Microbial Contaminants of Cord Blood Units Identified by 16S rRNA Sequencing and by API Test System, and Antibiotic Sensitivity Profiling.

Over a period of ten months a total of 5618 cord blood units (CBU) were screened for microbial contamination under routine conditions. The antibiotic resistance profile for all isolates was also examined using ATB strips. The detection rate for culture positive units was 7.5%, corresponding to 422 samples.16S rRNA sequence analysis and identification with API test system were used to identify the culturable aerobic, microaerophilic and anaerobic bacteria from CBUs. From these samples we recovered 485 isolates (84 operational taxonomic units, OTUs) assigned to the classes Bacteroidia, Actinobacteria, Clostridia, Bacilli, Betaproteobacteria and primarily to the Gammaproteobacteria. Sixty-nine OTUs, corresponding to 447 isolates, showed 16S rRNA sequence similarities above 99.0% with known cultured bacteria. However, 14 OTUs had 16S rRNA sequence similarities between 95 and 99% in support of genus level identification and one OTU with 16S rRNA sequence similarity of 90.3% supporting a family level identification only. The phenotypic identification formed 29 OTUs that could be identified to the species level and 9 OTUs that could be identified to the genus level by API test system. We failed to obtain identification for 14 OTUs, while 32 OTUs comprised organisms producing mixed identifications. Forty-two OTUs covered species not included in the API system databases. The API test system Rapid ID 32 Strep and Rapid ID 32 E showed the highest proportion of identifications to the species level, the lowest ratio of unidentified results and the highest agreement to the results of 16S rRNA assignments. Isolates affiliated to the Bacilli and Bacteroidia showed the highest antibiotic multi-resistance indices and microorganisms of the Clostridia displayed the most antibiotic sensitive phenotypes.

Palleronia abyssalis sp. nov., isolated from the deep Mediterranean Sea and the emended description of the genus Palleronia and of the species Palleronia marisminoris.

Three strains designated 221-F1(T), 221-F2 and 3030-F1 were isolated from the Matapan Vavilov Deep canyon, also known as Calypso Deep in the Eastern Mediterranean Sea, at a depth of 4,908 m. Based on 16S rRNA gene sequence analysis these strains were found to be most closely related to Palleronia marisminoris and Hwanghaeicola aestuarii, with 16S rRNA gene pairwise sequence similarity of 95.3 and 94.7 % respectively, belonging to the family Rhodobacteraceae. The strains were observed to be red-pigmented and to form non-motile cocci or pleomorphic cells. The cells were found to stain Gram-negative, to be strictly aerobic, oxidase and catalase positive. Strains 221-F1(T), 221-F2 and 3030-F1 were found to be mesophilic and to grow in medium containing up to 13 % NaCl. The major polar lipids of the three strains were identified as diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, an unidentified glycolipid and an unidentified aminolipid. Ubiquinone 10 (U-10) was found to be the major respiratory quinone. The DNA G+C content of strain 221-F1(T) was determined to be 64.7 mol%. Based on phylogenetic, physiological and biochemical characteristics we describe a new species represented by strain 221-F1(T) (=CECT 8504(T) = LMG 27977(T)) for which we propose the name Palleronia abyssalis sp. nov. We also propose to emend the description of the genus Palleronia and the species P. marisminoris to reflect new results obtained in this study.

Halorhabdus tiamatea: proteogenomics and glycosidase activity measurements identify the first cultivated euryarchaeon from a deep-sea anoxic brine lake as potential polysaccharide degrader.

Euryarchaea from the genus Halorhabdus have been found in hypersaline habitats worldwide, yet are represented by only two isolates: Halorhabdus utahensis AX-2(T) from the shallow Great Salt Lake of Utah, and Halorhabdus tiamatea SARL4B(T) from the Shaban deep-sea hypersaline anoxic lake (DHAL) in the Red Sea. We sequenced the H. tiamatea genome to elucidate its niche adaptations. Among sequenced archaea, H. tiamatea features the highest number of glycoside hydrolases, the majority of which were expressed in proteome experiments. Annotations and glycosidase activity measurements suggested an adaptation towards recalcitrant algal and plant-derived hemicelluloses. Glycosidase activities were higher at 2% than at 0% or 5% oxygen, supporting a preference for low-oxygen conditions. Likewise, proteomics indicated quinone-mediated electron transport at 2% oxygen, but a notable stress response at 5% oxygen. Halorhabdus tiamatea furthermore encodes proteins characteristic for thermophiles and light-dependent enzymes (e.g. bacteriorhodopsin), suggesting that H. tiamatea evolution was mostly not governed by a cold, dark, anoxic deep-sea habitat. Using enrichment and metagenomics, we could demonstrate presence of similar glycoside hydrolase-rich Halorhabdus members in the Mediterranean DHAL Medee, which supports that Halorhabdus species can occupy a distinct niche as polysaccharide degraders in hypersaline environments.

Natrinema salaciae sp. nov., a halophilic archaeon isolated from the deep, hypersaline anoxic Lake Medee in the Eastern Mediterranean Sea.

Two halophilic archaea, strains MDB25(T) and MDB20, were isolated from a sample of the brine from Lake Medee, at a depth of 3050 m, in the Mediterranean Sea. Cells of the organisms were Gram-negative, non-motile and pleomorphic, and colonies were red pigmented. Strains MDB25(T) and MDB20 showed optimum growth at 45°C, in 2.6-3.4M NaCl and at pH 7.0-8.0. The major polar lipids of the two strains were phosphatidylglycerol (PG1 and PG2), phosphatidylglycerol phosphate methyl ester (PGP-Me) and mannose-2,6-dissulfate (1→2)-glucose glycerol diether (S(2)-DGD). Menaquinone MK-8 and MK-8(H(2)) were the major respiratory quinones. The DNA G+C content of strain MDB25(T) was 63.0%. The strains were facultatively anaerobic but grew better under aerobic conditions, nitrate served as electron acceptor. Analysis of the almost complete 16S rRNA gene sequence indicated that the strains MDB25(T) and MDB20 represented a member of the genus Natrinema in the family Halobacteriaceae. Both strains formed a distinct cluster and were most closely related to Natrinema ejinorense JCM 13890(T) and Haloterrigena longa JCM 13562(T) (98.0% and 97.9% sequence similarity, respectively). Based on 16S rRNA gene sequence analysis, DNA-DNA hybridization results, physiological and biochemical characteristics we describe a new species represented by strain MDB25(T) (=DSM 25055(T) =JCM 17869(T)) for which we propose the name Natrinema salaciae sp. nov.

Description of Idiomarina insulisalsae sp. nov., isolated from the soil of a sea salt evaporation pond, proposal to transfer the species of the genus Pseudidiomarina to the genus Idiomarina and emended description of the genus Idiomarina.

A halophilic, aerobic Gram-negative bacterium, designated strain CVS-6(T), was isolated from a sea salt evaporation pond on the Island of Sal in the Cape Verde Archipelago. Phylogenetic analysis of the 16S rRNA gene sequence revealed a clear affiliation of the organism with members of the family Idiomarinaceae. Sequence similarities between CVS-6(T) and the type strains of the species of the genera Pseudidiomarina and Idiomarina ranged from 93.7% to 96.9%. The major isoprenoid quinone was ubiquinone 8 (Q-8). The major cellular fatty acids were 15:0 iso (21.8%), 17:0 iso (12.5%), 17:1 iso omega9c (10.7%), and 16:1 omega7c (10.6%). The DNA G+C content was 51.6 mol%. The species represented by strain CVS-6(T) could be distinguished from the species of the genera Pseudidiomarina and Idiomarina; however, it was not possible to distinguish both genera from each other using the phenotypic or chemotaxonomic characteristics examined. Consequently, we propose that the species classified in the genus Pseudidiomarina should be transferred to the genus Idiomarina. We also propose that, on the basis of physiological and biochemical characteristics, strain CVS-6(T) (=LMG 23123=CIP 108836) represents a new species which we name Idiomarina insulisalsae.

A new lineage of halophilic, wall-less, contractile bacteria from a brine-filled deep of the Red Sea.

A novel strictly anaerobic bacterium designated strain SSD-17B(T) was isolated from the hypersaline brine-sediment interface of the Shaban Deep, Red Sea. Cells were pleomorphic but usually consisted of a central coccoid body with one or two "tentacle-like" protrusions. These protrusions actively alternated between a straight, relaxed form and a contracted, corkscrew-like one. A peptidoglycan layer was not detected by electron microscopy. The organism forms "fried-egg"-like colonies on MM-X medium. The organism is strictly anaerobic and halophilic and has an optimum temperature for growth of about 30 to 37 degrees C and an optimum pH of about 7. Nitrate and nitrite are reduced; lactate is a fermentation product. The fatty acid profile is dominated by straight saturated and unsaturated chain compounds. Menaquinone 4 is the major respiratory quinone. Phylogenetic analysis demonstrated strain SSD-17B(T) represents a novel and distinct lineage within the radiation of the domain Bacteria. The branching position of strain SSD-17B(T) was equidistant to the taxa considered to be representative lineages of the phyla Firmicutes and Tenericutes (with its sole class Mollicutes). The phenotypic and phylogenetic data clearly show the distinctiveness of this unusual bacterium, and we therefore propose that strain SSD-17B(T) (= DSM 18853 = JCM 14575) represents a new genus and a new species, for which we recommend the name Haloplasma contractile gen. nov., sp. nov. We are also of the opinion that the organism represents a new order-level taxon, for which we propose the name Haloplasmatales.

Halorhabdus tiamatea sp. nov., a non-pigmented, extremely halophilic archaeon from a deep-sea, hypersaline anoxic basin of the Red Sea, and emended description of the genus Halorhabdus.

An extremely halophilic archaeon was isolated from a sample of the brine-sediment interface of the Shaban Deep in the northern Red Sea. Phylogenetic analysis of the 16S rRNA gene sequence revealed a close proximity to Halorhabdus utahensis (99.3%), the sole species of the genus Halorhabdus. Strain SARL4B(T) formed non-pigmented colonies and showed optimum growth at 45 degrees C, in 27% (w/v) NaCl and at pH 6.5-7.0. This organism utilized a few complex substrates, such as yeast extract and starch, for growth. Strain SARL4B(T) grew under anaerobic and microaerophilic conditions but grew extremely poorly under aerobic conditions. The ether lipids were diphytanyl derivatives. The DNA G+C content of the type strain was 61.7 mol%. On the basis of the phylogenetic data and physiological and biochemical characteristics, strain SARL4B(T) represents a novel species of the genus Halorhabdus, for which the name Halorhabdus tiamatea is proposed. The type strain is SARL4B(T) (=DSM 18392(T)=JCM 14471(T)). An emended description of the genus Halorhabdus is also proposed.

Bacillus isabeliae sp. nov., a halophilic bacterium isolated from a sea salt evaporation pond.

A low-G+C, Gram-positive isolate, designated strain CVS-8(T), was isolated from a sea salt evaporation pond on the Island of Sal in the Cape Verde Archipelago. This organism was found to be a catalase- and oxidase-positive, non-motile, spore-forming, aerobic, curved rod-shaped organism with an optimum growth temperature of about 35-37 degrees C and an optimum pH between 7.5 and 8.0. Optimal growth occurred in media containing 4-6% (w/v) NaCl and no growth occurred in medium without NaCl. The cell-wall peptidoglycan was of the A1gamma type with meso-diaminopimelic acid, the major respiratory quinone was MK-7, the major fatty acids were iso-15:0, 16:0, anteiso-15:0 and iso-16:0 and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified aminoglycophospholipid. The G+C content of the DNA was 37.9 mol%. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain CVS-8(T) represented a novel species of the genus Bacillus, the highest levels of sequence similarity (mean pairwise similarity values of approximately 97.5 %) being found with respect to the type strains of Bacillus shackletonii and Bacillus acidicola. On the basis of the phylogenetic, physiological and biochemical data, strain CVS-8(T) represents a novel species of the genus Bacillus, for which the name Bacillus isabeliae sp. nov. is proposed. The type strain is CVS-8(T) (=LMG 22838(T)=CIP 108578(T)).

Salirhabdus euzebyi gen. nov., sp. nov., a Gram-positive, halotolerant bacterium isolated from a sea salt evaporation pond.

A low-G+C, Gram-positive bacterium, designated CVS-14(T), was recovered from a sea salt evaporation pond on the island of Sal in the Cape Verde Archipelago. This organism was catalase- and oxidase-positive. Cells were motile, spore-forming aerobic rods, with an optimum growth temperature of about 35-40 degrees C and optimum pH between 7.0 and 8.5. Optimal growth occurred in media containing 4-6 % (w/v) NaCl, although the organism was able to grow in medium without added NaCl and in medium containing 16 % NaCl. The cell-wall peptidoglycan was of A1 gamma type and the major respiratory quinone was menaquinone 7 (MK-7). Major fatty acids were iso-15 : 0, anteiso-15 : 0, iso-17 : 0 and anteiso-17 : 0. The DNA G+C content was 37.0 mol%. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain CVS-14(T) formed a distinct new branch within the radiation of the moderately halophilic bacilli group, forming a separate lineage from species of the genera Salinibacillus, Paucisalibacillus, Oceanobacillus, Lentibacillus and Virgibacillus. Strain CVS-14(T) showed 16S rRNA gene pairwise similarity values of approximately 95 % with species of the genus Salinibacillus. On the basis of morphological, physiological, chemotaxonomic and phylogenetic characteristics, strain CVS-14(T) is considered to represent a novel species in a new genus, for which the name Salirhabdus euzebyi gen. nov., sp. nov. is proposed. The type strain is CVS-14(T) (=LMG 22839(T)=CIP 108577(T)).