Stable population structure in Europe since the Iron Age, despite high mobility.

Ancient DNA research in the past decade has revealed that European population structure changed dramatically in the prehistoric period (14,000-3000 years before present, YBP), reflecting the widespread introduction of Neolithic farmer and Bronze Age Steppe ancestries. However, little is known about how population structure changed from the historical period onward (3000 YBP - present). To address this, we collected whole genomes from 204 individuals from Europe and the Mediterranean, many of which are the first historical period genomes from their region (e.g. Armenia and France). We found that most regions show remarkable inter-individual heterogeneity. At least 7% of historical individuals carry ancestry uncommon in the region where they were sampled, some indicating cross-Mediterranean contacts. Despite this high level of mobility, overall population structure across western Eurasia is relatively stable through the historical period up to the present, mirroring geography. We show that, under standard population genetics models with local panmixia, the observed level of dispersal would lead to a collapse of population structure. Persistent population structure thus suggests a lower effective migration rate than indicated by the observed dispersal. We hypothesize that this phenomenon can be explained by extensive transient dispersal arising from drastically improved transportation networks and the Roman Empire's mobilization of people for trade, labor, and military. This work highlights the utility of ancient DNA in elucidating finer scale human population dynamics in recent history.

Y-Chromosome Genetic Analysis of Modern Polish Population.

The study presents a full analysis of the Y-chromosome variability of the modern male Polish population. It is the first study of the Polish population to be conducted with such a large set of data (2,705 individuals), which includes genetic information from inhabitants of all voivodeships, i.e., the first administrative level, in the country and the vast majority of its counties, i.e., the second level. In addition, the available data were divided into clusters corresponding to more natural geographic regions. Genetic analysis included the estimation of F ST distances, the visualization with the use of multidimensional scaling plots and analysis of molecular variance. Y-chromosome binary haplogroups were classified and visualized with the use of interpolation maps. Results showed that the level of differentiation within Polish population is quite low, but some differences were indicated. It was confirmed that the Polish population is characterized by a high degree of homogeneity, with only slight genetic differences being observed at the regional level. The use of regional clustering as an alternative to counties and voivodeships provided a more detailed view of the genetic structure of the population. Those regional differences identified in the present study highlighted the need for additional division of the population by cultural and ethnic criteria in such studies rather than just by geographical or administrative regionalization.

PPE51 Is Involved in the Uptake of Disaccharides by Mycobacterium tuberculosis.

We have recently found that selected thio-disaccharides possess bactericidal effects against Mycobacterium tuberculosis but not against Escherichia coli or Staphylococcus aureus. Here, we selected spontaneous mutants displaying resistance against the investigated thio-glycoside. According to next-generation sequencing, four of six analyzed mutants which were resistant to high concentrations of the tested chemical carried nonsynonymous mutations in the gene encoding the PPE51 protein. The complementation of these mutants with an intact ppe51 gene returned their sensitivity to the wild-type level. The uptake of tritiated thio-glycoside was significantly more abundant in wild-type Mycobacterium tuberculosis compared to the strain carrying the mutated ppe51 gene. The ppe51 mutations or CRISPR-Cas9-mediated downregulation of PPE51 expression affected the growth of mutant strains on minimal media supplemented with disaccharides (maltose or lactose) but not with glycerol or glucose as the sole carbon and energy source. Taking the above into account, we postulate that PPE51 participates in the uptake of disaccharides by tubercle bacilli.

1H-Benzo[d]Imidazole Derivatives Affect MmpL3 in Mycobacterium tuberculosis.

1H-benzo[d]imidazole derivatives exhibit antitubercular activity in vitro at a nanomolar range of concentrations and are not toxic to human cells, but their mode of action remains unknown. Here, we showed that these compounds are active against intracellular Mycobacterium tuberculosis To identify their target, we selected drug-resistant M. tuberculosis mutants and then used whole-genome sequencing to unravel mutations in the essential mmpL3 gene, which encodes the integral membrane protein that catalyzes the export of trehalose monomycolate, a precursor of the mycobacterial outer membrane component trehalose dimycolate (TDM), as well as mycolic acids bound to arabinogalactan. The drug-resistant phenotype was also observed in the parental strain overexpressing the mmpL3 alleles carrying the mutations identified in the resistors. However, no cross-resistance was observed between 1H-benzo[d]imidazole derivatives and SQ109, another MmpL3 inhibitor, or other first-line antitubercular drugs. Metabolic labeling and quantitative thin-layer chromatography (TLC) analysis of radiolabeled lipids from M. tuberculosis cultures treated with the benzoimidazoles indicated an inhibition of trehalose dimycolate (TDM) synthesis, as well as reduced levels of mycolylated arabinogalactan, in agreement with the inhibition of MmpL3 activity. Overall, this study emphasizes the pronounced activity of 1H-benzo[d]imidazole derivatives in interfering with mycolic acid metabolism and their potential for therapeutic application in the fight against tuberculosis.

Screening methods for detection of ancient Mycobacterium tuberculosis complex fingerprints in next-generation sequencing data derived from skeletal samples.

BACKGROUND: Recent advances in ancient DNA studies, especially in increasing isolated DNA yields and quality, have opened the possibility of analysis of ancient host microbiome. However, such pitfalls as spurious identification of pathogens based on fragmentary data or environmental contamination could lead to incorrect epidaemiological conclusions. Within the Mycobacterium genus, Mycobacterium tuberculosis complex members responsible for tuberculosis share up to ∼99% genomic sequence identity, while other more distantly related Mycobacteria other than M. tuberculosis can be causative agents for pulmonary diseases or soil dwellers. Therefore, reliable determination of species complex is crucial for interpretation of sequencing results. RESULTS: Here we present a novel bioinformatical approach, used for screening of ancient tuberculosis in sequencing data, derived from 28 individuals (dated 4400-4000 and 3100-2900 BC) from central Poland. We demonstrate that cost-effective next-generation screening sequencing data (∼20M reads per sample) could yield enough information to provide statistically supported identification of probable ancient disease cases. CONCLUSIONS: Application of appropriate bioinformatic tools, including an unbiased selection of genomic alignment targets for species specificity, makes it possible to extract valid data from full-sample sequencing results (without subjective targeted enrichment procedures). This approach broadens the potential scope of palaeoepidaemiology both to older, suboptimally preserved samples and to pathogens with difficult intrageneric taxonomy.

Genomic Insights Into the Mycobacterium kansasii Complex: An Update.

Only very recently, has it been proposed that the hitherto existing Mycobacterium kansasii subtypes (I-VI) should be elevated, each, to a species rank. Consequently, the former M. kansasii subtypes have been denominated as Mycobacterium kansasii (former type I), Mycobacterium persicum (II), Mycobacterium pseudokansasii (III), Mycobacterium innocens (V), and Mycobacterium attenuatum (VI). The present work extends the recently published findings by using a three-pronged computational strategy, based on the alignment fraction-average nucleotide identity, genome-to-genome distance, and core-genome phylogeny, yet essentially independent and much larger sample, and thus delivers a more refined and complete picture of the M. kansasii complex. Furthermore, five canonical taxonomic markers were used, i.e., 16S rRNA, hsp65, rpoB, and tuf genes, as well as the 16S-23S rRNA intergenic spacer region (ITS). The three major methods produced highly concordant results, corroborating the view that each M. kansasii subtype does represent a distinct species. This work not only consolidates the position of five of the currently erected species, but also provides a description of the sixth one, i.e., Mycobacterium ostraviense sp. nov. to replace the former subtype IV. By showing a close genetic relatedness, a monophyletic origin, and overlapping phenotypes, our findings support the recognition of the M. kansasii complex (MKC), accommodating all M. kansasii-derived species and Mycobacterium gastri. None of the most commonly used taxonomic markers was shown to accurately distinguish all the MKC species. Likewise, no species-specific phenotypic characteristics were found allowing for species differentiation within the complex, except the non-photochromogenicity of M. gastri. To distinguish, most reliably, between the MKC species, and between M. kansasii and M. persicum in particular, whole-genome-based approaches should be applied. In the absence of clear differences in the distribution of the virulence-associated region of difference 1 genes among the M. kansasii-derived species, the pathogenic potential of each of these species can only be speculatively assessed based on their prevalence among the clinically relevant population. Large-scale molecular epidemiological studies are needed to provide a better understanding of the clinical significance and pathobiology of the MKC species. The results of the in vitro drug susceptibility profiling emphasize the priority of rifampicin administration in the treatment of MKC-induced infections, while undermining the use of ethambutol, due to a high resistance to this drug.

Molecular typing of Mycobacterium kansasii using pulsed-field gel electrophoresis and a newly designed variable-number tandem repeat analysis.

Molecular epidemiological studies of Mycobacterium kansasii are hampered by the lack of highly-discriminatory genotyping modalities. The purpose of this study was to design a new, high-resolution fingerprinting method for M. kansasii. Complete genome sequence of the M. kansasii ATCC 12478 reference strain was searched for satellite-like repetitive DNA elements comprising tandem repeats. A total of 24 variable-number tandem repeat (VNTR) loci were identified with potential discriminatory capacity. Of these, 17 were used to study polymorphism among 67 M. kansasii strains representing six subtypes (I-VI). The results of VNTR typing were compared with those of pulsed-field gel electrophoresis (PFGE) with AsnI digestion. Six VNTRs i.e. (VNTR 1, 2, 8, 14, 20 and 23) allow to differentiate analyzed strains with the same discriminatory capacities as use of a 17-loci panel. VNTR typing and PFGE in conjunction revealed 45 distinct patterns, including 11 clusters with 33 isolates and 34 unique patterns. The Hunter-Gaston's discriminatory index was 0.95 and 0.66 for PFGE and VNTR typing respectively, and 0.97 for the two methods combined. In conclusion, this study delivers a new typing scheme, based on VNTR polymorphism, and recommends it as a first-line test prior to PFGE analysis in a two-step typing strategy for M. kansasii.

Draft Genome Sequences of Mycobacterium kansasii Clinical Strains.

Mycobacterium kansasii is a nontuberculous mycobacterial (NTM) pathogen, frequently isolated from clinical samples and responsible for a large part of NTM infections in the human population. Here, we report the draft genome sequences of 12 M. kansasii strains isolated from clinical and host-associated sources from the Netherlands, Germany, and Poland.

In silico Analysis of Virulence Associated Genes in Genomes of Escherichia Coli Strains Causing Colibacillosis in Poultry.

INTRODUCTION: Colibacillosis - the most common disease of poultry, is caused mainly by avian pathogenic Escherichia coli (APEC). However, thus far, no pattern to the molecular basis of the pathogenicity of these bacteria has been established beyond dispute. In this study, genomes of APEC were investigated to ascribe importance and explore the distribution of 16 genes recognised as their virulence factors. MATERIAL AND METHODS: A total of 14 pathogenic for poultry E. coli strains were isolated, and their DNA was sequenced, assembled de novo, and annotated. Amino acid sequences from these bacteria and an additional 16 freely available APEC amino acid sequences were analysed with the DIFFIND tool to define their virulence factors. RESULTS: The DIFFIND tool enabled quick, reliable, and convenient assessment of the differences between compared amino acid sequences from bacterial genomes. The presence of 16 protein sequences indicated as pathogenicity factors in poultry resulted in the generation of a heatmap which categorises genomes in terms of the existence and similarity of the analysed protein sequences. CONCLUSION: The proposed method of detection of virulence factors using the capabilities of the DIFFIND tool may be useful in the analysis of similarities of E. coli and other sequences deriving from bacteria. Phylogenetic analysis resulted in reliable segregation of 30 APEC strains into five main clusters containing various virulence associated genes (VAGs).

Draft Genome Sequences of Mycobacterium kansasii Strains 1010001454, 1010001458, 1010001468, 1010001493, 1010001495, and 1010001469, Isolated from Environmental Sources.

Mycobacterium kansasii belongs to the nontuberculous mycobacteria (NTM) and causes opportunistic infections with both pulmonary and extrapulmonary manifestations. Here, we report the draft genome sequences of six environmental M. kansasii strains, designated 1010001495 (type I), 1010001469 (type II), 1010001468 (type III), 1010001458 (type IV), 1010001454 (type V), and 1010001493 (type V), originally isolated in five different European countries.