Photobacterium Species Research Articles

Acute nitrite exposure causes gut microbiota dysbacteriosis and proliferation of pathogenic Photobacterium in shrimp

Nitrite exposure has become a significant concern in the aquaculture industry, posing a severe threat to aquatic animals such as shrimp. While studies have reported the adverse effects of nitrite on shrimp growth, the part played by the gut microbiota in shrimp mortality resulting from nitrite exposure is poorly understood. Here, the effects of nitrite on shrimp gut bacterial community were investigated using 16S rRNA amplicon sequencing, bacterial isolation, genomic analysis, and infection experiments. Compared to the control_healthy group, changes in the bacterial composition of the nitrite_dead group were associated with reduced abundance of specific beneficial bacteria and increased abundance of certain pathogenic bacteria. Notably, members of the Photobacterium genus were found to be significantly enriched in the nitrite_dead group. Genomic analysis of a representative Photobacterium strain (LvS-8n3) revealed a variety of genes encoding bacterial toxins, including hemolysin, adhesin, and phospholipase. Furthermore, it was also found that LvS-8n3 exhibits strong pathogenicity, probably due to its high production of pathogenic factors and the ability to utilize nitrite for proliferation. Therefore, the proliferation of pathogenic Photobacterium species appears pivotal for driving shrimp mortality caused by nitrite exposure. These findings provide novel insights into the disease mechanism in shrimp under conditions of environmental change.

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-42T (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 45.6mol%. 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 82885T).

Distinct coral environments shape the dynamic of planktonic Vibrio spp.

BackgroundCoral reefs are one of the most biodiverse and productive ecosystems, providing habitat for a vast of species. Reef-building scleractinian corals with a symbiotic microbiome, including bacteria, archaea, viruses and eukaryotic microbes, are referred to coral holobionts. Among them, coral diseases, mainly caused by Vibrio spp., have significantly contributed to the loss of coral cover and diversity. Habitat filtering across the globe has led to a variety structure of marine bacterial communities. Coral species, quantity and characteristics are significant differences between the Xisha Islands and Daya Bay (Guangdong Province). Thus, the Vibrio communities may be distinct between coral rich and poor areas.ResultsThrough comparison of Vibrio dynamics between coral-rich (Xisha Islands) and coral-poor (Daya Bay) locations, we uncovered differences in Vibrio abundance, diversity, community composition and assembly mechanisms associated with corals. The higher abundance of Vibrio in coral rich areas may indicate a strong interaction between vibrios and corals. V. campbellii, Paraphotobacterium marinum and V. caribbeanicus were widely distributed in both coral rich and poor areas, likely indicating weak species specificity in the coral-stimulated growth of Vibrio. Random-forest prediction revealed Vibrio species and Photobacterium species as potential microbial indicators in the coral rich and coral poor areas, respectively. Ecological drift rather than selection governed the Vibrio community assembly in the Xisha Islands. Comparatively, homogenizing selection was more important for the Daya Bay community, which may reflect a role of habitat filtration.ConclusionThis study revealed the different distribution pattern and assembly mechanism of Vibrio spp. between coral rich and poor areas, providing the background data for the research of Vibrio community in coral reef areas and may help the protection of coral reef at the biological level. The main reasons for the difference were different number and species of corals, environmental (e.g., temperature) and spatial factors. It reflected the strong interaction between Vibrio and corals, and provided a new perspective for the investigation of Vibrio in coral reef ecosystem.

Isolation, characterization, mathematical modeling and assessment of a novel Photobacterium sp. EAH3, as toxicity biosensor

Marine ecosystems are full of unique microbial phenomena, among them bioluminescence that could be harnessed for different ecological functions. This study was conducted to isolate luminous bacteria and utilize their glowing properties as a potential biosensor for toxicity assessment designs. It is worth noting that this study is the first report on the isolation of an Octopus associated Photobacterium species from Alexandria, Egypt. Based on light intensity, strain Photobacterium sp. EAH3 was selected and identified using 16S-rRNA gene sequencing. The luminescence production was further optimized using experimental. Among eight tested variables, the Plackett-Burman design showed that yeast extract, glycerol, peptone, and NaCl were the most important factors affecting luminescence. The central composite design calculated the optimum concentration of these variables in the optimized medium, which enhanced the bioluminescence activity by 1.39 folds. The employment of Photobacterium sp. EAH3 as a biosensor for the toxicity assessment of some solvents, hydrocarbons and heavy metals was examined using bioluminescence inhibition assay. According to the IC50 results, the highest toxicity values recorded were 0.004 M, 4.15 ppm and 1.4 ppm for isoamyl alcohol, Hg2+ and catechol, respectively. Moreover, our study sheds light on futuristic methodology for monitoring toxicity in real polluted environmental and effluents' samples reflecting its promising efficiency.

Development of Fluorescent Bacteria with Lux and Riboflavin Genes

Lumazine protein from marine luminescent bacteria of Photobacterium species bind with very high affinity to the fluorescent chromophore 6,7-dimethyl-8-ribitylumazine. The light emission of bacterial luminescent systems is used as a sensitive, rapid, and safe assay for an ever-increasing number of biological systems. Plasmid pRFN4, containing the genes encoding riboflavin from the rib operon of Bacillus subtilis, was designed for the overproduction of lumazine. To construct fluorescent bacteria for use as microbial sensors, novel recombinant plasmids (pRFN4-Pp N-lumP and pRFN4-Pp luxLP N-lumP) were constructed by amplifying the DNA encoding the N-lumP gene (luxL) from P. phosphoreum and the promoter region (luxLP) present upstream of the lux operon of the gene by PCR and ligating into the pRFN4-Pp N-lumP plasmid. A new recombinant plasmid, pRFN4-Pp luxLP-N-lumP, was constructed with the expectation that the fluorescence intensity would be further increased when transformed into Escherichia coli. When this plasmid was transformed into E. coli 43R, the fluorescence intensity of transformants was 500 times greater than that of E. coli alone. As a result, the recombinant plasmid in which the gene encoding N-LumP and DNA containing the lux promoter exhibited expression that was so high as to show fluorescence in single E. coli cells. The fluorescent bacterial systems developed in the present study using lux and riboflavin genes can be utilized in the future as biosensors with high sensitivity and rapid analysis times.

Exemplifying ‘pathobiome’ concept through case study: Co-infection with Vibrio harveyi, Photobacterium damsela and Cryptocaryon irritans in Salema (Sarpa salpa)

A pathobiome approach has been revealed as a set of organisms (i.e. prokaryotic, eukaryotic, and virus associations) in the aquatic environment that interact with the host (fish) to cause disease. The approach of the one pathogen–one disease is not wholly satisfactory to comment on the impairment of health status and disease process in fish. To exemplify the pathobiome concept, we present a fish disease in which more than one pathogen and possible synergistic interaction of inadequate water quality. In this case, the heavy mortalities were observed in Salema (Sarpa salpa) from an exhibition aquaria. Bacterial identification with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF MS) showed the pathogen bacteria species of vibrio and photobacterium. In the parasitic examination, the invasion of marine ciliata, Cryptocaryon irritans was observed in the diseased fish. Moreover, poor water quality was considered as contributing factor to disease emergence in this case. The co-existence of pathogen Vibrio species (Vibrio harveyi) and Photobacterium damsela with its subspecies (subsp. damsela and subsp. piscicida) as well as marine ciliata, C. irritans for a description of a disease picture in a marine fish species are reported for the first time, providing an example of a pathobiome paradigm.

Pathogenicity and virulence of bacterial strains associated with summer mortality in marine mussels (Perna canaliculus).

The occurrence of pathogenic bacteria has emerged as a plausible key component of summer mortalities in mussels. In the current research, four bacterial isolates retrieved from moribund Greenshell࣪ mussels, Perna canaliculus, from a previous summer mortality event, were tentatively identified as Vibrio and Photobacterium species using morpho-biochemical characterization and MALDI-TOF MS and confirmed as V. celticus, P. swingsii, P. rosenbergii, and P. proteolyticum using whole genome sequencing. These isolates were utilized in a laboratory challenge where mussels were injected with cell concentrations ranging from 105 to 109 CFU/mussel. Of the investigated isolates, P. swingsii induced the highest mortality. Additionally, results from quantitative polymerase chain reaction analysis, focusing on known virulence genes were detected in all isolates grown under laboratory conditions. Photobacterium rosenbergii and P. swingsii showed the highest expression levels of these virulence determinants. These results indicate that Photobacterium spp. could be a significant pathogen of P. canaliculus, with possible importance during summer mortality events. By implementing screening methods to detect and monitor Photobacterium concentrations in farmed mussel populations, a better understanding of the host-pathogen relationship can be obtained, aiding the development of a resilient industry in a changing environment.

Isolation and Identification of Luminescent Bacteria in Deep Sea Marine Organisms from Sicilian Waters (Mediterranean Sea)

Luminescent bacteria are a fascinating component of marine microbial communities, often related to the light emissions in deep sea marine organisms. They are mainly affiliated with specific phylogenetic groups, such as Photobacterium, Vibrio, and Photorhabdus, and are sometimes involved in symbiotic relationships. However, the luminescence of some marine organisms remains a poorly understood process, and it is not always certain whether their luminescence is attributable to associated luminescent bacteria. In this study, for the first time, luminescent bacteria were isolated from two deep sea organisms, namely, the cephalopod Neorossia caroli and the teleost Chlorophthalmus agassizi. The isolation was carried out on glycerol-supplemented medium, and the search for the luxAB gene was performed on all isolates as a complementary tool to the culture-dependent techniques to detect bioluminescence by molecular approach. The optimum of salinity, temperature, and pH was evaluated by physiological tests for all isolates. The production of extracellular polymeric substances was also preliminarily screened. A total of 24 luminescent isolates were obtained, with an abundance from C. agassizi specimens. All the isolates were taxonomically characterized and were related to different species of Photobacterium, with the exception of Vibrio sp. CLD11 that was from C. agassizi. The luxAB gene was detected in about the 90% of the analysed strains.

Comparative genomics of Photobacterium species from terrestrial and marine habitats.

Photobacterium (P.) is a genus widely studied in regards to its association with and ubiquitous presence in marine environments. However, certain species (P. phosphoreum, P. carnosum, P. iliopiscarium) have been recently described to colonize and spoil raw meats without a marine link. We have studied 27 strains from meat as well as 26 strains from marine environments in order to probe for intraspecies marine/terrestrial subpopulations and identify distinct genomic features acquired by environmental adaptation. We have conducted phylogenetic analysis (MLSA, ANI, fur, codon usage), search of plasmids (plasmidSPADES), phages (PHASTER), CRISPR-cas operons (CRISPR-finder) and secondary metabolites gene clusters (antiSMASH, BAGEL), in addition to a targeted gene search for specific pathways (e.g. TCA cycle, pentose phosphate, respiratory chain) and elements relevant for growth, adaptation and competition (substrate utilization, motility, bioluminescence, sodium and iron transport). P. carnosum appears as a conserved single clade, with one isolate from MAP fish clustering apart that doesn't, however, show distinct features that could indicate different adaptation. The species harbors genes for a wide carbon source utilization (glycogen/starch, maltose, pullulan, fucose) for colonization of diverse niches in its genome. P. phosphoreum is represented by two different clades on the phylogenetic analyses not correlating to their origin or distribution of other features analyzed that can be divided into two novel subspecies based on genome-wide values. A more diverse antimicrobial activity (sactipeptides, microcins), production of secondary metabolites (siderophores and arylpolyenes), stress response and adaptation (bioluminescence, sodium transporters, catalase, high affinity for oxygen cytochrome cbb3 oxidase, DMSO reductase and proton translocating NADH dehydrogenase) is predicted compared to the other species. P. iliopiscarium was divided into two clades based on source of isolation correlating with phylogeny and distribution of several traits. The species shows traits common to the other two species, similar carbon utilization/transport gene conservation as P. carnosum for the meat-isolated strains, and predicted utilization of marine-common DMSO and flagellar cluster for the sea-isolated strains. Results additionally suggest that photobacteria are highly prone to horizontal acquisition/loss of genetic material and genetic transduction, and that it might be a strategy for increasing the frequency of strain- or species-specific features that offers a growth/competition advantage.

Influence of the packaging atmosphere and presence of co-contaminants on the growth of photobacteria on chicken meat

Fresh meat is commonly packaged in modified atmosphere to decelerate spoilage processes. The applied gas mixture affects the growth of spoilage organisms and selectively shapes the spoilage community. In this study, we investigated the impact of O2 and CO2 on the growth of Photobacterium (P.) phosphoreum and P. carnosum strains in situ on chicken meat by packaging under different modified atmospheres (air, 70% O2/30% CO2, 70% N2/30% CO2, 100% N2). Combination of 70% O2 and 30% CO2 resulted in significant growth reduction of the analyzed strains, suggesting inhibitory effects of both gases in combination. In contrast, 30% CO2 alone had only a minor effect and photobacteria are supposed to have a growth advantage over other meat spoilers in this atmosphere. Additionally, single growth of the strains in the different atmospheres was compared when challenged with the presence of Pseudomonas (Ps.) fragi or Brochothrix (B.) thermosphacta as prominent co-contaminants in different ratios (10:1, 1:1, 1:10). Presence of co-contaminants resulted in increased cell numbers of P. carnosum TMW2.2149 but reduced or unchanged cell numbers of P. phosphoreum TMW2.2103 in most packaging atmospheres. The initial ratio of photobacteria and co-contaminants defined the relative abundance during storage but did not change the type of the interaction. Our results suggest either a commensalistic (P. carnosum) or competitive interaction (P. phosphoreum) of photobacteria and co-contaminants on modified atmosphere packaged chicken, respectively. Furthermore, in a mix comprising seven prominent spoilers, strains of both Photobacterium species prevailed as a constant part of the spoilage microbiome during 7 days of refrigerated storage on chicken meat packaged under O2/CO2 atmosphere.

Diverse Horizontally-Acquired Gene Clusters Confer Sucrose Utilization to Different Lineages of the Marine Pathogen Photobacterium damselae subsp. damselae.

The ability to metabolize sucrose is a variable trait within the family Vibrionaceae. The marine bacterium Photobacterium damselae subsp. damselae (Pdd), pathogenic for marine animals and humans, is generally described as negative for sucrose utilization (Scr−). Previous studies have reported sucrose-utilizing isolates (Scr+), but the genetic basis of this variable phenotype remains uncharacterized. Here, we carried out the genome sequencing of five Scr+ and two Scr− Pdd isolates and conducted a comparative genomics analysis with sixteen additional Pdd genomes sequenced in previous studies. We identified two different versions of a four-gene cluster (scr cluster) exclusive of Scr+ isolates encoding a PTS system sucrose-specific IIBC component (scrA), a fructokinase (scrK), a sucrose-6-phosphate hydrolase (scrB), and a sucrose operon repressor (scrR). A scrA deletion mutant did not ferment sucrose and was impaired for growth with sucrose as carbon source. Comparative genomics analyses suggested that scr clusters were acquired by horizontal transfer by different lineages of Pdd and were inserted into a recombination hot-spot in the Pdd genome. The incongruence of phylogenies based on housekeeping genes and on scr genes revealed that phylogenetically diverse gene clusters for sucrose utilization have undergone extensive horizontal transfer among species of Vibrio and Photobacterium.

Comparative Analysis of CRISPR-Cas Systems in Vibrio and Photobacterium Genomes of High Influence in Aquaculture Production

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs), together with cas genes, constitute the systems CRISPR-Cas, a novel type of innate defense system in prokaryotes for the destruction of exogenous elements (plasmids or phages). The genus Vibrio and Photobacterium represents one of the main responsible infectious agents in aquaculture and cause huge losses for the shrimp industry. The few bioinformatic investigations on CRISPR/Cas systems in the Vibrionaceae family are mainly oriented to clinical species. Therefore, the objective of this research was to make a comparative analysis of CRISPR-Cas systems in Vibrio and Photobacterium genomes of strong influence in aquaculture production. A total of 69 complete genomes across the family were studied. Seven CRISPR loci were identified in 4 species (V. parahaemolyticus, V. harveyi, V. vulnificus, and Photobacterium damselae). The CRISPR/Cas systems were found to correspond to subtypes I-E, I-F, and III-D. Only 47% of the spacers presented homology with bacteriophages. The results showed a low presence of CRISPR structures in the species of Vibrio and Photobacterium with immunity to specific phages. These results are interesting if sensitivity is considered, and it can be explored for the development of strategies such as phagotherapy in shrimp farming.

Use of bacteriophage vB_Pd_PDCC-1 as biological control agent of Photobacterium damselae subsp. damselae during hatching of longfin yellowtail (Seriola rivoliana) eggs.

This study describes the effect of phage therapy on hatching of longfin yellowtail (Seriola rivoliana) eggs challenged with Photobacterium damselae subsp. damselae. A lytic phage (vB_Pd_PDCC-1) against P. damselae subsp. damselae was isolated and characterized. The use of phage vB_Pd_PDCC-1 increased the hatching rate of eggs, and reduced presumptive Vibrio species to non-detectable numbers, even in non-disinfected eggs. High-throughput 16S rRNA gene sequencing analysis revealed that phage vB_Pd_PDCC-1 caused significant changes in the composition and structure of the associated microbiota, allowing that members (e.g. those belonging to the family Vibrionaceae) of the class Gammaproteobacteria to be displaced by members of the class Alphaproteobacteria. To the best of our knowledge, this represents the first study evaluating phage therapy to control potential negative effects of P. damselae subsp. damselae during hatching of longfin yellowtail eggs. The Seriola genus includes several important commercial fish species due to its rapid growth and easy adaptability to confinement conditions. However, bacterial infections (especially those caused by Vibrio and Photobacterium species) are among the main limiting factors for the intensification of marine fish aquaculture, particularly during early development stages. Therefore, the use of phages, which are natural killers of bacteria, represents a promising strategy to reduce the mortality of farmed organisms caused by pathogenic bacteria.

Genomic and phenomic analysis of a marine bacterium, Photobacterium marinum J15

Photobacterium species are widely distributed in the marine environment. The overall metabolism of this genus remains largely unknown. In order to improve our knowledge on this bacterium, the relationship between the genome and phenome of the Photobacterium isolate was analyzed. The cream colored, Gram-negative, rod-shaped and motile bacterial strain, J15, was isolated from marine water of Tanjung Pelepas, Johor, Malaysia. The 5,684,538 bp genome of strain J15 comprised 3 contigs (2 chromosomes and 1 plasmid) with G + C content of 46.39 % and contained 4924 protein-coding genes including 180 tRNAs and 40 rRNAs. The phenotypic microarray (PM) as analyzed using BIOLOG showed the utilization of; i) 93 of the 190 carbon sources tested, where 61 compounds were used efficiently; ii) 41 of the 95 nitrogen sources tested, where 22 compounds were used efficiently; and iii) 3 of the 94 phosphorous and sulphur sources tested. Furthermore, high tolerance to osmotic stress, basic pH and toxic compounds as well as resistance to antibiotics of strain J15 were determined by BIOLOG PM. The ANI and kSNP analyses revealed that strain J15 to be the same species with Photobacterium marinum AK15 with ANI value of 96.93 % and bootstrapping value of 100 in kSNP. Based on the ANI and kSNP analyses, strain J15 was identified as P. marinum J15.

Virulence properties of three new Photobacterium species affecting cultured fish.

Several virulence factors of three new Photobacterium species: Photobacterium toruni, Photobacterium malacitanum and Photobacterium andalusiense associated with diseases of cultured redbanded seabream (Pagrus auriga) were studied. The exoenzymatic activities, adherence and cytotoxic capabilities, and iron-uptake mechanisms were determined both in bacterial extracellular products (ECP) and whole bacterial cells. The histopathology damages provoked on redbanded seabream by the ECP was also studied. The highest exoenzymatic activities of the ECP were alkaline- and acid-phosphatase, phosphohydrolase and lipase. The ECP were strongly lethal for fish at 4-96h post-inoculation (p.i). Histological changes were evident at 96 hpi of ECP, affecting head kidney, splenic parenchyma and heart. Cytotoxicity assays, on three fish lines and one human cell line, were conducted using whole bacterial cells and their ECP. The new species tested were cytotoxic only for fish cell lines using whole bacterial cells. Bacterial adherence showed an adherence index moderate on CHSE-214 cell line. All strains showed variable haemolytic activity, and were able to grow under iron-limiting conditions, although the CAS reactivitiy was very low. However, all strains produced high amounts of extracelullar citrate that could be used as iron carrier, and use haem as iron source, except the P. toruni strains because a deletion in the genomic region encoding this ability in all Vibrionaceae members. The toxic activity of the bacterial ECPs was thermolabile, and not associated with their thermoresistant lipopolysaccharide content. The virulence of the strains tested could not be related to the haemolytic activity. Iron uptake could be based on the use of endogenous citrate as iron carrier and P. toruni lacks the ability to use haem as iron source. The study analyses for the first time the virulence properties of three new species of Photobacterium pathogenic for fish.

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).

Analysis of cultivable aerobic bacterial community composition and screening for facultative sulfate-reducing bacteria in marine corrosive steel

Anaerobic, aerobic, and facultative bacteria are all present in corrosive environments. However, as previous studies to address corrosion in the marine environment have largely focused on anaerobic bacteria, limited attention has been paid to the composition and function of aerobic and facultative bacteria in this process. For analysis in this study, ten samples were collected from rust layers on steel plates that had been immersed in seawater for different periods (i.e., six months and eight years) at Sanya and Xiamen, China. The cultivable aerobic bacterial community structure as well as the number of sulfate-reducing bacteria (SRB) were analyzed in both cases, while the proportion of facultative SRB among the isolated aerobic bacteria in each sample was also evaluated using a novel approach. Bacterial abundance results show that the proportions are related to sea location and immersion time; abundances of culturable aerobic bacteria (CAB) and SRB from Sanya were greater in most corrosion samples than those from Xiamen, and abundances of both bacterial groups were greater in samples immersed for six months than for eight years. A total of 213 isolates were obtained from all samples in terms of CAB community composition, and a phylogenetic analysis revealed that the taxa comprised four phyla and 31 genera. Bacterial species composition is related to marine location; the results show that Firmicutes and Proteobacteria were the dominant phyla, accounting for 98.13% of the total, while Bacillus and Vibrio were the dominant genera, accounting for 53.06% of the total. An additional six facultative SRB strains were also screened from the isolates obtained and were found to encompass the genus Vibrio (four strains), Staphylococcus (one strain), and Photobacterium (one strain). It is noteworthy that mentions of Photobacterium species have so far been absent from the literature, both in terms of its membership of the SRB group and its relationship to corrosion.

Biogenic Amine Production by and Phylogenetic Analysis of 23 Photobacterium Species

Photobacterium species are members of the bacterial communities typically associated with scombrotoxin-forming fish. Reclassification and discovery of new Photobacterium species has caused confusion as to which species are capable of biogenic amine production. We analyzed histamine, cadaverine, and putrescine production by 104 Photobacterium strains representing 23 species. The presence of the genes for histidine decarboxylase ( hdc), lysine decarboxylase ( ldc), and ornithine decarboxylase ( odc) was determined by real-time or conventional PCR and whole genome sequencing. Significant histamine production (>200 ppm) was detected in five Photobacterium species: P. angustum, P. aquimaris, P. kishitanii, P. damselae, and P. phosphoreum. The hdc gene was detected in all of these histamine-producing species except P. phosphoreum. Cadaverine was produced by eight Photobacterium species: P. angustum, P. aquimaris, P. damselae, P. iliopiscarium, P. kishitanii, P. leiognathi, P. mandapamensis, and P. phosphoreum. Putrescine was produced by six Photobacterium species: P. angustum, P. aquimaris, P. kishitanii, P. leiognathi, P. mandapamensis, and Photobacterium sp. Cadaverine production correlated closely with the presence of the ldc gene, but putrescine production did not correlate closely with the presence of the odc gene. Characterization of the biogenic amine production by Photobacterium species will allow identification of these marine bacteria and help ensure that current guidelines account for mitigation of these bacteria.

Description of New and Amended Clades of the Genus Photobacterium.

Phylogenetic relationships between species in the genus Photobacterium have been poorly studied despite pathogenic and ecological relevance of some of its members. This is the first phylogenetic study that includes new species of Photobacterium (validated or not) that have not been included in any of the previously described clades, using 16S rRNA sequences and multilocus sequence analysis (MLSA) in concatenated sequences of gyrB, gapA, topA, ftsZ and mreB housekeeping genes. Sequence analysis has been implemented using Maximum-parsimony (MP), Neighbour-joining (NJ) and Maximum likelihood (ML) treeing methods and the predicted evolutionary relationship between the Photobacterium clades was established on the basis of bootstrap values of >75% for 16S rRNA sequences and MLSA. We have grouped 22 species of the genus Photobacterium into the following 5 clades: Phosphoreum (comprises P. aquimaris, “P. carnosum,” P. iliopiscarium, P. kishitanii, P. phosphoreum, “P. piscicola” and “P. toruni”); clade Profundum (composed of P. aestuarii, P. alginatilyticum, P. frigidiphilum, P. indicum, P. jeanii, P. lipolyticum, “P. marinum,” and P. profundum); clade Damselae (two subspecies of P. damselae, damselae and piscicida); and two new clades: clade Ganghwense (includes P. aphoticum, P. aquae, P. galatheae, P. ganghwense, P. halotolerans, P. panuliri and P. proteolyticum); and clade Leiognathi (composed by P. angustum, P. leiognathi subsp. leiognathi and “P. leiognathi subsp. mandapamensis”). Two additional clades, Rosenbergii and Swingsii, were formed using a phylogenetic method based on 16S rRNA gene, although they are not confirmed by any MLSA methods. Only P. aplysiae could not be included in none of the established clade, constituting an orphan clade.

Photobacterium carnosum sp. nov., isolated from spoiled modified atmosphere packaged poultry meat

Analysis of spoilage-associated microbiota of modified-atmosphere packaged poultry meat revealed four different bacterial isolates that could not be assigned to known species. They showed a Gram-negative staining behavior, were facultatively aerobic, non-motile with variable cell morphology. Phylogenetic analysis of 16S rDNA and gyrB, rpoD and recA revealed a distinct lineage within the genus Photobacterium with Photobacterium (P.) iliopiscarium DSM 9896T, P. phosphoreum DSM 15556T, P. kishitanii DSM 19954T, P. piscicola LMG 27681T and P. aquimaris DSM 23343T as closest relatives.The designated type strain TMW 2.2021T is non-luminous and grew at 0–20°C (optimum 10–15°C), within pH 5.0–8.5 (optimum 6–8) and in the presence of 0.5–3% (w/v) NaCl (optimum 1%). Major cellular fatty acids of TMW 2.2021T were summed feature 3 (C16:1ω7c/iso-C15 3-OH), C16:0, C18:1ω7c and summed feature 2 (C12:0 aldehyde and C10.928 unknown). Quinone analysis revealed Q-8 as sole respiratory ubiquinone. The genome of TMW 2.2021T has a size of 4.56Mb and a G+C content of 38.49mol%. The ANI value between TMW 2.2021T and the type strain of closest relative P. iliopiscarium DSM 9896T was 91.43%. Fingerprinting on the base of M13-RAPD-PCR band pattern and MALDI-TOF MS profiles allowed intraspecies differentiation between our isolates but also supported their distinct lineage to a novel species. Based on phylogenetic, genomic, phenotypic and chemotaxonomic data, strain TMW 2.2021T and further strains represent a novel species of the genus Photobacterium, for which the name Photobacterium carnosum sp. nov. is proposed. The type strain is TMW 2.2021T (=DSM 105454T=CECT 9394T).