The metabolic impact of zinc oxide on porcine intestinal cells and enterotoxigenic Escherichia coli K88

Pharmacological levels of zinc oxide (ZnO) incorporated into the post-weaning piglet diet reduce the incidence of diarrhoea caused by enterotoxigenic Escherichia coli (ETEC) K88. The mechanism for this is not understood. Here, Intestinal Porcine Epithelial Cells (IPEC) J2 were used as an in vitro model of the porcine intestine. ZnO reduced IPEC J2 viability at concentrations >= 200 mu M, and ETEC adhesion to the host cell was unaffected by ZnO. Characterisation of the metabolism of IPEC J2 cells and ETEC established the effects of ZnO treatment on the metabolic profile of both. Although 100 mu M ZnO did not inhibit growth of either host or pathogen in fully supplemented media, metabolic profiles were significantly altered. Glucose and mannose were essential energy sources for IPEC J2 cells in the presence of ZnO, as the ability to utilise other sources was compromised. The increase in specificity of requirements to support respiration in ETEC was more pronounced, in particular the need for cysteine as a nitrogen source. These findings indicate that ZnO impacts on both host cell and pathogen metabolism and may provide insight into the mechanism for diarrhoea reduction. (C) 2010 Elsevier B.V. All rights reserved.

Persistence of a wild type Escherichia coli and its multiple antibiotic-resistant (MAR) derivatives in the abattoir and on chilled pig carcasses

The aim of this study was to evaluate the ability of an Escherichia coli with the multiple antibiotic resistance (MAR) phenotype to withstand the stresses of slaughter compared to an isogenic progenitor strain. A wild type E. coli isolate (345-2RifC) of porcine origin was used to derive 3 isogenic MAR mutants. Escherichia coli 345-2RifC and its MAR derivatives were inoculated into separate groups of pigs. Once colonisation was established, the pigs were slaughtered and persistence of the E. coli strains in the abattoir environment and on the pig carcasses was monitored and compared. No significant difference (P>0.05) was detected between the shedding of the different E. coli strains from the live pigs. Both the parent strain and its MAR derivatives persisted in the abattoir environment, however the parent strain was recovered from 6 of the 13 locations sampled while the MAR derivatives were recovered from 11 of 13 and the number of MAR E. coil recovered was 10-fold higher than the parent strain at half of the locations. The parent strain was not recovered from any of the 6 chilled carcasses whereas the MAR derivatives were recovered from 3 out of 5 (P<0.001). This study demonstrates that the expression of MAR in 345-2RifC increased its ability to survive the stresses of the slaughter and chilling processes. Therefore in E. coli, MAR can give a selective advantage, compared to non-MAR strains, for persistence on chilled carcasses thereby facilitating transit of these strains through the food chain. (C) 2010 Elsevier B.V. All rights reserved.

A 96-well plate fluorescence assay for assessment of cellular permeability and active efflux in Salmonella enterica serovar Typhimurium and Escherichia coli

Multiply antibiotic-resistant (MAR) mutants of Escherichia coli and Salmonella enterica are characterized by reduced susceptibility to several unrelated antibiotics, biocides and other xenobiotics. Porin loss and/or active efflux have been identified as a key mechanisms of MAR. A single rapid test was developed for MAR. The intracellular accumulation of the fluorescent probe Hoechst (H) 33342 (bisbenzimide) by MAR mutants and those with defined disruptions in efflux pump and porin genes was determined in 96-well plate format. The accumulation of H33342 was significantly (P < 0.0001) reduced in MAR mutants of S. enterica serovar Typhimurium (n = 4) and E. coli (n = 3) by 41 +/- 8% and 17.3 +/- 7.2%, respectively, compared with their parental strains, which was reversed by the transmembrane proton gradient-collapsing agent carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) and the efflux pump inhibitor phenylalanine-arginine-beta-naphthylamide (PA beta N). The accumulation of H33342 was significantly reduced in mutants of Salmonella Typhimurium with defined disruptions in genes encoding the porins OmpC, OmpF, OmpX and OmpW, but increased in those with disruptions in efflux pump components TolC, AcrB and AcrF. Reduced accumulation of H33342 in three other MAR mutants of Salmonella Typhimurium correlated with the expression of porin and efflux pump proteins. The intracellular accumulation of H33342 provided a sensitive and specific test for MAR that is cheap and relatively rapid. Differential sensitivity to CCCP and PA beta N provided a further means to phenotypically identify MAR mutants and the role of active efflux in each strain.

Characterisation of Escherichia fergusonii isolates from farm animals using an Escherichia coli virulence gene array and tissue culture adherence assays

Escherichia fergusonii has been associated with a wide variety of intestinal and extra-intestinal infections in both humans and animals but, despite strong circumstantial evidence, the degree to which the organism is responsible for the pathologies identified remains uncertain. Thirty isolates of E fergusonii collected between 2003 and 2004 were screened using an Escherichia coli virulence gene array to test for the presence of homologous virulence genes in E. fergusonii. The iss (increased serum survival) gene was present in 13/30 (43%) of the test strains and the prfB (P-related fimbriae regulatory) and ireA (siderophore receptor IreA) genes were also detected jointly in 3/30 (10%) strains. No known virulence genes were detected in 14/30 (47%) of strains. Following confirmatory PCR and sequence analysis, the E. fergusonii prfB, iss and ireA genes shared a high degree of sequence similarity to their counterparts in E. coli, and a particular resemblance was noted with the E. coli strain APEC O1 pathogenicity island. In tissue culture adherence assays, nine E. fergusonii isolates associated with HEp-2 cells with a 'localised adherence' or 'diffuse adherence' phenotype, and they proved to be moderately invasive. The E fergusonii isolates in this study possess both some phenotypic and genotypic features linked to known pathotypes of E coli, and support existing evidence that strains of E fergusonii may act as an opportunistic pathogens, although their specific virulence factors may need to be explored. Crown Copyright (c) 2008 Published by Elsevier Ltd. All rights reserved.

An investigation of the expression and adhesin function of H7 flagella in the interaction of Escherichia coli O157: H7 with bovine intestinal epithelium

Enterohaemorrhagic Escherichia coli O157 : H7 is a bacterial pathogen that can cause haemorrhagic colitis and haemolytic uremic syndrome. In the primary reservoir host, cattle, the terminal rectum is the principal site of E. coli O157 colonization. In this study, bovine terminal rectal primary epithelial cells were used to examine the role of H7 flagella in epithelial adherence. Binding of a fliC(H7) mutant O157 strain to rectal epithelium was significantly reduced as was binding of the flagellated wild-type strain following incubation with H7-specific antibodies. Complementation of fliC(H7) mutant O157 strain with fliC(H7) restored the adherence to wild-type levels; however, complementation with fliC(H6) did not restore it. High-resolution ultrastructural and imunofluorescence studies demonstrated the presence of abundant flagella forming physical contact points with the rectal epithelium. Binding to terminal rectal epithelium was specific to H7 by comparison with other flagellin types tested. In-cell Western assays confirmed temporal expression of flagella during O157 interaction with epithelium, early expression was suppressed during the later stages of microcolony and attaching and effacing lesion formation. H7 flagella are expressed in vivo by individual bacteria in contact with rectal mucosa. Our data demonstrate that the H7 flagellum acts as an adhesin to bovine intestinal epithelium and its involvement in this crucial initiating step for colonization indicates that H7 flagella could be an important target in intervention strategies.

Escherichia coli O157:H7 colonization in small domestic ruminants

Enterohaemorrhagic Escherichia coli O157:H7 was first implicated in human disease in the early 1980s, with ruminants cited as the primary reservoirs. Preliminary studies indicated cattle to be the sole source of E. coli O157:H7 outbreaks in humans; however, further epidemiological studies soon demonstrated that E. coli O157:H7 was widespread in other food sources and that a number of transmission routes existed. More recently, small domestic ruminants (sheep and goats) have emerged as important sources of E. coli O157:H7 human infection, particularly with the widespread popularity of petting farms and the increased use of sheep and goat food products, including unpasteurized cheeses. Although the colonization and persistence characteristics of E. coli O157:H7 in the bovine host have been studied intensively, this is not the case for small ruminants. Despite many similarities to the bovine host, the pathobiology of E. coli O157:H7 in small domestic ruminants does appear to differ significantly from that described in cattle. This review aims to critically review the current knowledge regarding colonization and persistence of E. coli O157:H7 in small domestic ruminants, including comparisons with the bovine host where appropriate.

Intermittent Escherichia coli O157:H7 colonisation at the terminal rectum mucosa of conventionally-reared lambs

In cattle, the lymphoid rich regions of the rectal-anal mucosa at the terminal rectum are the preferred site for Escherichia coli O157:H7 colonisation. All cattle infected by rectal swab administration demonstrate long-term E. coli O157:H7 colonisation, whereas orally challenged cattle do not demonstrate long-term E. coli O157:H7 colonisation in all animals. Oral, but not rectal challenge of sheep with E. coli O157:H7 has been reported, but an exact site for colonisation in sheep is unknown. To determine if E. coli O157:H7 can effectively colonise the ovine terminal rectum, in vitro organ culture (IVOC) was initiated. Albeit sparsely, large, densely packed E. coli O157:H7 micro-colonies were observed on the mucosa of ovine and control bovine terminal rectum explants. After necropsy of orally inoculated lambs, bacterial enumeration of the proximal and distal gastrointestinal tract did suggest a preference for E. coli O157:H7 colonisation at the ovine terminal rectum, albeit for both lymphoid rich and non-lymphoid sites. As reported for cattle, rectal inoculation studies were then conducted to determine if all lambs would demonstrate persistent colonisation at the terminal rectum. After necropsy of E. coli O157:H7 rectally inoculated lambs, most animals were not colonised at gastrointestinal sites proximal to the rectum, however, large densely packed micro-colonies of E. coli O157:H7 were observed on the ovine terminal rectum mucosa. Nevertheless, at the end point of the study (day 14), only one lamb had E. coli O157:H7 micro-colonies associated with the terminal rectum mucosa. A comparison of E. coli O157:H7 shedding yielded a similar pattern of persistence between rectally and orally inoculated lambs. The inability of E. coli O157:H7 to effectively colonise the terminal rectum mucosa of all rectally inoculated sheep in the long term, suggests that E. coli O157:H7 may colonise this site, but less effectively than reported previously for cattle.

Exposure of Escherichia coli and Salmonella enterica serovar Typhimurium to triclosan induces a species-specific response, including drug detoxification

Objectives: The use of triclosan within various environments has been linked to the development of multiple drug resistance (MDR) through the increased expression of efflux pumps such as AcrAB-ToIC. In this work, we investigate the effect of triclosan exposure in order to ascertain the response of two species to the presence of this widely used biocide. Methods: The transcriptomes of Salmonella enterica serovar Typhimurium SL1344 and Escherichia coli K-12 MG1655 after exposure to the MIC of triclosan (0.12 mg/L) were determined in microarray experiments. Phenotypic validation of the transcriptomic data included RT-PCR, ability to form a biofilm and motility assays. Results: Despite important differences in the triclosan-dependent transcriptomes of the two species, increased expression of efflux pump component genes was seen in both. Increased expression of soxS was observed in Salmonella Typhimurium, however, within E. coli, decreased expression was seen. Expression of fabBAGI in Salmonella Typhimurium was decreased, whereas in E. coli expression of fabABFH was increased. Increased expression of ompR and genes within this regulon (e.g. ompC, csgD and ssrA) was seen in the transcriptome of Salmonella Typhimurium. An unexpected response of E. coli was the differential expression of genes within operons involved in iron homeostasis; these included fhu, fep and ent. Conclusions: These data indicate that whilst a core response to triclosan exposure exists, the differential transcriptome of each species was different. This suggests that E. coli K-12 should not be considered the paradigm for the Enterobacteriaceae when exploring the effects of antimicrobial agents.

Interaction between attaching-effacing Escherichia coli O26:K60 and O157:H7 in young lambs

Recent surveys have shown that Escherichia coli O26 is prevalent in ruminants compared with E. coli O157. These serogroups share common colonisation factors and we hypothesised that prior colonisation by E. coli O26 may show reduced colonisation by E. coli O157. To test this hypothesis, strains of E. coli O26:K6O and O157:H7 were tested in competitive in vitro and in vivo studies. Using an established 6-week-old lamb model, an experimental group of lambs was dosed orally with E. coli O26:K6O and then E. coli O157:147 four days later. The faecal shedding of O26:K6O and O157:H7 organisms from this experimental group was compared with that from animals dosed with either O26:K6O alone or O157:H7 alone. Shedding data indicated that counts for O157:H7 were unaffected by the competition from O26:K6O, whereas the O26:K6O counts were lower when competing with O157:H7. (C) 2008 Elsevier Ltd. All rights reserved.

Genome scale reconstruction of a Salmonella metabolic model comparison of similarity and differences with a commensal Escherichia coli strain

Salmonella are closely related to commensal Escherichia coli but have gained virulence factors enabling them to behave as enteric pathogens. Less well studied are the similarities and differences that exist between the metabolic properties of these organisms that may contribute toward niche adaptation of Salmonella pathogens. To address this, we have constructed a genome scale Salmonella metabolic model (iMA945). The model comprises 945 open reading frames or genes, 1964 reactions, and 1036 metabolites. There was significant overlap with genes present in E. coli MG1655 model iAF1260. In silico growth predictions were simulated using the model on different carbon, nitrogen, phosphorous, and sulfur sources. These were compared with substrate utilization data gathered from high throughput phenotyping microarrays revealing good agreement. Of the compounds tested, the majority were utilizable by both Salmonella and E. coli. Nevertheless a number of differences were identified both between Salmonella and E. coli and also within the Salmonella strains included. These differences provide valuable insight into differences between a commensal and a closely related pathogen and within different pathogenic strains opening new avenues for future explorations.

Genetic diversity among Escherichia coli O157 : H7 isolates and identification of genes linked to human infections

An Escherichia coli oligonucleotide microarray based on three sequenced genomes was validated for comparative genomic microarray hybridization and used to study the diversity of E. coli O157 isolates from human infections and food and animal sources. Among 26 test strains, 24 (including both Shiga toxin [Stx]-positive and -negative strains) were found to be related to the two sequenced E. coli O157:117 strains, EDL933 and Sakai. However, these strains showed much greater genetic diversity than those reported previously, and most of them could not be categorized as either lineage I or H. Some genes were found more often in isolates from human than from nonhuman sources; e.g., ECs1202 and ECs2976, associated with stx2AB and stx1AB, were in all isolates from human sources but in only 40% of those from nonhuman sources. Some (but not all) lineage I-specific or -dominant genes were also more frequently associated with isolates from human. The results suggested that it might be more effective to concentrate our efforts on finding markers that are directly related to infection rather than those specific to certain lineages. In addition, two Stx-negative O157 cattle isolates (one confirmed to be 117) were significantly different from other Stx-positive and -negative E. coli O157:117 strains and were more similar to MG1655 in their gene content. This work demonstrates that not all E. coli O157:117 strains belong to the same clonal group, and those that were similar to E. coli K-12 might be less virulent.

A high prevalence of antimicrobial resistant Escherichia coli isolated from pigs and a low prevalence of antimicrobial resistant E-coli from cattle and sheep in Great Britain at slaughter

The incidence of antimicrobial resistance and expressed and unexpressed resistance genes among commensal Escherichia coli isolated from healthy farm animals at slaughter in Great Britain was investigated. The prevalence of antimicrobial resistance among the isolates varied according to the animal species; of 836 isolates from cattle tested only 5.7% were resistant to one or more antimicrobials, while only 3.0% of 836 isolates from sheep were resistant to one or more agents. However, 92.1% of 2480 isolates from pigs were resistant to at least one antimicrobial. Among isolates from pigs, resistance to some antimicrobials such as tetracycline (78.7%), sulphonamide (66.9%) and streptomycin (37.5%) was found to be common, but relatively rare to other agents such as amikacin (0.1%), ceftazidime ( 0.1%) and coamoxiclav (0.2%). The isolates had a diverse range of resistance gene profiles, with tet(B), sul2 and strAB identified most frequently. Seven out of 615 isolates investigated carried unexpressed resistance genes. One trimethoprim-susceptible isolate carried a complete dfrA17 gene but lacked a promoter for it. However, in the remaining six streptomycin-susceptible isolates, one of which carried strAB while the others carried aadA, no mutations or deletions in gene or promoter sequences were identified to account for susceptibility. The data indicate that antimicrobial resistance in E. coli of animal origin is due to a broad range of acquired genes.

Escherichia coli isolates from extraintestinal organs of livestock animals harbour diverse virulence genes and belong to multiple genetic lineages

Escherichia coli, the most common cause of bacteraemia in humans in the UK, can also cause serious diseases in animals. However the population structure, virulence and antimicrobial resistance genes of those from extraintestinal organs of livestock animals are poorly characterised. The aims of this study were to investigate the diversity of these isolates from livestock animals and to understand if there was any correlation between the virulence and antimicrobial resistance genes and the genetic backbone of the bacteria and if these isolates were similar to those isolated from humans. Here 39 E. coli isolates from liver (n=31), spleen (n=5) and blood (n=3) of cattle (n=34), sheep (n=3), chicken (n=1) and pig (n=1) were assigned to 19 serogroups with O8 being the most common (n=7), followed by O101, O20 (both n=3) and O153 (n=2). They belong to 29 multi-locus sequence types, 20 clonal complexes with ST23 (n=7), ST10 (n=6), ST117 and ST155 (both n=3) being most common and were distributed among phylogenetic group A (n=16), B1 (n=12), B2 (n=2) and D (n=9). The pattern of a subset of putative virulence genes was different in almost all isolates. No correlation between serogroups, animal hosts, MLST types, virulence and antimicrobial resistance genes was identified. The distributions of clonal complexes and virulence genes were similar to other extraintestinal or commensal E. coli from humans and other animals, suggesting a zoonotic potential. The diverse and various combinations of virulence genes implied that the infections were caused by different mechanisms and infection control will be challenging.

Assessing the microbial oxidative stress mechanism of ozone treatment through the responses of Escherichia coli mutants

Aims: To investigate the effect of the oxidative stress of ozone on the microbial inactivation, cell membrane integrity and permeability and morphology changes of Escherichia coli. Methods and Results: Escherichia coli BW 25113 and its isogenic mutants in soxR, soxS, oxyR, rpoS and dnaK genes were treated with ozone at a concentration of 6 lg ml)1 for a period up to 240 s. A significant effect of ozone exposure on microbial inactivation was observed. After ozonation, minor effects on the cell membrane integrity and permeability were observed, while scanning electron microscopy analysis showed slightly altered cell surface structure. Conclusions: The results of this study suggest that cell lysis was not the major mechanism of microbial inactivation. The deletion of oxidative stress–related genes resulted in increased susceptibility of E. coli cells to ozone treatment, implying that they play an important role for protection against the radicals produced by ozone. However, DnaK that has previously been shown to protect against oxidative stress did not protect against ozone treatment in this study. Furthermore, RpoS was important for the survival against ozone. Significance and Impact of the Study: This study provides important information about the role of oxidative stress in the responses of E. coli during ozonation.