Young-Hyo Chang

Molecular analysis of the luminal- and mucosal-associated intestinal microbiota in diarrhea-predominant irritable bowel syndrome.

Alterations in the intestinal microbiota have been suggested as an etiological factor in the pathogenesis of irritable bowel syndrome (IBS). This study used a molecular fingerprinting technique to compare the composition and biodiversity of the microbiota within fecal and mucosal niches between patients with diarrhea-predominant IBS (D-IBS) and healthy controls. Terminal-restriction fragment (T-RF) length polymorphism (T-RFLP) fingerprinting of the bacterial 16S rRNA gene was used to perform microbial community composition analyses on fecal and mucosal samples from patients with D-IBS (n = 16) and healthy controls (n = 21). Molecular fingerprinting of the microbiota from fecal and colonic mucosal samples revealed differences in the contribution of T-RFs to the microbiota between D-IBS patients and healthy controls. Further analysis revealed a significantly lower (1.2-fold) biodiversity of microbes within fecal samples from D-IBS patients than healthy controls (P = 0.008). No difference in biodiversity in mucosal samples was detected between D-IBS patients and healthy controls. Multivariate analysis of T-RFLP profiles demonstrated distinct microbial communities between luminal and mucosal niches in all samples. Our findings of compositional differences in the luminal- and mucosal-associated microbiota between D-IBS patients and healthy controls and diminished microbial biodiversity in D-IBS fecal samples further support the hypothesis that alterations in the intestinal microbiota may have an etiological role in the pathogenesis of D-IBS and suggest that luminal and mucosal niches need to be investigated.

Selection of a potential probiotic Lactobacillus strain and subsequent in vivo studies

The probiotic potential of a Lactobacillus strain, isolated from pig faeces, was assessed as a probiotic in piglets. The strain was examined for resistance to pH 2.0, 0.5% oxgall and antibiotics, and antimicrobial activities against enteric pathogenic bacteria. The probiotic strain, L. reuteri BSA131, was administered through the feed to 25 1-month-old Landrace piglets. The piglets were divided into five groups of five piglets each and fed with different diets for 28 days. The daily consumption of L. reuteriBSA131 was assigned into two groups by the concentration of 106 or 108 freeze-dried bacteria. Fecal samples were collected before, during, and after consumption. Lactobacilli and enterobacteria cell counts were determined in the fecal samples. The liveweight gains and feed consumption of the piglets were recorded daily. This study showed that strain BSA 131 enhanced liveweight gains and feed conversion rates in piglets. It also showed a significant increase in lactobacilli cell counts and decreases in enterobacterial numbers in the fecal samples. Strain BSA 131 was considered to be a potential probiotic for piglets, especially after weaning.

Comamonas koreensis sp. nov., a non-motile species from wetland in Woopo, Korea

A bacterial strain, designated YH12T, was isolated from a wetland sample collected from Woopo, Republic of Korea, and characterized using a polyphasic approach. Analysis of 16S rDNA indicated that the isolate formed a monophyletic clade with the members of the genus Comamonas. The closest phylogenetic relative among the valid species was Comamonas testosteroni, with 96.6% 16S rDNA similarity. The chemotaxonomic properties of the wetland isolate supported its membership of the genus Comamonas, as it contained ubiquinone Q-8 as a major respiratory quinone and hexadecanoic, methylene-hexadecanoic and octadecenoic acids as major cellular fatty acids. The G+C content of the DNA was 66 mol%. The isolate is a gram-negative, non-pigmented, rod-shaped, oxidase- and catalase-positive, non-motile, non-endospore-forming and non-fermentative bacterium. The phenotypic properties of the isolate were compared with those of the type strains of Comamonas terrigena, C. testosteroni and Delftia acidovorans. A number of tests, including motility, can differentiate our isolate from related taxa. On the basis of the 16S rDNA phylogenetic, chemotaxonomic and phenotypic evidence given in this study, it is proposed that strain YH12T (= KCTC 12005T = IMSNU 11158T) be assigned as the type strain of a novel species of the genus Comamonas, Comamonas koreensis sp. nov.

Sporolactobacillus vineae sp. nov., a spore-forming lactic acid bacterium isolated from vineyard soil

Two spore-forming, facultatively anaerobic, lactic acid bacteria, strains SL153(T) and SL1153, were isolated from vineyard soil in Korea. Cells of both strains were slightly curved, Gram-positive, motile rods that measured between 1 and 4 mum in length and were approximately 0.5 mum in diameter. Strains SL153(T) and SL1153 fermented glucose, fructose, mannose and sorbitol, but were negative for nitrate reduction, catalase and oxidase. The predominant cellular fatty acids of the two isolates were iso-C(15 : 0), anteiso-C(15 : 0) and anteiso-C(17 : 0). meso-Diaminopimelic acid, glucose, mannose and galactose were determined in their whole-cell hydrolysates. 16S rRNA gene sequences from the two strains were almost identical (99.9 %) and they could be placed in the genus Sporolactobacillus according to phylogenetic analysis. The species most closely related to SL153(T) were Sporolactobacillus inulinus and Sporolactobacillus terrae with 16S rRNA gene similarities of 95.7 and 95.5 %, respectively, with the type strains. Levels of DNA-DNA relatedness between strain SL153(T) and the type strains of S. inulinus, S. terrae and Sporolactobacillus kofuensis were 18.5, 18.0 and 17.0 %, respectively. On the basis of the phylogenetic (16S rRNA gene), chemotaxonomic and phenotypic evidence given in this study, it is proposed that strains SL153(T) and SL1153 should be assigned to the genus Sporolactobacillus as representatives of the novel species Sporolactobacillus vineae sp. nov. The type strain is SL153(T) (=KCTC 5376(T)=JCM 14637(T)).

Description of Lysinibacillus sinduriensis sp. nov., and transfer of Bacillus massiliensis and Bacillus odysseyi to the genus Lysinibacillus as Lysinibacillus massiliensis comb. nov. and Lysinibacillus odysseyi comb. nov. with emended description of the genus Lysinibacillus.

A Gram-positive, rod-shaped, endospore-forming bacterium, designated strain BLB-1(T), was isolated from samples of tidal flat sediment from the Yellow Sea. 16S rRNA gene sequence analysis demonstrated that the isolate belonged to the Bacillus rRNA group 2 and was closely related to Bacillus massiliensis CIP 108446(T) (97.4%), Bacillus odysseyi ATCC PTA-4993(T) (96.7%), Lysinibacillus fusiformis DSM 2898(T) (96.2%) and Lysinibacillus boronitolerans DSM 17140(T) (95.9%). Sequence similarities with related species in other genera, including Caryophanon, Sporosarcina and Solibacillus, were <96.1%. Chemotaxonomic data supported the affiliation of strain BLB-1(T) with the genus Lysinibacillus. The major menaquinone was MK-7, the cell-wall sugars were glucose and xylose, the cell-wall peptidoglycan type was A4α (L-Lys-D-Asp), the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and several unknown phospholipids, and the major fatty acids were anteiso-C(15:0) (35.6%), iso-C(15:0) (25.6%) and anteiso-C(17:0) (16.5%). The most closely related species, Bacillus massiliensis and Bacillus odysseyi, were also assigned to this genus based on phylogenetic analysis and phenotypic data. The results of DNA-DNA hybridizations and phenotypic tests supported the differentiation of all three taxa from species of the genus Lysinibacillus with validly published names. Thus, strain BLB-1(T) ( = KCTC 13296(T)  = JCM 15800(T)) represents a novel species, for which the name Lysinibacillus sinduriensis sp. nov. is proposed. It is also proposed that Bacillus massiliensis CIP 108446(T) ( =4400831(T) = CCUG49529(T)  =KCTC 13178(T)) and Bacillus odysseyi NBRC 100172(T) ( =34hs-1(T)  =ATCC PTA-4993(T)  =NRRL B-30641(T)  =DSM 18869(T)  =CIP 108263(T)  =KCTC 3961(T)) be transferred to the genus Lysinibacillus as Lysinibacillus massiliensis comb. nov. and Lysinibacillus odysseyi comb. nov., respectively.

Comparison of rpoB gene sequencing, 16S rRNA gene sequencing, gyrB multiplex PCR, and the VITEK2 system for identification of Acinetobacter clinical isolates

Since accurate identification of species is necessary for proper treatment of Acinetobacter infections, we compared the performances of 4 bacterial identification methods using 167 Acinetobacter clinical isolates to identify the best identification method. To secure more non-baumannii Acinetobacter (NBA) strains as target strains, we first identified Acinetobacter baumannii in a total of 495 Acinetobacter clinical isolates identified using the VITEK 2 system. Because 371 of 495 strains were identified as A. baumannii using gyrB multiplex 1 PCR and blaOXA51-like PCR, we performed rpoB gene sequencing and 16S rRNA gene sequencing on remaining 124 strains belonging to NBA and 52 strains of A. baumannii. For identification of Acinetobacter at the species level, the accuracy rates of rpoB gene sequencing, 16S rRNA gene sequencing, gyrB multiplex PCR, and the VITEK 2 were 98.2%, 93.4%, 77.2%, and 35.9%, respectively. The gyrB multiplex PCR seems to be very useful for the detection of ACB complex because its concordance rates to the final identification of strains of ACB complex were 100%. Both the rpoB gene sequencing and the 16S rRNA gene sequencing may be useful in identifying Acinetobacter.

Paenibacillus tyraminigenes sp. nov. isolated from Myeolchi-jeotgal, a traditional Korean salted and fermented anchovy

A bacterial strain H3029, a gram-positive, rod-shaped, oxidase-negative, endospore-forming bacterium that characteristically produces tyramine from tyrosine, was isolated from a Myeolchi-jeotgal, a traditional Korean salted and fermented anchovy (Engraulis japonicus). The H3029 strain showed a high ability to produce 4140 microg/ml of tyramine from the culture broth containing 5000 microg/ml tyrosine. On the other hand, the strain produced a relatively low level of other putrefactive amines, at 973 microg/ml of putrescine and 147 microg/ml of cadaverine from the media, with each 5000 microg/ml of ornithine hydrochloride and lysine hydrochloride. The H3029 strain produced no detectable level of histamine (detection limit of 4 microg/ml) from the media containing 5000 microg/ml of histidine hydrochloride. Meanwhile, tyramine, the main product of the strain, showed the antimicrobial activity at the level of over 1 mg/disk against Staphylococcus aureus by agar diffusion test, and the mutagenicity in Ames test at 0.1 mg/plate using Salmonella typhimurium TA98 and TA1535. On the basis of the polyphasic taxonomic study, the H3029(T) strain was assigned a novel species of the genus Paenibacillus as Paenibacillus tyraminigenes sp. nov. The type strain of which is strain H3029(T) (=KCTC 10694BP(T)).

Bacillus manliponensis sp. nov., a new member of the Bacillus cereus group isolated from foreshore tidal flat sediment

A Gram-positive, endospore-forming, new Bacillus species, strain BL4-6T, was isolated from tidal flat sediment of the Yellow Sea. Strain BL4-6T is a straight rod, with motility by peritrichate flagella. The cell wall contains meso-diaminopimelic acid, and the major respiratory quinone is menaquinone-7. The major fatty acids are iso-C15:0 and summed feature 3 (containing C16:1 ω7c/iso-C15:0 2OH, and/or iso-C15:0 2OH/C16:1 ω7c). Cells are catalase-positive and oxidase-negative. The G+C content of the genomic DNA is 38.0 mol%. Based on a comparative 16S rRNA gene sequence analysis, the isolate belongs to the genus Bacillus, forms a clade with the Bacillus cereus group, and is closely related to Bacillus mycoides (98.5%), Bacillus cereus (98.5%), Bacillus anthracis (98.4%), Bacillus thuringiensis (98.4%), Bacillus weihenstephanensis (98.1%), and Bacillus pseudomycoides (97.5%). The isolate showed less than 85% similarity of the gyrA gene sequence and below 95% similarity of the rpoB gene sequence to the members of this group. DNA-DNA relatedness between strain BL4-6T and B. cereus group was found to be in a range of 22.8–42.3%, and thus BL4-6T represents a unique species. On the basis of these studies, strain BL4-6T (=KCTC 13319T =JCM 15802T) is proposed to represent the type strain of a novel species, Bacillus manliponensis sp. nov.

Bacillus gaemokensis sp. nov., isolated from foreshore tidal flat sediment from the Yellow Sea.

A Gram-positive, rod-shaped, endospore-forming organism, strain BL3-6T, was isolated from tidal flat sediments of the Yellow Sea in the region of Tae-An. A 16S rRNA gene sequence analysis demonstrated that this isolate belongs to the Bacillus cereus group, and is closely related to Bacillus mycoides (99.0% similarity), Bacillus thuringiensis (99.0%), Bacillus weihenstephanensis (99.0%), Bacillus cereus (98.9%), Bacillus anthracis (98.8%), and Bacillus pseudomycoides (98.1%). The phylogenetic distance from any validly described Bacillus species outside the Bacillus cereus group was less than 95.6%. The DNA G+C content of the strain was 39.4 mol% and the major respiratory quinone was menaquinone-7. The major cellular fatty acids were iso-C14:0 (17.8%), iso-C16:0 (15.8%), and iso-C12:0 (11.3%). The diagnostic amino acid of the cell wall was meso-diaminopimelic acid and the major cell wall sugar was galactose. The results of DNA-DNA hybridization (<55.6%) and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain BL3-6T from the published Bacillus species. BL3-6T therefore represents a new species, for which the name Bacillus gaemokensis sp. nov. is proposed, with the type strain BL3-6T (=KCTC 13318T =JCM 15801T).

A real‐time PCR assay for detection and quantification of Lactococcus garvieae

Aims:  To develop a rapid, sensitive, specific tool for the detection and quantification of Lactococcus garvieae in food and environmental samples.