A 2-year national genomic screening in the Czech Republic identified a notable prevalence of the New Delhi metallo-β-lactamase 5 (NDM-5)-producing Escherichia coli sequence type 38 (ST38) in the city of Brno. Forty-two ST38 E. coli isolates harbored the blaNDM-5 gene on the chromosome. Virulence factors confirmed the persistence of these isolates through biofilm formation. Single Nucleotide Polymorphisms (SNPs)-based phylogeny and CRISPR assay typing showed minimal genomic variations, implying a clonally driven outbreak. Results suggest that this high-risk clone may impose a nationwide problem.

• MeSH
• antibakteriální látky farmakologie   MeSH
• beta-laktamasy * genetika   MeSH
• biofilmy růst a vývoj   MeSH
• epidemický výskyt choroby * MeSH
• Escherichia coli * genetika   účinky léků   izolace a purifikace   enzymologie   MeSH
• faktory virulence genetika   MeSH
• fylogeneze MeSH
• genom bakteriální MeSH
• genomika metody   MeSH
• infekce vyvolané Escherichia coli * mikrobiologie   epidemiologie   MeSH
• jednonukleotidový polymorfismus * MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH

• MeSH
• bakteriální proteiny * genetika   MeSH
• beta-laktamasy * genetika   analýza   MeSH
• laktony metabolismus   MeSH
• lidé MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice * metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• dopisy MeSH

BACKGROUND: Sepsis is a common worldwide health condition with high mortality. It is caused by a dysregulated immune response to the pathogen. Severe infections resulting in sepsis can be also determined by monitoring several bloodstream biomarkers, one of them being pro-hormone procalcitonin (PCT). PCT concentration in the bloodstream correlates well with sepsis and in severe cases increases up to a thousand times from the healthy physiological values in a short time. In this study, we developed a rapid technique for PCT detection by MALDI-TOF mass spectrometry, that uses in-situ enrichment directly on the specialized immuno MALDI chips that are utilized as MALDI plates. The method's ability to detect PCT was confirmed by comparing the results with LC-MS bottom-up workflow. The new method detects intact PCT by its m/z and uncovers its alternations in septic serum. METHODS: The MALDI chips used for the detection of PCT were prepared by ambient ion soft landing of anti-PCT antibody on an ITO glass slide. The chips were used for the development of the rapid MALDI-TOF MS method. A parallel method based on affinity enrichment on magnetic beads followed by LC-MS/MS data-dependent peptide microsequencing was used to prove PCT presence in the sample. All samples were also tested by ELISA to determine PCT concentration prior to analyzing them by mass spectrometry methods. RESULTS: The MALDI chip method was optimized using recombinant PCT spiked into the human serum. The PCT detection limit was 10 ng/mL. The optimized method was used to analyze 13 sera from patients suffering sepsis. The PCT results were confirmed by LC-MS/MS. The measurement of the intact PCT by the MALDI chip method revealed that sera of patients with severe sepsis have other forms of PCT present, which show post-processing of the primary sequence by cleavage of PCT, resulting in the formation of N and C termini fragments. CONCLUSIONS: Procalcitonin from human serum was successfully enriched and detected using immunoaffinity MALDI chips. The intact PCT was characterized in 13 septic patients. The method is more specific compared to non-MS-based immunoaffinity techniques and allows observation of different variants of PCT in septic patients.

• Publikační typ
• časopisecké články MeSH

The resistance to carbapenems is usually mediated by enzymes hydrolyzing β-lactam ring. Recently, an alternative way of the modification of the antibiotic, a β-lactone formation by OXA-48-like enzymes, in some carbapenems was identified. We focused our study on a deep analysis of OXA-48-like-producing Enterobacterales, especially strains showing poor hydrolytic activity. In this study, well characterized 74 isolates of Enterobacterales resistant to carbapenems were used. Carbapenemase activity was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), liquid chromatography/mass spectrometry (LC-MS), Carba-NP test and modified Carbapenem Inactivation Method (mCIM). As meropenem-derived β-lactone possesses the same molecular weight as native meropenem (MW 383.46 g/mol), β-lactonization cannot be directly detected by MALDI-TOF MS. In the spectra, however, the peaks of m/z = 340.5 and 362.5 representing decarboxylated β-lactone and its sodium adduct were detected in 25 out of 35 OXA-48-like producers. In the rest 10 isolates, decarboxylated hydrolytic product (m/z = 358.5) and its sodium adduct (m/z = 380.5) have been detected. The peak of m/z = 362.5 was detected in 3 strains co-producing OXA-48-like and NDM-1 carbapenemases. The respective signal was identified in no strain producing class A or class B carbapenemase alone showing its specificity for OXA-48-like carbapenemases. Using LC-MS, we were able to identify meropenem-derived β-lactone directly according to the different retention time. All strains with a predominant β-lactone production showed negative results of Carba NP test. In this study, we have demonstrated that the strains producing OXA-48-like carbapenemases showing false-negative results using Carba NP test and MALDI-TOF MS preferentially produced meropenem-derived β-lactone. We also identified β-lactone-specific peak in MALDI-TOF MS spectra and demonstrated the ability of LC-MS to detect meropenem-derived β-lactone.

• MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální proteiny * analýza   MeSH
• beta-laktamasy analýza   MeSH
• Enterobacteriaceae * MeSH
• karbapenemy farmakologie   MeSH
• meropenem farmakologie   MeSH
• mikrobiální testy citlivosti MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• Publikační typ
• časopisecké články MeSH

The aim of the present study is to describe the ongoing spread of the KPC-producing strains, which is evolving to an epidemic in Czech hospitals. During the period of 2018-2019, a total of 108 KPC-producing Enterobacterales were recovered from 20 hospitals. Analysis of long-read sequencing data revealed the presence of several types of blaKPC-carrying plasmids; 19 out of 25 blaKPC-carrying plasmids could be assigned to R (n = 12), N (n = 5), C (n = 1) and P6 (n = 1) incompatibility (Inc) groups. Five of the remaining blaKPC-carrying plasmids were multireplicon, while one plasmid couldn't be typed. Additionally, phylogenetic analysis confirmed the spread of blaKPC-carrying plasmids among different clones of diverse Enterobacterales species. Our findings demonstrated that the increased prevalence of KPC-producing isolates was due to plasmids spreading among different species. In some districts, the local dissemination of IncR and IncN plasmids was observed. Additionally, the ongoing evolution of blaKPC-carrying plasmids, through genetic rearrangements, favours the preservation and further dissemination of these mobile genetic elements. Therefore, the situation should be monitored, and immediate infection control should be implemented in hospitals reporting KPC-producing strains.

• MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální léková rezistence * MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• beta-laktamasy metabolismus   MeSH
• epidemie MeSH
• infekce bakteriemi rodu Klebsiella epidemiologie   mikrobiologie   MeSH
• Klebsiella pneumoniae izolace a purifikace   metabolismus   MeSH
• lidé MeSH
• nemocnice statistika a číselné údaje   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika 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.

• 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

Carbapenemase-mediated resistance to carbapenems in Enterobacteriaceae has become the main challenge in the treatment and prevention of infections recently. The partially unnoticed spread of OXA-48-type carbapenemase producers is usually assigned to low minimum inhibitory concentrations (MICs) of carbapenems that OXA-48-producing isolates often display. Therefore, there is an urgent need of specific and sensitive methods for isolation and detection of OXA-48 producers in clinical microbiology diagnostics. The influence of bicarbonates on carbapenem MICs against carbapenemase-producing Enterobacteriaceae was tested. We also checked whether the addition of bicarbonates to liquid media supplemented with meropenem may facilitate the selective enrichment of various carbapenemase producers in cultures. Furthermore, the sensitivity of carbapenemase confirmation by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) and spectrophotometric hydrolysis assays upon the addition of NH4HCO3 was examined. The addition of NaHCO3 significantly increased MICs of ertapenem and meropenem for OXA-48 producers. Furthermore, liquid media supplemented with NaHCO3 and meropenem were reliable for the selective enrichment of carbapenemase producers. The presence of NH4HCO3 in buffers used in the spectrophotometric and MALDI-TOF MS carbapenemase detection increased the sensitivity of that assay. Our results demonstrate that bicarbonates in media or reaction buffers can enhance the sensitivity of screening methods and diagnostic tests for carbapenemase producers.

• MeSH
• bakteriální proteiny analýza   sekrece   MeSH
• beta-laktamasy analýza   sekrece   MeSH
• Enterobacteriaceae enzymologie   izolace a purifikace   MeSH
• enterobakteriální infekce diagnóza   mikrobiologie   MeSH
• hydrogenuhličitany diagnostické užití   MeSH
• kultivační média chemie   MeSH
• mikrobiální testy citlivosti metody   MeSH
• pufry MeSH
• senzitivita a specificita MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH

Spread of carbapenemase-producing bacteria has been described all over the world. This phenomenon may be accelerated by many factors, including wars and natural disasters. In this report, we described an NDM-1-producing Klebsiella pneumonia ST11 recovered from a patient injured during the Maidan revolution in Ukraine. To our knowledge, this is the first report of a carbapenemase-producing Enterobacteriaceae in Ukraine and one of several reports describing wound colonization/infection of humans injured during war.

• Publikační typ
• časopisecké články MeSH

A total of 2,683 nonrepetitive Escherichia coli isolates were collected from microbiological laboratories covering all regions of the Czech Republic, during April 2011. Antimicrobial susceptibility patterns of E. coli were assessed. All 38 cefotaxime-resistant (CTX-R) isolates were found to be extended-spectrum β-lactamase (ESBL)-positive by the double-disc synergy test. Thirty-two of those isolates produced enzymes of CTX-M-1 family, five of CTX-M-9 family, and one isolate both CTX-M types. Genotyping by multilocus sequence typing classified all ESBL-producing isolates into 13 sequence types (STs). ST131 was the most prevalent and was exclusively correlated with E. coli belonging to the more-virulent phylogroup B2. blaCTX-M-15 and blaCTX-M-9-like genes were mainly carried by plasmids belonging to the IncF group, while replicon I1 was predominant among CTX-M-1-encoding plasmids. Additionally, 63% of the ESBL-producing isolates were also resistant to ciprofloxacin. Sequence analysis of quinolone resistance-determining regions of gyrA and parC revealed the presence of amino acid substitutions in 22 out of 23 ciprofloxacin-resistant isolates. The acc(6')-Ib-cr and qnrB1 plasmid-mediated quinolone resistance genes were also detected in some of the isolates. This is the first report on the emergence and spread of CTX-M-producing E. coli in the community of the Czech Republic, indicating the high prevalence of ST131 clone among CTX-M producers.

• MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální geny MeSH
• beta-laktamasy genetika   MeSH
• buněčné klony MeSH
• diskové difúzní antimikrobiální testy MeSH
• dítě MeSH
• DNA bakterií genetika   MeSH
• dospělí MeSH
• Escherichia coli účinky léků   enzymologie   genetika   izolace a purifikace   patogenita   MeSH
• fluorochinolony farmakologie   MeSH
• fylogeneze MeSH
• infekce vyvolané Escherichia coli epidemiologie   mikrobiologie   MeSH
• infekce získané v komunitě epidemiologie   mikrobiologie   MeSH
• kojenec MeSH
• konjugace genetická MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mnohočetná bakteriální léková rezistence genetika   MeSH
• multilokusová sekvenční typizace MeSH
• plazmidy genetika   MeSH
• předškolní dítě MeSH
• prevalence MeSH
• proteiny z Escherichia coli genetika   MeSH
• pulzní gelová elektroforéza MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• virulence genetika   MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH

Cíl: Jedním z faktorů ohrožujících současnou medicínu je šíření multirezistentních gramnegativních bakterií. V článku jsou shrnuty výsledky 6měsíční prevalenční studie zaměřené na kmeny K. pneumoniae a E. coli provedené v českých nemocnicích v rámci projektu EuSCAPE (European Survey on Carbapenemase-Producing Bacteria). Metodika: Do studie bylo zapojeno 10 nemocnic ze všech krajů České republiky. Během sledovaného období (1. listopad 2013 až 30. duben 2014) bylo shromažďováno deset prvozáchytů K. pneumoniae a E. coli necitlivých ke karba-penemům z klinických vzorků (krev, dolní cesty dýchací, moč, punktát, stěry z ran). Zároveň byl deponován první citlivý izolát zachycený od téhož pacienta a vybraného bakteriálního druhu. Byla stanovena citlivost k 15 různým antibiotikům podle metodiky EUCAST. Karbapenemázy byly identifikovány metodou MALDI-TOF MS hydrolýzy meropenemu. Kmeny produkující karbapenemázy byly podrobeny multilokusové sekvenační typizaci a byly identifikovány geny karpapenemáz. Výsledky: Během sledovaného období bylo shromážděno 30 izolátů (K. pneumoniae n = 28, E. coli n = 2) necitlivých ke karbapenemům. U pacienta přeloženého z Ukrajiny po zranění během revoluce na Majdanu byl identifikován sekvenční typ (ST) 11 K. pneumoniae produkující karbapenemázu NDM-1. Druhý izolát produkující karbapenemázu produkoval enzym OXA-48. Jednalo se o kmen K. pneumoniae ST101 identifikovaný u pacienta přijatého z komunity. Závěr: Tato prevalenční studie opět potvrdila nízkou prevalenci enterobakterií produkujících karbapenemázy (CPE) v České republice. Zároveň opět ukazuje na riziko spojené s importem CPE ze zemí s vysokou prevalencí nebo neznámou epidemiologickou situací..

Objective: One of the most important threats of current medicine is the spread of multiresistant Gram-negative bacteria. We report here data from a six-month prevalence study on carbapenemase-producing K. pneumoniae and E. coli performed in Czech hospitals participating on European Survey on Carbapenemase-Producing Enterobacteriaceae (EuSCAPE). Methods: Ten hospitals covering all regions of the Czech Republic were selected. During the study period (1st November 2013 to 30th April 2014), first ten carbapenem non-susceptible isolates of K. pneumoniae or E. coli isolated from non-surveillance specimens (i.e., blood, lower respiratory tract secretions, urine, puncture fluids, and wound secretions) of single successive patients were collected. Successive carbapenem-susceptible isolates of the same species were also preserved as controls. Susceptibility to 15 antibiotics was determined using EUCAST recommendations. Carbapenemase activity was detected by MALDI-TOF MS meropenem hydrolysis assay. Positive isolates were subjected for molecular typing (multi-locus sequence typing, identification of carbapenemase gene). Results: During the study period, thirty non-susceptible isolates (K. pneumoniae n = 28, E. coli n = 2) were identified in 5 hospitals. Only two of them were confirmed to be carbapenemase producers. A NDM-1-producing K. pneumoniae ST11 was recovered from a patient, transferred from Ukraine, being injured during a Maidan revolution. The second isolate, an OXA-48-producing K. pneumoniae, belonging to ST101, was recovered from a patient admitted to a hospital for an ischemic stroke. Conclusions: This study again confirmed that the Czech Republic still belongs to the countries with low prevalence of carbapenemase-producing Enterobacteriaceae (CPE). Cases of CPE are usually restricted to an import from high-prevalence countries or countries with unknown epidemiological situation.

• Klíčová slova
• karbapenemázy, MALDI-TOF,
• MeSH
• bakteriální léková rezistence MeSH
• bakteriální proteiny genetika   MeSH
• beta-laktamasy genetika   MeSH
• beta-laktamová rezistence * MeSH
• Enterobacteriaceae MeSH
• Escherichia coli * genetika   izolace a purifikace   MeSH
• genetické lokusy MeSH
• Klebsiella pneumoniae * genetika   izolace a purifikace   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• nemocnice MeSH
• průřezové studie MeSH
• sekvenční analýza MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• multicentrická studie MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH