Cíl práce: Zjištění klonálních charakteristik souboru kmenů Streptococcus pneumoniae (S. pneumoniae) působících invazivní pneu-mokokové onemocnění (IPO) v České republice (ČR) v roce 2017. Porovnání klonálního členění kmenů probíhalo v Národní referenční laboratoři pro streptokokové nákazy (NRL) použitím rutinně využívané metody multilokusové sekvenační typizace (Multilocus Sequence Typing, MLST) a nově zavedené metody multilokusové analýzy tandemových repetic (Multiple-Locus Variable number tandem repeat Analysis, MLVA). Materiál a metodika: Studován byl soubor 87 kmenů S. pneumoniae, vybraný z izolátů doručených v rámci programu surveillance IPO v roce 2017 do NRL z celé ČR. Výběr byl veden přes sérotypy S. pneumoniae tak, aby soubor zahrnoval izoláty sérotypů zastoupených v pneumokokové konjugované 13valentní vakcíně (sérotypy 1, 4, 9V) a izoláty sérotypů nevakcinačních, vyskytujících se však v ČR ve značném zastoupení (sérotypy 8, 9N, 22F). Pro studium byla použita metoda MLST, která je světově rozšířeným standardem klonální charakterizace pneumokokových izolátů a využívá sekvenování souboru genových oblastí a metoda MLVA, která charakterizuje izoláty podle počtu tandemově repetitivních úseků v intergenových oblastech. Výsledky: MLST analýza ukázala a potvrdila vysokou míru klonální homogenity izolátů S. pneumoniae sérotypů 1, 9N, 9V, 22F a značnou genetickou proměnlivost izolátů sérotypů 4 a 8. Mezi klonálním členěním, poskytnutým oběma metodami, byla rámcová korelace. V porovnání s metodou MLST poskytovala metoda MLVA detailnější klonální odlišení. U izolátů s nově zjištěnými MLVA profily je žádoucí přiřazení nového MLVA typu (MT). Tato webová podpora MLVA schématu S. pneumonie však ustupuje do pozadí a neposkytuje aktuálně všechny služby oproti webové podpoře pro MLST charakterizaci. Závěry: Metoda MLST je a nadále zůstává u izolátů S. pneumoniae standardem při klonální charakterizaci původců IPO při provádění surveillance na úrovni místní i mezinárodní. MLST charakteristiky izolátů jsou využity při sledování klonální proměnlivosti národními i nadnárodními orgány ochrany veřejného zdraví. Metoda MLVA rutinně používána není, lze ji však použít jako doplňkovou pro zrychlené zjištění klonální příbuznosti izolátů ze situací lokálního charakteru, například ohniskových výskytů infekce. Zde může snáze zachytit vznik a šíření virulentní klonální varianty.
Aim: To determine clonal characteristics of Streptococcus pneumoniae (S. pneumoniae) strains causing invasive pneumococcal disease (IPD) in the Czech Republic (CR) in 2017. Clonal assignment of strains was performed in the National Reference Laboratory for Streptococcal Infections (NRL) by the routinely used method, multilocus sequence typing (MLST), and a newly introduced method, multiple-locus variable number tandem repeat analysis (MLVA). Material and method: The study strains were 87 isolates of S. pneumoniae selected from those referred to the NRL within the IPD surveillance programme from all over the CR in 2017. The study set covers S. pneumoniae isolates of both pneumococcal 13-valent conjugate vaccine serotypes (1, 4, and 9V) and non-vaccine serotypes (8, 9N, and 22F) widely spread in the CR. The study methods were MLST, the standard method used worldwide for the characterisation of pneumococcal isolates based on sequencing of a set of gene regions, and MLVA, which allows to characterise isolates based on the number of tandem repeats in intergenic regions. Results: MLST revealed and confirmed a high level of clonal homogeneity of S. pneumoniae isolates of serotypes 1, 9N, 9V, and 22F and a considerable genetic variability of serotype 4 and 8 isolates. There was a general correlation between the MLST and MLVA clonal complex assignments. In comparison with MLST, MLVA has superior clonal discriminatory power. Isolates with the newly determined MLVA profiles should be assigned to new MLVA types (MT). Nevertheless, the new web support of the MLVA scheme for S. pneumoniae is less relevant as it does not provide services comparable to those available from the web support for MLST characterisation. Conclusions: MLST continues to be the standard method for clonal characterisation of S. pneumoniae isolates from IPD for the purposes of both national and international surveillance. MLST characteristics of isolates are helpful in the study of clonal variability conducted by both national and transnational public health protection authorities. MLVA is not routinely used but can serve as a complementary method for rapid identification of clonal relatedness between isolates, e.g. those from local outbreaks. It is more suitable for the detection of emergence and spread of a virulent clonal variant.
Pro klonální analýzu kmenů Streptococcus pneumoniae z invazivního pneumokokového onemocnění byla v NRLpro streptokokové nákazy SZÚ zavedena a ověřena metoda Multiple-Locus Variable number tandem repeat Ana-lysis (MLVA). Oproti rutinně používané metodě Multilocus Sequence Typing (MLST) je výhodou metody MLVAsnadnost a rychlost provedení i nižší nákladnost. Rozlišení klonů je u obou metod obdobné. Standardem klonálníanalýzy pro molekulární surveillance původců závažných pneumokokových infekcí je však MLST, MLVA můžebýt doplňkem pro klonální porovnání izolátů z lokálních epidemiologických situací.
The NRL for Streptococcal Infections implemented and tested Multiple-Locus Variable number tandem repeatAnalysis (MLVA) for clonal analysis of Streptococcus pneumoniae strains from invasive pneumococcal disease.As compared to the routinely used Multilocus Sequence Typing (MLST) method, MLVA has the advantage of beingeasier, faster, and less expensive to perform. Both methods have similar resolution. Nevertheless, the standardmethod for clonal analysis in molecular surveillance of causative agents of severe pneumococcal infections isMLST, and MLVA can be used as a complementary method for clonal comparison of local isolates.
- MeSH
- DNA, Bacterial genetics MeSH
- Minisatellite Repeats genetics MeSH
- Multilocus Sequence Typing * methods MeSH
- Pneumococcal Infections diagnosis epidemiology MeSH
- Streptococcus pneumoniae * genetics isolation & purification MeSH
- Bacterial Typing Techniques methods MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Keywords
- Salmonella enteritidis fagotyp 8 MLVA profil 2-9-7-3-2,
- MeSH
- European Union MeSH
- Salmonella enteritidis * genetics MeSH
- Geographicals
- Czech Republic MeSH
Investigating genetically-structured diversity in pathogen populations over time is important to better understand disease maintenance and spread. Herd-level surveillance of Mycobacterium bovis genotypes (multi-locus VNTR analysis types, MLVA types) from all culture-confirmed bovine tuberculosis (TB) herd cases was undertaken in Northern Ireland (NI), generating an unparalleled, longitudinal, population-level 14-year survey for this pathogen. Across this population, 295 genetically-distinct M. bovis MLVA types were identified in the 19,717 M. bovis isolates surveyed. Of these, the most frequent was MLVA type 002 (23.0%); 151 MLVA types were represented more than once, in groups ranging from 2 to 4438 isolates. Only 23 MLVA types were isolated in all 14 years. Investigating inter-annual frequency of M. bovis MLVA types, examples of statistically-significant expansions (MLVA types 002, 004, 006, 009 and 027), contractions (MLVA types 001, 007 and 011) and maintenance (MLVA types 003 and 005) were disclosed, during a period of fluctuating bovine TB herd-level incidence at the NI scale. The fixed period frequency distribution of MLVA types remained highly right-skewed. Novel VNTR copy number variant MLVA types (N = 242; an average of 17 per annum) were identified throughout the survey. The MLVA type distribution in the landscape was not random; MLVA types showed statistically-significant geographical localization and strong spatial associations with Divisional Veterinary Office (DVO) regions. There was also evidence of differential risk of particular MLVA types across breeds (Holstein/Friesian vs. other), age-class, and sex and some evidence of an association between the number of animals testing positive for bovine TB during the disclosing test and particular MLVA types, although there was substantial variation.
- MeSH
- Breeding MeSH
- Genotyping Techniques veterinary MeSH
- Longitudinal Studies MeSH
- Minisatellite Repeats * MeSH
- Multilocus Sequence Typing veterinary MeSH
- Mycobacterium bovis classification genetics growth & development isolation & purification MeSH
- Risk Factors MeSH
- Cattle MeSH
- Population Surveillance MeSH
- Tuberculosis, Bovine diagnosis epidemiology MeSH
- DNA Copy Number Variations MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Cattle MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Northern Ireland MeSH
Cíl práce: Cílem práce je Multiple-locus variable number tandem repeat analýza (MLVA) souboru kmenů B. pertussis ze sbírky Národní referenční laboratoře pro pertusi a difterii (NRL/DIPE) Státního zdravotního ústavu (SZÚ) Praha. Jedná se o kmeny získané ze vzorků klinického materiálu převážně na území ČR v téměř padesátiletém období 1967–2015 (červen). Je porovnávána genetická diverzita a zastoupení detekovaných MLVA typů (MT) ve třech obdobích lišících se vakcinační strategií a trendem výskytu pertuse. Na podkladě získaných výsledků je posouzena vhodnost použití metody MLVA pro analýzu epidemických výskytů B. pertussis v ČR. Materiál a metodiky: Vzorky DNA extrahované z kmenů B. pertussis studovaného souboru byly analyzovány metodou MLVA podle standardizovaného postupu. Data byla zpracována pomocí algoritmu eBURST, pro statistické zpracování byl zvolen výpočet Simpsonova indexu diverzity (DI). Data byla zpracována pro celý soubor a též odděleně pro skupiny kmenů z tří období: 1967–1980, 1990–2007 a 2008–2015 (červen). Výsledky: V souboru bylo detekováno 14 různých MT, z toho 3 dosud nezjištěné. Nejčetnější byly v celém souboru MT27 a MT29. MT29 převládal v období 1967–1980, MT27 v obdobích 1990–2007 a 2008–2015 (červen). Hodnota DI byla nejnižší (0,49) pro období 2008–2015 (červen), pro dvě předchozí období byly hodnoty vyšší a vzájemně srovnatelné (0,667 pro období 1967–1980 a 0,654 pro období 1990–2007). Závěr: MLVA analýzou souboru kmenů B. pertussis izolovaných z klinického materiálu na území ČR v období 1967–2015 (červen) byl prokázán pokles genetické diverzity ve studované populaci a změny v zastoupení a četnosti jednotlivých MT. Tyto změny lze charakterizovat jako postupný nárůst výskytu celosvětově rozšířených MT v České republice na úkor MT regionálně unikátních. Převažujícím MT, stejně jako ve většině geografických oblastí s dlouhodobě proočkovanou populací, je MT27. Výsledky MLVA analýzy souboru 136 kmenů B. pertussis mohou být podkladem pro účelné využití metody k molekulárně epidemiologickému zpracování souborů menšího rozsahu.
Aim: To perform multiple-locus variable number tandem repeat analysis (MLVA) of B. pertussis strains from the collection of the National Reference Laboratory for Diphtheria and Pertussis (NRL/DIPE), National Institute of Public Health (NIPH), Prague. The study strains were isolated from clinical specimens collected mostly in the Czech Republic over a nearly 50-year period from 1967 to 2015 (June). The isolates from three periods characterized by different vaccination strategies and trends in pertussis are compared for genetic diversity and distribution of MLVA types (MT). Based on the results obtained, the suitability for use of MLVA in the analysis of epidemic outbreaks of B. pertussis in the Czech Republic is considered. Material and methods: DNA samples extracted from B. pertussis strains included in the present study were examined by MLVA using the standard protocol. Data were processed by means of the eBURST algorithm and the calculation of the Simpson diversity index (DI) was used for the statistical analysis. Data were analyzed as a whole and also separately for strains from the three periods: 1967–1980, 1990–2007, and 2008–2015 (June). Results: Fourteen different MT were detected in the study strains, with three of them not being reported before. The most common MTs were MT27 and MT29. MT29 was predominant in 1967–1980 while MT27 was the most prevalent in 1990–2007 and 2008–2015 (June). The DI was the lowest (0.49) in 2008–2015 (June), and comparably higher DIs were calculated for the two previous periods (i.e. 0.667 for 1967–1980 and 0.654 for 1990–2007). Conclusion: MLVA revealed a decrease in genetic diversity and shifts in MT distribution of B. pertussis strains isolated from clinical specimens in the Czech Republic from 1967 to 2015 (June). These shifts in the Czech Republic can be characterized as a progressive increase in global MTs at the expense of the locally unique ones. The most common MT, similarly to other geographical areas with long-term high vaccination coverage, is MT27. The results of MLVA of 136 B. pertussis strains can provide a background for using this method in molecular epidemiological analysis of smaller groups of strains.
- MeSH
- Vaccines, Acellular immunology MeSH
- Genes, Bacterial genetics MeSH
- Bordetella pertussis * genetics MeSH
- Time Factors MeSH
- DNA, Bacterial genetics MeSH
- Genetic Variation genetics MeSH
- Mass Vaccination MeSH
- Humans MeSH
- Minisatellite Repeats * genetics MeSH
- Multilocus Sequence Typing * MeSH
- Whooping Cough epidemiology microbiology prevention & control MeSH
- Pertussis Vaccine immunology MeSH
- Tandem Repeat Sequences genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Czech Republic MeSH
- Czechoslovakia MeSH
PURPOSE: To characterise and compare twenty-eight Finnish Clostridium difficile RT027-like isolates, selected based on the presence of 18 bp deletion in the tcdC gene and toxin gene profile (A, B, binary), with eleven RT027 isolates from different Finnish geographical areas and time periods. METHODS: Twenty-eight C. difficile RT027-like isolates and 11 RT027 comparative strains were characterised by capillary-electrophoresis (CE) ribotyping, multi-locus variable tandem-repeats analysis (MLVA), multi-locus sequence typing (MLST), and sequencing of tcdC and gyrA gene fragments. Susceptibility to moxifloxacin was determined by E-test. RESULTS: Of 28 RT027-like isolates, seven RTs (016, 034, 075, 080, 153, 176 and 328), three WEBRIBO types (411, 475, AI-78) and three new profiles (F1-F3) were identified. MLVA revealed six clonal complexes (RTs 016, 027, 176 and F3). MLST showed eleven sequence types (1, 41, 47, 67, 95, 191,192, 223, 229, 264 and new ST). Twenty-two isolates (RTs 016, 080, 176, 328, F1, F2, F3 and WRTAI-78) carried Δ117 in the tcdC gene. Isolates of RTs 016, 027 and 176 were moxifloxacin resistant and harboured Thr82Ile in the GyrA. CONCLUSION: Our results show a high diversity within 28 Finnish RT027-like C. difficile isolates, with twelve CE-ribotyping profiles and eleven STs. MLVA revealed the regional spread of RTs 016, 027, 176 and F3. The presence of Δ117 in the tcdC gene in eight non-027 RTs highlights the importance of careful interpretation of the results from molecular systems targeting this site in the genome of C. difficile and the need of strain typing for epidemiological purposes.
- MeSH
- ADP Ribose Transferases genetics MeSH
- Anti-Bacterial Agents pharmacology MeSH
- Drug Resistance, Bacterial MeSH
- Bacterial Proteins genetics MeSH
- Bacterial Toxins analysis genetics MeSH
- Clostridioides difficile classification drug effects genetics isolation & purification MeSH
- DNA, Bacterial analysis MeSH
- DNA Gyrase genetics MeSH
- Adult MeSH
- Enterotoxins genetics MeSH
- Fluoroquinolones pharmacology MeSH
- Genotype MeSH
- Clostridium Infections epidemiology microbiology MeSH
- Middle Aged MeSH
- Humans MeSH
- Microbial Sensitivity Tests MeSH
- Minisatellite Repeats genetics MeSH
- Young Adult MeSH
- Molecular Epidemiology MeSH
- Moxifloxacin MeSH
- Multilocus Sequence Typing methods MeSH
- Polymerase Chain Reaction methods MeSH
- Repressor Proteins genetics MeSH
- Ribotyping methods MeSH
- Base Sequence MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Bacterial Typing Techniques * MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Male MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Finland MeSH
In 2011-2012, a survey was performed in three regional hospitals in the Czech Republic to determine the incidence of Clostridium difficile infections (CDIs) and to characterize bacterial isolates. C. difficile isolates were characterized by PCR ribotyping, toxin genes detection, multiple-locus variable-number tandem-repeat analysis (MLVA), and antimicrobial susceptibility testing to fidaxomicin, vancomycin, metronidazole, clindamycin, LFF571, and moxifloxacin using agar dilution method. The incidence of CDI in three studied hospitals was 145, 146, and 24 cases per 100,000 inhabitants in 2011 and 177, 258, and 67 cases per 100,000 inhabitants in 2012. A total of 64 isolates of C. difficile was available for molecular typing and antimicrobial susceptibility testing. 60.9% of the isolates were classified as ribotype 176. All 41 isolates of ribotypes 176 and 078 were positive for the presence of binary toxin genes. Ribotype 176 also carried 18-bp deletion in the regulatory gene tcdC. Tested isolates of C. difficile were fully susceptible to vancomycin and metronidazole, whereas 65.1% of the isolates were resistant to moxifloxacin. MLVA results indicated that isolates from three different hospitals were genetically related, suggesting transmission between healthcare facilities.
- MeSH
- Anti-Bacterial Agents pharmacology MeSH
- Bacterial Toxins analysis genetics MeSH
- Clostridioides difficile classification genetics isolation & purification MeSH
- Disk Diffusion Antimicrobial Tests MeSH
- Incidence MeSH
- Clostridium Infections epidemiology microbiology MeSH
- Humans MeSH
- Minisatellite Repeats MeSH
- Molecular Typing * MeSH
- Hospitals MeSH
- Ribotyping MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Czech Republic epidemiology MeSH
AIM: To perform a retrospective analysis of the high occurrence of Clostridium difficile infection in the surgical department of a Czech tertiary care hospital and to identify weaknesses in C. difficile infection (CDI) prevention and control policies. METHODS: Clinical and epidemiological data on eleven CDI cases were collected. C. difficile isolates were characterized by capillary electrophoresis ribotyping, multilocus variable tandem repeat analysis (MLVA), gyrA gene fragment sequencing, and erm(B) fragment PCR amplification. Antibiotic susceptibility to metronidazole, vancomycin, ciprofloxacin, moxifloxacin, and clindamycin was tested. FINDINGS: Eleven CDI cases were caused by C. difficile PCR ribotype 001 strains. These strains revealed two different MLVA profiles with 11 tandem repeat differences. All isolates were susceptible to metronidazole and vancomycin and resistant to ciprofloxacin (MIC ≥32 mg/L), moxifloxacin (MIC ≥32 mg/L), and clindamycin (MIC ≥256 mg/L). All isolates revealed amino acid substitution Thr82Ile, in the GyrA and were erm(B) negative. CONCLUSION: Two fluoroquinolone and clindamycin-resistant C. difficile PCR ribotype 001 strain clusters occurred at one of the surgical departments of a tertiary care hospital. Ineffective decontamination with suboptimal concentration and time of exposure of sporicidal disinfectants may have resulted in C. difficile transmission.
- MeSH
- Anti-Bacterial Agents pharmacology MeSH
- Tertiary Care Centers MeSH
- Ciprofloxacin pharmacology MeSH
- Clostridioides difficile classification drug effects genetics isolation & purification MeSH
- DNA, Bacterial genetics metabolism MeSH
- DNA Gyrase genetics metabolism MeSH
- Electrophoresis, Capillary MeSH
- Gene Expression MeSH
- Fluoroquinolones pharmacology MeSH
- Clindamycin pharmacology MeSH
- Clostridium Infections drug therapy microbiology MeSH
- Humans MeSH
- Methyltransferases genetics metabolism MeSH
- Metronidazole pharmacology MeSH
- Microbial Sensitivity Tests MeSH
- Minisatellite Repeats MeSH
- Drug Resistance, Multiple, Bacterial genetics MeSH
- Multilocus Sequence Typing MeSH
- Retrospective Studies MeSH
- Ribotyping MeSH
- Aged MeSH
- Amino Acid Substitution MeSH
- Vancomycin pharmacology MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Czech Republic MeSH
We aimed to characterize Clostridioides difficile isolates cultured during a six-month single-center study from stool samples of patients with C. difficile infection (CDI) genotyped by the Xpert®C. difficile/Epi assay by polymerase chain reaction (PCR) ribotyping, toxin genes' detection and multi-locus variable number tandem repeats analysis (MLVA). The susceptibility to metronidazole, vancomycin and moxifloxacin was determined by agar dilution. In addition, the presence of Thr82Ile in the GyrA and a single nucleotide deletion at position (Δ117) in the tcdC gene were investigated. Between January 1 and June 30, 2016, of 114 CDIs, 75 cases were genotyped as presumptive PCR ribotype (RT) 027 infections using a commercial assay. C. difficile isolates cultured from presumptive RT027 stool samples belonged to RT176. These isolates carried genes for toxin A (tcdA), B (tcdB), binary (cdtA/B) and had Δ117 in the tcdC gene. Using MLVA, the 71/75 isolates clustered into two clonal complexes (CCs). Of these, 39 isolates (54.9%) were from patients hospitalized in acute care and 32 isolates (45.1%) were isolated from patients hospitalized in the long-term care department. All isolates were susceptible to metronidazole and vancomycin, and 105 isolates were resistant to moxifloxacin (92%) carrying Thr83Ile in the GyrA. An outbreak of RT176 CDIs, suspected as RT027, was recognized in a Slovakian hospital. In order to monitor the emergence and spread of RT027-variants, the identification of a presumptive RT027 CDI should be confirmed at a strain level by PCR ribotyping.
- Publication type
- Journal Article MeSH
Methicillin-resistant Staphylococcus aureus (MRSA) belonging to the multilocus sequence type clonal complex 59 (MLST CC59) is the predominant community-associated MRSA clone in Asia. This clone, which is primarily linked with the spa type t437, has so far only been reported in low numbers among large epidemiological studies in Europe. Nevertheless, the overall numbers identified in some Northern European reference laboratories have increased during the past decade. To determine whether the S. aureus t437 clone is present in other European countries, and to assess its genetic diversity across Europe, we analysed 147 S. aureus t437 isolates from 11 European countries collected over a period of 11 years using multiple locus variable number tandem repeat fingerprinting/analysis (MLVF/MLVA) and MLST. Additionally 16 S. aureus t437 isolates from healthy carriers and patients from China were included. Most isolates were shown to be monophyletic with 98% of the isolates belonging to the single MLVA complex 621, to which nearly all included isolates from China also belonged. More importantly, all MLST-typed isolates belonged to CC59. Our study implies that the European S. aureus t437 population represents a genetically tight cluster, irrespective of the year, country and site of isolation. This underpins the view that S. aureus CC59 has been introduced into several European countries, not being restricted to particular geographical regions or specific host environments. The European S. aureus t437 isolates thus bear the general hallmarks of a high-risk clone.
- MeSH
- Community-Acquired Infections epidemiology microbiology MeSH
- Humans MeSH
- Methicillin-Resistant Staphylococcus aureus classification genetics isolation & purification MeSH
- Minisatellite Repeats * MeSH
- Molecular Epidemiology MeSH
- Multilocus Sequence Typing * MeSH
- Staphylococcal Infections epidemiology microbiology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Asia MeSH
- Europe MeSH