Dataset Information

Phenotypic and Molecular Characterization of Brucella microti-Like Bacteria From a Domestic Marsh Frog (Pelophylax ridibundus).

ABSTRACT: Several Brucella isolates have been described in wild-caught and "exotic" amphibians from various continents and identified as B. inopinata-like strains. On the basis of epidemiological investigations conducted in June 2017 in France in a farm producing domestic frogs (Pelophylax ridibundus) for human consumption of frog's legs, potentially pathogenic bacteria were isolated from adults showing lesions (joint and subcutaneous abscesses). The bacteria were initially misidentified as Ochrobactrum anthropi using a commercial identification system, prior to being identified as Brucella spp. by MALDI-TOF assay. Classical phenotypic identification confirmed the Brucella genus, but did not make it possible to conclude unequivocally on species determination. Conventional and innovative bacteriological and molecular methods concluded that the investigated strain was very close to B. microti species, and not B. inopinata-like strains, as expected. The methods included growth kinetic, antimicrobial susceptibility testing, RT-PCR, Bruce-Ladder, Suis-Ladder, RFLP-PCR, AMOS-ERY, MLVA-16, the ectoine system, 16S rRNA and recA sequence analyses, the LPS pattern, in silico MLST-21, comparative whole-genome analyses (including average nucleotide identity ANI and whole-genome SNP analysis) and HRM-PCR assays. Minor polyphasic discrepancies, especially phage lysis and A-dominant agglutination patterns, as well as, small molecular divergences suggest the investigated strain should be considered a B. microti-like strain, raising concerns about its environmental persistence and unknown animal pathogenic and zoonotic potential as for other B. microti strains described to date.

SUBMITTER: Jay M

PROVIDER: S-EPMC6249338 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Phenotypic and Molecular Characterization of Brucella microti-Like Bacteria From a Domestic Marsh Frog (Pelophylax ridibundus).

Jaý Maryne M Girault Guillaume G Perrot Ludivine L Taunay Benoit B Vuilmet Thomas T Rossignol Frédérique F Pitel Pierre-Hugues PH Picard Elodie E Ponsart Claire C Mick Virginie V

Frontiers in veterinary science 20181115

Several Brucella isolates have been described in wild-caught and "exotic" amphibians from various continents and identified as B. inopinata-like strains. On the basis of epidemiological investigations conducted in June 2017 in France in a farm producing domestic frogs (Pelophylax ridibundus) for human consumption of frog's legs, potentially pathogenic bacteria were isolated from adults showing lesions (joint and subcutaneous abscesses). The bacteria were initially misidentif ...[more]

PMID: 30498697

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Project description:BackgroundBrucella microti was first isolated from common vole (Microtus arvalis) in the Czech Republic in Central Europe in 2007. As B. microti is the only Brucella species known to live in soil, its distribution, ecology, zoonotic potential, and genomic organization is of particular interest. The present paper is the first to report the isolation of B. microti from a wild boar (Sus scrofa), which is also the first isolation of this bacterial species in Hungary.ResultsThe B. microti isolate was cultured, after enrichment in Brucella-selective broth, from the submandibular lymph node of a female wild boar that was taken by hunters in Hungary near the Austrian border in September 2014. Histological and immunohistological examinations of the lymph node sections with B. abortus-, B. suis- and B. canis-specific sera gave negative results. The isolate did not require CO2 for growth, was oxidase, catalase, and urease positive, H2S negative, grew well in the presence of 20 μg/ml basic fuchsin and thionin, and had brownish pigmentation after three days of incubation. It gave strong positive agglutination with anti-A and anti-M but had a negative reaction with anti-R monospecific sera. The API 20 NE test identified it as Ochrobactrum anthropi with 99.9% identity, and it showed B. microti-specific banding pattern in the Bruce- and Suis-ladder multiplex PCR systems. Whole genome re-sequencing identified 30 SNPs in orthologous loci when compared to the B. microti reference genome available in GenBank, and the MLVA analysis yielded a unique profile.ConclusionsGiven that the female wild boar did not develop any clinical disease, we hypothesize that this host species only harboured the bacterium, serving as a possible reservoir capable of maintaining and spreading this pathogen. The infectious source could have been either a rodent, a carcass that had been eaten or infection occurred via the boar rooting in soil. The low number of discovered SNPs suggests an unexpectedly high level of genetic homogeneity in this Brucella species.

Project description:BackgroundUsing a combination of pyrosequencing and conventional Sanger sequencing, the complete genome sequence of the recently described novel Brucella species, Brucella microti, was determined. B. microti is a member of the genus Brucella within the Alphaproteobacteria, which consists of medically important highly pathogenic facultative intracellular bacteria. In contrast to all other Brucella species, B. microti is a fast growing and biochemically very active microorganism with a phenotype more similar to that of Ochrobactrum, a facultative human pathogen. The atypical phenotype of B. microti prompted us to look for genomic differences compared to other Brucella species and to look for similarities with Ochrobactrum.ResultsThe genome is composed of two circular chromosomes of 2,117,050 and 1,220,319 base pairs. Unexpectedly, we found that the genome sequence of B. microti is almost identical to that of Brucella suis 1330 with an overall sequence identity of 99.84% in aligned regions. The most significant structural difference between the two genomes is a bacteriophage-related 11,742 base pairs insert only present in B. microti. However, this insert is unlikely to have any phenotypical consequence. Only four protein coding genes are shared between B. microti and Ochrobactrum anthropi but impaired in other sequenced Brucella. The most noticeable difference between B. microti and other Brucella species was found in the sequence of the 23S ribosomal RNA gene. This unusual variation could have pleiotropic effects and explain the fast growth of B. microti.ConclusionContrary to expectations from the phenotypic analysis, the genome sequence of B. microti is highly similar to that of known Brucella species, and is remotely related to the one of O. anthropi. How the few differences in gene content between B. microti and B. suis 1330 could result in vastly different phenotypes remains to be elucidated. This unexpected finding will complicate the task of identifying virulence determinants in the Brucella genus. The genome sequence of B. microti will serve as a model for differential expression analysis and complementation studies. Our results also raise some concerns about the importance given to phenotypical traits in the definition of bacterial species.

Project description:Water frogs of the genus Pelophylax (previous Rana) species have been much studied in Europe for their outstanding reproductive mechanism in which sympatric hybridization between genetically distinct parental species produces diverse genetic forms of viable hybrid animals. The most common hybrid is P. esculentus that carries the genomes of both parental species, P. ridibundus and P. lessonae, but usually transfers the whole genome of only one parent to its offsprings (hybridogenesis). The evolutionary cost of transfer of the intact genome and hence the hemiclonal reproduction is the depletion of heterozygosity in the hybrid populations. Pelophylax esculentus presents an excellent example of the long-term sustained hybridization and hemiclonal reproduction in which the effects of the low genetic diversity are balanced through the novel mutations and periodic recombinations. In this study, we analyzed the mitochondrial (mt) and microsatellites DNA variations in hybrid Pelophylax populations from southern parts of the Pannonian Basin and a north-south transect of the Balkan Peninsula, which are home for a variety of Pelophylax genetic lineages. The mtDNA haplotypes found in this study corresponded to P. ridibundus and P. epeiroticus of the Balkan - Anatolian lineage (ridibundus-bedriagae) and to P. lessonae and a divergent lessonae haplotype of the lessonae lineage. The mtDNA genomes showed considerable intraspecific variation and geographic differentiation. The Balkan wide distributed P. ridibundus was found in all studied populations and its nuclear genome, along with either the lessonae or the endemic epeiroticus genome, in all hybrids. An unexpected finding was that the hybrid populations were invariably heteroplasmic, that is, they contained the mtDNA of both parental species. We discussed the possibility that such extensive heteroplasmy is a result of hybridization and it comes from regular leakage of the paternal mtDNA from a sperm of one species that fertilizes eggs of another. In this case, the mechanisms that protect the egg from heterospecific fertilization and further from the presence of sperm mtDNA could become compromised due to their differences and divergence at both, mitochondrial and nuclear DNA. The heteroplasmy once retained in the fertilized egg could be transmitted by hybrid backcrossing to the progeny and maintained in a population over generations. The role of interspecies and heteroplasmic hybrid animals due to their genomic diversity and better fitness compare to the parental species might be of the special importance in adaptations to miscellaneous and isolated environments at the Balkan Peninsula.

Dataset Information

Phenotypic and Molecular Characterization of Brucella microti-Like Bacteria From a Domestic Marsh Frog (Pelophylax ridibundus).

Publications

Phenotypic and Molecular Characterization of <i>Brucella microti</i>-Like Bacteria From a Domestic Marsh Frog (<i>Pelophylax ridibundus</i>).

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