Classification of brown pigmented aeromonads isolated from river water

Nine Aeromonas strains having a brown exopigment were isolated during the microbiological examination of river water. These brown-pigmented aeromonads were characterized by the phenotyping, fatty-acid methyl-ester analysis and ribotyping. All methods identically confirmed that the group of brown-pigmented aeromonads is quite heterogeneous. Apart from the Aeromonas media taxon, the brown-pigmented aeromonads in river water were represented also by strains of A. allosaccharophila and A. salmonicida subsp. pectinolytica.     

Functional Tn5393-Like Transposon in the R Plasmid pRAS2 from the Fish Pathogen Aeromonas salmonicida subspecies salmonicida Isolated in Norway

Tn5393c containing strA-strB was identified as part of R plasmid pRAS2 from the fish pathogen Aeromonas salmonicida subsp. salmonicida. This is the first time an intact and active transposon in the Tn5393 family has been reported in an ecological niche other than an agricultural habitat.     

Aeromonas piscicola sp. nov., isolated from diseased fish

Four Aeromonas strains (S1.2^T, EO-0505, TC1 and TI 1.1) isolated from moribund fish in Spain showed a restriction fragment length polymorphism (RFLP) pattern related to strains of Aeromonas salmonicida and Aeromonas bestiarum but their specific taxonomic position was unclear. Multilocus sequence analysis (MLSA) of housekeeping genes rpoD, gyrB, recA and dnaJ confirmed the allocation of these isolates to an unknown genetic lineage within the genus Aeromonas with A. salmonicida, A. bestiarum and Aeromonas popoffii as the phylogenetically nearest neighbours. Furthermore, a strain biochemically labelled as Aeromonas hydrophila (AH-3), showing a pattern of A. bestiarum based on 16S rDNA-RFLP, also clustered with the unknown genetic lineage. The genes rpoD and gyrB proved to be the best phylogenetic markers for differentiating these isolates from their neighbouring species. Useful phenotypic features for differentiating the novel species from other known Aeromonas species included their ability to hydrolyze elastin, produce acid from l-arabinose and salicin, and their inability to produce acid from lactose and use l-lactate as a sole carbon source. A polyphasic approach using phenotypic characterization, phylogenetic analysis of the 16S rRNA gene and of four housekeeping genes, as well as DNA–DNA hybridization studies and an analysis of the protein profiles by MALDI-TOF-MS, showed that these strains represented a novel species for which the name Aeromonas piscicola sp. nov. is proposed with isolate S1.2^T (=CECT 7443^T, =LMG 24783^T) as the type strain.     

Identification and Molecular Characterization of the Homogentisate Pathway Responsible for Pyomelanin Production,the Major Melanin Constituents in Aeromonas media WS

The pigmentation of many Aeromonas species has been thought to be due to the production of a L-DOPA (L-3,4-dihydroxyphenylalanine) based melanin. However, in this study we found that although L-DOPA synthesis occurs in the high-melanin-yielding Aeromonas media strain WS, it plays a minor, if any, role in pigmentation. Instead, the pigmentation of A. media strain WS is due to the production of pyomelanin through HGA (homogentisate). Gene products of phhA (encodes phenylalanine hydroxylase), tyrB and aspC (both encode aromatic amino acid aminotransferase), and hppD (encodes 4-hydroxyphenylpyruvate dioxygenase) constitute a linear pathway of converting phenylalanine to HGA and disruption of any one of these genes impairs or blocks pigmentation of A. media strain WS. This HGA biosynthesis pathway is widely distributed in Aeromonas, but HGA is only detectable in the cultures of pigmented Aeromonas species. Heterologous expression of HppD from both pigmented and non-pigmented Aeromonas species in E. coli leads to the production of pyomelanin and thus pigmentation, suggesting that most Aeromonas species have the critical enzymes to produce pyomelanin through HGA. Taken together, we have identified a widely conserved biosynthesis pathway of HGA based pyomelanin in Aeromonas that may be responsible for pigmentation of many Aeromonas species.     

Proposal to assign Aeromonas diversa sp. nov. as a novel species designation for Aeromonas group 501

The Aeromonas group 501, also named Aeromonas sp. HG13, is taxonomically close to A. schubertii. Results obtained in previous studies, including DNA–DNA hybridization and DNA fingerprinting, suggest that Aeromonas group 501 could constitute a different Aeromonas species. In this work we have performed a polyphasic study with the two strains comprising the Aeromonas sp. HG13 in order to propose a formal species name. They could be differentiated from A. schubertii by the indole and lysine decarboxylase tests and the utilization of l-lactate. Phenotypically, both strains were also easily separated from the other Aeromonas species. Sequence analysis of the 16S rRNA gene showed high sequence similarities (>97%) between Aeromonas group 501 and all Aeromonas species. Nevertheless, sequence divergences of cpn60, dnaJ, gyrB and rpoD genes were higher than the intraspecific threshold values established for each gene (3.5%, 3.3%, 2.3% and 2.6%, respectively), while sequence divergences between strains CDC 2478-85^T and CDC 2555-87 were low (0.6–1.1%). The DNA G+C content of the type strain was 62.2 mol%. Phenotypic and genotypic evidence strongly suggests that the Aeromonas group 501 is a novel species of the genus Aeromonas, for which the name Aeromonas diversa sp. nov. is proposed. The type strain is CDC 2478-85^T (=CECT 4254^T=ATCC 43946^T=LMG 17321^T).     

A proposal to unify two subspecies of Staphylococcus equorum: Staphylococcus equorum subsp. equorum and Staphylococcus equorum subsp. linens

Twelve isolates from jeotgal, a Korean high-salt-fermented seafood, identified as Staphylococcus equorum were compared by phenotypic and genotypic methods to determine their precise taxonomic identities at the subspecies level. Four strains and three strains had complete 16S rRNA gene sequence matches with S. equorum subsp. equorum DSM 20674^T and S. equorum subsp. linens DSM 15097^T, respectively. Five strains showed 99.9 % identity with the sequences of both type strains. In our DNA–DNA hybridization analyses among two type strains and two isolates, the similarities were over 72 % and were higher than the similarities presented at the subspecies proposal. Physiological characteristics such as sugar utilization, β-galactosidase activity, novobiocin resistance and salt tolerance, which were adopted for subspecies separation, could not be applied to assign the isolates to a taxonomic unit. Antibiotic susceptibility, hemolytic activity, biofilm formation and protein profiles did not present markers to divide the isolates into either of the subspecies. Multilocus sequence typing of the sequences of the 16S rRNA gene and five housekeeping genes did not produce any coherent relationship among the isolates and type strains. Repetitive element-PCR fingerprinting using ERIC (enterobacterial repetitive intergenic consensus) primers classified 12 isolates to three genotypes, and the genotypes of both type strains coincided with two isolates expressing different characteristics. Based on these phenotypic and genotypic analyses results, we propose to unify the present two subspecies of S. equorum into one species, S. equorum.     

The AsaP1 peptidase of Aeromonas salmonicida subsp. achromogenes is a highly conserved deuterolysin metalloprotease (family M35) and a major virulence factor

Infections by the bacterium Aeromonas salmonicida subsp. achromogenes cause significant disease in a number of fish species. In this study, we showed that AsaP1, a toxic 19-kDa metallopeptidase produced by A. salmonicida subsp. achromogenes, belongs to the group of extracellular peptidases (Aeromonas type) (MEROPS ID M35.003) of the deuterolysin family of zinc-dependent aspzincin endopeptidases. The structural gene of AsaP1 was sequenced and found to be highly conserved among gram-negative bacteria. An isogenic ΔasaP1 A. salmonicida subsp. achromogenes strain was constructed, and its ability to infect fish was compared with that of the wild-type (wt) strain. The ΔasaP1 strain was found to infect Arctic charr, Atlantic salmon, and Atlantic cod, but its virulence was decreased relative to that of the wt strain. The 50% lethal dose of the AsaP1 mutant was 10-fold higher in charr and 5-fold higher in salmon than that of the wt strain. The pathology induced by the AsaP1-deficient strain was also different from that of the wt strain. Furthermore, the mutant established significant bacterial colonization in all observed organs without any signs of a host response in the infected tissue. AsaP1 is therefore the first member of the M35 family that has been shown to be a bacterial virulence factor.     

Indirect evidence of alteration in the expression of the rDNA genes in interspecific hybrids betweenDrosophila melanogaster andDrosophila simulans

Crosses betweenDrosophila melanogaster females andD. simulans males produce viable hybrid females, while males are lethal. These males are rescued if they carry theD. simulans Lhr gene. This paper reports that females of the wild-typeD. melanogaster population Staket do not produce viable hybrid males when crossed withD. simulans Lhr males, a phenomenon which we designate as the Staket phenotype. The agent responsible for this phenomenon was found to be the StaketX chromosome (X ^mel ,Stk). Analysis of the Staket phenotype showed that it is suppressed by extra copies ofD. melanogaster rDNA genes and that theX ^mel ,Stk chromosome manifests a weak bobbed phenotype inD. melanogaster X ^mel ,Stk/0 males. The numbers of functional rDNA genes inX ^mel ,Stk andX ^mel ,y w (control) chromosomes were found not to differ significantly. Thus a reduction in rDNA gene number cannot account for the weak bobbedX ^mel ,Stk phenotype let alone the Staket phenotype. The rRNA precursor molecules transcribed from theX ^mel ,Stk rDNA genes seem to be correctly processed in both intraspecific (melanogaster) and interspecific (melanogaster-simulans) conditions. It is therefore suggested that theX ^mel ,Stk rDNA genes are inefficiently transcribed in themelanogaster-simulans hybrids.     

Melanin Production by Rhizobium Strains

Different Rhizobium and Bradyrhizobium strains were screened for their ability to produce melanin. Pigment producers (Mel⁺) were found among strains of R. leguminosarum biovars viceae, trifolii, and phaseoli, R. meliloti, and R. fredii; none of 19 Bradyrhizobium strains examined gave a positive response. Melanin production and nod genes were plasmid borne in R. leguminosarum biovar trifolii RS24. In R. leguminosarum biovar phaseoli CFN42 and R. meliloti GR015, mel genes were located in the respective symbiotic plasmids. In R. fredii USDA 205, melanin production correlated with the presence of its smallest indigenous plasmid.     

Comparison of phenotypical and genetic identification of Aeromonas strains isolated from diseased fish

Phenotypicaly identified Aeromonas strains (n=119) recovered mainly from diseased fish were genetically re-identified and the concordance between the results was analysed. Molecular characterization based on the GCAT genus specific gene showed that only 90 (75.6%) strains belonged to the genus Aeromonas. The 16S rDNA-RFLP method identified correctly most of the strains with the exception of a few that belonged to A. bestiarum, A. salmonicida or A. piscicola. Separation of these 3 species was correctly assessed with the rpoD gene sequences, which revealed that 5 strains with the RFLP pattern of A. salmonicida belonged to A. piscicola, as did 1 strain with the pattern of A. bestiarum. Correct phenotypic identification occurred in only 32 (35.5%) of the 90 strains. Only 14 (21.8%) of the 64 phenotypically identified A. hydrophila strains belonged to this species. However, coincident results were obtained in 88% (15/17) of the genetically identified A. salmonicida strains. Phenotypic tests were re-evaluated on the 90 genetically characterized Aeromonas strains and there were contradictions in the species A. sobria for a number of previously published species-specific traits. After genetic identification, the prevailing species were A. sobria, A. salmonicida, A. bestiarum, A. hydrophila, A. piscicola and A. media but we could also identify a new isolate of the recently described species A. tecta. This work emphasizes the need to rely on the 16S rDNA-RFLP method and sequencing of housekeeping genes such as rpoD for the correct identification of Aeromonas strains.     

Molecular Basis of Sulfonamide and Trimethoprim Resistance in Fish-Pathogenic Aeromonas Isolates

Sulfonamide-trimethoprim-resistant Aeromonas salmonicida and motile Aeromonas spp. from diseased fish of the GERM-Vet study carried the sul1 gene together with mostly cassette-borne trimethoprim resistance genes, including the novel gene dfrA28. The seven dfrA and dfrB genes identified were located mostly in class 1 integrons which commonly harbored other gene cassettes.     

Staphylococcus petrasii sp. nov. including S. petrasii subsp. petrasii subsp. nov. and S. petrasii subsp. croceilyticus subsp. nov., isolated from human clinical specimens and human ear infections

Thirteen coagulase-negative, oxidase-negative, and novobiocin-susceptible staphylococci were isolated from human clinical specimens. The isolates were differentiated from known staphylococcal species on the basis of 16S rRNA, hsp60, rpoB, dnaJ, tuf, and gap gene sequencing, automated ribotyping, (GTG)₅-PCR fingerprinting, and MALDI-TOF MS analysis. Phylogenetic analysis based on the 16S rRNA gene sequence indicated phylogenetic relatedness of the analyzed strains to Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus devriesei, and Staphylococcus lugdunensis. DNA–DNA hybridization experiments between representative strains CCM 8418^T, CCM 8421^T, and the closest phylogenetic neighbors confirmed that the isolates represent novel Staphylococcus species, for which the name Staphylococcus petrasii sp. nov. is proposed. Genotypic and phenotypic analyses unambiguously split the strains into two closely related subclusters. Based on the results, two novel subspecies S. petrasii subsp. petrasii subsp. nov. and S. petrasii subsp. croceilyticus subsp. nov. are proposed, with type strains CCM 8418^T (=CCUG 62727^T) and CCM 8421^T (=CCUG 62728^T), respectively.     

Construction of a new cloning vector utilizing a cryptic plasmid and the highly expressed melanin-synthesizing gene operon from Streptomyces castaneoglobisporus

Streptomyces castaneoglobisporus HUT6202 overproduces a diffusible melanin pigment and harbors a cryptic 7.4-kb plasmid, pHY6202. We constructed a Streptomyces cloning vector, pSY10CMM, consisting of the HUT6202 rep gene, the thiostrepton resistance gene and an operon encoding the synthesis of melanin pigment, abbreviated mel, from S. castaneoglobisporus. The vector, which has SphI and BamHI sites as cloning sites with insertional inactivation of the mel, is a more convenient cloning vector than commonly used pIJ702, because of its broad host range for antibiotic-producing Streptomyces strains and its much greater production of diffusible melanin pigment.     

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.     

Isolation,Characterization, and Antifungal Susceptibility of Melanin-Deficient Mutants of <Emphasis Type="Italic">Scedosporium prolificans</Emphasis>

Scedosporium prolificans mutants lacking the ability to synthesize melanin were selected after ultraviolet light (UV) irradiation. UV exposure of S. prolificans conidia resulted in a high frequency of melanin-deficient (mel⁻) mutants. Stable and non-stable morphological variants were found in the population: reversion of the mutant phenotype was always to the wild-type phenotype. Based on their morphological differences, these variants were classified into five different groups that were phenotypically characterized. The mel⁻ mutants plus the wild-type strain were examined for in vitro susceptibility to antifungal agents with different and/or the same mechanism of action. There was no apparent difference in minimum inhibitory concentrations when comparing the wild-type and the mel⁻ mutants. Therefore, melanin does not appear to confer protection against the more important antifungal agents in S. prolificans. Received: 30 April 2002 / Accepted: 10 July 2002     

Draft Genome Sequence of High-Melanin-Yielding Aeromonas media Strain WS

We sequenced the genome of the high-melanin-yielding Aeromonas media strain WS and then analyzed genes potentially involved in melanin formation. The 4.2-Mb draft genome carries multiple genes responsible for pyomelanin synthesis and other candidate genes identified in our separate study, which have no homolog in other strains of Aeromonas species.     

Indigenous Bacteria in Hemolymph and Tissues of Marine Bivalves at Low Temperatures

Hemolymph and soft tissues of Pacific oysters (Crassostrea gigas) kept in sand-filtered seawater at temperatures between 1 and 8°C were normally found to contain bacteria, with viable counts (CFU) in hemolymph in the range 1.4 × 10² to 5.6 × 10² bacteria per ml. Pseudomonas, Alteromonas, Vibrio, and Aeromonas organisms dominated, with a smaller variety of morphologically different unidentified strains. Hemolymph and soft tissues of horse mussels (Modiolus modiolus), locally collected from a 6- to 10-m depth in the sea at temperatures between 4 and 6°C, also contained bacteria. The CFU in horse mussel hemolymph was of the same magnitude as that in oysters (mean, 2.6 × 10⁴), and the bacterial flora was dominated by Pseudomonas (61.3%), Vibrio (27.0%), and Aeromonas (11.7%) organisms. In soft tissues of horse mussels, a mean CFU of 2.9 × 10⁴ bacteria per g was found, with Vibrio (38.5%), Pseudomonas (33.0%), and Aeromonas (28.5%) constituting the major genera. After the challenge of oysters in seawater at 4°C to the psychrotrophic fish pathogen Vibrio salmonicida (strains NCIMB 2245 from Scotland and TEO 84001 from Norway) and a commensal Aeromonas sp. isolated from oysters, the viable count in hemolymph increased 1,000-fold to about 10⁵ bacteria per ml. In soft tissues, about a 1,000-fold increase in CFU to 6 × 10⁷ was observed. V. salmonicida NCIMB 2245 invaded hemolymph and soft tissues after 14 days and dominated these compartments after 41 days, whereas strain TEO 84001 did not invade soft tissues to the same extent. Challenge with V. salmonicida NCIMB 2245 resulted in 100% mortality, whereas about 50% of the oysters survived challenge with the Norwegian strain, TEO 84001. The commensal Aeromonas sp. invaded hemolymph and soft tissues and caused 100% mortality. Oyster hemolymph contained agglutinins for Vibrio anguillarum but not for V. salmonicida, whereas we did not find agglutinins for either of these bacteria in horse mussels. Agglutinins for horse and human erythrocytes were found in hemolymph from both animals. We found no differences in agglutinin titers in oysters from different Norwegian locations, and long-term challenge with bacteria in seawater did not result in changes of agglutinin activity. These studies demonstrate that bacteria exist in hemolymph and soft tissues of marine bivalves at temperatures below 8°C. Increased bacterial numbers in seawater at 4°C result in augmented invasion of bacteria in hemolymph and soft tissues. V. salmonicida, a bacterium pathogenic for fish at low temperatures, invades bivalve hemolymph and soft tissues, and thus bivalves may serve as a reservoir for pathogens of fish at low seawater temperatures.     

Fusobacterium nucleatum subsp. fusiforme Gharbia and Shah 1992 is a Later Synonym of Fusobacterium nucleatum subsp. vincentii Dzink et al. 1990

On the basis of the DNA–DNA hybridization patterns and phenotypic characteristics, Fusobacterium nucleatum was classified into five subspecies. Previous studies have suggested that F. nucleatum subsp. vincentii is genetically similar to F. nucleatum subsp. fusiforme. The aim of this study was to investigate the possibility of classifying these two subspecies into a single subspecies by phylogenetic analysis using a single sequence (24,715 bp) concatenated 22 housekeeping genes of eight F. nucleatum strains including type strains of five F. nucleatum subspecies. The phylogenetic analysis indicated that F. nucleatum subsp. vincentii and F. nucleatum subsp. fusiforme were clustered in the same group and each strain of other F. nucleatum subspecies were also separated into the same cluster. These results suggested that F. nucleatum subsp. fusiforme and F. nucleatum subsp. vincentii can be classified into a single subspecies. F. nucleatum subsp. vincentii was early published name; therefore, F. nucleatum subsp. fusiforme Gharbia and Shah 1992 can be regarded as a later synonym of F. nucleatum subsp. vincentii Dzink et al. 1990.