Sledovat ovlivnění či průběh genové exprese je v mnoha studiích velmi důležitou součástí výzkumu. V současné době dochází k rozvoji mnoha metod usnadňujících sledování regulace genové exprese, jedním z takových příkladů je využití tzv. genových reportérových testů (z angl. gene reporter assays). Tyto systémy představují rozsáhlý soubor nástrojů ke studiu regulačních sekvencí promotorů, zesilovačů a transkripčních faktorů. Existuje celá řada testů využívajících reportérové buňky pro stanovení biologické aktivity studovaných sloučenin. Cílem tohoto přehledového článku je představit přípravu reportérových plasmidů, což je vždy prvním krokem u testů využívajících reportérové geny. Následně budou popsány nejčastější druhy reportérových testů a představeny příklady jejich použití v testování in vitro.
Monitoring the influence or process of gene expression is a very important part of research in many studies. Currently, many methods are being developed to facilitate the monitoring of gene expression regulation, the use of gene reporter assays being one of the examples. These systems represent an extensive set of tools to study the regulatory sequences of promoters, enhancers, and transcription factors. There are several assays using reporter cells to determine the biological activity of the compounds studied. The aim of this review article is to present the preparation of reporter plasmids, which is always the first step in assays using reporter genes. Subsequently, the most common types of reporter test are described, and examples of their use in in vitro testing are presented.
- MeSH
- DNA analýza genetika MeSH
- lidé MeSH
- luciferasy analýza genetika MeSH
- luminiscenční měření * metody MeSH
- MFC-7 buňky MeSH
- plazmidy analýza genetika MeSH
- protein BRCA1 genetika MeSH
- reportérové geny MeSH
- reprodukovatelnost výsledků MeSH
- transfekce * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- dopisy MeSH
- práce podpořená grantem MeSH
- MeSH
- bakteriální proteiny genetika MeSH
- beta-laktamasy genetika MeSH
- Enterobacter aerogenes genetika izolace a purifikace MeSH
- enterobakteriální infekce mikrobiologie veterinární MeSH
- nemoci ptáků mikrobiologie MeSH
- plazmidy analýza MeSH
- vrány * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- dopisy MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Kanada MeSH
OBJECTIVES: The aac(6')-Ih gene encoding aminoglycoside 6'-N-acetyltransferase type I subtype h [AAC(6')-Ih] is plasmid-borne in Acinetobacter baumannii where it confers high-level amikacin resistance, but its origin remains unknown. We searched for the gene in the genomes of a collection of 133 Acinetobacter spp. and studied its species specificity, expression and dissemination. METHODS: Gene copy number was determined by quantitative PCR, expression by quantitative RT-PCR, MIC by microdilution and transfer by plasmid mobilization. RESULTS: The aac(6')-Ih gene was present in the chromosome of the two Acinetobacter gyllenbergii of the collection and was detected in all seven A. gyllenbergii clinical isolates. They had indistinguishable flanking regions indicating that the gene was intrinsic to this species. A. baumannii PIS Aba23 promoters were provided by insertion of ISAba23, which disrupted the Pnative promoter in A. gyllenbergii. Both types of promoters were similarly potent in Escherichia coli and A. baumannii. Aminoglycoside MICs for A. baumannii harbouring pIP1858 were higher than for A. gyllenbergii due to gene dosage. The non-self-transferable plasmid could be mobilized to other A. baumannii cells by the broad host range plasmid RP4. CONCLUSIONS: We have found the origin of aac(6')-Ih in A. gyllenbergii, a species isolated, although rarely, in humans, and documented that dissemination of this gene is restricted to the Acinetobacter genus.
- MeSH
- acetyltransferasy genetika metabolismus MeSH
- Acinetobacter baumannii klasifikace účinky léků enzymologie genetika MeSH
- aminoglykosidy metabolismus farmakologie MeSH
- antibakteriální látky metabolismus farmakologie MeSH
- bakteriální léková rezistence * MeSH
- genová dávka MeSH
- infekce bakteriemi rodu Acinetobacter mikrobiologie MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- plazmidy analýza MeSH
- přenos genů horizontální MeSH
- promotorové oblasti (genetika) MeSH
- transpozibilní elementy DNA MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Recent years have seen rising concerns over increasing antibiotic resistance of the gonorrhea-causing bacterium, Neisseria gonorrhoeae. This is especially true for third-generation cephalosporins, which are currently recommended for the treatment of such infections. Therefore, susceptibility to these antibiotics should be monitored internationally to the greatest extent possible. The susceptibility of N. gonorrhoeae strains to ceftriaxone and penicillin, as well as production of beta-lactamase by the Cefinase test was determined. Moreover, the presence and type of penicillinase plasmids were determined by PCR. All strains were susceptible to ceftriaxone, the minimal inhibitory concentration (MIC) values ranged from 0.002 to 0.125 mg/L; MIC50 was =0.016 mg/L and MIC90 was =0.064 mg/L. As much as 7.7 % of the strains demonstrated ceftriaxone MIC of 0.125 mg/L. For penicillin, the MICs ranged from 0.064 to 32 mg/L; MIC50 was =0.5 mg/L and MIC90 was =4 mg/L. It was shown that only 1.5 % of the strains were sensitive to penicillin according to The European Committee on Antimicrobial Susceptibility Testing (EUCAST). Among the penicillin-resistant strains, six (30.0 %) produced penicillinase. The MICs of penicillin were substantially higher for penicillinase-producing than for penicillin-resistant, penicillinase-negative strains. MICs of ceftriaxone for penicillinase-producing strains were low (0.002-0.016 mg/L). Three of the penicillinase-producing strains possessed plasmids of African type (50 %) and three Toronto/Rio type (50 %). An increase of the proportion of beta-lactamase-positive strains in the last years as well as emergence of strains with elevated MIC of ceftriaxone indicate a need to constantly monitor N. gonorrhoeae strains for their susceptibility to beta-lactam antibiotics, as well as for their ability to produce beta-lactamases.
- MeSH
- antibakteriální látky farmakologie MeSH
- cefalosporiny metabolismus MeSH
- ceftriaxon farmakologie MeSH
- gonorea mikrobiologie MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- Neisseria gonorrhoeae účinky léků genetika izolace a purifikace MeSH
- penicilinasa sekrece MeSH
- peniciliny farmakologie MeSH
- plazmidy analýza MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Polsko MeSH
OBJECTIVES: To describe the prevalence and diversity of IncX plasmids with antibiotic resistance genes in Enterobacteriaceae and to identify the most disseminated lineages of the plasmid family. METHODS: IncX plasmids were screened in 1894 Enterobacteriaceae isolates resistant to cefotaxime (2 mg/L) or with reduced susceptibility to ciprofloxacin (0.05 mg/L) obtained from various sources in five continents using PCR. IncX plasmid-harbouring isolates were identified using MALDI-TOF or biochemical tests, and screened for antibiotic resistance genes using PCR and sequencing; their clonality was determined by PFGE. Horizontal transfer of plasmids was tested using transformation and conjugation. IncX plasmids were characterized by S1-nuclease and PFGE, RFLP and hybridization. RESULTS: A total of 164 Escherichia coli isolates (8.7%, n = 1894) carried at least one IncX subgroup. Seven isolates harboured two distinct subgroups. IncX1 subgroup was found in 93 isolates, followed by IncX2 (35 isolates), IncX4 (28) and IncX3 (15). IncX4 plasmids were not transferred horizontally as single plasmids and therefore excluded from further analysis. The most disseminated lineages of IncX plasmids included IncX1 harbouring qnrS1 and blaTEM-1,-135 found in 36 E. coli from different sources in Europe and Australia and IncX2 carrying qnrS1 and tet(A) detected in nine E. coli from wildlife in Europe. IncX3 plasmids harboured predominantly blaSHV-12 and qnrS1 or qnrB7. CONCLUSIONS: IncX plasmids were widely distributed in E. coli from wildlife in Europe and were predominantly associated with fluoroquinolone resistance genes. Plasmids showing indistinguishable restriction profiles were identified in E. coli from different sources and countries suggesting wide dissemination of certain plasmid lineages.
- MeSH
- bakteriální geny * MeSH
- bakteriální léková rezistence * MeSH
- bakteriální transformace MeSH
- beta-laktamy farmakologie MeSH
- celosvětové zdraví MeSH
- divoká zvířata MeSH
- Escherichia coli účinky léků genetika izolace a purifikace MeSH
- fluorochinolony farmakologie MeSH
- genetická variace MeSH
- genotyp MeSH
- infekce vyvolané Escherichia coli mikrobiologie veterinární MeSH
- konjugace genetická MeSH
- lidé MeSH
- molekulární typizace MeSH
- plazmidy analýza klasifikace MeSH
- polymerázová řetězová reakce MeSH
- přenos genů horizontální MeSH
- prevalence MeSH
- pulzní gelová elektroforéza MeSH
- sekvenční analýza DNA MeSH
- shluková analýza MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
OBJECTIVES: The objective of this study was to perform a multinational survey of patients' colonization by metallo-β-lactamase (MBL)-producing Enterobacteriaceae, including their molecular characterization. METHODS: Patients in 18 hospital units across Europe and Israel (n = 17 945) were screened between mid-2008 and mid-2011. MBL-producing isolates were typed by PFGE and MLST. MBL genes were amplified and sequenced within their integrons. Plasmids with MBL genes were analysed by nuclease S1 plus hybridization profiling, mating and transformation assays, and by PCR-based replicon typing. RESULTS: Ninety-one patients in nine centres (six countries), including 62 patients in two Greek ICUs, carried 94 non-duplicate MBL-producing organisms. Klebsiella pneumoniae isolates from Greece dominated (n = 57) and belonged mainly to ST147, ST36 and ST383. All but one of the isolates expressed VIM-1-type MBLs. Isolates of Greek origins produced five enzymes, including new VIM-39, encoded by class 1 integrons of four types. In-e541-like elements prevailed, comprising six variants located on IncR, IncFIIK, IncR + FIIK, IncR + A/C or non-typeable plasmids. The other group were new In4873 and In4863, being the first In416-like elements identified in Greece, which were present on IncA/C or non-typeable plasmids. Isolates from other countries produced only VIM-1 and the major integron was In916, identified in 16 organisms from France, Italy and Spain. In916 was carried by four plasmid types, including IncA/C, IncFIIK and IncHI2. Other integrons included a new element, In3103, in Spain and In110 identified only in Latvia. CONCLUSIONS: This study provided fully comparable data on the occurrence and molecular characteristics of VIM-producing Enterobacteriaceae in a group of hospital units across Europe, documenting recent changes in their epidemiology.
- MeSH
- bakteriální geny MeSH
- beta-laktamasy sekrece MeSH
- DNA bakterií chemie genetika MeSH
- Enterobacteriaceae klasifikace enzymologie genetika izolace a purifikace MeSH
- enterobakteriální infekce epidemiologie mikrobiologie MeSH
- jednotky intenzivní péče MeSH
- lidé MeSH
- molekulární epidemiologie MeSH
- multilokusová sekvenční typizace MeSH
- nemocnice MeSH
- plazmidy analýza MeSH
- polymerázová řetězová reakce MeSH
- přenašečství epidemiologie mikrobiologie MeSH
- pulzní gelová elektroforéza MeSH
- sekvenční analýza DNA MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Evropa MeSH
Electrochemistry of nucleic acid is at present a booming field producing about 800 papers published per year. First papers in this field were published in 1958–1961 in Brno (Czech Republic) showing that purine and pyrimidine base residues in single-stranded DNA and RNA were reduced at Hg electrodes and the guanine residue produced an anodic signal when cyclic modes were used. The reduction sites of the base residues in native double-stranded (ds) DNA are hidden in the interior of the dsDNA molecule, which made their reduction difficult. At that time oscillographic polarography (ac chronopotentiometry) showed excellent sensitivity to changes in DNA structure and allowed to investigate DNA denaturation and hybridization. Later on also other electrochemical methods and electrodes were applied. In the following three decades basic principles were found which are at present used in the development of DNA hybridization sensors.
- MeSH
- chromozomy chemie MeSH
- dějiny 20. století MeSH
- dějiny 21. století MeSH
- DNA * analýza chemie MeSH
- elektrochemické techniky dějiny metody MeSH
- elektrochemie * dějiny metody MeSH
- plazmidy analýza chemie MeSH
- polarografie dějiny metody MeSH
- potenciometrie dějiny metody MeSH
- RNA transferová analýza chemie MeSH
- RNA virová analýza chemie MeSH
- Check Tag
- dějiny 20. století MeSH
- dějiny 21. století MeSH
- Publikační typ
- historické články MeSH
- práce podpořená grantem MeSH
OBJECTIVES: Faecal Escherichia coli strains were isolated from great cormorants (Phalacrocorax carbo) and mallards (Anas platyrhynchos), which are commonly occurring waterbirds in Europe, and studied for resistance to cephalosporins and fluoroquinolones. METHODS: Cloacal swabs or faeces from great cormorants and mallards in Central Europe were cultivated to isolate Escherichia coli strains with extended-spectrum β-lactamase (ESBL) and plasmid-mediated quinolone resistance (PMQR) genes. RESULTS: Ten ESBL-producing E. coli with the bla(CTX-M-15) or bla(CTX-M-27) gene were isolated from eight great cormorants (1.6%, n = 499). The bla(CTX-M) genes were harboured by plasmids of F and I1 incompatibility groups. CTX-M-27-producing isolates were identified as the epidemiologically important B2-O25b-ST131 clone. No ESBL-producing E. coli was isolated from 305 mallards. Eight E. coli isolates with PMQR genes [six aac(6')-Ib-cr and two qnrS1] were detected in six great cormorants (1.2%). Seventeen strains with qnrS1 were detected in 17 mallards (6%). The PMQR genes were located on plasmids of incompatibility groups F, N or X2. ESBL and PMQR genes were found on conjugative plasmids, enabling the horizontal spread of resistance. CONCLUSIONS: Both great cormorants and mallards can spread epidemiologically important antimicrobial-resistant E. coli isolates to water bodies throughout Europe.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální léková rezistence MeSH
- beta-laktamasy sekrece MeSH
- cefalosporiny farmakologie MeSH
- chinolony farmakologie MeSH
- Escherichia coli enzymologie genetika izolace a purifikace MeSH
- feces mikrobiologie MeSH
- kloaka mikrobiologie MeSH
- plazmidy analýza MeSH
- ptáci mikrobiologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Evropa MeSH
Cíl práce: Cílem naší studie bylo stanovení výskytu qnr genů u ESBL-pozitivních izolátů Klebsiella pneumoniae získaných z klinického materiálu pacientů hospitalizovaných na jednotkách intenzivní péče několika klinik Fakultní nemocnice Olomouc. Materiál a metody: Do studie bylo zařazeno 100 ESBL-pozitivních izolátů Klebsiella pneumoniae získaných od pacientů hospitalizovaných v období od 1. 1. 2008 do 31. 1. 2011. Geny kódující jednotlivé typy beta-laktamáz blaTEM, blaSHV, blaCTX-M a geny qnr byly detekovány prostřednictvím PCR (polymerázové řetězové reakce). Identita, resp. podobnost izolátů byla analyzována porovnáním restrikčních profilů pomocí PFGE (pulzní gelové elektroforézy). Výsledky a závěr: Gen blaSHV byl detekován u 99 ze 100 izolátů Klebsiella pneumoniae, což odpovídá publikovaným údajům, které předpokládají umístění genu blaSHV-1 na chromozomu jmenovaného druhu. U 77 izolátů byl rovněž zachycen gen blaCTX-M. Přítomnost genu qnr byla odhalena u 56 izolátů, většina z tohoto počtu (76,8 %) nesla současně geny pro beta-laktamázy CTX-M a TEM. Ve všech případech se jednalo o variantu qnrB. Byly identifikovány plasmidy patřící do devíti různých inkompatibilních skupin, nejčastěji ze skupiny IncFII (52 izolátů). Byla prokázána vysoká míra rezistence nejen k beta-laktamovým antibiotikům, ale současně i k aminoglykosidům, tetracyklinu a kotrimoxazolu. PFGE odhalila 65 jedinečných kmenů a několik skupin izolátů se shodným restrikčním profilem. Přestože klinický význam qnr genů zatím není zcela jednoznačně definován, jejich výskyt u ESBL-pozitivních enterobakterií je významný.
Background: The study aimed at determining the presence of qnr genes in ESBL-positive strains of Klebsiella pneumoniae isolated from clinical material obtained from patients hospitalized in several intensive care units in the University Hospital Olomouc. Material and methods: The study comprised 100 ESBL-producing Klebsiella pneumoniae isolates from clinical samples obtained from patients hospitalized between 1 January 2008 and 31 January 2011. blaTEM, blaSHV, blaCTX-M and qnr genes were detected by polymerase chain reaction (PCR). To compare the similarity or identity of the isolates, pulsed-field gel electrophoresis (PFGE) of DNA fragments was used. Results and Conclusion: The blaSHV gene was detected in 99 from 100 isolates of Klebsiella pneumoniae, which is in accordance with the published data that suppose the chromosomal position of the blaSHV-1 gene in this species. The blaCTX-M gene was detected in 77 isolates. The qnr genes were revealed in 56 isolates and majority (76.8 %) of these qnr-positive bacteria carried also the genes encoding the beta-lactamases CTX-M and TEM. In all cases, the qnrB variant was detected. Plasmids from nine different incompatibility groups were identified, in most cases from the IncFII group (52 isolates). Antibiotic susceptibility tests revealed high frequency of resistance not only to beta-lactams, but also to aminoglycosides, tetracycline and sulfamethoxazole/trimethoprim. PFGE detected 65 different strains and several groups of isolates with the same restriction profile. Although the clinical significance of the qnr genes is not well established, their presence in ESBL-positive Enterobacteriaceae is not negligible and it should be kept in mind.
- MeSH
- bakteriální léková rezistence genetika MeSH
- bakteriologické techniky MeSH
- beta-laktamasy genetika izolace a purifikace MeSH
- beta-laktamová rezistence genetika MeSH
- DNA bakterií genetika MeSH
- financování organizované MeSH
- Klebsiella pneumoniae genetika izolace a purifikace patogenita MeSH
- kohortové studie MeSH
- kultivační média chemie MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- mnohočetná léková rezistence genetika MeSH
- pacienti hospitalizovaní MeSH
- plazmidy analýza MeSH
- polymerázová řetězová reakce MeSH
- pulzní gelová elektroforéza MeSH
- Check Tag
- lidé MeSH
