Transposition Behavior Revealed by High-Resolution Description of Pseudomonas Aeruginosa Saltovirus Integration Sites.

Transposable phages, also called saltoviruses, of which the Escherichia coli phage Mu is the reference, are temperate phages that multiply their genome through replicative transposition at multiple sites in their host chromosome. The viral genome is packaged together with host DNA at both ends. In the present work, genome sequencing of three Pseudomonas aeruginosa transposable phages, HW12, 2P1, and Ab30, incidentally gave us access to the location of thousands of replicative integration sites and revealed the existence of a variable number of hotspots. Taking advantage of deep sequencing, we then designed an experiment to study 13,000,000 transposon integration sites of bacteriophage Ab30. The investigation revealed the presence of 42 transposition hotspots adjacent to bacterial interspersed mosaic elements (BIME) accounting for 5% of all transposition sites. The rest of the sites appeared widely distributed with the exception of coldspots associated with low G-C content segments, including the putative O-antigen biosynthesis cluster. Surprisingly, 0.4% of the transposition events occurred in a copy of the phage genome itself, indicating that the previously described immunity against such events is slightly leaky. This observation allowed drawing an image of the phage chromosome supercoiling into four loops.

Correction: Comparison of French and Worldwide Bacillus anthracis Strains Favors a Recent, Post-Columbian Origin of the Predominant North-American Clade.

[This corrects the article DOI: 10.1371/journal.pone.0146216.].

Comparison of French and Worldwide Bacillus anthracis Strains Favors a Recent, Post-Columbian Origin of the Predominant North-American Clade.

BACKGROUND: Bacillus anthracis, the highly dangerous zoonotic bacterial pathogen species is currently composed of three genetic groups, called A, B and C. Group A is represented worldwide whereas group B is present essentially in Western Europe and Southern Africa. Only three strains from group C have been reported. This knowledge is derived from the genotyping of more than 2000 strains collected worldwide. Strains from both group A and group B are present in France. Previous investigations showed that the majority of sporadic French strains belong to the so-called A.Br.011/009 group A clade and define a very remarkable polytomy with six branches. Here we explore the significance of this polytomy by comparing the French B. anthracis lineages to worldwide lineages. We take advantage of whole genome sequence data previously determined for 122 French strains and 45 strains of various origins. RESULTS: A total of 6690 SNPs was identified among the available dataset and used to draw the phylogeny. The phylogeny of the French B group strains which belongs to B.Br.CNEVA indicates an expansion from the south-east part of France (the Alps) towards the south-west (Massif-Central and Pyrenees). The relatively small group A strains belonging to A.Br.001/002 results from at least two independent introductions. Strikingly, the data clearly demonstrates that the currently predominant B. anthracis lineage in North America, called WNA for Western North American, is derived from one branch of the A.Br.011/009 polytomy predominant in France. CONCLUSIONS/SIGNIFICANCE: The present work extends the range of observed substitution rate heterogeneity within B. anthracis, in agreement with its ecology and in contrast with some other pathogens. The population structure of the six branches A.Br.011/009 polytomy identified in France, diversity of branch length, and comparison with the WNA lineage, suggests that WNA is of post-Columbian and west European origin, with France as a likely source. Furthermore, it is tempting to speculate that the polytomy's most recent common ancestor -MRCA- dates back to the Hundred Years' war between France and England started in the mid-fourteenth century. These events were associated in France with deadly epidemics and major economic and social changes.

Investigation of a Large Collection of Pseudomonas aeruginosa Bacteriophages Collected from a Single Environmental Source in Abidjan, Côte d'Ivoire.

Twenty two distinct bacteriophages were isolated from sewage water from five locations in the city of Abidjan, Côte d'Ivoire over a two-year period, using a collection of Pseudomonas aeruginosa strains with diverse genotypes. The phages were characterized by their virulence spectrum on a panel of selected P. aeruginosa strains from cystic fibrosis patients and by whole genome sequencing. Twelve virions representing the observed diversity were visualised by electron microscopy. The combined observations showed that 17 phages, distributed into seven genera, were virulent, and that five phages were related to temperate phages belonging to three genera. Some showed similarity with known phages only at the protein level. The vast majority of the genetic variations among virulent phages from the same genus resulted from seemingly non-random horizontal transfer events, inside a population of P. aeruginosa phages with limited diversity. This suggests the existence of a single environmental reservoir or ecotype in which continuous selection is taking place. In contrast, mostly point mutations were observed among phages potentially capable of lysogenisation. This is the first study of P. aeruginosa phage diversity in an African city and it shows that a large variety of phage species can be recovered in a limited geographical site at least when different bacterial strains are used. The relative temporal and spatial stability of the Abidjan phage population might reflect equilibrium in the microbial community from which they are released.

Draft Genome Sequence of the Biowarfare Simulant Bacillus atrophaeus Strain 930029.

We report here the draft genome sequence of Bacillus atrophaeus strain 930029. Strain 930029 shows evidence of drift, based on a comparison to the corresponding source strain publicly available today.

Yersinia pseudotuberculosis ST42 (O:1) Strain Misidentified as Yersinia pestis by Mass Spectrometry Analysis.

We report here the draft sequence of strain CEB14_0017, alias HIAD_DUP, recovered from a human patient and initially identified as Yersinia pestis by mass spectrometry analysis. Genotyping based on tandem repeat polymorphism assigned the strain to Yersinia pseudotuberculosis sequence type 42 (ST42). The total assembly length is 4,894,739 bp.

High-throughput sequencing of Bacillus anthracis in France: investigating genome diversity and population structure using whole-genome SNP discovery.

BACKGROUND: Single nucleotide polymorphisms (SNPs) are ideal signatures for subtyping monomorphic pathogens such as Bacillus anthracis. Here we report the use of next-generation sequencing technology to investigate the historical, geographic and genetic diversity of Bacillus anthracis in France. 122 strains isolated over a 60-years period throughout the country were whole-genome sequenced and comparative analyses were carried out with a focus on SNPs discovery to discriminate regional sub-groups of strains. RESULTS: A total of 1581 chromosomal SNPs precisely establish the phylogenetic relationships existing between the French strains. Phylogeography patterns within the three canSNP sub-lineages present in France (i.e. B.Br.CNEVA, A.Br.011/009 and A.Br.001/002) were observed. One of the more remarkable findings was the identification of a variety of genotypes within the A.Br.011/009 sub-group that are persisting in the different regions of France. The 560 SNPs defining the A.Br.011/009- affiliated French strains split the Trans-Eurasian sub-group into six distinct branches without any intermediate nodes. Distinct sub-branches, with some geographic clustering, were resolved. The 345 SNPs defining the major B.Br CNEVA sub-lineage clustered three main phylogeographic clades, the Alps, the Pyrenees, and the Massif Central, with a small Saône-et-Loire sub-cluster nested within the latter group. The French strains affiliated to the minor A.Br.001/002 group were characterized by 226 SNPs. All recent isolates collected from the Doubs department were closely related. Identification of SNPs from whole-genome sequences facilitates high-resolution strain tracking and provides the level of discrimination required for outbreak investigations. Eight diagnostic SNPs, representative of the main French-specific phylogeographic clusters, were therefore selected and developed into high-resolution melting SNP discriminative assays. CONCLUSIONS: This work has established one of the most accurate phylogenetic reconstruction of B. anthracis population structure in a country. An extensive next-generation sequencing (NGS) dataset of 122 French strains have been created that allowed the identification of novel diagnostic SNPs useful to rapidly determine the geographic origin of any strain found in France.

A novel Pseudomonas aeruginosa bacteriophage, Ab31, a chimera formed from temperate phage PAJU2 and P. putida lytic phage AF: characteristics and mechanism of bacterial resistance.

A novel temperate bacteriophage of Pseudomonas aeruginosa, phage vB_PaeP_Tr60_Ab31 (alias Ab31) is described. Its genome is composed of structural genes related to those of lytic P. putida phage AF, and regulatory genes similar to those of temperate phage PAJU2. The virion structure resembles that of phage AF and other lytic Podoviridae (S. enterica Epsilon 15 and E. coli phiv10) with similar tail spikes. Ab31 was able to infect P. aeruginosa strain PA14 and two genetically related strains called Tr60 and Tr162, out of 35 diverse strains from cystic fibrosis patients. Analysis of resistant host variants revealed different phenotypes, including induction of pigment and alginate overproduction. Whole genome sequencing of resistant variants highlighted the existence of a large deletion of 234 kbp in two strains, encompassing a cluster of genes required for the production of CupA fimbriae. Stable lysogens formed by Ab31 in strain Tr60, permitted the identification of the insertion site. During colonization of the lung in cystic fibrosis patients, P. aeruginosa adapts by modifying its genome. We suggest that bacteriophages such as Ab31 may play an important role in this adaptation by selecting for bacterial characteristics that favor persistence of bacteria in the lung.

Progenitor "Mycobacterium canettii" clone responsible for lymph node tuberculosis epidemic, Djibouti.

"Mycobacterium canettii," an opportunistic human pathogen living in an unknown environmental reservoir, is the progenitor species from which Mycobacterium tuberculosis emerged. Since its discovery in 1969, most of the ≈70 known M. canettii strains were isolated in the Republic of Djibouti, frequently from expatriate children and adults. We show here, by whole-genome sequencing, that most strains collected from February 2010 through March 2013, and associated with 2 outbreaks of lymph node tuberculosis in children, belong to a unique epidemic clone within M. canettii. Evolution of this clone, which has been recovered regularly since 1983, may mimic the birth of M. tuberculosis. Thus, recognizing this organism and identifying its reservoir are clinically important.

Draft Genome Sequences of Two Yersinia pseudotuberculosis ST43 (O:1b) Strains, B-7194 and B-7195.

We report the first draft genome sequences of two Yersinia pseudotuberculosis sequence type 43 (ST43) (O:1b) strains, B-7194 and B-7195, isolated in Russia. The total lengths of the assemblies are 4,427,121 bp and 4,608,472 bp, and 3,819 and 4,018 coding sequences, respectively, were predicted within the genomes.

The susceptibility of Pseudomonas aeruginosa strains from cystic fibrosis patients to bacteriophages.

Phage therapy may become a complement to antibiotics in the treatment of chronic Pseudomonas aeruginosa infection. To design efficient therapeutic cocktails, the genetic diversity of the species and the spectrum of susceptibility to bacteriophages must be investigated. Bacterial strains showing high levels of phage resistance need to be identified in order to decipher the underlying mechanisms. Here we have selected genetically diverse P. aeruginosa strains from cystic fibrosis patients and tested their susceptibility to a large collection of phages. Based on plaque morphology and restriction profiles, six different phages were purified from "pyophage", a commercial cocktail directed against five different bacterial species, including P. aeruginosa. Characterization of these phages by electron microscopy and sequencing of genome fragments showed that they belong to 4 different genera. Among 47 P. aeruginosa strains, 13 were not lysed by any of the isolated phages individually or by pyophage. We isolated two new phages that could lyse some of these strains, and their genomes were sequenced. The presence/absence of a CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats and Crisper associated genes) was investigated to evaluate the role of the system in phage resistance. Altogether, the results show that some P. aeruginosa strains cannot support the growth of any of the tested phages belonging to 5 different genera, and suggest that the CRISPR-Cas system is not a major defence mechanism against these lytic phages.

Draft Genome Sequences of Five Yersinia pseudotuberculosis ST19 Isolates and One Isolate Variant.

We report the first draft genome sequences of five Yersinia pseudotuberculosis isolates of sequence type (ST) 19 and of a variant from one of the five isolates. The total length of assemblies ranged from 4,226,485 bp to 4,274,148 bp, including between 3,808 and 3,843 predicted coding sequences.

Significance of the identification in the Horn of Africa of an exceptionally deep branching Mycobacterium tuberculosis clade.

Molecular and phylogeographic studies have led to the definition within the Mycobacterium tuberculosis complex (MTBC) of a number of geotypes and ecotypes showing a preferential geographic location or host preference. The MTBC is thought to have emerged in Africa, most likely the Horn of Africa, and to have spread worldwide with human migrations. Under this assumption, there is a possibility that unknown deep branching lineages are present in this region. We genotyped by spoligotyping and multiple locus variable number of tandem repeats (VNTR) analysis (MLVA) 435 MTBC isolates recovered from patients. Four hundred and eleven isolates were collected in the Republic of Djibouti over a 12 year period, with the other 24 isolates originating from neighbouring countries. All major M. tuberculosis lineages were identified, with only two M. africanum and one M. bovis isolates. Upon comparison with typing data of worldwide origin we observed that several isolates showed clustering characteristics compatible with new deep branching. Whole genome sequencing (WGS) of seven isolates and comparison with available WGS data from 38 genomes distributed in the different lineages confirms the identification of ancestral nodes for several clades and most importantly of one new lineage, here referred to as lineage 7. Investigation of specific deletions confirms the novelty of this lineage, and analysis of its precise phylogenetic position indicates that the other three superlineages constituting the MTBC emerged independently but within a relatively short timeframe from the Horn of Africa. The availability of such strains compared to the predominant lineages and sharing very ancient ancestry will open new avenues for identifying some of the genetic factors responsible for the success of the modern lineages. Additional deep branching lineages may be readily and efficiently identified by large-scale MLVA screening of isolates from sub-Saharan African countries followed by WGS analysis of a few selected isolates.