Distribution of 141 <i>S. agalactiae</i> isolates from adult (ACSF and AB) and neonatal (NCFS and NB) patients with invasive disease between prophage DNA groups, on the basis of PCR evaluations of the prophage content of isolates.

<p>Jaccard analysis generated a dendrogram of similarity values for the 10 prophage sequences described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020256#pone-0020256-t001" target="_blank">table 1</a> (SYSTAT 12 software). Five major prophage DNA groups were defined (groups A to E). The mean number of prophage DNA fragments amplified from strains by PCR and the mean number of absolute deviations (Avedev) were calculated for each prophage DNA group. ^a anatomic origin of isolates; ^b serotype of isolates; ST, sequence-type; CC, clonal complex; NT, nontypeable.</p

Distribution of the SLVs of the major clonal complexes as a function of difference in the number of nucleotides (one or more) with respect to the sequence of the founder sequence type.

<p>Distribution of the SLVs of the major clonal complexes as a function of difference in the number of nucleotides (one or more) with respect to the sequence of the founder sequence type.</p

Genetic diversity and sequence type (ST) distribution, determined by MLST [<b>10</b>], of 142 <i>S. agalactiae</i> isolates from cases of adult (AI) and neonatal (NI) invasive disease.

<p>We show the phylogenetic network applied to 43 parsimonious-informative sites from a total of 3,456 nucleotides generated with the neighbour-net algorithm for the 142 strains studied (<a href="http://splitstree.org/" target="_blank">http://splitstree.org/</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020256#pone.0020256-Huson1" target="_blank">[29]</a>. Strains were grouped into clonal complexes (CCs) with eBURST software (<a href="http://eburst.mlst.net/" target="_blank">http://eburst.mlst.net/</a>). Columns indicate the percentages of AI and NI strains in each CC. Recombination (R) and mutation (M) rates, based on MLST data, were evaluated as described by Feil <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020256#pone.0020256-Feil1" target="_blank">[31]</a>. The estimated recombination-mutation ratio (R/M) varied as a function of the CC to which the strain belonged.</p

PCR primers and amplicon sizes for prophage screening.

<p>PCR primers and amplicon sizes for prophage screening.</p

Serotype of <i>S. agalactiae</i> isolates from the various STs and CCs implicated in adult and neonatal invasive infections.

a<p>NT, nontypable.</p

Distribution of the <i>S. agalactiae</i> isolates of various origins, serotypes, sequence types, and clonal complexes between prophage DNA groups, as displayed by SYSTAT 12 software^a.

a<p>NP no prophage amplification; NT, nontypable.</p

CC, ST of <i>S. agalactiae</i> isolates from adult and neonatal invasive infections.

<p>CC, ST of <i>S. agalactiae</i> isolates from adult and neonatal invasive infections.</p

Oligonucleotide primers.

<p>Oligonucleotide primers.</p

Binding ability to immobilized human fibrinogen of the wild type (WT) L1 strain, and of isogenic mutant and complemented strains for <i>rgfAC</i>, <i>fbsA</i>, and <i>fbsB</i> genes.

<p>Flat bottomed 96-well polystyrene plates were coated with 21 nM human fibrinogen and 5×10⁶ to 5×10⁸ CFU per ml were added for 90 min at 37°C. Binding ability was calculated from the ratio between the number of bound bacteria and the number of bacteria present in the inoculum. Each experiment was performed at least three times. Boxes are means and bars are standard deviation of the means. The binding values of the mutant strains were significantly lower, at a <i>P</i> value of <0.001, than the values of the L1WT strain and of the corresponding complemented strains carrying <i>rgfAC</i>, <i>fbsA</i>, and <i>fbsB</i> genes on the pP1 plasmid.</p

Properties of <i>ΔrgfAC</i> mutant strains.

<p>(A) Fold change in transcription levels of <i>fbsA</i> (filled boxes) and <i>fbsB</i> (open boxes) genes in the isogenic <i>ΔrgfAC</i> mutants as compared to the wild type L1, L2, and L50 strains (WT). The amount of transcripts of each gene was normalized to the amount of <i>gyrA</i> transcripts and expressed relative to the level of transcription in corresponding WT strain. Each experiment was performed at least three times. Boxes are means and bars are standard deviation of the means. (B) Binding ability to immobilized human fibrinogen of the isogenic <i>ΔrgfAC</i> mutants (open boxes) and the WT strains (filled boxes). Flat bottomed 96-well polystyrene plates were coated with 21 nM human fibrinogen and 5×10⁶ to 5×10⁸ CFU per ml were added for 90 min at 37°C. Binding ability was calculated from the ratio between the number of bound bacteria and the number of bacteria present in the inoculum. The level of fibrinogen binding of WT strains is arbitrarily reported as 100 and the fibrinogen-binding levels of the isogenic mutants are relative values. Each experiment was performed at least three times. Boxes are means and bars are standard deviation of the means. * indicates that the binding values of the mutant strains were significantly lower than the values of the corresponding WT strains, at a <i>P</i> value of <0.001.</p