The prevalence of enterohaemorrhagic
Escherichia coli (EHEC) O157 in poultry is considered minimal compared with other species, especially ruminants. However, deliberate inoculation studies have ...shown that poultry are readily and persistently infected by this organism but that the mechanism of colonisation is independent of intimin, a recognised factor in host–EHEC interactions in mammalian species, and may be dependent upon flagella. Few strains of EHEC O157 have been tested in poultry and here 1-day-old and 6-week-old chicks were inoculated with seven non-toxigenic
E. coli O157 strains in separate experiments. Persistence was measured semi-quantitatively by bacteriological assessment of
E. coli O157 cultured from cloacal swabs (shedding score). In the 1-day-old chick model that was monitored for 43 days, all seven strains established well after inoculation. In the 6-week-old chicken model, one strain established and gave consistently high shedding for the duration of the experiment (156 days). Whereas of the remaining six strains, two persisted for 113 days, two persisted for 43 days, one persisted for 22 days and one strain was never detected.
Ruminants harbour both O157:H7 and non-O157 Attaching Effacing
Escherichia coli (AEEC) strains but to date only non-O157 AEEC have been shown to induce attaching effacing lesions in naturally ...infected animals. However, O157 may induce lesions in deliberate oral inoculation studies and persistence is considered dependent upon the bacterially encoded locus for enterocyte effacement. In concurrent infections in ruminants it is unclear whether non-O157 AEEC contribute either positively or negatively to the persistence of
E. coli O157:H7. To investigate this, and prior to animal studies,
E. coli O157:H7 NCTC 12900, a non-toxigenic strain that persists in conventionally reared sheep, and non-toxigenic AEEC O26:K60 isolates of sheep origin were tested for adherence to HEp-2 tissue culture alone and in competition one with another. Applied together, both strains adhered in similar numbers but lower than when either was applied separately. Pre-incubation of tissue culture with either one strain reduced significantly (
P < 0.05) the extent of adherence of the strain that was applied second. It was particularly noticeable that AEEC O26 when applied first reduced adherence and inhibited microcolony formation, as demonstrated by confocal microscopy, of
E. coli O157:H7. The possibility that prior colonisation of a ruminant by non-O157 AEEC such as O26 may antagonise O157 colonisation and persistence in ruminants is discussed.
Enterohaemorrhagic Escherichia coli O157 : H7 infections of man have been associated with consumption of unpasteurized goat's milk and direct contact with kid goats on petting farms, yet little is ...known about colonization of goats with this organism. To assess the contribution of flagella and intimin of E. coli O157 : H7 in colonization of the goat, 8-week-old conventionally reared goats were inoculated orally in separate experiments with 1x10(10) c.f.u. of a non-verotoxigenic strain of E. coli O157 : H7 (strain NCTC 12900 Nal(r)), an aflagellate derivative (DMB1) and an intimin-deficient derivative (DMB2). At 24 h after inoculation, the three E. coli O157 : H7 strains were shed at approximately 5x10(4) c.f.u. (g faeces)(-1) from all animals. Significantly fewer intimin-deficient bacteria were shed only on days 2 (P = 0.003) and 4 (P = 0.014), whereas from day 7 to 29 there were no differences. Tissues from three animals inoculated with wild-type E. coli O157 : H7 strain NCTC 12900 Nal(r) were sampled at 24, 48 and 96 h after inoculation and the organism was cultured from the large intestine of all three animals and from the duodenum and ileum of the animal examined at 96 h. Tissues were examined histologically but attaching-effacing (AE) lesions were not observed at any intestinal site of the animals examined at 24 or 48 h. However, the animal examined at 96 h, which had uniquely shed approximately 1x10(7) E. coli O157 : H7 (g faeces)(-1) for the preceding 3 days, showed a heavy, diffuse infection with cryptosporidia and abundant, multifocal AE lesions in the distal colon, rectum and at the recto-anal junction. These AE lesions were confirmed by immunohistochemistry to be associated with E. coli O157 : H7.
Conventional aldehyde based fixatives produce good morphological preservation. However, owing to their cross-linking mechanism of action, epitope loss may occur during fixation compromising the ...tissue for subsequent immunohistochemical (IHC) analysis. IHC is an important tool for characterizing antigen, cytokine and cytomorphological markers. The increasing use of mouse models for study of pathogenesis has highlighted the need to investigate alternative fixatives. In the study reported here, tissue samples from RIII mice with immune mediated lesions, Mycobacterium bovis infected mice, and uninfected control mice were fixed in either zinc salt fixative or buffered formalin, then tested for IHC using a panel of antibodies (CD3, CD4, CD8, CD45, CD54, F4/80, Interferon-gamma and MIP2). Zinc salt fixation preserved processing-sensitive murine cell markers (CD4, CD8 and CD54) and improved the intensity of immunolabeling for CD45, F4/80 and CD3. Buffered formalin failed to preserve any of the processing-sensitive murine epitopes for demonstration by subsequent IHC.
Spores from a number of different Bacillus species are currently being used as human and animal probiotics, although their mechanisms of action remain poorly understood. Here we describe the ...isolation of 237 presumptive gut-associated Bacillus spp. isolates that were obtained by heat and ethanol treatment of fecal material from organically reared broilers followed by aerobic plating. Thirty-one representative isolates were characterized according to their morphological, physiological, and biochemical properties as well as partial 16S rRNA gene sequences and screening for the presence of plasmid DNA. The Bacillus species identified included B. subtilis, B. pumilus, B. licheniformis, B. clausii, B. megaterium, B. firmus, and species of the B. cereus group, whereas a number of our isolates could not be classified. Intrinsic properties of potential importance for survival in the gut that could be advantageous for spore-forming probiotics were further investigated for seven isolates belonging to five different species. All isolates sporulated efficiently in the laboratory, and the resulting spores were tolerant to simulated gastrointestinal tract conditions. They also exhibited antimicrobial activity against a broad spectrum of bacteria, including food spoilage and pathogenic organisms such as Bacillus spp., Clostridium perfringens, Staphylococcus aureus, and Listeria monocytogenes. Importantly, the isolates were susceptible to most of the antibiotics tested, arguing that they would not act as donors for resistance determinants if introduced in the form of probiotic preparations. Together, our results suggest that some of the sporeformers isolated in this study have the potential to persist in or transiently associate with the complex gut ecosystem.
Objectives AcrA can function as the periplasmic adaptor protein (PAP) in several RND tripartite efflux pumps, of which AcrAB-TolC is considered the most important. This system confers innate multiple ...antibiotic resistance. Disruption of acrB or tolC impairs the ability of Salmonella Typhimurium to colonize and persist in the host. The aim of this study was to investigate the role of AcrA alone in multidrug resistance and pathogenicity. Methods The acrA gene was inactivated in Salmonella Typhimurium SL1344 by insertion of the aph gene and this mutant complemented with pWKS30acrA. The antimicrobial susceptibility of the mutant to six antibiotics as well as various dyes and detergents was determined. In addition, efflux activity was quantified. The ability of the mutant to adhere to, and invade, tissue culture cells in vitro was measured. Results Following disruption of acrA, RT–PCR and western blotting confirmed that acrB/AcrB was still expressed when acrA was disrupted. The acrA mutant was hypersusceptible to antibiotics, dyes and detergents. In some cases, lower MICs were seen than for the acrB or tolC mutants. Efflux of the fluorescent dye Hoechst H33342 was less than in wild-type following disruption of acrA. acrA was also required for adherence to, and invasion of, tissue culture cells. Conclusions Inactivation of acrA conferred a phenotype distinct to that of acrB::aph and tolC::aph. These data indicate a role for AcrA distinct to that of other protein partners in both efflux of substrates and virulence.
Four conventionally reared goats aged 6 days were inoculated orally with approximately 10
10 colony-forming units (cfu) of a non-verotoxigenic strain of
Escherichia coli O157:H7. All remained ...clinically normal. Tissues were sampled under terminal anaesthesia at 24 (two animals), 48 and 72
h post-inoculation (hpi).
E. coli O157:H7 was cultured from the ileum, caecum, colon and rectum of all animals, but the number of bacteria recovered at these sites varied between animals. Attaching-effacing (AE) lesions associated with O157 organisms, as confirmed by immunolabelling, were observed in the ileum of one of the two animals examined at 24
hpi, and in the ileum, caecum and proximal colon of an animal examined at 72
hpi.
E. coli O157 organisms were detected at ≥10
5
cfu/g of tissue at these sites. In addition, AE lesions associated with unidentified bacteria were observed at various sites in the large bowel of the same animals. Lesions containing both
E. coli O157 and unidentified bacteria (non-O157) were not observed. Non-O157 AE lesions were also observed in the large bowel of one of two uninoculated control animals. This indicated that three (one control and two inoculated) animals were colonized with an unidentified AE organism before the commencement of the experiment. The O157-associated AE lesions were observed only in animals colonized by non-O157 AE organisms and this raises questions about individual host susceptibility to AE lesions and whether non-O157 AE organisms influence colonization by
E. coli O157.
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