Enterohemorrhagic E. Coli Isolates Research Articles

Molecular Characterization and Plasmid Curing of Multidrug Resistant Enterohemorrhagic Escherichia coli Isolated from Some Fecal and Water Samples in Port Harcourt Metropolis

Public health is at risk due to the fact that enterohemorrhagic E. coli (EHEC) is becoming resistant to most antibiotics that demonstrates widespread misuse of antibiotics. The study was aimed at molecularly identify and perform plasmid curing with extracts of Morinda citrifolia (MC) and Xylopia aethiopica (XA) on multidrug resistant EHEC isolates that are obtained from some fecal and water samples in Port Harcourt, Rivers State. After isolation of E. coli, eleven (11) E. coli isolates were identified as EHEC. The Molecular Identification of seven (7) most resistant isolates using PCR confirmed 100% EHEC isolates. Of the 7 resistant EHEC isolates, the AmpC Gene and blaTEM Gene were detected in 71.4% and 42.9% respectively. Results of plasmid curing test revealed that the extracts of Xylopia aethiopica, Morinda citrifolia and Acridine Orange used as the control reduced the total percentage of multiple antibiotic resistant EHEC isolates from 35.61% to 10.61%, 18.18% and 16.67%, respectively. The extracts had the ability to cure plasmids, with X. aethiopica extract being the most successful against resistant EHEC isolates. In conclusion, this investigation revealed the genetic makeup of EHEC strains, detected their resistance genes and demonstrated the curing of the plasmids of resistant EHEC isolates found in fecal and water samples from the study area, both of which constitute a serious risk to public health.

High Prevalence of Antibiotic Resistant Escherichia coli Serotype O157: H7 among Pastoral Communities in Rural Uganda

Background: Non-prescribed use of antimicrobials in Agriculture incurs a transfer risk of resistant pathogens to humans, complicating treatment. The aim of this study was to determine the potential of Zoonotic E. coli to serve as drivers of antimicrobial resistance (AMR) among animals and humans in pastoralist communities in Kasese district, so as to protect the community.
 Materials and Methods: A laboratory based cross-sectional study was done using archived E. coli isolates previously obtained from humans in pastoralist communities of Kasese district, Uganda. Recovery of the isolates was done by conventional culture, and Identification by biochemical methods, serotyping and PCR. Kirby Bauer disc diffusion method was adopted for AMR profiling. Isolates were screened for resistance mechanisms including Extended Spectrum β-lactamase (ESBL), Carbapenemases and AmpC production using disc diffusion based methods.
 Results: The prevalence of Enterohemorrhagic E. coli (EHEC) was 16% (28/180). These EHEC isolates belonged to phylogroups; B1 (94%, 26/28), B2 (3%, 1/28) and A (3%, 1/28). All the 28 EHEC isolates possessed the virulence gene stx1, 26 of the 28 EHEC isolates contained the virulence gene stx2e, but none of the 28 possessed the virulence gene stx2. Highest resistance was seen to Cotrimoxazole (89%, 25/28), Tetracycline (71%, 20/28), Ampicillin (65%, 18/28) and Nitrofurantoin (28%, 8/28), these are the most commonly used antimicrobials in the agricultural sector in Uganda. Minimal resistance was observed to the antimicrobials that are commonly used in human medicine especially β-lactams, β-lactam+inhibitors and Carbapenems. Of the 28 zoonotic E. coli isolates, 17%, (5/28) were ESBL positive and among these 1 (3%, 1/28) was a Carbapenemase producer.
 Conclusion: There is a high prevalence of highly pathogenic, drug resistant E. coli O157:H7 among humans in pastoralist communities in Uganda. We suspect that these were acquired from animals because they mostly contained vero toxin gene vt2e which is animal specific, predominantly swine. Also majority of these EHEC isolates belonged to phylo-group B1 which has been documented to inhabit domestic animals. We recommend future studies to investigate relatedness of drug resistant isolates from humans and animals to ascertain the zoonotic spread of resistant enterohemorrhagic E. coli in pastoralist communities.

Human Microbiota Mice: New Animal Models for Enteric Escherichia coli Infections

Introduction: Conventional mice are resistant to infection with human enteric bacterial pathogens such as Enterohemorrhagic E.coli (EHEC) and Entertoxigenic E. colli (ETEC). This is, in part, because of host species-specific bacterial virulence factors, but also because of colonization resistance conferred by the normal microbiota of the mouse gut. Our aim was to determine whether gut microbiome composition influences infectivity of mice with either human EHEC or ETEC isolates Methods: C57BL/6'mice with either a mouse microbiome (MMB), or stably colonized with a human gut microbiome (HMB) were gavaged with EHEC strain 86-24 (Stx1, Stx2) or ETEC strain H10407 (ST, LT). Two groups were pretreated with streptomycin (SM) to disrupt their microbiota (MMBsm and HMBsm) before gavage. Mice were observed for weight, signs of infection and fecal shedding of the challenge strain, then euthanized to measure fluid secretion into total gut, small intestine (SI), cecum (CE) and colon (CO) by gut/carcass wt. ratios, and intestinal colonization by 86-24 or H10407 in small intestine and cecum. To define microbiome composition, DNA from fecal pellets was analysed for 16srDNA sequences. Results: For EHEC, a colonic pathogen, secretion was significantly increased over controls in HMB in total gut, SI and CO but was not increased in MMB without SM pretreatment. Secretion was increased to the same degree in SM treated HMbsm and SMBsm mice. Fecal shedding was increased in HMB, but not MMB, mice, as well as in both HMBsm and MMBsm SM-treated mice mice. Colonization was increased in CE and SI of HMB mice vs. MMB mice, and was equally increased in the CE and SI of both MMBsm and HMBsm treated with SM. For ETEC, a small intestinal pathogen, secretion, fecal shedding and colonization of intestinal segments was not observed in either MMB or MMB mice, unless they were treated with streptomycin. The development of intestinal secretion, colonization and fecal shedding in HMB mice challenged with EHEC 86-24 without SM pretreatment occurred without major changes in microbiome composition. In MMB mice increased secretion, colonization and shedding of either EHEC 86-24 or ETEC h10407 required SM treatment which induced major microbiome alteration.s. Conclusion: The presence of a human microbiota in C57BL/6 mice (HMB) rendered the “humanized” mice susceptible to EHEC infection without the need for microbiome disruption with streptomycin. In contrast, a human microbiota was not sufficient to induce susceptibility to infection by ETEC. which required pretreatment with streptomycin. Increased secretion colonization and shedding following EHEC or ETEC challenge of conventional C57BL/6 (MMB) mice was only seen after microbiome disruption with streptomycin. We conclude that a human microbiota in C57BL/6 mice renders them susceptible to infection by the colonic pathogen EHEC 86-24, but not by the small intestinal pathogen ETEC H10407.

Lambda Red-mediated Recombineering in the Attaching and Effacing Pathogen Escherichia albertii.

BackgroundThe ability to introduce site-specific mutations in bacterial pathogens is essential towards understanding their molecular mechanisms of pathogenicity. This has been greatly facilitated by the genetic engineering technique of recombineering. In recombineering, linear double- or single-stranded DNA molecules with two terminal homology arms are electroporated into hyperrecombinogenic bacteria that express a phage-encoded recombinase. The recombinase catalyzes the replacement of the endogenous allele with the exogenous allele to generate selectable or screenable recombinants. In particular, lambda red recombinase has been instrumental in engineering mutations to characterize the virulence arsenal of the attaching and effacing (A/E) pathogens enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and Citrobacter rodentium. Escherichia albertii is another member of this taxon; however, the virulence of E. albertii remains cryptic despite accumulating evidence that E. albertii is an emerging pathogen. Multiple retrospective studies have reported that a substantial number of EPEC and EHEC isolates (~15 %) that were previously incriminated in human outbreaks actually belong to the E. albertii lineage. Thus, there is increased urgency to reliably identify and rapidly engineer mutations in E. albertii to systematically characterize its virulence determinants. To the best of our knowledge not a single chromosomal gene has been altered by targeted mutagenesis in E. albertii since it was first isolated almost 25 years ago. This is disconcerting because an E. albertii outbreak could cause significant morbidity and mortality owing to our inadequate understanding of its virulence program.ResultsIn this report we describe a modified lambda red recombineering protocol to mutagenize E. albertii. As proof of principle, we successfully deleted three distinct virulence-associated genetic loci – ler, grlRA, and hfq – and replaced each wild type allele by a mutant allele with an encodable drug resistance cassette bracketed by FRT sites. Subsequently, the FRT-site flanked drug resistance marker was evicted by FLP-dependent site-specific recombination to generate excisants containing a solitary FRT site.ConclusionsOur protocol will enable researchers to construct marked and unmarked genome-wide mutations in E. albertii, which, in turn, will illuminate its molecular mechanisms of pathogenicity and aid in developing appropriate preventative and therapeutic approaches to combat E. albertii outbreaks.

Shiga Toxin (Verotoxin)-Producing Escherichia coli in Japan.

A series of outbreaks of infection with Shiga toxin (verocytotoxin)-producing Escherichia coli or enterohemorrhagic E. coli (EHEC) O157:H7 occurred in Japan in 1996, the largest outbreak occurring in primary schools in Sakai City, Osaka Prefecture, where more than 7,500 cases were reported. Although the reason for the sudden increase in the number of reports of EHEC isolates in 1996 is not known, the number of reports has grown to more than 3,000 cases per year since 1996, from an average of 105 reports each year during the previous 5-year period (1991-1995). Despite control measures instituted since 1996, including designating Shiga toxin-producing E. coli infection as a notifiable disease, and nationwide surveillance effectively monitoring the disease, the number of reports remains high, around 3,800 cases per year. Serogroup O157 predominates over other EHEC serogroups, but isolation frequency of non-O157 EHEC has gone up slightly over the past few years. Non-O157 EHEC has recently caused outbreaks where consumption of a raw beef dish was the source of the infection, and some fatal cases occurred. Laboratory surveillance comprised prefectural and municipal public health institutes, and the National Institute of Infectious Diseases has contributed to finding not only multiprefectural outbreaks but recognizing sporadic cases that could have been missed as an outbreak without the aid of molecular subtyping of EHEC isolates. This short overview presents recent information on the surveillance of EHEC infections in Japan.

Sensitive and specific detection of enteropathogenic and enterohemorrhagic Escherichia coli using recombinant anti-intimin antibody by immunofluorescence assay

The main and common virulence factor expressed by enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) is intimin, a 94-kDa outer membrane protein, which is a product of the eae gene, and, thus, an excellent target for the detection of these pathogens. Among the methods for detection of virulence factor expression, immunoassays can be considered the first alternative to either animal use or in vitro culture cells assays, for which polyclonal and/or monoclonal antibodies are raised. In the present work, we evaluated the sensitivity and specificity of an intimin recombinant antibody (scFv-intimin) using immunofluorescence assay. The scFv-intimin detected typical EPEC, atypical EPEC, and EHEC isolates (100% sensitivity) with no detection of eae− isolates (100% specificity). Thus, immunofluorescence is an effective and rapid method, and scFv-intimin, an excellent tool for the diagnosis of diarrhea caused by EPEC and EHEC and also can be employed in case–control epidemiological surveys.

Prevalence, Biogenesis, and Functionality of the Serine Protease Autotransporter EspP

Enterohemorrhagic E. coli (EHEC) causes severe diseases in humans worldwide. One of its virulence factors is EspP, which belongs to the serine protease autotransporters of Enterobacteriaceae (SPATE) family. In this review we recapitulate the current data on prevalence, biogenesis, structural properties and functionality. EspP has been used to investigate mechanistic details of autotransport, and recent studies indicate that this transport mechanism is not autonomous but rather dependent on additional factors. Currently, five subtypes have been identified (EspPα-EspPε), with EspPα being associated with highly virulent EHEC serotypes and isolates from patients with severe disease. EspPα has been shown to degrade major proteins of the complement cascade, namely C3 and C5 and probably interferes with hemostasis by cleavage of coagulation factor V. Furthermore, EspPα is believed to contribute to biofilm formation perhaps by polymerization to rope-like structures. Together with the proteolytic activity, EspPα might ameliorate host colonization and interfere with host response.

Molecular characterization of diarrheagenic Escherichia coli isolated from meat products sold at Mansoura city, Egypt

This study was carried out to determine the serological characteristics and virulence-associated genes of 32 Escherichia coli strains isolated from different meat products. Serotyping of somatic (O) and flagellar (H) antigens reveled that 3 strains were typed into 2 serogroups; O121:H19 (2 strains) and O148:H8 (1 strain), while the other 29 strains were not agglutinated with any serum. For molecular characterization, multiplex PCR has been performed by combining seven primer pairs specific for enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), and enteroinvasive E. coli (EIEC). The targets selected for each group were the genes encoding heat-labile (LT) and heat-stable (ST) toxins for ETEC isolates, eae, stx1 and stx2 for EHEC isolates, eae and bfpA for EPEC isolates, and ipaH for EIEC isolates. This facilitates simultaneous identification of the four different categories of diarrheagenic E. coli in a single reaction. Amongst the 32 E. coli strains tested, eleven (37.5%) were potentially diarrheagenic. Five of which (15.63%) were ETEC, three (9.38%) were EHEC, two (6.26%) were EPEC, and one strain (3.13%) was EIEC. The results appear to indicate that virulence gene-carrying E. coli strains are a normal part of intestinal bacterial populations that may be present among high numbers of E. coli contaminating meat products which do not necessarily correlate with disease.

Plasmid and chromosomal elements involved in the pathogenesis of attaching and effacing Escherichia coli

Attaching and effacing (A/E) intestinal lesions are produced by enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and RDEC-1, a pathogen of weanling rabbits. We recently identified a chromosomal locus (eae[E. coli A/E]) which is required for A/E activity in a wild-type EPEC strain. Sequences homologous to those of an eae gene probe were detected in EPEC, RDEC-1, and EHEC isolates. We report here that the eae gene is chromosomally encoded in all EPEC and EHEC strains tested and in RDEC-1. In addition, the eae probe was found to be 100% sensitive and 98% specific in detecting E. coli of EPEC serogroups that demonstrate A/E activity. Ten percent of E. coli of EPEC serogroups that hybridized with the eae probe and produced A/E activity did not hybridize with the EAF (EPEC adherence factor) probe, a plasmid-associated diagnostic probe which is currently used to identify EPEC. In addition to A/E factors, plasmid-associated adhesins also contribute to the pathogenesis of EPEC and RDEC-1. To further investigate the role of plasmid-associated adherence, a hybrid RDEC-1-EPEC strain containing the adherence plasmid of an EPEC strain in the A/E background of RDEC-1 was constructed. This hybrid strain, unlike the parent RDEC-1 strain, produced A/E lesions on human tissue culture cells, which suggests that the EPEC adherence plasmid provides tissue specificity to the hybrid strain and that the A/E factors of RDEC-1 are not host restricted.

Production and characterization of a monoclonal antibody specific for enterohemorrhagic Escherichia coli of serotypes O157:H7 and O26:H11

A monoclonal antibody (MAb 4E8C12) specific for Escherichia coli O157:H7 and O26:H11 was produced by immunizing BALB/c mice with a rough strain of E. coli O157:H7. The antibody reacted strongly by a direct enzyme-linked immunosorbent assay with each of 36 strains of E. coli O157:H7. No cross-reactivity was observed with strains of Salmonella spp., Yersinia enterocolitica, Shigella dysenteriae, Proteus spp., Escherichia hermanii, Klebsiella pneumoniae, Campylobacter jejuni, Serratia marcescens, Citrobacter spp., Enterobacter cloacae, Hafnia alvei, Aeromonas hydrophila, and all except five strains of E. coli other than serotype O157:H7 (including strains of serotype O157 but not H7). The E. coli strains (all of serotype O26:H11) that reacted with the antibody were enterohemorrhagic E. coli (EHEC) that were isolated from patients with hemolytic uremic syndrome or hemorrhagic colitis and produced verotoxin similar to that of E. coli O157:H7. MAb 4E8C12 belongs to the subclass immunoglobulin G2a and has a kappa light chain. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins of E. coli of different serotypes followed by Western immunoblot analysis revealed that MAb 4E8C12 reacted specifically with two proteins of EHEC strains of serotypes O157:H7 and O26:H11 with apparent molecular weights of 5,000 to 6,000. These proteins appeared to be markers specific for EHEC strains of serotypes O157:H7 and O26:H11. This MAb, because of its specificity, may be a useful reagent of an immunoassay for the rapid detection of these types of EHEC isolates in clinical and food specimens.