• Publikační typ
• abstrakty MeSH

Sekvenování genů 16S, 18S a ITS regionů se stalo jedním z nejdůležitějších nástrojů v molekulární diagnostice, zejména v oblasti mikrobiologie, patologie a soudního lékařství. Výše zmíněné geny, obsahující konzervované i variabilní oblasti, jsou hojně využívány pro taxonomické zařazení bakterií a eukaryot. Sekvenování 16S rDNA umožňuje detekci bakteriálních infekcí, zatímco sekvenace ITS regionů a 18S rDNA je využívána při identifikaci mykotických, případně parazitárních infekcí, a to především v případech, kdy tradiční metody selhávají. Tento článek se zaměřuje na rozšířené možnosti těchto metod, jejich uplatnění v klinické diagnostice a výzkumu, zkoumá výhody a nevýhody, a diskutuje potenciální budoucí vývoj v oblasti technologie sekvenování nové generace (NGS).

Gene sequencing of 16S, 18S, and ITS regions is a crucial tool in molecular diagnostics, especially in microbiology, pathology and forensic medicine. These genes contain conserved and variable regions and are widely used for the taxonomic classification of bacteria and eukaryotes. Sequencing of 16S rDNA helps detect bacterial infections, while sequencing of ITS regions and 18S rDNA is used to identify fungal or parasitic infections, especially when traditional methods are ineffective. This article focuses on the expanded possibilities of these methods, their application in clinical diagnostics and research, their advantages and disadvantages, and discusses potential future developments in the field of next-generation sequencing (NGS) technology.

• Klíčová slova
• Internal Transcribed Spacers, 16S rDNA, 18S rDNA,
• MeSH
• diagnostické techniky molekulární klasifikace   metody   MeSH
• DNA bakterií analýza   genetika   klasifikace   MeSH
• DNA analýza   genetika   klasifikace   ultrastruktura   MeSH
• infekční nemoci * diagnóza   genetika   MeSH
• lidé MeSH
• vysoce účinné nukleotidové sekvenování * klasifikace   metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH

Genetic diversity among 43 petroleum hydrocarbon-degrading Pseudomonas belonging to four different species and the type strain Pseudomonas aeruginosa MTCC1034 was assessed by using restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR)-amplified 16S-23S rDNA intergenic spacer regions (ISRs) polymorphism. PCR amplification from all Pseudomonas species yielded almost identical ISR amplicons of "?" 800 bp and in nested PCR of "?" 550 bp. The RFLP analysis with MboI and AluI revealed considerable intraspecific variations within the Pseudomonas species. The dendrogram constructed on the basis of the PCR-RFLP patterns of 16S-23S rDNA intergenic spacer regions differentiated all the species into seven different clusters.

• MeSH
• DNA bakterií genetika   MeSH
• fylogeneze MeSH
• mezerníky ribozomální DNA genetika   MeSH
• polymerázová řetězová reakce metody   MeSH
• polymorfismus délky restrikčních fragmentů MeSH
• Pseudomonas klasifikace   genetika   izolace a purifikace   metabolismus   MeSH
• RNA ribozomální 16S genetika   MeSH
• RNA ribozomální 23S genetika   MeSH
• ropa mikrobiologie   MeSH
• techniky typizace bakterií metody   MeSH
• uhlovodíky metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH

• MeSH
• Diptera genetika   klasifikace   MeSH
• fylogeneze MeSH
• ribozomální DNA genetika   MeSH
• RNA ribozomální 16S genetika   MeSH
• RNA ribozomální 18S genetika   MeSH
• vývojová biologie MeSH

BACKGROUND: Distribution and evolutionary history of resistance genes in environmental actinobacteria provide information on intensity of antibiosis and evolution of specific secondary metabolic pathways at a given site. To this day, actinobacteria producing biologically active compounds were isolated mostly from soil but only a limited range of soil environments were commonly sampled. Consequently, soil remains an unexplored environment in search for novel producers and related evolutionary questions. RESULTS: Ninety actinobacteria strains isolated at contrasting soil sites were characterized phylogenetically by 16S rRNA gene, for presence of erm and ABC transporter resistance genes and antibiotic production. An analogous analysis was performed in silico with 246 and 31 strains from Integrated Microbial Genomes (JGI_IMG) database selected by the presence of ABC transporter genes and erm genes, respectively. In the isolates, distances of erm gene sequences were significantly correlated to phylogenetic distances based on 16S rRNA genes, while ABC transporter gene distances were not. The phylogenetic distance of isolates was significantly correlated to soil pH and organic matter content of isolation sites. In the analysis of JGI_IMG datasets the correlation between phylogeny of resistance genes and the strain phylogeny based on 16S rRNA genes or five housekeeping genes was observed for both the erm genes and ABC transporter genes in both actinobacteria and streptomycetes. However, in the analysis of sequences from genomes where both resistance genes occurred together the correlation was observed for both ABC transporter and erm genes in actinobacteria but in streptomycetes only in the erm gene. CONCLUSIONS: The type of erm resistance gene sequences was influenced by linkage to 16S rRNA gene sequences and site characteristics. The phylogeny of ABC transporter gene was correlated to 16S rRNA genes mainly above the genus level. The results support the concept of new specific secondary metabolite scaffolds occurring more likely in taxonomically distant producers but suggest that the antibiotic selection of gene pools is also influenced by site conditions.

• MeSH
• ABC transportéry genetika   MeSH
• Actinobacteria klasifikace   účinky léků   genetika   izolace a purifikace   MeSH
• antibakteriální látky biosyntéza   MeSH
• bakteriální léková rezistence * MeSH
• fylogeneze * MeSH
• geny rRNA MeSH
• methyltransferasy genetika   MeSH
• molekulární sekvence - údaje MeSH
• půdní mikrobiologie MeSH
• ribozomální DNA chemie   genetika   MeSH
• RNA ribozomální 16S genetika   MeSH
• sekvenční analýza DNA MeSH
• shluková analýza MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

A total of 151 bacterial isolates were recovered from different developmental stages (larvae, nymphs and adults) of field-collected ticks (67 strains from Ixodes ricinus, 38 from Dermacentor reticulatus, 46 from Haemaphysalis concinna). Microorganisms were identified by means of 16S rRNA gene sequencing. Almost 87 % of the strains belonged to G(+) bacteria with predominantly occurring genera Bacillus and Paenibacillus. Other G(+) strains included Arthrobacter, Corynebacterium, Frigoribacterium, Kocuria, Microbacterium, Micrococcus, Plantibacter, Rhodococcus, Rothia, and Staphylococcus. G(-) strains occurred less frequently, comprising genera Advenella, Pseudomonas, Rahnella, Stenotrophomonas, and Xanthomonas. Several strains of medical importance were found, namely Advenella incenata, Corynebacterium aurimucosum, Microbacterium oxydans, M. schleiferi, Staphylococcus spp., and Stenotrophomonas maltophilia. Data on cultivable microbial diversity in Eurasian tick species D. reticulatus and H. concinna are given, along with the extension of present knowledge concerning bacterial flora of I. ricinus.

• MeSH
• arachnida jako vektory mikrobiologie   parazitologie   růst a vývoj   MeSH
• Bacteria genetika   izolace a purifikace   klasifikace   MeSH
• DNA bakterií genetika   MeSH
• financování organizované MeSH
• fylogeneze MeSH
• infestace klíšťaty parazitologie   MeSH
• Ixodidae mikrobiologie   parazitologie   růst a vývoj   MeSH
• molekulární sekvence - údaje MeSH
• obratlovci parazitologie   MeSH
• ribozomální DNA genetika   MeSH
• RNA ribozomální 16S genetika   MeSH
• techniky typizace bakterií MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH

The aging population and increased incidence of severe bacterial infection can lead to sepsis. Interest to early identification of endangered patients and identification of pathogen do not always confirm the infection. To use biomarkers can help in early identification of infection and opportunity to start therapy timeously. All biomarkers were defined in 33 out of 96 patients. Thirty-two (97 %) patients had bacterial infection and 1 (3 %) patient had systemic inflammatory response syndrome (SIRS) without infection. PCR confirmed the infection in 27 cases and blood cultures in 8. Area under curve (AUC) for CD64 was 1.00, meanwhile other biomarkers showed 2-fold smaller AUC for positive infection. CD64 index was associated with bacterial infection (p<0.001) and could be used to confirm assessment of SIRS severity (p=0.037). As regards to our results, limited to only 33 patients, CD64 index served as a good parameter to predict bacterial infection and determine severity. The use of broad range 16S ribosomal RNA (rRNA) PCR proved to be an excellent tool to confirm bloodstream infection. The CD64 index had the highest AUC, which exceeded all the others, and could be used to predict the outcome of broad range 16S rRNA PCR from whole blood. However, C-reactive protein (CRP), procalcitonin (PCT) and sCD14 are much easier and faster to measure, but the values could be elevated in other clinical assessments.

• MeSH
• Bacteria genetika   izolace a purifikace   MeSH
• bakteriemie diagnóza   MeSH
• biologické markery analýza   MeSH
• diagnostické testy rutinní metody   MeSH
• DNA bakterií genetika   MeSH
• lidé MeSH
• neutrofily chemie   MeSH
• polymerázová řetězová reakce MeSH
• receptory IgG analýza   MeSH
• ribozomální DNA chemie   genetika   MeSH
• RNA ribozomální 16S genetika   MeSH
• ROC křivka MeSH
• stupeň závažnosti nemoci * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH

The genes of ribosomal RNA are the most popular and frequently used markers for bacterial phylogeny and reconstruction of insect-symbiont coevolution. In primary symbionts, such as Buchnera and Wigglesworthia, genome economization leads to the establishment of a single copy of these sequences. In phylogenetic studies, they provide sufficient information and yield phylogenetic trees congruent with host evolution. In contrast, other symbiotic lineages (e.g., the genus Arsenophonus) carry a higher number of rRNA copies in their genomes, which may have serious consequences for phylogenetic inference. In this study, we show that in Arsenophonus triatominarum the degree of heterogeneity can affect reconstruction of phylogenetic relationships and mask possible coevolution between the symbiont and its host. Phylogenetic arrangement of individual rRNA copies was used, together with a calculation of their divergence time, to demonstrate that the incongruent 16S rDNA trees and low nucleotide diversity in the secondary symbiont could be reconciled with the coevolutionary scenario.

• MeSH
• DNA bakterií genetika   chemie   MeSH
• Enterobacteriaceae genetika   izolace a purifikace   klasifikace   MeSH
• financování organizované MeSH
• fylogeneze MeSH
• mezerníky ribozomální DNA genetika   chemie   MeSH
• molekulární evoluce MeSH
• molekulární sekvence - údaje MeSH
• polymorfismus genetický MeSH
• RNA ribozomální 16S genetika   MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• sekvenční seřazení MeSH
• Triatoma mikrobiologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH

PCR-primers were designed for identification of strictly anaerobic bacteria of the genus Zymophilus based on genus-specific sequences of the 16S-23S rDNA internal transcribed spacer region. The specificity of the primers was tested against 37 brewery-related non-target microorganisms that could potentially occur in the same brewery specimens. None DNA was amplified from any of the non-Zymophilus strains tested including genera from the same family (Pectinatus, Megasphaera, Selenomonas), showing thus 100% specificity. PCR assay developed in this study allows an extension of the spectra of detected beer spoilage microorganisms in brewery laboratories.

• MeSH
• anaerobióza MeSH
• anaerobní bakterie klasifikace   genetika   metabolismus   MeSH
• intergenová DNA chemie   genetika   MeSH
• molekulární sekvence - údaje MeSH
• polymerázová řetězová reakce MeSH
• RNA ribozomální chemie   genetika   MeSH
• sekvence nukleotidů MeSH
• sekvenční seřazení MeSH
• senzitivita a specificita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Based on 16S rDNA analyses, the primary symbionts of sucking lice were found to form a polyphyletic assemblage of several distant lineages that have arisen several times within Enterobacteriaceae and at least once within Legionellaceae. Another independent lineage of endosymbiotic enterobacteria inhabits a sister group of the sucking lice, Rhynchophthirina. The inspection of 16S rDNA supports the symbiotic nature of the investigated bacteria; they display a typical trait of degenerative processes, an increased AT content (Adenine-Thymine content) in comparison with free-living bacteria. The calculation of divergence time between the closest anopluran and rhynchophthirine symbionts further support their independent origin. The results shown here, together with evidence from other groups, indicate that the significance of primary symbionts for blood-feeding insects should be reconsidered.

• MeSH
• Bacteria genetika   klasifikace   MeSH
• financování organizované MeSH
• fyziologie bakterií MeSH
• mitochondriální DNA MeSH
• Phthiraptera fyziologie   genetika   mikrobiologie   MeSH
• ribozomální DNA genetika   MeSH
• RNA ribozomální 16S genetika   MeSH
• sekvence nukleotidů MeSH
• symbióza MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH