Distribution of packaged bacterial reads from C7-25 mitomycin C-induced phages.

<p>A) Mapping of the bacterial reads along the PAO1_Or genome. The blue horizontal arrow represents the prophage. B) Organization of the phiC725A prophage and close-up of the 800kb left flanking region; the distribution of the packaged bacterial sequence reads is shown. The collection of arrows underneath the graph and starting from the right at the tentative prophage <i>pac</i> site within the blue bar representing the phage genome, correspond to coverage maximums. The first such maximum is located approximately 25 kb from the left of the blue bar. The spacing between the arrows is approximately 72kb, compared to the 65 kb of phage genome suggesting that the terminal redundancy represents 10% of the phage size. The blue, black and yellow lines represent the minimum, average and maximum read coverage observed respectively.</p

Electron microscopy observation of phage phiC725A.

<p>The black bar represents 200nm.</p

Genomic organization of phiC725A.

<p>A) Distribution of the predicted CDSs along the phage genome. CDSs are shown as horizontal arrows. Position 1 in the sequence is defined in keeping with phage F116. The different colors correspond to the putative function: red, DNA replication, modification and repair; orange, terminase; green, morphogenesis and packaging; light green, putative RNA polymerase; pink, lysis; blue, lysogeny control; yellow, unidentified. The vertical arrow indicates the putative localization of the <i>pac</i> site. The <i>att</i> site towards the right end is in the TCTCTCCGTCCGCACCA orientation, i.e. the reverse orientation with respect to the PAO1Or host sequence orientation. B) Mapping of the sequencing reads showing an excess at the level of the terminase subunits genes, with a progressive decrease towards the right end.</p

Table_2_Genotypic Expansion Within the Population Structure of Classical Brucella Species Revealed by MLVA16 Typing of 1404 Brucella Isolates From Different Animal and Geographic Origins, 1974–2006.XLSX

<p>Previous studies have shown the usefulness of MLVA16 as a rapid molecular identification and classification method for Brucella species and biovars including recently described novel Brucella species from wildlife. Most studies were conducted on a limited number of strains from limited geographic/host origins. The objective of this study was to assess genetic diversity of Brucella spp. by MLVA16 on a larger scale. Thus, 1404 animal or human isolates collected from all parts of the world over a period of 32 years (1974-2006) were investigated. Selection of the 1404 strains was done among the approximately 4000 strains collection of the BCCN (Brucella Culture Collection Nouzilly), based on classical biotyping and on the animal/human/geographic origin over the time period considered. MLVA16 was performed on extracted DNAs using high throughput capillary electrophoresis. The 16 loci were amplified in four multiplex PCR reactions. This large scale study firstly confirmed the accuracy of MLVA16 typing for Brucella species and biovar identification and its congruence with the recently described Extended Multilocus Sequence Analysis. In addition, it allowed identifying novel MLVA11 (based upon 11 slowly evolving VNTRs) genotypes representing an increase of 15% relative to the previously known Brucella MLVA11 genotypes. Cluster analysis showed that among the MLVA16 genotypes some were genetically more distant from the major classical clades. For example new major clusters of B. abortus biovar 3 isolated from cattle in Sub-Saharan Africa were identified. For other classical species and biovars this study indicated also genotypic expansion within the population structure of classical Brucella species. MLVA proves to be a powerful tool to rapidly assess genetic diversity of bacterial populations on a large scale, as here on a large collection of strains of the genomically homogeneous genus Brucella. The highly discriminatory power of MLVA appears of particular interest as a first step for selection of Brucella strains for whole-genome sequencing. The MLVA data of this study were added to the public Brucella MLVA database at http://microbesgenotyping.i2bc.paris-saclay.fr. Current version Brucella_4_3 comprises typing data from more than 5000 strains including in silico data analysis of public whole genome sequence datasets.</p

Table_1_Genotypic Expansion Within the Population Structure of Classical Brucella Species Revealed by MLVA16 Typing of 1404 Brucella Isolates From Different Animal and Geographic Origins, 1974–2006.XLSX

<p>Previous studies have shown the usefulness of MLVA16 as a rapid molecular identification and classification method for Brucella species and biovars including recently described novel Brucella species from wildlife. Most studies were conducted on a limited number of strains from limited geographic/host origins. The objective of this study was to assess genetic diversity of Brucella spp. by MLVA16 on a larger scale. Thus, 1404 animal or human isolates collected from all parts of the world over a period of 32 years (1974-2006) were investigated. Selection of the 1404 strains was done among the approximately 4000 strains collection of the BCCN (Brucella Culture Collection Nouzilly), based on classical biotyping and on the animal/human/geographic origin over the time period considered. MLVA16 was performed on extracted DNAs using high throughput capillary electrophoresis. The 16 loci were amplified in four multiplex PCR reactions. This large scale study firstly confirmed the accuracy of MLVA16 typing for Brucella species and biovar identification and its congruence with the recently described Extended Multilocus Sequence Analysis. In addition, it allowed identifying novel MLVA11 (based upon 11 slowly evolving VNTRs) genotypes representing an increase of 15% relative to the previously known Brucella MLVA11 genotypes. Cluster analysis showed that among the MLVA16 genotypes some were genetically more distant from the major classical clades. For example new major clusters of B. abortus biovar 3 isolated from cattle in Sub-Saharan Africa were identified. For other classical species and biovars this study indicated also genotypic expansion within the population structure of classical Brucella species. MLVA proves to be a powerful tool to rapidly assess genetic diversity of bacterial populations on a large scale, as here on a large collection of strains of the genomically homogeneous genus Brucella. The highly discriminatory power of MLVA appears of particular interest as a first step for selection of Brucella strains for whole-genome sequencing. The MLVA data of this study were added to the public Brucella MLVA database at http://microbesgenotyping.i2bc.paris-saclay.fr. Current version Brucella_4_3 comprises typing data from more than 5000 strains including in silico data analysis of public whole genome sequence datasets.</p

Image_1_Genotypic Expansion Within the Population Structure of Classical Brucella Species Revealed by MLVA16 Typing of 1404 Brucella Isolates From Different Animal and Geographic Origins, 1974–2006.PDF

<p>Previous studies have shown the usefulness of MLVA16 as a rapid molecular identification and classification method for Brucella species and biovars including recently described novel Brucella species from wildlife. Most studies were conducted on a limited number of strains from limited geographic/host origins. The objective of this study was to assess genetic diversity of Brucella spp. by MLVA16 on a larger scale. Thus, 1404 animal or human isolates collected from all parts of the world over a period of 32 years (1974-2006) were investigated. Selection of the 1404 strains was done among the approximately 4000 strains collection of the BCCN (Brucella Culture Collection Nouzilly), based on classical biotyping and on the animal/human/geographic origin over the time period considered. MLVA16 was performed on extracted DNAs using high throughput capillary electrophoresis. The 16 loci were amplified in four multiplex PCR reactions. This large scale study firstly confirmed the accuracy of MLVA16 typing for Brucella species and biovar identification and its congruence with the recently described Extended Multilocus Sequence Analysis. In addition, it allowed identifying novel MLVA11 (based upon 11 slowly evolving VNTRs) genotypes representing an increase of 15% relative to the previously known Brucella MLVA11 genotypes. Cluster analysis showed that among the MLVA16 genotypes some were genetically more distant from the major classical clades. For example new major clusters of B. abortus biovar 3 isolated from cattle in Sub-Saharan Africa were identified. For other classical species and biovars this study indicated also genotypic expansion within the population structure of classical Brucella species. MLVA proves to be a powerful tool to rapidly assess genetic diversity of bacterial populations on a large scale, as here on a large collection of strains of the genomically homogeneous genus Brucella. The highly discriminatory power of MLVA appears of particular interest as a first step for selection of Brucella strains for whole-genome sequencing. The MLVA data of this study were added to the public Brucella MLVA database at http://microbesgenotyping.i2bc.paris-saclay.fr. Current version Brucella_4_3 comprises typing data from more than 5000 strains including in silico data analysis of public whole genome sequence datasets.</p

Diversity of <em>Acinetobacter baumannii</em> in Four French Military Hospitals, as Assessed by Multiple Locus Variable Number of Tandem Repeats Analysis

<div><h3>Background</h3><p>Infections by <em>A. calcoaceticus</em>-<em>A. baumannii</em> (ACB) complex isolates represent a serious threat for wounded and burn patients. Three international multidrug-resistant (MDR) clones (EU clone I-III) are responsible for a large proportion of nosocomial infections with <em>A. baumannii</em> but other emerging strains with high epidemic potential also occur.</p> <h3>Methodology/Principal Findings</h3><p>We automatized a Multiple locus variable number of tandem repeats (VNTR) analysis (MLVA) protocol and used it to investigate the genetic diversity of 136 ACB isolates from four military hospitals and one childrens hospital. <em>Acinetobacter</em> sp other than <em>baumannii</em> isolates represented 22.6% (31/137) with a majority being <em>A. pittii</em>. The genotyping protocol designed for <em>A.baumannii</em> was also efficient to cluster <em>A. pittii</em> isolates. Fifty-five percent of <em>A. baumannii</em> isolates belonged to the two international clones I and II, and we identified new clones which members were found in the different hospitals. Analysis of two CRISPR-cas systems helped define two clonal complexes and provided phylogenetic information to help trace back their emergence.</p> <h3>Conclusions/Significance</h3><p>The increasing occurrence of <em>A. baumannii</em> infections in the hospital calls for measures to rapidly characterize the isolates and identify emerging clones. The automatized MLVA protocol can be the instrument for such surveys. In addition, the investigation of CRISPR/cas systems may give important keys to understand the evolution of some highly successful clonal complexes.</p> </div

Polymorphism of the AYE CRISPR locus in <i>A. baumannii</i>.

<p>A) Schematic representation of the CRISPR locus in three sequenced genomes and at the growing end of isolates A28, P054 and P065. Arrows show the position of primers used to amplify a portion of the locus. Specific spacers are shown with grey boxes. B) Sequence of the growing end of strain AB307 and isolate A28. The sequence flanking the last DR is in italics.</p

Genetic diversity of 56 <i>A. baumannii</i> isolates.

<p>The legend is that of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044597#pone-0044597-g001" target="_blank">Figure 1</a>.</p

Oligonucleotides used for the multiplex PCR reactions.

a<p>The forward primer is labeled with one of three dyes D2, D3 and D4. The allele range for each reaction is indicated showing that amplicons with the same dye cannot overlap.</p>b<p>Values referred to the genome of strain ATCC 17978 and considered for allele assignment.</p>c<p>The allele range for each reaction is indicated showing that amplicons with the same dye cannot overlap. Values obtained with <i>A. baumannii</i> isolates.</p