Identification of lactic acid bacteria from fermented tea leaves (miang) in Thailand and proposals of Lactobacillus thailandensis sp. nov., Lactobacillus camelliae sp. nov., and Pediococcus siamensis sp. nov

Eighteen rod-shaped homofermentatives, six heterofermentatives, and a coccal homofermentative lactic acid bacteria were isolated from fermented tea leaves (miang) produced in the northern part of Thailand. The isolates were placed in a monophyletic cluster consisting of Lactobacillus and Pediococcus species. They were divided into seven groups by phenotypic and chemotaxonomic characteristics, DNA-DNA similarity, and 16S rRNA gene sequences. Groups I to VI belonged to Lactobacillus and Group VII to Pediococcus. All of the strains tested produced DL-lactic acid but those in Group IV produced L-lactic acid. The strains tested in Groups I, II and V had meso-diaminopimelic acid in the cell wall. Six strains in Group I were identified as Lactobacillus pantheris; five strains in Group II as Lactobacillus pentosus; and four strains in Group V as Lactobacillus suebicus. Two strains in Group VI showed high DNA-DNA similarity for each other and MCH4-2 was closest to Lactobacillus fermentum CECT 562(T) with 99.5% of 16S rRNA gene sequence similarity. Five strains in Group III are proposed as Lactobacillus thailandensis sp. nov., and MCH5-2(T) (BCC 21235(T)=JCM 13996(T)=NRIC 0671(T)=PCU 272(T)) is the type strain which has 49 mol% G+C of DNA. Two strains in Group IV are proposed as Lactobacillus camelliae sp. nov., and the type strain is MCH3-1(T) (BCC 21233(T)=JCM 13995(T)=NRIC 0672(T)=PCU 273(T)) which has 51.9 mol% G+C of DNA. One strain in Group VII is proposed as Pediococcus siamensis sp. nov., and MCH3-2(T) (BCC 21234(T)=JCM 13997(T)=NRIC 0675(T)=PCU 274(T)) is the type strain which has 42 mol% G+C of DNA.     

Lactobacillus oligofermentans sp. nov., associated with spoilage of modified-atmosphere-packaged poultry products

Unidentified lactic acid bacterium (LAB) isolates which had mainly been detected in spoiled, marinated, modified atmosphere packaged (MAP) broiler meat products during two previous studies, were identified and analyzed for their phenotypic properties and the capability to produce biogenic amines. To establish the taxonomic position of these isolates, 16S rRNA gene sequence analysis, numerical analysis of ribopatterns, and DNA-DNA hybridization experiments were done. Unexpectedly for a meat-spoilage-associated LAB, the strains utilized glucose very weakly. According to the API 50 CHL test, arabinose and xylose were the only carbohydrates strongly fermented. None of the six strains tested for production of histamine, tyramine, tryptamine, phenylethylamine, putrescine, and cadaverine were able to produce these main meat-associated biogenic amines in vitro. The polyphasic taxonomy approach showed that these strains represent a new Lactobacillus species. The six isolates sequenced for the 16S rRNA encoding genes shared the highest similarity (95.0 to 96.3%) with the sequence of the Lactobacillus durianis type strain. In the phylogenetic tree, these isolates formed a distinct cluster within the Lactobacillus reuteri group, which also includes L. durianis. Numerical analyses of HindIII-EcoRI ribotypes placed all isolates together in a cluster with seven subclusters well separated from the L. reuteri group reference strains. The DNA-DNA hybridization levels between Lactobacillus sp. nov. isolates varied from 67 to 96%, and low hybridization levels (3 to 15%) were obtained with the L. durianis type strain confirming that these isolates belong to the same species different from L. durianis. The name Lactobacillus oligofermentans sp. nov. is proposed, with strain LMG 22743T (also known as DSM 15707T or AMKR18T) as the type strain.     

Eight new species of the genus Micromonospora, Micromonospora citrea sp. nov., Micromonospora echinaurantiaca sp. nov., Micromonospora echinofusca sp. nov. Micromonospora fulviviridis sp. nov., Micromonospora inyonensis sp. nov., Micromonospora peucetia sp. nov., Micromonospora sagamiensis sp. nov., and Micromonospora viridifaciens sp. nov

A previous phylogenetic study on type strains of the genus Micromonospora and Micromonospora species bearing non-validly published names has pointed towards the species status of several of latter strains. Subsequent studies on morphological, cultural, chemotaxonomic, metabolic, and genomic properties, and on whole cell mass spectrometric analyses by matrix adsorbed laser desorption/ionization time-of-flight (MALDI-TOF) confirmed the species status, leading to the proposal of eight new Micromonospora species: Micromonospora citrea sp. nov., type strain DSM 43903T, Micromonospora echinaurantiaca sp. nov., type strain DSM 43904T, Micromonospora echinofusca sp. nov., type strain DSM 43913T, Micromonospora fulviviridis sp. nov., type strain DSM 43906T, Micromonospora inyonensis sp. nov., type strain DSM 46123T, Micromonospora peucetia sp. nov., type strain DSM 43363T, Micromonospora sagamiensis sp. nov., type strain DSM 43912T and Micromonospora viridifaciens sp. nov., type strain DSM 43909T.     

Taxonomic study of the Lactobacillus acidophilus group, with recognition of Lactobacillus gallinarum sp. nov. and Lactobacillus johnsonii sp. nov. and synonymy of Lactobacillus acidophilus group A3 (Johnson et al. 1980) with the type strain of Lactobacillus amylovorus (Nakamura 1981).

Biochemical properties and DNA-DNA reassociation studies of Lactobacillus acidophilus strains isolated from humans and animals indicate that these include six genomospecies. Two new species can be differentiated from the established species of the genus Lactobacillus: L. gallinarum sp. nov. (type strain, ATCC 33199) and L. johnsonii sp. nov. (type strain, ATCC 33200). Furthermore, it was clarified that L. acidophilus group A3 (Johnson et al. 1980) is synonymous with L. amylovorus.     

Candida alocasiicola sp. nov., Candida hainanensis sp. nov., Candida heveicola sp. nov. and Candida musiphila sp. nov., novel anamorphic, ascomycetous yeast species isolated from plants

In a taxonomic study on the ascomycetous yeasts isolated from plant materials collected in tropical forests in Yunnan and Hainan Provinces, southern China, four strains isolated from tree sap (YJ2E(T)) and flowers (YF9E(T), YWZH3C(T) and YYF2A(T)) were revealed to represent four undescribed yeast species. Molecular phylogenetic analysis based on the large subunit (26S) rRNA gene D1/D2 domain sequences showed that strain YJ2E(T) was located in a clade together with Candida haemulonii and C. pseudohaemulonii. Strain YF9E(T) was most closely related to C. azyma and strain YWZH3C(T) to C. sorbophila and C. spandovensis. Strain YYF2A(T) was clustered in a clade containing small-spored Metschnikowia species and related anamorphic Candida species. The new strains differed from their closely related described species by more than 10% mismatches in the D1/D2 domain. No sexual states were observed for the four strains on various sporulation media. The new species are therefore assigned to the genus Candida and described as Candida alocasiicola sp. nov. (type strain, YF9E(T) = AS 2.3484(T) = CBS 10702(T)), Candida hainanensis sp. nov. (type strain, YYF2A(T) = AS 2.3478(T) = CBS 10696(T)), Candida heveicola sp. nov. (type strain, YJ2E(T) = AS 2.3483(T) = CBS 10701(T)) and Candida musiphila sp. nov. (type strain, YWZH3C(T) = AS 2.3479(T) = CBS 10697(T)).     

Lactobacillus tucceti sp. nov., a new lactic acid bacterium isolated from sausage

Following the application of several molecular techniques strain R 19c, isolated from sausage by Reuter in 1970 and deposited at the DSMZ as Lactobacillus sp., has been identified as pertaining to a new species. It showed singular ISR-DdeI and ISR-HaeIII profiles that allowed its differentiation from 68 lactic acid bacteria reference strains analyzed. Phylogenetic analysis based on 16S rRNA gene sequences places this strain in the genus Lactobacillus within the Lactobacillus alimentarius group. Species L. versmoldensis is the closest phylogenetic neighbor with 96.3% sequence similarity. DNA-DNA hybridization experiments confirmed the independent status at species level of this strain. Species-specific primers for PCR detection of this new species have been developed. Phenotypically it can be distinguished from the closest relative L. versmoldensis by several traits such as the peptidoglycan type (L-Lys-Gly-D-Asp), acid production from L-rhamnose, D-mannitol and L-fucose and its inability to ferment d-galactose, d-melibiose and d-sucrose. The name Lactobacillus tucceti sp. nov. is proposed with strain R 19c(T) (=DSM 20183(T)= CECT 5920(T)) as the type strain.     

Mobilicoccus pelagius gen. nov., sp. nov. and Piscicoccus intestinalis gen. nov., sp. nov., two new members of the family Dermatophilaceae, and reclassification of Dermatophilus chelonae (Masters et al. 1995) as Austwickia chelonae gen. nov., comb. nov

Two Gram-positive bacteria, designated strains Aji5-31(T) and Ngc37-23(T), were isolated from the intestinal tracts of fishes. 16S rRNA gene sequence analysis indicated that both strains were related to the members of the family Dermatophilaceae, with 95.6-96.9% 16S rRNA gene sequence similarities. The family Dermatophilaceae contains 2 genera and 3 species: Dermatophilus congolensis, Dermatophilus chelonae and Kineosphaera limosa. However, it has been suggested that the taxonomic position of D. chelonae should be reinvestigated using a polyphasic approach, because the chemotaxonomic characteristics are not known (Stackebrandt, 2006; Stackebrandt and Schumann, 2000). Our present study revealed that strains Aji5-31(T), Ngc37-23(T) and D. chelonae NBRC 105200(T) should be separated from the other members of the family Dermatophilaceae on the basis of the following characteristics: the predominant menaquinone of strain Aji5-31(T) is MK-8(H(2)), strain Ngc37-23(T) possesses iso- branched fatty acids as major components, and the menaquinone composition of D. chelonae is MK-8(H(4)), MK-8 and MK-8(H(2)) (5 : 3 : 2, respectively). On the basis of these distinctive phenotypic characteristics and phylogenetic analysis results, it is proposed that strains Aji5-31(T) and Ngc37-23(T) be classified as two novel genera and species of the family Dermatophilaceae. The names are Mobilicoccus pelagius gen. nov., sp. nov. and Piscicoccus intestinalis gen. nov., sp. nov., and the type strains are Aji5-31(T) (=NBRC 104925(T) =DSM 22762(T)) and Ngc37-23(T) (=NBRC 104926(T) =DSM 22761(T)), respectively. In addition, D. chelonae should be reassigned to a new genus of the family Dermatophilaceae with the name Austwickia chelonae gen. nov., comb. nov.     

Stappia alba sp. nov., isolated from Mediterranean oysters

Three bacterial strains isolated from oysters recovered at the Spanish Mediterranean coast have been phenotypically and genetically characterized. The results of the phylogenetic analysis based on almost complete 16S rDNA sequences clustered all three strains together with 99.9% average sequence similarity and situated them in the neighbourhood of the genera Stappia, Roseibium and Pannonibacter, Stappia aggregata being their closest neighbour with sequence similarities between 98.8% and 98.9%. DNA-DNA hybridization experiments using DNA of strains 5OM6T and S. aggregata CECT 4269T as reference DNAs confirmed the independent status at species level of the oyster isolates. Phenotypically, they can be distinguished from the closest relatives by the ionic requirements, growth temperatures and use of carbon compounds. We propose these oyster strains constitute a new species of Stappia, for which the name Stappia alba sp. nov. has been chosen, and strain 5OM6T (= CECT 5095T = CIP 108402T) as its type strain.     

Characterization of Leuconostoc gasicomitatum sp. nov., associated with spoiled raw tomato-marinated broiler meat strips packaged under modified-atmosphere conditions

Lactic acid bacteria (LAB) associated with gaseous spoilage of modified-atmosphere-packaged, raw, tomato-marinated broiler meat strips were identified on the basis of a restriction fragment length polymorphism (RFLP) (ribotyping) database containing DNAs coding for 16S and 23S rRNAs (rDNAs). A mixed LAB population dominated by a Leuconostoc species resembling Leuconostoc gelidum caused the spoilage of the product. Lactobacillus sakei, Lactobacillus curvatus, and a gram-positive rod phenotypically similar to heterofermentative Lactobacillus species were the other main organisms detected. An increase in pH together with the extreme bulging of packages suggested a rare LAB spoilage type called "protein swell." This spoilage is characterized by excessive production of gas due to amino acid decarboxylation, and the rise in pH is attributed to the subsequent deamination of amino acids. Protein swell has not previously been associated with any kind of meat product. A polyphasic approach, including classical phenotyping, whole-cell protein electrophoresis, 16 and 23S rDNA RFLP, 16S rDNA sequence analysis, and DNA-DNA reassociation analysis, was used for the identification of the dominant Leuconostoc species. In addition to the RFLP analysis, phenotyping, whole-cell protein analysis, and 16S rDNA sequence homology indicated that L. gelidum was most similar to the spoilage-associated species. The two spoilage strains studied possessed 98.8 and 99.0% 16S rDNA sequence homology with the L. gelidum type strain. DNA-DNA reassociation, however, clearly distinguished the two species. The same strains showed only 22 and 34% hybridization with the L. gelidum type strain. These results warrant a separate species status, and we propose the name Leuconostoc gasicomitatum sp. nov. for this spoilage-associated Leuconostoc species.     

Lactobacillus oligofermentans sp. nov., Associated with Spoilage of Modified-Atmosphere-Packaged Poultry Products

Unidentified lactic acid bacterium (LAB) isolates which had mainly been detected in spoiled, marinated, modified atmosphere packaged (MAP) broiler meat products during two previous studies, were identified and analyzed for their phenotypic properties and the capability to produce biogenic amines. To establish the taxonomic position of these isolates, 16S rRNA gene sequence analysis, numerical analysis of ribopatterns, and DNA-DNA hybridization experiments were done. Unexpectedly for a meat-spoilage-associated LAB, the strains utilized glucose very weakly. According to the API 50 CHL test, arabinose and xylose were the only carbohydrates strongly fermented. None of the six strains tested for production of histamine, tyramine, tryptamine, phenylethylamine, putrescine, and cadaverine were able to produce these main meat-associated biogenic amines in vitro. The polyphasic taxonomy approach showed that these strains represent a new Lactobacillus species. The six isolates sequenced for the 16S rRNA encoding genes shared the highest similarity (95.0 to 96.3%) with the sequence of the Lactobacillus durianis type strain. In the phylogenetic tree, these isolates formed a distinct cluster within the Lactobacillus reuteri group, which also includes L. durianis. Numerical analyses of HindIII-EcoRI ribotypes placed all isolates together in a cluster with seven subclusters well separated from the L. reuteri group reference strains. The DNA-DNA hybridization levels between Lactobacillus sp. nov. isolates varied from 67 to 96%, and low hybridization levels (3 to 15%) were obtained with the L. durianis type strain confirming that these isolates belong to the same species different from L. durianis. The name Lactobacillus oligofermentans sp. nov. is proposed, with strain LMG 22743^T (also known as DSM 15707^T or AMKR18^T) as the type strain.     

Candida easanensis sp. nov., Candida pattaniensis sp. nov. and Candida nakhonratchasimensis sp. nov., three new species of yeasts isolated from insect frass in Thailand

Six strains of anamorphic yeasts isolated from insect frass collected in several regions of Thailand were assigned to the genus Candida based on the conventional taxonomic criteria used for yeast classification. These strains have Q-7 as the major ubiquinone and are suggested to have close relationships to the genus Pichia. Three strains, ST-225, ST-228 and ST-229, have identical nucleotide sequences in the D1/D2 domain of 26S rDNA and are closely related to Pichia japonica, but differ by six nucleotides (1.1% ) from this species. These three strains are considered to represent a single new species, which is described as Candida easanensis sp. nov. Two strains, ST-311 and ST-320, have identical sequences in the D1/D2 domain and resemble Pichia veronae and Pichia fabianii but differ from them by nine nucleotides (1.6%) in D1/D2 sequences. The two strains are described as Candida pattaniensis sp. nov. The remaining strain, ST-37, is related to Pichia americana and Pichia bimundalis but differs by six(1.1%) and seven (1.2%) nucleotides from these species, respectively. This strain is described as Candida nakhonratchasimensis sp. nov.     

Bifidobacterium reuteri sp. nov., Bifidobacterium callitrichos sp. nov., Bifidobacterium saguini sp. nov., Bifidobacterium stellenboschense sp. nov. and Bifidobacterium biavatii sp. nov. isolated from faeces of common marmoset (Callithrix jacchus) and red-handed tamarin (Saguinus midas)

Five strains of bifidobacteria were isolated from faeces of a common marmoset (Callithrix jacchus) and a red-handed tamarin (Saguinus midas). The five isolates clustered inside the phylogenetic group of the genus Bifidobacterium but did not show high sequence similarities between the isolates and to known species in the genus by phylogenetic analysis based on 16S rRNA gene sequences. Sequence analyses of dnaJ1 and hsp60 also indicated their independent phylogenetic positions to each other in the Bifidobacterium cluster. DNA G+C contents of the species ranged from 57.3 to 66.3 mol%, which is within the values recorded for Bifidobacterium species. All isolates showed fructose-6-phosphate phosphoketolase activity. Based on the data provided, the five isolates represent five novel species, for which the names Bifidobacterium reuteri sp. nov. (type strain: AFB22-1(T) = JCM 17295(T) = DSM 23975(T)), Bifidobacterium callitrichos sp. nov. (type strain: AFB22-5(T) = JCM 17296(T) = DSM 23973(T)), Bifidobacterium saguini sp. nov. (type strain: AFB23-1(T) = JCM 17297(T) = DSM 23967(T)), Bifidobacterium stellenboschense sp. nov. (type strain: AFB23-3(T) = JCM 17298(T) = DSM 23968(T)) and Bifidobacterium biavatii sp. nov. (type strain: AFB23-4(T) = JCM 17299(T) = DSM 23969(T)) are proposed.     

Systematic study of the genus Acetobacter with descriptions of Acetobacter indonesiensis sp. nov., Acetobacter tropicalis sp. nov., Acetobacter orleanensis (Henneberg 1906) comb. nov., Acetobacter lovaniensis (Frateur 1950) comb. nov., and Acetobacter estunensis (Carr 1958) comb. nov

Thirty-one Acetobacter strains obtained from culture collections and 45 Acetobacter strains isolated from Indonesian sources were investigated for their phenotypic characteristics, ubiquinone systems, DNA base compositions, and levels of DNA-DNA relatedness. Of 31 reference strains, six showed the presence of ubiquinone 10 (Q-10). These strains were eliminated from the genus Acetobacter. The other 25 reference strains and 45 Indonesian isolates were subjected to a systematic study and separated into 8 distinct groups on the basis of DNA-DNA relatedness. The known species, Acetobacter aceti, A. pasteurianus, and A. peroxydans are retained for three of these groups. New combinations, A. orleanensis (Henneberg 1906) comb. nov., A. lovaniensis (Frateur 1950) comb. nov., and A. estunensis (Carr 1958) comb. nov. are proposed for three other groups. Two new species, A. indonesiensis sp. nov. and A. tropicalis sp. nov. are proposed for the remaining two. No Indonesian isolates were identified as A. aceti, A. estunensis, and A. peroxydans. Phylogenetic analysis on the basis of 16S rDNA sequences was carried out for representative strains from each of the groups. This supported that the eight species belonged to the genus Acetobacter. Several strains previously assigned to the species of A. aceti and A. pasteurianus were scattered over the different species. It is evident that the value of DNA-DNA relatedness between strains comprising a new species should be determined for the establishment of the species. Thus current bacterial species without data of DNA-DNA relatedness should be reexamined for the stability of bacterial nomenclature.     

Hydrogenophaga intermedia sp. nov., a 4-aminobenzenesulfonate degrading organism

The taxonomic status of a gram-negative, oxidase positive rod (strain S1) able to degrade 4-aminobenzenesulfonate was studied using a polyphasic approach. Chemotaxonomic investigations of quinones and polar lipids established the allocation of this strain to the beta-subclass of the Proteobacteria and revealed similarities to Hydrogenophaga palleronii. 16S rRNA sequence comparisons demonstrated that this strain clusters phylogenetically with H. palleronii and H. taeniospiralis, but clearly represents a new species. The fatty acid patterns and substrate utilization profile displayed similarity to the characteristics of the four validly published species of Hydrogenophaga, although clear differentiating characters were also observed. No close similarities between the type strains of H. palleronii and H. taeniospiralis were detected in hybridization experiments with the genomic DNAs. On basis of these results, the new species Hydrogenophaga intermedia sp. nov. is proposed, with the type strain S1T (= DSM 5680).     

Leifsonia gen. nov., a genus for 2,4-diaminobutyric acid-containing actinomycetes to accommodate "Corynebacterium aquaticum" Leifson 1962 and Clavibacter xyli subsp. cynodontis Davis et al. 1984

"Corynebacterium aquaticum" was first proposed by Leifson in 1962 but not included in the approved lists of bacterial names in 1980. This species has been left from reclassification of the genus Corynebacterium because of the unusual chemotaxonomic characteristics such as 2,4-diaminobutyric acid (DAB) in the peptidoglycan and menaquinones of MK-10 and MK-11. A close relationship of "C. aquaticum" to the genera Agromyces and Rathayibacter has been pointed out from the viewpoint of chemotaxonomic profiles and phylogeny based on the 16S rDNA sequences. An analysis of DAB isomers of the peptidoglycan distinguished "C. aquaticum" clearly from these genera by possessing both L-DAB and D-DAB. We also found that the type strain of Clavibacter xyli subsp. cynodontis and two strains of amine-decomposing bacteria showed the similar chemotaxonomic features and formed a cluster with "C. aquaticum" in the phylogenetic tree based on 16S rDNA sequences in the family Microbacteriaceae. Considering these results, we propose a new genus Leifsonia to accommodate the four strains. The four species, Leifsonia aquatica sp. nov., nom. rev., comb. nov. (type species, type strain=JCM 1368), Leifsonia shinshuensis sp. nov. (type strain=DB102=JCM 10591), Leifsonia naganoensis sp. nov. (type strain=DB103=JCM 10592), and Leifsonia cynodontis comb. nov. (type strain=JCM 9733=ICMP 8790), were proposed here for the strains.     

Candida dajiaensis sp. nov., Candida yuanshanicus sp. nov., Candida jianshihensis sp. nov., and Candida sanyiensis sp. nov., four anamorphic, ascomycetous yeast species isolated from soil in Taiwan

Nine anamorphic, ascomycetous yeast strains belonging to the Pichia anomala clade were recovered from forest soil in 2006 in Taiwan. The nine yeast strains represent four novel yeast species based on the sequences of their D1/D2 domain of the large subunit (LSU) rRNA gene and their physiological characteristics. The scientific names of Candida dajiaensis sp. nov., Candida yuanshanicus sp. nov., Candida jianshihensis sp. nov., and Candida sanyiensis sp. nov. are proposed for these novel yeast species. The type strains are C. dajiaensis SM11S03(T) (=CBS 10590(T)=BCRC 23099(T)), C. yuanshanicus SY3S02(T) (=CBS 10589(T)=BCRC 23100(T)), C. jianshihensis SM8S04(T) (=CBS 10591(T)=BCRC 23096(T)), and C. sanyiensis SA1S06(T) (=CBS 10592(T)=BCRC 23094(T)). Sequence analysis of the D1/D2 of the LSU rRNA gene revealed that the three species, C. dajiaensis, C. yuanshanicus and Pichia onychis, shared a separate branch in the phylogenetic tree, C. jianshihensis is phylogenetically related to Candida ulmi and Pichia alni, and the phylogenetically closest relative of C. sanyiensis is Pichia populi.     

New species and new combinations in the yeast genera Kregervanrija gen. nov., Saturnispora and Candida

Analysis of nucleotide sequences from the domains 1 and 2 of the large-subunit rDNA demonstrated species of the Pichia fluxuum clade to be phylogenetically isolated, and domains 1 and 2 and internal transcribed spacer rDNA sequence analyses of strains phenotypically identified as P. fluxuum resulted in the discovery of three new species. From this work, the following new genus, new species, and new combinations are proposed: Kregervanrija gen. nov.; type species Kregervanrija fluxuum comb. nov. (type strain NRRL YB-4273, CBS 2287); Kregervanrija delftensis comb. nov. (type strain NRRL Y-7119, CBS 2614); Kregervanrija pseudodelftensis sp. nov. (type strain NRRL Y-5494, CBS 10105); Saturnispora besseyi comb. nov. (NRRL YB-4711, CBS 6343); Saturnispora mendoncae sp. nov. (type strain NRRL Y-11515, CBS 5620); and Candida abiesophila sp. nov. (type strain NRRL Y-11514, CBS 5366).