Characteristics of a new cellulolytic Clostridium sp. isolated from pig intestinal tract.

Gram-positive, spore-forming, motile, cellulolytic rods were isolated from 10(7) dilutions of pig fecal samples. The pigs had previously been fed pure cultures of the ruminal cellulolytic organism Clostridium longisporum. Isolates formed terminal to subterminal spores, and a fermentable carbohydrate was required for growth. Besides cellulose, the isolates utilized cellobiose, glycogen, maltose, and starch. However, glucose, fructose, sucrose, pectin, and xylose were not used as energy sources. Major fermentation products included formate and butyrate. The isolates did not digest proteins from gelatin or milk. Unlike C. longisporum, which has limited ability to degrade cell wall components from grasses (switchgrass, bromegrass, and ryegrass), the swine isolates were equally effective in degrading these components from both alfalfa and grasses. The extent of degradation was equal to or better than that observed with the predominant ruminal cellulolytic organisms. On the basis of morphology, motility, spore formation, fermentation products, and the ability to hydrolyze cellulose, the isolates are considered to be a new species of the genus Clostridium. It is unclear whether C. longisporum played a role in the establishment or occurrence of this newly described cellulolytic species. This is the first report of a cellulolytic Clostridium sp. isolated from the pig intestinal tract.     

Addition of cellulolytic clostridia to the bovine rumen and pig intestinal tract.

Studies were conducted to determine whether intestinal cellulolytic bacteria could be introduced into the bovine rumen or pig large intestine. In the first study, the ruminal fluid of three cows was evacuated and replaced with 20 liters of buffer and 6 liters of the ruminal or swine cellulolytic organism Clostridium longisporum or Clostridium herbivorans, respectively. The introduced organisms were the predominant cellulolytic bacterium in the fluid (> 10(7) cells ml-1) at 0 h. C. longisporum was still the predominant cellulolytic organism after 5 h, at 0.55 x 10(7) cells ml-1; however, after 24 h the count of C. longisporum decreased to 0.05 x 10(7) cells ml-1 compared with 2.8 x 10(7) cells ml-1 for the total cellulolytic organisms. After 48 h, C. longisporum was no longer detectable. C. herbivorans was identified in only one of the three cows after 24 h and was not detected at 72 h. In a second study, when C. longisporum (50 ml; 10(7) cells ml-1) was infused into the terminal ileum of seven pigs, it was not recovered when fecal samples were evaluated at 24, 48, or 72 h after infusion. These studies emphasize the competition that must be overcome to successfully introduce organisms into an intestinal ecosystem. Furthermore, these studies suggest that C. longisporum is a transient organism in the bovine rumen; however, C. herbivorans is part of the normal intestinal flora of some pigs, although the role that it plays in fiber degradation in these pigs is unclear.     

Two cellulolytic Clostridium species: Clostridium cellulosi sp. nov. and Clostridium cellulofermentans sp. nov.

Two cellulolytic clostridia, one thermophilic and the other mesophilic, were isolated and characterized. Cells of the thermophile are gram-negative rods that are motile with lophotrichous flagella and spherical terminal endospores which swell the cells. The optimum growth temperature is 55 to 60 degrees C, with a range of 40 to 65 degrees C. The deoxyribonucleic acid composition is 35 mol% G + C. The name Clostridium cellulosi sp. nov. is proposed. The type strain is AS 1.1777. Cells of the mesophile are gram negative and motile with peritrichous flagella and terminal oval or spherical spores which swell the cells. The deoxyribonucleic acid composition is 34 mol% G + C. The name Clostridium cellulofermentans sp. nov. is proposed. The type strain is AS 1.1775. Both C. cellulosi AS 1.1777 and C. cellulofermentans AS 1.1775 are deposited in the China Committee for Culture Collection of Microorganisms, Institute of Microbiology, Academia Sinica, Beijing, People's Republic of China.     

Lactobacillus alvi sp. nov., isolated from the intestinal tract of chicken

Strain R54(T) was isolated from the gizzard of hens. The isolate was Gram-positive, facultative anaerobic, gas-forming, catalase-negative, nonmotile, nonspore-forming and short-rod-shaped. The optimal temperature for growth was 40 °C and the DNA G+C content was 42.7 mol%. The 16S rRNA gene sequences similarity showed that strain R54(T) was most closely related to Lactobacillus ingluviei LMG 20380(T) (97.5%), followed by Lactobacillus coleohominis CIP 106820(T) (96.1%), Lactobacillus secaliphilus DSM 17896(T) (95.6%) and Lactobacillus gastricus LMG 22113(T) (95.4%). The DNA-DNA relatedness between strain R54(T) and L. ingluviei LMG 20380(T) , was 43.3%. The predominant cellular fatty acids of strain R54(T) were C(18:1 ω9c) (64.9%) and C(16:0) (20.0%), and the major polar lipid group was phospholipids. On the basis of polyphasic taxonomy approach, strain R54(T) represents a novel species of the genus Lactobacillus, for which the name Lactobacillus alvi sp. nov. is proposed (type strain R54(T) = KCCM 90099(T) = JCM 17644(T)).     

Cellobiose-oxidizing enzyme from a newly isolated cellulolytic bacterium Cytophaga sp. LX-7

Summary The cellobiose oxidizing enzyme of the newly isolated cellulolytic bacterium Cytophaga sp. LX-7 was produced extracellularly when grown on cellulose or other saccharides, which was previously noted only in fungi. The enzyme could use not only cellobiose, maltose, glucose and other saccharides but also cellulose as substrates, and use dichlorophenol indophenol and oxygen as electron acceptors.     

Clostridium quinii sp. nov., a new saccharolytic anaerobic bacterium isolated from granular sludge

A new species of sporulating saccharolytic anaerobe, designated as Clostridium quinii sp. nov., is described. A gram-positive strain BS1, was isolated from the granular metanogenic sludge (UASB) from a waste-water treatment plant at a sugar refinery. The strain exhibits a series of morphological stages, developing from a spore to a small rod to a motile rod (peritrichous flagella) in the exponential growth phase, and then swelling to form cigar-shaped cells, exhibiting tumbling movements, in the late exponential growth phase before finally becoming large nonmotile ovoid cells in the stationary phase. Swelling occurs as a result of glucose being taken up and stored as a glycogen-like substance. The main fermentation products when growing on glucose is H₂, CO₂, formate, acetate and ethanol as well as small amounts of butyrate during exponential growth. Lactate is formed during the stationary phase, when glucose is abundant. Optimal conditions for growth is 40–45°C and pH of around 7.4. The type strain BS1 contains 28.0% mol G+C.     

Clostridium aldrichii sp. nov., a cellulolytic mesophile inhabiting a wood-fermenting anaerobic digester

An anaerobic, mesophilic, spore-forming, cellulolytic bacterium was repeatedly isolated from a wood-fermenting anaerobic digester. Cells of this organism were gram-positive rods, motile with a bundle of polar flagella, and formed subterminal oblong spores. The colonies in agar had an irregular shape with many platelike structures and were greyish white. Cellulose, xylan, and cellobiose served as substrates for growth. Acetate, propionate, butyrate, isobutyrate, isovalerate, lactate, succinate, H2, and CO2 were products of cellobiose fermentation. The optimal temperature and pH for growth were 35 degrees C and 7, respectively. The DNA composition was 40 mol% G + C. The name Clostridium aldrichii sp. nov. is proposed. The type strain is P-1 (= OGI 112, = ATCC 49358).     

Identification and properties of an alpha-amylase from a strain of Eubacterium sp. isolated from the rat intestinal tract.

1. A bacterial amylase was isolated from the intestinal content of monoxenic rats inoculated with Eubacterium sp. B86. 2. Affinity chromatography on cross-linked starch allowed its separation from rat endogenous amylases. 3. The bacterial enzyme was characterized by its pI, molecular weight and action pattern. It behaves as a typical endo-amylase (alpha-amylase).     

Antimicrobial resistance of Enterococcus sp. isolated from the intestinal tract of patients from a university hospital in Brazil

This study reports the results about antimicrobial resistance of Enterococcus spp. isolated from intestinal tract of patients from a university hospital in Brazil. The identification of strains at species level was performed by conventional biochemical tests, API 20 Strep (bioMérieux), and polymerase chain reaction assay. The species distribution was E. faecium (34%), followed by E. faecalis (33%), E. gallinarum (23.7%), E. casseliflavus (5.2%), E. avium (1%), and E. hirae (1%). Intrinsic resistance to vancomycin characterized by presence of vanC genes was found in E. gallinarum and E. casseliflavus. The high prevalence of VanC phenotype enterococci is very important because these species have been reported as causing a wide variety of infections. Vancomycin-resistant E. faecium or E. faecalis were not found and no one isolate of these species was a beta-lactamase producer. Thirteen clinical isolates of enterococci (13.4%) showed multiresistance patterns, which were defined by resistance to three classes of antibiotics plus resistance to at least one aminoglycoside (gentamicin and/or streptomycin). The resistance to several antimicrobials shown by enterococcal strains obtained in this study is of concern because of the decrease in the therapeutic options for treatment of infections caused by enterococci.     

Clostridium asparagiforme sp. nov., isolated from a human faecal sample

An obligatory anaerobic, Gram-positive, rod-shaped organism was isolated from faeces of a healthy human donor. It was characterized using biochemical, phenotypic and molecular taxonomic methods. The organism produced acetate, lactate, and ethanol as the major products of glucose fermentation. The G + C content was 53 mol%. Based on comparative 16S rRNA gene sequencing, the unidentified bacterium is a member of the Clostridium subphylum of the Gram-positive bacteria, and most closely related to species of the Clostridium coccoides cluster (rRNA cluster XIVa) [M.D. Collins et al., The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations, Int. J. Syst. Bacteriol. 44 (1994) 812-826]. Clostridium bolteae and Clostridium clostridioforme were identified as the most closely related described species. A 16S rRNA sequence divergence value of > 3% suggested that the isolate represents a new species. This was also supported by the gyrase-encoding gyrB gene sequences. Based on these findings, we propose the novel bacterium from human faeces to be classified as a new species, Clostridium asparagiforme. The type strain of C. asparagiforme is N6 (DSM 15981 and CCUG 48471).     

Clostridium grantii sp. nov., a new obligately anaerobic,alginolytic bacterium isolated from mullet gut

A gram-positive, motile, rod-shaped, strictly anaerobic, sporulating bacterium was isolated from an enrichment initiated with mullet gut contents. The organism grew optimally at 30°C and pH6.5, and at a salinity of 1–10³. Out of a variety of polysaccharides tested as growth substrates, only alginate supported growth in either semidefined or complex culture medium. The organism also grew on a variety of mono- and disaccharides. Moles product per 100mol of alginate monomer degraded were: acetate, 186; ethanol, 19; formate, 54; and CO₂, 0.19. Moles product per 100mol of hexose in cellobiose or glucose degraded were: acetate, 135; ethanol,61; formate, 63: and CO₂, 61. Hydrogen was not detectable during the incubations (detection limit, <10^-5atm) and propionate, butyrate, lactate, or succinate were not produced as fermentation end products (<2 mol per 100 mol of monomer). The G+C content of DNA from the bacterium was 30.2±0.3 mol%, and the cell walls contained the peptidoglycan component meso-diaminopimelic acid. A phylogenetic analysis of the 16S rDNA sequence indicated that the organism grouped closely with members of the RNA-DNA homology group 1 of the genus Clostridium. However, it differed from other species of the genus with regard to morphology, growth temperature optimum, substrate range, and fermentation pattern and is therefore designated as a new species of Clostridium; the type strain is A-1 (DSM 8605).     

Acinetobacter apis sp. nov., isolated from the intestinal tract of a honey bee,Apis mellifera

A novel Gram-negative, obligate aerobic, non-motile, and both coccobacillus- and bacillus-shaped bacterium, designated strain HYN18^T, was isolated from the intestinal tract of a honey bee (Apis mellifera). The isolate was oxidasenegative and catalase-positive. Strain HYN18^T showed optimum growth at 25°C, pH 6–7, and in the presence of 1% (w/v) NaCl in trypticase soy broth medium. The isolate was negative for hydrolyses of starch, casein, gelatin and urea, indole production from tryptone and hemolysis on sheep blood agar. A phylogenetic analysis based on the 16S rRNA gene and rpoB gene sequence showed that strain HYN18^T was most closely related to Acinetobacter nectaris SAP 763.2^T and A. boissieri SAP 284.1^T with 98.3% and 98.1% similarity (16S rRNA gene), respectively, and 84.4% similarity with Acinetobacter nectaris SAP 763.2^T (rpoB gene). The major cellular fatty acids were summed features 3 (comprising C_16:1ω7c /C_16:1ω6c ), C_12:0 and C_16:0. The main isoprenoid quinone was ubiquinone-9 (Q-9). The polar lipids of strain HYN18^T were phosphatidylethanolamine, three unidentified lipids, an unidentified phospholipid and an unidentified glycolipid. The DNA G+C content was 40.6 mol%. DNADNA hybridization experiments indicated less than 33 ± 10% relatedness to the closest phylogenetic species, Acinetobacter nectaris SAP 763.2^T. Thus, the phenotypic, phylogenetic and genotypic analyses indicate that strain HYN18^T is a novel species within the genus Acinetobacter, for which the name Acinetobacter apis is proposed. The type strain is HYN18^T (=KACC 16906^T =JCM 18575^T).     

Clostridium bolteae sp. nov., isolated from human sources

Seven obligately anaerobic, gram-positive, rod-shaped, spore-forming organisms isolated from human sources were characterized using phenotypic and molecular taxonomic methods. Comparative 16S rRNA gene sequencing showed that the strains were genetically highly related to each other (displaying >99% sequence similarity) and represent a previously unknown sub-line within the Clostridium coccoides rRNA group of organisms. Strains of the unidentified bacterium used carbohydrate as fermentable substrates, producing acetic acid and lactic acid as the major products of glucose metabolism. The closest described species to the novel bacterium corresponded to Clostridium clostridioforme, although a 16S rRNA sequence divergence of 3% demonstrated they represent different species. Genomic DNA-DNA pairing studies confirmed the separateness of the unknown species and Clostridium clostridioforme. Based on phenotypic and phylogenetic evidence, it is therefore proposed that the unknown bacterium, be classified as Clostridium bolteae sp. nov. The type strain of Clostridium bolteae is WAL 16351T (= ATCC(T) = BAA-613T, CCUG(T) = 46953T).     

Clostridium bartlettii sp. nov., isolated from human faeces

Seven obligately anaerobic, Gram-positive, rod-shaped, spore-forming organisms isolated from human faecal specimens were characterized using phenotypic and molecular taxonomic methods. Strains of the unidentified bacterium used carbohydrates as fermentable substrates, producing acetic acid, isovaleric acid and phenylacetic acid (PAA) as the major products of glucose metabolism, and possessed a G +C content of approximately 29.8 mol%. Comparative 16S rRNA gene sequencing showed that the 7 strains were genetically highly related to each other (displaying >99.5% sequence similarity) and represent a previously unknown sub-line within the Clostridium Cluster XI. The closest described species to the novel bacterium is Clostridium glycolicum, although a 16S rRNA sequence divergence of 4% demonstrates that they represent different species. Genomic DNA-DNA pairing studies confirmed the separateness of the unknown species and C. glycolicum (30.6% similarity between the proposed type strain of the novel species, WAL 16138, and C. glycolicum ATCC 14880(T)). Based on morphologic, phenotypic and phylogenetic evidence, it is therefore proposed that the unknown bacterium be classified as C. bartlettii sp. nov. The type strain of C. bartlettii is WAL 16138(T) (= ATCCBAA-827(T)=CCUG48940(T)).     

Paenibacillus pinihumi sp. nov., a cellulolytic bacterium isolated from the rhizosphere of Pinus densiflora

A novel cellulolytic bacterium, strain S23^T, was isolated from the rhizosphere of the pine trees in Daejeon, Republic of Korea. This isolate was Gram-positive, strictly aerobic, rod-shaped, catalase-negative, oxidase-positive, motile by means of peritrichous flagella, and tested positive for alkaline phosphatase, esterase lipase, leucine arylamidase, α-galactosidase, and β-galactosidase activities. The DNA G+C content was 49.5 mol%. The main cellular fatty acids were anteiso-C_15:0 (51.9%), iso-C_16:0 (14.7%), and iso-C_15:0 (13.2%). The major isoprenoid quinone was menaquinone 7 (MK-7). Diagnostic diamino acid in the cell-wall pepti-doglycan was meso-diaminopimelic acid. Comparative 16S rRNA gene sequence analysis showed that this strain clustered with Paenibacillus species. The 16S rRNA gene sequence similarity values between S23^T and other Paenibacillus species were between 89.9% and 95.9%, and S23^T was most closely related to Paenibacillus tarimensis SA-7-6^T. On the basis of phylogenetic and phenotypic properties of strain S23^T, the isolate is considered as a novel species belonging to the genus Paenibacillus. Therefore, the name, Paenibacillus pinihumi sp. nov., is proposed for the rhizosphere isolate; the type strain is S23^T (=KCTC 13695^T =KACC 14199^T =JCM 16419^T)     

Characterization of cellulolytic enzymes and bioH2 production from anaerobic thermophilic Clostridium sp. TCW1

A thermophilic anaerobic bacterium Clostridium sp. TCW1 was isolated from dairy cow dung and was used to produce hydrogen from cellulosic feedstock. Extracellular cellulolytic enzymes produced from TCW1 strain were identified as endoglucanases (45, 53 and 70 kDa), exoglucanase (70 kDa), xylanases (53 and 60 kDa), and β-glucosidase (45 kDa). The endoglucanase and xylanase were more abundant. The optimal conditions for H₂ production and enzyme production of the TCW1 strain were the same (60 °C, initial pH 7, agitation rate of 200 rpm). Ten cellulosic feedstock, including pure or natural cellulosic materials, were used as feedstock for hydrogen production by Clostridium strain TCW1 under optimal culture conditions. Using filter paper at 5.0 g/L resulted in the most effective hydrogen production performance, achieving a H₂ production rate and yield of 57.7 ml/h/L and 2.03 mol H₂/mol hexose, respectively. Production of cellulolytic enzyme activities was positively correlated with the efficiency of dark-H₂ fermentation.     

Clostridium glycyrrhizinilyticum sp. nov., a glycyrrhizin-hydrolysing bacterium isolated from human faeces

Screening of faecal bacteria for glycyrrhetic acid (GA) production by hydrolysing of glycyrrhizin (GL) resulted in the isolation of two strains, designated ZM35T and ZM38. Strains ZM35T and ZM38 were Gram-positive, obligate anaerobic, non-spore-forming and rod-shaped bacteria. Analysis of the 16S rRNA gene sequences indicated that strains ZM35T and ZM38 belonged to cluster XIVa of the genus Clostridium. The 16S rRNA gene sequences of strains ZM35T and ZM38 were identical. Strain ZM35T exhibited approximately 94% to 95% identity with the validly described species, Clostridium oroticum(94.5%), Eubacterium contortum(93.8%), Ruminococcus gnavus(94.5%) and R. torques(95.1%). In an experiment of DNA-DNA hybridization, it was confirmed that strains ZM35T and ZM38 were the same species. The guanine-plus-cytosine (G+C) content of strain ZM35T is 45.7 mol%. Based on the phylogenetic and phenotypic findings, we propose that strains ZM35T and ZM38 be assigned to a novel species named Clostridium glycyrrhizinilyticum. The type strain is ZM35T (=JCM 13368T=DSM 17593T).     

Bacillus xiamenensis sp. nov., isolated from intestinal tract contents of a flathead mullet (Mugil cephalus)

A taxonomic study was carried out on strain HYC-10^T, which was isolated from the intestinal tract contents of a flathead mullet, Mugil cephalus, captured from the sea off Xiamen Island, China. The bacterium was observed to be Gram positive, oxidase and catalase positive, rod shaped, and motile by subpolar flagella. The bacterium was found to grow at salinities of 0–12 % and at temperatures of 8–45 °C. The isolate was found to hydrolyze aesculin and gelatin, but was unable to reduce nitrate to nitrite. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain HYC-10^T belongs to the genus Bacillus, with highest sequence similarity (99.3 %) to Bacillus aerophilus 28K^T, Bacillus stratosphericus 41KF2a^T and Bacillus altitudinis DSM 21631^T, followed by Bacillus safensis DSM 19292^T (99.5 %) and Bacillus pumilus DSM 27^T (99.5 %), while the sequence similarities to others were all below 97.6 %. The genomic ANIm values between strain HYC-10^T and three type strains (B. altitudinis DSM 21631^T, B. safensis DSM 19292^T and B. pumilus DSM 27^T) were determined to range from 89.11 to 91.53 %. The DNA–DNA hybridization estimate values between strain HYC-10^T and the three type strains were from 36.60 to 44.00 %. The principal fatty acids identified were iso-C_15:0 (39.1 %), anteiso-C_15:0 (22.7 %), iso-C_17:0 (13.1 %), C_16:0 (6.1 %), anteiso-C_17:0 (5.8 %) and iso-C_16:0 (5.1 %). The G+C content of the chromosomal DNA was determined from the draft genome sequence to be 41.3 mol%. The respiratory quinone was determined to be MK-7 (100 %). Phosphatidylglycerol, diphosphatidylglycerol, aminoglycolipid, two glycolipids and two unknown phospholipids were found to be present. The combined genotypic and phenotypic data show that strain HYC-10^T represents a novel species of the genus Bacillus, for which the name Bacillus xiamenensis sp. nov. is proposed, with the type strain HYC-10^T (=CGMCC NO.1.12326^T = LMG 27143^T = MCCC 1A00008^T).