Multilocus analysis
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"Candidatus Neoehrlichia mikurensis" is the tick-borne agent of neoehrlichiosis, an infectious disease that primarily affects immunocompromised patients. So far, the genetic variability of "Ca. Neoehrlichia" has been studied only by comparing 16S rRNA genes and groEL operon sequences. We describe the development and use of a multilocus sequence analysis (MLSA) protocol to characterize the genetic diversity of clinical "Ca. Neoehrlichia" strains in Europe and their relatedness to other species within the Anaplasmataceae family. Six genes were selected: ftsZ, clpB, gatB, lipA, groEL, and 16S rRNA. Each MLSA locus was amplified by real-time PCR, and the PCR products were sequenced. Phylogenetic trees of MLSA locus relatedness were constructed from aligned sequences. Blood samples from 12 patients with confirmed "Ca. Neoehrlichia" infection from Sweden (n = 9), the Czech Republic (n = 2), and Germany (n = 1) were analyzed with the MLSA protocol. Three of the Swedish strains exhibited identical lipA sequences, while the lipA sequences of the strains from the other nine patients were identical to each other. One of the Czech strains had one differing nucleotide in the clpB sequence from the sequences of the other 11 strains. All 12 strains had identical sequences for the genes 16S rRNA, ftsZ, gatB, and groEL. According to the MLSA, among the Anaplasmataceae, "Ca. Neoehrlichia" is most closely related to Ehrlichia ruminantium, less so to Anaplasma phagocytophilum, and least to Wolbachia endosymbionts. To conclude, three sequence types of infectious "Ca. Neoehrlichia" were identified: one in the west of Sweden, one in the Czech Republic, and one spread throughout Europe.
- MeSH
- Anaplasmataceae klasifikace genetika izolace a purifikace MeSH
- esenciální geny MeSH
- fylogeneze MeSH
- genetická variace * MeSH
- genotyp * MeSH
- infekce bakteriemi čeledi Anaplasmataceae epidemiologie mikrobiologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- molekulární epidemiologie metody MeSH
- multilokusová sekvenční typizace metody MeSH
- RNA ribozomální 16S genetika MeSH
- senioři MeSH
- shluková analýza MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
- Německo MeSH
- Švédsko MeSH
To date, genotyping data on giardiasis have not been available in the Czech Republic. In this study, we characterized 47 human isolates of Giardia intestinalis from symptomatic as well as asymptomatic giardiasis cases. Genomic DNA from trophozoites was tested by PCR-sequence analysis at three loci (β-giardin, glutamate dehydrogenase and triose phosphate isomerase). Sequence analysis showed assemblages A and B in 41 (87.2%) and six (12.8%) isolates, respectively. Two of the 41 assemblage A samples were genotyped as sub-assemblage AI, and 39 were genotyped as sub-assemblage AII. Four previously identified multilocus genotypes (MLGs: AI-1, AII-1, AII-4 and AII-9) and six likely novel variations of MLGs were found. In agreement with previous studies, sequences from assemblage B isolates were characterized by a large genetic variability and by the presence of heterogeneous positions, which prevent the definition of MLGs. This study also investigated whether there was a relationship between the assemblage and clinical data (including drug resistance). However, due to the large number of genotypes and the relatively small number of samples, no significant associations with the clinical data were found.
- MeSH
- dítě MeSH
- dospělí MeSH
- genotyp MeSH
- Giardia klasifikace enzymologie genetika MeSH
- giardiáza parazitologie MeSH
- glutamátdehydrogenasa genetika MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- multilokusová sekvenční typizace MeSH
- předškolní dítě MeSH
- protozoální proteiny genetika MeSH
- sekvenční analýza DNA MeSH
- triózafosfátizomeráza genetika MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
V článku jsou prezentovány první výsledky charakterizace 29 invazivních kmenů Streptococcuspyogenes, izolovaných v České republice v první polovině roku 2003, metodou multilokusové sekvenačnítypizace (MLST). U žádného z 16 přítomných emm typů nebyla mezi kmeny našeho souboruzjištěna variabilita sekvenčních typů (ST). První výsledky MLST ukazují, že populace kmenůzpůsobujících závažná onemocnění v České republice je odlišná od kmenů izolovaných v zahraničí.U sedmi kmenů byly popsány nové sekvenční typy: ST134, ST308, ST336, 5T340, obsahující novékombinace známých alel a ST341 se třemi dosud nepopsanými alelami (gki 91, murI 65 a yqiL 60),zjištěný u třech kmenů. Nově popsané sekvenční typy byly registrovány v celosvětové databáziMLST S. pyogenes.
First results of multilocus sequence typing (MLST) for characterization of 29 invasive Streptococcuspyogenes strains isolated in the Czech Republic in the first half of 2003 are presented. None of 16emm types detected among the study strains showed sequence type (ST) variability. The MLSTresults are indicative of differences between the strains causing serious diseases in the CzechRepublic and those isolated in other countries. In seven strains, four new STs with known alleles innew combinations, ST134, ST308, ST336, ST340, and one new ST with three as yet undescribed alleles(gki 91, murI 65 and yqiL 60), ST341, were described. These newly described STs were submitted tothe web-based reference MLST database for S. pyogenes.
- MeSH
- alely MeSH
- finanční podpora výzkumu jako téma MeSH
- klonování DNA MeSH
- lidé MeSH
- sekvenční analýza MeSH
- Streptococcus pyogenes izolace a purifikace klasifikace patogenita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- databáze MeSH
- přehledy MeSH
- srovnávací studie MeSH
- Geografické názvy
- Česká republika MeSH
Given the great biological importance and high diversity of temperate Staphylococcus aureus bacteriophages, a method is needed for the description of their genomic structure. Here we have updated a multiplex PCR strategy for the complex characterization of S. aureus phages of the family Siphoviridae. Based on the comparative genomic analysis of the available phage sequences, a multilocus PCR strategy for typing the major modules of the phage genome was designed. The genomic modules were classified on the basis of the genes for integrase (10 types), anti-repressor (five types), replication proteins polA, dnaC and dnaD (four types), dUTPase (four types), portal protein (eight types), tail appendices (four types) and endolysin (four types) corresponding to the integrase locus, lysogeny control region, and modules for DNA replication, transcription regulation, packaging, tail appendices and lysis respectively. The nine PCR assays designed for the above sequences were shown to be capable to identify the bacteriophage gene pool present both in the phage and bacterial genomes and their extensive mosaic structure. The established multiplex PCR-based multilocus diagnostic scheme is convenient for rapid and reliable phage and prophage classification and for the study of bacteriophage evolution.
- MeSH
- DNA virů genetika MeSH
- genom virový MeSH
- multilokusová sekvenční typizace MeSH
- polymerázová řetězová reakce metody MeSH
- profágy klasifikace genetika MeSH
- Siphoviridae klasifikace genetika MeSH
- srovnávací genomová hybridizace MeSH
- Staphylococcus aureus genetika virologie MeSH
- virové proteiny genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Treponema pallidum strains are closely related at the genome level but cause distinct diseases. Subspecies pallidum (TPA) is the causative agent of syphilis, subspecies pertenue (TPE) causes yaws while subspecies endemicum (TEN) causes bejel (endemic syphilis). Compared to the majority of treponemal genomic regions, several chromosomal loci were found to be more diverse. To assess genetic variability in diverse genomic positions, we have selected (based on published genomic data) and sequenced five variable loci, TP0304, TP0346, TP0488, TP0515 and TP0558, in 19 reference Treponema pallidum strains including all T. pallidum subspecies (TPA, TPE and TEN). Results of this multilocus analysis divided syphilitic isolates into two groups: SS14-like and Nichols-like. The SS14-like group is comprised of SS14, Grady, Mexico A and Philadelphia 1 strains. The Nichols-like group consisted of strains Nichols, Bal 73-1, DAL-1, MN-3, Philadelphia 2, Haiti B and Madras. The TP0558 locus was selected for further studies because it clearly distinguished between the SS14- and Nichols-like groups and because the phylogenetic tree derived from the TP0558 locus showed the same clustering pattern as the tree constructed from whole genome sequences. In addition, TP0558 was shown as the only tested locus that evolved under negative selection within TPA strains. Sequencing of a short fragment (573bp) of the TP0558 locus in a set of 25 clinical isolates from 22 patients collected in the Czech Republic during 2012-2013 revealed that clinical isolates follow the SS14- and Nichols-like distribution.
- MeSH
- dospělí MeSH
- genotyp MeSH
- lidé středního věku MeSH
- lidé MeSH
- molekulární epidemiologie MeSH
- multilokusová sekvenční typizace MeSH
- novorozenec MeSH
- shluková analýza MeSH
- syfilis epidemiologie mikrobiologie MeSH
- Treponema pallidum klasifikace genetika izolace a purifikace MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
Cronobacter spp. are bacterial pathogens that affect children and immunocompromised adults. In this study, we used multilocus sequence typing (MLST) to determine sequence types (STs) in 11 Cronobacter spp. strains isolated from retail foods, 29 strains from dust samples obtained from vacuum cleaners, and 4 clinical isolates. Using biochemical tests, species-specific polymerase chain reaction, and MLST analysis, 36 strains were identified as Cronobacter sakazakii, and 6 were identified as Cronobacter malonaticus. In addition, one strain that originated from retail food and one from a dust sample from a vacuum cleaner were identified on the basis of MLST analysis as Cronobacter dublinensis and Cronobacter turicensis, respectively. Cronobacter spp. strains isolated from the retail foods were assigned to eight different MLST sequence types, seven of which were newly identified. The strains isolated from the dust samples were assigned to 7 known STs and 14 unknown STs. Three clinical isolates and one household dust isolate were assigned to ST4, which is the predominant ST associated with neonatal meningitis. One clinical isolate was classified based on MLST analysis as Cronobacter malonaticus and belonged to an as-yet-unknown ST. Three strains isolated from the household dust samples were assigned to ST1, which is another clinically significant ST. It can be concluded that Cronobacter spp. strains of different origin are genetically quite variable. The recovery of C. sakazakii strains belonging to ST1 and ST4 from the dust samples suggests the possibility that contamination could occur during food preparation. All of the novel STs and alleles for C. sakazakii, C. malonaticus, C. dublinensis, and C. turicensis determined in this study were deposited in the Cronobacter MLST database available online ( http://pubmlst.org/cronobacter/).
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- Cronobacter sakazakii klasifikace izolace a purifikace metabolismus MeSH
- Cronobacter klasifikace izolace a purifikace metabolismus MeSH
- enterobakteriální infekce mikrobiologie MeSH
- fylogeneze MeSH
- lidé MeSH
- mikrobiologie životního prostředí * MeSH
- molekulární typizace MeSH
- multilokusová sekvenční typizace MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- potravinářská mikrobiologie * MeSH
- prach * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
Hybridization and introgression can impact the evolution of natural populations. Several wild canid species hybridize in nature, sometimes originating new taxa. However, hybridization with free-ranging dogs is threatening the genetic integrity of grey wolf populations (Canis lupus), or even the survival of endangered species (e.g., the Ethiopian wolf C. simensis). Efficient molecular tools to assess hybridization rates are essential in wolf conservation strategies. We evaluated the power of biparental and uniparental markers (39 autosomal and 4 Y-linked microsatellites, a melanistic deletion at the β-defensin CBD103 gene, the hypervariable domain of the mtDNA control-region) to identify the multilocus admixture patterns in wolf x dog hybrids. We used empirical data from 2 hybrid groups with different histories: 30 presumptive natural hybrids from Italy and 73 Czechoslovakian wolfdogs of known hybrid origin, as well as simulated data. We assessed the efficiency of various marker combinations and reference samples in admixture analyses using 69 dogs of different breeds and 99 wolves from Italy, Balkans and Carpathian Mountains. Results confirmed the occurrence of hybrids in Italy, some of them showing anomalous phenotypic traits and exogenous mtDNA or Y-chromosome introgression. Hybridization was mostly attributable to village dogs and not strictly patrilineal. The melanistic β-defensin deletion was found only in Italian dogs and in putative hybrids. The 24 most divergent microsatellites (largest wolf-dog FST values) were equally or more informative than the entire panel of 39 loci. A smaller panel of 12 microsatellites increased risks to identify false admixed individuals. The frequency of F1 and F2 was lower than backcrosses or introgressed individuals, suggesting hybridization already occurred some generations in the past, during early phases of wolf expansion from their historical core areas. Empirical and simulated data indicated the identification of the past generation backcrosses is always uncertain, and a larger number of ancestry-informative markers is needed.
- MeSH
- beta-defensiny genetika MeSH
- chromozom Y MeSH
- genetická variace MeSH
- genetické markery * MeSH
- genotyp MeSH
- hybridizace genetická * MeSH
- mikrosatelitní repetice MeSH
- mitochondriální DNA MeSH
- molekulární evoluce MeSH
- multilokusová sekvenční typizace * MeSH
- populační genetika MeSH
- psi MeSH
- shluková analýza MeSH
- vlci MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- psi MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Itálie MeSH
The aim of the study was to propose a multilocus calibrator containing seven specific sequences of SSTR1-SSTR5, D2R, ER1 genes and the GUS house-keeping reference gene. For preparation, gene synthesis and gene assembly technique were used. The experimental group consisted of 44 pituitary adenomas with overproduction of growth hormone leading to acromegaly. The gene expression was investigated using the real-time PCR. We revealed highest numbers of SSTR2 and D2R transcripts, lower numbers of SSTR1, SSTR3, SSTR5 and ER1 trans¬cripts, and the lowest transcription of the SSTR4 gene. The use of the multicalibrator ensured a high comparability of the results obtained for the tested genes.
- MeSH
- akromegalie * etiologie genetika MeSH
- biopsie MeSH
- dospělí MeSH
- exprese genu MeSH
- genetická transkripce MeSH
- insulinu podobný růstový faktor I sekrece MeSH
- kalibrace * MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé středního věku MeSH
- lidé MeSH
- multilokusová sekvenční typizace MeSH
- nádory hypofýzy * genetika MeSH
- receptory dopaminové * analýza MeSH
- receptory somatostatinu * analýza MeSH
- růstový hormon sekrece MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- klinická studie MeSH
- práce podpořená grantem MeSH
The MLST scheme currently used for Enterococcus faecium typing was designed in 2002 and is based on putative gene functions and Enterococcus faecalis gene sequences available at that time. As a result, the original MLST scheme does not correspond to the real genetic relatedness of E. faecium strains and often clusters genetically distant strains to the same sequence types (ST). Nevertheless, typing has a significant impact on the subsequent epidemiological conclusions and introduction of appropriate epidemiological measures, thus it is crucial to use a more accurate MLST scheme. Based on the genome analysis of 1,843 E. faecium isolates, a new scheme, consisting of 8 highly discriminative loci, was created in this study. These strains were divided into 421 STs using the new MLST scheme, as opposed to 223 STs assigned by the original MLST scheme. The proposed MLST has a discriminatory power of D = 0.983 (CI95% 0.981 to 0.984), compared to the original scheme's D = 0.919 (CI95% 0.911 to 0.927). Moreover, we identified new clonal complexes with our newly designed MLST scheme. The scheme proposed here is available within the PubMLST database. Although whole genome sequencing availability has increased rapidly, MLST remains an integral part of clinical epidemiology, mainly due to its high standardization and excellent robustness. In this study, we proposed and validated a new MLST scheme for E. faecium, which is based on genome-wide data and thus reflects the tested isolates' more accurate genetic similarity. IMPORTANCE Enterococcus faecium is one of the most important pathogens causing health care associated infections. One of the main reasons for its clinical importance is a rapidly spreading resistance to vancomycin and linezolid, which significantly complicates antibiotic treatment of infections caused by such resistant strains. Monitoring the spread and relationships between resistant strains causing severe conditions represents an important tool for implementing appropriate preventive measures. Therefore, there is an urgent need to establish a robust method enabling strain monitoring and comparison at the local, national, and global level. Unfortunately, the current, extensively used MLST scheme does not reflect the real genetic relatedness between individual strains and thus does not provide sufficient discriminatory power. This can lead directly to incorrect epidemiological measures due to insufficient accuracy and biased results.
