomized (1:1:1:1) to receive B7A following an overnight fast at doses of 108 or 109 colony forming units (cfu) or a 90-minute fast at doses of 109 or 1010 cfu. A second cohort included naïve and rechallenged subjects who had moderate-severe diarrhea and were given the target regimen. Immune responses to important ETEC antigens were assessed. RESULTS: Among subjects receiving 108 cfu of B7A, overnight fast, or 109 cfu, 90-minute fast, 42.9% (3/7) had moderate-severe diarrhea. Higher attack rates (71.4%; 5/7) occurred in subjects receiving 109 cfu, overnight fast, or 1010 cfu, 90-minute fast. Upon rechallenge with 109 cfu of B7A, overnight fast, 5/11 (45.5%) had moderate-severe diarrhea; the attack rate among concurrently challenge naïve subjects was 57.9% (11/19). Anti-CS6, O148 LPS and LT responses were modest across all groups. CONCLUSIONS: An overnight fast enabled a reduction in the B7A inoculum dose; however, the attack rate was inconsistent and protection upon rechallenge was minimal.

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.

Clinical aspects of heat-labile and heat-stable toxin-producing enterotoxigenic Escherichia coli: A prospective study among Finnish travellers.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is a major pathogen causing travellers' diarrhoea (TD) among visitors to low- and middle-income countries (LMIC). Scant data are available on rates of travel-acquired ETEC producing heat-labile (LT) and/or heat-stable (ST) toxin or its subtypes, STh (human) and STp (porcine) in various geographic regions, and on clinical pictures associated with each toxin. METHODS: Using qPCR, we analysed LT, STh, and STp in stools positive for ETEC in a prospective study among 103 Finnish travellers visiting LMIC. They filled in questionnaires and provided stool samples before and after travel. We scrutinized geographic distribution of LT, STh, and STp ETEC findings, and association between these different ETEC subtypes and moderate/severe TD. RESULTS: Among the 103 stool samples positive for ETEC toxins, the rate for LT was 76%, for STh 26%, and STp 41%. The rate for LT-only was 44%, for STh-only 6%, STp-only 16%, LT + STh 10%, LT + STp 15%, STh + STp 3%, and LT + STh + STp 8%. Findings varied by destination; the rates of LT, STh, and STp were 79%, 21%, and 57%, respectively, in Southern Asia (n = 14); 85%, 10%, and 20% in South-eastern Asia (n = 20); 84%, 13%, and 29% in Eastern Africa (n = 31); and 56%, 50%, and 63% in Western Africa (n = 32), respectively. Of travellers positive for LT, STh, and STp, 83%, 100%, and 88%, encountered TD; 35%, 55%, and 41% reported moderate/severe TD. STh was associated with moderate/severe TD. CONCLUSIONS: Toxin findings varied by destination; multiple toxins were commonly detected. Moderate/severe TD was reported most frequently by subjects with STh-ETEC.

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.

An interlaboratory study on the detection methods for enterotoxigenic Escherichia coli in vegetables using enterotoxin gene screening and selective agars for ETEC-specific isolation.

Enterotoxigenic Escherichia coli (ETEC) causes acute diarrhea and is transmitted through contaminated food and water; however, systematic procedures for its specific detection in foods have not been established. To establish an efficient detection method for ETEC in food, an interlaboratory study using ETEC O148 and O159 as representative serogroups was first conducted with 13 participating laboratories. A series of tests including enrichment, real-time PCR assays, plating on selective agars, and concentration by immunomagnetic separation followed by plating onto selective agar (IMS-plating methods) were employed. This study particularly focused on the detection efficiencies of real-time PCR assays for enterotoxin genes (sth, stp, and lt), IMS-plating methods, and direct plating onto sorbitol MacConkey agar and CHROMagar STEC medium, supplemented with tobramycin, which is a novel modification in the preparation of a selective agar. Cucumber and leek samples inoculated with ETEC O148 and O159, either at 4-7 CFU/25 g (low levels) or at 21-37 CFU/25 g (high levels) were used as samples with uninoculated samples used as controls. At high inoculation levels, the sensitivities of sth, stp, and lt detection, direct-plating, and IMS-plating methods in cucumber inoculated with O148 and in both foods inoculated with O159 were 100%. In leek inoculated with high levels of O148, the sensitivities of sth, stp, and lt detection, direct-plating, and the IMS-plating method were 76.9%, 64.1%, and 74.4%, respectively. At low inoculation levels, the sensitivities of sth, stp, and lt detection, direct plating, and IMS-plating method in cucumber inoculated with O148 and in both foods inoculated with O159 were in the range of 87.2-97.4%. In leek inoculated with low levels of O148, the sensitivities of sth, stp, and lt detection, direct plating, and the IMS-plating method were 59.0%, 33.3%, and 38.5%, respectively. Thus, ETEC in food contaminated with more than 21 CFU/25 g were detected at high rate (over 74%) using real-time PCR assays and IMS-plating onto selective agar. Therefore, screening sth, stp, and lt genes followed by isolation of STEC using the IMS-plating method may be an efficient method for ETEC detection.

In vitro mechanism of antibacterial action of a citrus essential oil on an enterotoxigenic Escherichia coli and Lactobacillus rhamnosus.

AIM: This study investigated the in vitro mechanism of action of a commercial citrus EO, Brazilian orange terpenes (BOT), on an enterotoxigenic Escherichia coli (ETEC) isolated from pig gut and on Lactobacillus rhamnosus. METHODS AND RESULTS: Firstly, bacteria were exposed sequentially to BOT every 3 h (three times) at sub-minimal inhibitory concentrations and results showed that sequential exposure to BOT provoked a higher reduction of bacteria viability than a single exposure and the reduction of ETEC viability was higher compared to that of L. rhamnosus. Then, evaluation of the BOT effects on the cell membrane permeability and integrity, indicated that BOT increased the membrane permeability and caused disruptive effects on the integrity of bacterial cells as reflected by an increase of the relative electric conductivity and the release of essential cell constituents. Interestingly, BOT effects were more pronounced on the ETEC than on L. rhamnosus. This was ratified by scanning electron microscopy, which showed more noticeable morphological damages and disturbances on ETEC cells than on the L. rhamnosus cells. Limonene was detected as the major compound in BOT by polar/nonpolar GC-MS (78·65%/79·38%). CONCLUSIONS: Results revealed that the probable mechanism of the selective antibacterial action of the citrus EO, BOT, can be described as altering more remarkable the permeability and integrity of the cytoplasmic membrane as well as the external structure of ETEC cells than L. rhamnosus cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information about the mechanism of antibacterial action displayed by a citrus EO, a by-product of the citrus processing industry, as a natural alternative to antibiotics used in pig production sector to combat pathogens such as ETECs.

An Extended-Spectrum Beta-Lactamase-Producing Hybrid Shiga-Toxigenic and Enterotoxigenic Escherichia coli Strain Isolated from a Piglet with Diarrheal Disease in Northeast China.

Shiga-toxigenic Escherichia coli (STEC) and enterotoxigenic E. coli (ETEC) can cause diarrhea in piglets. This is the first report and complete genome sequence of an extended-spectrum ß-lactamase-producing hybrid STEC/ETEC strain isolated from a piglet with diarrhea on a swine farm in China. We investigated the virulence genes and phylogenetic diversity with publicly available E. coli genomes. Both E. coli strains S17-13 and S17-20 harbored multiple virulence genes, mainly including stx2e and eastA genes. Other important virulence genes (estIa, estIb, fedABCDEF, and hlyABCD) were located in the plasmid p1713-1 of S17-13, which could be transferred from E. coli S17-13 to S17-20 by conjugation. The presence of virulence genes associated with different pathogroups (STEC or ETEC) confirmed the hybrid status of E. coli strain S17-13. Phylogenetic analysis showed that STEC/ETEC S17-13, STEC S17-20, avian pathogenic E. coli (APEC) O78, and APEC ACN001 are located in the same evolutionary branch, indicating that they may originate from a common ancestor. It is crucial to understand the phylogeny of pathogenic bacteria to evaluate how they have evolved and to monitor the emergence of potential new pathogens. The emergence of novel hybrid E. coli strains presents a new public health risk. More attention must be paid to these hybrid pathogens during typing and epidemiological surveillance of E. coli infections, which challenges the traditional diagnostics of E. coli infections.

Transmission of antimicrobial resistant non-O157 Escherichia coli at the interface of animal-fresh produce in sustainable farming environments.

The interaction of typical host adapted enteric bacterial pathogens with fresh produce grown in fields is complex. These interactions can be more pronounced in co-managed or sustainable farms where animal operations are, by design, close to fresh produce, and growers frequently move between the two production environments. The primary objectives of this study were to 1) determine the transmission of STEC or enteric pathogens from small and large animal herds or operations to fresh produce on sustainable farms in TN and NC, 2) identify the possible sources that impact transmission of AMR E. coli, specifically STEC on these systems, and 3) WGS to characterize recovered E. coli from these sources. Samples were collected from raw and composted manure, environment, and produce sources. The serotype, virulence, and genotypic resistance profile were determined using the assembled genome sequences sequenced by Illumina technology. Broth microdilution was used to determine the antimicrobial susceptibility of each isolate against a panel of fourteen antimicrobials. The prevalence of E. coli increased during the summer season for all sources tested. ParSNP trees generated demonstrated that the transmission of AMR E. coli is occurring between animal feeding operations and fresh produce. Ten isolates were identified as serotype O45, a serotype that is associated with the "Big Six" group that is frequently linked with foodborne outbreaks caused by non-O157 E. coli. However, these isolates did not possess the stx gene. The highest frequency of resistance was detected against streptomycin (n = 225), ampicillin (n = 190) and sulfisoxazole FIS (n = 140). A total of 35 (13.7%) isolates from two TN farms were positive for the blaCMY (n = 5) and blaTEM (n = 32) genes. The results of this study show the potential of AMR E. coli transmission between animal feeding operations and fresh produce, and more studies are recommended to study this interaction and prevent dissemination in sustainable farming systems.

Wild griffon vultures (Gyps fulvus) fed at supplementary feeding stations: Potential carriers of pig pathogens and pig-derived antimicrobial resistance?

The carriage of two important pathogens of pigs, that is enterotoxigenic Escherichia coli (ETEC) and Clostridioides difficile, was investigated in 104 cloacal samples from wild griffon vultures (Gyps fulvus) fed on pig carcasses at supplementary feeding stations (SFS), along with their level of antimicrobial resistance (AMR). E. coli was isolated from 90 (86.5%) samples, but no ETEC was detected, likely because ETEC fimbriae confer the species specificity of the pathogen. Resistance to at least one antimicrobial agent was detected in 89.9% of E. coli isolates, with AMR levels being extremely high (>70%) for tetracycline and streptomycin and very high (>50%) for ampicillin and sulfamethoxazole-trimethoprim. Resistance to other critically important antimicrobials such as colistin and extended-spectrum cephalosporins was 2.2% and 1.1%, respectively, and was encoded by the mcr-1 and blaSHV-12 genes. Multidrug resistance was displayed by 80% of the resistant E. coli, and blaSHV-12 gene shared plasmid with other AMR genes. In general, resistance patterns in E. coli from vultures mirrored those found in pigs. Clostridioides difficile was detected in three samples (2.9%); two of them belonged to PCR ribotype 078 and one to PCR ribotype 126, both commonly found in pigs. All C. difficile isolates were characterized by a moderate-to-high level of resistance to fluoroquinolones and macrolides but susceptible to metronidazole or vancomycin, similar to what is usually found in C. difficile isolates from pigs. Thus, vultures may contribute somewhat to the environmental dissemination of some pig pathogens through their acquisition from pig carcasses and, more importantly, of AMR for antibiotics of critical importance for humans. However, the role of vultures would likely be much lesser than that of disposing pig carcasses at the SFS. The monitoring of AMR, and particularly of colistin-resistant and ESBL-producing E. coli, should be considered in pig farms used as sources of carcasses for SFS.

Prevalence, Antimicrobial Resistance, and Pathogenic Potential of Enterotoxigenic and Enteropathogenic Escherichia coli Associated with Acute Diarrheal Patients in Tangail, Bangladesh.

In this study, the prevalence and antimicrobial resistance of enterotoxigenic Escherichia coli (ETEC) and enteropathogenic Escherichia coli (EPEC) were investigated. Altogether 100 stool samples were collected from diarrheal patients attending the Sheikh Hasina Medical College and Hospital, Tangail, Bangladesh, during the period from March 1 to May 30, 2018. In vivo pathogenic potential of ETEC and EPEC using a Caenorhabditis elegans infection model was investigated. Among 100 diarrheal patients, 31% were positive for both ETEC and EPEC strains, 23% were lt positive for ETEC strains, and 8% were bfpA positive for EPEC strains. It was detected that 82.60%, 65.21%, 73.91%, 78.26%, 47.82%, 60.86%, and 47.82% of ETEC strains were resistant to amoxicillin-clavulanic acid (AMC), tetracycline (TE), nalidixic acid (NA), azithromycin, ciprofloxacin, ampicillin (AMP), and erythromycin (E), respectively. Whereas it was detected that 87.5% strains were resistant to AMC, AMP, and E, 75% were resistant to TE and NA, respectively. Both strains developed multidrug resistance to commonly prescribed antibiotics. EPEC showed higher pathogenicity than ETEC as 67.75% and 60% of C. elegans died after 18 h postinfection with EPEC and ETEC, respectively. The high rate of antimicrobial resistance of EPEC and ETEC highlights the necessity for the prudent use of antimicrobials in Bangladesh.

Description of Enteropathic Escherichia coli Species in Pediatric Patients at a Quaternary Children's Hospital.

BACKGROUND: The epidemiology, demographics, clinical presentations, and outcomes associated with enteroaggregative Escherichia coli (EAEC), enteropathogenic E. coli (EPEC), and enterotoxigenic E. coli (ETEC) pathotypes in US children are not well understood. METHODS: This study was a retrospective chart review of all pediatric patients with a stool sample submitted to the Children's Hospital Colorado clinical microbiology laboratory for testing with the BioFire FilmArray Gastrointestinal Pathogen Panel from October 2015 through October 2017. RESULTS: During the study period, 5692 patient stool samples were submitted; 679 (13%) were positive for EAEC, EPEC, or ETEC. Of note, 163/232 (70%) patients with EAEC, 282/493 (57%) with EPEC, and 49/58 (85%) with ETEC had detection of at least 1 other pathogen. Of all E. coli-positive stool samples, only 158/679 (23%) were from low-risk patients who were singly infected with EAEC, EPEC, or ETEC. In this cohort, most cases were associated with acute diarrhea (50%), abdominal pain (61%), and/or cramping (49%) and presented without fever (14%), emesis (28%), or lethargy (7%). Thirteen (8%) of these 158 patients received antibiotics at the time of their initial presentation to care. Of the 145 patients who did not receive antibiotics at their initial visit, 23 (16%) returned to care due to persistence of symptoms. CONCLUSIONS: Our results suggest that the majority of patients singly infected with EAEC, EPEC, or ETEC present with mild, self-limited, gastrointestinal (GI) complaints. Further research is needed to determine what role these pathogens might play in children who present with chronic or inflammatory GI symptoms.

[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.