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Microb Genom ; 3(10): e000133, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-29177091


Yersinia pseudotuberculosis is a Gram-negative intestinal pathogen of humans and has been responsible for several nationwide gastrointestinal outbreaks. Large-scale population genomic studies have been performed on the other human pathogenic species of the genus Yersinia, Yersinia pestis and Yersinia enterocolitica allowing a high-resolution understanding of the ecology, evolution and dissemination of these pathogens. However, to date no purpose-designed large-scale global population genomic analysis of Y. pseudotuberculosis has been performed. Here we present analyses of the genomes of 134 strains of Y. pseudotuberculosis isolated from around the world, from multiple ecosystems since the 1960s. Our data display a phylogeographic split within the population, with an Asian ancestry and subsequent dispersal of successful clonal lineages into Europe and the rest of the world. These lineages can be differentiated by CRISPR cluster arrays, and we show that the lineages are limited with respect to inter-lineage genetic exchange. This restriction of genetic exchange maintains the discrete lineage structure in the population despite co-existence of lineages for thousands of years in multiple countries. Our data highlights how CRISPR can be informative of the evolutionary trajectory of bacterial lineages, and merits further study across bacteria.

Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Infecções por Yersinia pseudotuberculosis/microbiologia , Yersinia pseudotuberculosis/classificação , Yersinia pseudotuberculosis/genética , Animais , Biblioteca Gênica , Humanos , Filogenia , Sequenciamento Completo do Genoma
Vector Borne Zoonotic Dis ; 17(2): 123-133, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27854567


Voles (Arvicolinae, Rodentia) are known carriers of zoonotic bacteria such as Bartonella spp. and Francisella tularensis. However, apart from F. tularensis, the bacterial microbiome of voles has not previously been determined in Finland and rarely elsewhere. Therefore, we studied liver samples from 61 voles using 16S ribosomal RNA gene PCR analysis, followed by Sanger sequencing. Twenty-three of these samples were also studied with tag-encoded pyrosequencing. The samples originated from 21 field voles (Microtus agrestis), 37 tundra voles (Microtus oeconomus), and 3 bank voles (Myodes glareolus). With the more conventional 16S rDNA PCR analysis, 90 (33%) of the recovered 269 sequence types could be identified to genus level, including Bartonella, Francisella, Mycoplasma, Anaplasma, and Acinetobacter in 31, 15, 9, 9, and 9 sequences, respectively. Seventy-five (28%) matched best with sequences of uncultured bacteria, of which 40/75 could be classified to the order Clostridiales and, more specifically, to families Lachnospiraceae and Ruminococcaceae. Pyrosequencing from 23 samples revealed comparable and similar results: clinically relevant bacterial families such as Mycoplasmataceae, Bartonellaceae, Anaplasmataceae, and Francisellaceae were recognized. These analyses revealed significant bacterial diversity in vole livers, consisting of distinct and constant sequence patterns reflecting bacteria found in the intestinal gut, but including some known zoonotic pathogens as well. The molecular bacterial sequence types determined with the two different techniques shared major similarities and verified remarkable congruency between the methods.

Arvicolinae/microbiologia , Bactérias/genética , Bactérias/isolamento & purificação , Genoma Bacteriano , Metagenômica , Animais , Bactérias/classificação , Finlândia
J Microbiol Methods ; 128: 69-73, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27435532


Bacillus spp. include human pathogens such as Bacillus anthracis, the causative agent of anthrax and a biothreat agent. Bacillus spp. form spores that are physically highly resistant and may remain active over sample handling. We tested four commercial DNA extraction kits (QIAamp DNA Mini Kit, RTP Pathogen Kit, ZR Fungal/Bacterial DNA MiniPrep, and genesig Easy DNA/RNA Extraction kit) for sample inactivation and DNA recovery from two powders (icing sugar and potato flour) spiked with Bacillus thuringiensis spores. The DNA was analysed using a B. thuringiensis-specific real-time PCR assay. The detection limit was 3×10(1)CFU of spiked B. thuringiensis spores with the QIAamp DNA Mini, RTP Pathogen, and genesig Easy DNA/RNA Extraction kits, and 3×10(3)CFU with the ZR Fungal/Bacterial DNA MiniPrep kit. The results showed that manual extraction kits are effective and safe for fast and easy DNA extraction from powder samples even in field conditions. Adding a DNA filtration step to the extraction protocol ensures the removal of Bacillus spp. spores from DNA samples without affecting sensitivity.

Bacillus thuringiensis/isolamento & purificação , DNA Bacteriano/isolamento & purificação , Kit de Reagentes para Diagnóstico , Esporos Bacterianos/isolamento & purificação , Limite de Detecção , Pós/química
Environ Microbiol ; 17(11): 4306-21, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25712141


The clustered regularly interspaced short palindromic repeat - CRISPR-associated genes (CRISPR-Cas) system is used by bacteria and archaea against invading conjugative plasmids or bacteriophages. Central to this immunity system are genomic CRISPR loci that contain fragments of invading DNA. These are maintained as spacers in the CRISPR loci between direct repeats and the spacer composition in any bacterium reflects its evolutionary history. We analysed the CRISPR locus sequences of 335 Yersinia pseudotuberculosis complex strains. Altogether 1902 different spacer sequences were identified and these were used to generate a database for the spacer sequences. Only ∼10% of the spacer sequences found matching sequences. In addition, surprisingly few spacers were shared by Yersinia pestis and Y. pseudotuberculosis strains. Interestingly, 32 different protospacers were present in the conjugative plasmid pYptb32953. The corresponding spacers were identified from 35 different Y. pseudotuberculosis strains indicating that these strains had encountered pYptb32953 earlier. In conjugation experiments, pYptb32953-specific spacers generally prevented conjugation with spacer-positive and spacer-free strains. However, some strains with one to four spacers were invaded by pYptb32953 and some spacer-free strains were fully resistant. Also some spacer-positive strains were intermediate resistant to conjugation. This suggests that one or more other defence systems are determining conjugation efficiency independent of the CRISPR-Cas system.

Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/imunologia , Conjugação Genética/imunologia , Bases de Dados de Ácidos Nucleicos , Plasmídeos/imunologia , Yersinia pseudotuberculosis/genética , Bacteriófagos/imunologia , Sequência de Bases , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Genômica , Dados de Sequência Molecular , Plasmídeos/genética , Yersinia pestis/genética , Yersinia pseudotuberculosis/classificação
J Virol ; 86(23): 12625-42, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22973030


The bacteriophage vB_YecM-ϕR1-37 (ϕR1-37) is a lytic yersiniophage that can propagate naturally in different Yersinia species carrying the correct lipopolysaccharide receptor. This large-tailed phage has deoxyuridine (dU) instead of thymidine in its DNA. In this study, we determined the genomic sequence of phage ϕR1-37, mapped parts of the phage transcriptome, characterized the phage particle proteome, and characterized the virion structure by cryo-electron microscopy and image reconstruction. The 262,391-bp genome of ϕR1-37 is one of the largest sequenced phage genomes, and it contains 367 putative open reading frames (ORFs) and 5 tRNA genes. Mass-spectrometric analysis identified 69 phage particle structural proteins with the genes scattered throughout the genome. A total of 269 of the ORFs (73%) lack homologues in sequence databases. Based on terminator and promoter sequences identified from the intergenic regions, the phage genome was predicted to consist of 40 to 60 transcriptional units. Image reconstruction revealed that the ϕR1-37 capsid consists of hexameric capsomers arranged on a T=27 lattice similar to the bacteriophage ϕKZ. The tail of ϕR1-37 has a contractile sheath. We conclude that phage ϕR1-37 is a representative of a novel phage type that carries the dU-containing genome in a ϕKZ-like head.

Bacteriófagos/química , Bacteriófagos/genética , Genoma Viral/genética , Modelos Moleculares , Proteoma/genética , Vírion/química , Yersinia enterocolitica/virologia , Sequência de Bases , Northern Blotting , Southern Blotting , Biologia Computacional , Microscopia Crioeletrônica , Primers do DNA/genética , Processamento de Imagem Assistida por Computador , Espectrometria de Massas , Dados de Sequência Molecular , Análise de Sequência de DNA