Molecular characterisation of the gut microflora of healthy and inflammatory bowel disease cats using fluorescence in situ hybridisation with special reference to Desulfovibrio spp

Inflammatory bowel disease (IBD) is a common cause of chronic large bowel diarrhoea in cats. Although the aetiology of IBD is unknown, an immune-mediated response to a luminal antigen is thought to be involved. As knowledge concerning the colonic microflora of cats is limited and requires further investigation, the purpose of this study was to determine the presence of specific bacterial groups in normal and IBD cats, and the potential role they play in the health of the host. Total bacterial populations, Bacteroides spp., Bifidobacterium spp., Clostridium histolyticum subgp., Lactobacillus-Enterococcus subgp. and Desulfovibrio spp. were enumerated in 34 healthy cats and 11 IBD cats using fluorescence in situ hybridisation. The study is one of the first to show the presence of Desulfovibrio in cats. Total bacteria, Bifidobacterium spp. and Bacteroides spp. counts were all significantly higher in healthy cats when compared with IBD cats, whereas Desulfovibrio spp. (producers of toxic sulphides) numbers were found to be significantly higher in colitic cats. The information obtained from this study suggests that modulation of bacterial flora by increasing bifidobacteria and decreasing Desulfovibrio spp. may be beneficial to cats with IBD. Dietary intervention may be an important aspect of their treatment.

Permanent changes in growth and physiology of Bacillus subtilis ToC46 after loss of a recombinant plasmid

The physiology and growth of plasmid-bearing Bacillus subtilis carrying plasmid pPFF1, the non-transformed host, and cells after loss of the plasmid (so-called plasmid-cured cells) were investigated. It was found that, following plasmid loss, cells exhibited phenotypic characteristics different from those of the non-transformed host strains. Compared to plasmid-bearing cells and non-transformed host cells, an approximate 25% increase in the maximum specific growth rate and a more rapid increase in total RNA per unit cell mass were observed in plasmid-cured cells. The total enthalpy associated with irreversible denaturation events was determined in whole cells by differential scanning calorimetry. This showed higher enthalpies for plasmid-cured cells compared with the non-transformed host, which suggests increased ribosome numbers. The result from cellular DNA hybridisation suggests that there was no direct evidence of plasmid integration into the host chromosome. (C) 2004 Elsevier Inc. All rights reserved.

Use of a continuous culture fermentation system to investigate the effect of GanedenBC30 (Bacillus coagulans GBI-30, 6086) supplementation on pathogen survival in the human gut microbiota

Single-stage continuous fermentation systems were employed to examine the effects of GanedenBC30 supplementation on the human gastrointestinal microbiota in relation to pathogen challenge in vitro. Denaturing gradient gel electrophoresis analysis demonstrated that GanedenBC30 supplementation modified the microbial profiles in the fermentation systems compared with controls, with profiles clustering according to treatment. Overall, GanedenBC30 supplementation did not elicit major changes in bacterial population counts in vitro, although notably higher Bcoa191 counts were seen following probiotic supplementation (compared to the controls). Pathogen challenge did not elicit significant modification of the microbial counts in vitro, although notably higher Clit135 counts were seen in the control system post-Clostridium difficile challenge than in the corresponding GanedenBC30-supplemented systems. Sporulation appears to be associated with the anti-microbial activity of GanedenBC30, suggesting that a bi-modal lifecycle of GanedenBC30 in vivo may lead to anti-microbial activity in distal regions of the gastrointestinal tract.

Bacillus coagulans as a probiotic

Probiotics are live microbial feed additions that improve human or animal health. Their activities are towards improving the composition of the gastrointestinal microbiota in a manner that reduces the risk of disorder. In some cases, probiotics are also used therapeutically. Most probiotics use lactobacilli or bifidobacteria as the main constituents. These produce lactic acid as well as other anti-pathogenic attributes. Traditionally, probiotics are incorporated in dairy products (yoghurts or fermented drinks) or in lyophilised form. Because of stability and viability factors, heated products are not usually a target for probiotic use. This is because they are temperature sensitive. However, a spore-forming genus would have the ability to overcome this limitation. Here, we discuss evidence for the spore-forming Gram-positive bacterium Bacillus coagulans as a probiotic.

Recognition of greater diversity of Bacillus species and related bacteria in human faeces

In a study looking at the culturable, aerobic Actinobacteria associated with the human gastrointestinal tract, the vast majority of isolates obtained from dried human faeces belonged to the genus Bacillus and related bacteria. A total of 124 isolates were recovered from the faeces of 10 healthy adult donors. 16S rRNA gene sequence analyses showed the majority belonged to the families Bacillaceae (n = 81) and Paenibacillaceae (n = 3), with Bacillus species isolated from all donors. Isolates tentatively identified as Bacillus clausii (n = 32) and B. licheniformis (n = 28) were recovered most frequently, with the genera Lysinibacillus, Ureibacillus, Oceanobacillus, Ornithinibacillus and Virgibacillus represented in some donors. Phenotypic data confirmed the identities of isolates belonging to well-characterized species. Representatives of the phylum Actinobacteria were recovered in much lower numbers (n = 11). Many of the bacilli exhibited antimicrobial activity against one or more strains of Clostridium difficile, C. perfringens, Listeria monocytogenes and Staphylococcus aureus, with some (n = 12) found to have no detectable cytopathic effect on HEp-2 cells. This study has revealed greater diversity within gut-associated aerobic spore-formers than previous studies, and suggests that bacilli with potential as probiotics could be isolated from the human gut.

Strigolactone analogues induce suicidal seed germination of Striga spp. in soil

Striga hermonthica and Striga asiatica are obligate root parasites that cause serious problems in the production of staple cereal crops in Africa. Because of the high levels of infestation, there is an urgent need to control these weeds. A potentially useful control option is depletion of the soil seed bank by suicidal germination, which involves germination of the seeds in the absence of host plants. Suicidal germination is often mentioned in the literature, but not considered realistic, because of the alleged untimely decomposition of the stimulants in the soil, despite the fact that some encouraging results were reported around 1980. The alleged instability has prevented active research in this direction for the past 20–25 years. Five newly designed synthetic germination stimulants were investigated as candidates for suicidal germination. An important issue is the persistence of these stimulants in soil. Packets with Striga spp. seeds were put in pots with soil and then treated with aqueous solutions of the stimulants. All five compounds induced germination under these conditions, with percentages varying between 18% and 98% depending on stimulant and species. There were no noticeable signs of decomposition of the stimulants. The best performing stimulant is derived from 1-tetralone. We conclude that synthetic strigolactones analogues have excellent prospects for use in combating parasitic weeds. Further testing will be needed to evaluate whether such prospects can be realised in the field.

Bacillus subtilis spores germinate in the chicken gastrointestinal tract

A number of poultry probiotics contain bacterial spores. In this study, orally administered spores of Bacillus subtilis germinated in the gastrointestinal (GI) tracts of chicks. Furthermore, 20 h after spores were administered, vegetative cells outnumbered spores throughout the GI tract. This demonstrates that spore-based probiotics may function in this host through metabolically active mechanisms.

Bacillus subtilis spores competitively exclude Escherichia coli O78:K80 in poultry

Newly hatched specific pathogen-free chicks were dosed with a suspension of Bacillus subtilis spores prior to challenge with Escherichia coli O78:K80, a known virulent strain associated with avian colibacillosis. 24 h later. A single oral inoculum of 2.5 x 10(8) spores was sufficient to suppress all aspects of E. coli O78:K80 infection. Colonisation of deep organs was reduced by a factor of over 2 log(10) whilst colonisation of the intestine, as measured by direct caecal count, was reduced over 3 log(10). Shedding of E. coli O78:K80 was measured by semi-quantitative cloacal swabbing and was reduced significantly for the: duration of the experiment, 35 days. B, subtilis persisted in the intestine although with decreasing numbers over the same period. Challenge with the same dose 5 days after pre-dosing with spores overcame any suppressive effect of the spores. Crown Copyright (C) 2001 Published by Elsevier Science B.V. All rights reserved.

Competitive exclusion by Bacillus subtilis spores of Salmonella enterica serotype Enteritidis and Clostridium perfringens in young chickens

Cost effective control of avian diseases and food borne pathogens remains a high priority for all sectors of the poultry industry with cleansing and disinfection, vaccination and competitive exclusion approaches being used widely. Previous studies showed that Bacillus subtilis PY79(hr) was an effective competitive exclusion agent for use in poultry to control avian pathogenic Escherichia coli serotype O78:K80. Here we report experiments that were undertaken to test the efficacy of B. subtilis PY79(hr) in the control of Salmonella enterica serotype Enteritidis and Clostridium perfringens in young chickens. To do this, 1-day-old and 20-day-old specific pathogen free (SPF) chicks were dosed with a suspension of B. subtilis spores prior to challenge with S. Enteritidis (S1400) and C. perfringens, respectively. For both challenge models, a single oral inoculum of 1 x 10(9) spores given 24 h prior to challenge was sufficient to suppress colonisation and persistence of both S. Enteritidis and C perfringens. In particular, the faecal shedding of S. Enteritidis, as measured by a semi-quantitative cloacal swabbing technique, was reduced significantly for the 36 days duration of the experiment. B. subtilis persisted in the intestine although with decreasing numbers over the same period. These data add further evidence that B. subtilis spores may be effective agents in the control of avian diseases and food borne pathogens.

Prevalence of multiple antibiotic resistance in 443 Campylobacter spp. isolated from humans and animals

Aims: In view of recent findings that a multidrug efflux pump CmeABC exists in Campylobacter jejuni, 391 C. jejuni and 52 Campylobacter coli of human and animal origin were examined for a multidrug resistance phenotype. Materials and methods: The MICs of ampicillin, chloramphenicol, ciprofloxacin, erythromycin, kanamycin, tetracycline, cetrimide, triclosan, acridine orange, paraquat and ethidium bromide were determined. Resistance to organic solvents and the effect of salicylate (known inducer of the marRAB operon in Escherichia coli and Salmonella) were also examined. Results: Two C. coli and 13 C. jejuni isolates, mainly from pigs or poultry, were resistant to three or more antibiotics and 12 of these strains had reduced susceptibility to acridine orange and/or ethidium bromide. Strains (n=20) that were less susceptible to acridine orange, ethidium bromide and triclosan were significantly more resistant (P<0.05) to ampicillin, chloramphenicol, ciprofloxacin, erythromycin, nalidixic acid and tetracycline, with two- to four-fold increases in MIC values compared with strains (n=20) most susceptible to acridine orange, ethidium bromide and triclosan. Growth of strains with 1 mM salicylate caused a small (up to two-fold) but statistically significant (Pless than or equal to0.005) increase in the MICs of chloramphenicol, ciprofloxacin, erythromycin and tetracycline. Conclusions: These data indicate that multiple antibiotic resistant (MAR)-like Campylobacter strains occur and it may be postulated that these may overexpress cmeABC or another efflux system.

Sources and spread of thermophilic Campylobacter spp. during partial depopulation of broiler chicken flocks

The practice of partial depopulation or ‘thinning’, i.e. early removal of a proportion of birds from a commercial broiler flock, is a reported risk factor for Campylobacter colonization of residual birds because of the difficulty in maintaining biosecurity during the process. Therefore, the effect of this practice was studied in detail for 51 target flocks, each at a different growing farm belonging to one of seven major poultry companies throughout the United Kingdom. On 21 of these farms, the target flock was already colonized by Campylobacter and at slaughter all cecal samples examined were positive, with a mean of log10 8 cfu / g. A further 27 flocks became positive within 2 – 6 days of the start of thinning and had similarly high levels of cecal carriage at slaughter. Just prior to the thinning process, Campylobacter could be isolated frequently from the farm driveways, transport vehicles, equipment and personnel. Strains from seven such farms on which flocks became colonized after thinning were examined by PFGE typing. The study demonstrated an association between strains occurring at specific sampling sites and those isolated subsequently from the thinned flocks. There were also indications that particular strains had spread from one farm to another, when the farms were jointly company-owned and served by the same bird-catching teams and / or vehicles. The results highlighted the need for better hygiene control in relation to catching equipment and personnel, and more effective cleaning and disinfection of vehicles, and bird-transport crates.

Bacillus subtilis natto: a non-toxic source of poly-γ-glutamic acid that could be used as a cryoprotectant for probiotic bacteria

It is common practice to freeze dry probiotic bacteria to improve their shelf life. However, the freeze drying process itself can be detrimental to their viability. The viability of probiotics could be maintained if they are administered within a microbially produced biodegradable polymer - poly-γ-glutamic acid (γ-PGA) - matrix. Although the antifreeze activity of γ-PGA is well known, it has not been used for maintaining the viability of probiotic bacteria during freeze drying. The aim of this study was to test the effect of γ-PGA (produced by B. subtilis natto ATCC 15245) on the viability of probiotic bacteria during freeze drying and to test the toxigenic potential of B. subtilis natto. 10% γ-PGA was found to protect Lactobacillus paracasei significantly better than 10% sucrose, whereas it showed comparable cryoprotectant activity to sucrose when it was used to protect Bifidobacterium breve and Bifidobacterium longum. Although γ-PGA is known to be non-toxic, it is crucial to ascertain the toxigenic potential of its source, B. subtilis natto. Presence of six genes that are known to encode for toxins were investigated: three component hemolysin (hbl D/A), three component non-haemolytic enterotoxin (nheB), B. cereus enterotoxin T (bceT), enterotoxin FM (entFM), sphingomyelinase (sph) and phosphatidylcholine-specific phospholipase (piplc). From our investigations, none of these six genes were present in B. subtilis natto. Moreover, haemolytic and lecithinase activities were found to be absent. Our work contributes a biodegradable polymer from a non-toxic source for the cryoprotection of probiotic bacteria, thus improving their survival during the manufacturing process.