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Genome-based phylogeny has been proposed to be more accurate than phylogeny based in a few genes as MLST-based phylogeny. However, much is not always better. Here we analyzed 368 complete genomes corresponding to 9 bacterial species in order to address intraspecific phylogeny. The studied species were: Burkholderia pseudomallei, Campylobacter jejuni, Chlamydia trachomatis, Helicobacter pylori, Klebsiella pneumoniae, Listeria monocytogenes, Salmonella enterica, Staphylococcus aureus and Streptococcus pyogenes. The intra-specific phylogenies were inferred using the complete genome sequences of different strains of these species and their MLST schemes. A supermatrix approach was used to infer maximum likelihood phylogenies in both cases. The phylogenetic incongruence between the supermatrix-based genome or MLST tree and individual trees (constructed from genome fragments or MLST genes, respectively) was analyzed. In supermatrix-based trees for genomes, most branches showed a high branch support; however, a high number of branches also showed high percentage of topologically incongruent individual trees. Interestingly, genome and MLST trees showed similar levels of incongruence in the phylogeny for each bacteria specie. Both genome and MLST approaches showed that C. trachomatis and S. aureus have a tree-like evolutionary history (low levels of internal incongruence). Instead, B. pseudomallei and S. pyogenes show high levels of incongruence (network-like evolutionary story) probably caused by HGT (horizontal gene transfer). Concluding, our analysis showed that: high branch supports obtained in genome phylogenies could be an artifact probably caused by data size; MLST is valid to address intraspecific phylogenetic structure; and, each species has its own evolutionary history, which could be affected by HGT to different extents., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Phylogenetic relationships among major lineages of Trypanosoma cruzi are still debatable. Particularly, it is controversial the origin of two main lineages: TcIII and TcIV. Some authors proposed that these lineages have been the result of an ancient hybridization between TcI and TcII, and this was one of the most accepted evolutionary models in the scientific community for several years. In the present paper we analyse several genomes of T. cruzi in order to examine if there is evidence supporting that TcIII is an ancient TcI/TcII hybrid. Our results show that TcIII is mainly related to TcI and not to TcII and there is few evidence of mosaicism for TcIII. Our results challenge the hypothesis of the ancient TcI/TcII hybridization., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Leishmaniasis is a vector-borne protozoan infection affecting over 350 million people around the world. In Argentina cutaneous leishmaniasis is endemic in nine provinces and visceral leishmaniasis is spreading from autochthonous transmission foci in seven provinces. However, there is limited information about the diversity of the parasite in this country. Implementation of molecular strategies for parasite typing, particularly multilocus sequence typing (MLST), represents an improved approach for genetic variability and population dynamics analyses. We selected six loci as candidates implemented in reference strains and Argentinean isolates. Phylogenetic analysis showed high correlation with taxonomic classification of the parasite. Autochthonous Leishmania (Viannia) braziliensis showed higher genetic diversity than L. (Leishmania) infantum but low support was obtained for intra-L. braziliensis complex variants suggesting the need of new loci that contribute to phylogenetic resolution for an improved MLST or nested-MLST scheme. This study represents the first characterization of genetic variability of Leishmania spp. in Argentina., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The transmission cycles of Trypanosoma cruzi in the Gran Chaco are complex networks involving domestic and wild components, whose interrelationships are not well understood. Knowing the circuit of transmission of the different Discrete Typing Units (DTUs) of T. cruzi in the complex environment of the Chaco region is relevant to understanding how the different components (reservoirs, vectors, ecotopes) interact. In the present study we identified the DTUs infecting humans and dogs in two rural areas of the Gran Chaco in Argentina, using molecular methods which avoid parasite culture. Blood samples of humans and dogs were typified by PCR-DNA blotting and hybridization assays with five specific DNA probes (TcI, TcII, TcIII, TcV and TcVI). PCR analyses were performed on seropositive human and dog samples and showed the presence of T. cruzi DNA in 41.7% (98/235) and 53% (35/66) samples, respectively. The identification of infective DTUs was determined in 83.6% (82/98) and 91.4% (32/35) in human and dog samples, respectively. Single infections (36.7% - 36/98) and a previously not detected high proportion of mixed infections (47.9% - 47/98) were found. In a 15.3% (15/98) of samples the infecting DTU was not identified. Among the single infections TcV was the most prevalent DTU (30.6% - 30/98) in human samples; while TcVI (42.8% - 15/35) showed the highest prevalence in dog samples. TcV/TcVI was the most prevalent mixed infection in humans (32.6% - 32/98); and TcI/TcVI (14.3% - 5/35) in dogs. Significant associations between TcV with humans and TcVI with dogs were detected. For the first time, the presence of TcIII was detected in humans from this region. The occurrence of one human infected whit TcIII (a principally wild DTU) could be suggested the emergence of this, in domestic cycles in the Gran Chaco., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Trypanosoma cruzi has been historically classified as a species with preponderant clonal evolution (PCE). However, with the advent of highly polymorphic markers and studies at geographically reduced scales, the PCE in T. cruzi was challenged. In fact, some studies have suggested that recombination in T. cruzi lineage I (TcI) is much more frequent than previously believed. Further analyses of TcI populations from different geographical regions of Latin America are needed to examine this hypothesis. In the present study, we contribute to this topic by analyzing the population structure of TcI from a restricted geographical area in the Chaco region, Argentina. We analyzed TcI isolates from different hosts and vectors using a Multilocus Sequence Typing (MLST) approach. These isolates were previously characterized by sequencing the spliced leader intergenic region (SL-IR). Low levels of incongruence and well-supported clusters for MLST dataset were obtained from the analyses. Moreover, high linkage disequilibrium was found and five repeated and overrepresented genotypes were detected. In addition, a good correspondence between SL-IR and MLST was observed which is expected under PCE. However, recombination is not ruled out because five out of 28 pairs of loci were incompatible with strict clonality and one possible genetic exchange event was detected. Overall, our results represent evidence of PCE in TcI from the study area. Finally, considering our findings we discuss the scenario for the genetic structure of TcI., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Leptospirosis is a neglected zoonosis of global importance. Several multilocus sequence typing (MLST) methods have been developed for Leptospira spp., the causative agent of leptospirosis. In this study we reassessed the most commonly used MLST schemes in a set of worldwide isolates, in order to select the loci that achieve the maximum power of discrimination for typing Leptospira spp. Global eBURST algorithm was used to detect clonal complexes among STs and phylogenetic relationships among concatenated and individual sequences were inferred through maximum likelihood (ML) analysis. The evaluation of 12 loci combined to type a subset of strains rendered 57 different STs. Seven of these loci were selected into a final scheme upon studying the number of alleles and polymorphisms, the typing efficiency, the discriminatory power and the ratio dN/dS per nucleotide site for each locus. This new 7-locus scheme was applied to a wider collection of worldwide strains. The ML tree constructed from concatenated sequences of the 7 loci identified 6 major clusters corresponding to 6 Leptospira species. Global eBURST established 8 CCs, which showed that genotypes were clearly related by geographic origin and host. ST52 and ST47, represented mostly by Argentinian isolates, grouped the higher number of isolates. These isolates were serotyped as serogroups Pomona and Icterohaemorrhagiae, showing a unidirectional correlation in which the isolates with the same ST belong to the same serogroup. In summary, this scheme combines the best loci from the most widely used MLST schemes for Leptospira spp. and supports worldwide strains classification. The Argentinian isolates exhibited congruence between allelic profile and serogroup, providing an alternative to serological methods., (Published by Elsevier B.V.)

Multi-locus sequence typing (MLST) is a frequently used genotyping method whose goal is the unambiguous assignment of microorganisms to genetic clusters. MLST typically involves analysis of DNA sequence results generated from several house-keeping gene loci. MLST remains the gold standard for molecular typing of many bacterial pathogens. Eukaryotic pathogens have also been the subject of MLST, however, few tools are available to deal with diploid sequence data. Here we present novel software for MLST data analysis tailored towards diploid Eukaryotes: MLSTest. This software meets various methods used in MLST and introduces some novel methodologies for the evaluation of the data set. In addition to construction of allelic profiles and basic clustering analysis, the MLSTest looks for network structures that suggest genetic exchange in BURST graphs. Additionally, it uses several simple methods for tree construction with the advantage of managing heterozygous or three-state sites. Additionally, the software analyses whether concatenation of fragments from different genes is suitable for the data set using different tests (bionj-incongruence length difference test, Templeton test). It evaluates how the incongruence is distributed across the tree using a variation of the localized incongruence length difference test based on a modified neighbour joining algorithm. We tested the last method in simulated datasets. We showed that is conservative (adequate type I error rate) and moderately to highly powerful as well as useful to localize incongruences in two bacterial and two eukaryotic MLST datasets. MLSTest was also designed for developing MLST schemes. It thus has tools to optimize locus combinations and to reduce the number of targets required for typing. MLSTest also analyses whether the discriminatory power of the typing scheme is increased by including more loci. We evaluated the software over simulated and real datasets from bacterial and eukaryotic microorganisms. The software is freely available at http://www.ipe.unsa.edu.ar/software., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A Multilocus Sequence Typing (MLST) scheme was designed and applied to a set of 20 Trypanosoma cruzi stocks belonging to three main discrete typing units (T. cruzi I, V and VI) from a geographically restricted Chagas disease endemic area in Argentina, 12 reference strains comprising two from each of the six main discrete typing units of the parasite (T. cruzi I-VI), and one T. cruzi marinkellei strain. DNA fragments (≅400-bp) from 10 housekeeping genes were sequenced. A total of 4178 bp were analyzed for each stock. In all, 154 polymorphic sites were identified. Ninety-five sites were heterozygous in at least one analyzed stock. Seventeen diploid sequence types were identified from 32 studied T. cruzi stocks (including the reference strains). All stocks were correctly assigned to their corresponding discrete typing units. We propose this MLST scheme as provisional, with scope for improvement by studying new gene targets on a more diverse sample of stocks, in order to define an optimized MLST scheme for T. cruzi. This approach is an excellent candidate to become the gold standard for T. cruzi genetic typing. We suggest that MLST will have a strong impact on molecular epidemiological studies of Chagas disease and the phylogenetics of its causative agent., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Internal and geographical clustering within Trypanosoma cruzi I (TcI) has been recently revealed by using Multilocus Microsatellite Typing and sequencing of the Spliced-Leader Intergenic Region (SL-IR). In the present work, 14 isolates and 11 laboratory-cloned stocks obtained from a geographically restricted area in Chaco Province, Argentina, were analyzed by PCR and sequencing of SL-IR. We were able to differentiate 8 different genotypes that clustered into 4 groups. One of these groups was classified within the formerly described haplotype A and another one within the recently described SL-IR group E. Both were phylogenetically well-supported. In contrast, none of the stocks from the Chaco province were grouped within the cluster previously named haplotype D despite the fact that they shared a similar microsatellite motif in the SL-IR. No evidence of recombination or gene conversion within these stocks was found. On the other hand, multiple ambiguous alignments in the microsatellite region of SL-IR, affecting the tree topology and relationships among groups were detected. Finally, since there are multiple copies of the SL-IR, and they are arranged in tandem, we discuss how molecular processes affecting this kind of sequences could mislead phylogenetic inference., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The intergenic region of spliced-leader (SL-IR) genes from 105 Trypanosoma cruzi I (Tc I) infected biological samples, culture isolates and stocks from 11 endemic countries, from Argentina to the USA were characterised, allowing identification of 76 genotypes with 54 polymorphic sites from 123 aligned sequences. On the basis of the microsatellite motif proposed by Herrera et al. (2007) to define four haplotypes in Colombia, we could classify these genotypes into four distinct Tc I SL-IR groups, three corresponding to the former haplotypes Ia (11 genotypes), Ib (11 genotypes) and Id (35 genotypes); and one novel group, Ie (19 genotypes). Genotypes harbouring the Tc Ic motif were not detected in our study. Tc Ia was associated with domestic cycles in southern and northern South America and sylvatic cycles in Central and North America. Tc Ib was found in all transmission cycles from Colombia. Tc Id was identified in all transmission cycles from Argentina and Colombia, including Chagas cardiomyopathy patients, sylvatic Brazilian samples and human cases from French Guiana, Panama and Venezuela. Tc Ie gathered five samples from domestic Triatoma infestans from northern Argentina, nine samples from wild Mepraia spinolai and Mepraia gajardoi and two chagasic patients from Chile and one from a Bolivian patient with chagasic reactivation. Mixed infections by Tc Ia+Tc Id, Tc Ia+Tc Ie and Tc Id+Tc Ie were detected in vector faeces and isolates from human and vector samples. In addition, Tc Ia and Tc Id were identified in different tissues from a heart transplanted Chagas cardiomyopathy patient with reactivation, denoting histotropism. Trypanosoma cruzi I SL-IR genotypes from parasites infecting Triatoma gerstaeckeri and Didelphis virginiana from USA, T. infestans from Paraguay, Rhodnius nasutus and Rhodnius neglectus from Brazil and M. spinolai and M. gajardoi from Chile are to our knowledge described for the first time., (Copyright © 2010 Australian Society for Parasitology Inc. All rights reserved.)