Histidine decarboxylases and their role in accumulation of histamine in tuna and dried saury

Histamine-producing bacteria (HPB) such as Photobacterium phosphoreum and Raoultella planticola possess histidine decarboxylase (HDC), which converts histidine into histamine. Histamine fish poisoning (HFP) is attributable to the ingestion of fish containing high levels of histamine produced by HPB. Because freezing greatly decreases the histamine-producing ability of HPB, especially of P. phosphoreum, it has been speculated that HFP is caused by HDC itself from HPB cells autolyzing during frozen storage, even when HPB survive frozen storage. Here we constructed recombinant HDCs of P. phosphoreum, Photobacterium damselae, R. planticola, and Morganella morganii and investigated the ability of HDCs to produce sufficient histamine to cause HFP. To elucidate the character of these HDCs, we examined the specific activity of each recombinant HDC at various temperatures, pH levels, and NaCl concentrations. Further, we also investigated the stability of each HDC under different conditions (in reaction buffer, tuna, and dried saury) at various temperatures. P. damselae HDC readily produced sufficient histamine to cause HFP in fish samples. We consider that if HDC is implicated as an independent cause of HFP in frozen-thawed fish, the most likely causative agent is HDC of P. damselae.     

Photobacterium angustum and Photobacterium kishitanii,Psychrotrophic High-Level Histamine-Producing Bacteria Indigenous to Tuna

Scombrotoxin fish poisoning (SFP) remains the main contributor of fish poisoning incidents in the United States, despite efforts to control its spread. Psychrotrophic histamine-producing bacteria (HPB) indigenous to scombrotoxin-forming fish may contribute to the incidence of SFP. We examined the gills, skin, and anal vents of yellowfin (n = 3), skipjack (n = 1), and albacore (n = 6) tuna for the presence of indigenous HPB. Thirteen HPB strains were isolated from the anal vent samples from albacore (n = 3) and yellowfin (n = 2) tuna. Four of these isolates were identified as Photobacterium kishitanii and nine isolates as Photobacterium angustum; these isolates produced 560 to 603 and 1,582 to 2,338 ppm histamine in marine broth containing 1% histidine (25°C for 48 h), respectively. The optimum growth temperatures and salt concentrations were 26 to 27°C and 1% salt for P. kishitanii and 30 to 32°C and 2% salt for P. angustum in Luria 70% seawater (LSW-70). The optimum activity of the HDC enzyme was at 15 to 30°C for both species. At 5°C, P. kishitanii and P. angustum had growth rates of 0.1 and 0.2 h⁻¹, respectively, and the activities of histidine decarboxylase (HDC) enzymes were 71% and 63%, respectively. These results show that indigenous HPB in tuna are capable of growing at elevated and refrigeration temperatures. These findings demonstrate the need to examine the relationships between the rate of histamine production at refrigeration temperatures, seafood shelf life, and regulatory limits.     

Klebsiella pneumoniae Produces No Histamine: Raoultella planticola and Raoultella ornithinolytica Strains Are Histamine Producers

Histamine fish poisoning is caused by histamine-producing bacteria (HPB). Klebsiella pneumoniae and Klebsiella oxytoca are the best-known HPB in fish. However, 22 strains of HPB from fish first identified as K. pneumoniae or K. oxytoca by commercialized systems were later correctly identified as Raoultella planticola (formerly Klebsiella planticola) by additional tests. Similarly, five strains of Raoultella ornithinolytica (formerly Klebsiella ornithinolytica) were isolated from fish as new HPB. R. planticola and R. ornithinolytica strains were equal in their histamine-producing capabilities and were determined to possess the hdc genes, encoding histidine decarboxylase. On the other hand, a collection of 61 strains of K. pneumoniae and 18 strains of K. oxytoca produced no histamine.     

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.     

Genomic and Phylogenetic Characterization of Luminous Bacteria Symbiotic with the Deep-Sea Fish Chlorophthalmus albatrossis (Aulopiformes: Chlorophthalmidae)

Bacteria forming light-organ symbiosis with deep-sea chlorophthalmid fishes (Aulopiformes: Chlorophthalmidae) are considered to belong to the species Photobacterium phosphoreum. The identification of these bacteria as P. phosphoreum, however, was based exclusively on phenotypic traits, which may not discriminate between phenetically similar but evolutionarily distinct luminous bacteria. Therefore, to test the species identification of chlorophthalmid symbionts, we carried out a genomotypic (repetitive element palindromic PCR genomic profiling) and phylogenetic analysis on strains isolated from the perirectal light organ of Chlorophthalmus albatrossis. Sequence analysis of the 16S rRNA gene of 10 strains from 5 fish specimens placed these bacteria in a cluster related to but phylogenetically distinct from the type strain of P. phosphoreum, ATCC 11040^T, and the type strain of Photobacterium iliopiscarium, ATCC 51760^T. Analysis of gyrB resolved the C. albatrossis strains as a strongly supported clade distinct from P. phosphoreum and P. iliopiscarium. Genomic profiling of 109 strains from the 5 C. albatrossis specimens revealed a high level of similarity among strains but allowed identification of genomotypically different types from each fish. Representatives of each type were then analyzed phylogenetically, using sequence of the luxABFE genes. As with gyrB, analysis of luxABFE resolved the C. albatrossis strains as a robustly supported clade distinct from P. phosphoreum. Furthermore, other strains of luminous bacteria reported as P. phosphoreum, i.e., NCIMB 844, from the skin of Merluccius capensis (Merlucciidae), NZ-11D, from the light organ of Nezumia aequalis (Macrouridae), and pjapo.1.1, from the light organ of Physiculus japonicus (Moridae), grouped phylogenetically by gyrB and luxABFE with the C. albatrossis strains, not with ATCC 11040^T. These results demonstrate that luminous bacteria symbiotic with C. albatrossis, together with certain other strains of luminous bacteria, form a clade, designated the kishitanii clade, that is related to but evolutionarily distinct from P. phosphoreum. Members of the kishitanii clade may constitute the major or sole bioluminescent symbiont of several families of deep-sea luminous fishes.     

Regulation of hdc expression and HDC activity by enological factors in lactic acid bacteria

Aims: The aim of this work was to study the influence of enological factors on the histidine decarboxylase gene (hdc) expression and on histidine decarboxylase enzyme (HDC) activity in Lactobacillus hilgardii, Pediococcus parvulus and Oenococcus oeni. Methods and Results: Cell extracts and whole cells were used. Glucose, fructose, malic acid and citric acid diminished the hdc expression. Ethanol did not increase hdc expression or activity in cells, but increased HDC activity. Temperature and pH had effect on the activity of HDC but not on hdc expression. Tartaric acid and l -lactic acid, and sulphur dioxide (SO₂) had no effect on enzyme synthesis and activity. Bacterial species differ in the relative enzymatic activity but all the factors affected similarly to L. hilgardii, P. parvulus and O. oeni. Conclusions: The hdc gene expression was lowered by glucose, fructose, malic acid, and citric acid, whereas ethanol enhanced the HDC enzyme activity. The conditions that normally occur during malolactic fermentation and later on, could favour histamine production. SO₂ could prevent bacterial growth, but does not diminish the HDC enzyme activity. Significance and Impact of the Study: Information on hdc expression and HDC activity can contribute to the prevention of histamine formation during wine production and storage.     

A Transmissible Plasmid-Borne Pathogenicity Island Confers Piscibactin Biosynthesis in the Fish Pathogen Photobacterium damselae subsp. piscicida

The fish pathogen Photobacterium damselae subsp. piscicida produces the siderophore piscibactin. A gene cluster that resembles the Yersinia high-pathogenicity island (HPI) encodes piscibactin biosynthesis. Here, we report that this HPI-like cluster is part of a hitherto-uncharacterized 68-kb plasmid dubbed pPHDP70. This plasmid lacks homologs of genes that mediate conjugation, but we found that it could be transferred at low frequencies from P. damselae subsp. piscicida to a mollusk pathogenic Vibrio alginolyticus strain and to other Gram-negative bacteria, likely dependent on the conjugative functions of the coresident plasmid pPHDP60. Following its conjugative transfer, pPHDP70 restored the capacity of a vibrioferrin mutant of V. alginolyticus to grow under low-iron conditions, and piscibactin became detectable in its supernatant. Thus, pPHDP70 appears to harbor all the genes required for piscibactin biosynthesis and transport. P. damselae subsp. piscicida strains cured of pPHDP70 no longer produced piscibactin, had impaired growth under iron-limited conditions, and exhibited markedly decreased virulence in fish. Collectively, our findings highlight the importance of pPHDP70, with its capacity for piscibactin-mediated iron acquisition, in the virulence of P. damselae subsp. piscicida. Horizontal transmission of this plasmid-borne piscibactin synthesis gene cluster in the marine environment may facilitate the emergence of new pathogens.     

Isolation and Identification of Photobacterium phosphoreum from an Unexpected Niche: Migrating Salmon

Six luminous bacteria were isolated from migrating salmon in the Yukon River, Alaska. All isolates were identified as Photobacterium phosphoreum. Previous studies suggest that P. phosphoreum is an exclusively marine bacterium, while our Alaskan isolates are from salmon which migrated up to 1,228 km from the marine environment.     

Variation in 16S-23S rRNA Intergenic Spacer Regions in Photobacterium damselae: a Mosaic-Like Structure

Phenotypically, Photobacterium damselae subsp. piscicida and P. damselae subsp. damselae are easily distinguished. However, their 16S rRNA gene sequences are identical, and attempts to discriminate these two subspecies by molecular tools are hampered by their high level of DNA-DNA similarity. The 16S-23S rRNA internal transcribed spacers (ITS) were sequenced in two strains of Photobacterium damselae subsp. piscicida and two strains of P. damselae subsp. damselae to determine the level of molecular diversity in this DNA region. A total of 17 different ITS variants, ranging from 803 to 296 bp were found, some of which were subspecies or strain specific. The largest ITS contained four tRNA genes (tDNAs) coding for tRNA^Glu(UUC), tRNA^Lys(UUU), tRNA^Val(UAC), and tRNA^Ala(GGC). Five amplicons contained tRNA^Glu(UUC) combined with two additional tRNA genes, including tRNA^Lys(UUU), tRNA^Val(UAC), or tRNA^Ala(UGC). Five amplicons contained tRNA^Ile(GAU) and tRNA^Ala(UGC). Two amplicons contained tRNA^Glu(UUC) and tRNA^Ala(UGC). Two different isoacceptor tRNA^Ala genes (GGC and UGC anticodons) were found. The five smallest amplicons contained no tRNA genes. The tRNA-gene combinations tRNA^Glu(UUC)-tRNA^Val(UAC)-tRNA^Ala(UGC) and tRNA^Glu(UUC)-tRNA^Ala(UGC) have not been previously reported in bacterial ITS regions. The number of copies of the ribosomal operon (rrn) in the P. damselae chromosome ranged from at least 9 to 12. For ITS variants coexisting in two strains of different subspecies or in strains of the same subspecies, nucleotide substitution percentages ranged from 0 to 2%. The main source of variation between ITS variants was due to different combinations of DNA sequence blocks, constituting a mosaic-like structure.     

Changes in Histamine Content in Fish Products for Animal Feeds and in Aqueous Solution by Gamma-Irradiation

The irradiation effects on histamine in fish meals and fish solubles for animal feeds and in aqueous solution were investigated. The amount of histamine in fish meal (0.4mg/100g) was not changed by up to 5.0 Mrad (50kGy), while the histamine content in fish soluble (45.7 mg/100 g) decreased ca. 16% at 5.0 Mrad. There occurred no accumulation of histamine by irradiation or during storage.

The G values of histamine formation from histidine solution and histamine decomposition by irradiation in deoxygenated neutral solutions were 0.077 and 1.90, respectively. Oxygen inhibited the formation of histamine from histidine but it had little effect on histamine decomposition. Nitrous oxide accelerated the decomposition of histamine and inhibited the accumulation of histamine in histidine solution by irradiation. These results show that there is no problem of histamine accumulation in feedstuffs caused by irradiation since the decomposition of histamine is preferred to its formation.     

Luminous Enteric Bacteria of Marine Fishes: a Study of Their Distribution, Densities, and Dispersion

Three taxa of luminous bacteria (Photobacterium fischeri, P. phosphoreum, and Beneckea spp.) were found in the enteric microbial populations of 22 species of surface- and midwater-dwelling fishes. These bacteria often occurred in concentrations ranging between 10⁵ and 10⁷ colony-forming units per ml of enteric contents. By using a genetically marked strain, it was determined that luminous cells entering the fish during ingestion of seawater or contaminated particles traversed the alimentary tract and survived the digestive processes. After excretion, luminous bacteria proliferated extensively on the fecal material and became distributed into the surrounding seawater. Thus, this enteric habitat may serve as an enrichment of viable cells entering the planktonic luminous population.     

Distribution and Identification of Luminous Bacteria from the Sargasso Sea

Vibrio fischeri and Lucibacterium harveyi constituted 75 of the 83 luminous bacteria isolated from Sargasso Sea surface waters. Photobacterium leiognathi and Photobacterium phosphoreum constituted the remainder of the isolates. Luminescent bacteria were recovered at concentrations of 1 to 63 cells per 100 ml from water samples collected at depths of 160 to 320 m. Two water samples collected at the thermocline yielded larger numbers of viable, aerobic heterotrophic and luminous bacteria. Luminescent bacteria were not recovered from surface microlayer samples. The species distribution of the luminous bacteria reflected previously recognized growth patterns; i.e., L. harveyi and V. fischeri were predominant in the upper, warm waters (only one isolate of P. phosphoreum was obtained from surface tropical waters).     

Tetrodecamycin: An unusual and interesting tetronate antibiotic

The tetrodecamycins are a group of secondary metabolites that are characterized by the presence of a tetronate ring in their structure. Originally discovered for their antibiotic activity against Photobacterium damselae ssp. piscicida, the causative agent of pseudotuberculosis in fish, this family of molecules has also been shown to have potent antibiotic activity against methicillin-resistant Staphylococcus aureus. Due to their small size and highly cyclized nature, they represent an unusual member of the much larger group of bioactive molecules called the tetronates. Herein, we review what is known about the mechanism of action of these molecules and also present a hypothesis for their biosynthesis. A deeper understanding of the tetrodecamycins will provide a more holistic view of the tetronate-family, provide new chemical probes of bacterial biology, and may provide therapeutic lead molecules.     

Conditions that influence bacterial luminescence in the presence of blood serum

Conditions that influence the luminescence of natural and recombinant luminescent bacteria in the presence of blood serum were studied. In general, blood serum quenched the luminescence of the marine Photobacterium phosphoreum and the recombinant Escherichia coli strains harboring the luminescent system genes of Photobacterium leiognathi, but enhanced the luminescence of the soil bacterium Photorhabdus luminescens Zm1 and the recombinant E. coli strain harboring the lux operon of P. luminescens Zm1. The quenching effect of blood serum increased with its concentration and the time and temperature of incubation. The components of blood serum that determine the degree and specificity of its action on bacterial luminescence were identified.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 191–197.Original Russian Text Copyright © 2005 by Deryabin, Polyakov.     

Nonbioluminescent Strains of Photobacterium phosphoreum Produce the Cell-to-Cell Communication Signal N-(3-Hydroxyoctanoyl)homoserine Lactone

Bioluminescence is a common phenotype in marine bacteria, such as Vibrio and Photobacterium species, and can be quorum regulated by N-acylated homoserine lactones (AHLs). We extracted a molecule that induced a bacterial AHL monitor (Agrobacterium tumefaciens NT1 [pZLR4]) from packed cod fillets, which spoil due to growth of Photobacterium phosphoreum. Interestingly, AHLs were produced by 13 nonbioluminescent strains of P. phosphoreum isolated from the product. Of 177 strains of P. phosphoreum (including 18 isolates from this study), none of 74 bioluminescent strains elicited a reaction in the AHL monitor, whereas 48 of 103 nonbioluminescent strains did produce AHLs. AHLs were also detected in Aeromonas spp., but not in Shewanella strains. Thin-layer chromatographic profiles of cod extracts and P. phosphoreum culture supernatants identified a molecule similar in relative mobility (R_f value) and shape to N-(3-hydroxyoctanoyl)homoserine lactone, and the presence of this molecule in culture supernatants from a nonbioluminescent strain of P. phosphoreum was confirmed by high-performance liquid chromatography-positive electrospray high-resolution mass spectrometry. Bioluminescence (in a non-AHL-producing strain of P. phosphoreum) was strongly up-regulated during growth, whereas AHL production in a nonbioluminescent strain of P. phosphoreum appeared constitutive. AHLs apparently did not influence bioluminescence, as the addition of neither synthetic AHLs nor supernatants delayed or reduced this phenotype in luminescent strains of P. phosphoreum. The phenotypes of nonbioluminescent P. phosphoreum strains regulated by AHLs remains to be elucidated.     

Immobilization ofPhotobacterium phosphoreum for monitoring of toxic substances

A new sensing system based on the immobilization of luminescent bacteria,Photobacterium phosphoreum, was proposed for continuous real-time monitoring of pollutants. The response curves demonstrate thatPhotobacterium phosphoreum immobilized on the strontium alginate was very sensitive to seven reference chemicals used. The significant inhibitory concentrations for bioluminescence emission were 5 ppm for Pb(NO₃)₂, NiCl₂, CdCl₂, 50 ppm for NaAsO₂, 0.1 ppm for HgCl₂, 0.5 ppm for pentachlorophenol and less than 5 ppm for SDS, respectively. The alginate mixed-cells (AMC) retained their luminescence during experimental period (29 days) under storage condition of ? 80°C. The variables affecting performance of continuous flow through monitoring (CFTM) were optimized in order to ensure stability and efficiency. The flow through cell with strontium-alginate immobilized luminescent bacteria was tested with salicylate and 4-nitrophenol and a rapid response of luminescence was recorded by time drivemode in bioluminescence spectrometer after exposure to both toxicants.     

Growth and bioluminescence of luminous bacteria under the action of aflatoxin B1 before and after its treatment with nanodiamonds

The effect of aflatoxin B1 on growth and luminescence of marine luminous bacteria P. phosphoreum and recombinant E. coli Z905 cells was investigated. The bidirectional effect of aflatoxin B1 on the studied bacterial species was detected—an inhibition of luminescence in P. phosphoreum and its stimulation in E. coli. It was shown that aflatoxin B1 influences the cell luminescence in the freshly grown cultures and bacteria restored after lyophilization. It was detected that the effect of aflatoxin B1 was graded after interaction with the modified nanodiamond (MND) of detonation synthesis. After mycotoxin’s treatment with MND, it does not cause significant changes in bacterial luminescence. The possibilities for the use of P. phosphoreum and E. coli bacteria in the bioluminescent monitoring of aflatoxin B1 and the use of MND for mycotoxin deactivation are discussed.     

Expression,Purification, and Characterization of the Recombinant Putative Periplasmic Hemin-Binding Protein (HutB) of Photobacterium damselae subsp. piscicida

HutB, the periplasmic hemin binding protein of Photobacterium damselae subsp. piscicida, was produced as a recombinant protein. UV-Vis spectrophotometrical analysis showed absorption spectral changes in hemin upon mixing it with the recombinant protein, indicating complex formation. Spectrophotometric titration of HutB with hemin showed saturation at a heme/HutB ratio of 1:1 and a binding affinity (K _d) of 10 μM.     

我国华南沿海海水养殖鱼类病原菌美人鱼发光杆菌(Photobacterium damselae)分离株的毒力基因和耐药性分析

[目的] 为探讨我国华南沿海海水养殖鱼类病原菌美人鱼发光杆菌的非典型毒力基因和耐药性的时空变化,解析影响其毒力和耐药性变化的可能环境因素,为该菌所引起的病害防控提供建议。[方法] 本研究以分离自我国广东和海南沿海患病海水鱼的35株美人鱼发光杆菌为研究对象,利用普通PCR扩增技术,分析5个非典型毒力基因在菌株中的分布情况,并采用纸片扩散法（K-B）分析菌株对15种抗生素的耐药性。[结果] 19株菌含有1–2个被检测的非典型毒力基因,尤其是hlyA和vvh的检出率均高于20%。35株菌多重耐药指数为0.00–0.67,表现出27种耐药谱,多重耐药率（菌株耐抗生素种类>3）达到60.00%,尤其对万古霉素、阿莫西林、麦迪霉素和利福平的耐药率均高于50%,但对庆大霉素、诺氟沙星、环丙沙星、氯霉素和氟苯尼考的耐药率均低于10%。非典型毒力基因含量和耐药性,呈现一定的随年份增加而增强以及海南>广东的时空差异,尤其是耐药性中的谱型丰富度、多重耐药率、某一菌株的最多耐药数量以及多重耐药指数,海南（分别是1.00,69.23%,10,0.32）均大于广东（分别是0.82,54.55%,9,0.25）。[结论] 美人鱼发光菌的非典型毒力基因可能是通过水平基因转移获得,海南与广东区域美人鱼发光菌毒力基因与耐药的差异主要受到温度和抗生素使用的影响。