V článku jsou prezentovány první výsledky charakterizace 29 invazivních kmenů Streptococcuspyogenes, izolovaných v České republice v první polovině roku 2003, metodou multilokusové sekvenačnítypizace (MLST). U žádného z 16 přítomných emm typů nebyla mezi kmeny našeho souboruzjištěna variabilita sekvenčních typů (ST). První výsledky MLST ukazují, že populace kmenůzpůsobujících závažná onemocnění v České republice je odlišná od kmenů izolovaných v zahraničí.U sedmi kmenů byly popsány nové sekvenční typy: ST134, ST308, ST336, 5T340, obsahující novékombinace známých alel a ST341 se třemi dosud nepopsanými alelami (gki 91, murI 65 a yqiL 60),zjištěný u třech kmenů. Nově popsané sekvenční typy byly registrovány v celosvětové databáziMLST S. pyogenes.
First results of multilocus sequence typing (MLST) for characterization of 29 invasive Streptococcuspyogenes strains isolated in the Czech Republic in the first half of 2003 are presented. None of 16emm types detected among the study strains showed sequence type (ST) variability. The MLSTresults are indicative of differences between the strains causing serious diseases in the CzechRepublic and those isolated in other countries. In seven strains, four new STs with known alleles innew combinations, ST134, ST308, ST336, ST340, and one new ST with three as yet undescribed alleles(gki 91, murI 65 and yqiL 60), ST341, were described. These newly described STs were submitted tothe web-based reference MLST database for S. pyogenes.
- MeSH
- Alleles MeSH
- Research Support as Topic MeSH
- Cloning, Molecular MeSH
- Humans MeSH
- Sequence Analysis MeSH
- Streptococcus pyogenes isolation & purification classification pathogenicity MeSH
- Check Tag
- Humans MeSH
- Publication type
- Database MeSH
- Review MeSH
- Comparative Study MeSH
- Geographicals
- Czech Republic MeSH
"Candidatus Neoehrlichia mikurensis" is the tick-borne agent of neoehrlichiosis, an infectious disease that primarily affects immunocompromised patients. So far, the genetic variability of "Ca. Neoehrlichia" has been studied only by comparing 16S rRNA genes and groEL operon sequences. We describe the development and use of a multilocus sequence analysis (MLSA) protocol to characterize the genetic diversity of clinical "Ca. Neoehrlichia" strains in Europe and their relatedness to other species within the Anaplasmataceae family. Six genes were selected: ftsZ, clpB, gatB, lipA, groEL, and 16S rRNA. Each MLSA locus was amplified by real-time PCR, and the PCR products were sequenced. Phylogenetic trees of MLSA locus relatedness were constructed from aligned sequences. Blood samples from 12 patients with confirmed "Ca. Neoehrlichia" infection from Sweden (n = 9), the Czech Republic (n = 2), and Germany (n = 1) were analyzed with the MLSA protocol. Three of the Swedish strains exhibited identical lipA sequences, while the lipA sequences of the strains from the other nine patients were identical to each other. One of the Czech strains had one differing nucleotide in the clpB sequence from the sequences of the other 11 strains. All 12 strains had identical sequences for the genes 16S rRNA, ftsZ, gatB, and groEL. According to the MLSA, among the Anaplasmataceae, "Ca. Neoehrlichia" is most closely related to Ehrlichia ruminantium, less so to Anaplasma phagocytophilum, and least to Wolbachia endosymbionts. To conclude, three sequence types of infectious "Ca. Neoehrlichia" were identified: one in the west of Sweden, one in the Czech Republic, and one spread throughout Europe.
- MeSH
- Anaplasmataceae classification genetics isolation & purification MeSH
- Genes, Essential MeSH
- Phylogeny MeSH
- Genetic Variation * MeSH
- Genotype * MeSH
- Anaplasmataceae Infections epidemiology microbiology MeSH
- Middle Aged MeSH
- Humans MeSH
- Molecular Epidemiology methods MeSH
- Multilocus Sequence Typing methods MeSH
- RNA, Ribosomal, 16S genetics MeSH
- Aged MeSH
- Cluster Analysis MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Czech Republic MeSH
- Germany MeSH
- Sweden MeSH
The MLST scheme currently used for Enterococcus faecium typing was designed in 2002 and is based on putative gene functions and Enterococcus faecalis gene sequences available at that time. As a result, the original MLST scheme does not correspond to the real genetic relatedness of E. faecium strains and often clusters genetically distant strains to the same sequence types (ST). Nevertheless, typing has a significant impact on the subsequent epidemiological conclusions and introduction of appropriate epidemiological measures, thus it is crucial to use a more accurate MLST scheme. Based on the genome analysis of 1,843 E. faecium isolates, a new scheme, consisting of 8 highly discriminative loci, was created in this study. These strains were divided into 421 STs using the new MLST scheme, as opposed to 223 STs assigned by the original MLST scheme. The proposed MLST has a discriminatory power of D = 0.983 (CI95% 0.981 to 0.984), compared to the original scheme's D = 0.919 (CI95% 0.911 to 0.927). Moreover, we identified new clonal complexes with our newly designed MLST scheme. The scheme proposed here is available within the PubMLST database. Although whole genome sequencing availability has increased rapidly, MLST remains an integral part of clinical epidemiology, mainly due to its high standardization and excellent robustness. In this study, we proposed and validated a new MLST scheme for E. faecium, which is based on genome-wide data and thus reflects the tested isolates' more accurate genetic similarity. IMPORTANCE Enterococcus faecium is one of the most important pathogens causing health care associated infections. One of the main reasons for its clinical importance is a rapidly spreading resistance to vancomycin and linezolid, which significantly complicates antibiotic treatment of infections caused by such resistant strains. Monitoring the spread and relationships between resistant strains causing severe conditions represents an important tool for implementing appropriate preventive measures. Therefore, there is an urgent need to establish a robust method enabling strain monitoring and comparison at the local, national, and global level. Unfortunately, the current, extensively used MLST scheme does not reflect the real genetic relatedness between individual strains and thus does not provide sufficient discriminatory power. This can lead directly to incorrect epidemiological measures due to insufficient accuracy and biased results.
- MeSH
- Anti-Bacterial Agents MeSH
- Enterococcus faecium * genetics MeSH
- Gram-Positive Bacterial Infections * epidemiology MeSH
- Humans MeSH
- Multilocus Sequence Typing methods MeSH
- Whole Genome Sequencing MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
BACKGROUND: Out of 20 spirochete species from Borrelia burgdorferi sensu lato (s.l.) complex recognized to date some are considered to have a limited distribution, while others are worldwide dispersed. Among those are Borrelia burgdorferi sensu stricto (s.s.) and Borrelia bissettii which are distributed both in North America and in Europe. While B. burgdorferi s.s. is recognized as a cause of Lyme borreliosis worldwide, involvement of B. bissettii in human Lyme disease was not so definite yet. FINDINGS: Multilocus sequence typing of spirochete isolates originating from residents of Georgia and Florida, USA, revealed the presence of two Borrelia burgdorferi sensu stricto strains highly similar to those from endemic Lyme borreliosis regions of the northeastern United States, and an unusual strain that differed from any previously described in Europe or North America. Based on phylogenetic analysis of eight chromosomally located housekeeping genes divergent strain clustered between Borrelia bissettii and Borrelia carolinensis, two species from the B.burgdorferi s.l. complex, widely distributed among the multiple hosts and vector ticks in the southeastern United States. The genetic distance analysis showed a close relationship of the diverged strain to B. bissettii. CONCLUSIONS: Here, we present the analysis of the first North American human originated live spirochete strain that revealed close relatedness to B. bissettii. The potential of B. bissettii to cause human disease, even if it is infrequent, is of importance for clinicians due to the extensive range of its geographic distribution.
- MeSH
- Borrelia burgdorferi Group MeSH
- Borrelia burgdorferi MeSH
- Borrelia classification genetics isolation & purification MeSH
- Genes, Essential MeSH
- Genotype MeSH
- Humans MeSH
- Lyme Disease diagnosis microbiology MeSH
- Molecular Sequence Data MeSH
- Multilocus Sequence Typing * MeSH
- Sequence Analysis, DNA MeSH
- Cluster Analysis MeSH
- Spirochaetales MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Florida MeSH
- Georgia MeSH
To date, genotyping data on giardiasis have not been available in the Czech Republic. In this study, we characterized 47 human isolates of Giardia intestinalis from symptomatic as well as asymptomatic giardiasis cases. Genomic DNA from trophozoites was tested by PCR-sequence analysis at three loci (β-giardin, glutamate dehydrogenase and triose phosphate isomerase). Sequence analysis showed assemblages A and B in 41 (87.2%) and six (12.8%) isolates, respectively. Two of the 41 assemblage A samples were genotyped as sub-assemblage AI, and 39 were genotyped as sub-assemblage AII. Four previously identified multilocus genotypes (MLGs: AI-1, AII-1, AII-4 and AII-9) and six likely novel variations of MLGs were found. In agreement with previous studies, sequences from assemblage B isolates were characterized by a large genetic variability and by the presence of heterogeneous positions, which prevent the definition of MLGs. This study also investigated whether there was a relationship between the assemblage and clinical data (including drug resistance). However, due to the large number of genotypes and the relatively small number of samples, no significant associations with the clinical data were found.
- MeSH
- Child MeSH
- Adult MeSH
- Genotype MeSH
- Giardia classification enzymology genetics MeSH
- Giardiasis parasitology MeSH
- Glutamate Dehydrogenase genetics MeSH
- Middle Aged MeSH
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Multilocus Sequence Typing MeSH
- Child, Preschool MeSH
- Protozoan Proteins genetics MeSH
- Sequence Analysis, DNA MeSH
- Triose-Phosphate Isomerase genetics MeSH
- Check Tag
- Child MeSH
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Male MeSH
- Child, Preschool MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Cronobacter spp. are bacterial pathogens that affect children and immunocompromised adults. In this study, we used multilocus sequence typing (MLST) to determine sequence types (STs) in 11 Cronobacter spp. strains isolated from retail foods, 29 strains from dust samples obtained from vacuum cleaners, and 4 clinical isolates. Using biochemical tests, species-specific polymerase chain reaction, and MLST analysis, 36 strains were identified as Cronobacter sakazakii, and 6 were identified as Cronobacter malonaticus. In addition, one strain that originated from retail food and one from a dust sample from a vacuum cleaner were identified on the basis of MLST analysis as Cronobacter dublinensis and Cronobacter turicensis, respectively. Cronobacter spp. strains isolated from the retail foods were assigned to eight different MLST sequence types, seven of which were newly identified. The strains isolated from the dust samples were assigned to 7 known STs and 14 unknown STs. Three clinical isolates and one household dust isolate were assigned to ST4, which is the predominant ST associated with neonatal meningitis. One clinical isolate was classified based on MLST analysis as Cronobacter malonaticus and belonged to an as-yet-unknown ST. Three strains isolated from the household dust samples were assigned to ST1, which is another clinically significant ST. It can be concluded that Cronobacter spp. strains of different origin are genetically quite variable. The recovery of C. sakazakii strains belonging to ST1 and ST4 from the dust samples suggests the possibility that contamination could occur during food preparation. All of the novel STs and alleles for C. sakazakii, C. malonaticus, C. dublinensis, and C. turicensis determined in this study were deposited in the Cronobacter MLST database available online ( http://pubmlst.org/cronobacter/).
- MeSH
- Bacterial Proteins genetics metabolism MeSH
- Cronobacter sakazakii classification isolation & purification metabolism MeSH
- Cronobacter classification isolation & purification metabolism MeSH
- Enterobacteriaceae Infections microbiology MeSH
- Phylogeny MeSH
- Humans MeSH
- Environmental Microbiology * MeSH
- Molecular Typing MeSH
- Multilocus Sequence Typing MeSH
- Reverse Transcriptase Polymerase Chain Reaction MeSH
- Food Microbiology * MeSH
- Dust * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Czech Republic MeSH
Syphilis is an important public health problem and an increasing incidence has been noted in recent years. Characterization of strain diversity through molecular data plays a critical role in the epidemiological understanding of this re-emergence. We here propose a new high-resolution multilocus sequence typing (MLST) scheme for Treponema pallidum subsp. pallidum (TPA). We analyzed 30 complete and draft TPA genomes obtained directly from clinical samples or from rabbit propagated strains to identify suitable typing loci and tested the new scheme on 120 clinical samples collected in Switzerland and France. Our analyses yielded three loci with high discriminatory power: TP0136, TP0548, and TP0705. Together with analysis of the 23S rRNA gene mutations for macrolide resistance, we propose these loci as MLST for TPA. Among clinical samples, 23 allelic profiles as well as a high percentage (80% samples) of macrolide resistance were revealed. The new MLST has higher discriminatory power compared to previous typing schemes, enabling distinction of TPA from other treponemal bacteria, distinction between the two main TPA clades (Nichols and SS14), and differentiation of strains within these clades.
- MeSH
- Alleles MeSH
- Anti-Bacterial Agents pharmacology MeSH
- DNA, Bacterial genetics MeSH
- Phylogeny MeSH
- Genome, Bacterial MeSH
- Genotype MeSH
- Globus Pallidus MeSH
- Polymorphism, Single Nucleotide MeSH
- Macrolides pharmacology MeSH
- Multilocus Sequence Typing methods MeSH
- RNA, Ribosomal, 23S genetics MeSH
- Sequence Analysis, DNA methods MeSH
- Syphilis epidemiology MeSH
- Treponema pallidum genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- France MeSH
- Switzerland MeSH
Cystic fibrosis (CF) patients in the Czech Republic suffered in the late 1990s from an epidemic with ST32 strain of Burkholderia cepacia complex (Bcc). Cohort segregation of Bcc and of ST32 positive patients was introduced in 1999 and 2002, respectively. We performed a study to evaluate the molecular epidemiology of Bcc infection after implementation of these infection control measures. Patients attending the Prague CF adult Centre from 2000 to 2015 were included in the present study. Demographic data and microbial statuses were collected from patient records. All Bcc isolates were analyzed using multilocus sequence typing (MLST). The prevalences of epidemic strain ST32 and of other Bcc strains were calculated. Ninety out of 227 CF patients were infected with Bcc during the study period. The prevalence of ST32 cases significantly decreased from 46.5% in 2000-2001 to 10.4% in 2014-2015 (P < 0.001) due to occurrence of only one new case in 2003, as well as to the death of 72% of ST32-infected patients. Conversely, there was a significant increase in prevalence of other Bcc strains, which rose from 0 to 14.9% (P = 0.015) and of transient infections. A micro-epidemic of infection with ST630 strain was observed in 2014 in lung transplant patients hospitalized in intensive care unit. The prevalence of epidemic strain ST32 decreased, whereas that of non-clonal strains of Bcc increased. Routine use of MLST allowed early detection of new and potentially epidemic strains.
- MeSH
- Burkholderia cepacia complex classification genetics isolation & purification MeSH
- Cystic Fibrosis epidemiology microbiology MeSH
- Burkholderia Infections epidemiology microbiology MeSH
- Humans MeSH
- Molecular Epidemiology MeSH
- Multilocus Sequence Typing MeSH
- Prevalence MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Czech Republic epidemiology MeSH
BACKGROUND: The increased prevalence of syphilis in Cuba prompted us to map the circulating Treponema pallidum subsp. pallidum allelic profiles in this geographic region. METHODS: Samples were collected from 2012 to 2017, from 83 male patients with ulcers or skin lesions, and were examined using multilocus sequence typing. Additionally, we analyzed the 23S rDNA and 16S rDNA regions for the presence of possible mutations leading to macrolide and tetracycline resistance. RESULTS: Among 94% of fully typed strains, we found 7 different allelic profiles, of which 4 had not been previously described. More than 87% of patients were infected with the T. pallidum SS14-like group and only 8.2% with T. pallidum Nichols-like group. As in other countries, the 1.3.1 allelic profile (ie, SS14-like) was the most common. In addition, 1 of the newly described allelic profiles represents T. pallidum strains that arose by recombination events between members of different T. pallidum subgroups. More than 90% of patients were infected with treponemes harboring the A2058G mutation. However, we found no potential tetracycline-resistant T. pallidum mutations. CONCLUSIONS: Our results suggest that, in Cuba, tetracycline antibiotics could be used to treat syphilis in penicillin-allergic patients instead of macrolides.
- MeSH
- Alleles MeSH
- Anti-Bacterial Agents MeSH
- Drug Resistance, Bacterial genetics MeSH
- Adult MeSH
- Humans MeSH
- Macrolides therapeutic use MeSH
- Multilocus Sequence Typing MeSH
- Mutation MeSH
- Syphilis drug therapy microbiology MeSH
- Bacterial Typing Techniques MeSH
- Tetracycline therapeutic use MeSH
- Treponema pallidum classification genetics MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Male MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Cuba MeSH
