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16S rRNA gene sequence analysis of Photobacterium damselae and nested PCR method for rapid detection of the causative agent of fish pasteurellosis.

ABSTRACT: The causative agent of fish pasteurellosis, the organism formerly known as Pasteurella piscicida, has been reclassified as Photobacterium damselae subsp. piscicida on the basis of 16S rRNA gene sequence comparisons and chromosomal DNA-DNA hybridization data; thus, this organism belongs to the same species as Photobacterium damselae subsp. damselae (formerly Vibrio damselae). Since reassignment of P. damselae subsp. piscicida was based on only two strains, one objective of the present work was to confirm the taxonomic position of this fish pathogen by sequencing the 16S rRNA genes of 26 strains having different geographic and host origins. In addition, a nested PCR protocol for detection of P. damselae based on 16S rRNA was developed. This PCR protocol was validated by testing 35 target and 24 nontarget pure cultures, and the detection limits obtained ranged from 1 pg to 10 fg of DNA (200 to 20 cells). A similar level of sensitivity was observed when the PCR protocol was applied to fish tissues spiked with bacteria. The PCR approach described in this paper allows detection of the pathogen in mixed plate cultures obtained from asymptomatic fish suspected to be carriers of P. damselae subsp. piscicida, in which growth of this bacterium cannot be visualized. Our results indicate that the selective primers which we designed represent a powerful tool for sensitive and specific detection of fish pasteurellosis.

SUBMITTER: Osorio CR

PROVIDER: S-EPMC91440 | biostudies-literature | 1999 Jul

REPOSITORIES: biostudies-literature

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16S rRNA gene sequence analysis of Photobacterium damselae and nested PCR method for rapid detection of the causative agent of fish pasteurellosis.

Osorio C R CR Collins M D MD Toranzo A E AE Barja J L JL Romalde J L JL

Applied and environmental microbiology 19990701 7

The causative agent of fish pasteurellosis, the organism formerly known as Pasteurella piscicida, has been reclassified as Photobacterium damselae subsp. piscicida on the basis of 16S rRNA gene sequence comparisons and chromosomal DNA-DNA hybridization data; thus, this organism belongs to the same species as Photobacterium damselae subsp. damselae (formerly Vibrio damselae). Since reassignment of P. damselae subsp. piscicida was based on only two strains, one objective of the present work was to ...[more]

PMID: 10388687

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Project description:UnlabelledPhotobacterium damselae subsp. damselae is considered to be an emerging pathogen of marine fish of importance in aquaculture, with a notable increase in its geographical distribution during the last several years. In this study, we carried out for the first time to our knowledge a genetic and pathobiological characterization of 14 strains isolated from sea bass (Dicentrarchus labrax) reared in the Southeastern Black Sea, where high mortalities were observed at two aquaculture farms during the summer and autumn of 2011. Heterogeneity was evidenced among strains in phenotypical traits, such as sucrose fermentation, motility, and hemolysis. Although 11 of 14 isolates were hemolytic, we found that all of the isolates lacked the pPHDD1 virulence plasmid that encodes the phospholipase-D damselysin (Dly) and the pore-forming toxin PhlyP, two hemolysins previously reported to constitute major virulence factors for turbot. Subsequent PCR and sequencing analyses demonstrated that the 11 hemolytic isolates harbored a complete hlyAch gene, a chromosome I-borne gene that encodes HlyAch hemolysin, whereas the three nonhemolytic isolates contained hlyAch pseudogenes caused by insertion sequence elements. Virulence challenges with two representative strains revealed that, albeit less virulent than the pPHDD1-harboring strain RM-71, the plasmidless hlyAch-positive and hlyAch-negative Black Sea isolates were pathogenic for sea bass. A phylogenetic analysis based on the toxR gene sequence uncovered a greater diversity in the isolates, indicating that the presence of this pathogen in the Black Sea was not caused by the introduction and spread of a single virulent clone but by the proliferation of different clones.ImportanceThe geographical distribution of marine bacterial pathogens is undergoing a worldwide increase. In particular, bacteria of the group vibrios are increasingly being isolated as the causative agents of disease in novel species of cultivated fish in areas where they had not been previously reported. Here we characterize for the first time to our knowledge a collection of isolates of the fish and human pathogen Photobacterium damselae subsp. damselae from diseased sea bass reared in the Black Sea. We uncovered great genetic diversity in the Black Sea isolates of this pathogen, suggesting a multiclonal origin. We also demonstrate for the first time that these isolates bear pathogenic potential for sea bass cultures by virulence challenges.

Project description:Photobacterium damselae subsp. damselae is a pathogen of marine animals, including fish of importance in aquaculture. The virulence plasmid pPHDD1, characteristic of highly hemolytic isolates, encodes the hemolysins damselysin (Dly) and phobalysin (PhlyP). Strains lacking pPHDD1 constitute the vast majority of the isolates from fish outbreaks, but genetic studies to identify virulence factors in plasmidless strains are scarce. Here, we show that the chromosome I-encoded hemolysin PhlyC plays roles in virulence and cell toxicity in pPHDD1-negative isolates of this pathogen. By combining the analyses of whole genomes and of gene deletion mutants, we identified two hitherto uncharacterized chromosomal loci encoding a phospholipase (PlpV) and a collagenase (ColP). PlpV was ubiquitous in the subspecies and exerted hemolytic activity against fish erythrocytes, which was enhanced in the presence of lecithin. ColP was restricted to a fraction of the isolates and was responsible for the collagen-degrading activity in this subspecies. Consistent with the presence of signal peptides in PlpV and ColP sequences, mutants for the type II secretion system (T2SS) genes epsL and pilD exhibited impairments in phospholipase and collagenase activities. Sea bass virulence experiments and cell culture assays demonstrated major contributions of PhlyC and PlpV to virulence and toxicity.IMPORTANCE This study constitutes genetic and genomic analyses of plasmidless strains of an emerging pathogen in marine aquaculture, Photobacterium damselae subsp. damselae To date, studies on the genetic basis of virulence were restricted to the pPHDD1 plasmid-encoded toxins Dly and PhlyP. However, the vast majority of the recent isolates of this pathogen from fish farm outbreaks lack this plasmid. Here we demonstrate that the plasmidless strains produce two hitherto uncharacterized ubiquitous toxins encoded in chromosome I, namely, the hemolysin PhlyC and the phospholipase PlpV. We report the main roles of these two toxins in fish virulence and in cell toxicity. Our results constitute the basis for a better understanding of the virulence of a widespread marine pathogen.

Project description:BackgroundSequencing of the PCR-amplified 16S rRNA gene has become a common approach to microbial community investigations in the fields of human health and environmental sciences. This approach, however, is difficult when the amount of DNA is too low to be amplified by standard PCR. Nested PCR can be employed as it can amplify samples with DNA concentration several-fold lower than standard PCR. However, potential biases with nested PCRs that could affect measurement of community structure have received little attention.ResultsIn this study, we used 17 DNAs extracted from vaginal swabs and 12 DNAs extracted from stool samples to study the influence of nested PCR amplification of the 16S rRNA gene on the estimation of microbial community structure using Illumina MiSeq sequencing. Nested and standard PCR methods were compared on alpha- and beta-diversity metrics and relative abundances of bacterial genera. The effects of number of cycles in the first round of PCR (10 vs. 20) and microbial diversity (relatively low in vagina vs. high in stool) were also investigated. Vaginal swab samples showed no significant difference in alpha diversity or community structure between nested PCR and standard PCR (one round of 40 cycles). Stool samples showed significant differences in alpha diversity (except Shannon's index) and relative abundance of 13 genera between nested PCR with 20 cycles in the first round and standard PCR (P<0.01), but not between nested PCR with 10 cycles in the first round and standard PCR. Operational taxonomic units (OTUs) that had low relative abundance (sum of relative abundance <0.167) accounted for most of the distortion (>27% of total OTUs in stool).ConclusionsNested PCR introduced bias in estimated diversity and community structure. The bias was more significant for communities with relatively higher diversity and when more cycles were applied in the first round of PCR. We conclude that nested PCR could be used when standard PCR does not work. However, rare taxa detected by nested PCR should be validated by other technologies.

Dataset Information

16S rRNA gene sequence analysis of Photobacterium damselae and nested PCR method for rapid detection of the causative agent of fish pasteurellosis.

Publications

16S rRNA gene sequence analysis of Photobacterium damselae and nested PCR method for rapid detection of the causative agent of fish pasteurellosis.

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