Faecal Supernatants Research Articles

Changes in signalling from faecal neuroactive metabolites following dietary modulation of IBS pain

ObjectiveDietary therapies for irritable bowel syndrome (IBS) have received increasing interest but predicting which patients will benefit remains a challenge due to a lack of mechanistic insight. We recently found...

Strain-specific impacts of probiotics are a significant driver of gut microbiome development in very preterm infants

The development of the gut microbiome from birth plays important roles in short- and long-term health, but factors influencing preterm gut microbiome development are poorly understood. In the present study, we use metagenomic sequencing to analyse 1,431 longitudinal stool samples from 123 very preterm infants (<32 weeks’ gestation) who did not develop intestinal disease or sepsis over a study period of 10 years. During the study period, one cohort had no probiotic exposure whereas two cohorts were given different probiotic products: Infloran (Bifidobacterium bifidum and Lactobacillus acidophilus) or Labinic (B. bifidum, B. longum subsp. infantis and L. acidophilus). Mothers’ own milk, breast milk fortifier, antibiotics and probiotics were significantly associated with the gut microbiome, with probiotics being the most significant factor. Probiotics drove microbiome transition into different preterm gut community types (PGCTs), each enriched in a different Bifidobacterium sp. and significantly associated with increased postnatal age. Functional analyses identified stool metabolites associated with PGCTs and, in preterm-derived organoids, sterile faecal supernatants impacted intestinal, organoid monolayer, gene expression in a PGCT-specific manner. The present study identifies specific influencers of gut microbiome development in very preterm infants, some of which overlap with those impacting term infants. The results highlight the importance of strain-specific differences in probiotic products and their impact on host interactions in the preterm gut.

OP0245 MICROBIOTA-INDUCED INTESTINAL BARRIER DYSFUNCTION PRECEDES THE ONSET OF ARTHRITIS AND ALLOWS THE SHUTTLING OF IMMUNE CELLS FROM THE GUT THE JOINTS

Background:While it is known that microbial dysbiosis is associated with the onset of rheumatoid arthritis, mechanistic insights how it facilitates the development of arthritis remained largely elusive to date. It is especially interesting how microbial dysbiosis affects the transition from asymptomatic autoimmunity to arthritis. We speculated that a breakdown of intestinal barrier function caused by microbial dysbiosis allows immune cells to shuttle from the gut to the joints.Objectives:To test whether intestinal barrier function is impaired before the onset of human RA and experimental arthritis and to seek for evidence that immune cells from the gut migrate to the joints.Methods:In a longitudinal cohort of RA-at risk individuals markers of disturbed intestinal barrier function, such as zonulin, were analysed and linked to RA onset. Furthermore, new-onset RA patients were assessed for gut leakiness and their intestinal biopsies for the expression of tight junction proteins and immune cell infiltration. In the murine model of collagen-induced arthritis, sequential analysis of intestinal dysbiosis, intestinal barrier function and arthritis onset was carried out. Additionally, barrier function was assessed on intestinal organoids exposed to faecal supernatants from eu- and dysbiotic mice with and without inhibition of zonulin. Furthermore, three types of interventions restoring intestinal barrier function were carried out for testing their effects on the inhibition of arthritis onset. Finally, photo- converted cells from the gut were traced in the joints to test for cellular trafficking from one to the other compartment.Results:Zonulin, a potent regulator for intestinal tight junctions, was elevated in autoimmune mice and men before the onset of arthritis and predicted the onset of human RA. Intestinal barrier functions as well as epithelial tight junctions were decreased before the onset of experimental arthritis and at onset of human RA. In mice, induction of autoimmunity was followed by rapid intestinal dysbiosis followed by gut leakiness before arthritis started. Faecal supernatants of arthritic mice induce epithelial barrier dysfunction in intestinal organoids in zonulin dependent manner. Restoration of the intestinal barrier in the pre-phase of arthritis using butyrate, CB1R agonist or zonulin antagonist larazotide inhibited the development of arthritis. Finally, using photoconvertible mice, gut-borne immune cells were identified that homed to the joints when barrier function was impaired.Conclusion:In summary, these data show the intestinal barrier dysfunction precedes the onset of RA and allows the trafficking of immune cells from the gut to the joints. Targeting of intestinal tight junction function may therefore allow preventing the onset of RA.Acknowledgments:Funded by the DFG-FOR2886 PANDORA, DFG–CRC118, Staedtler foundation, Johannes und Frieda Marohn-Stiftung, Else Kröner-Fresenius foundation, Interdisciplinary Centre for Clinical Research, Erlangen (IZKF), BMBF-MASCARA and the IMI funded projectRTCure.Disclosure of Interests:Mario Zaiss: None declared, Narges Taijc: None declared, Kerstin Sarter: None declared, Vugar Azizov: None declared, laura Bucci: None declared, Yubin Luo: None declared, Juan de Dios Cañete: None declared, francesco ciccia Grant/research support from: pfizer, novartis, roche, Consultant of: pfizer, novartis, lilly, abbvie, Speakers bureau: pfizer, novartis, lilly, abbvie, Georg Schett Speakers bureau: AbbVie, BMS, Celgene, Janssen, Eli Lilly, Novartis, Roche and UCB

Faecal Proteases from Pouchitis Patients Activate Protease Activating Receptor-2 to Disrupt the Epithelial Barrier.

The pathogenesis of pouch inflammation may involve epithelial barrier disruption. We investigated whether faecal proteolytic activity is increased during pouchitis and results in epithelial barrier dysfunction through protease activating receptor [PAR] activation, and assessed whether the intestinal microbiome may be the source of the proteases. Faecal samples were measured for protease activity using a fluorescein isothiocyanate [FITC]-casein florescence assay. Caco-2 cell monolayers were exposed to faecal supernatants to assess permeability to FITC-dextran. Tight junction protein integrity and PAR activation were assessed by immunoblot and immunofluorescence. A truncated PAR2 protein in Caco-2 cells was achieved by stable transfection using CRISPR/Cas9 plasmid. PAR2 activation in pouch biopsies was examined using antibodies directed to the N-terminus of the protein. Microbial composition was analysed based on 16S rRNA gene sequence analysis. Ten pouchitis patients, six normal pouch [NP] patients and nine healthy controls [HC] were recruited. The pouchitis patients exhibited a 5.19- and 5.35-fold higher faecal protease [FP] activity [p ≤ 0.05] compared to the NP and HC participants, respectively. The genus Haemophilus was positively associated with FP activity [R = 0.718, false discovery rate < 0.1]. Faecal supernatants from pouchitis patients activated PAR2 on Caco-2 monolayers, disrupted tight junction proteins and increased epithelial permeability. PAR2 truncation in Caco-2 abrogated faecal protease-mediated permeability. Pouch biopsies obtained from pouchitis patients, but not from NP patients, displayed PAR2 activation. Protease-producing bacteria may increase faecal proteolytic activity that results in pouch inflammation through disruption of tight junction proteins and increased epithelial permeability in a PAR2-dependent manner. This mechanism may initiate or propagate pouch inflammation.

Serine proteases as luminal mediators of intestinal barrier dysfunction and symptom severity in IBS

ObjectiveThe intestinal lumen contains several proteases. Our aim was to determine the role of faecal proteases in mediating barrier dysfunction and symptoms in IBS.Design39 patients with IBS and 25 healthy...

New Micro-amount of Virion Enrichment Technique (MiVET) to detect influenza A virus in the duck faeces.

Transboundary animal diseases, including highly pathogenic avian influenza, cause vast economic losses throughout the world. While it is important to identify the sources and propagation routes of the spread, such strategies are often hindered by incomplete epidemiological evidence. Isolation/detection of micro-amounts of pathogens from environmental samples is rarely successful due to the very low contamination level. This paper describes the development of the micro-amount of virion enrichment technique (MiVET), a simple and highly sensitive method that combines the use of a complex comprising a polyclonal antibody and protein G-coated magnetic beads for virion capture, and simple sodium dodecyl benzenesulfonate (SDBS) elution for low volume samples. The performance of the MiVET was evaluated using avian influenza A viruses (AIVs) in artificially spiked samples by real-time reverse transcription polymerase chain reaction (rRT-PCR). Four AIVs, H3N2, H4N2, H5N2 and H7N7, were used to artificially spike 50ml of phosphate-buffered saline (PBS) and 1ml of 10%-25% duck faecal supernatants. The MiVET system successfully concentrated AIVs in both PBS and faecal samples with at least 2 and 1 log greater efficacy, respectively, than conventional RNA extraction methods. The MiVET could be completed in <30min from the beginning of sample preparation to final RNA extraction. The MiVET effectively prevented the effects of inhibitors in faecal samples, and did not require special equipment. This is the first report of this novel type of system, which is expected to be useful for the detection of micro-amounts of various veterinary and human viruses to elucidate their circulation dynamics in the environment, and for rapid and sensitive diagnosis with greater detection power.

Preclinical Development of a Novel, Orally-Administered Anti-Tumour Necrosis Factor Domain Antibody for the Treatment of Inflammatory Bowel Disease

TNFα is an important cytokine in inflammatory bowel disease. V565 is a novel anti-TNFα domain antibody developed for oral administration in IBD patients, derived from a llama domain antibody and engineered to enhance intestinal protease resistance. V565 activity was evaluated in TNFα-TNFα receptor-binding ELISAs as well as TNFα responsive cellular assays and demonstrated neutralisation of both soluble and membrane TNFα with potencies similar to those of adalimumab. Although sensitive to pepsin, V565 retained activity after lengthy incubations with trypsin, chymotrypsin, and pancreatin, as well as mouse small intestinal and human ileal and faecal supernatants. In orally dosed naïve and DSS colitis mice, high V565 concentrations were observed in intestinal contents and faeces and immunostaining revealed V565 localisation in mouse colon tissue. V565 was detected by ELISA in post-dose serum of colitis mice, but not naïve mice, demonstrating penetration of disrupted epithelium. In an ex vivo human IBD tissue culture model, V565 inhibition of tissue phosphoprotein levels and production of inflammatory cytokine biomarkers was similar to infliximab, demonstrating efficacy when present at the disease site. Taken together, results of these studies provide confidence that oral V565 dosing will be therapeutic in IBD patients where the mucosal epithelial barrier is compromised.

CrAssphage is not associated with diarrhoea and has high genetic diversity.

crAssphage is a newly discovered gut bacteriophage. However, its pathogenicity and molecular epidemiology in humans are as yet unclear. In this study, we investigated the association between crAssphage and diarrhoea, as well as the molecular epidemiology of crAssphage in Chinese patients from our hospital. Our results indicated that there were no significant differences in the crAssphage-positive ratio and viral loads in faecal supernatants between adults with diarrhoea and healthy adults. Of infants and children with diarrhoea, 2·8% were found to be crAssphage-positive, including two infants aged <1 month. Markedly, of all confirmed crAssphage-positive strains, 100% had the ORF00039 deletion and 77·8% had low identity of ORF00018 compared to crAssphage (GenBank accession no. NC_024711, designated genotype 1). Thus, crAssphage was not associated with diarrhoea and most strains of crAssphage in Chinese patients (designated genotype 2) were characterized by the ORF00039 deletion and low identity of ORF00018.

A microbiological assay to estimate the antimicrobial activity of parenteral tildipirosin against foodborne pathogens and commensals in the colon of beef cattle and pigs.

Tildipirosin (TIP) is a novel 16-membered-ring macrolide authorized for the treatment of bovine and swine respiratory disease. The pH dependency of macrolide antimicrobial activity is well known. Considering that the pH in the colon contents of growing beef cattle and pigs is usually below pH 7.0, the minimum inhibitory concentrations (MIC) of TIP against foodborne bacterial pathogens such as Campylobacter (C.) coli, C. jejuni and Salmonella enterica and commensal species including Enterococcus (E.) faecalis, E. faecium and Escherichia coli were determined under standard (pH 7.3 ± 1) or neutral as well as slightly acidic conditions. A decrease in pH from 7.3 to 6.7 resulted in an increase in MICs of TIP. Except for the MICs > 256 μg/mL observed in the resistant subpopulation of the C. coli and the Enterococcus species, the MIC ranges increased from 2-8 μg/mL to 64-> 256 μg/mL for Salmonella enterica and E. coli, from 8-16 μg/mL to 32-128 μg/mL for the two Campylobacter species, and from 4-32 μg/mL to 128-> 256 μg/mL for both Enterococcus species. To estimate the antimicrobial activity of TIP in the colon contents of livestock during recommended usage of the parenterally administered TIP (Zuprevo(®) ), and to compare this with the increased MICs at the slightly acidic colonic pH, we developed and validated a microbiological assay for TIP and used this to test incurred faecal samples collected from cattle and pigs. Microbiological activity of luminal TIP was determined in aqueous supernatants from diluted faeces, using standard curves produced from TIP-spiked faecal supernatants. The limit of quantification (LOQ) for TIP was 1 μg/mL (ppm). In a cattle study (n = 14), 3 of 28 faecal samples collected 24 and 48 h post-treatment were found to contain TIP above the LOQ (concentrations of 1.3-1.8 ppm). In another cattle study (n = 12) with faecal samples collected at 8, 24 and 48 h post-treatment, TIP concentrations were above the LOQ in 4 of the 8 h samples (1.2-2.6 ppm) and one of the 24-h samples (1.3 ppm). In a pig study (n = 12) with faecal samples collected 24, 48 and 72 h post-treatment, only one sample contained TIP above the LOQ (concentration 1.5 ppm). In another pig study (n = 12), with samples collected at 8, 24 48 and 96 h post-treatment, TIP concentrations were above the LOQ in one 8-h sample (1.1 ppm) and two 24-h samples (2.3 and 2.5 ppm). None of the 48-h and 96-h samples from these 4 studies contained measurable TIP concentrations. Thus, in cattle and pigs, only a small fraction of faecal samples collected up to 24 h postdosing contained measurable microbiologically active TIP, with its maximum limited to 2.6 μg/mL. This is several log2 dilution steps below the MICs of TIP against foodborne pathogens and commensals collected under acidic conditions comparable with those in the colonic contents and may explain a lack of intestinal dysbacteriosis with parenteral tildipirosin in livestock.

Cost-effective screening of pooled faecal specimens from patients with nosocomial diarrhoea for Clostridium perfringens enterotoxin

Purpose:Clostridium perfringens is a significant cause of nosocomial AAD. The prevalence of C. perfringens enterotoxin (CPE)-positive stool specimens in hospitalised patients is very low in the Indian setting making the diagnostics very expensive. Therefore, a cost-effective diagnostic approach to screen faecal specimens for CPE was devised. Materials and Methods: Faecal specimens from 540 hospitalised patients with various ailments and from 340 healthy subjects were investigated for CPE. An aliquot of pooled faecal supernatants was made by mixing 100 μl each of 10 specimens to be tested. Each aliquot was investigated for the presence of CPE by an enzyme immunoassay. A repetition of the assay was done with individual specimens of the pooled aliquots from each positive well as seen visually by colour development. Results: Of the 540 patient specimens tested, 405 (75%) patients were on antibiotics, the predominant ones being cephalosporins, penicillin, quinolones, aminoglycosides, etc. During the time of sampling, diarrhoea was present in 481 (89%), abdomen pain in 203 (37.6%) and fever in 242 (44.8%) patients. C. perfringens enterotoxin was positive in nine wells of the 540 pooled test specimens whereas all of the pooled 340 control samples were negative. Repeat of individual specimens comprising the nine wells with positive samples helped to identify the individual patients positive for CPE. Conclusion: Only two CPE kits were needed for a total of 880 faecal specimens tested. The cost-effective diagnostic approach to screen faecal specimens for CPE, as described herein will help to save institutional resources.

Increased faecal serine protease activity in diarrhoeic IBS patients: a colonic lumenal factor impairing colonic permeability and sensitivity

Objectives: Diarrhoea-predominant irritable bowel syndrome (IBS-D) is characterised by elevated colonic lumenal serine protease activity. The aims of this study were (1) to investigate the origin of this elevated serine...

Dog echinococcosis in northern Spain: Comparison of coproantigen and serum antibody assays with coprological exam

A large sheep-dog population from the province of Álava (northern Spain) has been investigated in order to determine the prevalence of the cestode parasite Echinococcus granulosus. Worms were detected in 14.0% of 721 dog faecal supernatants by coproantigen ELISA, and in 9.1% of 754 dog serum samples by serum antibody ELISA. A weak but statistically significant correlation (Spearman's ρ = 0.103, 95% CI: 0.023–0.178) between the two immunoassay results was found. In addition, eggs of the family Taeniidae were detected in 10.3% of 726 faecal samples examined by coproparasitological (flotation and sedimentation) tests. The overall E. granulosus infection rate, based on a Bayesian latent class model that accounts for the imperfect sensitivities and specificities of all diagnostic tests used, was estimated to be 8.0% (95% credible interval: 5.4–11.4%), corroborating that sheep-dog is the dog class most vulnerable to acquiring the infection. Dog sex did not influence the prevalence of E. granulosus, independently of the diagnostic test used or the dog region of origin. No significant linear correlation was found between the coproantigen ELISA OD values and the dog age (Spearman's ρ = −0.049, 95% CI: −0.234 to 0.135), suggesting that there were no differences in prevalence of E. granulosus between old and young dogs. The obtained results highlight the importance of initiating a control program based on regular treatment of the sheep-dogs with praziquantel in the province of Álava.

Echinococcus granulosus coproantigens: chromatographic fractionation and characterization.

Dogs infected with adult tapeworms of Echinococcus granulosus release antigens (coproantigens) in faeces which can be detected by a capture ELISA. Supernatants prepared from E. granulosus-infected dog faecal samples were fractionated by size-exclusion fast protein liquid chromatography (FPLC) on a Superose-6 column. Coproantigen ELISA and Western blotting were used to demonstrate the immunoreactivity of eluted fractions. Two main FPLC peaks of antigenic activity were detected and designated as fraction F1 and fraction F2 with approximate relative molecular weights > 670 kDa, and in the range of 146 to 440 kDa respectively. These two antigenic fractions (F1 and F2) fractionated from infected dog faeces were heat stable and largely protease-insensitive, but were highly sensitive to sodium periodate treatment, which strongly suggested the involvement of carbohydrates. Capture IgG antibodies against E. granulosus proglottis somatic extracts, detected a molecule with an approximate molecular weight of 155 kDa in fraction F2 after immunoblotting. The 155 kDa antigen could be completely ablated by sodium periodate treatment, but not after protease or lipase treatment. A surface tegument preparation of adult E. granulosus tapeworms contained large amounts of antigen that corresponded in size range and antigenicity to that observed in the FPLC fraction F2. There was also a peak of antigenic activity at > 670 kDa corresponding to fraction F1 from a culture derived excretory-secretory (E-S) adult tapeworm preparation. The involvement of carbohydrate moieties in coproantigen activity present in the FPLC fractions F1 and F2 from faecal supernatants of E. granulosus-infected dogs was confirmed by lectin-binding assays and exoglycosidase treatment, which showed that alpha-D-mannose and/or alpha-D-glucose, beta-galactose and N-acetyl-beta-glucosamine residues were the most important carbohydrate components in putative coproantigens present in both fractions. N-acetyl-beta-glucosamine and sialic acid residues were also contained in coproantigen molecules present in fraction F2. These results suggested that coproantigens detected in faeces of E. granulosus-infected dogs are large molecular weight molecules that may be derived from the carbohydrate-rich surface glycocalyx of adult worms, and are shed, released or secreted during the life-span of the tapeworm.

Partial characterisation of carbohydrate-rich Echinococcus granulosus coproantigens

Coproantigen ELISA based tests for diagnosis of canine echinococcosis provide high specificity and sensitivity. However, the antigenic molecules present in faeces from infected dogs have not yet been characterised. While initial attempts to determine the molecular weights of Echinococcus granulosus coproantigens by SDS–PAGE and Western blotting with coproantigen reactive capture antibodies were equivocal, they suggested presence of a significant carbohydrate component. Periodate treatment of coproantigen positive faecal supernatants resulted in a significant reduction (53%) in ELISA activity, suggesting that carbohydrates are involved in the antigenic structure of E. granulosus coproantigens. Protease treatment of antigenic molecules resulted in an 11% reduction in absorbance in ELISA, indicating that protein components were also present which affected by enzyme activity. Lectin-binding ELISA assays indicated strong affinity of E. granulosus coproantigens to concanavalin agglutinin and Lens culinaris agglutinin, and moderate binding to wheat-germ agglutinin and peanut agglutinin. No binding was detectable to Ulex europaensis agglutinin-I, Bandeiraea simplicifolia or Dolichos biflorus agglutinin. These data indicate that E. granulosus coproantigens from infected dog faeces possibly contained α- d-mannose and/or α- d-glucose, β-galactose and N-acetyl-β-glucosamine residues. To verify the role of carbohydrate moieties in coproantigens, faecal samples were treated with exoglycosidase and tested in the coproantigen ELISA. Treatment with β-galactosidase or N-acetyl-β-glucosamine reduced ELISA activity by 44 and 30%, respectively. Incubation with a panel of other specific exoglycosidases including α-galactosidase as well as α- l-fucosidase, α-mannosidase, β-mannosidase, α-glucosidase, β-glucosidase, β- fructosidase, or neuraminidase, did not alter coproantigen detection in ELISA. The results indicate that coproantigens present in faeces from E. granulosus naturally infected dogs were highly glycosylated and contain β- galactose and N-acetyl-β-glucosamine. The putative relationship of antigenic molecules with the tapeworm glycocalyx is discussed.

Kinetics of Taenia solium antibodies and antigens in experimental taeniosis.

Two groups of hamsters were infected with Taenia solium cysticerci, one of which was suppressed with methyl-prednisolone acetate on the day of infection and every 14 days thereafter. The other did not receive steroid treatment. Faecal and serum samples were taken prior to infection and then at weekly intervals. Parasite circulating- and coproantigens were detected by a capture ELISA with rabbit polyclonal antibodies against T. solium tapeworms. IgG antibodies in serum and in faecal supernatants were detected by ELISA with excretory-secretory products of T. solium adults recovered from hamsters. Infections remained up to 17 weeks in suppressed hamsters, but after week 11 no tapeworms were found in non-suppressed hosts. T. solium coproantigens in both groups of hamsters were positive from the 1st week post-infection (wpi) until the tapeworms were rejected. Circulating antigens were detected only in non-suppressed hamsters from the 3rd wpi until 1 week before T. solium was eliminated. All infected hamsters developed serum IgG antibodies against tapeworms which were detected from the 2nd wpi and decreased slowly after T. solium expulsion. Specific IgG in faecal supernatants was detected from the 3rd wpi only in non-suppressed hamsters. When suppression was stopped, coproantibodies could also be detected. The presence of IgG antibodies indicates that tapeworms induced an immune response in the experimental host and that when hamsters were suppressed with corticosteroids the immune response was impaired and did not allow the detection of IgG coproantibodies. This indicates, in addition, that the passage of T. solium antigens from the small intestine to the circulation was blocked.

Coproantigen detection in dogs experimentally and naturally infected with Echinococcus granulosus by a monoclonal antibody-based enzyme-linked immunosorbent assay

A sandwich ELISA for the detection of Echinococcus granulosus coproantigen in formalin and heat-treated faecal supernatants of dogs was developed. The assay used affinity-purified polyclonal antibodies obtained from rabbits hyperimmunised with E. granulosus excretory/secretory antigens and biotiaylated monoclonal antibody EmA9 produced against adult E. multilocularis somatic extract. The test was sensitive to 7 ng and 2.3 ng of E. granulosus protein and carbohydrate/ml of faecal supernatant, respectively. Thirteen helminth-free dogs were infected with different amounts of E. granulosus protoscoleces and the presence of coproantigen was monitored during the prepatent period until day 35 post-infection, when they were necropsied. Faecal antigen levels started to rise above the normal range between days 10 and 20 post-infection, and typically peaked at the end of the experiment. All the dogs, bearing from 3 to 67 700 worms, showed positive values in the ELISA during the prepatent period. One dog experimentally infected with Taenia hydatigena metacestode and harbouring three worms, tested positive only after the prepatent period at day 52. The test was applied to 98 stray dogs. The ELISA detected all of four dogs naturally infected with E. granulosus, two dogs with patent infections of T. hydatigena and two dogs with no cestode infections, sbowing a sensitivity of 100% and a specificity of 96%.

Distribution and characteristics of verocytotoxigenic Escherichia coli isolated from Ontario dairy cattle.

Faecal swabs obtained from a random sample of 1131 cows and 659 calves on 100 southern Ontario dairy farms were examined for verocytotoxigenic Escherichia coli (VTEC) using a Vero cell assay. Five isolates from each positive culture were tested similarly. Positive colonies were examined with DNA probes for Shiga-like toxin I (SLT-I) and SLT-II sequences. Probe-negative colonies were tested for neutralization of verocytotoxicity using anti-SLT-I and anti-SLT-IIv antisera. Colonies showing no neutralization response were examined in a polymerase chain reaction procedure. Colonies positive by any test were confirmed to be E. coli biochemically, serotyped, biotyped and tested for antimicrobial resistance. Faecal culture supernatants which were positive in the Vero cell assay, but culture negative, were examined using the neutralization assay. Animals were classified positive by faecal culture supernatant or by positive VTEC isolate. The prevalence rates of VTEC infection in cows and calves were estimated to be 9.5 and 24.7%, respectively. The proportion of animals infected on each farm ranged from 0 to 60% for cows and 0 to 100% for calves. Of 206 VTEC isolates identified, few were of serotypes which have been isolated from humans and none were E. coli O 157.H7.

The effect of ceftriaxone on the anaerobic bacterial flora and the bacterial enzymatic activity in the intestinal tract

The normal flora of the intestinal tract, mainly consisting of anaerobic bacteria, protects the host against colonization by pathogenic microorganisms. Antimicrobial treatment with ceftriaxone may influence the colonic microflora and as a consequence, the protective effect. Ten healthy volunteers received 1 g of ceftriaxone intramuscularly for five days. This resulted in a significant decrease (p less than 0.05) of the mean cultural counts (+/- SEM) of total anaerobes from 10.67 (0.11) (prior to treatment) to 9.02 (0.45) and 8.97 (0.46) at days 3 and 5, respectively (during treatment). After treatment (days 10 and 15-19), the cultural counts of anaerobes returned to 10.17 (0.16) and 10.44 (0.18), respectively. Bacterial enzymes may serve as an indicator of protective microflora. beta- aspartylpeptidase and deoxycholate hydrolase activity was determined in faecal supernatants of the volunteers and compared with anaerobic culturing. Both enzymatic activities show a significant correlation with the total number of anaerobes present at day 3 of deftriaxone treatment. At day 5 and 8 only beta-aspartylpeptidase showed significant correlations with cultural counts of total anaerobes, Bacteroides spp. or bifidobacteria. At day 15 to 19 (ten to 14 days after treatment) beta-aspartylpeptidase showed only a significant correlation with the number of Bacteroides spp. This indicates that changes in the indigenous bacterial flora during and shortly after treatment with ceftriaxone can be monitored by determination of beta-aspartylpeptidase. Recovery of the intestinal flora is difficult to assess in this manner.

Coproantigens in gut tapeworm infections: Hymenolepis diminuta in rats.

A capture enzyme-linked immunosorbent assay (ELISA) for the detection of Hymenolepis diminuta coproantigen in detergent-solubilised faecal supernatants was developed. The assay was sensitive to 400 ng H. diminuta protein/ml faecal supernatant and to 30 ng/ml in PBS 0.3% Tween. Faecal antigen levels started to rise on day 7 post infection and peaked on day 20. Following drug treatment of infected rats with 10 mg/kg praziquantel, there was an initial steep rise in faecal antigen levels, followed by a decline to background levels for uninfected animals by day 7 post treatment. When assayed against faecal supernatants prepared from H. microstoma, H. citelli, Diphyllobothrium ditrenum, Necator americanus, Strongyloides ratti and Nematospiroides dubius infections as well as uninfected animals, all results from these groups were 2 SD below those for animals infected with H. diminuta. The application of such a test to important intestinal cestodiases in man is discussed.

Inactivation of aztreonam by faecal supernatants of healthy volunteers as determined by HPLC.

The enzymatic inactivation of aztreonam to its biologically inactive open ring form, by faecal enzyme preparations from 12 volunteers, was investigated. The percentage inactivation was determined by a high-performance liquid chromatography assay at months 0, 6 and 24. Faecal enzyme preparations from five volunteers could inactivate aztreonam by more than 50% within 20 h at one or more of these sampling times. Monitoring of aztreonam inactivation over a shorter period of time (14 days) in three of the volunteers showed that aztreonam inactivation cannot be predicted.