Recombination Blurs Phylogenetic Groups Routine Assignment in <i>Escherichia coli</i>: Setting the Record Straight

<div><p>The characterization of population structures plays a main role for understanding outbreaks and the dynamics of bacterial spreading. In <i>Escherichia coli</i>, the widely used combination of multiplex-PCR scheme together with goeBURST has some limitations. The purpose of this study is to show that the combination of different phylogenetic approaches based on concatenated sequences of MLST genes results in a more precise assignment of <i>E. coli</i> phylogenetic groups, complete understanding of population structure and reconstruction of ancestral clones. A collection of 80 <i>Escherichia coli</i> strains of different origins was analyzed following the Clermont and Doumith's multiplex-PCR schemes. Doumith's multiplex-PCR showed only 1.7% of misassignment, whereas Clermont's-2000 protocol reached 14.0%, although the discrepancies reached 30% and 38.7% respectively when recombinant C, F and E phylogroups were considered. Therefore, correct phylogroup attribution is highly variable and depends on the clonal composition of the sample. As far as population structure of these <i>E. coli</i> strains, including 48 <i>E. coli</i> genomes from GenBank, goeBURST provides a quite dispersed population structure; whereas NeighborNet approach reveals a complex population structure. MLST-based eBURST can infer different founder genotypes, for instance ST23/ST88 could be detected as the founder genotypes for STC23; however, phylogenetic reconstructions might suggest ST410 as the ancestor clone and several evolutionary trajectories with different founders. To improve our routine understanding of <i>E. coli</i> molecular epidemiology, we propose a strategy based on three successive steps; first, to discriminate three main groups A/B1/C, D/F/E and B2 following Doumith's protocol; second, visualization of population structure based on MLST genes according to goeBURST, using NeighborNet to establish more complex relationships among STs; and third, to perform, a cost-free characterization of evolutionary trajectories in variants emerging along the clonal expansion using parsimony methods of phylogenetic analysis.</p></div

<i>E. coli</i> population structure of our collection visualized by using goeBURST in combination with Con-MLST phylogenetic analysis.

<p>Up: Four phylogroups identified based on multiplex PCR techniques. Down: Seven phylogroups based on Con-MLST phylogenetic analysis.</p

Phylogenetic tree based on concatenated MLST-genes (Con-MLST) using BEAST v1.5.4 program.

<p>Phylogroups were established with posterior probability >0.95. Discrepancies between multiplex PCR and phylogeny are shown as *(discrepancies using Clermont's protocol) and # (discrepancies using Doumith's protocol). The discrepancies affecting members belonging to non-detected phylogroups (C, F and E) using multiplex PCRs are shown close to the character defining the phylogroup. Forty-eight sequences of reference strains downloaded from GenBank were used in the analysis, but one strain for each phylogroup is shown.</p

Network phylogenetic analysis based on Con-MLST obtained with NeighborNet algorithm in SplitsTree v.4.

<p>This representation allows inferring more complex interactions among the strains than goeBURST. The main phylogroups are differentiated in coloured circles. Members belonging to phylogroup C are located in two positions in the tree as two different patterns of recombination between B1 and A phylogroups were observed (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105395#pone-0105395-g005" target="_blank">Figure 5</a>).</p

MLST genes recombination frequencies inferred in the different <i>E. coli</i> phylogenetic groups.

<p>Recombination frequency was inferred as n° of phylogenetic incongruences with respect to total number of strains in each phylogroup. ^a<i>purA</i> gene belongs to phylogroup A in seven strains and in eight of them belongs to phylogroup B1. ^b<i>purA</i> gen belongs to phylogroup D in five strains and in six of them belongs to phylogroup B2.</p

Consensus tree based on Con-MLST; overprinted, the concatenated trees of individual genes used in MLST analysis.

<p>Maximum clade credibility tree was generated after burning 10% samples with posterior probability limit >0.5 using TreeAnnotator. Each segment in the figure corresponds to the seven genes used in MLST scheme in the following order: <i>adk-icd-fumC-recA-mdh-gyrB-purA</i>, being <i>adk</i> gene the inner segment. Recombination events in the different individual genes are shown. Phylogroups (A, B1, D, B2; U = unknown) were established with posterior probability >0.95. This approximation infers that most of the novel lineages could be the result of recombination events.</p

Genetic maps of Tn<i>1546</i> variants.

<p>Tn<i>1546</i> variants are represented as previously described by Novais et <i>al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060589#pone.0060589-Novais1" target="_blank">[27]</a> although grouped differently and specific types have been further explored (PP10, PP30): Tn<i>1546</i> prototype A corresponds to the original sequence described by Arthur <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060589#pone.0060589-Arthur1" target="_blank">[77]</a> and D corresponds to Tn<i>1546</i> variants from animals. Tn<i>1546</i> variants with IS<i>Ef1</i> within <i>vanX-vanY</i> intergenic region (PP2a, PP4, PP5, PP9, PP24) and Tn<i>1546</i> variants with IS<i>1216</i> insertions at different positions (PP10, PP2b, PP13, PP15, PP16, PP17, PP20, PP23, PP27, PP30, X) are represented. The positions of genes and open reading frames and the direction of transcription are depicted with open arrows. IS elements are represented by triangles; other sequences are designated by rectangles. DNA insertions are represented highlighting the first nucleotide upstream and downstream from the insertion sites whenever known. Deletions are indicated by dots and discontinuous lines indicate sequences that were not characterized. (^a) DNA sequence with homology to ORF3 (unknown protein product) and ORF1 (replication protein) of pEFNP1 plasmid (GenBank accession number AB038522). (^b) DNA sequence with no match to any sequence available in GenBank. (*) PP23 was identified in an isolate susceptible to teicoplanin; this variant contained an insertion in the <i>vanY</i> gene that would affect the transcription of <i>vanZ</i> and it might explain the susceptibility to this glycopeptide as previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060589#pone.0060589-Novais1" target="_blank">[27]</a>. (**) PP30 was identified in an ST78 isolate susceptible to both glycopeptides (MIC against vancomycin and teicoplanin of 4 mg/L) carrying <i>vanA</i>-Tn<i>1546</i>. This variant contained alterations within the <i>vanS-vanH</i> intergenic region (an IS<i>1216</i> insertion), which is involved in the expression and regulation of the resistance to vancomycin, and it constitutes the first description of a <i>vanA</i> isolate phenotypically susceptible to vancomycin in Portugal.</p

Plasmids identified in this study.

<p>Abbreviations: RFLP, restriction fragment length polymorphism; ST, sequence type; NI, not identified.</p>a<p>Plasmid type RFLP_12 (Rep_{17.2/pRUM-like} + Rep_2/pRE25/pEF1 + Rel_6/pEF1) contains a partial sequence of the replication gene of the RCR plasmid pEFNP1 (GenBank accession number AB038522), suggesting the integration of this RCR plasmid on the mobile element carrying Tn<i>1546</i> involving truncation of the rep_{14/pRI1/pEFNP1}.</p>b<p>Plasmid types RFLP_3, _4, _5, _6 and _13 (Rep_{17.2/pRUM-like} + Rel_6/pEF1 and eventually containing Rep_1/pIP501, Rep_2/pRE25/pEF1 or TAI_nc18) shared common bands and were identified in the same or different clonal backgrounds in different cities for extended periods of time.</p>c<p>Plasmids types RFLP 8, _9 and _10 also shared a variable number of common bands.</p>d<p>Plasmids showing patterns related to RFLP_27 (75–85 kb; rep_9/pAD1 + rel_5/pAD1 + rep_1/pIP501 + <i>par</i>_pAD1 and/or rep_2/pRE25/pEF1) initially recovered from the widespread ST6-CC2 Efs clone in Coimbra in 1996 and other Efs (ST55 and ST159) and Efm clones contained similar IS<i>Ef1</i>-Tn<i>1546</i> variants (PP-2a, PP-4, PP-9). Other highly related mosaic Inc18-pAD1-related plasmids carrying IS<i>1216</i>-Tn<i>1546</i> were recovered from ST6 VREfs and ST80 VREfm isolates (type ˝II_Efs ˝, rep_9/pAD1 +rel_5/pAD1 + <i>par</i>_pAD1 + rep_2/pRE25/pEF1<i>versus</i> type ˝II_Efm ˝, rep_9/pAD1 + rep_2/pRE25/pEF1).</p

Restriction fragment length polymorphism patterns of plasmids showing RFLP_5 profiles after digestion with <i>Cla</i>I (I) and <i>Eco</i>RI (II) restriction enzymes (New England Biolabs Inc, UK).

<p>Lane 1, RFLP_5 (PFGE H78, ST18 Efm,); lane 2, RFLP_5 (PFGE H72, ST18 Efm); lane 3, RFLP 5.2 (PFGE H100, ST280 Efm), lane 4, RFLP 5 (PFGE H78, ST18 Efm); lane 5, RFLP_5 (PFGE H78, ST18 Efm); lane 6, RFLP_5.2’ (PFGE H132, ST132 Efm).</p

Effect of the different 13-valent pneumococcal conjugate vaccination uptakes on the invasive pneumococcal disease in children: Analysis of a hospital-based and population-based surveillance study in Madrid, Spain, 2007-2015

<div><p>In the Community of Madrid, the 13-valent pneumococcal conjugate vaccine (PCV13) replaced the 7-valent (PCV7) in the fully government-funded Regional Immunization Program (RIP) in May, 2010, but was later excluded in May, 2012, and included again in January, 2015. These unique changes allowed us to assess the impact of the different pneumococcal vaccination policies on PCV13 uptake in infants and on the incidence rate (IR) of invasive pneumococcal disease (IPD) in children <15 years old. In this prospective, active, surveillance study, we estimated PCV13 uptakes, IR and incidence rate ratios (IRR) for total IPD and for IPD caused by PCV13- and non-PCV13 serotypes in children <15 years, stratified by age, in four periods with different vaccination policies: fully government-funded PCV7 vaccination, fully government-funded PCV13, mixed public/private funding and only private funding. Vaccine uptakes reached 95% in periods with public-funded pneumococcal vaccination, but fell to 67% in the private funding period. Overall, IR of IPD decreased by 68% (p<0.001) in 2014–15, due to 93% reduction in the IR of PCV13-type IPD (p<0.001) without significant changes in non-PCV13-type IPD. A fully government-funded PCV13 vaccination program lead to high vaccine uptake and dramatic reductions in both overall and PCV13-type IPD IR. When this program was switched to private PCV13 vaccination, there was a fall in vaccine coverage and stagnation in the decline of PCV13-type IPD with data suggesting a weakening of herd immunity.</p></div