Molecular and in silico analyses for detection of Shiga toxin-producing Escherichia coli (STEC) and highly pathogenic enterohemorrhagic Escherichia coli (EHEC) using genetic markers located on plasmid, O Island 57 and O Island 71.

BACKGROUND: Due to the diversity of Shiga toxin-producing Escherichia coli (STEC) isolates, detecting highly pathogenic strains in foodstuffs is challenging. Currently, reference protocols for STEC rely on the molecular detection of eae and the stx1 and/or stx2 genes, followed by the detection of serogroup-specific wzx or wzy genes related to the top 7 serogroups. However, these screening methods do not distinguish between samples in which a STEC possessing both determinants are present and those containing two or more organisms, each containing one of these genes. This study aimed to evaluate ecf1, Z2098, Z2099, and nleA genes as single markers and their combinations (ecf1/Z2098, ecf1/Z2099, ecf1/nleA, Z2098/Z2099, Z2098/nleA, and Z2099/nleA) as genetic markers to detect potentially pathogenic STEC by the polymerase chain reaction (PCR) in 96 animal samples, as well as in 52 whole genome sequences of human samples via in silico PCR analyses. RESULTS: In animal isolates, Z2098 and Z2098/Z2099 showed a strong association with the detected top 7 isolates, with 100% and 69.2% of them testing positive, respectively. In human isolates, Z2099 was detected in 95% of the top 7 HUS isolates, while Z2098/Z2099 and ecf1/Z2099 were detected in 87.5% of the top 7 HUS isolates. CONCLUSIONS: Overall, using a single gene marker, Z2098, Z2099, and ecf1 are sensitive targets for screening the top 7 STEC isolates, and the combination of Z2098/Z2099 offers a more targeted initial screening method to detect the top 7 STEC isolates. Detecting non-top 7 STEC in both animal and human samples proved challenging due to inconsistent characteristics associated with the genetic markers studied.

High frequency of multidrug-resistant Escherichia coli from cattle in the Cerrado and Pantanal biomes of Brazil.

The indiscriminate use of antimicrobials has led to the emergence of resistant bacteria, especially pathogenic strains of Escherichia coli, which are associated with diseases in animals and humans. The aim of the present study was to characterize E. coli isolates in calves with regards to the presence of virulence genes and investigate the resistance of the isolates to different antimicrobials. Between 2021 and 2023, 456 fecal samples were collected from calves in the Pantanal and Cerrado biomes of the state of Mato Grosso do Sul, Brazil. All samples were subjected to microbiological analysis and disc diffusion antibiogram testing. The polymerase chain reaction method was used to detect virulence genes. Bacterial growth was found in 451 of the 456 samples and biochemically identified as Escherichia coli. All 451 isolates (100 %) exhibited some phenotypic resistance to antimicrobials and 67.62 % exhibited multidrug resistance. The frequency of multidrug-resistant isolates in the Cerrado biome was significantly higher than that in the Pantanal biome (p = 0.0001). In the Cerrado, the most common pathotype was Shiga toxin-producing Escherichia coli (STEC) (28 %), followed by toxigenic Escherichia coli (ETEC) (11 %), enterohemorrhagic Escherichia coli (EHEC) (8 %) and enteropathogenic Escherichia coli (EPEC) (2 %). In most cases, the concomitant occurrence of pathotypes was more common, the most frequent of which were ETEC + STEC (33 %), ETEC + EHEC (15 %) and ETEC + EPEC (3 %). The STEC pathotype (30 %) was also found more frequently in the Pantanal, followed by EHEC (12 %), ETEC (9 %) and EPEC (6 %). The STEC pathotype had a significantly higher frequency of multidrug resistance (p = 0.0486) compared to the other pathotypes identified. The frequency of resistance was lower in strains from the Pantanal biome compared to those from the Cerrado biome. Although some factors are discussed in this paper, it is necessary to clarify the reasons for this difference and the possible impacts of these findings on both animal and human health in the region.

ENTEROHEMORRHAGIC ESCHERICHIA COLI LEADING TO HAEMOLYTIC UREMIC SYNDROME - CASE STUDY AND REVIEW.

Escherichia coli is a gram-negative bacillus and considered to be the normal pathogen of intestinal and extraintestinal manifestations depending upon the strain. A variety of strains exist that are responsible for causing myriads of clinical presentation. E.coli O157: H7 being the most common and severe bacterial pathogen is the leading cause of bloody diarrhea. EHEC (Enterohemorrhagic E.coli) is responsible for causing severe complications like HC (Hemorrhagic colitis). Herein, we present the case of a young girl with E.coli O157:H7 infection and review the related literature. A previously healthy 37-year-old female presented with bloody diarrhea, fever, headache, and lower abdominal pain. As per history she had eaten a hamburger, denied any recent travel and absence of inflammatory bowel disease or bloody stools in family history. Physical examination revealed normal vital signs and the physical findings were unremarkable except for severe abdominal pain. Her stool was hem-occult positive. The complete blood count was within normal limits except neutrophilia and leukocytosis. An abdominal ultrasound showed thickened bowel loops consistent with colitis. First week of her hospital course, she continued to have bloody diarrhea and severe abdominal pain. Her final stool submitted to the laboratory on day 7 was consistent with a blood clot, following her developed low urine output and hematuria, with a serum creatinine of 2.1 mg/dl on day 5. Her renal symptoms were treated with fluids. She was given supportive treatment, and her platelet count and hemoglobin were stabilized. In early stages of bloody diarrhea, parental hydration plays a major role in accelerating volume expansion. Rapid stool analysis for these bacteria can alert specialists to deal with severe complications like HUS.

Proportions and Serogroups of Enterohemorrhagic Shiga Toxin-producing Escherichia coli in Feces of Fed and Cull Beef and Cull Dairy Cattle at Harvest.

Cattle are considered a primary reservoir of Shiga toxin (stx)-producing Escherichia coli that cause enterohemorrhagic disease (EHEC), and contaminated beef products are one vehicle of transmission to humans. However, animals entering the beef harvest process originate from differing production systems: feedlots, dairies, and beef breeding herds. The objective of this study was to determine if fed cattle, cull dairy, and or cull beef cattle carry differing proportions and serogroups of EHEC at harvest. Feces were collected via rectoanal mucosal swabs (RAMSs) from 1,039 fed cattle, 1,058 cull dairy cattle, and 1,018 cull beef cattle at harvest plants in seven U.S. states (CA, GA, NE, PA, TX, WA, and WI). The proportion of the stx gene in feces of fed cattle (99.04%) was not significantly different (P > 0.05) than in the feces of cull dairy (92.06%) and cull beef (91.85%) cattle. When two additional factors predictive of EHEC (intimin and ecf1 genes) were considered, EHEC was significantly greater (P < 0.05) in fed cattle (77.29%) than in cull dairy (47.54%) and cull beef (38.51%) cattle. The presence of E. coli O157:H7 and five common non-O157 EHEC of serogroups O26, O103, O111, O121, and O145 was determined using molecular analysis for single nucleotide polymorphisms (SNPs) followed by culture isolation. SNP analysis identified 23.48%, 17.67%, and 10.81% and culture isolation confirmed 2.98%, 3.31%, and 3.00% of fed, cull dairy, and cull beef cattle feces to contain one of these EHEC, respectively. The most common serogroups confirmed by culture isolation were O157, O103, and O26. Potential EHEC of fourteen other serogroups were isolated as well, from 4.86%, 2.46%, and 2.01% of fed, cull dairy, and cull beef cattle feces, respectively; with the most common being serogroups O177, O74, O98, and O84. The identification of particular EHEC serogroups in different types of cattle at harvest may offer opportunities to improve food safety risk management.

Improved Molecular Diagnosis and Culture of the Emerging Heteropathotype Enterohemorrhagic Escherichia coli O80:H2 Using Its Non-Melibiose-Fermenting and Antibiotic-Resistance Properties.

Enterohemorrhagic Escherichia coli (EHEC) O80:H2, belonging to sequence type ST301, is among the main causes of hemolytic and uremic syndrome in Europe, a major concern in young children. Aside from the usual intimin and Shiga toxin virulence factors (VFs), this emerging serotype possesses a mosaic plasmid combining extra-intestinal VF- and antibiotic resistance-encoding genes. This hybrid pathotype can be involved in invasive infections, a rare occurrence in EHEC infections. Here, we aimed to optimize its detection, improve its clinical diagnosis, and identify its currently unknown reservoir. O80:H2 EHEC strains isolated in France between 2010 and 2018 were phenotypically and genetically analyzed and compared with non-O80 strains. The specificity and sensitivity of a PCR test and a culture medium designed, based on the molecular and phenotypic signatures of O80:H2 EHEC, were assessed on a collection of strains and stool samples. O80:H2 biotype analysis showed that none of the strains (n = 137) fermented melibiose versus 5% of non-O80 EHEC (n = 19/352). This loss of metabolic function is due to deletion of the entire melibiose operon associated with the insertion of a 70-pb sequence (70mel), a genetic scar shared by all ST301 strains. This metabolic hallmark was used to develop a real-time PCR test (100% sensitivity, 98.3% specificity) and a melibiose-based culture medium including antibiotics, characterized by 85% specificity and sensitivity for clinical specimens. These new tools may facilitate the diagnosis of this atypical clone, help the food industry to identify the reservoir and improve our epidemiological knowledge of this threatening and emerging clone.

Evaluation of the eazyplex® EHEC complete (Amplex) assay for the differentiation of stx1 and stx2 positivity in stool samples.

The performance of the eazyplex® EHEC complete (Amplex) for the detection of Shiga toxin genes in stool samples was evaluated. The assay performed well in distinguishing between stx1 and stx2 but suboptimal sensitivity may limit its use to complementary testing rather than primary diagnosis of Shiga toxin-producing Escherichia coli infections.

Frequency of five Escherichia Coli pathotypes in Iranian adults and children with acute diarrhea.

BACKGROUND: Knowledge about the distribution of Escherichia Coli (E. coli) pathotypes in Iran is limited. This nation-wide survey aims to provide a comprehensive description of the distribution of five pathogenic E. coli in Iran. METHODS: Stool samples were collected from 1,306 acute diarrhea cases from 15 provinces (2013-2014). E. coli-positive cultures underwent PCR testing for the detection of STEC, ETEC, EPEC, EAEC, and EIEC pathotypes. Pathotype frequency by province, age-group, and season was estimated. RESULTS: 979 diarrhea samples (75.0%) were culture-positive for E. coli (95% CI: 72.6, 77.3%), and 659 (50.5%) were pathogenic E. coli (95% CI: 47.8, 53.2%). STEC was the most frequent pathotype (35.4%). ETEC (14.0%) and EPEC (13.1%) were the second and the third most frequent pathotypes, respectively. EAEC (4.3%) and EIEC (0.3%) were not highly prevalent. Fars (88.7%) and Khorasan-e-Razavi (34.8%) provinces had the highest and lowest frequencies, respectively. E. coli pathotypes were more frequent in warmer than cooler seasons, showed the highest frequency among children under five years of age (73%), and had no significant association with participants' gender. CONCLUSIONS: Diarrheagenic E. coli may be an important cause of acute diarrhea in adults and children in Iran. STEC and ETEC seem to be widespread in the country with a peak in warmer seasons, impacting the recommended use of seasonal STEC and ETEC vaccines, especially in high-risk groups. Monitoring the incidence of E. coli pathotypes, serotypes, and antibiotic resistance over time is highly recommended for evaluation of interventions.

Genetic characterization of Shigatoxigenic and enteropathogenic Escherichia coli O80:H2 from diarrhoeic and septicaemic calves and relatedness to human Shigatoxigenic E. coli O80:H2.

AIM: The purpose of this work was to identify and genetically characterize enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) O80:H2 from diarrhoeic and septicaemic calves in Belgium and to comparing them with human EHEC after whole genome sequencing. METHODS AND RESULTS: Ten EHEC and 21 EPEC O80 identified by PCR between 2009 and 2018 from faeces, intestinal content and a kidney of diarrhoeic or septicaemic calves were genome sequenced and compared to 19 human EHEC identified between 2008 and 2019. They all belonged to the O80:H2 serotype and ST301, harboured the eaeξ gene, and 23 of the 29 EHEC contained the stx2d gene. Phylogenetically, they were distributed in two major sub-lineages: one comprised a majority of bovine EPEC whereas the second one comprised a majority of stx2d bovine and human EHEC. CONCLUSIONS: Not only EPEC but also EHEC O80:H2 are present in diarrhoeic and septicaemic calves in Belgium and are genetically related to human EHEC. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings support the need to assess cattle as potential source of contamination of humans by EHEC O80:H2 and to understand the evolution of bovine and human EHEC and EPEC O80:H2.

Low frequency of enterohemorrhagic, enteroinvasive and diffusely adherent Escherichia coli in children under 5 years in rural Mozambique: a case-control study.

BACKGROUND: Diarrheagenic Escherichia coli (DEC) are among the leading pathogens associated with endemic diarrhea in low income countries. Yet, few epidemiological studies have focused the contribution of enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC) and diffusely adherent E. coli (DAEC). METHODS: We assessed the contribution of EHEC, EIEC and DAEC isolated from stool samples from a case-control study conducted in children aged < 5 years in Southern Mozambique between December 2007 and November 2012. The isolates were screened by conventional PCR targeting stx1 and stx2 (EHEC), ial and ipaH (EIEC), and daaE (DAEC) genes. RESULTS: We analyzed 297 samples from cases with less-severe diarrhea (LSD) matched to 297 controls, and 89 samples from cases with moderate-to-severe diarrhea (MSD) matched to 222 controls, collected between November 3, 2011 and November 2, 2012. DEC were more common among LSD cases (2.7%, [8/297] of cases vs. 1.3% [4/297] of controls; p = 0.243]) than in MSD cases (0%, [0/89] of cases vs. 0.4%, [1/222] of controls; p = 1.000). Detailed analysis revealed low frequency of EHEC, DAEC or EIEC and no association with diarrhea in all age strata. Although the low frequency, EIEC was predominant in LSD cases aged 24-59 months (4.1% for cases vs. 0% for controls), followed by DAEC in similar frequency for cases and controls in infants (1.9%) and lastly EHEC from one control. Analysis of a subset of samples from previous period (December 10, 2007 and October 31, 2011) showed high frequency of DEC in controls compared to MSD cases (16.2%, [25/154] vs. 11.9%, [14/118], p = 0.383, respectively). Among these, DAEC predominated, being detected in 7.7% of cases vs. 17.6% of controls aged 24-59 months, followed by EIEC in 7.7% of cases vs. 5.9% of controls for the same age category, although no association was observed. EHEC was detected in one sample from cases and two from controls. CONCLUSIONS: Our data suggests that although EHEC, DAEC and EIEC are less frequent in endemic diarrhea in rural Mozambique, attention should be given to their transmission dynamics (e.g. the role on sporadic or epidemic diarrhea) considering that the role of asymptomatic individuals as source of dissemination remains unknown.

Overall changes in the transcriptome of Escherichia coli O26:H11 induced by a subinhibitory concentration of ciprofloxacin.

AIMS: The goal was to explore the effects of subinhibitory concentration (SIC) (0·5 MIC = 20 µg l-1 ) of ciprofloxacin on the transcriptome of enterohaemorrhagic Escherichia coli O26:H11 isolate by 60 minutes of exposure. MATERIALS AND RESULTS: We used a combination of comparative genomic and transcriptomic (RNAseq) analyses. The whole genome of the E. coli O26:H11 #30934 strain of bovine origin was sequenced and assembled. This genome was next used as reference for the differential gene expression analysis. A whole-genome-based analysis of 36 publicly available E. coli O26:H11 genomes was performed to define the core and the accessory transcriptome of E. coli O26:H11. Using RNAseq and RT-qPCR analysis we observed overexpression of the SOS response and of T3SS effectors, together with the inhibition of specific motility-associated genes. Among the large set of transposases present, only three were activated, suggesting moderate transposition of genes with low doses of ciprofloxacin. Our results illustrated that transcriptional repressors, such as the CopG family protein, belonging to the core genome of E. coli O26:H11, are altered in response to fluoroquinolone exposure. The gene ontology enrichment analysis showed SIC of ciprofloxacin induced binding functions and catalytic activities, including mostly transferase and hydrolase proteins. The amino acid pathways involved in metabolic processes were significantly enhanced after the treatment. CONCLUSIONS: Although the core genome of E. coli O26:H11 constituted only 54·5% of the whole genome, we demonstrated that most differentially expressed genes were associated with the core genome of E. coli O26:H11, and that effects on the mobile genetic element, phage, and plasmid-related genes were rare. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time the effect of low dose of ciprofloxacin on the core transcriptome of E. coli O26:H11 was described. The effects on the main biological functions and protein classes including transcriptional regulators were illustrated.