Heterologously expressed and purified azoreductase enzyme from facultative Klebsiella pneumoniae was used to degrade sulphonated azo dye. Methyl orange (MO) was used as the model dye to study the azo dye decolorization potential of the purified enzyme at different conditions. The enzyme had maximum activity at 40 °C and pH 8.0. The enzyme was observed to be thermo-stable as some enzyme activity was retained even at 80 °C. The apparent kinetic parameters, i.e., appKm and appVmax, for azoreductase using MO as a substrate were found to be 17.18 μM and 0.08/min, respectively. The purified enzyme was able to decolorize approximately 83% of MO (20 μM) within 10 min in the presence of NADH. Thus, efficient decolorization of MO was observed by the purified enzyme. The recombinant enzyme was purified approximately 18-fold with 46% yield at the end of four steps of the purification process. Enzyme was present in a tetrameric structure as confirmed by the volume at which protein was eluted in gel filtration chromatography, and the monomeric molecular mass of enzyme was found to be 23 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The dye degradation efficiency of azoreductase cloned from Klebsiella pneumoniae and purified from recombinant Escherichia coli was observed to be much higher as compared with the efficiencies of the reported azoreductases from other bacterial strains. In the present study, we report the purification and characterization of the azoreductase cloned from Klebsiella pneumoniae and expressed in Escherichia coli.

• MeSH
• azosloučeniny metabolismus   MeSH
• bakteriální proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• barvicí látky metabolismus   MeSH
• biodegradace MeSH
• Escherichia coli genetika   metabolismus   MeSH
• kinetika MeSH
• Klebsiella pneumoniae enzymologie   genetika   MeSH
• koncentrace vodíkových iontů MeSH
• molekulová hmotnost MeSH
• nitroreduktasy chemie   genetika   izolace a purifikace   metabolismus   MeSH
• rekombinantní proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• azoreductase MeSH Prohlížeč
• azosloučeniny MeSH
• bakteriální proteiny MeSH
• barvicí látky MeSH
• nitroreduktasy MeSH
• rekombinantní proteiny MeSH

Oxacillinases (OXA) have been mostly described in Enterobacteriaceae, Acinetobacter, and Pseudomonas species. Recent years have witnessed an increased prevalence of intrinsic and/or acquired β-lactamase-producing Acinetobacter in food-producing animals. This study was conducted to assess the prevalence of OXA among selected bacterial species and to characterize these enzymes by in silico analysis. Screening of OXA was performed by PCR amplification using specific pairs of oligonucleotides. Overall, 40 pairs of primers were designed, of which 6 were experimentally tested in vitro. Among 49 bacterial isolates examined, the presence of blaOXA-1-like genes was confirmed in 20 cases (41%; 19 times in Klebsiella pneumoniae and once in Enterobacter cloacae). No OXA were found in animal isolates. The study results confirmed the specificity of the designed oligonucleotide pairs. Furthermore, the designed primers were found to possess the ability to specifically detect 90.2% of all OXA. These facts suggest that the in silico and in vitro tested primers could be used for single or multiplex PCR to screen for the presence of OXA in various bacteria, as well as to monitor their spread. At the same time, the presence of conserved characteristic amino acids and motifs was confirmed by in silico analysis of sequences of representative members of OXA.

• Klíčová slova
• PCR, antibiotic resistance, oxacillinases, primers,
• MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• beta-laktamasy genetika   metabolismus   MeSH
• DNA primery chemická syntéza   metabolismus   MeSH
• Enterobacter cloacae klasifikace   účinky léků   enzymologie   genetika   MeSH
• Escherichia coli klasifikace   účinky léků   enzymologie   genetika   MeSH
• exprese genu MeSH
• fylogeneze MeSH
• gramnegativní bakteriální infekce diagnóza   epidemiologie   mikrobiologie   veterinární   MeSH
• Klebsiella pneumoniae klasifikace   účinky léků   enzymologie   genetika   MeSH
• kur domácí mikrobiologie   MeSH
• lidé MeSH
• maso mikrobiologie   MeSH
• mikrobiální testy citlivosti MeSH
• multiplexová polymerázová řetězová reakce metody   MeSH
• peniciliny farmakologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika epidemiologie   MeSH
• Názvy látek
• antibakteriální látky MeSH
• bakteriální proteiny MeSH
• beta-laktamasy MeSH
• DNA primery MeSH
• oxacillinase MeSH Prohlížeč
• peniciliny MeSH

Klebsiella pneumoniae infections have always been an important problem in public health, but today, the increasing resistance of these bacteria to antibiotics due to β-lactamases production has renewed interest in K. pneumoniae infections. The aim of the study was to present a case of a neurosurgical patient with multidrug-resistant K. pneumoniae ST11 infection after craniectomy. Four K. pneumoniae isolates from various clinical materials of the patient undergone identification and susceptibility testing with the Vitek2 system. Tests for β-lactamases production were performed according to EUCAST guidelines. Strains were analyzed for bla genes responsible for β-lactamase production (blaTEM, blaSHV, blaCTX-M, blaVIM, blaIMP, blaNDM, blaKPC, blaOXA-48) using PCR. Moreover, the genetic relatedness of these isolates was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). All tested strain presented multidrug resistance. The highest susceptibility was observed for imipenem, meropenem, and ertapenem. The strain isolated from the nervous system was ESBL-positive with blaSHV-11, blaTEM-1, and blaCTX-M-15 genes. Additionally, the strain from urine was blaKPC-3-positive. Molecular typing revealed that all strains belonged to the same clone and identified two PFGE profiles. The analysis of MLST allelic profile showed that tested K. pneumoniae strains belonged to ST11. Identification of ST11 K. pneumoniae as etiological factor of infection unfavorably impacts on prognosis among neurosurgical patient after craniectomy.

• MeSH
• antibakteriální látky farmakologie   terapeutické užití   MeSH
• beta-laktamasy genetika   MeSH
• fatální výsledek MeSH
• infekce bakteriemi rodu Klebsiella diagnostické zobrazování   farmakoterapie   mikrobiologie   MeSH
• Klebsiella pneumoniae klasifikace   účinky léků   enzymologie   MeSH
• kraniotomie škodlivé účinky   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• mladý dospělý MeSH
• mnohočetná bakteriální léková rezistence * MeSH
• multilokusová sekvenční typizace MeSH
• počítačová rentgenová tomografie MeSH
• pulzní gelová elektroforéza MeSH
• techniky typizace bakterií MeSH
• Check Tag
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• Názvy látek
• antibakteriální látky MeSH
• beta-laktamasy MeSH

Bacterial microcompartments (BMCs) are prokaryotic organelles consisting of a protein shell and an encapsulated enzymatic core. BMCs are involved in several biochemical processes, such as choline, glycerol and ethanolamine degradation and carbon fixation. Since non-native enzymes can also be encapsulated in BMCs, an improved understanding of BMC shell assembly and encapsulation processes could be useful for synthetic biology applications. Here we report the isolation and recombinant expression of BMC structural genes from the Klebsiella pneumoniae GRM2 locus, the investigation of mechanisms behind encapsulation of the core enzymes, and the characterization of shell particles by cryo-EM. We conclude that the enzymatic core is encapsulated in a hierarchical manner and that the CutC choline lyase may play a secondary role as an adaptor protein. We also present a cryo-EM structure of a pT = 4 quasi-symmetric icosahedral shell particle at 3.3 Å resolution, and demonstrate variability among the minor shell forms.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• cholin metabolismus   MeSH
• elektronová kryomikroskopie MeSH
• genetické lokusy MeSH
• Klebsiella pneumoniae cytologie   enzymologie   genetika   ultrastruktura   MeSH
• lyasy genetika   metabolismus   MeSH
• organely enzymologie   ultrastruktura   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• syntetická biologie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• cholin MeSH
• lyasy MeSH
• rekombinantní proteiny MeSH

Studying bacterial population diversity is important to understand healthcare associated infections' epidemiology and has a significant impact on dealing with multidrug resistant bacterial outbreaks. We characterised the extended-spectrum beta-lactamase producing K. pneumoniae (ESBLp KPN) population in our hospital using mini-MLST. Then we used whole genome sequencing (WGS) to compare selected isolates belonging to the most prevalent melting types (MelTs) and the colonization/infection pair isolates collected from one patient to study the ESBLp KPN population's genetic diversity. A total of 922 ESBLp KPN isolates collected between 7/2016 and 5/2018 were divided into 38 MelTs using mini-MLST with only 6 MelTs forming 82.8% of all isolates. For WGS, 14 isolates from the most prominent MelTs collected in the monitored period and 10 isolates belonging to the same MelTs collected in our hospital in 2014 were randomly selected. Resistome, virulome and ST were MelT specific and stable over time. A maximum of 23 SNV per core genome and 58 SNV per core and accessory genome were found. To determine the SNV relatedness cut-off values, 22 isolates representing colonization/infection pair samples obtained from 11 different patients were analysed by WGS with a maximum of 22 SNV in the core genome and 40 SNV in the core and accessory genome within pairs. The mini-MLST showed its potential for real-time epidemiology in clinical practice. However, for outbreak evaluation in a low diversity bacterial population, mini-MLST should be combined with more sensitive methods like WGS. Our findings showed there were only minimal differences within the core and accessory genome in the low diversity hospital population and gene based SNV analysis does not have enough discriminatory power to differentiate isolate relatedness. Thus, intergenic regions and mobile elements should be incorporated into the analysis scheme to increase discriminatory power.

• MeSH
• bakteriální proteiny genetika   MeSH
• beta-laktamasy genetika   metabolismus   MeSH
• dítě MeSH
• DNA bakterií genetika   MeSH
• dospělí MeSH
• infekce bakteriemi rodu Klebsiella enzymologie   epidemiologie   genetika   mikrobiologie   MeSH
• infekce spojené se zdravotní péčí enzymologie   epidemiologie   genetika   mikrobiologie   MeSH
• Klebsiella pneumoniae enzymologie   genetika   izolace a purifikace   MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mnohočetná bakteriální léková rezistence * MeSH
• multilokusová sekvenční typizace * MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• sekvenování celého genomu * MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• beta-laktamasy MeSH
• DNA bakterií MeSH

This study evaluated the carriage of AmpC and extended-spectrum beta-lactamase (ESBL) genes and associated plasmids in faecal bacteria of Canadian corvids. Faecal samples from 449 birds in five roosting sites across Canada were analyzed using selective media, screening for AmpC and ESBL genes by PCR, and sequencing. Genomic relatedness was determined by PFGE and MLST. Plasmid mobility was studied by conjugation and transformation experiments, followed by plasmid typing. In total, 96 (21%, n = 449) cefotaxime-resistant Escherichia coli and three (0.7%) Klebsiella pneumoniae isolates were identified. ESBL genes blaCTX-M-1 (n = 3), blaCTX-M-14 (n = 2), blaCTX-M-32 (n = 2) and blaCTX-M-124 (n = 1) were detected in eight E. coli isolates, whereas blaSHV-2 (2) was found in two K. pneumoniae. E. coli isolates contained blaCMY-2 (n = 83) and blaCMY-42 (n = 1). The high genetic diversity of the isolates and presence of clinically important E. coli ST69 (n = 1), ST117 (n = 7) and ST131 (n = 1) was revealed. AmpC genes were predominantly carried by plasmids of incompatibility groups I1 (45 plasmids), A/C (10) and K (7). The plasmid IncI1/ST12 was most common and found in diverse E. coli STs in all sites. Highly diverse E. coli isolates containing AmpC and ESBL genes, including clinically important clones and emerging plasmids, are in circulation throughout Canadian wildlife.

• MeSH
• bakteriální proteiny genetika   MeSH
• beta-laktamasy genetika   MeSH
• Escherichia coli klasifikace   enzymologie   genetika   izolace a purifikace   MeSH
• feces mikrobiologie   MeSH
• Klebsiella pneumoniae enzymologie   genetika   izolace a purifikace   MeSH
• multilokusová sekvenční typizace MeSH
• plazmidy genetika   MeSH
• vrány mikrobiologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Kanada MeSH
• Názvy látek
• AmpC beta-lactamases MeSH Prohlížeč
• bakteriální proteiny MeSH
• beta-laktamasy MeSH

Ten Enterobacteriaceae isolates collected in a Czech hospital carried blaKPC-positive plasmids of different sizes (∼30, ∼45, and ∼80 kb). Sequencing revealed three types of plasmids (A to C) with the Tn4401a transposon. Type A plasmids comprised an IncR backbone and a KPC-2-encoding multidrug resistance (MDR) region. Type B plasmids were derivatives of type A plasmids carrying an IncN3-like segment, while type C plasmids were IncP6 plasmids sharing the same KPC-2-encoding MDR region with type A and B plasmids.

• Klíčová slova
• Citrobacter freundii, Illumina sequencing, IncR, ST18, Tn4401a,
• MeSH
• antibakteriální látky terapeutické užití   MeSH
• beta-laktamasy genetika   metabolismus   MeSH
• Citrobacter freundii účinky léků   enzymologie   genetika   izolace a purifikace   MeSH
• enterobakteriální infekce farmakoterapie   epidemiologie   mikrobiologie   MeSH
• Escherichia coli účinky léků   enzymologie   genetika   izolace a purifikace   MeSH
• exprese genu MeSH
• izoenzymy genetika   metabolismus   MeSH
• karbapenemy terapeutické užití   MeSH
• Klebsiella pneumoniae účinky léků   enzymologie   genetika   izolace a purifikace   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• mnohočetná bakteriální léková rezistence genetika   MeSH
• Morganella morganii účinky léků   enzymologie   genetika   izolace a purifikace   MeSH
• nemocnice MeSH
• otevřené čtecí rámce MeSH
• plazmidy chemie   klasifikace   metabolismus   MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• transpozibilní elementy DNA MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika epidemiologie   MeSH
• Názvy látek
• antibakteriální látky MeSH
• beta-lactamase KPC-2 MeSH Prohlížeč
• beta-laktamasy MeSH
• izoenzymy MeSH
• karbapenemy MeSH
• transpozibilní elementy DNA MeSH

The aim of this study was to characterize the first cases and outbreaks of OXA-48-like-producing Enterobacteriaceae recovered from hospital settings in the Czech Republic. From 2013 to 2015, 22 Klebsiella pneumoniae isolates, 3 Escherichia coli isolates, and 1 Enterobacter cloacae isolate producing OXA-48-like carbapenemases were isolated from 20 patients. Four of the patients were colonized or infected by two or three different OXA-48-like producers. The K. pneumoniae isolates were classified into nine sequence types (STs), with ST101 being predominant (n = 8). The E. coli isolates were of different STs, while the E. cloacae isolate belonged to ST109. Twenty-four isolates carried blaOXA-48, while two isolates carried blaOXA-181 or blaOXA-232 Almost all isolates (n = 22) carried blaOXA-48-positive plasmids of a similar size (∼60 kb), except the two isolates producing OXA-181 or OXA-232. In an ST45 K. pneumoniae isolate and an ST38 E. coli isolate, S1 nuclease profiling plus hybridization indicated a chromosomal location of blaOXA-48 Sequencing showed that the majority of blaOXA-48-carrying plasmids exhibited high degrees of identity with the pOXA-48-like plasmid pE71T. Additionally, two novel pE71T derivatives, pOXA-48_30715 and pOXA-48_30891, were observed. The blaOXA-181-carrying plasmid was identical to the IncX3 plasmid pOXA181_EC14828, while the blaOXA-232-carrying plasmid was a ColE2-type plasmid, being a novel derivative of pOXA-232. Finally, sequencing data showed that the ST45 K. pneumoniae and ST38 E. coli isolates harbored the IS1R-based composite transposon Tn6237 containing blaOXA-48 integrated into their chromosomes. These findings underlined that the horizontal transfer of pOXA-48-like plasmids has played a major role in the dissemination of blaOXA-48 in the Czech Republic. In combination with the difficulties with their detection, OXA-48 producers constitute an important public threat.

• Klíčová slova
• ColE2-like, IncL, IncX3, Klebsiella pneumoniae, OXA-181, OXA-232, Tn1999.2, Tn1999.5,
• MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální chromozomy genetika   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• beta-laktamasy genetika   metabolismus   MeSH
• Enterobacteriaceae účinky léků   enzymologie   genetika   MeSH
• Escherichia coli účinky léků   enzymologie   genetika   MeSH
• Klebsiella pneumoniae účinky léků   enzymologie   genetika   MeSH
• mikrobiální testy citlivosti MeSH
• plazmidy genetika   MeSH
• přenos genů horizontální genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• antibakteriální látky MeSH
• bakteriální proteiny MeSH
• beta-laktamasy MeSH
• carbapenemase MeSH Prohlížeč

OBJECTIVES: Global dissemination of KPC-type carbapenemases is mainly associated with the spread of high-risk clones of Klebsiella pneumoniae and of KPC-encoding plasmids. In this study, we explored the population structure of KPC-encoding plasmids from the recent epidemics of KPC-producing K. pneumoniae (KPC-Kp) in Greece and Italy, the two major European endemic settings. METHODS: Thirty-four non-replicate clinical strains of KPC-Kp representative of the early phases (2008-11) of the Greek (n = 22) and Italian (n = 12) epidemics were studied. Isolates were typed by MLST, and blaKPC-carrying plasmids were characterized by S1 profiling, PCR-based replicon typing and RFLP. Transfer experiments by conjugation or transformation were carried out with Escherichia coli recipients. Eleven plasmids, representative of all different restriction profiles, were completely sequenced. RESULTS: The representative Greek strains belonged to 14 sequence types (STs), with a predominance of ST258. The representative Italian strains belonged to three STs, with a predominance of clonal complex 258 (ST258, ST512). The 34 strains carried plasmids of variable size (78-166 kb), either with blaKPC-2 or blaKPC-3 gene embedded in a Tn4401a transposon. Plasmids from Greek strains were mostly of a single RFLP type (A) and resembled the archetypal pKpQIL KPC-encoding plasmid, while plasmids from Italian strains belonged to a more heterogeneous population, showing five RFLP profiles (A, C-F). Types A and C resembled pKpQIL or deletion derivatives thereof, while types D-F included plasmids with hybrid structures between pKpQIL, pKPN3 and pKPN101-IT. CONCLUSIONS: pKpQIL-like plasmids played a major role in the dissemination of blaKPC in Greece and Italy, but evolved with different dynamics in these endemic settings.

• MeSH
• bakteriální léková rezistence genetika   MeSH
• bakteriální proteiny genetika   MeSH
• beta-laktamasy genetika   MeSH
• endemické nemoci MeSH
• epidemie MeSH
• Escherichia coli genetika   MeSH
• infekce bakteriemi rodu Klebsiella epidemiologie   mikrobiologie   MeSH
• karbapenemy farmakologie   MeSH
• Klebsiella pneumoniae účinky léků   enzymologie   genetika   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• multilokusová sekvenční typizace metody   MeSH
• plazmidy genetika   MeSH
• polymorfismus délky restrikčních fragmentů MeSH
• pulzní gelová elektroforéza MeSH
• sekvenční analýza DNA MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Itálie epidemiologie   MeSH
• Řecko epidemiologie   MeSH
• Názvy látek
• bakteriální proteiny MeSH
• beta-laktamasy MeSH
• carbapenemase MeSH Prohlížeč
• karbapenemy MeSH

Minim typing is derived from the multi-locus sequence typing (MLST). It targets the same genes, but sequencing is replaced by high resolution melt analysis. Typing can be performed by analysing six loci (6MelT), four loci (4MelT) or using data from four loci plus sequencing the tonB gene (HybridMelT). The aim of this study was to evaluate Minim typing to discriminate extended-spectrum beta-lactamase producing Klebsiella pneumoniae (ESBL-KLPN) isolates at our hospital. In total, 380 isolates were analyzed. The obtained alleles were assigned according to both the 6MelT and 4MelT typing scheme. In 97 isolates, the tonB gene was sequenced to enable HybridMelT typing. We found that the presented method is suitable to quickly monitor isolates of ESBL-KLPN; results are obtained in less than 2 hours and at a lower cost than MLST. We identified a local ESBL-KLPN outbreak and a comparison of colonizing and invasive isolates revealed a long term colonization of patients with the same strain.

• Klíčová slova
• ESBL, High-resolution melt analysis, Klebsiella pneumoniae, Minim typing, Multi-locus sequence typing,
• MeSH
• beta-laktamasy metabolismus   MeSH
• časové faktory MeSH
• centra terciární péče MeSH
• infekce bakteriemi rodu Klebsiella mikrobiologie   MeSH
• Klebsiella pneumoniae klasifikace   enzymologie   izolace a purifikace   MeSH
• lidé MeSH
• molekulární typizace metody   MeSH
• polymerázová řetězová reakce MeSH
• přenašečství mikrobiologie   MeSH
• sekvenční analýza DNA MeSH
• tranzitní teplota MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• beta-laktamasy MeSH