Mycobacterium tuberculosis Latin–American–Mediterranean family in Bulgaria

AbstractIntroductionTuberculosis (TB) remains an important public health issue for Bulgaria. Although a number of new cases are showing a steady decline (44/100,000 in 2000, 40/100,000 in 2005), the TB incidence rate in Bulgaria is still sufficiently high (26.7/100,000 in 2013). The current population structure of Mycobacterium tuberculosis is clonal. Certain genetic families of this species have justly attracted more attention due to their global dissemination and/or remarkable pathogenic properties. Beijing, Haarlem, and LAM are the most known examples. The latter family LAM (Latin–American–Mediterranean) was shown in an increasing number of studies to possess increased transmissibility, hence the importance of its rapid detection and correct estimation of its prevalence in a population. Spoligotyping signature of LAM is absence of signals 21–24 and 33–36, although abridged spoligo-profiles with long blocks of deleted spacers result in an uncertain definition of such strains. The use of other molecular markers may be helpful. This study aimed to evaluate the prevalence of LAM strains among M. tuberculosis strains in Bulgaria based on the use of different molecular markers.Materials and methodsM. tuberculosis isolates were randomly selected among M. tuberculosis strains isolated from newly diagnosed, adult, pulmonary TB patients in different regions of Bulgaria from December 2004 to March 2006. Spoligotyping was used to analyze a variation in the DR locus. The spoligotyping patterns were compared with the international database SITVIT_WEB (Institut Pasteur de Guadeloupe). Analysis of the IS6110 element specific for the LAM genetic family was performed as described previously (Marais et al., 2006).ResultsA study sample included 133 strains from different regions of the country and characterized in previous publications (Valcheva et al., 2008, 2010). Application of the published rules for the definition of the major spoligotype clades and comparison with SITVIT_WEB global database permitted this study to assign most of the 133 strains to the known spoligotype families. All available strains (n=101) were tested for LAM-specific IS6110 insertion. Comparison of results by different methods identified 3 groups of strains. The first group included 11 strains with 2 amplified bands which present an apparent discrepancy; further study is warranted. The second group included 86 strains with a single amplified band, specific for the absence of the IS6110 insertion in the target locus, hence indicative of the other than LAM family. The third group included 4 strains with a LAM-specific band only.ConclusionsApplication of the LAM-specific PCR revealed double-sided discrepancies when the obtained results were compared with those obtained by spoligotyping. As a whole, a phylogenetic family of 38 strains was revised or questioned: 27 strains were shown not to belong to LAM, while 11 more strains showed apparently discrepant results that question the global utility of such PCR or at least highlight an importance of using multiple markers for molecular detection of the LAM family of M. tuberculosis.AcknowledgementsDr. Nadya Markova, Prof. Olga Narvskaya, and Prof. Nalin Rastogi are gratefully acknowledged for kind support and guidance

HLA DNA Sequence Variation among Human Populations: Molecular Signatures of Demographic and Selective Events

Molecular differences between HLA alleles vary up to 57 nucleotides within the peptide binding coding region of human Major Histocompatibility Complex (MHC) genes, but it is still unclear whether this variation results from a stochastic process or from selective constraints related to functional differences among HLA molecules. Although HLA alleles are generally treated as equidistant molecular units in population genetic studies, DNA sequence diversity among populations is also crucial to interpret the observed HLA polymorphism. In this study, we used a large dataset of 2,062 DNA sequences defined for the different HLA alleles to analyze nucleotide diversity of seven HLA genes in 23,500 individuals of about 200 populations spread worldwide. We first analyzed the HLA molecular structure and diversity of these populations in relation to geographic variation and we further investigated possible departures from selective neutrality through Tajima's tests and mismatch distributions. All results were compared to those obtained by classical approaches applied to HLA allele frequencies

Tracing the Evolution of Competence in Haemophilus influenzae

Natural competence is the genetically encoded ability of some bacteria to take up DNA from the environment. Although most of the incoming DNA is degraded, occasionally intact homologous fragments can recombine with the chromosome, displacing one resident strand. This potential to use DNA as a source of both nutrients and genetic novelty has important implications for the ecology and evolution of competent bacteria. However, it is not known how frequently competence changes during evolution, or whether non-competent strains can persist for long periods of time. We have previously studied competence in H. influenzae and found that both the amount of DNA taken up and the amount recombined varies extensively between different strains. In addition, several strains are unable to become competent, suggesting that competence has been lost at least once. To investigate how many times competence has increased or decreased during the divergence of these strains, we inferred the evolutionary relationships of strains using the largest datasets currently available. However, despite the use of three datasets and multiple inference methods, few nodes were resolved with high support, perhaps due to extensive mixing by recombination. Tracing the evolution of competence in those clades that were well supported identified changes in DNA uptake and/or transformation in most strains. The recency of these events suggests that competence has changed frequently during evolution but the poor support of basal relationships precludes the determination of whether non-competent strains can persist for long periods of time. In some strains, changes in transformation have occurred that cannot be due to changes in DNA uptake, suggesting that selection can act on transformation independent of DNA uptake

Multi-Locus Sequence Typing of a Geographically and Temporally Diverse Sample of the Highly Clonal Human Pathogen Bartonella quintana

Bartonella quintana is a re-emerging pathogen and the causative agent of a variety of disease manifestations in humans including trench fever. Various typing methods have been developed for B. quintana, but these tend to be limited by poor resolution and, in the case of gel-based methods, a lack of portability. Multilocus sequence typing (MLST) has been used to study the molecular epidemiology of a large number of pathogens, including B. henselae, a close relative of B. quintana. We developed a MLST scheme for B. quintana based on the 7 MLST loci employed for B. henselae with two additional loci to cover underrepresented regions of the B. quintana chromosome. A total of 16 B. quintana isolates spanning over 60 years and three continents were characterized. Allelic variation was detected in five of the nine loci. Although only 8/4270 (0.002%) of the nucleotide sites examined were variable over all loci, these polymorphisms resolved the 16 isolates into seven sequence types (STs). We also demonstrate that MLST can be applied on uncultured isolates by direct PCR from cardiac valve tissue, and suggest this method presents a promising approach for epidemiological studies in this highly clonal organism. Phylogenetic and clustering analyses suggest that two of the seven STs form a distinct lineage within the population

Inactivation of DNA-Binding Response Regulator Sak189 Abrogates β-Antigen Expression and Affects Virulence of Streptococcus agalactiae

BACKGROUND: Streptococcus agalactiae is able to colonize numerous tissues employing different mechanisms of gene regulation, particularly via two-component regulatory systems. These systems sense the environmental stimuli and regulate expression of the genes including virulence genes. Recently, the novel two-component regulatory system Sak188/Sak189 was identified. In S. agalactiae genome, it was adjacent to the bac gene encoding for beta-antigen, an important virulence factor. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the sak188 and sak189 genes were inactivated, and the functional role of Sak188/Sak189 two-component system in regulation of the beta-antigen expression was investigated. It was demonstrated that both transcription of bac gene and expression of encoded beta-antigen were controlled by Sak189 response regulator, but not Sak188 histidine kinase. It was also found that the regulation occurred at transcriptional level. Finally, insertional inactivation of sak189 gene, but not sak188 gene, significantly affected virulent properties of S. agalactiae. CONCLUSIONS/SIGNIFICANCE: Sak189 response regulator is necessary for activation of bac gene transcription. It also controls the virulent properties of S. agalactiae. Given that the primary functional role of Sak188/Sak189 two-component systems is a control of bac gene transcription, this system can be annotated as BgrR/S (bacgene regulatory system)

A Real-Time PCR Array for Hierarchical Identification of Francisella Isolates

A robust, rapid and flexible real-time PCR assay for hierarchical genetic typing of clinical and environmental isolates of Francisella is presented. Typing markers were found by multiple genome and gene comparisons, from which 23 canonical single nucleotide polymorphisms (canSNPs) and 11 canonical insertion-deletion mutations (canINDELs) were selected to provide phylogenetic guidelines for classification from genus to isolate level. The specificity of the developed assay, which uses 68 wells of a 96-well real-time PCR format with a detection limit of 100 pg DNA, was assessed using 62 Francisella isolates of diverse genetic and geographical origins. It was then successfully used for typing 14 F. tularensis subsp. holarctica isolates obtained from tularemia patients in Sweden in 2008 and five more genetically diverse Francisella isolates of global origins. When applied to human ulcer specimens for direct pathogen detection the results were incomplete due to scarcity of DNA, but sufficient markers were identified to detect fine-resolution differences among F. tularensis subsp. holarctica isolates causing infection in the patients. In contrast to other real-time PCR assays for Francisella, which are typically designed for specific detection of a species, subspecies, or strain, this type of assay can be easily tailored to provide appropriate phylogenetic and/or geographical resolution to meet the objectives of the analysis

A PubMed-Wide Associational Study of Infectious Diseases

Background: Computational discovery is playing an ever-greater role in supporting the processes of knowledge synthesis. A significant proportion of the more than 18 million manuscripts indexed in the PubMed database describe infectious disease syndromes and various infectious agents. This study is the first attempt to integrate online repositories of text-based publications and microbial genome databases in order to explore the dynamics of relationships between pathogens and infectious diseases. Methodology/Principal Findings: Herein we demonstrate how the knowledge space of infectious diseases can be computationally represented and quantified, and tracked over time. The knowledge space is explored by mapping of the infectious disease literature, looking at dynamics of literature deposition, zooming in from pathogen to genome level and searching for new associations. Syndromic signatures for different pathogens can be created to enable a new and clinically focussed reclassification of the microbial world. Examples of syndrome and pathogen networks illustrate how multilevel network representations of the relationships between infectious syndromes, pathogens and pathogen genomes can illuminate unexpected biological similarities in disease pathogenesis and epidemiology. Conclusions/Significance: This new approach based on text and data mining can support the discovery of previously hidden associations between diseases and microbial pathogens, clinically relevant reclassification of pathogeni

Complex Population Structure of Lyme Borreliosis Group Spirochete Borrelia garinii in Subarctic Eurasia

Borrelia garinii, a causative agent of Lyme borreliosis in Europe and Asia, is naturally maintained in marine and terrestrial enzootic cycles, which primarily involve birds, including seabirds and migratory passerines. These bird groups associate with, correspondingly, Ixodes uriae and Ixodes ricinus ticks, of which the latter species may bite and transmit the infection to humans. Studies of the overlap between these two natural cycles of B. garinii have been limited, in part due to the absence of representative collections of this spirochete's samples, as well as of the lack of reliable measure of the genetic heterogeneity of its strains. As a prerequisite for understanding the epidemiological correlates of the complex maintenance of B. garinii, the present study sought to assess the diversity and phylogenetic relationships of this species' strains from its natural hosts and patients with Lyme borreliosis from subarctic Eurasia. We used sequence typing of the partial rrs-rrl intergenic spacer (IGS) of archived and prospective samples of B. garinii from I. uriae ticks collected predominantly on Commander Islands in North Pacific, as well as on the islands in northern Sweden and arctic Norway. We also typed B. garinii samples from patients with Lyme borreliosis and I. ricinus ticks infesting migratory birds in southern Sweden, or found questing in selected sites on the islands in the Baltic Sea and Lithuania. Fifty-two (68%) of 77 B. garinii samples representing wide geographical range and associated with I. ricinus and infection of humans contributed 12 (60%) of total 20 identified IGS variants. In contrast, the remaining 25 (32%) samples recovered from I. uriae ticks from a few islands accounted for as many as 10 (50%) IGS types, suggesting greater local diversity of B. garinii maintained by seabirds and their ticks. Two IGS variants of the spirochete in common for both tick species were found in I. ricinus larvae from migratory birds, an indication that B. garinii strains are exchanged between different ecological niches. Notably, B. garinii variants associated with I. uriae ticks were found in each of the six clusters, representing two phylogenetic lineages of this species identified among the studied samples. Our findings suggest that B. garinii in subarctic Eurasia comprises two partially overlapping populations with different levels of genetic heterogeneity, presumably, due to distinctive selective pressures on the spirochete in its marine and terrestrial enzootic cycles