High prevalence of heteroresistance in Staphylococcus aureus is caused by a multitude of mutations in core genes.

Heteroresistance (HR) is an enigmatic phenotype where, in a main population of susceptible cells, small subpopulations of resistant cells exist. This is a cause for concern, as this small subpopulation is difficult to detect by standard antibiotic susceptibility tests, and upon antibiotic exposure the resistant subpopulation may increase in frequency and potentially lead to treatment complications or failure. Here, we determined the prevalence and mechanisms of HR for 40 clinical Staphylococcus aureus isolates, against 6 clinically important antibiotics: daptomycin, gentamicin, linezolid, oxacillin, teicoplanin, and vancomycin. High frequencies of HR were observed for gentamicin (69.2%), oxacillin (27%), daptomycin (25.6%), and teicoplanin (15.4%) while none of the isolates showed HR toward linezolid or vancomycin. Point mutations in various chromosomal core genes, including those involved in membrane and peptidoglycan/teichoic acid biosynthesis and transport, tRNA charging, menaquinone and chorismite biosynthesis and cyclic-di-AMP biosynthesis, were the mechanisms responsible for generating the resistant subpopulations. This finding is in contrast to gram-negative bacteria, where increased copy number of bona fide resistance genes via tandem gene amplification is the most prevalent mechanism. This difference can be explained by the observation that S. aureus has a low content of resistance genes and absence of the repeat sequences that allow tandem gene amplification of these genes as compared to gram-negative species.

Host Adaptation in Legionellales Is 1.9 Ga, Coincident with Eukaryogenesis.

Bacteria adapting to living in a host cell caused the most salient events in the evolution of eukaryotes, namely the seminal fusion with an archaeon, and the emergence of both mitochondrion and chloroplast. A bacterial clade that may hold the key to understanding these events is the deep-branching gammaproteobacterial order Legionellales-containing among others Coxiella and Legionella-of which all known members grow inside eukaryotic cells. Here, by analyzing 35 novel Legionellales genomes mainly acquired through metagenomics, we show that this group is much more diverse than previously thought, and that key host-adaptation events took place very early in its evolution. Crucial virulence factors like the Type IVB secretion (Dot/Icm) system and two shared effector proteins were gained in the last Legionellales common ancestor (LLCA). Many metabolic gene families were lost in LLCA and its immediate descendants, including functions directly and indirectly related to molybdenum metabolism. On the other hand, genome sizes increased in the ancestors of the Legionella genus. We estimate that LLCA lived approximately 1.89 Ga, probably predating the last eukaryotic common ancestor by approximately 0.4-1.0 Gy. These elements strongly indicate that host adaptation arose only once in Legionellales, and that these bacteria were using advanced molecular machinery to exploit and manipulate host cells early in eukaryogenesis.

Morphological and molecular differentiation of Diplostomum spp. metacercariae from brain of minnows (Phoxinus phoxinus L.) in four populations of northern Europe and East Asia.

Metacercariae of trematodes from the genus Diplostomum are major helminth pathogens of freshwater fish, infecting the eye or the brain. The taxonomy of the genus Diplostomum is complicated, and has recently been based mainly on the molecular markers. In this study, we report the results of the morphological and molecular genetic analysis of diplostomid metacercaria from the brain of the minnow Phoxinus phoxinus from three populations in Fennoscandia (Northern Europe) and one population in Mongolia (East Asia). We obtained the data on the polymorphism of the partial mitochondrial cox1 gene and ribosomal ITS1-5.8S-ITS2 region of these parasites. РСА-based morphological analysis revealed that the parasites in the Asian and the European groups of Diplostomum sp. were distinctly different. Metacercariae from the brain of Mongolian minnows were much larger than those from the brain of Fennoscandian minnows but had much fewer excretory granules. Considering that the two study regions were separated by a distance of about 4500 km, we also tested the genetic homogeneity of their host, the minnow, using the mitochondrial cytb gene. It was shown that Diplostomum-infected minnows from Mongolia and Fennoscandia represented two previously unknown separate phylogenetic lineages of the genus Phoxinus. Both molecular and morphological analysis demonstrated that the parasites from Fennoscandia belonged the species Diplostomum phoxini, while the parasites from Mongolia belonged to a separate species, Diplostomum sp. MТ.Each of the two studied Diplostomum spp. was associated with a specific, and previously unknown, genealogical lineage of its second intermediate host, P. phoxinus.

Contribution of Podoviridae and Myoviridae bacteriophages to the effectiveness of anti-staphylococcal therapeutic cocktails.

Bacteriophage therapy is considered one of the most promising therapeutic approaches against multi-drug resistant bacterial infections. Infections caused by Staphylococcus aureus are very efficiently controlled with therapeutic bacteriophage cocktails, containing a number of individual phages infecting a majority of known pathogenic S. aureus strains. We assessed the contribution of individual bacteriophages comprising a therapeutic bacteriophage cocktail against S. aureus in order to optimize its composition. Two lytic bacteriophages vB_SauM-515A1 (Myoviridae) and vB_SauP-436A (Podoviridae) were isolated from the commercial therapeutic cocktail produced by Microgen (Russia). Host ranges of the phages were established on the panel of 75 S. aureus strains. Phage vB_SauM-515A1 lysed 85.3% and vB_SauP-436A lysed 68.0% of the strains, however, vB_SauP-436A was active against four strains resistant to vB_SauM-515A1, as well as to the therapeutic cocktail per se. Suboptimal results of the therapeutic cocktail application were due to extremely low vB_SauP-436A1 content in this composition. Optimization of the phage titers led to an increase in overall cocktail efficiency. Thus, one of the effective ways to optimize the phage cocktails design was demonstrated and realized by using bacteriophages of different families and lytic spectra.

Author Correction: System OMICs analysis of Mycobacterium tuberculosis Beijing B0/W148 cluster.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Metabolic Changes of Mycobacterium tuberculosis during the Anti-Tuberculosis Therapy.

Tuberculosis, caused by Mycobacterium tuberculosis complex bacteria, remains one of the most pressing health problems. Despite the general trend towards reduction of the disease incidence rate, the situation remains extremely tense due to the distribution of the resistant forms. Most often, these strains emerge through the intra-host microevolution of the pathogen during treatment failure. In the present study, the focus was on three serial clinical isolates of Mycobacterium tuberculosis Beijing B0/W148 cluster from one patient with pulmonary tuberculosis, to evaluate their changes in metabolism during anti-tuberculosis therapy. Using whole genome sequencing (WGS), 9 polymorphisms were determined, which occurred in a stepwise or transient manner during treatment and were linked to the resistance (GyrA D94A; inhA t-8a) or virulence. The effect of the inhA t-8a mutation was confirmed on both proteomic and transcriptomic levels. Additionally, the amount of RpsL protein, which is a target of anti-tuberculosis drugs, was reduced. At the systemic level, profound changes in metabolism, linked to the evolution of the pathogen in the host and the effects of therapy, were documented. An overabundance of the FAS-II system proteins (HtdX, HtdY) and expression changes in the virulence factors have been observed at the RNA and protein levels.

System OMICs analysis of Mycobacterium tuberculosis Beijing B0/W148 cluster.

Mycobacterium tuberculosis Beijing B0/W148 is one of the most widely distributed clusters in the Russian Federation and in some countries of the former Soviet Union. Recent studies have improved our understanding of the reasons for the "success" of the cluster but this area remains incompletely studied. Here, we focused on the system omics analysis of the RUS_B0 strain belonging to the Beijing B0/W148 cluster. Completed genome sequence of RUS_B0 (CP020093.1) and a collection of WGS for 394 cluster strains were used to describe the main genetic features of the population. In turn, proteome and transcriptome studies allowed to confirm the genomic data and to identify a number of finds that have not previously been described. Our results demonstrated that expression of the whiB6 which contains cluster-specific polymorphism (a151c) increased almost 40 times in RUS_B0. Additionally, the level of ethA transcripts in RUS_B0 was increased by more than 7 times compared to the H37Rv. Start sites for 10 genes were corrected based on the combination of proteomic and transcriptomic data. Additionally, based on the omics approach, we identified 5 new genes. In summary, our analysis allowed us to summarize the available results and also to obtain fundamentally new data.

The role of IS6110 in micro- and macroevolution of Mycobacterium tuberculosis lineage 2.

The insertion sequence 6110 (IS6110) is the most studied transposable element in the Mycobacterium tuberculosis complex species. The element plays a significant role in genome plasticity of this important human pathogen, but still many causes and consequences of its transposition have not been fully studied. Here, we analyzed insertion sites for 902 Mycobacterium tuberculosis lineage 2 strains using whole-genome sequencing data. In total, 17,972 insertions were found, corresponding to 827 independent positions in the genome of the reference strain H37Rv. To trace the history of IS6110 expansion since proto-Beijing strains up to modern sublineages, we looked at the distribution of IS6110 across the genome-wide SNP-based phylogenetic tree. This analysis demonstrated a stepwise transposition of IS6110 that occurs by «copy-and-paste¼ mechanism. Additionally, we detected evolutionary-scale and sublineage-specific integration sites, which can be used for typing and for understanding the reasons for the success of the lineage. A significant part of such insertions affected the genes that are essential for the pathogen. Finally, we identified and confirmed deletions that occurred between differently oriented elements, which is uncommon for this family of insertion elements and appears to be another mechanism of genome variability.

MGERT: a pipeline to retrieve coding sequences of mobile genetic elements from genome assemblies.

BACKGROUND: Genomes of eukaryotes are inhabited by myriads of mobile genetic elements (MGEs) - transposons and retrotransposons - which play a great role in genome plasticity and evolution. A lot of computational tools were developed to annotate them either in genomic assemblies or raw reads using de novo or homology-based approaches. But there has been no pipeline enabling users to get coding and flanking sequences of MGEs suitable for a downstream analysis from genome assemblies. RESULTS: We developed a new pipeline, MGERT (Mobile Genetic Elements Retrieving Tool), that automates all the steps necessary to obtain protein-coding sequences of mobile genetic elements from genomic assemblies even if no previous knowledge on MGE content of a particular genome is available. CONCLUSIONS: Using MGERT, researchers can easily find MGEs, their coding and flanking sequences in the genome of interest. Thus, this pipeline helps researchers to focus on the biological analysis of MGEs rather than excessive scripting and pipelining.

Simple Assay for Detection of the Central Asia Outbreak Clade of the Mycobacterium tuberculosis Beijing Genotype.

The Central Asia outbreak (CAO) clade is a branch of the Mycobacterium tuberculosis Beijing genotype that is associated with multidrug resistance, increased transmissibility, and epidemic spread in parts of the former Soviet Union. Furthermore, migration flows bring these strains far beyond their areas of origin. We aimed to find a specific molecular marker of the Beijing CAO clade and develop a simple and affordable method for its detection. Based on the bioinformatics analysis of the large M. tuberculosis whole-genome sequencing (WGS) data set (n = 1,398), we identified an IS6110 insertion in the Rv1359-Rv1360 intergenic region as a specific molecular marker of the CAO clade. We further designed and optimized a multiplex PCR method to detect this insertion. The method was validated in silico with the recently published WGS data set from Central Asia (n = 277) and experimentally with M. tuberculosis isolates from European and Asian parts of Russia, the former Soviet Union, and East Asia (n = 319). The developed molecular assay may be recommended for rapid screening of retrospective collections and for prospective surveillance when comprehensive but expensive WGS is not available or practical. The assay may be especially useful in high multidrug-resistant tuberculosis (MDR-TB) burden countries of the former Soviet Union and in countries with respective immigrant communities.

Proteogenomic analysis of Mycobacterium tuberculosis Beijing B0/W148 cluster strains.

Nowadays proteomics is one of the major instruments for editing and correcting annotation of genomic information. The correct genome annotation is necessary for omics studies of clinically relevant pathogens like Mycobacterium tuberculosis as well as for the progress in drug design and in silico biology. Here, we focused on the proteogenomic analysis of W-148 strain belonging to the Beijing B0/W148 cluster. This cluster, also known as a "successful" clone possesses unique pathogenic properties and has a unique genome organization. Taking into account high similarity of cluster strains at the genomic level we analyzed MS/MS dataset obtained for 63 clinical isolates of Beijing B0/W148. Based on H37Rv and W-148 annotations we identified 2546 proteins representing more than 60% of total proteome. A set of peptides (n = 404) specific for W-148 was found when compared with H37Rv. Start sites for 32 genes were corrected based on the combination of LC-MS/MS proteomic data with genomic six-frame translation. Additionally, we have shown the presence of peptides related to 10 genes earlier known as "pseudogenes". SIGNIFICANCE: Mycobacterium tuberculosis is one of the most dangerous pathogens. Phylogenetically, it may be divided into major lineages and among them, lineage 2 (predominantly Beijing genotype) one of the most successful lineages with an increasing prevalence in the global population. At the same time, strains of the Beijing B0/W148 cluster, a "successful" clone of Mycobacterium tuberculosis possess even more interesting features. Only one complete genome of this cluster, W-148, present in the NCBI database (CP012090.1) and it demonstrates a number of significant differences from the well-known reference genome H37Rv. For the W-148 strain many genes are annotated as "pseudo" and no attempts were made to correct this. Thereby, in this study, we have conducted a proteomic analysis of the cluster strains and corrected current genome annotation. We hope that the data obtained will help to increase the quality of identifications in proteomic and transcriptomic analysis of M. tuberculosis Beijing B0/W148 cluster strain in subsequent studies.

Multiple interspecific hybridization and microsatellite mutations provide clonal diversity in the parthenogenetic rock lizard Darevskia armeniaca.

BACKGROUND: The parthenogenetic Caucasian rock lizard Darevskia armeniaca, like most other parthenogenetic vertebrate species, originated through interspecific hybridization between the closely related sexual Darevskia mixta and Darevskia valentini. Darevskia armeniaca was shown to consist of one widespread allozyme clone and a few rare ones, but notwithstanding the origin of clonal diversity remains unclear. We conduct genomic analysis of D. armeniaca and its parental sexual species using microsatellite and SNP markers to identify the origin of parthenogenetic clonal lineages. RESULTS: Four microsatellite-containing loci were genotyped for 111 specimens of D. armeniaca, 17 D. valentini, and four D. mixta. For these species, a total of 47 alleles were isolated and sequenced. Analysis of the data revealed 13 genotypes or presumptive clones in parthenogenetic D. armeniaca, including one widespread clone, two apparently geographically restricted clones, and ten rare clones. Comparisons of genotype-specific markers in D. armeniaca with those of its parental species revealed three founder-events including a common and two rare clones. All other clones appeared to have originated via post-formation microsatellite mutations in the course of evolutionary history of D. armeniaca. CONCLUSION: Our new approach to microsatellite genotyping reveals allele-specific microsatellite and SNP markers for each locus studied. Interspecies comparison of these markers identifies alleles inherited by parthenospecies from parental species, and provides new information on origin and evolution of clonal diversity in D. armeniaca. SNP analyses reveal at least three interspecific origins of D. armeniaca, and microsatellite mutations in these initial clones give rise to new clones. Thus, we first establish multiple origins of D. armeniaca. Our study identifies the most effective molecular markers for elucidating the origins of clonal diversity in other unisexual species that arose via interspecific hybridization.

The complete mitochondrial genome of the causative agent of the human cercarial dermatitis, the visceral bird schistosome species Trichobilharzia szidati (platyhelminthes: Trematoda: Schistosomatidae).

We report the first complete mitochondrial genome of visceral bird schistosome Trichobilharzia szidati (Platyhelminthes, Trematoda: Schistosomatidae). The circular genome is 14293 bp in length and contains 12 protein-coding genes, 12S and 16S rRNAs genes, 22 tRNAs and one non-coding region (202 bp) (accession number MF136777). Phylogenetic relationships based on 12 protein-coding gene sequences (PCG) of mitogenomes of a number of trematode and cestode species have shown that T. szidati is the closest genetic relative to nasal bird schistosome T. regenti. The complete mitogenome sequence of T. szidati may serve as a resource for comparative mitogenomics and trematode evolution studies.