Bacteroides Distasonis Research Articles

Occurrence and activity of human intestinal bacteria involved in the conversion of dietary lignans

The human intestinal microbiota is necessary for the production of enterolignans from the dietary lignan secoisolariciresinol diglucoside (SDG). However, little is known about the bacteria that contribute to SDG conversion. Therefore, we aimed at describing the occurrence and activity of SDG metabolising bacteria. The data showed differences in conversion efficiency between SDG deglycosylating species, but SDG was completely deglycosylated within 20 h by five of six strains. The strain Clostridium sp. SDG-Mt85-3Db showed the highest initial rate of SDG deglycosylation. Furthermore, we found that Bacteroides distasonis and B. fragilis made up 0.5% and 3.3% of total faecal bacteria, respectively. However, Clostridium sp. SDG-Mt85-3Db was detected within the dominant microbiota of only two out of 20 faecal samples. Bacteria involved in the demethylation step of SDG conversion also demethylated a variety of compounds other than SDG. In particular, Peptostreptococcus productus demethylated the lignans pinoresinol, lariciresinol and matairesinol. Finally, Eggerthella lenta catalysed the reduction of pinoresinol and lariciresinol to secoisolariciresinol.

16S–23S rRNA gene internal transcribed spacer sequences for analysis of the phylogenetic relationships among species of the genus Porphyromonas

The 16S-23S rRNA gene internal transcribed spacer (ITS) regions of 11 reference strains of Porphyromonas species, together with Bacteroides distasonis and Tannerella forsythensis, were analysed to examine interspecies relationships. Compared with the phylogenetic tree generated using 16S rRNA gene sequences, the resolution of the ITS sequence-based tree was higher, but species positioning and clustering were similar with both approaches. The recent separation of Porphyromonas gulae and Porphyromonas gingivalis into distinct species was confirmed by the ITS data. In addition, analysis of the ITS sequences of 24 clinical isolates of Porphyromonas asaccharolytica plus the type strain ATCC 25260(T) divided the sequences into two clusters, of which one was alpha-fucosidase-positive (like the type strain) while the other was alpha-fucosidase-negative. The latter resembled the previously studied unusual extra-oral isolates of 'Porphyromonas endodontalis-like organisms' (PELOs) which could therefore be called 'Porphyromonas asaccharolytica-like organisms' (PALOs), based on the genetic identification. Moreover, the proposal of alpha-fucosidase-negative P. asaccharolytica strains as a new species should also be considered.

Antimicrobial susceptibility patterns of competitive exclusion bacteria applied to newly hatched chickens

Competitive exclusion (CE) products are mixtures of obligate and facultative anaerobic bacteria applied to poultry hatchlings for prevention of Salmonella colonization. These mixtures have the potential to introduce bacteria with undesirable antimicrobial drug resistance traits into the human food supply. Antimicrobial drug susceptibilities of 27 obligate and facultative anaerobes isolated from a commercial CE product were evaluated with a microdilution minimal inhibitory concentration (MIC) assay. Bacteroides distasonis and Bacteroides fragilis isolates were resistant to tetracycline and other antimicrobial drugs. An Escherichia coli isolate was resistant to four antimicrobial drugs: erythromycin, penicillin, vancomycin, and tylosin. Erythromycin-resistant enterococci and vancomycin-resistant Lactococcus lactis isolates in the CE product were detected. These findings suggest that more work needs to be done to assess the potential effects of CE product use in poultry on the food supply.

Presence of the cfxA gene in Bacteroides distasonis

In this study we investigated the presence of the cfxA gene (encoding a class A β-lactamase) in 73 strains of the Bacteroides fragilis group belonging to the species B. distasonis (34), B. vulgatus (14), B. thetaiotaomicron (8), B. merdae (6), B. caccae (9) and B. ovatus (2) isolated from human intestinal microflora of healthy children and adults. Employing specific primers to the cfxA gene, a 312-bp amplified fragment was obtained in 2 strains of B. vulgatus and 9 strains, the majority from children, of B. distasonis. The expression of this enzyme was analysed by determining the MICs to cefoxitin and cefotaxime and values varied from 2 to >256 μg/ml of both cefoxitin and cefotaxime. Sequence analysis of the amplicons corresponding to the cfxA gene from B. distasonis and B. vulgatus revealed identical sequences between these isolates and high similarity with other β-lactamase genes of anaerobes such as cfxA of B. vulgatus (99%) and cfxA2 of Prevotella intermedia (99%), both sequences of which deposited in Genbank under accession numbers U38243 and AF118110, respectively. However, a fragment obtained from a B. distasonis strain (EC17-4) showed a unique RFLP profile and 87% nucleotide similarity with cfxA and cfxA2 genes. These results seem to suggest a dissemination of these resistance determinants among Bacteroides species.

Growth inhibition of intestinal bacteria and mutagenicity of 2-, 3-, 4-aminobiphenyls, benzidine, and biphenyl

2-Aminobiphenyl (2-ABP), 3-aminobiphenyl (3-ABP) and 4-aminobiphenyl (4-ABP), but not benzidine (Bz) and biphenyl (Bp), were found to be inhibitory to the growth of human intestinal bacteria Bifidobacterium infantis ATCC 15697, B. bifidium ATCC 11863, Clostridium perfringens ATCC 13124, Escherichia coli ATCC 25922, E. coli ATCC 35218, Enterobacter cloacae ATCC 13047 and Salmonella typhimurium TA98, TA100, YG1041 at 10–200 μg/ml in culture broth. Bacteroides distasonis ATCC 8503 , B. fragilis ATCC 25285, B. theataiotaomicron ATCC 29741, C. paraputrificum ATCC 26780, C. clostridiiforme ATCC 25537, Lactobacillus acidophilus ATCC 4356 and Enterococcus faecium ATCC 19434 were not inhibited by the above mentioned compounds in concentrations up to 200 μg/ml. The Ames Salmonella/microsome assay was employed to test the mutagenicity of the above-mentioned compounds using strains TA98 and TA100 in the presence and absence of Aroclor 1254-induced rat S9 mix. It was found that 4-ABP was mutagenic to both TA98 and TA100, and Bz was mutagenic to TA98 in the presence of rat S9 mix. 2-Aminobiphenyl, 3-ABP, and Bp were not mutagenic to both strains tested. 2-Aminobiphenyl and 3-ABP are chemical isomers of 4-ABP and are as strong as 4-ABP in inhibiting the growth of intestinal bacteria but not as mutagenic as 4-ABP. Evidence suggested that the mechanism of growth inhibition is not involved with the interaction of DNA that causes mutations, but rather on the electron transport system of these organisms.

Enumeration of Bacteroides Species in Human Faeces by Fluorescent in situ Hybridisation Combined with Flow Cytometry Using 16S rRNA Probes

Bacteroides is a predominant group of the faecal microbiota in healthy adults. To investigate the species composition of Bacteroides by fluorescent in situ hybridisation (FISH) combined with flow cytometry, we developed five species-specific probes targeting the 16S rRNA. Probes were designed to identify cells belonging to Bacteroides distasonis, B. fragilis, B. ovatus, B. vulgatus and B. putredinis. The species-specificity of the probes was assessed against a collection of reference strains from the Cytophaga-Flavobacterium-Bacteroides group. The results of the FISH experiments showed that the probes were specific as they only detected strains of the target species. Determining the fluorescence intensity of each probe relative to that of the EUB 338 probe (domain bacteria) further showed that each species probe easily accessed the targeted site. The probes were applied to quantify the Bacteroides species in faeces collected from 20 healthy adults. All five species were detected in the faecal samples. Cells hybridised with Bfra 998 were the most frequent as they were observed in 90% of individuals (18/20 samples, mean proportion of 3.9 +/- 2.2%). The cells hybridised with Bvulg 1017 were observed in 85% of individuals (17/20 samples) and represented with a mean proportion of 4.2 +/- 6.1%, the most abundant Bacteroides species in human faeces. Cells hybridising with probes for B. ovatus, B. distasonis and B. putredinis were less frequently detected. The large distribution of B. vulgatus and B. fragilis in human faeces is in accordance with previous reports based on culture or molecular studies. This work showed that fluorescent in situ hybridisation is a tool appropriate for a high-resolution analysis of the species composition of complex ecosystems and especially of the Bacteroides group within the faecal microbiota.

Outer-membrane pore-forming proteins in gram-negative anaerobic bacteria.

The outer-membrane proteins (OMPs) of bacteria function as the dynamic interface between the bacterium and its surroundings and are involved in maintenance of cell structure, binding a variety of substances, adhesion to other cells, and regulation of transport of both nutrients and bactericidal agents. There is a vast amount of information about aerobic OMPs and their roles in immunogenicity, virulence, and antimicrobial resistance. Knowledge about OMPs in anaerobic bacteria is much sparser. Genetic data present in data banks regarding aerobic porins are not readily helpful in identifying or analyzing anaerobic porins because of the large phylogenetic distance between the aerobic and anaerobic organisms. We recently identified and sequenced the genes for both a porin protein complex and an OmpA protein in Bacteroides fragilis, and the data are summarized here. Also, recent information is presented about similar OMPs found in other gram-negative anaerobic bacteria, including Bacteroides thetaiotaomicron, Bacteroides distasonis, Porphyromonas, and Fusobacterium.

National survey on the susceptibility of Bacteroides Fragilis Group: report and analysis of trends for 1997-2000.

The results of a multicenter US survey using the National Committee for Clinical Laboratory Standards currently recommended methodology for measuring in vitro susceptibility of 2673 isolates of Bacteroides fragilis group species were compared from 1997 to 2000. The test panel consisted of 14 antibiotics: 3 carbapenems, 3 beta-lactam-beta-lactamase inhibitors, 3 cephamycins, 2 fluoroquinolones, clindamycin, chloramphenicol, and metronidazole. Declines in the geometric mean minimum inhibitory concentrations were seen with imipenem, meropenem, ampicillin-sulbactam, and the cephamycins. Increased geometric means were observed with the fluoroquinolones and were usually accompanied by an increase in resistance rates. Bacteroides distasonis shows the highest resistance rates among beta-lactam antibiotics, whereas Bacteroides vulgatus shows the highest resistance levels among fluoroquinolones. B. fragilis shows the lowest resistance rates for all antibiotics. All strains were susceptible to chloramphenicol and metronidazole concentrations <8 microgram/mL. The data underscore the need for species identification and continued surveillance to monitor resistance patterns.

Design and evaluation of oligonucleotide-microarray method for the detection of human intestinal bacteria in fecal samples

An oligonucleotide-microarray method was developed for the detection of intestinal bacteria in fecal samples collected from human subjects. The 16S rDNA sequences of 20 predominant human intestinal bacterial species were used to design oligonucleotide probes. Three 40-mer oligonucleotides specific for each bacterial species (total 60 probes) were synthesized and applied to glass slides. Cyanine5 (CY5)-labeled 16S rDNAs were amplified by polymerase chain reaction (PCR) from human fecal samples or bacterial DNA using two universal primers and were hybridized to the oligo-microarray. The 20 intestinal bacterial species tested were Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides fragilis, Bacteroides distasonis, Clostridium clostridiiforme, Clostridium leptum, Fusobacterium prausnitzii, Peptostreptococcus productus, Ruminococcus obeum, Ruminococcus bromii, Ruminococcus callidus, Ruminococcus albus, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium infantis, Eubacterium biforme, Eubacterium aerofaciens, Lactobacillus acidophilus, Escherichia coli, and Enterococcus faecium. The two universal primers were able to amplify full size 16S rDNA from all of the 20 bacterial species tested. The hybridization results indicated that the oligo-microarray method developed in this study is a reliable method for the detection of predominant human intestinal bacteria in the fecal samples.

An In Vitro Assay To Evaluate Competitive Exclusion Products for Poultry

An in vitro assay was developed to measure the ability of competitive exclusion (CE) bacteria to protect Caco-2 and CRL-2117 epithelial cells from invasion by Salmonella Typhimurium. The proposed assay is needed to expedite the development of defined-flora CE products. The average significantly protective concentration of the commercial poultry-specific CE product Preempt was 4.05 log CFU/6.41 log human Caco-2 cells and 3.71 log CFU/6.89 log CFU chicken CRL-2117 cells. Enterococcus faecalis isolated from Preempt protected CRL-2117 cells, Escherichia coli isolates protected Caco-2 cells, Lactococcus lactis and Bacteroides distasonis isolates protected both cell lines, and three species of Lactobacillus isolates failed to protect either cell line. A defined mixture of 29 strains of bacteria similar to the constituents of Preempt protected both cell lines from Salmonella invasion at a concentration of 7.83 log CFU. The constituents of the defined CE culture were separated into mixtures of obligate (8.42 log CFU) and facultative (8.49 log CFU) anaerobes, which both protected the cell lines, suggesting that both types of bacteria were equally protective. Although not a substitute for in vivo testing, the in vitro CE assay is a rapid technique for the evaluation of bacterial mixtures for potential CE products.

Reclassification of Bacteroides forsythus (Tanner et al. 1986) as Tannerella forsythensis corrig., gen. nov., comb. nov.

The characteristics of the fusiform species Bacteroides forsythus, isolated from human periodontal pockets, were examined. 165 rDNA sequence analysis confirmed that B. forsythus was not a species within the genus Bacteroides sensu stricto. Although B. forsythus was phylogenetically related to Bacteroides distasonis and Bacteroides merdae in the phylogenetic tree, the ratio of anteiso-15:0 to iso-15:0 in whole-cell methanolysates of B. forsythus was different from those of B. distasonis, B. merdae and other Bacteroides species. B. forsythus did not grow on medium containing 20% bile, but members of the Bacteroides fragilis group did. B. forsythus was the only species tested that was trypsin-positive in API ZYM tests. The dehydrogenase enzyme pattern was of no use for the differentiation of B. forsythus and the B. fragilis group. On the basis of these data, a new genus, Tannerella, is proposed for Bacteroides forsythus, with one species, Tannerella forsythensis corrig., gen. nov., comb. nov. The type strain of Tannerella forsythensis is JCM 10827T (= ATCC 43037T).

Adhesive property of Bifidobacterium lactis LKM512 and predominant bacteria of intestinal microflora to human intestinal mucin.

The adhesive property to the intestinal mucin of Bifidobacterium lactis LKM512, B. longum, B. breve, B. bifidum, B. adolescentis, B. infantis, Bacteroides vulgatus, Bacteroides distasonis, Eubacterium aerofaciens, Clostridium perfringens, Escherichia coli, and Lactobacillus acidophilus were examined. Adhesive rate of LKM512 to the mucin was significantly (p < 0.05, 0.01, or 0.001) stronger than the other strains from 2 to 100 time. Though the adhesive property of many strains was almost same to the mucin of 20-year-old and 50-year-old generations, in case of 4-month-old was different. Adhesive inhibitory effect of C. perfringens to the mucin by LKM512 was examined. Under the condition that LKM512 was 108/ml and that C. perfringens was 106/ml, adhesion of C. perfringens to the mucin was inhibited at 99.6%, when LKM512 adhered in advance. There was the strong inhibition of adhesion at 74.0%, when C. perfringens adhered to mucin in advance. Thus, LKM512 can inhibit the adhesion of harmful bacteria to the intestinal mucin, the possibility of using as a probiotic strain has to be verified.

Synergic activity, for anaerobes, of trovafloxacin with clindamycin or metronidazole: chequerboard and time-kill methods.

Chequerboard titrations were used to test the activity of trovafloxacin, alone and in combination with clindamycin or metronidazole, against 156 Gram-positive or Gram-negative anaerobes, including 47 Bacteroides fragilis group, 36 Prevotella spp., 26 fusobacteria, 21 peptostreptococci and 26 clostridia. MIC50/MIC90 values (mg/L) of each drug alone against all 156 strains were: trovafloxacin, 0.5/1; clindamycin, 0.25/2; metronidazole, 1/2. Synergy (FIC indices </= 0.5) was seen in two strains with trovafloxacin plus clindamycin, and seven with trovafloxacin plus metronidazole. All other combinations were additive (FIC indices >0. 5-2.0); no antagonism (FIC indices >4.0) was seen. In addition, synergy was tested by time-kill methodology for each of the above combinations against 12 Gram-positive or Gram-negative strains. Results indicated that synergy (defined as a >/= 2 log(10) decrease in cfu/mL at 48 h compared with the more active drug alone) was found between trovafloxacin at or below the MIC and both clindamycin and metronidazole at or below the MIC in one strain each of Bacteroides fragilis, Bacteroides thetaiotaomicron, Prevotella intermedia, Fusobacterium varium, Peptostreptococcus asaccharolyticus and Clostridium bifermentans. Synergy between trovafloxacin (</=MIC) and metronidazole alone was seen in one strain each of Bacteroides distasonis, Prevotella bivia, Fusobacterium mortiferum, P. asaccharolyticus and C. bifermentans. In many cases of synergy, including those at the trovafloxacin MIC, regrowth after 48 h, which was commonly seen with trovafloxacin alone, was inhibited, and 99.9% killing was observed with the combination after 48 h, but not with trovafloxacin alone.

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.

Metabolism of Antioxidant in Lemon Fruit (Citrus limon BURM. f.) by Human Intestinal Bacteria

The metabolism of eriocitrin (eriodictyol 7-rutinoside), an antioxidant in lemon fruit (Citrus limon BURM. f.), was examined using intestinal bacteria. In the experiment, it was shown that eriocitrin was hydrolyzed to eriodictyol, its aglycon, by Bacteroides distasonis and Bacteroides uniformis. Eriodictyol was converted to 3,4-dihydroxyhydrocinnamic acid (3,4,-DHCA) and phloroglucinol (PHL) by Clostridium butyricum. The metabolic pathway of eriocitrin to eriodictyol and 3,4-DHCA was confirmed by using human feces. The antioxidative activity of eriocitrin metabolites, eriodictyol, 3,4-DHCA, and PHL, was examined using linoleic acid autoxidation and a rabbit erythrocyte membrane oxidation system. It was shown that eriodictyol exhibited stronger activity than α-tocopherol. The activities of 3,4-DHCA and PHL were weak, but they preserved the antioxidative activity. Keywords: Lemon fruit; Citrus limon; antioxidant; eriocitrin; flavanone; metabolism; intestinal bacteria

Polyphasic analysis of strains of the genus Capnocytophaga and Centers for Disease Control group DF-3.

A polyphasic approach was used to determine the relationships between well-characterized reference strains representing all seven Capnocytophaga species. One Centers for Disease Control (CDC) group DF-3 strain, a presumed relative of the genus Capnocytophaga, and 15 field isolates were included as well. Fourteen isolates were assigned to named Capnocytophaga species, all of which could be differentiated by means of whole-organism protein electrophoresis. A separate position was occupied by the CDC group DF-3 strain and by one field isolate representing a novel Capnocytophaga species. The phylogenetic position of each taxon was determined by means of 16S rRNA sequence analysis. A considerable genotypic heterogeneity within the genus Capnocytophaga was detected in spite of the minimal phenotypic differences. Comparative 16S rRNA sequence analysis revealed that CDC group DF-3 is not a close relative of the capnocytophagas but constitutes a separate genus that clusters together with Bacteroides forsythus and Bacteroides distasonis, two generically misclassified Bacteroides species. The degree of protein similarity correlated with our and published DNA-DNA binding values. Percentage 16S rRNA similarity values of greater than 97% did not guarantee conspecificity. All Capnocytophaga strains had very similar fatty acid contents characterized by significant amounts of 14:0, 15:0 iso (greater than 55%), 16:0, 16:0 3OH, and 17:0 iso 3OH. PCR-mediated DNA fingerprinting allowed discrimination of most species, although some strains could not be classified efficiently because of DNA polymorphisms.

PCR detection and quantitation of predominant anaerobic bacteria in human and animal fecal samples.

PCR procedures based on 16S rRNA gene sequences specific for 12 anaerobic bacteria that predominate in the human intestinal tract were developed and used for quantitative detection of these species in human (adult and baby) feces and animal (rat, mouse, cat, dog, monkey, and rabbit) feces. Fusobacterium prausnitzii, Peptostreptococcus productus, and Clostridium clostridiiforme had high PCR titers (the maximum dilutions for positive PCR results ranged from 10(-3) to 10(-8)) in all of the human and animal fecal samples tested. Bacteroides thetaiotaomicron, Bacteroides vulgatus, and Eubacterium limosum also showed higher PCR titers (10(-2) to 10(-6)) in adult human feces. The other bacteria tested, including Escherichia coli, Bifidobacterium adolescentis, Bifidobacterium longum, Lactobacillus acidophilus, Eubacterium biforme, and Bacteroides distasonis, were either at low PCR titers (less than 10(-2)) or not detected by PCR. The reported PCR procedure including the fecal sample preparation method is simplified and rapid and eliminates the DNA isolation steps.

Inactivation of tryptic activity by a human-derived strain of Bacteroides distasonis in the large intestines of gnotobiotic rats and mice.

Tryptic activity disappeared and trypsin was no longer detected with an antitrypsin antiserum in the large intestines of gnotobiotic rats and mice monoassociated with a human-derived strain of Bacteroides distasonis, whereas tryptic activity was not modified in the small intestines. This function was shown to be strain specific.

Age-dependent susceptibility to intraabdominal abscess formation

Infection remains a major cause of morbidity and mortality in the surgical neonate, with the risk of developing infectious complications decreasing as age increases. Developmental changes in the immune system, as well as transplacentally acquired immunity, likely play a role in this differential risk. The purpose of this study was to determine whether there is an age-related susceptibility to intraabdominal abscess formation. The authors used a mouse model in which the combination of an aerobe, anaerobe, and adjuvant routinely forms abscesses. Litters of at least six C57 BL 6 mice were used. The mice received 10 7 of Enterococcus faecalis and 10 7 Bacteroides distasonis or 10 7 B distasonis alone. The mice were given 8 μL/g of 50% wheat bran (40 mg/mL) and 50% bacteria. There were at least four litters for each experiment. Intraabdominal abscesses were counted after 7 days. Ten-day-old mice had an incidence of intraabdominal abscesses that was similar to that of the adults (81% v 91%). There was a significant decrease in intraperitoneal infection after 10 days, until weaning (37% and 38%; P < .05). The authors conclude that there are significant developmental changes in susceptibility to intraabdominal abscess formation, which may reflect changes in peritoneal defense mechanisms.

Design and evaluation of Bacteroides DNA probes for the specific detection of human fecal pollution.

Because Bacteroides spp. are obligate anaerobes that dominate the human fecal flora, and because some species may live only in the human intestine, these bacteria might be useful to distinguish human from nonhuman sources of fecal pollution. To test this hypothesis, PCR primers specific for 16S rRNA gene sequences of Bacteroides distasonis, B. thetaiotaomicron, and B. vulgatus were designed. Hybridization with species-specific internal probes was used to detect the intended PCR products. Extracts from 66 known Bacteroides strains, representing 10 related species, were used to confirm the specificity of these PCR-hybridization assays. To test for specificity in feces, procedures were developed to prepare DNA of sufficient purity for PCR. Extracts of feces from 9 humans and 70 nonhumans (cats, dogs, cattle, hogs, horses, sheep, goats, and chickens) were each analyzed with and without an internal positive control to verify that PCR amplification was not inhibited by substances in the extract. In addition, serial dilutions from each extract that tested positive were assayed to estimate the relative abundance of target Bacteroides spp. in the sample. Depending on the primer-probe set used, either 78 or 67% of the human fecal extracts tested had high levels of target DNA. On the other hand, only 7 to 11% of the nonhuman extracts tested had similarly high levels of target DNA. An additional 12 to 20% of the nonhuman extracts had levels of target DNA that were 100- to 1,000-fold lower than those found in humans.(ABSTRACT TRUNCATED AT 250 WORDS)