Úvod a cíl práce: Parodontitida, která u dospělé populace patří mezi onemocnění s hromadným výskytem, ve svých důsledcích představuje u osob starších než 35 let téměř stejně častý důvod ztráty zubu jako následky zubního kazu. Cílem této studie bylo zhodnotit využitelnost sekvenačních metod nové generace jako nástroje pro komplexní charakterizaci subgingiválního bakteriálního biofilmu u pacientů s pokročilou parodontitidou a pro sledování dynamiky změn složení subgingiválního bakteriálního biofilmu v průběhu terapie. Předpokládá se, že sekvenační metody nové generace poskytnou novou kvalitu sledování průběhu léčby a nabídnou parodontologům nástroj pro objektivní hodnocení účinnosti zvoleného terapeutického postupu a volbu další léčby. Metody: Do studie vstoupilo 43 pacientů s diagnózou pokročilé parodontitidy. Pacienti byli rozděleni do dvou podskupin a léčeni jednou ze dvou standardně schválených metod – pouze deep scaling a root planing (podskupina A – 20 pacientů) a deep scaling a root planing doplněný o antibiotickou terapii (podskupina B – 23 pacientů). Stav jejich parodontu byl hodnocen parodontálními indexy PPD (periodontal pocket depth), BOP (bleeding on probing), CAL (clinical attachment loss) před léčbou a dva týdny, tři měsíce a 18 měsíců po léčbě. Každému pacientovi byla ve shodných časových intervalech odebrána sada čtyř vzorků z nejhlubšího parodontálního chobotu pro stanovení složení mikrobiomu. Výsledky: V průběhu léčby došlo k významnému poklesu parodontálních indexů BOP, CAL a PPD u obou podskupin pacientů. V průběhu léčby došlo také k významnému poklesu indexu parodontálního rizika R/G. U podskupiny pacientů léčených deep scaling a root planing navíc s aplikací antibiotik (podskupina B) došlo 14 dní po léčbě k významně vyššímu poklesu R/G indexu v porovnání s podskupinou A léčenou pouze deep scaling a root planing. Tři měsíce a 18 měsíců po léčbě byly hodnoty indexu parodontálního rizika R/G u obou podskupin pacientů obdobné. Závěr: Taxonomická charakterizace mikrobiomu vyjádřená formou R/G indexu umožňuje monitorovat dynamické změny v průběhu léčby, na rozdíl od pouhého sledování klinických parametrů ukazuje jednoznačné rozdíly mezi skupinami s použitím a bez použití antibiotik v čase 14 dnů po terapii deep scaling a root planing. S ohledem na klinické parametry obou skupin, které se po třech ani 18 měsících v podstatě neliší, je ovšem na místě velmi pečlivě zvážit, zda je antibiotická podpora terapie deep scaling a root planing přínosem, neboť existuje nezanedbatelné riziko rozvoje a šíření rezistencí. Použití antibiotik v parodontologii by mělo být podloženo podrobným klinickým, mikrobiologickým a celkově medicínským vyšetřením.

Introduction, aim: Periodontitis, which is one of the most common diseases in the adult population, is almost as common cause of tooth loss in people over the age of 35 as it is due to tooth decay. The aim of this study was to evaluate the utility of new-generation sequencing methods as a tool for complex characterization of subgingival bacterial biofilm in patients with advanced periodontitis and for monitoring the dynamics of changes in the composition of subgingival bacterial biofilm during therapy. We assume that next-generation sequencing methods will provide a new quality of monitoring the course of treatment and offer periodontologists a tool for objective evaluation of the effectiveness of the chosen therapeutic procedure and choice of further treatment. Methods: The study included 43 patients with a diagnosis of advanced periodontitis. Patients were divided into two subgroups and treated with one of two standard approved methods – deep scaling and root planing only (subgroup A – 20 patients) and deep scaling and root planing supplemented with antibiotic therapy (subgroup B – 23 patients) – and their periodontal status was assessed by periodontal indices BOP (bleeding on probing), CAL (clinical attachment loss) a PPD (periodontal pocket depth) before treatment and two weeks, three months and 18 months after therapy. A set of four samples was taken from each deepest periodontal pocket at equal time intervals to determine the microbiome composition. Results: During treatment, there was a significant decrease in the periodontal indices PPD, BOP and CAL in both subgroups of patients. There was also a significant decrease in the periodontal risk index R/G during treatment. In addition, the subgroup of patients treated with deep scaling and root planing with antibiotics (subgroup B) had a significantly higher decrease in R/G index 14 days after treatment compared to subgroup A treated with deep scaling and root planing alone. Three months and 18 months after treatment, the periodontal risk index R/G values were similar in both subgroups of patients. Conclusion: Taxonomic characterization of the microbiome expressed in the form of R/G index allows to monitor dynamic changes during treatment, in contrast to mere monitoring of clinical parameters shows clear differences between groups with and without antibiotics at 14 days after deep scaling and root planing therapy. However, given the clinical parameters of the two groups, which do not differ significantly after three or 18 months, it is appropriate to carefully consider whether antibiotic support for deep scaling and root planing is beneficial, as there is a significant risk of developing and spreading resistance. The use of antibiotics in periodontology should be based on detailed clinical, microbiological and general medical examination.

• Klíčová slova
• orální mikrobiom,
• MeSH
• antibakteriální látky terapeutické užití   MeSH
• klinická studie jako téma MeSH
• lidé MeSH
• mikrobiota MeSH
• parodontitida * farmakoterapie   mikrobiologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

• Publikační typ
• abstrakt z konference MeSH

The lincomycin biosynthetic gene lmbX was deleted in Streptomyces lincolnensis ATCC 25466, and deletion of this gene led to abolition of lincomycin production. The results of complementation experiments proved the blockage in the biosynthesis of lincomycin precursor 4-propyl-L-proline. Feeding this mutant strain with precursor derivatives resulted in production of 4'-butyl-4'-depropyllincomycin and 4'-pentyl-4'-depropyllincomycin in high titers and without lincomycin contamination. Moreover, 4'-pentyl-4'-depropyllincomycin was found to be more active than lincomycin against clinical Staphylococcus isolates with genes determining low-level lincosamide resistance.

• MeSH
• antibakteriální látky farmakologie   chemie   metabolismus   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• lidé MeSH
• linkomycin analogy a deriváty   farmakologie   chemie   metabolismus   MeSH
• mikrobiální testy citlivosti MeSH
• molekulární struktura MeSH
• prolin analogy a deriváty   metabolismus   MeSH
• stafylokokové infekce mikrobiologie   MeSH
• Staphylococcus účinky léků   MeSH
• Streptomyces genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

In acetohydroxy acid synthase from Streptomyces cinnamonensis mutants affected in valine regulation, the impact of mutations on interactions between the catalytic and the regulatory subunits was examined using yeast two-hybrid system. Mutations in the catalytic and the regulatory subunits were projected into homology models of the respective proteins. Two changes in the catalytic subunit, E139A (alpha domain) and DeltaQ217 (beta domain), both located on the surface of the catalytic subunit dimer, lowered the interaction with the regulatory subunit. Three consecutive changes in the N-terminal part of the regulatory subunit were examined. Changes G16D and V17D in a loop and adjacent alpha-helix of ACT domain affected the interaction considerably, indicating that this region might be in contact with the catalytic subunit during allosteric regulation. In contrast, the adjacent mutation L18F did not influence the interaction at all. Thus, L18 might participate in valine binding or conformational change transfer within the regulatory subunits. Shortening of the regulatory subunit to 107 residues reduced the interaction essentially, suggesting that the C-terminal part of the regulatory subunit is also important for the catalytic subunit binding.

• MeSH
• acetolaktátsynthasa chemie   genetika   metabolismus   MeSH
• alosterická regulace MeSH
• sekundární struktura proteinů MeSH
• Streptomyces enzymologie   MeSH
• techniky dvojhybridového systému MeSH
• valin metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The incidence of potential periodontal pathogens (Aggregatibacter actinomycetemcomitans, formerly Actinobacillus actinomycetemcomitans, Tannerella forsythia, Porphyromonas gingivalis, Prevotella nigrescens, Prevotella intermedia and Capnocytophaga ochracea) was monitored in patients with chronic periodontitis and in healthy control subjects. Two types of studies were carried out in which the composition of the bacterial communities in different niches of the same oral cavity ecosystem was investigated. Fluctuation or at least pronounced quantitative changes in the incidence of individual species in time were documented in the long-term study as well as after the local administration of antibacterial drug Chlo-Site or Metronidazole. Even within two weeks, a turnover of the monitored bacteria in separate niches of the oral biotope can be detected. A relatively high incidence of the tested periopathogens in the clinically healthy teeth of patients implies that even the "healthy" niches in the periodontal biotope function as a dynamic reservoir of periopathogenic microorganisms. This should be kept in mind when a local application of antibacterial compounds is used in the therapy of periodontal disease.

• MeSH
• antibakteriální látky terapeutické užití   MeSH
• časové faktory MeSH
• dospělí MeSH
• financování organizované MeSH
• gramnegativní bakteriální infekce diagnóza   epidemiologie   farmakoterapie   mikrobiologie   MeSH
• gramnegativní bakterie izolace a purifikace   účinky léků   MeSH
• incidence MeSH
• lidé MeSH
• monitorování léčiv MeSH
• nemoci parodontu diagnóza   epidemiologie   farmakoterapie   mikrobiologie   MeSH
• studie případů a kontrol MeSH
• ústa mikrobiologie   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH

To follow the role of selected presumptive periodontal pathogens in the development of periodontal diseases. To application of the advanced DNA techniques for rapid, sensitive and specific detection of major periodontal pathogens.

DNA metodu použít pro identifikaci vybraných orálních mikroorganizmů patřících do skupiny parodontálních patogenů a pro sledování složení bakteriálního společenství v paradontálním chobotu, resp. gingiválním sulku. Sledovat stálost suspektních parodontálních patogenů při vzniku a rozvoji onemocnění. Molekulární techniky využít pro sledování účinnosti lokálních antibakteriálních intervencí při terapii onemocnění parodontu.

• MeSH
• genetické techniky trendy   využití   MeSH
• gingivální exsudát mikrobiologie   MeSH
• nemoci parodontu diagnóza   mikrobiologie   MeSH
• polymerázová řetězová reakce metody   využití   MeSH
• ústa mikrobiologie   MeSH
• zubní prevence MeSH

• Konspekt
• Stomatologie
• NLK Obory
• zubní lékařství
• biologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

Acetohydroxy-acid synthases (AHAS) of two mutant strains Streptomyces cinnamonensis ACB-NLR-2 and BVR-18 were chosen for this study for their apparent activation by valine, which regularly acts as an allosteric inhibitor. Sequencing the ilvB genes coding for the AHAS catalytic subunit revealed two distant changes in the mutants, DeltaQ217 and E139A, respectively. Homology modeling was used to propose the structural changes caused by those mutations. In the mutant strain ACB-NLR-2 (resistant to 2-amino-3-chlorobutyrate and norleucine), deletion of Q217 affected a helix in ss-domain, distant from the active center. As no mutation was found in the regulatory subunit of this strain, DeltaQ217 in IlvB was supposed to be responsible for the observed valine activation, probably via changed properties on the proposed regulatory-catalytic subunit interface. In mutant strain BVR-18 (resistant to 2-oxobutyrate), substitution E139A occurred in a conservative loop near the active center. In vitro AHAS activity assay with the enzyme reconstituted from the wild-type regulatory and BVR-18 catalytic subunits proved that the substitution in the catalytic subunit led to the apparent activation of AHAS by valine. We suggest that the conservative loop participated in a conformational change transfer to the active center during the allosteric regulation.

• MeSH
• acetolaktátsynthasa genetika   chemie   metabolismus   MeSH
• aktivace enzymů MeSH
• alosterická regulace imunologie   MeSH
• bakteriální proteiny genetika   chemie   metabolismus   MeSH
• bodová mutace MeSH
• katalytická doména imunologie   MeSH
• konformace proteinů MeSH
• missense mutace MeSH
• molekulární modely MeSH
• rekombinantní fúzní proteiny chemie   metabolismus   MeSH
• sekvenční homologie aminokyselin MeSH
• Streptomyces enzymologie   genetika   MeSH
• substituce aminokyselin MeSH
• valin metabolismus   MeSH
• vztahy mezi strukturou a aktivitou MeSH

A cosmid bearing an insert of 38 217 bp covering the gene cluster and its flanking regions of type strain Streptomyces lincolnensis ATCC 25466 was sequenced. Two relatively extensive sequence changes and several hundred point mutations were identified if compared with the previously published sequence of the lincomycin (Lin) industrial strain S. lincolnensis 78-11. Analysis of the cluster-flanking regions revealed its localization within the genome of the ATCC 25466 strain. The cluster-bearing cosmid was integrated into the chromosome of Lin non-producing strains S. coelicolor CH 999 and S. coelicolor M 145. The modified strains heterologously produced Lin but the level dropped to approximately 1-3% of the production in the ATCC 25466 strain.

• MeSH
• antibakteriální látky biosyntéza   chemie   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• biotechnologie MeSH
• bodová mutace MeSH
• genová knihovna MeSH
• kosmidy MeSH
• linkomycin biosyntéza   chemie   MeSH
• multigenová rodina MeSH
• sekvenční analýza DNA MeSH
• Streptomyces coelicolor genetika   metabolismus   MeSH
• Streptomyces genetika   metabolismus   růst a vývoj   MeSH

Mitochondrial processing peptidases are heterodimeric enzymes (alpha/betaMPP) that play an essential role in mitochondrial biogenesis by recognizing and cleaving the targeting presequences of nuclear-encoded mitochondrial proteins. The two subunits are paralogues that probably evolved by duplication of a gene for a monomeric metallopeptidase from the endosymbiotic ancestor of mitochondria. Here, we characterize the MPP-like proteins from two important human parasites that contain highly reduced versions of mitochondria, the mitosomes of Giardia intestinalis and the hydrogenosomes of Trichomonas vaginalis. Our biochemical characterization of recombinant proteins showed that, contrary to a recent report, the Trichomonas processing peptidase functions efficiently as an alpha/beta heterodimer. By contrast, and so far uniquely among eukaryotes, the Giardia processing peptidase functions as a monomer comprising a single betaMPP-like catalytic subunit. The structure and surface charge distribution of the Giardia processing peptidase predicted from a 3-D protein model appear to have co-evolved with the properties of Giardia mitosomal targeting sequences, which, unlike classic mitochondrial targeting signals, are typically short and impoverished in positively charged residues. The majority of hydrogenosomal presequences resemble those of mitosomes, but longer, positively charged mitochondrial-type presequences were also identified, consistent with the retention of the Trichomonas alphaMPP-like subunit. Our computational and experimental/functional analyses reveal that the divergent processing peptidases of Giardia mitosomes and Trichomonas hydrogenosomes evolved from the same ancestral heterodimeric alpha/betaMPP metallopeptidase as did the classic mitochondrial enzyme. The unique monomeric structure of the Giardia enzyme, and the co-evolving properties of the Giardia enzyme and substrate, provide a compelling example of the power of reductive evolution to shape parasite biology.

• MeSH
• down regulace genetika   MeSH
• fylogeneze MeSH
• genová dávka MeSH
• Giardia lamblia genetika   metabolismus   ultrastruktura   MeSH
• glycin fyziologie   genetika   chemie   MeSH
• metaloendopeptidasy genetika   chemie   metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• multimerizace proteinu MeSH
• organely metabolismus   MeSH
• podjednotky proteinů genetika   MeSH
• posttranslační úpravy proteinů genetika   MeSH
• proteinové domény bohaté na prolin fyziologie   genetika   MeSH
• řízená evoluce molekul MeSH
• sekvence aminokyselin MeSH
• transport proteinů MeSH
• Trichomonas vaginalis genetika   metabolismus   ultrastruktura   MeSH
• vodík metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH
• srovnávací studie MeSH

A new separation and quantification method using ultra-performance liquid chromatography (UPLC) with UV detection was developed for detection of lincomycin traces in fermentation broth of different Streptomyces spp. A similar high-performance liquid chromatography (HPLC) protocol was simultaneously developed for comparison purposes. Both methods were validated and showed a linear range of detector response for quantification of lincomycin in concentration from 3.125 to 1000.0 microgml(-1) with correlation coefficient 0.999 and recoveries ranging from 81.5 to 89.85% with precision < or =5%. Compared with the HPLC, the UPLC method offered high sample throughput and about 10 times lower consumption of solvents. The developed assays were used for determination of lincomycin production in genetically manipulated production strain Streptomyces lincolnensis and for determination of lincomycin production after heterologous expression of lincomycin biosynthetic gene cluster in non-producing strain Streptomyces coelicolor.

• MeSH
• chromatografie kapalinová metody   MeSH
• fermentace MeSH
• financování organizované MeSH
• geneticky modifikované organismy metabolismus   MeSH
• linkomycin analýza   MeSH
• senzitivita a specificita MeSH
• spektrofotometrie ultrafialová metody   MeSH
• Streptomyces genetika   metabolismus   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH