Mutation of toxB and a truncated version of the efa-1 gene in Escherichia coli O157 : H7 influences the expression and secretion of locus of enterocyte effacement-encoded proteins but not intestinal colonization in calves or sheep

Enterohemorrhagic Escherichia coli (EHEC) strains comprise a broad group of bacteria, some of which cause attaching and effacing (AE) lesions and enteritis in humans and animals. Non-O157:H7 EHEC strains contain the gene efa-1 (referred to in previous publications as efa1), which influences adherence to cultured epithelial cells. An almost identical gene in enteropathogenic E. coli (lifA) mediates the inhibition of lymphocyte proliferation and proinflammatory cytokine synthesis. We have shown previously that significantly lower numbers of EHEC 05 and 0111 efa-1 mutants are shed in feces following experimental infection in calves and that these mutants exhibit reduced adherence to intestinal epithelia compared with isogenic wild-type strains. E. coli O157:H7 strains lack efa-1 but encode a homolog on the pO157 plasmid (toxB/l7095) and contain a truncated version of the efa-1 gene (efa-1'/z4332 in O island 122 of the EDL933 chromosome). Here we report that E. coli O157:H7 toxB and efa-1' single and double mutants exhibit reduced adherence to cultured epithelial cells and show reduced expression and secretion of proteins encoded by the locus of enterocyte effacement (LEE), which plays a key role in the host-cell interactions of EHEC. The activity of LEE1, LEE4, and LEE5 promoters was not significantly altered in E. coli O157:H7 strains harboring toxB or efa-1' mutations, indicating that the effect on the expression of LEE-encoded secreted proteins occurs at a posttranscriptional level. Despite affecting type III secretion, mutation of toxB and efa-1' did not significantly affect the course of fecal shedding of E. coli O157:H7 following experimental inoculation of 10- to 14-day-old calves or 6-week-old sheep. Mutation of tir caused a significant reduction in fecal shedding of E. coli O157:H7 in calves, indicating that the formation of AE lesions is important for colonization of the bovine intestine.

A defined intestinal colonization microbiota for gnotobiotic pigs

Maximising the ability of piglets to survive exposure to pathogens is essential to reduce early piglet mortality, an important factor in efficient commercial pig production. Mortality rates can be influenced by many factors, including early colonization by microbial commensals. Here we describe the development of an intestinal microbiota, the Bristol microbiota, for use in gnotobiotic pigs and its influence on synthesis of systemic immunoglobulins. Such a microbiota will be of value in studies of the consequences of early microbial colonization on development of the intestinal immune system and subsequent susceptibility to disease. Gnotobiotic pig studies lack a well-established intestinal microbiota. The use of the Altered Schaedler Flora (ASF), a murine intestinal microbiota, to colonize the intestines of Caesarean-derived, gnotobiotic pigs prior to gut closure, resulted in unreliable colonization with most (but not all) strains of the ASF. Subsequently, a novel, simpler porcine microbiota was developed. The novel microbiota reliably colonized the length of the intestinal tract when administered to gnotobiotic piglets. No health problems were observed, and the novel microbiota induced a systemic increase in serum immunoglobulins, in particular IgA and IgM. The Bristol microbiota will be of value for highly controlled, reproducible experiments of the consequences of early microbial colonization on susceptibility to disease in neonatal piglets, and as a biomedical model for the impact of microbial colonization on development of the intestinal mucosa and immune system in neonates.

Role of intimin-Tir interactions and the Tir-cytoskeleton coupling protein in the colonization of calves and lambs by Escherichia coli O157:H7

Intimin facilitates intestinal colonization by enterohemorrhagic Escherichia coli O157:H7; however, the importance of intimin binding to its translocated receptor (Tir) as opposed to cellular coreceptors is unknown. The intimin-Tir interaction is needed for optimal actin assembly under adherent bacteria in vitro, a process which requires the Tir-cytoskeleton coupling protein (TccP/EspF(U)) in E. coli O157:H7. Here we report that E. coli O157:H7 tir mutants are at least as attenuated as isogenic eae mutants in calves and lambs, implying that the role of intimin in the colonization of reservoir hosts can be explained largely by its binding to Tir. Mutation of tccP uncoupled actin assembly from the intimin-Tir-mediated adherence of E. coli O157:H7 in vitro but did not impair intestinal colonization in calves and lambs, implying that pedestal formation may not be necessary for persistence. However, an E. coli O157:H7 tccP mutant induced typical attaching and effacing lesions in a bovine ligated ileal loop model of infection, suggesting that TccP-independent mechanisms of actin assembly may operate in vivo.

Environmental regulation and colonization attributes of the long polar fimbriae (LPF) of Escherichia coli O157:H7

The ability of Escherichia coli O157:H7 to colonize the intestinal epithelia is dependent on the expression of intimin and other adhesins. The chromosome of E. coli O157:H7 carries two loci encoding long polar fimbriae (LPF). These fimbriae mediate adherence to epithelial cells and are associated with colonization of the intestine. In order to increase our knowledge about the conditions controlling their expression and their role in colonization of an animal model, the environmental cues that promote expression of lpf genes and the role of E. coli O157:H7 LPF in intestinal colonization of lambs were investigated. We found that expression of lpf1 was regulated in response to growth phase, osmolarity, and pH; that lpf2 transcription was stimulated during late exponential growth and iron depletion; and that LPF impacts the ability of E. coli O157:H7 to persist in the intestine of infected 6-week-old lambs.

Characterization of two non-locus of enterocyte effacement-encoded type III-translocated effectors, NleC and NleD, in attaching and effacing pathogens

Intestinal colonization by enteropathogenic and enterohemorrhagic Escherichia coli requires the locus of enterocyte effacement-encoded type III secretion system. We report that NleC and NleD are translocated into host cells via this system. Deletion mutants induced attaching and effacing lesions in vitro, while infection of calves or lambs showed that neither gene was required for colonization.

In vitro studies on colonization resistance of the human gut microbiota to Candida albicans and the effects of tetracycline and Lactobacillus plantarum LPK

An anaerobic three-vessel continuous-flow culture system, which models the three major anatomical regions of the human colon, was used to study the persistence of Candida albicans in the presence of a faecal microbiota. During steady state conditions, overgrowth of C. albicans was prevented by commensal bacteria indigenous to the system. However antibiotics, such as tetracycline have the ability to disrupt the bacterial populations within the gut. Thus, colonization resistance can be compromised and overgrowth of undesirable microorganisms like C. albicans can then occur. In this study, growth of C. albicans was not observed in the presence of an established faecal microbiota. However, following the addition of tetracycline to the growth medium, significant growth of C. albicans occurred. A probiotic Lactobacillus plantarum LPK culture was added to the system to investigate whether this organism had any effects upon the Candida populations. Although C. albicans was not completely eradicated in the presence of this bacterium, cell counts were markedly reduced, indicating a compromised physiological function. This study shows that the normal gut flora can exert 'natural' resistance to C. albicans, however this may be diminished during antibiotic intake. The use of probiotics can help fortify natural resistance.

Lactobacilli antagonize the growth, motility, and adherence of brachyspira pilosicoli: a potential intervention against avian intestinal spirochetosis

Avian intestinal spirochetosis (AIS) results from the colonization of the ceca and colorectum of poultry by pathogenic Brachyspira species. The number of cases of AIS has increased since the 2006 European Union ban on the use of antibiotic growth promoters, which, together with emerging antimicrobial resistance in Brachyspira, has driven renewed interest in alternative intervention strategies. Probiotics have been reported as protecting livestock against infection with common enteric pathogens, and here we investigate which aspects of the biology of Brachyspira they antagonize in order to identify possible interventions against AIS. The cell-free supernatants (CFS) of two Lactobacillus strains, Lactobacillus reuteri LM1 and Lactobacillus salivarius LM2, suppressed the growth of Brachyspira pilosicoli B2904 in a pH-dependent manner. In in vitro adherence and invasion assays with HT29-16E three-dimensional (3D) cells and in a novel avian cecal in vitro organ culture (IVOC) model, the adherence and invasion of B. pilosicoli in epithelial cells were reduced significantly by the presence of lactobacilli (P < 0.001). In addition, live and heat-inactivated lactobacilli inhibited the motility of B. pilosicoli, and electron microscopic observations indicated that contact between the lactobacilli and Brachyspira was crucial in inhibiting both adherence and motility. These data suggest that motility is essential for B. pilosicoli to adhere to and invade the gut epithelium and that any interference of motility may be a useful tool for the development of control strategies.

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.

Colonization of 8-week-old conventionally reared goats by Escherichia coli O157:H7 after oral inoculation

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 1 X 10(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 In after inoculation, the three E coli O157 : H7 strains were shed at approximately 5 X 1 04 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 Nalr were sampled at 24,48 and 96 In 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 In. However, the animal examined at 96 h, which had uniquely shed approximately 1 x 10(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.

Restricting microbial exposure in early life negates the immune benefits associated with gut colonization in environments of high microbial diversity

Background: Acquisition of the intestinal microbiota in early life corresponds with the development of the mucosal immune system. Recent work on caesarean-delivered infants revealed that early microbial composition is influenced by birthing method and environment. Furthermore, we have confirmed that early-life environment strongly influences both the adult gut microbiota and development of the gut immune system. Here, we address the impact of limiting microbial exposure after initial colonization on the development of adult gut immunity. Methodology/Principal Findings: Piglets were born in indoor or outdoor rearing units, allowing natural colonization in the immediate period after birth, prior to transfer to high-health status isolators. Strikingly, gut closure and morphological development were strongly affected by isolator-rearing, independent of indoor or outdoor origins of piglets. Isolator-reared animals showed extensive vacuolation and disorganization of the gut epithelium, inferring that normal gut closure requires maturation factors present in maternal milk. Although morphological maturation and gut closure were delayed in isolatorreared animals, these hard-wired events occurred later in development. Type I IFN, IL-22, IL-23 and Th17 pathways were increased in indoor-isolator compared to outdoor-isolator animals during early life, indicating greater immune activation in pigs originating from indoor environments reflecting differences in the early microbiota. This difference was less apparent later in development due to enhanced immune activation and convergence of the microbiota in all isolator-reared animals. This correlated with elevation of Type I IFN pathways in both groups, although T cell pathways were still more affected in indoor-reared animals. Conclusions/Significance: Environmental factors, in particular microbial exposure, influence expression of a large number of immune-related genes. However, the homeostatic effects of microbial colonization in outdoor environments require sustained microbial exposure throughout development. Gut development in high-hygiene environments negatively impacts on normal succession of the gut microbiota and promotes innate immune activation which may impair immune homeostasis.

Evidence for systemic spread of the potentially zoonotic intestinal spirochaete Brachyspira pilosicoli in experimentally challenged laying chickens

Brachyspira pilosicoli is a potentially zoonotic anaerobic intestinal spirochaete that is one of several species causing avian intestinal spirochaetosis. The aim of this study was to develop a reproducible model of infection in point-of-lay chickens and compare the virulence of two strains of B. pilosicoli in a model using experimentally challenged laying chickens. Seventeen-week-old commercial laying chickens were experimentally challenged by oral gavage with either B. pilosicoli strain B2904 or CPSp1, following an oral dose of 10 % sodium bicarbonate to neutralize acidity in the crop. Approximately 80 % of the chickens became colonized and exhibited increased faecal moisture content, reduced weight gain and delayed onset of lay. Tissues sampled at post-mortem examination were analysed to produce a quantitative output on the number of spirochaetes present and hence, the extent of colonization. The liver and spleen were colonized, and novel histopathology was observed in these tissues. The infection model we report here has potential use in studies to improve our understanding of the mechanisms by which Brachyspira elicit disease in poultry and in testing novel intervention strategies.

New evidence for the antiquity of the intestinal parasite Trichuris (whipworm) in Europe

The whipworm, Trichuris trichiura L., is one of the most common human intestinal parasites worldwide, yet little is known of its origin and global spread. Archaeological records for this nematode have all been of Neolithic or later date, suggesting a possible association between the spread of pastoral farming and human acquisition of whipworm. This paper reports the discovery of eggs of the genus Trichuris in late Mesolithic deposits from south Wales, indicating that whipworm was present in Europe before the arrival of agriculture. This raises the possibility that human infection by Trichuris arose through contact with wild animals in parts of the landscape frequented by both human and animal groups.

Mixed culture fermentation studies on the effects of synbiotics on the human intestinal pathogens Campylobacter jejuni and Escherichia coli

Batch and continuous culture anaerobic fermentation systems, inoculated with human faeces, were utilised to investigate the antimicrobial actions of two probiotics, Lactobacillus plantartan 0407, combined with oligofructose and Bifidobacterium bifidum Bb12, combined with a mixture of oligofructose and xylo-oligosaccharides (50:50 w/w) against E coli and Campylobacter jejuni. In batch fermenters, both E coli and C jejuni were inhibited by the synbiotics, even when the culture pH was maintained at around neutral. In continuous culture C jejuni was inhibited but the synbiotic failed to inhibit E coli. Although no definitive answer in addressing the mechanisms underlying antimicrobial activity was derived, results suggested that acetate and lactate directly were conferring antagonistic action, rather than as a result of lowering culture pH. In the course of the study culturing and fluorescent in situ hybridisation (FISH) methodologies for the enumeration of bacterial populations were compared. Bifidobacterial populations were underestimated using plating techniques, suggesting the non-culturability of certain bifidobacterial species. (C) 2003 Elsevier Ltd. All rights reserved.

The effects of probiotics on the composition of the intestinal microbiota following antibiotic therapy

The effects of probiotic supplementation on the intestinal re-growth microbiota following antibiotic therapy were studied in a double-blind placebo-controlled study. In the placebo group, numbers of facultative anaerobes and enterobacteria increased significantly, and at day 35 the numbers were significantly higher in the placebo group than in the active group; in the active group, the numbers of bacteroides increased significantly. Although the numbers of enterococci in both groups did not change, in the placebo group the number of patients harbouring antibiotic-resistant enterococci post therapy increased significantly. There was no change in the incidence rate of antibiotic resistance among the patients in the probiotic group.