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An agar selective enumeration of necrotoxigenic Escherichia coli O55 (NTEC2) and probiotic E. coli Nissle 1917, using modified MacConkey agar, was developed to study bacterial interference between these E. coli strains in a gnotobiotic piglet model. Replacement of lactose with saccharose in the agar enables the direct visual enumeration of red colonies of E. coli O55 and yellow colonies of E. coli Nissle 1917 that are co-cultured in the same Petri dish. A total of 336 colonies (168 for each color) were subjected to strain-specific PCR identification with LNA probes. Sensitivity, specificity, and positive and negative predictive values were 96.43%, 95.83%, 95.86% and 96.41% respectively in E. coli O55, and 98.21%, 97.02%, 97.06% and 98.19% respectively in E. coli Nissle 1917. Color-based enumeration of both E. coli strains in colonic contents and mesenteric lymph nodes homogenates of gnotobiotic piglets demonstrated the applicability of this method for the gnotobiotic piglet model of enteric diseases., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background & objectives: Urinary tract infections (UTI) are a serious health problem affecting millions of people each year. Although appreciable work on various aspects of UTI including aetiology per se has been done, information on the emerging pathogens like necrotoxigenic Escherichia coli (NTEC) is largely lacking in India. In the present study E. coli isolates from patients with urinary tract infection from northeastern India were investigated for detection and characterization of NTEC. Methods: E. coli isolated and identified from urine samples of patients with UTI were serotyped. Antibiogram was determined by disc diffusion test. Plasmid profile was also determined. Virulence genes of NTEC (cnf1, cnf2, pap, aer, sfa, hly, afa) were detected by PCR assay. E.coli isolates carrying cnf gene (s) were identified as NTEC. Results: A total of 550 E. coli were isolated and tested for the presence of cnf genes. Of these, 84 (15.27%) belonged to NTEC. The cnf1 gene was present in 52 (61.9%) isolates, cnf2 in 23 (27.4%) and 9 (10.7%) carried both cnf1 and cnf2 genes. All the NTEC strains were found to harbour the pap and aer genes. Serogroup O4 was found to be the most common among the 12 serogroups identified amongst the NTEC isolates. Majority of the isolates (96.4%) were sensitive to furazolidone and were highly resistant to ampicillin. NTEC were found to harbour different numbers of plasmids (1 to 7). No association was observed between the number of plasmids and the antibiotic resistance of the isolates. Interpretation & conclusions: The results of the present study showed that about 15 per cent of E. coli isolates associated with UTI belonged to NTEC. More studies need to be done from other parts of the country. [ABSTRACT FROM AUTHOR]

Abstract: Necrotoxigenic Escherichia coli, or NTEC, are defined as E. coli producing the toxin known as cytotoxic necrotizing factor, or CNF. NTEC are responsible for various diseases of humans and animals, including urinary tract infection, septicemia, and diarrhea. A subgroup of NTEC known as NTEC-2 produce a variant of CNF (CNF-2) whose gene is located on a plasmid known as Vir. Because of its involvement in NTEC-2 pathogenesis and its broad distribution among production animals, a Vir plasmid from a bovine NTEC-2 strain was sequenced and analyzed. This plasmid was found to belong to the RepFIB and RepFIIA replicon types, and it totaled 138,682 base pairs in size. Within this plasmid was an approximately 60-kb pathogenicity island, defined by its possession of multiple virulence factors within distinct genetic regions of lower G+C content bounded by inverted repeats. Within this PAI were a variety of putative virulence factors, including F17b fimbrial genes, genes of a novel fimbrial operon, tibAC, hemolysins, and the cnf2 and cdt toxin-encoding genes. The occurrence of this plasmid''s virulence- and replication-associated genes was sought among a collection of 96 CNF-2-positive isolates. The most prevalent genes among this collection included repA (RepFIB), cnf2, an ompP homolog, and the tib-AC genes encoding for aggregation and biofilm formation. The Vir plasmid has evolved from an IncFIB ancestral backbone, with the RepFIB locus apparently driving the acquisition of its accessory virulence-associated elements via site-specific recombination. Overall, the completed sequence of a Vir plasmid increases our understanding of NTEC-2 pathogenomics and IncFIB plasmid evolution. [Copyright &y& Elsevier]

Abstract: The bacterial quality and safety of raw milk sources in Taif region (Western Saudi Arabia) were analyzed for the natural contamination of fecal coliform and Escherichia coli by standard most probable number method. The E. coli isolates were identified to species identity using API 20E and screened for markers of STEC (Stx1,Stx2), ETEC (ST,LT), (EaeA) and ExPEC (CNF1, CNF2, SfaS, PapA, CdtB, IutA, FyuA, TraT) using PCR assays. Thirty-three E. coli strains were recovered from raw milk sample sources, which were contaminated by fecal coliform. None of the investigated isolates reacted positively with the PapA, SfaS and CdtB, ST and LT-specific primers. Markers of STEC and EPEC were each detected in three strains. NTEC was detected in four strains. The most frequent virulence markers were TraT (17 strains), IutA (11 strains) and FyuA (8 strains). Results suggested a possibility of potential public health threat of E. coli originating from raw milk sources. [Copyright &y& Elsevier]