Bacterial diversity and antibiotic resistances of abundant aerobic culturable bacteria in input and output samples of 15 German biogas plants.

AIM: Determination of the diversities and antibiotic resistances of the most abundant aerobic culturable bacteria and their survival in 15 German biogas plants (BGPs). METHODS AND RESULTS: Microbiological standard media (complex and selective media) were applied to enumerate mesophilic and oligotrophic bacteria (Enterobacteriaceae, Salmonella, staphylococci and enterococci) in input and output samples of BGPs. Concentrations of aerobic culturable bacteria in outputs were mostly one to two orders of magnitude lower than in input samples. In total, 852 isolates from input and 902 from output samples were identified either by partial 16S rRNA gene sequencing or Matrix-assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) analysis. Antibiotic resistance screening of target families against veterinary relevant antibiotics resulted in similar resistance pattern in input and output samples. CONCLUSIONS: Potentially pathogenic bacteria were detected in output samples of BGPs, but in decreased abundance. The selectivity of the most applied clinical standard media was insufficient. SIGNIFICANCE AND IMPACT OF THE STUDY: Only little is known about the release of antibiotic resistant and potentially pathogenic bacteria from BGPs. However, this study indicates the detection of several potentially pathogenic bacteria in output samples, which showed several antibiotic resistances.

Chelativorans intermedius sp. nov. and proposal to reclassify Thermovum composti as Chelativorans composti comb. nov.

Two Gram-stain-negative, non-endospore-forming, rod-shaped bacteria, strains CC-MHSW-5(T) and A1392, were isolated from water of coastal hot springs located in Taiwan and China, respectively, and investigated for their taxonomic position. The two strains shared identical 16S rRNA gene sequences, a DNA-DNA hybridization value >80% and similar genomic DNA G+C contents (64.3 and 64.6 mol%), but showed different genomic fingerprint patterns generated by BOX-PCR and three random amplification polymorphic DNA PCRs. The strains shared highest 16S rRNA gene sequence similarities with the type strains of Chelativorans multitrophicus (96.7 and 96.1%), Thermovum composti (96.2 and 96.1%) and Chelativorans oligotrophicus (96.1 and 95.8%). Phylogenetic trees (based on 16S rRNA and recA gene sequence comparisons) showed a distinct clustering of both strains with the type strains of species of the genus Chelativorans and T. composti Nis3(T). The quinone systems of strains CC-MHSW-5(T) and Nis3(T) contained ubiquinone Q-10 as the major component. The major polyamine in both strains was sym-homospermidine. Putrescine, spermidine and, for strain CC-MHSW-5(T), spermine were found in minor concentrations. Their polar lipid profiles consisted of phosphatidylmonomethylethanolamine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine and diphosphatidylglycerol. The fatty acid profile contained major amounts of C18 : 1ω7c and C19 : 0 cyclo ω8c. On the basis of these results, the two strains are considered to represent a novel species of the genus Chelativorans , for which the name Chelativorans intermedius sp. nov. is proposed. The type strain is CC-MHSW-5(T) ( =CCM 8543(T) =LMG 28482(T) =DSM 29391(T) =CIP 110825(T)). Based on both genotypic and phenotypic characters, it is proposed that T. composti be reclassified within the genus Chelativorans as Chelativorans composti comb. nov.

Rhodococcus defluvii sp. nov., isolated from wastewater of a bioreactor and formal proposal to reclassify [Corynebacterium hoagii] and Rhodococcus equi as Rhodococcus hoagii comb. nov.

A Gram-stain-positive, non-endospore-forming rod-shaped bacterium, strain Ca11(T), was isolated from a bioreactor with extensive phosphorus removal and was studied in detail for its taxonomic allocation. 16S rRNA gene sequence analysis revealed closest sequence similarity of the strain to type strains of [Corynebacterium hoagii] and Rhodococcus equi (98.9%), Rhodococcus koreensis and Rhodococcus wratislaviensis (both 98.4%), Rhodococcus opacus and Rhodococcus canchipurensis (both 98.0%) followed by Rhodococcus kunmingensis and Rhodococcus imtechensis (97.7%). Phylogenetic trees showed a distinct clustering of strain Ca11(T) with the type strains of [C. hoagii], R. equi, and R. kunmingensis separate to all other species of the genus Rhodococcus. The quinone system of strain Ca11(T) was composed of dihydrogenated menaquinones with 8 (major amount) as well as 7 and 6 isoprenoid units [MK-8(H2), MK-7(H2), MK-6(H2)]. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside, one unknown phospholipid and an unidentified glycolipid. The fatty acid profile was similar to that reported for R. equi and contained major amounts of C16:0, C18:1ω9c and 10-methyl C18:0, supporting the allocation of the strain to the genus Rhodococcus. Physiological and biochemical characterization and DNA-DNA hybridization with type strains of the most closely related species allowed clear phenotypic and genotypic differentiation of the isolate. On the basis of these results, strain Ca11(T) ( = DSM 45893(T) = LMG 27563(T)) represents a novel species of the genus Rhodococcus, with the proposed name Rhodococcus defluvii sp. nov. In addition, a polyphasic taxonomic analysis of [Corynebacterium hoagii] DSM 20295(T) and Rhodococcus equi DSM 20307(T) indicated that the two strains belong to the same species, for which the name Rhodococcus hoagii comb. nov. takes priority, according to the Rules of the Bacteriological Code.

Falsiporphyromonas endometrii gen. nov., sp. nov., isolated from the post-partum bovine uterus, and emended description of the genus Porphyromonas Shah and Collins 1988.

Two black-pigmented, anaerobic bacterial strains, designated LMM 40(T) and LMM 41, were isolated from the bovine post-partum endometrium of two Holstein cows. The 16S rRNA gene sequences of the two strains were identical and showed the highest similarity to the 16S rRNA gene sequence of the type strain of Porphyromonas crevioricanis (90.2%) but only 85.1% 16S rRNA gene sequence similarity to the type strain of the type species of the genus Porphyromonas, Porphyromonas asaccharolytica. The major fatty acid profiles of the two strains were similar to those of species of the genus Porphyromonas, containing iso-C(15 : 0) as the major component and moderate amounts of anteiso-C(15 : 0), iso-C(13 : 0), C(15 : 0) and C(16 : 0). Hydroxylated fatty acids, such as iso-C(14 : 0) 3-OH, iso-C(16 : 0) 3-OH and iso-C(17 : 0) 3-OH, were also detected. The quinone profiles were dominated by the menaquinones MK-8 and MK-9, while spermidine was the major polyamine. The polar lipid profiles contained major amounts of phosphatidylethanolamine, an unidentified phospholipid, an unidentified aminophospholipid and two unidentified lipids and minor amounts of phosphatidylglycerol, an unidentified aminolipid, a second unidentified aminophospholipid and an unidentified glycolipid. The cell-wall peptidoglycan contained meso-diaminopimelic acid. The genomic DNA G+C contents of LMM 40(T) and LMM 41 were 40.7 and 41.3 mol%, respectively. Based on a polyphasic approach, including phylogenetic analysis, physiological and biochemical tests as well as metabolic fingerprinting, it is proposed that the two strains are members of a novel genus and species, for which the name Falsiporphyromonas endometrii gen. nov., sp. nov. is proposed. The type strain of Falsiporphyromonas endometrii is LMM 40(T) ( = DSM 27210(T) = CCUG 64267(T)). An emended description of the genus Porphyromonas is also presented.

Methylobacterium trifolii sp. nov. and Methylobacterium thuringiense sp. nov., methanol-utilizing, pink-pigmented bacteria isolated from leaf surfaces.

Three pink-pigmented, aerobic, Gram-stain-negative, rod-shaped and facultatively methylotrophic strains were isolated from the phyllosphere of Trifolium repens and Cerastium holosteoides. 16S rRNA gene sequence analysis support the affiliation of all strains to the genus Methylobacterium. The closest relatives of strains C34(T) and T5 were Methylobacterium gnaphalii 23e(T) (98.0 and 98.5 % sequence similarity, respectively) and Methylobacterium organophilum JCM 2833(T) (97.0 and 97.2 %, respectively). Strain TA73(T) showed the highest sequence similarities to Methylobacterium marchantiae JT1(T) and Methylobacterium bullatum F3.2(T) (both 97.9 %), followed by Methylobacterium phyllosphaerae CBMB27(T) and Methylobacterium brachiatum DSM 19569(T) (both 97.8 %), Methylobacterium cerastii C15(T) and Methylobacterium radiotolerans JCM 2831(T) (both 97.7 %). The major components in the fatty acid profiles were C18 : 1ω7c, C16 : 0 and one unknown fatty acid for strain TA73(T) and C18 : 1ω7c, C16 : 1ω7c/iso-C15 : 0 2-OH, C18 : 0 and C16 : 0 for strains C34(T) and T5. Physiological and biochemical analysis, including DNA-DNA hybridization, revealed clear differences between the investigated strains and their closest phylogenetic neighbours. DNA-DNA hybridization studies also showed high similarities between strains C34(T) and T5 (59.6-100 %). Therefore, the isolates represent two novel species within the genus Methylobacterium, for which the names Methylobacterium trifolii sp. nov. (type strain TA73(T) = LMG 25778(T) = CCM 7786(T)) and Methylobacterium thuringiense sp. nov. (type strain C34(T) = LMG 25777(T) = CCM 7787(T)) are proposed.

Flavobacterium cutihirudinis sp. nov., isolated from the skin of the medical leech Hirudo verbana.

A Gram-staining-negative, non-endospore-forming, yellow-pigmented strain (E89(T)) was isolated from the skin of the medical leech Hirudo verbana obtained from a leech farm located in Biebertal, Germany. 16S rRNA gene sequencing analysis showed that the isolate was grouped in the genus Flavobacterium. Strain E89(T) was most closely related to Flavobacterium chilense LM-09-Fp(T) (98.2 %), Flavobacterium chungangense CJ7(T) (98.1 %), and Flavobacterium oncorhynchi 631-08(T) (98.1 %). 16S rRNA gene sequence similarities to all other species of the genus Flavobacterium were ≤ 97.4 %. A menaquinone of the type MK-6 was found to be the predominant respiratory quinone and the polar lipid profile consisted of the major compounds phosphatidylethanolamine, phosphatidylserine, two unidentified aminolipids, one unknown phospholipid and two unknown lipids. The fatty acid profile was composed of iso-C15 : 0, C15 : 0, summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH) found in major amounts and several hydroxylated fatty acids in smaller amounts, among them iso-C15 : 0 3-OH and iso-C17 : 0 3-OH. All these data support the allocation of the isolate in the genus Flavobacterium. Physiological/biochemical characterization and DNA-DNA hybridizations with the type strains of the most closely related species allowed a clear phenotypic and genotypic differentiation of the strain. Based on these data, strain E89(T) represents a novel species of the genus Flavobacterium, for which the name Flavobacterium cutihirudinis sp. nov. is proposed. The type strain is E89(T) (= DSM 25795(T) = LMG 26922(T) = CIP 110374(T)).

Stappia taiwanensis sp. nov., isolated from a coastal thermal spring.

A beige-coloured, Gram-stain-negative, aerobic, non-motile moderately thermotolerant, rod-shaped organism, strain CC-SPIO-10-1(T), was isolated from a coastal hot spring of Green Island (Lutao), located off Taituang, Taiwan, on Marine Agar 2216. Based on 16S rRNA gene sequence analysis, this organism was grouped into the genus Stappia, showing 98.3 % sequence similarity to Stappia indica B106(T) and 98.2 % gene sequence similarity to Stappia stellulata IAM 12621(T). Ubiquinone Q-10 was the major respiratory quinone and C18 : 1ω7c and C18 : 1ω7c 11-methyl were detected as the major fatty acids. The hydroxylated fatty acid C18 : 0 3-OH was detected as well. Predominant polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, unidentified aminolipid AL1 and unidentified phospholipid PL1. Minor amounts of several unidentified lipids (PL2 and L1-L7) were present as well. The polyamine pattern contained the major compounds spermidine and spermine. Strain CC-SPIO-10-1(T) could be differentiated from the type strains of S. stellulata and S. indica by a set of biochemical tests. On the basis of the 16S rRNA gene sequence analysis and the chemotaxonomic and physiological data, it is concluded that strain CC-SPIO-10(T) represents a novel species of the genus Stappia for which the name Stappia taiwanensis sp. nov. is proposed. The type strain is CC-SPIO-10 (T) ( = CCUG 59208(T) = LMG 25538 (T) = CCM 7757(T)).

Ornithinimicrobium murale sp. nov., isolated from an indoor wall colonized by moulds.

A Gram-positive, non-spore-forming actinobacterium (01-Gi-040(T)) isolated from an indoor wall was studied to examine its taxonomic position. The isolate formed a very rudimentary substrate-mycelium that fragmented into rod-shaped to coccoid cells. On the basis of the 16S rRNA gene sequence similarity studies, strain 01-Gi-040(T) was shown to belong to the genus Ornithinimicrobium closely related to Ornithinimicrobium kibberense K22-20(T) (97.1 %), Ornithinimicrobium humiphilum DSM 12362(T) (96.2 %) and Ornithinimicrobium pekingense LW6(T) (96.1 %). A close relationship was also found with Arsenicicoccus bolidensis CCUG 47306(T) (95.9 %) and Arsenicicoccus piscis Kis4-19(T) (95.7 %) and a moderate relationship to the type strains of the genus Serinicoccus (94.0-94.1 %). The predominant menaquinone of strain 01-Gi-040(T) was MK-8(H(4)). The peptidoglycan contained ornithine as the diagnostic diamino acid. The polar lipid profile consisted of the lipids phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol, an unknown phospholipid, an unknown aminolipid and two unknown phosphoglycolipids. The major fatty acids iso-C(15 : 0), iso-C(16 : 0) and iso-C(17 : 0) were consistent with the fatty acid patterns reported for members of the genus Ornithinimicrobium. The results of DNA-DNA hybridizations, physiological and biochemical tests allowed phenotypic differentiation of strain 01-Gi-040(T) from the three recognized species of the genus Ornithinimicrobium. Strain 01-Gi-040(T) represents a novel species of the genus Ornithinimicrobium, for which we propose the name Ornithinimicrobium murale sp. nov., with the type strain 01-Gi-040(T) (= DSM 22056(T) = CCM 7610(T)).

Rhodococcus cerastii sp. nov. and Rhodococcus trifolii sp. nov., two novel species isolated from leaf surfaces.

Two Gram-positive, non-endospore-forming rods, strains C5(T) and T8(T), were isolated from the phyllospheres of Cerastium holosteoides and Trifolium repens, respectively, and were studied in detail for their taxonomic position. 16S rRNA gene sequence analysis allocated both isolates clearly to the genus Rhodococcus. Isolate C5(T) was most closely related to Rhodococcus fascians and Rhodococcus yunnanensis, showing 99.2 % gene sequence similarity to both species. Strain T8(T) revealed the highest 16S rRNA gene sequence similarity to Rhodococcus corynebacterioides (98.8 %) and Rhodococcus kroppenstedtii (98.6 %). The quinone system of both strains was composed of dihydrogenated menaquinones with eight (major amount) as well as nine, seven and six isoprenoid units (MK-8H2, MK-9H2 MK-7H2 MK-6H2).The polar lipid profiles of strains C5(T) and T8(T) consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside and one unknown phospholipid. Additionally, strain C5(T) contained one unknown glycolipid, and strain T8(T) three unknown aminolipids. The fatty acid profiles contained major amounts of C16 : 0, C18 : 1ω9c and 10-methyl C18 : 0, which supported the grouping of the two isolates in the genus Rhodococcus. Physiological/biochemical characterization and DNA-DNA hybridizations with the type strains of the most closely related species allowed a clear phenotypic and genotypic differentiation of both strains. For this reason, we propose strain C5(T) ( = LMG 26203(T)  = CCM 7906(T)) as the type strain of a novel species with the name Rhodococcus cerastii sp. nov., and strain T8(T) ( = LMG 26204(T)  = CCM 7905(T)) as the type strain of a second novel species with the name Rhodococcus trifolii sp. nov.

Ruegeria intermedia sp. nov., a moderately thermophilic bacterium isolated from a coastal hot spring.

A cream-coloured, Gram-negative, aerobic, non-motile moderately thermophilic, rod-to-irregular-shaped bacterium, CC-GIMAT-2(T), was isolated from a coastal hot spring of Green Island (Lutao), located off Taituang, Taiwan, on marine agar 2216. The 16S rRNA gene sequence analysis and subsequent comparisons showed that it was placed into the genus Ruegeria with 97.4 % similarity to Ruegeria lacuscaerulensis ITI-1157(T), and a lower sequence similarity to all other species of the genus Ruegeria. Reconstruction of phylogenetic trees indicated that strain CC-GIMAT-2(T) clustered within the genus Ruegeria. Robust tree topology for the genus Ruegeria including the new strain was only obtained by including all Rhodobacteraceae type strains but not if the analysis was limited to few selected taxa. The quinone system contained exclusively ubiquinone Q-10 and the fatty acid profile consisted mainly of C18 : 1ω7c, 11-methyl C18 : 1ω7c and C12 : 0 3-OH. The predominant polar lipids were diphosphatidylglycerol, phosphatidylglycerol and an unidentified aminolipid. Other lipids were detected in moderate to minor amounts. The characteristic feature of the polyamine pattern was the predominant triamine spermidine. On the basis of the 16S rRNA gene sequence analysis and of the chemotaxonomic and physiological data, strain CC-GIMAT-2(T) represents a novel species of the genus Ruegeria, for which the name Ruegeria intermedia sp. nov. is proposed. The type strain is CC-GIMAT-2(T) ( = CCUG 59209(T) = LMG 25539(T) = CCM 7758(T)).

Gordonia phosphorivorans sp. nov., isolated from a wastewater bioreactor with phosphorus removal.

Two Gram-stain-positive, non-endospore-forming actinobacteria (Ca8(T)and Ca14) were isolated from a bioreactor with extensive phosphorus removal. Based on 16S rRNA gene sequence similarity comparisons, strains Ca8(T) and Ca14 were shown to belong to the genus Gordonia and were most closely related to Gordonia hirsuta DSM 44140(T) (98.0 % sequence similarity) and Gordonia hydrophobica DSM 44015(T) (97.2 %). In comparison with the sequences of the type strains of all other species of the genus Gordonia tested, similarities were below 97 %. The quinone systems of the strains were determined to consist predominantly of MK-9H(2). The polar lipid profile for both organisms consisted of diphosphatidylglycerol, phosphatidylglycerol, phospatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside. Whole-organism hydrolysates contained meso-diaminopimelic acid as the diamino acid of the peptidoglycan; mycolic acids were detected as well. These chemotaxonomic traits and the major fatty acids, which were C(16 : 1)cis-9, C(16 : 0) and C(18 : 1) and tuberculostearic acid strongly supported the grouping of strains Ca8(T) and Ca14 into the genus Gordonia. The two strains showed a DNA-DNA similarity of 96 %. DNA-DNA hybridizations of strain Ca8(T) with G. hirsuta DSM 44140(T) and G. hydrophobica DSM 44015(T) resulted in values of 26.3 and 25.0 %, respectively. These results and those of the physiological and biochemical tests allowed a clear phenotypic differentiation of strains Ca8(T) and Ca14 from the most closely related species of the genus Gordonia. It is concluded that strains Ca8(T) and Ca14 represent a novel species, for which the name Gordonia phosphorivorans sp. nov. is proposed, with the type strain Ca8(T) (= DSM 45630(T) = CCUG 61533(T) = CCM 7957(T) = LMG 26648(T)).

Paracoccus rhizosphaerae sp. nov., isolated from the rhizosphere of the plant Crossostephium chinense (L.) Makino (Seremban).

A Gram-negative, coccoid-shaped bacterium, strain CC-CCM15-8(T), was isolated from a rhizosphere soil sample of the plant Crossostephium chinense (L.) Makino (Seremban) from Budai Township, Chiayi County, Taiwan. 16S rRNA gene sequence analysis clearly allocated strain CC-CCM15-8(T) to the Paracoccus cluster, showing highest similarities to the type strains of 'Paracoccus beibuensis' (98.8%), Paracoccus homiensis (97.6%), Paracoccus aestuarii (97.7%) and Paracoccus zeaxanthinifaciens (97.7%). The fatty acid profile, comprising C(18:1)ω7c as the major component and C(10:0) 3-OH as the characteristic hydroxylated fatty acid, supported the placement of strain CC-CCM15-8(T) within the genus Paracoccus. The polyamine pattern consisted of putrescine and spermidine as major components. Ubiqinone Q-10 was the major quinone type (95%); ubiquinone Q-9 was also detected (5%). The complex polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, and unidentified phospholipids, lipids and glycolipids. Levels of DNA-DNA relatedness between strain CC-CCM15-8(T) and 'P. beibuensis' LMG 25871(T), P. aestuarii DSM 19484(T), P. zeaxanthinifaciens LMG 21993(T) and P. homiensis KACC 11518(T) were 24.9% (34.8%, reciprocal analysis), 15.7% (17.5%), 17.7% (23.4%) and 16.0% (25.4%), respectively. Physiological and biochemical test results allowed the phenotypic differentiation of strain CC-CCM15-8(T) from its closest relatives in the genus Paracoccus. Based on the data presented, it is concluded that strain CC-CCM15-8(T) represents a novel species of the genus Paracoccus, for which the name Paracoccus rhizosphaerae sp. nov. is proposed. The type strain is CC-CCM15-8(T) (=LMG 26205(T)=CCM 7904(T)).

Microbacterium murale sp. nov., isolated from an indoor wall.

A Gram-positive rod, designated 01-Gi-001(T), was isolated from a wall colonized with moulds. The 16S rRNA gene sequence analysis clearly showed that the isolate belonged to the genus Microbacterium. On the basis of pairwise comparisons of 16S rRNA gene sequences, strain 01-Gi-001(T) was most closely related to Microbacterium hydrocarbonoxydans DSM 16089(T) (98.9% sequence similarity), Microbacterium profundi Shh49(T) (98.7%), Microbacterium phyllosphaerae DSM 13468(T) (98.3%) and Microbacterium foliorum DSM 12966(T) (98.1%). The diagnostic diamino acid of the peptidoglycan was ornithine. The major menaquinones detected were MK-13 and MK-12. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, one unknown phospholipid and one unknown glycolipid. The major fatty acids were anteiso-C(15:0), iso-C(16:0) and anteiso-C(17:0), which were in agreement with those reported for other members of the genus Microbacterium. Physiological and biochemical characteristics and DNA-DNA relatedness between strain 01-Gi-001(T) and the type strains of its closest phylogenetic neighbours showed clear differences. For this reason, strain 01-Gi-001(T) (=DSM 22178(T)=CCM 7640(T)) is proposed as the type strain of a novel species, Microbacterium murale sp. nov.

Microbulbifer taiwanensis sp. nov., isolated from coastal soil.

A Gram-negative, non-spore-forming rod (CC-LN1-12(T)) was isolated from coastal soil samples of Lutao Island (Green Island), Taiwan, and its taxonomic position was studied. 16S rRNA gene sequence analysis showed that isolate CC-LN1-12(T) was grouped into the Microbulbifer cluster, with the highest similarities to Microbulbifer okinawensis ABABA23(T) (97.9 %), Microbulbifer maritimus TF-17(T) (97.7 %) and Microbulbifer donghaiensis CN85(T) (97.7 %), similarities to all other species of the genus Microbulbifer were lower than 96.8 %. The polyamine pattern contained the major compounds spermidine and cadaverine. The fatty acid profile, comprising the major fatty acids iso-C(15 : 0), iso-C(17 : 1)ω9c, C(18 : 1)ω7c and iso-C(11 : 0) 3-OH as the major hydroxylated fatty acid, supported the affiliation of strain CC-LN1-12(T) to the genus Microbulbifer. DNA-DNA hybridizations between strain CC-LN1-12(T) and Microbulbifer okinawensis ABABA23(T), M. donghaiensis CN85(T) and M. maritimus JCM 12187(T) resulted in relatedness values of 21.5 % (14.3 %, reciprocal analysis), 35.9 % (48.5 %, reciprocal analysis) and 48.1 % (52.1 %, reciprocal analysis), respectively. From these data, as well as from physiological and biochemical tests, strain CC-LN1-12(T) could be clearly differentiated from the most closely related species of the genus Microbulbifer. It is concluded that strain CC-LN1-12(T) represents a novel species, for which the name Microbulbifer taiwanensis sp. nov. is proposed. The type strain is CC-LN1-12(T) ( = LMG 26125(T) = CCM 7856(T)).

Flavobacterium chilense sp. nov. and Flavobacterium araucananum sp. nov., isolated from farmed salmonid fish.

Three Gram-staining-negative non-endospore-forming strains were isolated from farmed fish in Chile: one (LM-09-Fp(T)) from a rainbow trout (Oncorhynchus mykiss) and the others (LM-19-Fp(T) and LM-20-Fp) from two Atlantic salmon (Salmo salar). Phylogenetic analyses based on 16S rRNA gene sequences indicated that all three isolates belonged to the genus Flavobacterium. In these analyses, strain LM-09-Fp(T) appeared most closely related to the type strains of Flavobacterium chungangense (98.5 % sequence similarity), Flavobacterium glaciei (98.2 %), Flavobacterium aquidurense (97.6 %), Flavobacterium saccharophilum (97.6 %) and Flavobacterium hercynium (97.6 %). The 16S rRNA gene sequences of strains LM-19-Fp(T) and LM-20-Fp were found to be identical and most similar to the corresponding sequences of the type strains of Flavobacterium aquidurense (98.6 %), Flavobacterium frigidimaris (98.5 %), Flavobacterium hercynium (97.9 %), Flavobacterium saccharophilum (97.7 %) and Flavobacterium pectinovorum (97.7 %). For each of the three novel strains, menaquinone (MK-6) was the predominant respiratory quinone and the major compounds in the polar lipid profile were phosphatidylethanolamine, an unidentified aminolipid, phosphatidylserine and two or three unknown lipids. The fatty acid profile of each strain, which comprised major amounts of iso-C(15:0), C(15:0) and summed feature 3 (C(16:1)ω7c and/or iso-C(15:0) 2-OH) as well as smaller amounts of various hydroxylated fatty acids (e.g. iso-C(16:0) 3-OH, iso-C(17:0) 3-OH, C(16:0) 3-OH and C(15:0) 3-OH), indicated that each belonged to the genus Flavobacterium. Based on their physiological and biochemical characteristics and the results of DNA-DNA hybridizations, which showed relatively low levels of relatedness between the novel strains and the most closely related Flavobacterium species, strain LM-09-Fp(T) ( = LMG 26360(T) = CCM 7940(T)) represents a novel species within the genus Flavobacterium, for which the name Flavobacterium chilense sp. nov. is proposed, and strains LM-19-Fp(T) ( = LMG 26359(T) = CCM 7939(T)) and LM-20-Fp ( = LMG 26331) represent a second novel species within the same genus, for which the name Flavobacterium araucananum sp. nov. is proposed.

Camelimonas abortus sp. nov., isolated from placental tissue of cattle.

A gram-negative, rod-shaped, non-spore-forming bacterium, isolated from placental tissue of a cow, was investigated for its taxonomic position. On the basis of 16S rRNA gene sequence similarities, strain UK34/07-5(T) was shown to belong to the class Alphaproteobacteria, closely related to the type strain of Camelimonas lactis (96.0 % sequence similarity). The polyamine pattern showed the major compound spermidine and moderate amounts of putrescine. The major quinone was ubiquinone Q-10. The polar lipid profile was composed of the major compounds phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol and phosphatidylmonomethylethanolamine and moderate amounts of diphosphatidylglycerol, three unidentified aminolipids and an unidentified phospholipid. The profile of major fatty acids, consisting of C(19 : 0) cyclo ω8c and C(18 : 1)ω7c, with C(18 : 0) 3-OH as the hydroxylated fatty acid, was very similar to that of C. lactis M 2040(T). The results of DNA-DNA hybridization and physiological and biochemical tests allowed both genotypic and phenotypic differentiation of the isolate from C. lactis. The relatively low 16S rRNA gene sequence similarity of 96.0 % to C. lactis M 2040(T) and marked differences in the polar lipid profiles as well as the results of physiological tests and the DNA-DNA hybridization data support the creation of a novel species, for which the name Camelimonas abortus sp. nov. is proposed, with the type strain UK34/07-5(T) ( = CIP 110303(T)  = CCUG 61094(T)  = DSM 24741(T)  = CCM 7941(T)).

Methylobacterium cerastii sp. nov., isolated from the leaf surface of Cerastium holosteoides.

Two Gram-stain-negative, non-endospore-forming, rod-like strains, designated C15T and C44, were isolated from the phyllosphere of Cerastium holosteoides and were studied in detail in order to assess their taxonomic position. 16S rRNA gene sequence analysis allocated both isolates clearly to the genus Methylobacterium. Both strains showed the highest 16S rRNA gene sequence similarity to Methylobacterium marchantiae JT1T (97.5 %) and Methylobacterium jeotgali S2R03-9T (97.4 %). The fatty acid profiles contained major amounts of C16:0, C18:1ω7c and C16:1ω7c/iso-C15:0 2-OH (summed feature 3), which supported the grouping of the isolates in the genus Methylobacterium. Physiological/biochemical characterization and DNA-DNA hybridizations with the type strains of the most closely related species allowed a clear phenotypic and genotypic differentiation of the strains. For this reason, we propose for strain C15T (=DSM 23679T=CCUG 60040T=CCM 7788T) a novel species with the name Methylobacterium cerastii sp. nov. Strain C44 (=DSM 23675=CCM 7789) is an additional strain of M. cerastii.

Dietzia aurantiaca sp. nov., isolated from a human clinical specimen.

A Gram-positive, coccoid, non-endospore-forming actinobacterium (strain CCUG 35676(T)) was isolated from cerebrospinal fluid from a 24-year-old woman in Gothenborg, Sweden. Based on pairwise 16S rRNA gene sequence similarity studies, strain CCUG 35676(T) was shown to belong to the genus Dietzia and was most closely related to the type strains of Dietzia aerolata (99.3%), Dietzia lutea (98.8%), Dietzia schimae (98.5%), Dietzia maris (98.5%), Dietzia alimentaria (98.3%) and Dietzia cercidiphylli (98.0%). The major menaquinone was MK-8(H(2)). Major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, an unidentified aminophospholipid (APL1), an unidentified phospholipid (PL1) and unidentified glycolipids (GL1 and GL3). Numerous other lipids were also detected. The fatty acid profile, comprising C(16:0), C(17:0,) C(18:1)ω9c and 10-methyl-C(18:0) as major fatty acids, supported the affiliation of strain CCUG 35676(T) to the genus Dietzia. On the basis of the results of physiological and biochemical tests and DNA-DNA hybridizations, a clear phenotypic and genotypic differentiation of strain CCUG 35676(T) from the most closely related Dietzia species is possible. Strain CCUG 35676(T) represents a novel species, for which the name Dietzia aurantiaca sp. nov. is proposed, with CCUG 35676(T) (=JCM 17645(T)) as the type strain.

Diversity and biogeography of selected phyllosphere bacteria with special emphasis on Methylobacterium spp.

On the basis of cultivation-dependent (isolation on mineral salt medium supplemented with 0.5% methanol) and -independent (DGGE analysis) methods, we investigated the influence of the host plant species Trifolium repens and Cerastium holosteoides, three geographic locations and the land-use types meadow, mown pasture and pasture on the abundance and community composition of selected phyllosphere bacteria with emphasis on Methylobacterium species. Methylobacterium abundance was significantly higher on leaves of T. repens (mean value 2.0×10(7) CFU PPFM per g leaf) than on leaves of C. holosteoides (mean value 2.0×10(6) CFU per g leaf). Leaves from the sampling site Schorfheide-Chorin showed slightly lower Methylobacterium numbers than leaves of the other sampling sites. Land-use and sampling period had no consistent influence on Methylobacterium community size. Methylobacterium community composition was very similar over both sampling periods, all three sampling sites, all land-use types and both plant species. Moreover, no relationship between geographic and genetic distance was observed. Community composition of selected Proteobacteria was influenced by plant species, geographic location and land-use. Often, differences in community composition could be observed between meadows, mown pastures and pastures but not between different kinds of meadows (cutted once versus three times) and mown pastures (fertilized versus non-fertilized). The results also indicate, that whether there are differences between land-use types or not strongly depends on the investigated host plant species and ecosystem. Besides Methylobacterium, representatives of Methylophilus were detected. The results indicate that Methylobacterium species are generally abundant and stable members of the phyllosphere community whereas other genera occur more occasionally, and that Methylobacterium clearly dominates the methylotrophic phyllosphere community.

Williamsia phyllosphaerae sp. nov., isolated from the surface of Trifolium repens leaves.

A Gram-positive-staining, non-endospore-forming actinobacterium, designated C7(T), was isolated from the leaf surface of Trifolium repens. On the basis of 16S rRNA gene sequence analysis, strain C7(T) was shown to belong to the genus Williamsia and was most closely related to Williamsia maris SJS0289/JS1(T) (98.0 % 16S rRNA gene sequence similarity), Williamsia deligens IMMIB RIV-956(T) (96.4 %) and Williamsia serinedens IMMIB SR-4(T) (95.7 %). The quinone system consisted predominantly of the menaquinones MK-9(H(2)), MK-8(H(2)) and MK-7(H(2)). The major components in the polar lipid profile were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol. Mycolic acids were present. These chemotaxonomic traits and the major fatty acids, which were C(16 : 1)ω7c, C(16 : 0), C(18 : 0), C(18 : 1)ω9c and tuberculostearic acid, supported the affiliation of strain C7(T) with the genus Williamsia. Physiological and biochemical analysis revealed clear differences between strain C7(T) and its closest phylogenetic neighbours. Therefore, strain C7(T) represents a novel species, for which the name Williamsia phyllosphaerae sp. nov. is proposed. The type strain is C7(T) ( = CCUG 60465(T) = CCM 7855(T)).