Clostridium hathewayi sp. nov., from human faeces

A strictly anoxic, Gram-positive, sporeforming, rod-shaped bacterium was isolated from a chemostat inoculated with human faeces. The bacterium used carbohydrate as fermentable substrates, producing acetate, ethanol, carbon dioxide and hydrogen as the major products of glucose metabolism, and possessed a G + C content of 50.7 to 50.9 mol%. Comparative 16S rRNA gene sequencing showed that the unidentified bacterium represents a previously unrecognised sub-line within the Clostridium coccoides rRNA group of organisms. The nearest relatives of the unknown bacterium corresponded to Clostridium algidixylanolyticum, C. aerotolerans, C. celerecrescens, C. indolis, C. sphenoides, C. methoxybenzovorans and C. xylanolyticum but 16S rRNA sequence divergence values of >4% demonstrated that it represents a novel species. Based on the presented findings a new species, Clostridium hathewayi, is described. The type strain of Clostridium hathewayi is DSM = 13479T (= CCUG 43506 T).     

Isolation, Culture Characteristics, and Identification of Anaerobic Bacteria from the Chicken Cecum

Studies on the anaerobic cecal microflora of the 5-week-old chicken were made to determine a suitable roll-tube medium for enumeration and isolation of the bacterial population, to determine effects of medium components on recovery of total anaerobes, and to identify the predominant bacterial groups. The total number of microorganisms in cecal contents determined by direct microscope cell counts varied (among six samples) from 3.83 x 10(10) to 7.64 x 10(10) per g. Comparison of different nonselective media indicated that 60% of the direct microscope count could be recovered with a rumen fluid medium (M98-5) and 45% with medium 10. Deletion of rumen fluid from M98-5 reduced the total anaerobic count by half. Colony counts were lower if chicken cecal extract was substituted for rumen fluid in M98-5. Supplementing medium 10 with liver, chicken fecal, or cecal extracts improved recovery of anaerobes slightly. Prereduced blood agar media were inferior to M98-5. At least 11 groups of bacteria were isolated from high dilutions (10(-9)) of cecal material. Data on morphology and physiological and fermentation characteristics of 90% of the 298 isolated strains indicated that these bacteria represented species of anaerobic gram-negative cocci, facultatively anaerobic cocci and streptococci, Peptostreptococcus, Propionibacterium, Eubacterium, Bacteroides, and Clostridium. The growth of many of these strains was enhanced by rumen fluid, yeast extract, and cecal extract additions to basal media. These studies indicate that some of the more numerous anaerobic bacteria present in chicken cecal digesta can be isolated and cultured when media and methods that have been developed for ruminal bacteria are employed.     

Chicken intestine microbiota following the administration of lupulone, a hop-based antimicrobial

The use of antibiotic growth promotants in poultry rearing is a public health concern due to antibiotic resistance in bacteria and the harborage of resistance genes. Lupulone, a hop β-acid from Humulus lupulus, has been considered as a potential feed additive growth promotant. Here, the effect of lupulone was evaluated for its effect on the microbiota of the chicken intestine. The intestinal microbiota of broilers was quantified after the addition of 125 mg L(-1) lupulone to water and challenge with Clostridium perfringens. Microbial DNA was extracted from the broiler midgut and cecal sections and bacterial groups were quantified using real-time PCR. The predominant cecal bacterial groups were Clostridium leptum subgroup 16S rRNA gene Cluster IV, Clostridium coccoides subgroup 16S rRNA gene Clusters XIVa and XIVb and Bacteroides, whereas Lactobacillus, the Enterobacteriaceae family and Enterococcus dominated the midgut. Lupulone at 125 mg L(-1) significantly decreased the C. perfringens subgroup 16S rRNA gene Cluster I, which contains several pathogenic species, in both the midgut and the cecum and Lactobacillus in the midgut. No significant changes were noted in the overall microbiota for the cecum or the midgut. Lupulone warrants further evaluation as a botanical agent to mitigate C. perfringens overgrowth in antibiotic-free reared poultry.     

Multiplex PCR for rapid differentiation of three species in the "Clostridium clostridioforme group"

Clostridium clostridioforme is a relatively antimicrobial resistant, phenotypically heterogeneous anaerobe that has been involved in a variety of infections. 16S rDNA sequencing analysis revealed three principal species in what has been called Clostridium clostridioforme - Clostridium bolteae, C. clostridioforme, and Clostridium hathewayi. Based on the 16S rDNA sequence information we obtained, we developed a cost-effective, timesaving one-step multiplex PCR assay for rapid and accurate differentiation of these three species. The established multiplex PCR identification scheme was applied to the identification of 88 clinical isolates that had previously been identified phenotypically as C. clostridioforme. The identification obtained from multiplex PCR assays showed 100% agreement with 16S rDNA sequencing identification. This scheme will permit more accurate assessment of the role of these three Clostridium species in infection and of the degree of antimicrobial resistance in each of the species.     

Diagnosis of spontaneous Clostridium spiroforme iota enterotoxemia in a barrier rabbit breeding colony

Five New Zealand White rabbits (Oryctolagus cuniculus) in a rigid barrier rabbit breeding colony developed acute diarrhea 1 week after weaning. Both Clostridium spiroforme and an iota-toxin were isolated from cecal and colon contents of all five rabbits. When pure isolates of C. spiroforme were administered to two normal healthy rabbits, the rabbits developed identical disease and shed both the organism and the iota-toxin. Results of this study suggested that C. spiroforme is an important enteric pathogen of weanling rabbits and the etiology of this diarrhea complex can be rapidly confirmed using four diagnostic criteria.     

16S rRNA-based analysis of microbiota from the cecum of broiler chickens.

The microbiota of the intestinal tract of chickens plays an important role in inhibiting the establishment of intestinal pathogens. Earlier culturing and microscopic examinations indicated that only a fraction of the bacteria in the cecum of chickens could be grown in the laboratory. Therefore, a survey of cecal bacteria was done by retrieval of 16S rRNA gene sequences from DNA isolated from the cecal content and the cecal mucosa. The ribosomal gene sequences were amplified with universal primers and cloned or subjected to temporal temperature gradient gel electrophoresis (TTGE). Partial 16S rRNA gene sequences were determined from the clones and from the major bands in TTGE gels. A total of 1,656 partial 16S rRNA gene sequences were obtained and compared to sequences in the GenBank. The comparison indicated that 243 different sequences were present in the samples. Overall, sequences representing 50 phylogenetic groups or subgroups of bacteria were found, but approximately 89% of the sequences represented just four phylogenetic groups (Clostridium leptum, Sporomusa sp., Clostridium coccoides, and enterics). Sequences of members of the Bacteroides group, the Bifidobacterium infantis subgroup, and of Pseudomonas sp. each accounted for less than 2% of the total. Sequences related to those from the Escherichia sp. subgroup and from Lactobacillus, Pseudomonas, and Bifidobacterium spp. were generally between 98 and 100% identical to sequences already deposited in the GenBank. Sequences most closely related to those of the other bacteria were generally 97% or less identical to those in the databases and therefore might be from currently unknown species. TTGE and random cloning indicated that certain phylogenetic subgroups were common to all birds analyzed, but sequence data from random cloning also provided evidence for qualitative and quantitative differences among the cecal microbiota of individual birds reared under very similar conditions.     

Studies on the Cecal Microflora of Commercial Broiler Chickens

A study was made of the cecal microflora isolated from broilers (5-week-old) reared under typical commercial husbandry conditions. Three hundred and twenty-five bacterial strains (randomly isolated from colonies representing 49 to 81% of the microscopic count) were isolated from cecal digesta of six animals on a rumen fluid roll tube medium (M98-5). Seventy-seven percent of these strains consisted of strict anaerobes: gram-negative, pleomorphic cocci (5.2%), Peptostreptococcus (1.5%), gram-positive rods (36.1% as Propionibacterium acnes and Eubacterium sp.), gram-negative rods (18.6% as Bacteroides clostridiiformis, B. hypermegas and B. fragilis) and sporeforming rods (15.7% as Clostridium sp.). Two types of facultatively anaerobic bacteria (gram-positive cocci and Escherichia coli) were also isolated and constituted 17.5% of the remaining flora. The distribution of the bacterial groups isolated from six cecal samples varied considerably. Data on the growth requirements of anaerobic strains indicated that many could be cultured in a simple medium consisting of an energy source, minerals, reducing agent, Trypticase, and yeast extract (or a vitamin mixture in place of yeast extract). The growth of some of these bacteria was also enhanced by CO(2) and rumen fluid. These preliminary data suggest that some of the more numerous anaerobes isolated from the chicken cecum may not require complex nutrients for growth and, in fact, may be nutritionally similar to rumen anaerobes.     

Absence of cecal secondary bile acids in gnotobiotic mice associated with two human intestinal bacteria with the ability to dehydroxylate bile acids in vitro.

Germ-free mice were orally inoculated with human intestinal 7alpha-dehydroxylating bacterial strains to evaluate their ability to transform bile acids in vivo. Three weeks after inoculation of the bacteria, cecal bile acids were examined. Among free-form bile acids, only beta-muricholic acid was detected in the cecal contents of gnotobiotic mice associated with Bacteroides distasonis strain K-5. No secondary bile acid was observed in the cecal contents of any of the gnotobiotic mice associated with 7alpha-dehydroxylating bacteria, Clostridium species strain TO-931 or Eubacterium species strain 36S.     

Production and characterization of monoclonal antibodies to a Veillonella species from a continuous-flow culture system of chicken cecal bacteria

Administering native intestinal flora to newly hatched chicks protects against cecal Salmonella colonization, and is known as competitive exclusion. Continuous-flow culture systems have been used to maintain defined competitive exclusion cultures. We have recently demonstrated that such a stable continuous-flow culture, CF3, contains 29 bacterial strains representing ten genera. Broiler chicks treated with CF3 are protected against Salmonella colonization of the ceca. Such protection is correlated with elevated concentrations of proprionic acid in the cecal contents of treated chicks. In this study we report on the preparation and characterization of monoclonal antibodies to one of the proprionic acid producing anaerobes contained in CF3, namely Veillonella CF3. Five different monoclonal antibodies were characterized with respect to: (1) isotype; (2)Veillonella specificity as judged by cross-reactivity profiles with other bacteria; (3) sensitivity as measured by the limit of detection of the number of colony forming units of Veillonella; and (4) antigen recognition of Veillonella by Western Blot analysis. These antibodies have been used to enumerate Veillonella in both the CF3 cultures and in the ceca of young chicks.     

Effect of Bacterial Contamination on Cecal Size and Cecal Contents of Gnotobiotic Rodents

In the present investigation the effect of various bacterial contaminations of gnotobiotic mice and rats on cecal size is presented. Of the species tested, Bacteroides oralis and Fusobacterium nucleatum did not establish in germ-free mice. Streptococcus mutans, Clostridium difficile, a Neisseria strain and two recent cecal isolates established, but failed to exert an effect upon the cecum of mice. A group K streptococcus and B. fragilis increased the cecal size apparently by increasing the levels of water-soluble protein, peptides, and carbohydrates in the cecal contents. Mixed ileal bacteria decreased the cecal size by preventing accumulation of soluble proteins and carbohydrates in the cecum. A Peptococcus strain caused a reduction by lowering the levels of insoluble material in the cecum. When this strain was combined with two Clostridium isolates and introduced into gnotobiotic rats, 50 to 65% cecal reduction was observed. This polycontamination did not decrease the per cent water of the cecal contents but caused lower levels of both soluble and insoluble material to accumulate in the cecum. No net nitrogen absorption from the distal small intestine occurred in either the germ-free or polycontaminated rats.     

Selective colonization of insoluble substrates by human faecal bacteria

Insoluble plant polysaccharides and endogenous mucin are important energy sources for human colonic microorganisms. The object of this study was to determine whether or not specific communities colonize these substrates. Using faecal samples from four individuals as inocula for an anaerobic in vitro continuous flow system, the colonization of wheat bran, high amylose starch and porcine gastric mucin was examined. Recovered substrates were extensively washed and the remaining tightly attached bacterial communities were identified using polymerase chain reaction-amplified 16S rRNA gene sequences and fluorescent in situ hybridization. The substrate had a major influence on the species of attached bacteria detected. Sequences retrieved from bran were dominated by clostridial cluster XIVa bacteria, including uncultured relatives of Clostridium hathewayi, Eubacterium rectale and Roseburia species. Bacteroides species were also detected. The most abundant sequences recovered from starch were related to the cultured species Ruminococcus bromii, Bifidobacterium adolescentis, Bifidobacterium breve and E. rectale. The most commonly recovered sequences from mucin were from Bifidobacterium bifidum and uncultured bacteria related to Ruminococcus lactaris. This study suggests that a specific subset of bacteria is likely to be the primary colonizers of particular insoluble colonic substrates. For a given substrate, however, the primary colonizing species may vary between host individuals.     

Phylogenetic analysis of cecal microbiota in chicken by the use of 16S rDNA clone libraries

The chicken cecum contains a great many bacteria, most of which are strict anaerobes. A strictly anaerobe culture-based method was used in the present study, in conjunction with the 16S rDNA clone library, to elucidate bacterial diversity and the phylogenetic relationship of cecal microbiota in the chicken. A comparative 16S rDNA sequence analysis of cultivated strains and retrieved clones from cecal contents was performed. Approximately 90% of the bacterial cells detected by microscopy did not form colonies on a medium 10 in plate-in-bottle. The 19 isolated strains yielded 11 distinct rDNA sequences, 58% of which were classified as low G + C gram-positive bacteria, 26% were related to Bacteroides spp., and 16% were classified as Proteobacteria. Based on the sequence analysis of 164 clones, 24% were identified to belong to 8 known species and 76% were considered to be 65 novel phylotypes. Approximately 94% of cloned sequences were classified into low G + C gram-positive bacteria, 4% were related to Bacteroides spp., and 2% were classified into Proteobacteria. Clostridium subcluster XIVa (38%), Clostridium cluster IV (13%), Lactobacillus spp. (24%), and Bacteroides spp. (4%) were the major groups constituting the cecal microbiota in chicken, in which the Clostridium subcluster XIVa was the most phylogenetically diverse group in chicken cecum. The 16S rDNA sequences of Lactobacillus acidophilus, L. crispatus, L. salivarius, and L. reuteri were the most frequently found in the Lactobacillus group in chicken cecum.     

Isolation and identification of a Paenibacillus polymyxa strain that coproduces a novel lantibiotic and polymyxin

A new bacterial strain, displaying potent antimicrobial properties against gram-negative and gram-positive pathogenic bacteria, was isolated from food. Based on its phenotypical and biochemical properties as well as its 16S rRNA gene sequence, the bacterium was identified as Paenibacillus polymyxa and it was designated as strain OSY-DF. The antimicrobials produced by this strain were isolated from the fermentation broth and subsequently analyzed by liquid chromatography-mass spectrometry. Two antimicrobials were found: a known antibiotic, polymyxin E1, which is active against gram-negative bacteria, and an unknown 2,983-Da compound showing activity against gram-positive bacteria. The latter was purified to homogeneity, and its antimicrobial potency and proteinaceous nature were confirmed. The antimicrobial peptide, designated paenibacillin, is active against a broad range of food-borne pathogenic and spoilage bacteria, including Bacillus spp., Clostridium sporogenes, Lactobacillus spp., Lactococcus lactis, Leuconostoc mesenteroides, Listeria spp., Pediococcus cerevisiae, Staphylococcus aureus, and Streptococcus agalactiae. Furthermore, it possesses the physico-chemical properties of an ideal antimicrobial agent in terms of water solubility, thermal resistance, and stability against acid/alkali (pH 2.0 to 9.0) treatment. Edman degradation, mass spectroscopy, and nuclear magnetic resonance were used to sequence native and chemically modified paenibacillin. While details of the tentative sequence need to be elucidated in future work, the peptide was unequivocally characterized as a novel lantibiotic, with a high degree of posttranslational modifications. The coproduction of polymyxin E1 and a lantibiotic is a finding that has not been reported earlier. The new strain and associated peptide are potentially useful in food and medical applications.     

Intestinal colonization of laboratory rats with Oxalobacter formigenes.

Six strains of Oxalobacter formigenes (anaerobic oxalate-degrading bacteria) were examined for their ability to colonize the gastrointestinal tracts of adult laboratory rats. These rats did not harbor O. formigenes. Strain OxCR6, isolated from the cecal contents of a laboratory rat that was naturally colonized by oxalate-degrading bacteria, colonized the ceca and colons of adult rats fed a diet that contained 4.5% sodium oxalate. Five days after rats were inoculated intragastrically with 10(9) viable cells of strain OxCR6, oxalate degradation rates in cecal and colonic contents increased by 19 and 40 times, respectively. Viable counts of strain OxCR6 from these rats averaged 10(8)/g (dry weight) of cecal contents. Strain OxCR6 was not detected in the cecal contents of inoculated rats fed diets that contained less than 3.0% sodium oxalate. Strains of O. formigenes isolated from the cecal contents of swine, guinea pigs, and wild rats and from human feces also colonized the ceca of laboratory rats; a ruminal strain failed to colonize the rat cecum.     

Isolation of members of the Haemophilus-Pasteurella-Actinobacillus group from feral rodents

46 feral rodents, including a common vole (Microtus arvalis), house mice (Mus musculus), muskrats (ondatra zibetica), house rats (Rattus rattus) and brown rats (R. norvegicus) were examined for bacteria of the Haemophilus-Pasteurella-Actinobacillus group. Haemophilus spp. (only M. musculus examined) were not obtained. All animal species were found contaminated by P. pneumotropica and/or Actinobacillus spp. Almost all M. musculus (96%) and most Rattus spp. (76%) were contaminated by P. pneumotropica and/or Actinobacillus spp. These bacteria were obtained most frequently from the upper respiratory tract, to a lesser extent from the lung and rarely from caecal contents. It is concluded that feral rodents might constitute an important source of contamination of laboratory rodents by members of the HPA-group.     

Microbial degradation of oxalate in the gastrointestinal tracts of rats

Rates of oxalate degradation by mixed bacterial populations in cecal contents from wild rats ranged from 2.5 to 20.6 mumol/g (dry weight) per h. The oxalate-degrading activity in cecal contents from three strains of laboratory rats (Long-Evans, Wistar, and Sprague-Dawley) from four commercial breeders was generally lower, ranging from 1.8 to 3.5 mumol/g (dry weight) of cecal contents per h. This activity did not increase when diets were supplemented with oxalate. When Sprague-Dawley rats from a fifth commercial breeder were fed an oxalate diet, rates of oxalate degradation in cecal contents increased from 2.0 to 23.1 mumol/g (dry weight) per h. Obligately anaerobic, oxalate-degrading bacteria, similar to ruminal strains of Oxalobacter formigenes, were isolated from the latter group of laboratory rats and from wild rats. Viable counts of these bacteria were as high as 10(8)/g (dry weight) of cecal contents, which was less than 0.1% of the total viable population. This report presents the first evidence for the presence of anaerobic oxalate-degrading bacteria in the cecal contents of rats and represents the first direct measurement of the concentration of these bacteria in the large bowel of monogastric animals. We propose that methods used for the maintenance of most commercial rat colonies often preclude the intestinal colonization of laboratory rats with anaerobic oxalate-degrading bacteria.     

Microbial degradation of oxalate in the gastrointestinal tracts of rats.

Rates of oxalate degradation by mixed bacterial populations in cecal contents from wild rats ranged from 2.5 to 20.6 mumol/g (dry weight) per h. The oxalate-degrading activity in cecal contents from three strains of laboratory rats (Long-Evans, Wistar, and Sprague-Dawley) from four commercial breeders was generally lower, ranging from 1.8 to 3.5 mumol/g (dry weight) of cecal contents per h. This activity did not increase when diets were supplemented with oxalate. When Sprague-Dawley rats from a fifth commercial breeder were fed an oxalate diet, rates of oxalate degradation in cecal contents increased from 2.0 to 23.1 mumol/g (dry weight) per h. Obligately anaerobic, oxalate-degrading bacteria, similar to ruminal strains of Oxalobacter formigenes, were isolated from the latter group of laboratory rats and from wild rats. Viable counts of these bacteria were as high as 10(8)/g (dry weight) of cecal contents, which was less than 0.1% of the total viable population. This report presents the first evidence for the presence of anaerobic oxalate-degrading bacteria in the cecal contents of rats and represents the first direct measurement of the concentration of these bacteria in the large bowel of monogastric animals. We propose that methods used for the maintenance of most commercial rat colonies often preclude the intestinal colonization of laboratory rats with anaerobic oxalate-degrading bacteria.     

16S rRNA-Based Analysis of Microbiota from the Cecum of Broiler Chickens

The microbiota of the intestinal tract of chickens plays an important role in inhibiting the establishment of intestinal pathogens. Earlier culturing and microscopic examinations indicated that only a fraction of the bacteria in the cecum of chickens could be grown in the laboratory. Therefore, a survey of cecal bacteria was done by retrieval of 16S rRNA gene sequences from DNA isolated from the cecal content and the cecal mucosa. The ribosomal gene sequences were amplified with universal primers and cloned or subjected to temporal temperature gradient gel electrophoresis (TTGE). Partial 16S rRNA gene sequences were determined from the clones and from the major bands in TTGE gels. A total of 1,656 partial 16S rRNA gene sequences were obtained and compared to sequences in the GenBank. The comparison indicated that 243 different sequences were present in the samples. Overall, sequences representing 50 phylogenetic groups or subgroups of bacteria were found, but approximately 89% of the sequences represented just four phylogenetic groups (Clostridium leptum, Sporomusa sp., Clostridium coccoides, and enterics). Sequences of members of the Bacteroides group, the Bifidobacterium infantis subgroup, and of Pseudomonas sp. each accounted for less than 2% of the total. Sequences related to those from the Escherichia sp. subgroup and from Lactobacillus, Pseudomonas, and Bifidobacterium spp. were generally between 98 and 100% identical to sequences already deposited in the GenBank. Sequences most closely related to those of the other bacteria were generally 97% or less identical to those in the databases and therefore might be from currently unknown species. TTGE and random cloning indicated that certain phylogenetic subgroups were common to all birds analyzed, but sequence data from random cloning also provided evidence for qualitative and quantitative differences among the cecal microbiota of individual birds reared under very similar conditions.     

Fatal septicemia due to Clostridium hathewayi and Campylobacter hominis

Clostridium hathewayi and Campylobacter hominis have not been previously reported in infection. We report a fatal case of septicemia, massive intravascular hemolysis, shock, and disseminated intravascular coagulation; both of these organisms were recovered on blood culture, although it seems likely that the C. hathewayi was responsible for the clinical picture and that the C. hominis was an incidental finding.     

Isolation and Genetic Characterization of Toxoplasma gondii from Black Bears (Ursus americanus), Bobcats (Lynx rufus), and Feral Cats (Felis catus) from Pennsylvania

Toxoplasma gondii infects virtually all warm‐blooded hosts worldwide. Recently, attention has been focused on the genetic diversity of the parasite to explain its pathogenicity in different hosts. It has been hypothesized that interaction between feral and domestic cycles of T. gondii may increase unusual genotypes in domestic cats and facilitate transmission of potentially more pathogenic genotypes to humans, domestic animals, and wildlife. In the present study, we tested black bear (Ursus americanus), bobcat (Lynx rufus), and feral cat (Felis catus) from the state of Pennsylvania for T. gondii infection. Antibodies to T. gondii were found in 32 (84.2%) of 38 bears, both bobcats, and 2 of 3 feral cats tested by the modified agglutination test (cut off titer 1:25). Hearts from seropositive animals were bioassayed in mice, and viable T. gondii was isolated from 3 of 32 bears, 2 of 2 bobcats, and 2 of 3 feral cats. DNA isolated from culture‐derived tachyzoites of these isolates was characterized using multilocus PCR‐RFLP markers. Three genotypes were revealed, including ToxoDB PCR‐RFLP genotype #1 or #3 (Type II, 1 isolate), #5 (Type 12, 3 isolates), and #216 (3 isolates), adding to the evidence of genetic diversity of T. gondii in wildlife in Pennsylvania. Pathogenicity of 3 T. gondii isolates (all #216, 1 from bear, and 2 from feral cat) was determined in outbred Swiss Webster mice; all three were virulent causing 100% mortality. Results indicated that highly mouse pathogenic strains of T. gondii are circulating in wildlife, and these strains may pose risk to infect human through consuming of game meat.