Isolation and identification of intestinal steroid-desulfating bacteria from rats and humans

We isolated 12 strictly anaerobic steroid-3-sulfate-desulfating strains from the intestinal floras of rats and humans. Two strains (S1 and S2) of the same atypical Clostridium species and an atypical Lactobacillus strain (termed R9) were obtained from rats. The human isolates were identified as Eubacterium cylindroides (two strains, H1 and H2), Peptococcus niger (two strains, H4 and H89), and Clostridium clostridiiforme. We also isolated, from different human fecal samples, four strains of phenotypically similar asaccharolytic Bacteroides strains, H6.2a, H6.2b, H65, and H175. Aryl steroid sulfatase activity for estrogen sulfates was present in all isolates. Alkyl steroid sulfatase activity for both 3 alpha- and 3 beta-sulfates was found only in P. niger H4. The same P. niger strain and Clostridium strains S1 and S2 also possessed bile acid sulfatase activity.     

Isolation and identification of intestinal steroid-desulfating bacteria from rats and humans.

We isolated 12 strictly anaerobic steroid-3-sulfate-desulfating strains from the intestinal floras of rats and humans. Two strains (S1 and S2) of the same atypical Clostridium species and an atypical Lactobacillus strain (termed R9) were obtained from rats. The human isolates were identified as Eubacterium cylindroides (two strains, H1 and H2), Peptococcus niger (two strains, H4 and H89), and Clostridium clostridiiforme. We also isolated, from different human fecal samples, four strains of phenotypically similar asaccharolytic Bacteroides strains, H6.2a, H6.2b, H65, and H175. Aryl steroid sulfatase activity for estrogen sulfates was present in all isolates. Alkyl steroid sulfatase activity for both 3 alpha- and 3 beta-sulfates was found only in P. niger H4. The same P. niger strain and Clostridium strains S1 and S2 also possessed bile acid sulfatase activity.     

Clostridium difficile and enterotoxigenic Bacteroides fragilis strains isolated from patients with antibiotic associated diarrhoea

From the fecal samples of 332 patients with a clinical diagnosis of antibiotic associated diarrhoea (AAD), 131 Clostridium difficile strains were isolated. For detection of toxin A in the isolated strains the enzymatic immunoassay was used. The cytopathic effect was determined on McCoy cell line. PCR was used for the detection of non-repeating and repeating sequences of toxin A gene and non-repeating sequences of toxin B gene. One hundred and six isolated C. difficile strains were TcdA(+)TcdB(+), 10 strains TcdA(-)TcB(+) and 15 were non-toxigenic TcdA(-)TcdB(-). Out of the same fecal samples 50 Bacteroides fragilis strains were isolated. All B. fragilis strains were tested in PCR reaction for fragilysine gene detection (bft). In 9 strains (18%) this gene was detected and the strains could be assumed as enterotoxigenic Bacteroides fragilis (ETBF). In 4 fecal samples toxigenic C. difficile (TcdA(+)TcdB(+)) was found simultaneously with ETBF. One sample contained C. difficile (TcdA(-)TcdB(+)) and ETBF. Out of 4 fecal samples only ETBF was isolated. The cytotoxicity of ETBF strains was tested on HT29/C1 human colon carcinoma cell line. The cytotoxicity titer in the range of 20 and 80 was observed.     

Urease assay and urease-producing species of anaerobes in the bovine rumen and human feces.

A growth medium and test were developed for rapid detection of urease in fermentative anaerobic bacteria. Using nonselective rumen fluid roll-tube agar medium and the new test, it was confirmed that Peptostreptococcus productus is often the most numerous urease-forming species in human feces. Also, some fecal strains of Ruminococcus albus, Clostridium innocuum, and Clostridium beijerinckii produced urease. Single strains of Fusobacterium prausnitzii, Coprococcus catus, and Streptococcus mitis that were strongly ureolytic on isolation later lost this ability. Urease activity was also detected in many strains of nonselectively isolated rumen species. They include Succinivibrio dextrinosolvens, Treponema sp., Ruminococcus bromii (not previously known to be present in the rumen), Butyrivibrio sp., Bifidobacterium sp., Bacteroides ruminicola, and P. productus. Most P. productus strains contain urease; however, the uniformity of this feature in the other species noted above is not known. The urease in many of these species was not detected if the growth medium contained 0.2% or more (each) yeast extract and Trypticase.     

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.     

栀子豉汤及拆方对六种人肠道菌的影响

目的研究栀子豉汤及栀子、淡豆豉对6种人肠道菌的影响。方法从特定人的粪便中分离培养肠杆菌、肠球菌、双歧杆菌、乳杆菌、产气荚膜梭菌和脆弱拟杆菌。分别将高、中、低浓度的栀子、淡豆豉及栀子豉汤药液加入选择性培养基中,以不加药液的培养基作为空白对照,将实验菌分别于厌氧或有氧条件下培养,计数菌落。结果不同浓度的栀子对肠杆菌的抑制率均为100.0%,淡豆豉在20%、50%、70%浓度分别使肠杆菌数量下调100.00%、53.83%和22.11%,而栀子豉汤可使肠杆菌数量维持在一定水平(104～106 CFU/g);两味药单独都表现了对肠球菌较强的抑制作用,而低、中浓度栀子豉汤则使肠球菌保持较稳定的数量;单味药及复方对产气荚膜梭菌和双歧杆菌敏感性较弱;栀子对脆弱拟杆菌抑制作用最强,而淡豆豉和栀子豉汤则可使其数量上调约20.00%;淡豆豉、栀子豉汤和高浓度栀子对乳杆菌非常敏感,低、中浓度栀子对乳杆菌数量仅有轻微下调作用。结论栀子豉汤比单独的栀子和淡豆豉更能维持肠道菌群的相对平衡。     

Characteristic of clinical strains of gram-negative obligate anaerobes

The aim of the study was to assess prevalence and antibiotic susceptibility profiles ofGram-negative strictly anaerobic bacteria isolated from clinical specimens taken from hospitalized patients in 2005-2006. Biochemical identification and antibiotic susceptibility were done in an automated system ATB Expression (bioMerieux sa). From 12262 specimens examined 867 strains of obligate anaerobes were isolated. Gram-negative strictly anaerobic bacteria were cultured in number of 138 strains (15,9%). All cultures were performed on Columbia agar and Schaedler agar media (bioMerieux sa) supplemented with 5% sheep blood and incubated at 37 degrees C for 48-120 h in 85% N2, 10% H2, 5% CO2. Most frequently isolated was Bacteroides spp. (41,3%). For this group beta-lactamase activity was evaluated by using nitrocefin disc test (Cefinase BBL, Becton Dickinson and Co., Cockeysville, MD, USA). Production of ESBLs was detected with the use of two disc diffusion methods: the double-disc synergy test (DDST) according to Jarlier et al. and the diagnostic disc (DD) test according to Appleton. ESBLs were produced by 5,3% strains of Bacteroides spp. For all Bacteroides spp. strains MIC values were determined by gradient diffusion method Etest (AB BIODISK, Sweden). ESBLs and MIC were performed on Wilkins-Chalgren solid medium supplemented with 5% sheep blood (Difco Lab., USA) and all plates were incubated at 35 degrees C for 48 hours in 85% N2, 10% H2, 5% CO2. Most Gram-negative obligate anaerobes isolated from clinical specimens are still susceptible to imipenem (100%), metronidazole (99,3%) and beta-lactam antibiotics with beta-lactamase inhibitors: piperacillin/tazobactam (99,3%), ticarcillin/clavulanate (99.3%), amoxicillin/clavulanate (97.8%).     

A note on ultra-violet red fluorescence of anaerobic bacteria in vitro

Anaerobes other than the Bacteroides melaninogenicus group isolated from clinical material produce an ultra-violet red fluorescence when grown under certain conditions in vitro. These organisms include other members of the genus Bacteroides as well as strains of some species of Clostridium, Bifidobacterium and Actinomyces. The major fluorescent pigment was identified as protoporphyrin IX. Factors necessary for the production of fluorescence are the presence of blood or haem and a fermentable carbohydrate during growth on a solid medium. Fluorescence intensity was related to the concentration of blood and fermentable carbohydrate present but was independant of inoculum size. Certain commercially available blood agar bases designed specifically for the isolation of fastidious anaerobes from clinical material which contain added carbohydrate were shown to induce fluorescence in certain organisms. This may lead to the misidentification of some anaerobes as B. melaninogenicus.     

A note on ultra-violet red fluorescence of anaerobic bacteria in vitro

Anaerobes other than the Bacteroides melaninogenicus group isolated from clinical material produce an ultra-violet red fluorescence when grown under certain conditions in vitro. These organisms include other members of the genus Bacteroides as well as strains of some species of Clostridium, Bifidobacterium and Actinomyces. The major fluorescent pigment was identified as protoporphyrin IX. Factors necessary for the production of fluorescence are the presence of blood or haem and a fermentable carbohydrate during growth on a solid medium. Fluorescence intensity was related to the concentration of blood and fermentable carbohydrate present but was independent of inoculum size. Certain commercially available blood agar bases designed specifically for the isolation of fastidious anaerobes from clinical material which contain added carbohydrate were shown to induce fluorescence in certain organisms. This may lead to the misidentification of some anaerobes as B. melaninogenicus.     

Isolation of toxigenic strains of Clostridium difficile and enterotoxin producing Bacteroides fragilis from fecal specimens of patients suspected of antibiotic associated diarrhoea

Fifty faecal samples from patients suspected of AAD (antibiotic associated diarrhoea) were studied for Clostridium difficile and enterotoxin producing Bacteroides fragilis (ETBF). Using TCD (Becton-Dickinson) and C. difficile Toxin A test (Oxoid) in 34% of specimens the presence of toxin A was detected. From all specimens 25 C. difficile strains were isolated. All isolated strains produced toxin B in vitro which was shown in Mc Coy cytotoxicity test. Eighteen strains only were toxin A positive in vitro. From all isolated C. difficile strains 28% were tox A (-) tox B (+). By means of PCR presence of toxin A and toxin B genes was tested directly in faecal samples and in strains. From the same 50 faecal samples 17 B. fragilis strains were isolated. Four of them produced the enterotoxin (fragilisin) which was detected on the HT 29/C1 cell line. Genes of fragilisin were found in strains and directly in faecal samples. Toxin producing C. difficile and B. fragilis (ETBF) together were found in 3 samples. From one faecal sample only ETBF was cultured.     

Investigation of arcobacters in meat and faecal samples of clinically healthy cattle in Turkey

AIMS: To investigate the presence of Arcobacter spp. in minced beef meat (n = 97) and rectal faecal samples (n = 200) collected from cattle immediately after slaughter at a local abattoir in Turkey. METHODS AND RESULTS: Meat samples were examined using three different isolation procedures (CAT-supplemented media, de Boer arcobacter isolation method and membrane filtration method), but only one method (CAT-supplemented media) was employed for faecal samples. The isolated Arcobacter strains were identified by genus- and species-(multiplex) specific PCR assays. Arcobacter spp. were isolated from 5 and 9.5% of meat and faecal samples respectively. Although the only Arcobacter sp. found in meat samples was Arcobacter butzleri, all three pathogenic species--A. butzleri, A. cryaerophilus and A. skirrowii--were detected in the rectal swabs. No Arcobacter was isolated when the de Boer method was used for minced meat samples but the same five meat samples were found positive for arcobacters when CAT-supplemented media and membrane filtration method were used. CONCLUSIONS: The membrane filtration method was found to be superior to the CAT-supplemented media, because it led to a reduction in competing microflora. However, the necessity for one filter and medium for each sample makes this method somewhat expensive. The multiplex-PCR (m-PCR) assay shortened significantly the time required for the identification of Arcobacter spp. and also removed the possibility of false positive results due to other campylobacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports the isolation of Arcobacter spp. in cattle for the first time in Turkey. The m-PCR assay enables the identification and differentiation of all arcobacters simultaneously in one-step PCR.     

Phylogeny of human intestinal bacteria that activate the dietary lignan secoisolariciresinol diglucoside

The human intestinal microbiota is essential for the conversion of the dietary lignan secoisolariciresinol diglucoside (SDG) via secoisolariciresinol (SECO) to the enterolignans enterodiol (ED) and enterolactone (EL). However, knowledge of the species that catalyse the underlying reactions is scant. Therefore, we focused our attention on the identification of intestinal bacteria involved in the conversion of SDG. Strains of Bacteroides distasonis, Bacteroides fragilis, Bacteroides ovatus and Clostridium cocleatum, as well as the newly isolated strain Clostridium sp. SDG-Mt85-3Db, deglycosylated SDG. Demethylation of SECO was catalysed by strains of Butyribacterium methylotrophicum, Eubacterium callanderi, Eubacterium limosum and Peptostreptococcus productus. Dehydroxylation of SECO was catalysed by strains of Clostridium scindens and Eggerthella lenta. Finally, the newly isolated strain ED-Mt61/PYG-s6 catalysed the dehydrogenation of ED to EL. The results indicate that the activation of SDG involves phylogenetically diverse bacteria, most of which are members of the dominant human intestinal microbiota.     

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.     

Selective medium for primary isolation of members of the tribeProteeae

A selectiveProteeae medium (SPM) for isolation and preliminary detection of species of generaProteus, Morganella, andProvidencia was evaluated. The SPM contains tryptose phosphate agar with phenolphthalein monophosphate (as substrate for phosphatase activity), bile salts and polymyxin B (as inhibitors). The selectivity of the SPM was tested by the ecometric method of quality assurance of culture media. Fourteen reference cultures of enterobacteria and fifty-four strains ofProteeae were tested for their absolute growth index (AGI). Ninety-five percent of testedProteeae strains display an AGI above 2.5. The detected phosphatase activity proved to be able to discriminate colonies of members of the tribeProteeae. The ability of SPM for primary isolation of members ofProteeae was tested on food and clinical material and 94 strains were isolated. In addition, the SPM was employed in routine practice of clinical microbiology. From 1016 clinical samples (stool, urine, vaginal and urethral swabs), 57 strains ofProteeae were detected by the SPM in contrast to 35 strains by the routine procedure. The difference amounts to nearly 40%.     

Azoreductase activity of anaerobic bacteria isolated from human intestinal microflora.

A plate assay was developed for the detection of anaerobic bacteria that produce azoreductases. With this plate assay, 10 strains of anaerobic bacteria capable of reducing azo dyes were isolated from human feces and identified as Eubacterium hadrum (2 strains), Eubacterium spp. (2 species), Clostridium clostridiiforme, a Butyrivibrio sp., a Bacteroides sp., Clostridium paraputrificum, Clostridium nexile, and a Clostridium sp. The average rate of reduction of Direct Blue 15 dye (a dimethoxybenzidine-based dye) in these strains ranged from 16 to 135 nmol of dye per min per mg of protein. The enzymes were inactivated by oxygen. In seven isolates, a flavin compound (riboflavin, flavin adenine dinucleotide, or flavin mononucleotide) was required for azoreductase activity. In the other three isolates and in Clostridium perfringens, no added flavin was required for activity. Nondenaturing polyacrylamide gel electrophoresis showed that each bacterium expressed only one azoreductase isozyme. At least three types of azoreductase enzyme were produced by the different isolates. All of the azoreductases were produced constitutively and released extracellularly.     

Azoreductase activity of anaerobic bacteria isolated from human intestinal microflora

A plate assay was developed for the detection of anaerobic bacteria that produce azoreductases. With this plate assay, 10 strains of anaerobic bacteria capable of reducing azo dyes were isolated from human feces and identified as Eubacterium hadrum (2 strains), Eubacterium spp. (2 species), Clostridium clostridiiforme, a Butyrivibrio sp., a Bacteroides sp., Clostridium paraputrificum, Clostridium nexile, and a Clostridium sp. The average rate of reduction of Direct Blue 15 dye (a dimethoxybenzidine-based dye) in these strains ranged from 16 to 135 nmol of dye per min per mg of protein. The enzymes were inactivated by oxygen. In seven isolates, a flavin compound (riboflavin, flavin adenine dinucleotide, or flavin mononucleotide) was required for azoreductase activity. In the other three isolates and in Clostridium perfringens, no added flavin was required for activity. Nondenaturing polyacrylamide gel electrophoresis showed that each bacterium expressed only one azoreductase isozyme. At least three types of azoreductase enzyme were produced by the different isolates. All of the azoreductases were produced constitutively and released extracellularly.     

Participation of gram-negative anaerobes in infections in hospitalized patients

The aim of the study was to assess the prevalence and antibiotic susceptibility profiles of strictly anaerobic Gram-negative bacteria isolated from clinical samples taken from hospitalized patients from 01.01.2007 to 31.12.2008. The specimens were cultured using media, incubated at 37 degrees C under anaerobic conditions. Biochemical identification and antibiotic susceptibility were done in an automated system ATB Expression (bioMerieux S.A, France). For selected strains of Bacteroides sp. sensitivity was determined using E-test (AB BIODISK, Sweden). Overall 1274 strains of obligate anaerobes were isolated. Gram-negative bacteria were cultured in number of 333 strains. Most frequently isolated was Bacteroides sp. (46,9%) and Prevotella sp. (29,7%). Isolated bacteria are still susceptible to imipenem (100%), metronidazole (100%) and beta-lactam antibiotics with beta-lactamase inhibitors: amoxicillin/clavulanate (97,8%) piperacillin/tazobactam (99,1%), ticarcillin/clavulanate (99,1%).     

Comparative fermentation studies of industrial strains belonging to four species of solvent-producing clostridia

Industrial and culture collection strains of solvent-producing clostridia, classified as Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharobutylicum, and Clostridium saccharoperbutylacetonicum were utilised in a comparative study of fermentation performance in a laboratory fermentation medium, a molasses fermentation medium, and a maize fermentation medium under standardised culture conditions. At least one representative strain was selected from each of the sub-groups within the four species. Preliminary evaluations were first undertaken for the three different fermentation media to determine the most appropriate media formulations, carbohydrate concentrations, and culture conditions for comparison of the solvent-producing ability of these strains. Standardised fermentation media and culture conditions were then selected for each of the comparative fermentation studies. These included TYA medium containing 4% glucose, a supplemented molasses medium containing 6% fermentable sugars, and a supplemented maize mash medium containing 8% maize. Additional comparative fermentation studies on industrial strains belonging to two species of solvent-producing clostridia were carried out in molasses containing higher concentrations of fermentable sugars, and the sugar concentrations supporting maximum levels of solvent production were determined. Although all the strains tested grew in the maize fermentation medium and degraded starch, only a few strains produced consistently high solvent levels. Optimum starch utilisation and solvent production was obtained at a maize concentration of 80 g/l. Pretreatment of the maize by milling or saccharification decreased the buffering capacity of the medium and resulted in decreased solvent production. Decreasing the time used to gelatinise the starch had little effect. Solvent yields and concentrations obtained in this study were compared with various published data in the scientific and patent literature and appeared to closely simulate the results obtained in the industrial fermentation process. The fermentation performances of individual strains could provide useful comparative data for the selection and development of strains for use on various commercial fermentation substrates.     

Comparison of fructooligosaccharide utilization by Lactobacillus and Bacteroides species

The utilization of 1-kestose (GF(2)) and nystose (GF(3)), the main components of fructooligosaccharides (FOS), by Lactobacillus and Bacteroides species was examined. Of seven Lactobacillus and five Bacteroides strains that utilized FOS, L. salivarius, L. rhamnosus, L. casei, and L. gasseri utilized only GF(2), whereas L. acidophilus and all the Bacteroides strains utilized both GF(2) and GF(3). Only the strains able to utilize both GF(2) and GF(3) had β-fructosidase activity in the culture supernatants. The culture supernatants of the Lactobacillus strains had higher β-fructosidase activity for GF(2) than for GF(3), whereas those of the Bacteroides strains had higher activity for GF(3) than for GF(2). Furthermore, β-fructosidase activity of the culture supernatants of the Lactobacillus cells grown in the GF(3) medium was much higher than that of the cells grown in the GF(2) medium, whereas the activity of the culture supernatants of the Bacteroides cells grown in the GF(3) medium was almost the same as that of the cells grown in the GF(2) medium. These results indicate that Lactobacillus species metabolize FOS in a different way from that of Bacteroides species.     

Cecal and fecal bacterial flora of the Mongolian gerbil and the chinchilla.

The Mongolian gerbil is being increasingly used as a laboratory animal and as a pet. Both chinchillas and gerbils are used as animal models for otitis media and other otic research. Previously, only incomplete information was available regarding the indigenous bacterial flora of the lower intestinal tracts of these coprophagic animals. Using the strict anaerobic methodology of the Virginia Polytechnic Institute Anaerobe Laboratory, we studied the predominant bacterial flora of the cecum and fecal pellets of the gerbil and the chinchilla and the bacterial flora of digesta pellets in the proximal colon. We found species of the following anaerobic genera in high dilutions of gerbil fecal pellets: Bifidobacterium, Clostridium, Propionibacterium, Lactobacillus, and Bacteroides. Only lactobacilli were found in high dilutions of digesta from the upper colon, although the cecum yielded Peptostreptococcus, Bifidobacterium, Clostridium, Lactobacillus, Propionibacterium, and Bacteroides species from high dilutions of cecal contents. The facultatively anaerobic and aerobic flora isolated consisted of species of Bacillus, Streptococcus, Staphylococcus, Acinetobacter, Alcaligenes, Escherichia, Pasteurella, and Pseudomonas plus several unidentifiable organisms. Species of Bifidobacterium, Bacteroides, Eubacterium, and anaerobic Lactobacillus were isolated from chinchillas.