Analysis of the genetic distribution among members of Clostridium botulinum group I using a novel multilocus sequence typing (MLST) assay
Jaran Strand OlsenHolger C. ScholzSilvia FilloVincent RamisseFlorigio ListaAnette Kjoshagen TrømborgTone AarskaugIngjerd ThraneJan Blatný
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Multilocus sequence typing
Clostridium botulinum
Botulinum neurotoxin
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Globally, over 800 000 children under five die each year from infectious diseases caused by Streptococcus pneumoniae. To understand genetic relatedness between isolates, study transmission routes, assess the impact of human interventions e.g. vaccines, and determine infection sources, genotyping methods are required. The 'gold standard' genotyping method, Multi-Locus Sequence Typing (MLST), is useful for long-term and global studies. Another genotyping method, Multi-Locus Variable Number of Tandem Repeat Analysis (MLVA), has emerged as a more discriminatory, inexpensive and faster technique; however there is no universally accepted method and it is currently suitable for short-term and localised epidemiology studies. Currently Australia has no national MLST database, nor has it adopted any MLVA method for short-term or localised studies. This study aims to improve S. pneumoniae genotyping methods by modifying the existing MLVA techniques to be more discriminatory, faster, cheaper and technically less demanding than previously published MLVA methods and MLST.Four different MLVA protocols, including a modified method, were applied to 317 isolates of serotyped invasive S. pneumoniae isolated from sterile body sites of Queensland children under 15 years from 2007-2012. MLST was applied to 202 isolates for comparison.The modified MLVA4 is significantly more discriminatory than the 'gold standard' MLST method. MLVA4 has similar discrimination compared to other MLVA techniques in this study). The failure to amplify particular loci in previous MLVA methods were minimised in MLVA4. Failure to amplify BOX-13 and Spneu19 were found to be serotype specific.We have modified a highly discriminatory MLVA technique for genotyping Queensland invasive S. pneumoniae. MLVA4 has the ability to enhance our understanding of the pneumococcal epidemiology and the changing genetics of the pneumococcus in localised and short-term studies.
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This study describes the comparison of three methods for genotyping of Mycobacterium tuberculosis, namely MIRU-VNTR (mycobacterial interspersed repetitive units-variable number of tandem repeats), spoligotyping and, for the first time, MLST (Multilocus Sequence Typing). In order to evaluate the discriminatory power of these methods, a total of 44 M. tuberculosis isolates obtained from sputum specimens of patients from Brazil were genotyped. Among the three methods, MLST showed the lowest discriminatory power compared to the other two techniques. MIRU-VNTR showed better discriminatory power when compared to spoligotyping, however, the combination of both methods provides the greatest level of discrimination and therefore this combination is the most useful genotyping tool to be applied to M. tuberculosis isolates.
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Many molecular typing methods have been employed as major tools in epidemiological investigation for identifying clonal relatedness of P.aeruginosa isolates. Having own application points and principles they have certain advantages and disadvantages. The aim of this study was the estimation of discriminatory power and concordance between four different typing methods: PFGE, MLVA, MLST and wgMLST. 58 P.aeruginosa genome were analyzed in silico to determine PFGE, MLVA, MLST and wgMLST types and their cluster/clonal complexes. These data were estimated in term of discriminatory power and concordance. All four typing approaches demonstrated high resolution power (Simpson's ID): wgMLST (1.0), PFGE (0.999), MLVA (0.997) and MLST (0.983). Concordance between PFGE, MLVA, MLST was weak/moderate and was no more 74.1%. WgMLST demonstrated high concordance between wgMLST clusters and clonal complex/groups/clusters determined by MLST (AR=0.938), PFGE (AR=0.952) and MLVA (AR=0.798) typing methods after choosing appropriate cut-off value for wgMLST. wgMLST with more than 5100 target genes showed the highest index diversity and high concordance with other typing methods.
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Multilocus sequence typing
Clostridium botulinum
Botulinum neurotoxin
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Molecular typing of Mycoplasma pneumoniae is an important tool for identifying grouped cases and investigating outbreaks. In the present study, we developed a new genotyping method based on single nucleotide polymorphisms (SNPs) selected from the whole-genome sequencing of eight M. pneumoniae strains, using the SNaPshot minisequencing assay. Eight SNPs, localized in housekeeping genes, predicted lipoproteins, and adhesin P1 genes were selected for genotyping. These SNPs were evaluated on 140 M. pneumoniae clinical isolates previously genotyped by multilocus variable-number tandem-repeat analysis (MLVA-5) and adhesin P1 typing. This method was also adapted for direct use with clinical samples and evaluated on 51 clinical specimens. The analysis of the clinical isolates using the SNP typing method showed nine distinct SNP types with a Hunter and Gaston diversity index (HGDI) of 0.836, which is higher than the HGDI of 0.583 retrieved for the MLVA-4 typing method, where the nonstable Mpn1 marker was removed. A strong correlation with the P1 adhesin gene typing results was observed. The congruence was poor between MLVA-5 and SNP typing, indicating distinct genotyping schemes. Combining the results increased the discriminatory power. This new typing method based on SNPs and the SNaPshot technology is a method for rapid M. pneumoniae typing directly from clinical specimens, which does not require any sequencing step. This method is based on stable markers and provides information distinct from but complementary to MLVA typing. The combined use of SNPs and MLVA typing provides powerful discrimination of strains.
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AIM Comparative molecular-genetic typing of Brucella strains isolated in Mongolia from different animal species as well as from humans. MATERIALS AND METHODS Twenty-one strains of Brucella isolated from different hosts in 7 provinces of Mongolia were typed. Conventional phenotypic methods, genotyping by PCR with primers for genus- and species-specific differentiating targets of Brucella genes as well as multiple locus variable number tandem repeats analysis (MLVA) with 12 pairs of primers bounding locus variable tandem repeats of different length (from 134 bp to 8 bp). RESULTS Phenotypic identification and genotyping by PCR using primers for differentiating DNA markers allowed to attribute 14 isolates to B. melitensis biovar 2, and 7 - to B. abortus biovar 3. By using the MLVA method, connection of MLVA genotypes of 9 Brucella isolates with their reservoir hosts (sheep, cows) was shown providing their circulation in Khentii, Bulqan, and Khubsgul provinces bordering with Russia. Nine isolates from different hosts (camel, yaks, goats, sheep) isolated in Ovorkhangai, Dundgovi, and Dornogovi provinces, which have not border with Russia, had closely related MLVA genotypes indicating an opportunity of migration of pathogenic Brucella species to not-typical hosts. CONCLUSION Molecular-genetic typing of Brucella isolated in Mongolia was done for the first time; levels of their genetic relation and diversity were demonstrated. Circulation of Brucella isolated with specific MLVA genotypes was connected to territories of specific Mongolian provinces. The study proved migration of Brucella to not-typical hosts. Comparative study of isolates circulating in frontier with Mongolia areas of Russia (Irkutsk region, Tyva and Buryat Republics) are necessary to perform.
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Staphylococcus aureus is an important pathogen causing both hospital and community-acquired infections. S. aureus strains are often resistant to commonly used antibiotics, thus development of techniques to rapidly identify and genotype is essential in order to control the dissemination of multidrug resistant strains of S. aureus in the environment, which could lead to an epidemic. Typing methods can be divided into those based on phenotypic and those based on genetic characteristics of the examined microorganism. The most reliable genotypic methods in S. aureus epidemiological studies were described. These would include the following: Plasmid Profile Analysis (PPA), Pulsed-Field Gel Electrophoresis (PFGE), Multi-Locus Sequence Typing (MLST), Spa Typing, Random Amplified Polymorphic DNA (RAPD), Amplified-Fragment Length Polymorphism (AFLP), Restriction Fragment Length Polymorphism (RFLP), Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA) and Whole Genome Sequencing (WGS). All of these methods have been compared in terms of their reproducibility, costs, time of analysis, differentiation potential and exemplary applications in epidemiological studies of S. aureus. Despite there being a variety of methods currently used for genotyping, not all of them meet the criteria set by different kinds of epidemiological investigations. Because of the large amount of generated data and decrease of costs, the most popular methods used for S. aureus genotyping are based on sequencing techniques.
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Mycoplasma hyorhinis is a swine pathogen bacterium, which causes significant economic losses. The infection spreads through direct contact between the animals. Powerful genotyping methods like PCR based multi-locus sequence typing (MLST) and multiple-locus variable-number tandem-repeat analysis (MLVA) are necessary to monitor the infections and to conduct epidemiological investigations; hence supporting the control of the disease. The aims of the present study were to examine M. hyorhinis isolates originating mainly from Hungary with MLST and MLVA developed in the study, and to compare the results of the two typing methods. To characterize 39 M. hyorhinis isolates and the type strain (NCTC 10,130), six house-keeping genes were selected for MLST and six tandem-repeat regions were chosen for MLVA. We were able to differentiate 31 sequence types and 37 genotypes within the 40 analyzed isolates by the MLST and the MLVA, respectively. With the combination of the two newly developed assays all examined isolates were distinguished with the exception of the ones originating from the same animal. The developed MLST assay provided a robust and high resolution phylogenetic tree, while the MLVA system is suitable for the differentiation of closely related isolates from the same farm, hence the assay is appropriate for epidemiologic studies.
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