The commonly-used DNA probe for diffusely-adherent Escherichia coli cross-reacts with a subset of enteroaggregative E. coli.

BACKGROUND: The roles of diffusely-adherent Escherichia coli (DAEC) and enteroaggregative E. coli (EAEC) in disease are not well understood, in part because of the limitations of diagnostic tests for each of these categories of diarrhoea-causing E. coli. A HEp-2 adherence assay is the Gold Standard for detecting both EAEC and DAEC but DNA probes with limited sensitivity are also employed. RESULTS: We demonstrate that the daaC probe, conventionally used to detect DAEC, cross-reacts with a subset of strains belonging to the EAEC category. The cross hybridization is due to 84% identity, at the nucleotide level, between the daaC locus and the aggregative adherence fimbriae II cluster gene, aafC, present in some EAEC strains. Because aaf-positive EAEC show a better association with diarrhoea than other EAEC, this specific cross-hybridization may have contributed to an over-estimation of the association of daaC with disease in some studies. We have developed a discriminatory PCR-RFLP protocol to delineate EAEC strains detected by the daaC probe in molecular epidemiological studies. CONCLUSIONS: A PCR-RFLP protocol described herein can be used to identify aaf-positive EAEC and daaC-positive DAEC and to delineate these two types of diarrhoeagenic E. coli, which both react with the daaC probe. This should help to improve current understanding and future investigations of DAEC and EAEC epidemiology.

Two plasmid-encoded genes of enteropathogenic Escherichia coli strain K798 promote invasion and survival within HEp-2 cells.

Enteropathogenic Escherichia coli (EPEC) are considered to be extracellular pathogens, inducing attaching and effacing lesions following their attachment to the surface of eukaryotic cells; however, in vitro and in vivo invasion by EPEC has been reported in several studies. A cloned 4.6 kb fragment of EPEC plasmid pLV501 has been shown to facilitate invasion of E. coli K-12, and here we further investigate the nature of this process. Two of the three complete open reading frames contained within the plasmid fragment have been cloned to E. coli, and in HEp-2 adherence assays both tniA2 and pecM were shown to be expressed during the first 3 h of infection from a plac promoter. Escherichia coli transformants carrying pecM alone or in combination with tniA2 were able to both survive intracellularly and escape eukaryotic cells to re-establish themselves within the medium, whereas those bacterial cells carrying tniA2 alone could not be isolated from within HEp-2 cells after 24 h of infection, but were present in the previously sterile medium surrounding the cells. Bacteria carrying pecM and tniA2 adhered to HEp-2 cells with sites of adhesion characterized by underlying actin polymerization. The invasive potential conferred by these genes may give EPEC strains a survival advantage during prolonged infection.

Long-term effects of hydrated lime and quicklime on the decay of human remains using pig cadavers as human body analogues: Field experiments.

An increased number of police enquiries involving human remains buried with lime have demonstrated the need for more research into the effect of different types of lime on cadaver decomposition and its micro-environment. This study follows previous studies by the authors who have investigated the effects of lime on the decay of human remains in laboratory conditions and 6 months of field experiments. Six pig carcasses (Sus scrofa), used as human body analogues, were buried without lime with hydrated lime (Ca(OH)2) and quicklime (CaO) in shallow graves in sandy-loam soil in Belgium and recovered after 17 and 42 months of burial. Analysis of the soil, lime and carcasses included entomology, pH, moisture content, microbial activity, histology and lime carbonation. The results of this study demonstrate that despite conflicting evidence in the literature, the extent of decomposition is slowed down by burial with both hydrated lime and quicklime. The more advanced the decay process, the more similar the degree of liquefaction between the limed and unlimed remains. The end result for each mode of burial will ultimately result in skeletonisation. This study has implications for the investigation of clandestine burials, for a better understanding of archaeological plaster burials and potentially for the interpretation of mass graves and management of mass disasters by humanitarian organisation and DVI teams.

Short-term effects of hydrated lime and quicklime on the decay of human remains using pig cadavers as human body analogues: Laboratory experiments.

Contradictions and misconceptions regarding the effect of lime on the decay of human remains have demonstrated the need for more research into the effect of different types of lime on cadaver decomposition. This study follows previous research by the authors who have investigated the effect of lime on the decomposition of human remains in burial environments. A further three pig carcasses (Sus scrofa), used as human body analogues, were observed and monitored for 78 days without lime, with hydrated lime (Ca(OH)2) and with quicklime (CaO) in the taphonomy laboratory at the University of Bradford. The results showed that in the early stages of decay, the unlimed and hydrated lime cadavers follow a similar pattern of changes. In contrast, the application of quicklime instigated an initial acceleration of decay. Microbial investigation demonstrated that the presence of lime does not eliminate all aerobic bacteria. The experiment also suggested that lime functions as a sink, buffering the carbon dioxide evolution. This study complements the field observations. It has implications for the investigation of time since death of limed remains. Knowledge of the effects of lime on decomposition processes is of interest to forensic pathologists, archaeologists, humanitarian organisations and those concerned with disposal of animal carcasses or human remains in mass disasters.

Characterization of a novel EAST-negative enteropathogenic E. coli strain implicated in a food-borne outbreak of diarrhoea in adults.

Enteropathogenic Escherichia coli (EPEC) is usually associated with outbreaks and sporadic cases of severe infantile diarrhoea in the developing world, and less commonly with sporadic cases in developed countries. Very little evidence indicates that EPEC is a food-borne pathogen for adults. In a previous study, two groups of adult travellers became ill, and eae(+) E. coli of serogroup O111 was isolated from affected individuals and epidemiologically linked to food consumption. Here the strain responsible was further investigated and characterized as an unusual atypical EPEC. PCR analysis of the designated type isolate showed the presence of the rorf1 and espB genes of the LEE pathogenicity island, which was inserted at the chromosomal selC locus. The isolate was negative for the enteroaggregative E. coli EAST-1 toxin present in other strains of EPEC associated with food-borne outbreaks. The strain adhered sparsely to HEp-2 cell monolayers in a diffuse manner, but fluorescent actin staining demonstrated that it was capable of inducing polymerization of actin at the sites of bacterial attachment. Strain P2583 is the first EAST-negative EPEC to be confirmed as a cause of outbreaks of infection in adults following the consumption of contaminated food or water.

IS3 profiling identifies the enterohaemorrhagic Escherichia coli O-island 62 in a distinct enteroaggregative E. coli lineage.

BACKGROUND: Enteroaggregative Escherichia coli (EAEC) are important diarrhoeal pathogens that are defined by a HEp-2 adherence assay performed in specialist laboratories. Multilocus sequence typing (MLST) has revealed that aggregative adherence is convergent, providing an explanation for why not all EAEC hybridize with the plasmid-derived probe for this category, designated CVD432. Some EAEC lineages are globally disseminated or more closely associated with disease. RESULTS: To identify genetic loci conserved within significant EAEC lineages, but absent from non-EAEC, IS3-based PCR profiles were generated for 22 well-characterised EAEC strains. Six bands that were conserved among, or missing from, specific EAEC lineages were cloned and sequenced. One band corresponded to the aggR gene, a plasmid-encoded regulator that has been used as a diagnostic target but predominantly detects EAEC bearing the plasmid already marked by CVD432. The sequence from a second band was homologous to an open-reading frame within the cryptic enterohaemorrhagic E. coli (EHEC) O157 genomic island, designated O-island 62. Screening of an additional 46 EAEC strains revealed that the EHEC O-island 62 was only present in those EAEC strains belonging to the ECOR phylogenetic group D, largely comprised of sequence type (ST) complexes 31, 38 and 394. CONCLUSIONS: The EAEC 042 gene orf1600, which lies within the EAEC equivalent of O-island 62 island, can be used as a marker for EAEC strains belonging to the ECOR phylogenetic group D. The discovery of EHEC O-island 62 in EAEC validates the genetic profiling approach for identifying conserved loci among phylogenetically related strains.

Genotoxicity and cytotoxicity of zinc oxide and titanium dioxide in HEp-2 cells.

AIMS: The rapidly growing industrial and medical use of nanomaterials, especially zinc oxide and titanium dioxide, has led to growing concerns about their toxicity. Accordingly, the intrinsic genotoxic and cytotoxic potential of these nanoparticles have been evaluated. MATERIALS & METHODS: Using a HEp-2 cell line, cytotoxicity was tested along with mitochondrial activity and neutral red uptake assays. The genotoxic potential was determined using the Comet and the cytokinesis-blocked micronucleus assays. In addition, tyrosine phosphorylation events were investigated. RESULTS & CONCLUSION: We found concentration- and time-dependent cytotoxicity and an increase in DNA and cytogenetic damage with increasing nanoparticle concentrations. Mainly for zinc oxide, genotoxicity was clearly associated with an increase in tyrosine phosphorylation. Our results suggest that both types of nanoparticles can be genotoxic over a range of concentrations without being cytotoxic.