Vagococcus entomophilus sp. nov., from the digestive tract of a wasp (Vespula vulgaris).

Three unknown Gram-stain-positive, catalase-negative, facultatively anaerobic and coccus-shaped strains of bacteria were isolated from the digestive tracts of wasps (Vespula vulgaris). Analysis of 16S rRNA gene sequences revealed that these strains had identical sequences and showed that Vagococcus salmoninarum, with 96.2% sequence similarity, was the closest phylogenetic neighbour. Further analyses based on hsp60 and pheS gene sequences of representatives of the family Enteroccocaceae and genotypic and phenotypic characterization using (GTG)5-PCR fingerprintings, EcoRI ribotyping, DNA G+C content, whole-cell protein profiling, cellular fatty acid profiles analysis and extensive biotyping confirmed that the investigated strains were representatives of a novel bacterial species within the genus Vagoccocus for which the name Vagoccocus entomophilus sp. nov. is proposed. The type strain is VOSTP2(T) ( = DSM 24756(T) = CCM 7946(T)).

Lactobacillus rodentium sp. nov., from the digestive tract of wild rodents.

Three strains of regular, long, Gram-stain-positive bacterial rods were isolated using TPY, M.R.S. and Rogosa agar under anaerobic conditions from the digestive tract of wild mice (Mus musculus). All 16S rRNA gene sequences of these isolates were most similar to sequences of Lactobacillus gasseri ATCC 33323T and Lactobacillus johnsonii ATCC 33200T (97.3% and 97.2% sequence similarities, respectively). The novel strains shared 99.2-99.6% 16S rRNA gene sequence similarities. Type strains of L. gasseri and L. johnsonii were also most related to the newly isolated strains according to rpoA (83.9-84.0% similarities), pheS (84.6-87.8%), atpA (86.2-87.7%), hsp60 (89.4-90.4%) and tuf (92.7-93.6%) gene sequence similarities. Phylogenetic studies based on 16S rRNA, hsp60, rpoA, atpA and pheS gene sequences, other genotypic and many phenotypic characteristics (results of API 50 CHL, Rapid ID 32A and API ZYM biochemical tests; cellular fatty acid profiles; cellular polar lipid profiles; end products of glucose fermentation) showed that these bacterial strains represent a novel species within the genus Lactobacillus. The name Lactobacillus rodentium sp. nov. is proposed to accommodate this group of new isolates. The type strain is MYMRS/TLU1T (=DSM 24759T=CCM 7945T).

Reclassification of Bifidobacterium stercoris Kim et al. 2010 as a later heterotypic synonym of Bifidobacterium adolescentis.

The taxonomic position of Bifidobacterium stercoris Eg1(T) ( = JCM 15918(T)) based on comparative 16S rRNA gene and hsp60 sequence analyses was found to be controversial, as the strain showed high similarity to the type strain of Bifidobacterium adolescentis, CCUG 18363(T). Therefore, the relationship between the two species was investigated by a taxonomic study that included, in addition to re-evaluation of the 16S rRNA gene sequence, determination of DNA-DNA binding and multilocus sequence analysis (MLSA) of housekeeping genes encoding the DNA-directed RNA polymerase B subunit (rpoC), putative xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (xfp), elongation factor EF-G (fusA), 50S ribosomal protein L2 (rplB) and DNA gyrase B subunit (gyrB). Comparative 16S rRNA gene sequence analysis showed relatively high similarity (98.9 %) between B. stercoris KCTC 5756(T) and B. adolescentis ATCC 15703(T). MLSA revealed close relatedness between B. stercoris KCTC 5756(T) and B. adolescentis CCUG 18363(T), with 99.3-100 % similarity between the rpoC, xfp, fusA, rplB and gyrB gene sequences. In addition, relatively high dnaJ1 gene sequence similarity of 97.7 % was found between the strains. Similar phenotypes and a high DNA-DNA binding value (78.9 %) confirmed that B. stercoris and B. adolescentis are synonymous. Based on these results, it is proposed that the species Bifidobacterium stercoris Kim et al. 2010 should be reclassified as a later heterotypic synonym of Bifidobacterium adolescentis Reuter 1963 (Approved Lists 1980).

Alloscardovia macacae sp. nov., isolated from the milk of a macaque (Macaca mulatta), emended description of the genus Alloscardovia and proposal of Alloscardovia criceti comb. nov.

A novel bacterial strain, designated M8(T), was isolated from milk of a female macaque bred in captivity. The strain was Gram-stain-positive, anaerobic, irregular coccoid-rod-shaped without catalase activity. Analysis of 16S rRNA gene sequence similarity revealed that the isolate was most closely related to Alloscardovia omnicolens CCUG 31649(T) (96.4%) and Metascardovia criceti OMB105(T) (96.6%). Sequences of hsp60, fusA, and xfp genes also confirmed that the strain was most closely related to the type strains of A. omnicolens and M. criceti. The isolate produced fructose-6-phosphate phosphoketolase which is in agreement with classification within the family Bifidobacteriaceae. The major fatty acids were C18 : 1ω9c (35.8%), C16 : 1 (6.2 %) and C14 : 0 (5.7 %). Polar lipid analysis revealed five different glycolipids, two unidentified phospholipids and diphosphatidylglycerol. The peptidoglycan was of the type A4α l-Lys-d-Asp with the presence of d(l)-alanine, d-glutamine, d-asparagine and l-lysine. The DNA G+C content of strain M8(T) was 50.1 mol%. On the basis of genetic, phylogenetic and phenotypic data, strain M8(T) represents a novel species of the genus Alloscardovia for which the name Alloscardovia macacae sp. nov. is proposed. The type strain is M8(T) ( = DSM 24762(T) = CCM 7944(T)). In addition, our results also revealed that Alloscardovia omnicolens DSM 21503(T) and Metascardovia criceti DSM 17774(T) do not belong to different genera within the family Bifidobacteriaceae. We therefore propose to reclassify Metascardovia criceti as Alloscardovia criceti comb. nov. An emended description of the genus Alloscardovia is also provided.

Oral administration of Parabacteroides distasonis antigens attenuates experimental murine colitis through modulation of immunity and microbiota composition.

Commensal bacteria have been shown to modulate the host mucosal immune system. Here, we report that oral treatment of BALB/c mice with components from the commensal, Parabacteroides distasonis, significantly reduces the severity of intestinal inflammation in murine models of acute and chronic colitis induced by dextran sulphate sodium (DSS). The membranous fraction of P. distasonis (mPd) prevented DSS-induced increases in several proinflammatory cytokines, increased mPd-specific serum antibodies and stabilized the intestinal microbial ecology. The anti-colitic effect of oral mPd was not observed in severe combined immunodeficient mice and probably involved induction of specific antibody responses and stabilization of the intestinal microbiota. Our results suggest that specific bacterial components derived from the commensal bacterium, P. distasonis, may be useful in the development of new therapeutic strategies for chronic inflammatory disorders such as inflammatory bowel disease.

Bifidobacterium actinocoloniiforme sp. nov. and Bifidobacterium bohemicum sp. nov., from the bumblebee digestive tract.

Our previous study, based primarily on PCR-denaturing gradient gel electrophoresis and 16S rRNA gene sequencing, focused on the isolation of four bifidobacterial groups from the digestive tract of three bumblebee species. In that study, we proposed that these isolated groups potentially represented novel species of the family Bifidobacteriaceae. One of the four, Bifidobacterium bombi, has been described recently. Strains representing two of the other groups have been classified as members of the genus Bifidobacterium on the basis of positive results for fructose-6-phosphate phosphoketolase activity and analysis of partial 16S rRNA and heat-shock protein 60 (hsp60) gene sequences. Analysis of 16S rRNA gene sequence similarities revealed that the isolates of the first group were affiliated to Bifidobacterium asteroides YIT 11866(T), B. indicum JCM 1302(T) and B. coryneforme ATCC 25911(T) (96.2, 96.0 and 95.9 % sequence similarity, respectively), together with other bifidobacteria showing lower sequence similarity. Additional representatives of the second group were found to be affiliated to Bifidobacterium minimum YIT 4097(T) and B. coryneforme ATCC 25911(T) (96.0 and 96.3 % sequence similarity) and also to other bifidobacteria with lower sequence similarity. These results indicate that the isolates of the two groups belong to novel species within the genus Bifidobacterium. This observation was further substantiated by the results of partial sequencing of hsp60. On the basis of phylogenetic and phenotypic analyses and analysis of 16S rRNA and partial hsp60 gene sequences, we propose two novel species, Bifidobacterium actinocoloniiforme sp. nov. (type strain LISLUCIII-P2(T)  = DSM 22766(T)  = CCM 7728(T)) and Bifidobacterium bohemicum sp. nov. (type strain JEMLUCVIII-4(T)  = DSM 22767(T)  = CCM 7729(T)).

Detection of possible AI-2-mediated quorum sensing system in commensal intestinal bacteria.

The Vibrio harveyi strain BB170-autoinducer bioassay was used to detect possible quorum sensing autoinducer-2 molecule (AI-2) in culture fluids of commensal intestinal bacteria. Culture fluids of Bacteroides vulgatus, Clostridium proteoclasticum, Escherichia coli, Eubacterium rectale, Lachnospira multipara, Pseudobutyrivibrio ruminis, Roseburia intestinalis, Ruminococcus albus and Ruminococcus flavefaciens contained AI-2-like molecules. The PCR bands from some of the tested strains could be also amplified using primers designed for the luxS gene. These findings suggest that AI-2 is present in the gastrointestinal tract; however, it has not yet been proved whether it is used for bacterial cell-to-cell communication.

Chitinolytic activity of the sheep rumen ciliate Diploplastron affine.

Supplementation of the rumen ciliate Diploplastron affine growth medium with commercial chitin stimulated growth of ciliates and the density of their population was positively correlated with chitin doses (r = 0.95; p < 0.01). The cell-free extracts prepared from bacteria-free ciliates degraded chitin to N-acetyl-D: -glucosamine and chitobiose. Three exochitinases, two endochitinases and two beta-N-acetylglucosaminidases were identified in the cell-free extract of protozoa. The molar mass of exochitinases was 80, 65 and 30 kDa, and endochitinases 75 and 50 kDa; the molar mass of one of the identified beta-N-acetylglucosaminidases was 45 kDa.

The intestinal microflora of childhood patients with indicated celiac disease.

The fecal short-chain fatty acids concentration was higher (154 +/- 46.9 mmol/L) in childhood patients than in healthy children (96.6 +/- 19.2 mmol/L). On the other hand, pH values were nonsignificantly lower in patients stool (6.78 +/- 0.75 vs. children 7.42 +/- 0.74). Using denaturing gradient gel electrophoresis specific for total bacteria, lactobacilli and bifidobacteria the microbial population was characterized in fecal samples and in duodenal biopsies. Bacteria adhering to duodenal biopsies were not dominating in stool samples. More than 50 % of detected bacterial species belonged to as yet uncultured strains.

Diversity of insect intestinal microflora.

The influence of geographic location, season, age, and part of the digestive tract on bacterial diversity was evaluated on intestinal microflora of honeybees, wasps, and cockroaches using DGGE analysis. PCR-DGGE analyses with universal bacterial primers targeting 200-bp region of the 16S rDNA gene afforded the profile of complex bacterial DNA; specific primers were used to determine the profile of bifidobacteria whose concentration in digestive tract was determined by real-time PCR. Selected PCR products were identified by sequencing. The microflora of the bees exhibited little variations among the hives from distant locations. Their bifidobacterial population formed 2.8-8.4 % of total bacteria and was very homogeneous. The total gut microflora of wasps was also homogeneous, only two samples being affected by the season or the location; on the other hand, wasp bifidobacterial population was very heterogeneous. Cockroaches showed the highest variations in microflora composition, the age and diet being the ultimate factors; bifidobacteria counts also varied among tested individuals (0.1-34.1 % of total bacteria). Our results suggest that nutrition habits are the strongest factor affecting the insect microflora, giving higher variations to omnivorous species.

Postnatal development of bacterial population in the gastrointestinal tract of calves.

Bacterial 16S rDNA from fecal samples of two calves were amplified by PCR and analyzed by denaturing gradient gel electrophoresis; selected bands were sequenced. Escherichia coli and Bifidobacterium animalis were the initial colonizers, followed by species closely related to the genera Bacteroides, Clostridium and Faecalibacterium. Change of diet was connected with shifts of bacterial population and with the occurrence of many bacterial species that have not been cultured up to now. The diet change corresponded with an alteration in a volatile-fatty-acid concentration in fecal samples.

Effect of fatty acids on growth of conjugated-linoleic-acids-producing bacteria in rumen.

Microorganisms with high activity of linoleic acid delta12-cis,delta11-trans-isomerase were isolated from the digestive tract of ruminants and characterized. The isolate with the highest isomerase activity was identified as Pseudobutyrivibrio ruminis. The susceptibility of this strain to 3 fatty acids added to the grow medium was determined. A significant inhibition of bacterial growth (during a 3-d period) by linoleic acid (0.1 %) and oleic acid (5 ppm) was observed; no inhibition was found in the presence of stearic acid.

Effect of gluten-free diet on microbes in the colon.

The effect of gluten-free diet (GFD) and chitosan was evaluated in healthy individuals; GFD remarkably influenced the structure of the gut bacterial population and its metabolism. Administration of GFD and chitosan (3 g daily) significantly changed composition and metabolism of the bacterial population. Chitosan stimulated the counts of fecal chitinolytic bacteria and decreased the body mass of treated persons.

Diet-dependent shifts in ruminal butyrate-producing bacteria.

The influence of a host's diet on Butyrivibrio and Pseudobutyrivibrio populations was investigated by competitive PCR. Specific primers were designed and competitive PCRs developed for both groups. Results (from 4 cows with different diets) suggested that high-fiber intake essentially increases the Butyrivibrio amounts in the rumen, whereas high-energy food additives lead to its suppression. The Pseudobutyrivibrio concentration also changed during the experiment but without any significant relation to the host's diet.

Detection of infant faecal bifidobacteria by enzymatic methods.

An enzyme-based assay was developed for the detection of bifidobacteria in infant faeces. Ninety-five samples from 51 breast-fed infants in the age between 3 and 276 days were investigated. Bifidobacteria and other bacterial groups were determined by cultivation and fluorescence in situ hybridisation (FISH). Faecal samples were examined for the activity of fructoso-6-phosphate phosphoketolase (F6PPK) and for other enzymatic reactions using the API-ZYM kit. Twenty-nine infants had high numbers of bifidobacteria (usually higher than 9 log CFU/g) in their faeces. Seventeen infants (35%) did not contain detectable amounts of bifidobacteria in their faecal samples. The remaining five individuals had low counts of bifidobacteria (3-6 log CFU/g). Most negative infants possessed major amounts of clostridia in their faecal flora. There were no significant differences among bifidobacterial counts obtained by cultivation and FISH, detection of F6PPK, alpha-galactosidase and alpha-glucosidase activities could routinely be used for the rapid and simple detection of bifidobacteria in infant faecal samples. Bifidobacterial colonies were identified using enzymatic tests and PCR procedure based on 16S rRNA gene sequences species-specific primers. In 14 samples, the identifications of individual isolates were compared with direct analyses of faeces using the nested PCR-denaturing gradient gel electrophoresis (nested DGGE) procedure. The results obtained in several cases are not identical. Bifidobacterium longum and Bifidobacterium breve were most frequently identified. Bifidobacteria-positive samples had high activities of alpha-galactosidase and alpha-glucosidase. On the contrary, negative samples missed either one or both of these enzymatic activities. While all positive samples tested showed distinctive fructose-6-phosphate phosphoketolase activity (F6PPK), none of the negative samples expressed F6PPK activity.

Cellulolytic enzymes of rumen anaerobic fungi Orpinomyces joyonii and Caecomyces communis.

The rumen anaerobic fungi Orpinomyces joyonii A4 and Caecomyces communis JB1 were grown on microcrystalline cellulose (MC) and alfalfa hay. The cellular distribution of cellulases produced by these organisms was monitored. Fungal cultures were separated into extracellular, intracellular and cell wall fractions and assayed for endoglucanase (EG) and beta-glucosidase activity. In both fungal isolates, EG activity was the highest in the extracellular fraction regardless of the substrate used. The beta-glucosidase activity produced by O. joyonii was mainly found in the cell wall fraction. On the contrary, the same enzyme activity in C. communis predominated in the extracellular fraction. The polycentric isolate A4 more efficiently utilized both substrates, produced more short chain fatty acids (up to 31 mmol/l) and showed higher total levels of EG (2744 nmol glucose/h/ml) than the monocentric strain JB1. On the other hand, beta-glucosidase (9033 nmol glucose/h/ml) activity was the highest in cultures of C. communis grown on cellulose. In cultures of O. joyonii grown on MC, the production of yellow affinity substance (YAS) with similar properties compared with yellow substance from Clostridium thermocellum was observed. This compound increased the adsorption of fungal cellulases to MC the temperature and pH range tested.

Adenocarcinoma cervicis uteri. An appraisal of treatment results for a period of 40 years.

A retrospective study was done in 316 patients with primary cervical adenocarcinoma treated at the Research Institute for Clinical and Experimental Oncology in Brno over a period of 40 years (1939-1978). The treatment results were compared to those in 2571 patients with epidermoid carcinomas of the uterine cervix treated at the Institute over the same period. The 5-year survival rate was significantly lower in adenocarcinoma patients (in Stage I patients, 77.8% vs. 84.5%, and, in the whole group, 60.8% vs. 70.3%). The assessment of the treatment results of this study has clearly showed that in cervical adenocarcinomas, surgery combined with radiotherapy was much more effective than radical radiotherapy alone (77.4% vs. 64.7%). In contrast to this, in epidermoid carcinomas the treatment results were better after radical radiotherapy (86.5% vs. 81.3%). Thus, in the prognosis of cervical adenocarcinomas the mass of the tumor, the size of the uterus, as well as the grading play a role. That means that primary cervical adenocarcinomas at early stages can be successfully treated by a combination of radical surgery and radiotherapy, while radiotherapy of advanced stages of this tumor is less successful.

Multiple tumors in gynecology.

Two large groups of patients, i.e. 1893 patients treated at the Institute for carcinoma cervicis uteri and 1184 patients with Ca corporis uteri were selected and evaluated for a follow-up study and statistical processing of tumor duplicity of gynecological origin. Double tumor in the group with Ca cervicis uteri was found in 88 women, i.e. 4.6%, and in that treated for Ca corporis uteri in 85, i.e. 7.2% of the patients. The most frequent combination in the two groups of carcinoma is breast cancer amounting to 28.4% in Ca cervicis uteri and to 35.3% in Ca corporis uteri. The second most frequent primary carcinoma in Ca cervicis uteri is dermal carcinoma--23.9%, and in Ca corporis uteri that of the digestive tract--21.1%. A statistical follow-up over 40 years indicates a rise in tumor duplicity in both groups of gynecological carcinoma--cervical and endometrial, with the number in the former having increased more than twofold.

Stage IIb, IIIb, and IVa carcinoma of the uterine cervix--results of treatment by radiotherapy in 649 patients.

During the period of January 1969 to December 1980, 649 patients have been treated by radical radiotherapy for Stage IIb, IIIb, and IVa carcinoma of the cervix uteri. This retrospective study was performed to assess therapeutical results in two groups of patients. Clinical staging and the methods of treatment were standard in both groups. Group I was treated by external irradiation of the pelvis minor with 60Co in combination with intracavitary radium administration. Group II patients were irradiated with a 42 MeV betatron according to the findings of lymphography, again in combination with radium brachytherapy. In Group I the 5-year survival rate was 59.2%, that in Group II was 66.7%. There was a statistically significant difference in the 5-year survival rate in Stage IIb patients of Group II (85.5%) against that in Group I (75.6%). The incidence of serious complications elicited by radiotherapy increased from 4.8% in Group I patients to 7.5% in Group II. Clinical stage, age at the time of diagnosis, findings of lymphography and tolerance to irradiation are prognostically important factors that influence the cure of the patients. On the basis of these findings, the possibilities of further therapeutic improvements are discussed.

Chitinolytic enzymes produced by ovine rumen bacteria.

Two strains of clostridia, isolated from the rumen fluid of sheep as potential antagonists toward anaerobic fungi showed a complete array of chitinolytic enzymes. Enzyme tests in cultures demonstrated endochitinase, exochitinase, N-acetyl-glucosaminidase, chitosanase and chitin deacetylase activities mainly in the extracellular fractions. In all samples, the highest was the activity of exochitinase (600-1100 nmol mL-1 h-1); the activity of endochitinase (280-500 nmol mL-1 h-1) was also significant. Chitinases were stimulated in the presence of reducing compounds and no dependence on cations was observed. In both strains different isoforms of chitinases of molar mass 36-96 kDa were detected. The chitinases from our isolates lyzed cell walls of anaerobic fungi in vitro and inhibited the activity of fungal beta-1,4-endoglucanases. Of the two bacteria examined, one was more effective in both antifungal effects.