Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
nestr.
Narůstající incidence bakterií rezistentních k antibiotikům poslední volby představuje jeden z nejvýznamnějších medicínských problémů, který komplikuje úspěšnou léčbu život ohrožujících infekcí. V rámci navrhovaného projektu bude provedena molekulárně-epidemiologická/genomická analýza Enterobacterales produkujících karbapenemázy (CPE) izolovaných z pacientů hospitalizovaných v českých nemocnicích a srovnání s bakteriální populací citlivou ke karbapenemům. S využitím nejmodernějších postupů celogenomového sekvenování a bioinformatické analýzy budou identifikovány jedinečné rysy CPE, vysoce rizikové klony se zvýšeným významem pro zdraví populace a mobilní genetické elementy spojené s šířením rezistence ke karbapenemům v nemocnicích. Komplexní genomická analýza umožní popsat genetickou strukturu sledovaných bakteriálních populací včetně její dynamiky prostřednictvím mikroevolučních změn během šíření nemocničních nákaz. Výsledky projektu budou bezprostředně využity pro zlepšení a zpřesnění diagnostiky infekčních nemocí a návrh účinnějších postupů v prevenci a kontrole šíření patogenů.; Growing incidence of bacteria resistant to last-line antibiotics represents one of the most important medical issue, complicating the successful treatment of life-threatening infections. In this project, molecular-epidemiological and genomic analysis of carbapenemase-producing Enterobacterales (CPE) isolated from patients in Czech hospitals and its comparison with the carbapenem-susceptible population will be performed. Whole genome sequencing and bioinformatics analysis will allow to identify specific features of CPE, high-risk clones with increased importance for public health and mobile genetic elements associated with the dissemination of resistance to carbapenems in hospitals. Complex genomic analysis will reveal genetic structure of the studied bacterial populations including their dynamics through microevolutionary changes in the clinical context of a hospital outbreak. The project outcomes will be used to improve the diagnostics of infectious diseases and to outline effective interventions for the prevention and control of the transmission of bacterial pathogens.
Resistance to ceftolozane/tazobactam (C/T) in Pseudomonas aeruginosa is a health concern. In this study, we conducted a whole-genome-based molecular characterization to correlate resistance patterns and β-lactamases with C/T resistance among multi-drug resistant P. aeruginosa clinical isolates. Resistance profiles for 25 P. aeruginosa clinical isolates were examined using disk diffusion assay. Minimal inhibitory concentrations (MIC) for C/T were determined by broth microdilution. Whole-genome sequencing was used to check for antimicrobial resistance determinants and reveal their genetic context. The clonal relatedness was evaluated using MLST, PFGE, and serotyping. All the isolates were resistant to C/T. At least two β-lactamases were detected in each with the blaOXA-4, blaOXA-10, blaOXA-50, and blaOXA-395 being the most common. blaIMP-15, blaNDM-1, or blaVIM-2, metallo-β-lactamases, were associated with C/T MIC >256 μg/mL. Eight AmpC variants were identified, and PDC-3 was the most common. We also determined the clonal relatedness of the isolates and showed that they grouped into 11 sequence types (STs) some corresponding to widespread clonal complexes (ST111, ST233, and ST357). C/T resistance was likely driven by the acquired OXA β-lactamases such as OXA-10, and OXA-50, ESBLs GES-1, GES-15, and VEB-1, and metallo- β-lactamases IMP-15, NDM-1, and VIM-2. Collectively, our results revealed C/T resistance determinants and patterns in multi-drug resistant P. aeruginosa clinical isolates. Surveillance programs should be implemented and maintained to better track and define resistance mechanisms and how they accumulate and interact.
BACKGROUND: VIM metallo-β-lactamases are enzymes characterized by the ability to hydrolyze all β-lactams. Usually, blaVIM-like genes are carried by class 1 integrons. In the Czech Republic, only sporadic cases of VIM-producing Enterobacterales have been reported in which those isolates carried the VIM-1 carbapenemase-encoding integron In110. However, during 2019-2020, an increased number was reported. Therefore, the aim of the current study was to characterize the genetic elements involved in the increased spread of blaVIM genes. MATERIALS AND METHODS: 32 VIM-producing Enterobacterales collected between 2019 and 2020 were subjected to: antimicrobial susceptibility testing, integron analysis, and short reads sequencing. Based on the results, 19 isolates were selected as representative and sequenced using Sequel I platform. RESULTS: The 32 VIM-producing isolates exhibited variations in the MICs of carbapenems. Based on short-read data, 26 of the 32 sequenced isolates harbored the blaVIM-1 allele while six isolates carried the blaVIM-4 gene. The most prevalent was the In110 integron (n = 24) and two isolates carried the In4873 class 1 integron. The blaVIM-4 allele was identified in class 1 integrons In1174 (n = 3), In416 (n = 1), In2143 (n = 1) and In2150. Long reads sequencing revealed that the blaVIM was carried by: pKPC-CAV1193-like (n = 6), HI1 (pNDM-CIT; n = 4), HI2 (n = 3), FIB (pECLA; n = 2) and N (n = 1) incompatibility groups. Two blaVIM-carrying plasmids could not be typed by the database, while another one was integrated into the chromosome. CONCLUSION: We observed the spread of VIM-encoding integrons, mainly of In110, among Enterobacterales isolated from Czech hospitals, but also an increased number of novel elements underlining the ongoing evolution.
- Publikační typ
- časopisecké články MeSH
Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
Nestr.
V poslední době dochází k celosvětovému šíření antibiotické rezistence. Cílem předkládaného projektu je studium mechanizmů klinicky významné antibiotické rezistence enterobakterií pomocí MALDI-TOF hmotnostní spektrometrie a Ramanovy spektroskopie na klinicky relevantním modelu sepse (prasečí model). Budou vyvinuty nové spektrometrické metody, které by měly napomoci k objasnění chování beta-laktamáz v bakteriální buňce během infekce. Tyto metody napomohou k ustavení vhodných interpretačních kritérií při vyšetření citlivosti na antibiotika. Běhemm řešení budou použity epidemicky úspěšné klony enterobakterií, které budou detailně charakterizovány pomocí celogenomové sekvenace. Zároveň bude vyvinut vhodný animální model, který umožní získat dostatečnou koncentraci bakterií v klinickém vzorku (např. peritonitida). Budou rovněž validovány metody fixace a transportu klinického materiálu do laboratoře tak, aby byly vhodné pro výše uvedené analýzy. Zmíněné techniky umožní lépe pochopit patogenezi infekcí způsobených bakteriemi produkujícími beta-laktamázy.; Antibiotic resistance is an emerging problem worldwide. The aim of the present project is the in vivo study of clinically important antibiotic resistance mechanisms in Enterobacteriaceae on clinically relevant model of sepsis (pig model) using MALDI-TOF mass spectrometry and Raman spectroscopy. Novel assays that should allow explanation of the behavior of beta-lactamases into the bacterial cell during an infection will be developed. These assays should help us to validate and set up proper interpretation criteria for susceptibility testing. In the project, successful clones carrying clinically important beta-lactam resistance mechanisms will be selected based on whole-genome sequencing data. Additionally, a proper infection model must be established, in order a sufficient number of bacteria to be obtained. Methods for proper fixation and transport of the bacteria will be developed and validated. Thus, these techniques will enable us to better understand the pathogenesis of beta-lactamase-producing bacteria.
- MeSH
- beta-laktamasy MeSH
- gramnegativní bakteriální infekce mikrobiologie MeSH
- hmotnostní spektrometrie metody MeSH
- lidé MeSH
- mikrobiologické techniky MeSH
- mikrobiota MeSH
- mnohočetná bakteriální léková rezistence MeSH
- modely nemocí na zvířatech MeSH
- prasata MeSH
- sekvenování celého genomu metody MeSH
- sepse mikrobiologie MeSH
- spektrální analýza metody MeSH
- Check Tag
- lidé MeSH
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- infekční lékařství
- farmacie a farmakologie
- mikrobiologie, lékařská mikrobiologie
- NLK Publikační typ
- závěrečné zprávy o řešení grantu AZV MZ ČR
The aim of this study was to report the characterization of the first mcr-positive Enterobacterales isolated from Czech hospitals. In 2019, one Citrobacter freundii and four Enterobacter isolates were recovered from Czech hospitals. The production of carbapenemases was examined by a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) imipenem hydrolysis assay. Additionally, bacteria were screened for the presence of carbapenemase-encoding genes and plasmid-mediated colistin resistance genes by PCR. To define the genetic units carrying mcr genes, the genomic DNAs of mcr-carrying clinical isolates were sequenced on the PacBio Sequel I platform. Results showed that all isolates carried blaVIM- and mcr-like genes. Analysis of whole-genome sequencing (WGS) data revealed that all isolates carried mcr-9-like alleles. Furthermore, the three sequence type 106 (ST106) Enterobacter hormaechei isolates harbored the blaVIM-1 gene, while the ST764 E. hormaechei and ST95 C. freundii included blaVIM-4 Analysis of plasmid sequences showed that, in all isolates, mcr-9 was carried on IncHI2 plasmids. Additionally, at least one multidrug resistance (MDR) region was identified in each mcr-9-carrying IncHI2 plasmid. The blaVIM-4 gene was found in the MDR regions of p48880_MCR_VIM and p51929_MCR_VIM. In the three remaining isolates, blaVIM-1 was localized on plasmids (∼55 kb) exhibiting repA-like sequences 99% identical to the respective gene of pKPC-CAV1193. In conclusion, to the best of our knowledge, these 5 isolates were the first mcr-9-positive bacteria of clinical origin identified in the Czech Republic. Additionally, the carriage of the blaVIM-1 on pKPC-CAV1193-like plasmids is described for the first time. Thus, our findings underline the ongoing evolution of mobile elements implicated in the dissemination of clinically important resistance determinants.IMPORTANCE Infections caused by carbapenemase-producing bacteria have led to the revival of polymyxins as the "last-resort" antibiotic. Since 2016, several reports describing the presence of plasmid-mediated colistin resistance genes, mcr, in different host species and geographic areas were published. Here, we report the first detection of Enterobacterales carrying mcr-9-like alleles isolated from Czech hospitals in 2019. Furthermore, the three ST106 Enterobacter hormaechei isolates harbored blaVIM-1, while the ST764 E. hormaechei and ST95 Citrobacter freundii isolates included blaVIM-4 Analysis of WGS data showed that, in all isolates, mcr-9 was carried on IncHI2 plasmids. blaVIM-4 was found in the MDR regions of IncHI2 plasmids, while blaVIM-1 was localized on pKPC-CAV1193-like plasmids, described here for the first time. These findings underline the ongoing evolution of mobile elements implicated in dissemination of clinically important resistance determinants. Thus, WGS characterization of MDR bacteria is crucial to unravel the mechanisms involved in dissemination of resistance mechanisms.
- MeSH
- antibakteriální látky farmakologie terapeutické užití MeSH
- bakteriální proteiny genetika MeSH
- beta-laktamasy genetika MeSH
- Enterobacter genetika izolace a purifikace MeSH
- lidé MeSH
- nemocnice MeSH
- plazmidy genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- multicentrická studie MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
INTRODUCTION: Tuberculosis is considered one of the most fatal diseases worldwide, with an estimation of 10.1 million cases. In this study, whole-genome sequencing was used to determine the genomic characterisation of 40 Mycobacterium tuberculosis isolates from patients with different nationalities hospitalised in the Czech Republic. MATERIALS AND METHODS: Susceptibility testing for first-line drugs was performed. DNA was sequenced using the Illumina MiSeq platform. Spoligotype single-nucleotide polymorphisms and mutations in antibiotic-resistant genes were detected, and phylogenetic analysis was performed. RESULTS: Samples showing phenotypic resistance to at least one drug were 12 to streptomycin, 11 to isoniazid, 7 to rifampicin, 6 to ethambutol and 5 to pyrazinamide. Phenotypic and genotypic profiles did not match in all cases, suggesting the presence of a novel mutation in some cases and a low expression of resistant genes in others. The presented phylogeny enables the correct assignation of M. tuberculosis lineages and sublineages. Our results suggest that the most dominant lineage in our samples was lineage 4 (33/40). CONCLUSION: To our knowledge, this is the first study using this approach to be done in the Czech Republic. Lineage 4 was the predominant lineage identified among our samples. Nevertheless, the dominance of Lineage 4 along with other lineages suggests that infections can originate from different sources.
- MeSH
- antituberkulotika * farmakologie MeSH
- fylogeneze MeSH
- lidé MeSH
- mnohočetná bakteriální léková rezistence * genetika MeSH
- multirezistentní tuberkulóza * MeSH
- mutace MeSH
- Mycobacterium tuberculosis * genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
Background: The spread of carbapenemase genes, such as blaNDM-1, in Proteus mirabilis poses a public health threat. The aim of the study was to characterize the genome and plasmids sequences of an NDM-1-positive strain (IBCRE14), which was isolated in 2019 from a catheterized patient hospitalized in Italy. Methods: Whole genome sequencing (WGS) of IBCRE14 was performed on extracted genomic DNA using Sequel I platform. Genome assembly was performed using "Microbial Assembly". Genomic analysis was conducted by uploading the contigs to ResFinder and PlasmidFinder databases from the Center for Genomic Epidemiology. Results: IBCRE14 had a genome size of 4,018,329 bp and harboured genes coding for resistance to aminoglycosides (aadA1), phenicol (cat), tetracycline (tetJ), and trimethoprim (dfrA1). A large plasmid (pIB_NDM_1) harboured antibiotic resistance genes against sulphonamide (sul1), trimethoprim (dfrA14), tetracycline (tetB), rifampicin (arr-2), aminoglycosides (aadA1, aph3-VI), and beta-lactams (blaOXA-10, blaNDM-1). Furthermore, a small plasmid (pIB_COL3M) harboured a qnrD1 gene coding for quinolone resistance. Conclusion: The ability to conjugate and the presence of a composite antibiotic resistance island suggests that pIB_NDM_1 could both acquire more resistance genes and easily disseminate. To our knowledge, this is the first report on an untypable plasmid harbouring blaNDM-1 in P. mirabilis, in Italy.
- Publikační typ
- časopisecké články MeSH
Here, we describe two plasmids carrying mcr-4.3 in two Acinetobacter baumannii strains isolated from imported food and a clinical sample. The comparative analysis of these plasmids, with two other plasmids reported in the NCBI database, highlighted the common origin of the plasmidic structure carrying mcr-4.3 This is the first case of the mcr-4.3 gene in a A. baumannii strain isolated from a clinical case in Europe. We hypothesize that food import is initiating the spread in Czech Republic.
- MeSH
- Acinetobacter baumannii genetika MeSH
- bakteriální léková rezistence genetika MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- peptidy genetika MeSH
- plazmidy genetika MeSH
- senioři nad 80 let MeSH
- Check Tag
- lidé MeSH
- senioři nad 80 let MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
- Evropa MeSH
OBJECTIVES: Contamination of fresh water with clinically important Gram-negative bacteria in Lebanon is being investigated in-depth, especially with evidence of dissemination into clinical settings. This study aimed to report the draft genome sequence of a Klebsiella pneumoniae strain with an integrated plasmid segment harbouring two antibiotic resistance islands (ARI). It is believed that this is the first report of plasmid antibiotic resistance islands integration in the genome of K. pneumoniae. METHODS: Whole genome sequencing of the isolate was performed using Sequel platform. The genome was assembled using HGAP4. Analysis was conducted by uploading the sequence to the online databases from the Center for Genomic Epidemiology. RESULTS: The strain had a newly assigned ST 3483 with a genome size of 5385844 bp. The investigation of the antibiotic resistance islands suggested integration of two DNA segments from a previously identified IncFIA plasmid. The results revealed that the integration could have been accomplished either as a single-step integration event, with the two segments being integrated as a whole transposon mediated by the flanking IS26, or through two separate integration events involving the two segments, but independently. CONCLUSION: The sequenced genome revealed interesting aspects related to antibiotic resistance dissemination. The ARI are more stable in the genome and the chance of losing it is less probable, with the possibility of the described transposon to re-integrate in other plasmids, facilitating the dissemination of such resistance determinants.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální léková rezistence genetika MeSH
- beta-laktamasy genetika MeSH
- délka genomu MeSH
- DNA bakterií genetika MeSH
- genom bakteriální MeSH
- genomové ostrovy genetika MeSH
- infekce bakteriemi rodu Klebsiella mikrobiologie MeSH
- Klebsiella pneumoniae genetika MeSH
- mikrobiologie vody MeSH
- odpadní vody mikrobiologie MeSH
- plazmidy genetika MeSH
- sekvenční analýza DNA MeSH
- sekvenování celého genomu MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Libanon MeSH
We report two KPC-producing Citrobacter freundii isolates from unrelated patients. In one case, blaKPC-2 was harbored on a novel variant of a Tn4401 transposon of an IncN plasmid conjugated together with a coresident IncA plasmid, whereas in the other one, blaKPC-3 was on a Tn4401a transposon located on an IncX3-IncA self-conjugative plasmid fusion. The interplay among plasmids carrying blaKPC and the coresident IncA plasmids offers new information on plasmids coresident within clinically relevant enterobacteria.
