Photobacterium pectinilyticum sp. nov., a novel bacterium isolated from surface seawater of Qingdao offshore.

A Gram-staining-negative, facultative aerobic, motile strain, designated strain ZSDE20T, was isolated from the surface seawater of Qingdao offshore. Phylogenetic analysis of the 16S rRNA gene of strain ZSDE20T, affiliated it to the genus Photobacterium. It was closely related to Photobacterium lutimaris DF-42 T (98.92% 16S rRNA gene sequence similarity). Growth occurred at 4-28ºC (optimum 28ºC), pH 1.0-7.0 (optimum 7.0) and in the presence of 1-7% (w/v) NaCl (optimum 3%). The dominant fatty acids were summed feature 3 (C16:1 ω7c or/and C16:1 ω6c, 34.23%), summed feature 8 (C18:1 ω7c and C18:1 ω6c, 10.36%) and C16:0 (20.05%). The polar lipids of strain ZSDE20T comprised phosphatidylethanolamine, phosphatidylcholine, lyso-phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol dimannoside, phosphatidylinositol mannosides and two unknown lipids. The major respiratory quinone was ubiquinone-8 (Q-8). The DNA G + C content of strain ZSDE20T was 45.6 mol%. Average nucleotide identity (ANI) values between ZSDE20T and its reference species were lower than the threshold for species delineation (95-96%); in silico DNA-DNA hybridization further showed that strain ZSDE20T had less than 70% similarity to its relatives. Based on the polyphasic evidences, strain ZSDE20T is proposed as representing a novel species of the genus Photobacterium, for which the name Photobacterium pectinilyticum sp. nov. is proposed. The type strain is ZSDE20T (= MCCC 1K06283T = KCTC 82885 T).

The strains of bioluminescent bacteria isolated from the White Sea finfishes: genera Photobacterium, Aliivibrio, Vibrio, Shewanella, and first luminous Kosakonia.

Bioluminescence is a spectacular feature of some prokaryotes. In the present work, we address the distribution of bioluminescence among bacteria isolated from the White Sea finfishes. Luminous bacteria are widely distributed throughout the World Ocean. Many strains have been isolated and described for tropical latitudes, while Nordic seas still remain quite a white spot in studying bioluminescence of bacteria. We describe the strains related to the two main genera of luminous bacteria, Photobacterium and Aliivibrio, as well as Shewanella and Vibrio. They are related to families Vibrionaceae and Shewanellaceae of the Gammaproteobacteria class. Here, we at the first time, report the bioluminescence of the Enterobacteriaceae Kosakonia cowanii. Moreover, we applied the polyphasic approach to identify and describe the isolated microorganisms. The data on sequencing, diversity of cell fine structure, and light emission spectra at room temperature on the solid medium are discussed. The bacteria are characterized by features in their light emission spectra. It may reflect possible molecular mechanisms of bioluminescence as well as features of bacterial composition. The obtained data expands the existing body of knowledge about the bioluminescence spread among the bacteria of Nordic latitudes and provides complex information that is crucial for their precise identification.

Photobacterium salinisoli sp. nov., isolated from a sulfonylurea herbicide-degrading consortium enriched with saline soil.

A Gram-stain-negative, aerobic, motile, rod-shaped bacterium, designated strain LAM9072T, was isolated from a sample of a sulfonylurea herbicide-degrading consortium enriched with saline soil. The optimal temperature and pH for the growth of strain LAM9072T were 35 °C and 7.0, respectively. Strain LAM9072T could grow in the presence of NaCl up to 9 % (w/v). Comparative analysis of the 16S rRNA gene sequences revealed that strain LAM9072T was closely related to members of the family Vibrionaceae, with the highest similarities to Photobacterium halotolerans MACL01T (97.7 %) and Photobacterium galatheae S2753T (97.7 %). Strain LAM9072T formed a distinct phylogenetic subclade within the genus Photobacterium in the 16S rRNA gene phylogenetic trees. The results of multi-locus sequence analysis revealed a distinct lineage with P. halotolerans MACL01T as its closest relative. The genomic G+C content was 50.2 mol%. The DNA-DNA hybridization values between strain LAM9072T and P. halotolerans LMG 22194T and P. galatheae LMG 28894T were 41.6 and 22.2 %, respectively. The average nucleotide identity values were 90.9 and 78.8 %, respectively, by comparing the draft genome sequences of strain LAM9072T and P. halotolerans LMG 22194T and P. galatheae LMG 28894T. The major fatty acids were summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c), C16 : 0 and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). Ubiquinone 8 was detected as the predominant respiratory quinone. The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, aminophospholipid and four unidentified lipids. Based on its phenotypic characteristics and the results of genotypic analyses, we propose that strain LAM9072T represents a novel species, for which the name Photobacteriumsalinisoli sp. nov. is proposed. The type strain is LAM9072T (=ACCC 19961T=JCM 30852T).

Photobacterium chitinilyticum sp. nov., a marine bacterium isolated from seawater at the bottom of the East China Sea.

A Gram-stain-negative, facultative aerobic, motile by a polar flagellum, rod-shaped strain, designated BEI247T, was isolated from seawater at the bottom of the East China Sea. Phylogenetic analysis of the 16S rRNA gene and whole genome data affiliated it with the genus Photobacterium. It was most closely related to Photobacterium alginatilyticum P03D4T (97.36 % 16S rRNA gene similarity). Multi-locus sequence analysis (MLSA) revealed a distinct lineage with P. alginatilyticum P03D4T as its closest relative. Strain BEI247T was found to have lower than 86.0 % similarities to the type strains of its most closely related species in MLSA, less than 82.3 % using genome average nucleotide identities, and less than 25.3 % in DNA-DNA relatedness studies. Growth occurred at 10-37 °C (optimum, 24 °C), pH 5.0-8.0 (pH 7.0) and in the presence of 1-5 % (w/v) NaCl (3 %). The dominant fatty acids were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and C16 : 0. The polar lipids of strain BEI247T comprised phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, two phospholipids and one unknown lipid. The major respiratory quinone was ubiquinone-8 (Q-8). The DNA G+C content of strain BEI247T was 46.45 mol%. On the basis of the polyphasic evidence, strain BEI247T is proposed as representing a novel species of the genus Photobacterium, for which the name Photobacterium chitinilyticum sp. nov. is proposed. The type strain is BEI247T (=JCM 32689T=MCCC 1K03517T=KCTC 62619T).

Molecular and phenotypic characterization of Photobacterium damselae among some marine fishes in Lake Temsah.

Photobacterium damselae species are one of the most devastating bacterial pathogens in mariculture worldwide. Some species of Photobacterium are pathogenic for marine animals and human. They are the causative agents of photobacteriosis, formerly known as pasteurellosis. A total of (202) marine fishes of three different species were represented as: seabass (Dicentrarchus labrax), seabream (Sparus aurata) and gray mullet (Mugil capitus) randomly collected from Lake Temsah at Ismailia governorate along the parallel Pelagic road to the lake in the governorate from August 2015 to July 2016. The clinical picture and gross lesions of the diseased fishes were recorded. Isolation and identification of suspected bacteria using traditional and molecular methods. Samples from affected organs were collected for studying the histopathological alterations of these pathogens. Fifty one fishes were found to be infected with Photobacterium damselae subsp. Piscicida. Seabass (Dicentrarchus labrax) was the most infected fish species (23), followed by seabream (Sparus aurata) (18) finally gray mullet (Mugil capitus) was (10). 91fishes were found to be infected with P. damselae subsp. damselae, seabass (Dicentrarchus labrax) was the most infected fish sp. (36), followed by seabream (Sparus aurata) (32), then gray mullet (Mugil capitus) (23). The results indicated that, the total prevalence of P. damselae subsp. piscicida in all examined species (25.24%), the highest seasonal prevalence was recorded in summer season (37.09%) followed by autumn (26%) then spring (20.37%) and winter (11.11%). On the other hand, the total prevalence of P. damselae subsp. damselae in all examined species (45.04%), the highest seasonal prevalence was recorded in summer season (67.74%) followed by autumn (52%) then spring (29.62%) and winter (19.44%). Molecular diagnosis with conventional PCR used to confirm the traditional isolation was applied by using specific primers of two genes (polycapsular saccharide gene and urease C gene). The histopathological studies of naturally infected marine fishes showed severe inflammatory reactions in different organs with accumulation of melanomacrophages and necrosis. The results confirm that P. damselae subspecies damsalea is the most prevalent pathogen between marine fishes, and seabass (Dicentrarchus labrax) was the highly affected marine fishes in this study.

Photobacterium alginatilyticum sp. nov., a marine bacterium isolated from bottom seawater.

A Gram-straining-negative, facultatively aerobic, rod-shaped strain, motile by a polar flagellum and designated P03D4T, was isolated from the bottom seawater of the East China Sea. Growth occurred at 10-50 °C (optimum 32 °C), pH 5.0-10.0 (optimum pH 6.0) and in the presence of 1-7 % (w/v) NaCl (optimum 3 %). Phylogenetic analysis based on 16S rRNA gene sequence placed P03D4T within the genus Photobacterium of the family Vibrionaceae in the class Gammaproteobacteria, and revealed that strain P03D4T was most closely related to Photobacterium frigidiphilum SL13T with 96.9 % sequence similarity and had sequence similarities with other species of the genus Photobacterium in the range 94.6-96.9 %. The dominant fatty acids were summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH) and C16 : 0. The polar lipids of strain P03D4T comprised phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine and one unknown lipid. The major respiratory quinone was ubiquinone-8 (Q-8). The DNA G+C content of strain P03D4T was 44.3 mol%. On the basis of the evidence from this polyphasic study, strain P03D4T is proposed as representing a novel species of the genus Photobacterium, for which the name Photobacterium alginatilyticum sp. nov. is proposed. The type strain is P03D4T (=KCTC 52365T=MCCC 1K03200T=CGMCC 1.15764T).

Photobacterium panuliri sp. nov., an alkalitolerant marine bacterium isolated from eggs of spiny lobster, Panulirus penicillatus from Andaman Sea.

A facultative anaerobe, alkalitolerant, gram-negative marine bacterium strain LBS5(T), was isolated from eggs carried on the pleopods of female spiny lobster (Panulirus penicillatus) in Andaman Sea from a depth of 3.5 m. Heterotrophic growth was observed at 15-38 °C and pH 5.5-11. Optimum growth occurred at 28 °C and pH 7.5. It can grow in the presence of 0.5-7 % NaCl (w/v), and the optimal NaCl required for growth was 2-4 %. 16S rRNA gene sequence analysis revealed the strain LBS5(T) belongs to the genus Photobacterium and showed 99.6 % similarity with P. aquae AE6(T), 98.2 % with P. aphoticum M46(T), 97 % with P. rosenbergii CC1(T), 96.9 % with P. lutimaris DF-42(T), and 96.6 % with P. halotolerans MACL01(T). The DNA-DNA similarities between strains LBS5(T) with other closely related strains were well below 70 %. The DNA G + C content was 50.52 (±0.9) mol%. The major fatty acids were C16:1w7c/w6c, C18:1w6c/w7c, C16:0, C15:0 iso, C16:0 10-methyl/17:1 iso w9c, C17:0 iso. Polar lipids included a phosphatidylglycerol, a diphosphatidylglycerol, a phosphatidylethanolamine, and one unidentified lipid. Based on the polyphasic evidences, strain LBS5(T) represents a novel species of the genus Photobacterium for which Photobacterium panuliri sp. nov. is proposed. The type strain is LBS5(T) (=DSM 27646(T) = LMG 27617(T) = JCM 19199(T)).

Photobacterium piscicola sp. nov., isolated from marine fish and spoiled packed cod.

Five isolates from marine fish (W3(T), WM, W1S, S2 and S3) and three isolates misclassified as Photobacterium phosphoreum, originating from spoiled modified atmosphere packed stored cod (NCIMB 13482 and NCIMB 13483) and the intestine of skate (NCIMB 192), were subjected to a polyphasic taxonomic study. Phylogenetic analysis of 16S rRNA gene sequences showed that the isolates were members of the genus Photobacterium. Sequence analysis using the gapA, gyrB, pyrH, recA and rpoA loci showed that these isolates formed a distinct branch in the genus Photobacterium, and were most closely related to Photobacterium aquimaris, Photobacterium kishitanii, Photobacterium phosphoreum and Photobacterium iliopiscarium. The luxA gene was present in isolates W3(T), WM, W1S, S2 and S3 but not in NCIMB 13482, NCIMB 13483 and NCIMB 192. AFLP and (GTG)5-PCR fingerprinting indicated that the eight isolates represented at least five distinct genotypes. DNA-DNA hybridizations revealed 89% relatedness between isolate W3(T) and NCIMB 192, and values below 70% with the type strains of the phylogenetically closest species, P. iliopiscarium LMG 19543(T), P. kishitanii LMG 23890(T), P. aquimaris LMG 26951(T) and P. phosphoreum LMG4233(T). The strains of this new taxon could also be distinguished from the latter species by phenotypic characteristics. Therefore, we propose to classify this new taxon as Photobacterium piscicola sp. nov., with W3(T) (=NCCB 100098(T)=LMG 27681(T)) as the type strain.

Photobacterium marinum sp. nov., a marine bacterium isolated from a sediment sample from Palk Bay, India.

The novel, cream colored, Gram-staining-negative, rod-shaped, motile bacteria, designated strains AK15(T) and AK18, were isolated from sediment samples collected from Palk Bay, India. Both strains were positive for arginine dihydrolase, lysine decarboxylase, oxidase, nitrate reduction and methyl red test. The major fatty acids were C16:0, C18:1 ω7c, C16:1 ω7c and/or C16:1 ω6c and/or iso-C15:0 2-OH (summed feature 3). Polar lipids content of strains AK15(T) and AK18 were found to bephosphatidylethanolamine (PE), two unidentified phospholipids (PL1 and PL2) and three unidentified lipids (L1-L3). The 16S rRNA gene sequence analysis indicated strains AK15(T) and AK18 as the members of the genus Photobacterium and closely related to the type strain Photobacterium jeanii with pair-wise sequence similarity of 96.7%. DNA-DNA hybridization between strain AK15(T) and AK18 showed a relatedness of 87%. Based on data from the current polyphasic study, strains AK15(T) and AK18 are proposed as novel species of the genus Photobacterium, for which the name Photobacterium marinum sp. nov. is proposed. The type strain of Photobacterium marinum is AK15(T) (=MTCC 11066(T)=DSM 25368(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)).

Evaluation of different DNA-based fingerprinting methods for typing Photobacterium damselae ssp. piscicida

This study evaluates the effectiveness of three different molecular techniques, repetitive extragenic palindromic PCR (REP-PCR), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) and the random amplified polymorphic DNA (RAPD-PCR) for rapid typing of Photobacterium damselae ssp. piscicida strains isolated from different species of marine fish and geographic areas. The results obtained by the three methods showed that RAPD and ERIC-PCR were more discriminative for suitable rapid typing of Ph. damselae ssp. piscicida than REP-PCR. The analysis of DNA banding patterns generated by both molecular methods (RAPD and ERIC-PCR) clearly separated the strains into two main groups that strongly correlated with their geographic origin. Moreover, the REP-PCR analysis was less reproducible than the RAPD and ERIC-PCR methods and does not allow the establishment of genetic groups. RAPD and ERIC-PCR constitute valuable tools for molecular typing of Ph. damselae ssp. piscicida strains, which can be used in epidemiological studies of photobacteriosis infections.

Photobacterium ganghwense sp. nov., a halophilic bacterium isolated from sea water.

A marine bacterial strain, designated FR1311T, was isolated from a sea-water sample from Ganghwa Island, South Korea. Cells were Gram-negative, facultatively anaerobic, catalase- and oxidase-positive, motile, oval or rod-shaped and halophilic (optimum sea-salt concentration for growth of 5-6 %). Phylogenetic analysis of its 16S rRNA gene sequence revealed that it represented a distinct line of descent within the genus Photobacterium. The major fatty acids were straight-chain saturated (C(16 : 0)) and monounsaturated fatty acids (C(16 : 1)omega7c and C(18 : 1)omega7c). The predominant respiratory lipoquinone was Q-8. The DNA G+C content was 44 mol%. The phenotypic features of strain FR1199T were similar to those of Photobacterium damselae subsp. damselae and Photobacterium damselae subsp. piscicida, but several physiological and chemotaxonomic properties readily distinguish the new isolate from them. On the basis of the polyphasic results revealed in this study, FR1311T is considered to be the type strain of a novel species, for which the name Photobacterium ganghwense sp. nov. is proposed. The type strain is FR1311T (=IMSNU 60287T = KCTC 12328T = JCM 12487T).

Photobacterium rosenbergii sp. nov. and Enterovibrio coralii sp. nov., vibrios associated with coral bleaching.

Six new Vibrio-like isolates originating from different species of bleached and healthy corals around Magnetic Island (Australia) were investigated using a polyphasic approach. Phylogenetic analyses based on 16S rRNA, recA and rpoA gene sequences split the isolates in two new groups. Strains LMG 22223(T), LMG 22224, LMG 22225, LMG 22226 and LMG 22227 were phylogenetic neighbours of Photobacterium leiognathi LMG 4228(T) (95.6 % 16S rRNA gene sequence similarity), whereas strain LMG 22228(T) was related to Enterovibrio norvegicus LMG 19839(T) (95.5 % 16S rRNA gene sequence similarity). The two new groups can be distinguished from closely related species on the basis of several phenotypic features, including fermentation of d-mannitol, melibiose and sucrose, and utilization of different compounds as carbon sources, arginine dihydrolase activity, nitrate reduction, resistance to the vibriostatic agent O/129 and the presence of fatty acids 15 : 0 iso and 17 : 0 iso. The names Photobacterium rosenbergii sp. nov. (type strain LMG 22223(T)=CBMAI 622(T)=CC1(T)) and Enterovibrio coralii sp. nov. (type strain LMG 22228(T)=CBMAI 623(T)=CC17(T)) are proposed to accommodate these new isolates. The G+C contents of the DNA of the two type strains are respectively 47.6 and 48.2 mol%.

Virulence of Photobacterium damselae subsp. piscicida in cultured cobia Rachycentron canadum.

An outbreak of serious mortality among the cultured cobia Rachycentron canadum (weighing 3 kg) characterized by the presence of whitish granulomatous deposits on the kidney, liver and spleen occurred in July of 2000 in Taiwan. A non-motile strain CP1 was isolated from kidney and/or liver on tryptic soy agar and/or brain heart infusion agar plates (both supplemented with 1% NaCl, w/v). This strain was characterized and identified as Photobacterium damselae subsp. piscicida using biochemical characteristics and Bionor mono-Pp tests. The bacterium and its extracellular products (ECP) were lethal to the cobia (weighing 10 g) with LD50 values of 1.03 x 10(4) colony forming units and 1.26 microg protein/g fish body weight, respectively. All the moribund/dead fish exhibited darkness in color with no gross or internal leasions. However, the bacteria could be reisolated from kidney and liver after bacterial challenge. The present results reveal that Ph. damselae subsp. piscicida is the causative agent of fish photobacteriosis in the cobia and the bacterium isolated from sub-adult cobia (chronic form) is virulent to young cobia causing acute form of the disease.

Strain characterization and classification of oxyphotobacteria in clone cultures on the basis of 16S rRNA sequences from the variable regions V6, V7, and V8.

A major problem in development of a polyphasic taxonomy is that the identification of oxyphotobacterial strains (cyanobacteria and prochlorophytes) in culture collections may be incorrect. We have therefore developed a diagnostic system using the DNA sequence polymorphism in the 16S rRNA regions V6 to V8 for individual strain characterization and identification. PCR primers amplifying V6 to V8 from oxyphotobacteria in unialgal cultures were constructed. Direct solid-phase or cyclic sequencing was used to determine the sequences from the amplified DNA. This survey includes 10 strains of Nostoc/Anabaena/Aphanizomenon (Nostoc category), 5 strains of Microcystis (Microcystis category), and 4 strains of Planktothrix (Planktothrix category). Fifteen additional strains of cyanobacteria and two strains of prochlorophytes were included such that the major phyletic groups were represented. One of the strains, Phormidium sp. NIVA-CYA 203, contained an 11-nucleotide insertion with no homology to other known 16S rRNA sequences. Based on parsimony and neighbor-joining trees, the phyletic relationships of the strains were investigated. Thirteen major branches were found, with Pseudanabaena limnetica NIVA-CYA 276/6 as the most divergent strain. The strain categories Nostoc, Planktothrix, and Microcystis were all monophyletic. The sequence polymorphism within Nostoc was higher than that in Planktothrix and Microcystis. Based on the sequence and phyletic information, group-specific PCR primers for the categories Nostoc, Planktothrix, and Microcystis were constructed. For the strains included in this work, the amplifications were specific for the relevant groups. By combination of magnetic solid-phase DNA isolation and group-specific PCR amplifications, an accurate method for characterization, classification and identification of oxyphotobacterial clone cultures has been developed.