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Bioinformatics Describes Novel Loci for High Resolution Discrimination of Leptospira Isolates  [PDF]
Gustavo M. Cerqueira,Alan J. A. McBride,Rudy A. Hartskeerl,Niyaz Ahmed,Odir A. Dellagostin,Marcus R. Eslab?o,Ana L. T. O. Nascimento
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0015335
Abstract: Leptospirosis is one of the most widespread zoonoses in the world and with over 260 pathogenic serovars there is an urgent need for a molecular system of classification. The development of multilocus sequence typing (MLST) schemes for Leptospira spp. is addressing this issue. The aim of this study was to identify loci with potential to enhance Leptospira strain discrimination by sequencing-based methods.
Multilocus Sequence Typing (MLST) for Characterization of Enterobacter cloacae  [PDF]
Tohru Miyoshi-Akiyama, Kayoko Hayakawa, Norio Ohmagari, Masahiro Shimojima, Teruo Kirikae
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0066358
Abstract: Enterobacter cloacae is an important emerging pathogen, which sometime causes respiratory infection, surgical site infection, urinary infection, sepsis, and outbreaks at neonatal units. We have developed a multilocus sequence typing (MLST) scheme utilizing seven housekeeping genes and evaluated the performance in 101 clinical isolates. The MLST scheme yielded 83 sequence types (ST) including 78 novel STs found in the clinical isolates. These findings supported the robustness of the MLST scheme developed in this study.
Conservation of the S10-spc-α Locus within Otherwise Highly Plastic Genomes Provides Phylogenetic Insight into the Genus Leptospira  [PDF]
Berta Victoria, Ahmed Ahmed, Richard L. Zuerner, Niyaz Ahmed, Dieter M. Bulach, Javier Quinteiro, Rudy A. Hartskeerl
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0002752
Abstract: S10-spc-α is a 17.5 kb cluster of 32 genes encoding ribosomal proteins. This locus has an unusual composition and organization in Leptospira interrogans. We demonstrate the highly conserved nature of this region among diverse Leptospira and show its utility as a phylogenetically informative region. Comparative analyses were performed by PCR using primer sets covering the whole locus. Correctly sized fragments were obtained by PCR from all L. interrogans strains tested for each primer set indicating that this locus is well conserved in this species. Few differences were detected in amplification profiles between different pathogenic species, indicating that the S10-spc-α locus is conserved among pathogenic Leptospira. In contrast, PCR analysis of this locus using DNA from saprophytic Leptospira species and species with an intermediate pathogenic capacity generated varied results. Sequence alignment of the S10-spc-α locus from two pathogenic species, L. interrogans and L. borgpetersenii, with the corresponding locus from the saprophyte L. biflexa serovar Patoc showed that genetic organization of this locus is well conserved within Leptospira. Multilocus sequence typing (MLST) of four conserved regions resulted in the construction of well-defined phylogenetic trees that help resolve questions about the interrelationships of pathogenic Leptospira. Based on the results of secY sequence analysis, we found that reliable species identification of pathogenic Leptospira is possible by comparative analysis of a 245 bp region commonly used as a target for diagnostic PCR for leptospirosis. Comparative analysis of Leptospira strains revealed that strain H6 previously classified as L. inadai actually belongs to the pathogenic species L. interrogans and that L. meyeri strain ICF phylogenetically co-localized with the pathogenic clusters. These findings demonstrate that the S10-spc-α locus is highly conserved throughout the genus and may be more useful in comparing evolution of the genus than loci studied previously.
Multilocus sequence typing method for identification and genotypic classification of pathogenic Leptospira species
Niyaz Ahmed, S Manjulata Devi, M de los á Valverde, P Vijayachari, Robert S Machang'u, William A Ellis, Rudy A Hartskeerl
Annals of Clinical Microbiology and Antimicrobials , 2006, DOI: 10.1186/1476-0711-5-28
Abstract: We for the first time report development of a robust MLST method for genotyping of Leptospira. Genotyping based on DNA sequence identity of 4 housekeeping genes and 2 candidate genes was analyzed in a set of 120 strains including 41 reference strains representing different geographical areas and from different sources. Of the six selected genes, adk, icdA and secY were significantly more variable whereas the LipL32 and LipL41 coding genes and the rrs2 gene were moderately variable. The phylogenetic tree clustered the isolates according to the genome-based species.The main advantages of MLST over other typing methods for leptospires include reproducibility, robustness, consistency and portability. The genetic relatedness of the leptospires can be better studied by the MLST approach and can be used for molecular epidemiological and evolutionary studies and population genetics.Leptospirosis is a zoonotic and an emerging infectious disease caused by the pathogenic Leptospira species and is identified in the recent years as a global public health problem because of its increased mortality and morbidity in different countries. Leptospirosis is frequently misdiagnosed as a result of its protean and non-specific presentation resembling many other febrile diseases, notably viral haemorrhagic fevers such as dengue [1]. There is, for certain, an underestimation of the leptospirosis problem due to lack of awareness and under-recognition through a lack of proper use of diagnostic tools.The common mode of transmission of the infection in humans is either by direct or indirect contact with the urine of infected animals and may lead to potential lethal disease. A unique feature of this organism is to parasitize in a wide variety of wild and domestic animals [2]. Traditionally, two species have been identified, i.e. Leptospira interrogans and L. biflexa for pathogenic and non-pathogenic leptospires, respectively. The serovar is the basic identifier, characterized on the basis of ser
A Dominant Clone of Leptospira interrogans Associated with an Outbreak of Human Leptospirosis in Thailand  [PDF]
Janjira Thaipadungpanit,Vanaporn Wuthiekanun,Wirongrong Chierakul,Lee D. Smythe,Wimol Petkanchanapong,Roongrueng Limpaiboon,Apichat Apiwatanaporn,Andrew T. Slack,Yupin Suputtamongkol,Nicholas J. White,Edward J. Feil,Nicholas P. J. Day,Sharon J. Peacock
PLOS Neglected Tropical Diseases , 2007, DOI: 10.1371/journal.pntd.0000056
Abstract: Background A sustained outbreak of leptospirosis occurred in northeast Thailand between 1999 and 2003, the basis for which was unknown. Methods and Findings A prospective study was conducted between 2000 and 2005 to identify patients with leptospirosis presenting to Udon Thani Hospital in northeast Thailand, and to isolate the causative organisms from blood. A multilocus sequence typing scheme was developed to genotype these pathogenic Leptospira. Additional typing was performed for Leptospira isolated from human cases in other Thai provinces over the same period, and from rodents captured in the northeast during 2004. Sequence types (STs) were compared with those of Leptospira drawn from a reference collection. Twelve STs were identified among 101 isolates from patients in Udon Thani. One of these (ST34) accounted for 77 (76%) of isolates. ST34 was Leptospira interrogans, serovar Autumnalis. 86% of human Leptospira isolates from Udon Thani corresponded to ST34 in 2000/2001, but this figure fell to 56% by 2005 as the outbreak waned (p = 0.01). ST34 represented 17/24 (71%) of human isolates from other Thai provinces, and 7/8 (88%) rodent isolates. By contrast, 59 STs were found among 76 reference strains, indicating a much more diverse population genetic structure; ST34 was not identified in this collection. Conclusions Development of an MLST scheme for Leptospira interrogans revealed that a single ecologically successful pathogenic clone of L. interrogans predominated in the rodent population, and was associated with a sustained outbreak of human leptospirosis in Thailand.
Optimized Multilocus Sequence Typing (MLST) Scheme for Trypanosoma cruzi  [PDF]
Patricio Diosque ,Nicolás Tomasini,Juan José Lauthier,Louisa Alexandra Messenger,María Mercedes Monje Rumi,Paula Gabriela Ragone,Anahí Maitén Alberti-D'Amato,Cecilia Pérez Brandán,Christian Barnabé,Michel Tibayrenc,Michael David Lewis,Martin Stephen Llewellyn,Michael Alexander Miles,Matthew Yeo
PLOS Neglected Tropical Diseases , 2014, DOI: 10.1371/journal.pntd.0003117
Abstract: Trypanosoma cruzi, the aetiological agent of Chagas disease possess extensive genetic diversity. This has led to the development of a plethora of molecular typing methods for the identification of both the known major genetic lineages and for more fine scale characterization of different multilocus genotypes within these major lineages. Whole genome sequencing applied to large sample sizes is not currently viable and multilocus enzyme electrophoresis, the previous gold standard for T. cruzi typing, is laborious and time consuming. In the present work, we present an optimized Multilocus Sequence Typing (MLST) scheme, based on the combined analysis of two recently proposed MLST approaches. Here, thirteen concatenated gene fragments were applied to a panel of T. cruzi reference strains encompassing all known genetic lineages. Concatenation of 13 fragments allowed assignment of all strains to the predicted Discrete Typing Units (DTUs), or near-clades, with the exception of one strain that was an outlier for TcV, due to apparent loss of heterozygosity in one fragment. Monophyly for all DTUs, along with robust bootstrap support, was restored when this fragment was subsequently excluded from the analysis. All possible combinations of loci were assessed against predefined criteria with the objective of selecting the most appropriate combination of between two and twelve fragments, for an optimized MLST scheme. The optimum combination consisted of 7 loci and discriminated between all reference strains in the panel, with the majority supported by robust bootstrap values. Additionally, a reduced panel of just 4 gene fragments displayed high bootstrap values for DTU assignment and discriminated 21 out of 25 genotypes. We propose that the seven-fragment MLST scheme could be used as a gold standard for T. cruzi typing, against which other typing approaches, particularly single locus approaches or systematic PCR assays based on amplicon size, could be compared.
A combined approach of VNTR and MLST analysis: improving molecular typing of Argentinean isolates of Leptospira interrogans
Caimi, Karina;Varni, Vanina;Melendez, Yamil;Koval, Ariel;Brihuega, Bibiana;Ruybal, Paula;
Memórias do Instituto Oswaldo Cruz , 2012, DOI: 10.1590/S0074-02762012000500011
Abstract: leptospirosis is an emerging infectious disease that has been identified as both a human and animal health problem worldwide. regular outbreaks associated with specific risk factors have been reported in argentina. however, there are no available data concerning the genetic population level for this pathogen. therefore, the aim of this work was to describe the genetic diversity of leptospira interrogans through the application of two molecular typing strategies: variable number of tandem repeats (vntr) and multilocus sequence typing (mlst). for this purpose, seven reference strains and 18 non-epidemiologically related isolates from diverse hosts and argentinean regions were analysed. among them, nine genotypes and seven sequence types (sts), including three unreported sts, were described using vntr and mlst, respectively. eburst analysis demonstrated that st37 was the most frequent and founder genotype of a clonal complex (ccs) containing stn1 and stn3, suggesting the importance of studying the serovars belonging to this cc in argentina. the data from maximum parsimony analysis, which combined both techniques, achieved intra-serovar discrimination, surmounted microscopic agglutination test discrepancies and increased the discriminatory power of each technique applied separately. this study is the first to combine both strategies for l. interrogans typing to generate a more comprehensive molecular genotyping of isolates from argentina in a global context.
Leptospira spp. strain identification by MALDI TOF MS is an equivalent tool to 16S rRNA gene sequencing and multi locus sequence typing (MLST)  [cached]
Rettinger Anna,Krupka Inke,Grünwald Karola,Dyachenko Viktor
BMC Microbiology , 2012, DOI: 10.1186/1471-2180-12-185
Abstract: Background In this study mass spectrometry was used for evaluating extracted leptospiral protein samples and results were compared with molecular typing methods. For this, an extraction protocol for Leptospira spp. was independently established in two separate laboratories. Reference spectra were created with 28 leptospiral strains, including pathogenic, non-pathogenic and intermediate strains. This set of spectra was then evaluated on the basis of measurements with well-defined, cultured leptospiral strains and with 16 field isolates of veterinary or human origin. To verify discriminating peaks for the applied pathogenic strains, statistical analysis of the protein spectra was performed using the software tool ClinProTools. In addition, a dendrogram of the reference spectra was compared with phylogenetic trees of the 16S rRNA gene sequences and multi locus sequence typing (MLST) analysis. Results Defined and reproducible protein spectra using MALDI-TOF MS were obtained for all leptospiral strains. Evaluation of the newly-built reference spectra database allowed reproducible identification at the species level for the defined leptospiral strains and the field isolates. Statistical analysis of three pathogenic genomospecies revealed peak differences at the species level and for certain serovars analyzed in this study. Specific peak patterns were reproducibly detected for the serovars Tarassovi, Saxkoebing, Pomona, Copenhageni, Australis, Icterohaemorrhagiae and Grippotyphosa. Analysis of the dendrograms of the MLST data, the 16S rRNA sequencing, and the MALDI-TOF MS reference spectra showed comparable clustering. Conclusions MALDI-TOF MS analysis is a fast and reliable method for species identification, although Leptospira organisms need to be produced in a time-consuming culture process. All leptospiral strains were identified, at least at the species level, using our described extraction protocol. Statistical analysis of the three genomospecies L. borgpetersenii, L. interrogans and L. kirschneri revealed distinctive, reproducible differentiating peaks for seven leptospiral strains which represent seven serovars. Results obtained by MALDI-TOF MS were confirmed by MLST and 16S rRNA gene sequencing.
A Rickettsiella Bacterium from the Hard Tick, Ixodes woodi: Molecular Taxonomy Combining Multilocus Sequence Typing (MLST) with Significance Testing  [PDF]
Andreas Leclerque, Regina G. Kleespies
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0038062
Abstract: Hard ticks (Acari: Ixodidae) are known to harbour intracellular bacteria from several phylogenetic groups that can develop both mutualistic and pathogenic relationships to the host. This is of particular importance for public health as tick derived bacteria can potentially be transmitted to mammals, including humans, where e.g. Rickettsia or Coxiella act as severe pathogens. Exact molecular taxonomic identification of tick associated prokaryotes is a necessary prerequisite of the investigation of their relationship to both the tick and possible vertebrate hosts. Previously, an intracellular bacterium had been isolated from a monosexual, parthenogenetically reproducing laboratory colony of females of the hard tick, Ixodes woodi Bishopp, and had preliminarily been characterized as a “Rickettsiella-related bacterium”. In the present molecular taxonomic study that is based on phylogenetic reconstruction from both 16 S ribosomal RNA and protein-encoding marker sequences complemented with likelihood-based significance testing, the bacterium from I. woodi has been identified as a strain of the taxonomic species Rickettsiella grylli. It is the first time that a multilocus sequence typing (MLST) approach based on a four genes comprising MLST scheme has been implemented in order to classify a Rickettsiella-like bacterium to this species. The study demonstrated that MLST holds potential for a better resolution of phylogenetic relationships within the genus Rickettsiella, but requires sequence determination from further Rickettsiella-like bacteria in order to complete the current still fragmentary picture of Rickettsiella systematics.
Multilocus Sequence Typing (MLST) for Lineage Assignment and High Resolution Diversity Studies in Trypanosoma cruzi  [PDF]
Matthew Yeo ,Isabel L. Mauricio,Louisa A. Messenger,Michael D. Lewis,Martin S. Llewellyn,Nidia Acosta,Tapan Bhattacharyya,Patricio Diosque,Hernan J. Carrasco,Michael A. Miles
PLOS Neglected Tropical Diseases , 2011, DOI: 10.1371/journal.pntd.0001049
Abstract: Background Multilocus sequence typing (MLST) is a powerful and highly discriminatory method for analysing pathogen population structure and epidemiology. Trypanosoma cruzi, the protozoan agent of American trypanosomiasis (Chagas disease), has remarkable genetic and ecological diversity. A standardised MLST protocol that is suitable for assignment of T. cruzi isolates to genetic lineage and for higher resolution diversity studies has not been developed. Methodology/Principal Findings We have sequenced and diplotyped nine single copy housekeeping genes and assessed their value as part of a systematic MLST scheme for T. cruzi. A minimum panel of four MLST targets (Met-III, RB19, TcGPXII, and DHFR-TS) was shown to provide unambiguous assignment of isolates to the six known T. cruzi lineages (Discrete Typing Units, DTUs TcI-TcVI). In addition, we recommend six MLST targets (Met-II, Met-III, RB19, TcMPX, DHFR-TS, and TR) for more in depth diversity studies on the basis that diploid sequence typing (DST) with this expanded panel distinguished 38 out of 39 reference isolates. Phylogenetic analysis implies a subdivision between North and South American TcIV isolates. Single Nucleotide Polymorphism (SNP) data revealed high levels of heterozygosity among DTUs TcI, TcIII, TcIV and, for three targets, putative corresponding homozygous and heterozygous loci within DTUs TcI and TcIII. Furthermore, individual gene trees gave incongruent topologies at inter- and intra-DTU levels, inconsistent with a model of strict clonality. Conclusions/Significance We demonstrate the value of systematic MLST diplotyping for describing inter-DTU relationships and for higher resolution diversity studies of T. cruzi, including presence of recombination events. The high levels of heterozygosity will facilitate future population genetics analysis based on MLST haplotypes.
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