Parvicella tangerina gen. nov., sp. nov. (Parvicellaceae fam. nov., Flavobacteriales), first cultured representative of the marine clade UBA10066, and Lysobacter luteus sp. nov., from activated sludge of a seawater-processing wastewater treatment plant.

Two strains isolated from a sample of activated sludge that was obtained from a seawater-based wastewater treatment plant on the southeastern Mediterranean coast of Spain have been characterized to achieve their taxonomic classification, since preliminary data suggested they could represent novel taxa. Given the uniqueness of this habitat, as this sort of plants are rare in the world and this one used seawater to process an influent containing intermediate products from amoxicillin synthesis, we also explored their ecology and the annotations of their genomic sequences. Analysis of their 16S rRNA gene sequences revealed that one of them, which was orange-pigmented, was distantly related to Vicingus serpentipes (family Vicingaceae) and to other representatives of neighbouring families in the order Flavobacteriales (class Flavobacteriia) by 88-89 % similarities; while the other strain, which was yellow-pigmented, was a putative new species of Lysobacter (family Xanthomonadaceae, order Xanthomonadales, class Gammaproteobacteria) with Lysobacter arseniciresistens as closest relative (97.3 % 16S rRNA sequence similarity to its type strain). Following a polyphasic taxonomic approach, including a genome-based phylogenetic analysis and a thorough phenotypic characterization, we propose the following novel taxa: Parvicella tangerina gen. nov., sp. nov. (whose type strain is AS29M-1T=CECT 30217T=LMG 32344T), Parvicellaceae fam. nov. (whose type genus is Parvicella), and Lysobacter luteus sp. nov. (whose type strain is AS29MT=CECT 30171T=LMG 32343T).

Chryseobacterium potabilaquae sp. nov., Chryseobacterium aquaeductus sp. nov. and Chryseobacterium fistulae sp. nov., from drinking water systems.

A polyphasic taxonomic study was conducted on three strains isolated from drinking water systems that had previously been deposited as Chryseobacterium species at the Spanish Type Culture Collection in order to complete their classification. Strains CECT 9293T, CECT 9390T and CECT 9393T were isolated from sites in Barcelona, Spain, in the framework of a project aimed at generating the first MALDI-TOF database specific for bacteria present in water for human consumption. Their partial 16S rRNA sequences showed that their closest relatives among the type strains of Chryseobacterium exhibited 98 % similarity or less, supporting their taxonomic novelty. At the same time, comparison between them revealed that strains CECT 9293T and CECT 9393T could perhaps be related at the species level as they shared 99.5 % similarity. However, whole genome sequencing was performed and the subsequent calculation of relatedness indices, average nucleotide identity and estimated DNA-DNA hybridization, ruled out that possibility and confirmed instead that each of the strains should be considered a separate species in the genus Chryseobacterium. Having clarified their status, we also performed phylogenomic analyses and searched for possible environmental or non-type material sequences that could be related to any of them at the species level. In parallel, the strains were characterized phenotypically and compared to their closest relatives to determine diagnostic traits to support their formal proposal. The proposed species are Chryseobacterium potabilaquae sp. nov. with the type strain CECT 9293T (=LMG 32084T), Chryseobacterium aquaeductus sp. nov. with the type strain CECT 9390T (=LMG 32085T) and Chryseobacterium fistulae sp. nov. with the type strain CECT 9393T (=LMG 32086T).

Mycobacterium avium subsp. paratuberculosis (Map) Fatty Acids Profile Is Strain-Dependent and Changes Upon Host Macrophages Infection.

Johne's disease is a chronic granulomatous enteritis of ruminants caused by the intracellular bacterium Mycobacterium avium subsp. paratuberculosis (Map). We previously demonstrated that Map isolates from sheep persisted within host macrophages in lower CFUs than cattle isolates after 7 days of infection. In the current study, we hypothesize that these phenotypic differences between Map isolates may be driven be the fatty acids (FAs) present on the phosphadidyl-1-myo-inositol mannosides of the Map cell wall that mediate recognition by the mannose receptors of host macrophages. FAs modifications may influence Map's envelope fluidity ultimately affecting pathogenicity. To test this hypothesis, we investigated the responses of two Map isolates from cattle (K10 isolate) and sheep (2349/06-1) to the bovine and ovine macrophage environment by measuring the FAs content of extracellular and intracellular bacteria. For this purpose, macrophages cell lines of bovine (BOMAC) and ovine (MOCL-4) origin were infected with the two isolates of Map for 4 days at 37°C. The relative FAs composition of the two isolates recovered from infected BOMAC and MOCL-4 cells was determined by gas chromatography and compared with that of extracellular bacteria and that of bacteria grown in Middlebrook 7H9 medium. Using this approach, we demonstrated that the FAs composition of extracellular and 7H9-grown bacteria was highly conserved within each Map isolate, and statistically different from that of intracellular bacteria. Analysis of FAs composition from extracellular bacteria enabled the distinction of the two Map strains based on the presence of the tuberculostearic acid (18:0 10Me) exclusively in the K10 strain of Map. In addition, significant differences in the content of Palmitic acid and cis-7 Palmitoleic acid between both isolates harvested from the extracellular environment were observed. Once the infection established itself in BOMAC and MOCL-4 cells, the FAs profiles of both Map isolates appeared conserved. Our results suggest that the FAs composition of Map might influence its recognition by macrophages and influence the survival of the bacillus within host macrophages.

Marinomonas blandensis sp. nov., a novel marine gammaproteobacterium.

A novel Gram-staining-negative, chemoorganotrophic, moderately halophilic, strictly aerobic bacterium, strain MED121T, was isolated from a seawater sample collected at the Blanes Bay Microbial Observatory in the north-western Mediterranean Sea. Analysis of its 16S rRNA gene sequence, retrieved from the whole-genome sequence, showed that this bacterium was most closely related to Marinomonas dokdonensis and other Marinomonas species (96.3 and 93.3-95.7 % sequence similarities, respectively), within the family Oceanospirillaceae. Strain MED121T was included into a whole-genome sequencing study and, subsequently, it was characterized using a polyphasic taxonomic approach. It was found to be oxidase and catalase positive, its cells are cocci to short rods, it does not ferment carbohydrates and does not reduce nitrate to nitrite or gas and it requires at least 2.5 % (w/v) marine salts and tolerates up to 7 % (w/v) salts. Its major cellular fatty acids in order of abundance are C16 : 1ω7c/C16 : 1ω6c, C18 : 1ω7c, C16 : 0 and C10 : 0 3-OH. Its genome had an approximate length of 5.1 million bases and a DNA G+C content equal to 40.9 mol%. Analysis of the annotated genes reveals the capacity for the synthesis of ubiquinone 8 (Q8) and the polar lipids phosphatidylglycerol and phosphatidylethanolamine, in agreement with other members of the genus. All the data collected supported the creation of a novel species to accommodate this bacterium, for which the name Marinomonas blandensis sp. nov. is proposed. The type strain is MED121T (=CECT 7076T=LMG 29722T).

Roseovarius albus sp. nov., a new Alphaproteobacterium isolated from the Mediterranean Sea.

Strain 4SM10(T), an aerobic marine, Gram-negative, heterotrophic and non pigmented bacterium isolated from seawater from Vinaroz in Castellón, Spain, was characterized using a polyphasic approach. Analysis of the 16S rRNA gene sequence placed the strain within the Roseobacter clade in the family Rhodobacteraceae. Phylogenetic analyses also showed that strain 4SM10(T) forms a stable clade with species of the genus Roseovarius, being related to Roseovarius nubinhibens ISM(T) and Roseovarius aestuarii SMK-122(T) at 97.5 and 97.4 % 16S rRNA sequence similarity, respectively. Average Nucleotide Identity (ANI) values, determined as a measure of overall genomic resemblance, confirmed that strain 4SM10(T) does not belong to the same species as R. aestuarii CECT 7745(T) and Roseovarius nubinhibens CECT 7750(T) displaying ANI values well below the 95 % boundary for genomic species. Strain 4SM10(T) requires Na(+) plus a divalent cation (either Mg(2+) or Ca(2+)) to grow, reduces nitrate to nitrite and uses a large number of amino acids and organic acids (but no carbohydrates) as sole carbon sources. Enzymatic activities displayed in API ZYM tests are alkaline phosphatase, leucine arylamidase and acid phosphatase. The major cellular fatty acids were identified as C18:1 ω7c and/or C18:1 ω6c (67.1 %). The DNA G+C content was determined to be 54.27 mol%. Based on the genotypic and phenotypic data obtained, the name Roseovarius albus sp. nov. is proposed for this novel taxon, with the type strain 4SM10(T) (=CECT 7450(T) = KCTC 22653(T)).

Description of Tropicibacter mediterraneus sp. nov. and Tropicibacter litoreus sp. nov.

Four strains (M15∅_3, M17(T), M49 and R37(T)) were isolated from Mediterranean seawater at Malvarrosa beach, Valencia, Spain. Together with an older preserved isolate (strain 2OM6) from cultured oysters at Vinaroz, Castellón, Spain, the strains were thoroughly characterized in a polyphasic study and were placed phylogenetically within the Roseobacter clade in the family Rhodobacteraceae. Highest 16S rRNA sequence similarities of the five strains to the types of any established species corresponded to Tropicibacter multivorans (95.8-96.4%), Phaeobacter inhibens (95.9-96.3%) and Phaeobacter gallaeciensis (95.9-96.2%). On the other hand, whole genome (ANI) and protein fingerprinting (MALDI-TOF) data proved: (i) non clonality among the strains, and (ii) the existence of two genospecies, one consisting of strains M15∅_3, M17(T), M49 and 2OM6 and another one consisting of strain R37(T). Phenotypic traits determined allow differentiating both genospecies from each other and from closely related taxa. In view of all data collected we propose to accommodate these isolates in two species as members of the genus Tropicibacter, Tropicibacter mediterraneus sp. nov. (type strain M17(T)=CECT 7615(T)=KCTC 23058(T)) and Tropicibacter litoreus sp. nov. (type strain R37(T)=CECT 7639(T)=KCTC 23353(T)).

Marinifilum flexuosum sp. nov., a new Bacteroidetes isolated from coastal Mediterranean Sea water and emended description of the genus Marinifilum Na et al., 2009.

A facultatively anaerobe, moderately halophilic, Gram-negative, filamentous, non motile and unpigmented bacterium, designated M30(T), was isolated from coastal Mediterranean Sea water in Valencia, Spain. Phylogenetic analysis based on 16S rRNA sequences placed this strain in the phylum "Bacteroidetes" with Marinifilum fragile JC2469(T) as its closest relative with 97% sequence similarity. Average nucleotide identity (ANI) values between both strains were far below the 95% threshold value for species delineation (about 89% using BLAST and about 90% using MUMmer). A comprehensive polyphasic study, including morphological, biochemical, physiological, chemotaxonomic and phylogenetic data, confirmed the independent species status of strain M30(T) within the genus Marinifilum, for which the name Marinifilum flexuosum sp. nov. is proposed. The type strain of Marinifilum flexuosum is M30(T) (=CECT 7448(T)=DSM 21950(T)).

Vibrio aestivus sp. nov. and Vibrio quintilis sp. nov., related to Marisflavi and Gazogenes clades, respectively.

Two new Vibrio species, Vibrio aestivus and Vibrio quintilis, are described after a polyphasic characterization of strains M22(T), M61 and M62(T), isolated from seawater collected off a beach on the East coast of Spain (Valencia). All three strains are Gram negative, mesophilic, slightly halophilic, fermentative rods. V. aestivus (M22(T)=CECT 7558(T)=CAIM 1861(T)=KCTC 23860(T) and M61=CECT 7559=CAIM 1862=KCTC 23861) is oxidase positive, reduces nitrates to nitrites, is negative for Voges Proskauer, arginine dihydrolase and indole and non hydrolytic on most substrates tested. The 16S rRNA gene sequences of M22(T) and M61 are most similar to Vibrio marisflavi (97.1-97.2%) but phylogenetic analysis using NJ, MP and ML methods display Vibrio stylophorae (96.2% similarity) as sibling species. The three species form a deep clade in the genus Vibrio. Average Nucleotide Identity (ANI) values, determined as a measure of overall genomic resemblance, confirmed that strains M22(T) and M61 are members of the same species, different to V. marisflavi CECT 7928(T). V. quintilis (M62(T)=CECT 7734(T)=CAIM 1863(T)=KCTC 23833(T)) is aerogenic, arginine dihydrolase and Voges Proskauer positive, oxidase negative and unable to reduce nitrate, traits shared by most species in the Gazogenes clade. It is unpigmented and does not grow on TCBS Agar. 16S rRNA gene similarities to its nearest species, Vibrio aerogenes and Vibrio mangrovi, are 97.6% and 96.0% respectively. Strain M62(T) and V. aerogenes CECT 7868(T) display ANI values well below the 95% boundary for genomic species.

Tropicibacter multivorans sp. nov., an aerobic alphaproteobacterium isolated from surface seawater.

Strain MD5T, an aerobic marine alphaproteobacterium, was isolated from Mediterranean seawater at Malvarrosa beach, Valencia, Spain. The strain was characterized in a polyphasic study and was placed phylogenetically within the Roseobacter clade in the family Rhodobacteraceae. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain MD5T is related to Tropicibacter naphthalenivorans C02T, Phaeobacter inhibens T5T, P. gallaeciensis BS107T and P. daeponensis TF-218T, with 96.9, 96.2, 96.1 and 96.1 % sequence similarity, respectively. Phylogenetic analyses also showed that strain MD5T forms a stable clade only with T. naphthalenivorans C02T. Strain MD5T requires Na+ plus a divalent cation (either Mg2+ or Ca2+) to grow, does not reduce nitrate to nitrite and uses a large number of carbohydrates as sole carbon sources. It is positive for ß-galactosidase and urease activities and aesculin hydrolysis. Enzyme activities displayed in the API ZYM strip were alkaline phosphatase, leucine arylamidase, acid phosphatase and α-glucosidase. Major cellular fatty acids were summed feature 8 (C18:1ω7c and/or C18:1ω6c; 70.9 %) and C16:0 (8.2 %). The results of physiological and biochemical tests allowed clear phenotypic differentiation of this isolate from the only described species of the genus Tropicibacter. It is evident from the genotypic and phenotypic data obtained that the strain should be classified in a novel species in the genus Tropicibacter. The name Tropicibacter multivorans sp. nov. is proposed, with the type strain MD5T (=CECT 7557T=KCTC 23350T).

Pseudomonas litoralis sp. nov., isolated from Mediterranean seawater.

Strains 2SM5(T) and 2SM6, two strictly aerobic chemo-organotrophic gammaproteobacteria, were isolated from Mediterranean seawater off the coast of Vinaroz, Castellón, Spain, in February, 1990. They were extensively characterized by a polyphasic study that placed them in the genus Pseudomonas. Phylogenetic analysis of 16S rRNA gene sequences showed that both strains shared 100 % sequence similarity and were closely related to members of the Pseudomonas pertucinogena clade, with less than 97.3 % similarity to strains of established species; Pseudomonas xiamenensis was the closest relative. Analysis of sequences of three housekeeping genes, rpoB, rpoD and gyrB, further confirmed the phylogenetic assignment of the Mediterranean isolates. Chemotaxonomic traits such as quinone and polar lipid composition also corroborated the placement of strains 2SM5(T) and 2SM6 in the gammaproteobacteria. Other phenotypic traits, including fatty acid composition, enabled clear differentiation of both isolates from other species of Pseudomonas. We therefore conclude that strains 2SM5(T) and 2SM6 represent a novel species of Pseudomonas, for which the name Pseudomonas litoralis is proposed; the type strain is 2SM5(T) ( = CECT 7670(T) = KCTC 23093(T)).

Photobacterium aphoticum sp. nov., isolated from coastal water.

A facultatively anaerobic marine gammaproteobacterium, designated strain M46(T), was isolated from Mediterranean seawater at Malvarrosa beach, Valencia, Spain. The strain was characterized by using a polyphasic approach and was found to be situated within the genus Photobacterium in the family Vibrionaceae. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain M46(T) was closely related to P. rosenbergii CECT 7644(T), P. halotolerans CECT 5860(T) and P. ganghwense CECT 7641(T), showing sequence similarities of 96.8, 96.4 and 96.2 %, respectively. According to the results of phylogenetic analyses based on recA and gyrB gene sequences, the most closely related taxon was P. ganghwense CECT 7641(T) with 87.4 and 85.0 % sequence similarity, respectively. Regardless of the gene used in phylogenetic analysis, strain M46(T) always formed a separate and stable clade containing these three species of the genus Photobacterium. Strain M46(T) was not luminescent and produced a diffusible brown pigment. It required NaCl to grow, reduced nitrate to nitrite and oxidized a small number of substrates in Biolog GN plates. Strain M46(T) was positive for arginine dihydrolase (ADH), ß-galactosidase, aesculin hydrolysis and DNase activity. In API ZYM tests, the novel strain was positive for alkaline phosphatase, leucine arylamidase and acidic phosphatase activities. The major cellular fatty acids were unsaturated C(18) and C(16), as in other members of the genus Photobacterium, but their relative amounts and the presence or absence of other fatty acids differentiated strain M46(T) from its closest relatives. Based on the results of this polyphasic taxonomic study, strain M46(T) represents a novel species of the genus Photobacterium, for which the name Photobacterium aphoticum is proposed. The type strain is M46(T) ( = CECT 7614(T)  = KCTC 23057(T)).