Národní referenční laboratoř pro mykobakterie (NRLM) prošla od roku 2019 výraznými personálními a metodickými změnami, především došlo k revizi používaných metod a k zavedení nových tak, aby portfolio činností NRLM odpovídalo nejnovějším evropským i světovým standardům a výsledky z NRLM bylo možné porovnávat a interpretovat na mezinárodní úrovni. V tomto článku tým NRLM prezentuje hlavní změny, ke kterým za uplynulé dva roky v laboratoři došlo jako stručný přehled aktuálních činností NRLM v oblasti diagnostiky M. tuberculosis a netuberkulózních mykobakterií. Blíže se pak věnujeme nově zavedeným metodám stanovení citlivosti na tuhém médiu Middlebrook 7H11 a stanovení minimální inhibiční koncentrace na tekutém médiu Middlebrook 7H9. Tato média jsou významná především pro stanovení citlivosti u nově zaváděných antibiotik, která se využívají v léčbě pacientů s multirezistentními kmeny M. tuberculosis.
The National Reference Laboratory for Mycobacteria (NRLM) has undergone significant personnel and methodological changes since 2019, in particular the revision of the methods used and the introduction of new ones, so that the NRLM’s portfolio of activities meets the latest European and global standards in order to generate internationally comparable and interpretable results. In this article, the NRLM team presents the main changes that have taken place in the laboratory over the past two years as a brief review of the current activities in the diagnosis of M. tuberculosis and non-tuberculous mycobacteria. A closer look is taken at the newly introduced methods of susceptibility testing on Middlebrook 7H11 solid medium and minimum inhibitory concentration testing on Middlebrook 7H9 liquid medium. These media are particularly important for susceptibility testing of newly introduced antibiotics intended for patients with multidrug-resistant TB.
Salicylanilides have proved their activity against tuberculosis (TB). One weak electron-withdrawing substituent is favored at the salicylic part, specially Cl or Br atoms at positions 4 or 5. On the other hand, the antimycobacterial activity of salicylanilides is negatively affected when a strong electron-withdrawing substituent (NO2) is present at the same positions. Herein we describe the synthesis and characterization of novel salicylanilides possessing two weak electron-withdrawing groups (halogen atoms) at their salicylic part and compare their antitubercular activity with their monohalogenated analogues. All dihalogenated derivatives proved to possess antitubercular activity at a very narrow micromolar range (MIC=1-4μM), similar with their most active monohalogenated analogues. More importantly, the most active final molecules were further screened against multidrug resistant strains and found to inhibit their growth at the range of 0.5-4μM.
- MeSH
- antituberkulotika chemická syntéza chemie farmakologie MeSH
- buňky Hep G2 MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- molekulární struktura MeSH
- multirezistentní tuberkulóza farmakoterapie MeSH
- Mycobacterium účinky léků růst a vývoj MeSH
- salicylanilidy chemická syntéza chemie farmakologie MeSH
- salicylany chemie farmakologie MeSH
- viabilita buněk účinky léků MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Chalcones, i.e., compounds with the chemical pattern of 1,3-diphenylprop-2-en-1-ones, exert a wide range of bio-activities, e.g., antioxidant, anti-inflammatory, anticancer, anti-infective etc. Our research group has been focused on pyrazine analogues of chalcones; several series have been synthesized and tested in vitro on antifungal and antimycobacterial activity. The highest potency was exhibited by derivatives with electron withdrawing groups (EWG) in positions 2 and 4 of the ring B. As halogens also have electron withdrawing properties, novel halogenated derivatives were prepared by Claisen-Schmidt condensation. All compounds were submitted for evaluation of their antifungal and antibacterial activity, including their antimycobacterial effect. In the antifungal assay against eight strains of selected fungi, growth inhibition of Candida glabrata and Trichophyton interdigitale (formerly T. mentagrophytes) was shown by non-alkylated derivatives with 2-bromo or 2-chloro substitution. In the panel of selected bacteria, 2-chloro derivatives showed the highest inhibitory effect on Staphylococcus sp. In addition, all products were also screened for their antimycobacterial activity against Mycobacterium tuberculosis H37RV My 331/88, M. kansasii My 235/80, M. avium 152/80 and M. smegmatis CCM 4622. Some of the examined compounds, inhibited growth of M. kansasii and M. smegmatis with minimum inhibitory concentrations (MICs) comparable with those of isoniazid.
- MeSH
- antiinfekční látky * chemická syntéza chemie farmakologie MeSH
- Candida glabrata růst a vývoj MeSH
- chalkon * chemická syntéza chemie farmakologie MeSH
- halogenované uhlovodíky * chemická syntéza chemie farmakologie MeSH
- Mycobacterium růst a vývoj MeSH
- pyraziny * chemická syntéza chemie farmakologie MeSH
- Trichophyton růst a vývoj MeSH
- Publikační typ
- časopisecké články MeSH
Nontuberculous mycobacteria (NTM) are ubiquitous organisms in all natural ecosystems, including water environments. Several of these species are potential pathogens which affect human health. NTM most commonly cause pulmonary, skin or soft tissue infections. Primary sludge obtained from the water treatment plants of four drinking water reservoirs were subjected to analysis for mycobacteria. Five decontamination methods (5% oxalic acid, modified Petroff, HCl-NaOH, N-acetyl-L-cysteine-sodium hydroxide and 0.05% cetylpyridinium chloride), three growth media (Herrold's egg yolk medium with and without the antibiotic cocktail PANTA and Löwenstein-Jensen medium with sodium pyruvate) and three incubation temperatures (25, 30 and 37 °C) for isolation of mycobacteria were compared in the analysis of 18 sludge samples. To evaluate examined methods, the overall positive, negative, and contamination rate, and these rates in respect to localities are taken into account. Statistical analysis demonstrated that the best combination for the recovery of mycobacteria with the minimum number of contaminating microorganisms is 5% oxalic acid decontamination cultured on Herrold's egg yolk medium with the antibiotic cocktail PANTA at 25 °C. The least suitable is N-acetyl-L-cysteine-sodium hydroxide decontamination cultured on Löwenstein-Jensen medium with sodium pyruvate at 25 °C. From 18 sludge samples we isolated 27 mycobacterial species or groups; Mycobacterium algericum, M. arabiense, M. heraklionense, M. minnesotense, M. moriokaense, M. salmoniphilum and M. vulneris were isolated from the natural water environment for the first time. Because the natural water environment is the main source of potentially pathogenic mycobacteria for humans, it is important to direct particular focus to newly described mycobacterial species.
- MeSH
- bakteriologické techniky metody MeSH
- čištění vody přístrojové vybavení metody MeSH
- dekontaminace metody MeSH
- kultivační média analýza metabolismus MeSH
- Mycobacterium genetika růst a vývoj izolace a purifikace MeSH
- odpadní vody mikrobiologie MeSH
- sladká voda mikrobiologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
New quaternary ammonium salt-type compounds with lipophilic cholesterol and terpene moieties were synthesized. The compounds showed promising antibacterial and antimycobacterial activities. Those compounds containing the cholesterol moiety showed significant activity against Staphylococcus aureus, Staphylococcus epidermidis, and Enterococcus faecium. On the contrary, the antimycobacterial activity increased with the presence of the terpene unit in the molecule.
- MeSH
- antiinfekční látky chemická syntéza farmakologie MeSH
- cholesterol analogy a deriváty chemická syntéza farmakologie MeSH
- Enterococcus faecium účinky léků růst a vývoj MeSH
- houby účinky léků růst a vývoj MeSH
- kvartérní amoniové sloučeniny chemická syntéza farmakologie MeSH
- mikrobiální testy citlivosti MeSH
- molekulární struktura MeSH
- Mycobacterium účinky léků růst a vývoj MeSH
- racionální návrh léčiv MeSH
- Staphylococcus aureus účinky léků růst a vývoj MeSH
- Staphylococcus epidermidis účinky léků růst a vývoj MeSH
- terpeny diagnostické užití farmakologie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
The environment is a reservoir of nontuberculous mycobacteria and is considered a source of infection for animals and humans. Mycobacteria can persist in different types of environments for a relatively long time. We have studied their possible internalization into plant tissue through intact, as well as damaged, root systems of different types of plants grown in vitro and under field conditions. The substrate into which plants were seeded was previously contaminated with different strains of Mycobacterium avium (10(8) to 10(10) cells/g of soil) and feces from animals with paratuberculosis. We detected M. avium subsp. avium, hominissuis, and paratuberculosis in the stems and leaves of the plants by both culture and real-time quantitative PCR. The presence of mycobacteria in the plant tissues was confirmed by microscopy. The concentration of mycobacteria found inside plant tissue was several orders of magnitude lower (up to 10(4) cells/g of tissue) than the initial concentration of mycobacteria present in the culture medium or substrate. These findings led us to the hypothesis that plants may play a role in the spread and transmission of mycobacteria to other organisms in the environment.
- MeSH
- bakteriologické techniky MeSH
- endocytóza * MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- listy rostlin mikrobiologie MeSH
- mikroskopie MeSH
- Mycobacterium genetika růst a vývoj fyziologie MeSH
- rostliny mikrobiologie MeSH
- stonky rostlin mikrobiologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Nontuberculous mycobacteria (NTM) are potentially pathogenic agents commonly found in natural ecosystems, while food is considered to be another source of NTM for humans. We investigated a total of 92 tissue samples of freshwater fish and fish products: fish directly obtained from ponds (n=25), retail fresh (n=23) and frozen fish (n=23) and smoked fish products (n=21). Culture examination for the presence of mycobacteria was positive in 11 (11.9%) from all the examined samples. The 15 obtained isolates were identified as Mycobacterium fortuitum (n=5), M. immunogenum (n=2), M. phocaicum/ mucogenicum (n=1), M. neoaurum (n=2), M. peregrinum (n=2), M. porcinum (n=1) and M. senegalense/houstonense/conceptionense (n=2). NTM DNA was found in one (4.0%) sample of fresh fish from ponds and in 60.9% and 91.3% of retail fresh and frozen fish, respectively. None of the smoked fish products contained NTM DNA. The results of our study suggest that freshwater fish and fish products, especially retail frozen fish, might be a reservoir of NTM for humans, and proper handling and treatment before consumption of such products is recommended.
- MeSH
- DNA analýza metabolismus MeSH
- lidé MeSH
- molekulární typizace MeSH
- mražené potraviny analýza ekonomika mikrobiologie MeSH
- Mycobacterium fortuitum klasifikace růst a vývoj izolace a purifikace metabolismus MeSH
- Mycobacterium klasifikace růst a vývoj izolace a purifikace metabolismus MeSH
- mykobakteriózy mikrobiologie MeSH
- potrava z moře (živočišná) analýza ekonomika mikrobiologie MeSH
- potraviny konzervované analýza ekonomika mikrobiologie MeSH
- rybí výrobky analýza ekonomika mikrobiologie MeSH
- ryby metabolismus mikrobiologie MeSH
- sladká voda MeSH
- zdroje nemoci MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
Design, results of in vitro antimycobacterial evaluation, and study of structure-activity relationships of various pyrazinecarboxylic acid reversible derivatives are presented. This review deals with some pyrazinamide analogues/prodrugs derived from Nphenylpyrazine- 2-carboxamides (1), arylaminopyrazine-2,5-dicarbonitriles (2), aryl/alkylsulphanylpyrazines (3,4), and aroylpyrazines (5) effecting >50% inhibition in the primary antimycobacterial screen. The promising pyrazine candidates for further antimycobacterial evaluation were discovered. Results give good view onto structure-activity relationships of these analogues and promise even better activity of new compounds prepared after some structure optimization experiments.
- MeSH
- antibakteriální látky škodlivé účinky chemie farmakologie MeSH
- buněčné linie MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- molekulární struktura MeSH
- Mycobacterium účinky léků růst a vývoj MeSH
- prekurzory léčiv škodlivé účinky chemie farmakologie MeSH
- pyrazinamid škodlivé účinky analogy a deriváty chemie farmakologie MeSH
- racionální návrh léčiv MeSH
- viabilita buněk účinky léků MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Častými „kontaminantami" zachycenými při mykobakteriologické kultivaci dekontaminovaných vzorků jsou bakterie řádu Actinomycetales. Jsou to obvykle bakterie klasifikované v čeledi Corynehacterineae rodů Corynebacteríum, Dietzia, Gordonia, Nocardia, Rhodococcus a Tsukamurella. Tyto bakterie často kolonizují dýchací cesty a za určitých podmínek mohou vyvolat život ohrožující onemocnění. U pacientů s těžkou imunodeficiencí jsou pravidelným vyvolavatelem život ohrožujících infekcí bakterie rodu Nocardia. Jsou to vláknité bakterie, vytvářející v kultuře vzdušné mycelium, jsou částečně acidorezistentní a jsou rezistentní na lysozym. Vyvolávají nokardiózu, tj. vzácné ale závažné onemocnění pacientů s různými typy imunodeficience. Diferenciálně diagnosticky je třeba odlišit rody Streptomyces, Actinomadura, Nocardiopsis a další půdní saprofyty, které nejsou acidorezistentní, jsou citlivé na lysozym a rychleji rostou. Často kolonizují dýchací cesty u pacientů s postižením plic, onemocnění vyvolávají velmi vzácně. Diagnostika aerobních aktinomycet a vyšetření citlivosti na antibiotika mají svá úskalí vyplývající z delší růstové doby, obtížné barvitelnosti a necharakteristických biochemických reakcí. K přesné identifikaci do rodu a species je nutná polyfázová identifikace izolatu s využitím metod molekulární mikrobiologie. Při včasné diagnóze bývají infekce vyvolané aerobními aktinomycetami dobře léčitelné cílenou terapií antibiotiky.
Frequent "contaminants" detected during mycobacterial culture of decontaminated samples are bacteria of the order Actinomycetales. These are usually bacteria classified as the family Corynebacterineae, genera Corynebacterium, Dietzia, Gordonia, Nocardia, Rhodococcus and Tsukamurella. These bacteria frequently colonize the airways and, under certain circumstances, they may cause life-threatening diseases. In severely immunocompromised patients, they regularly cause life-threatening infections with bacteria of the genus Nocardia. These filamentous bacteria, developing aerial mycelium in the culture, are partly acid-resistant and resistant to lysozyme. They cause nocardiosis, a rare but serious disease in patients with various types of immune deficiency. Differential diagnosis must distinguish between the genera Streptomyces, Actinomadura and Nocardiopsis and other soil saprophytes that are not acid-resistant, sensitive to lysozyme and faster growing. They frequently colonize the airways of patients with lung disease but very rarely cause diseases. The diagnosis of aerobic actinomycetes and determination of their sensitivity to antibiotics are problematic since they grow longer, are difficult to stain and are involved in atypical biochemical reactions. Precise identification of the genera and species requires polyphasic identification of isolates using molecular microbiology methods. If diagnosed early, infections caused by aerobic actinomycetes are easy to treat with targeted antibiotic therapy.
- Klíčová slova
- léčba, vyšetření citlivosti, identifikace, diagnostika, Tsukamurella, Gordonia, Dietzia, dekontaminované vzorky,
- MeSH
- Actinomycetales klasifikace růst a vývoj MeSH
- bakteriologické techniky MeSH
- Corynebacterium MeSH
- epidemiologie MeSH
- infekce bakteriemi řádu Actinomycetales diagnóza MeSH
- lidé MeSH
- Mycobacterium růst a vývoj MeSH
- Nocardia MeSH
- nokardióza MeSH
- Rhodococcus MeSH
- Streptomyces MeSH
- tuberkulóza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
Different methods for the detection of Mycobacterium avium ssp. avium (MAA) in naturally infected hens were compared. They included the conventional culture method (solid Herrold's and Stonebrink media and liquid Sula medium) and newly developed liquid culture systems, the manual mycobacteria growth indicator tube (M-MGIT) and the fully automated BACTEC MGIT 960 system (A-MGIT). 152 tissues originating from 15 naturally infected hens have been processed. The overall detection rates (percentage of positive cultures from the number of positive cultures determined by all the methods together) were 60, 70 and 76 % for the conventional media, M-MGIT and A-MGIT systems, respectively, the mean time of mycobacteria detection being 32.6, 17.6 and 14.6 d, respectively. The lowest contamination rate (2.0 %) was found in A-MGIT compared with M-MGIT (4.6 %) and conventional media (10.4 %).
- MeSH
- bakteriologické techniky veterinární MeSH
- diagnostické techniky a postupy veterinární MeSH
- financování organizované MeSH
- fluorescence MeSH
- kultivační média metabolismus MeSH
- kultivační techniky veterinární MeSH
- kur domácí MeSH
- Mycobacterium izolace a purifikace metabolismus růst a vývoj MeSH
- ptačí tuberkulóza diagnóza mikrobiologie MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
