Four genotyping schemes for phylogenetic analysis of Pseudomonas aeruginosa: comparison of their congruence with multi-locus sequence typing.

Several molecular typing schemes have been proposed to differentiate among isolates and clonal groups, and hence establish epidemiological or phylogenetic links. It has been widely accepted that multi-locus sequence typing (MLST) is the gold standard for phylogenetic typing/long-term epidemiological surveillance, but other recently described methods may be easier to carry out, especially in settings with limited access to DNA sequencing. Comparing the performance of such techniques to MLST is therefore of relevance. A study was therefore carried out with a collection of P. aeruginosa strains (n = 133) typed by four typing schemes: MLST, multiple-locus variable number tandem repeat analysis (MLVA), pulsed-field gel electrophoresis (PFGE) and the commercial DiversiLab microbial typing system (DL). The aim of this study was to compare the results of each typing method with MLST. The Simpson's indices of diversity were 0.989, 0.980, 0.961 and 0.906 respectively for PFGE, MLVA, DL and MLST. The congruence between techniques was measured by the adjusted Wallace index (W): this coefficient indicates the probability that a pair of isolates which is assigned to the same type by one typing method is also typed as identical by the other. In this context, the congruence between techniques was recorded as follow: MLVA-type to predict MLST-type (93%), PFGE to MLST (92%), DL to MLST (64.2%), PFGE to MLVA (63.5%) and PFGE to DL (61.7%). Conversely, for all above combinations, prediction was very poor. The congruence was increased at the clonal complex (CC) level. MLST is regarded the gold standard for phylogenetic classification of bacteria, but is rather laborious to carry out in many settings. Our data suggest that MLVA can predict the MLST-type with high accuracy, and even higher when studying the clonal complex level. Of the studied three techniques MLVA was therefore the best surrogate method to predict MLST

Number of genotypes, clonal complexes and singletons retrieved from the four genotyping schemes.

<p>Number of genotypes, clonal complexes and singletons retrieved from the four genotyping schemes.</p

Minimum Spanning trees (MSTs) of 133 <i>P. aeruginosa</i> isolates based on MLVA, MLST, PFGE and DiversiLab data.

<p>Each network represents its own genetic relatedness among isolates and displays its relative concordance against MLVA-CC and MLST-CC. A; Clustering of MLVA profile was done using the categorical coefficient. Each circle represents an MT, the size of which indicates the number of isolates with this particular type. Black lines connecting pairs of MTs indicate that they differ in one VNTR locus (thick lines), two VNTR locus (thin), or three to 15 VNTR locus (dashed). Grey zones surround MTs that belong to the same MLVA-CC. B; Clustering was done using MLST character data. Each circle represents an ST, the size of each circle indicates the number of isolates with this type. Black lines connecting pairs of STs indicate that they differ in one allele locus (thick lines), two alleles locus (thin), or three to seven (dashed). Grey zones surround STs that belong to the same MLST-CC. C; MST with permutation resampling with majority summary based on band matching class, each single node represents a distinct PFGE-type, the collapsed nodes represent closely related patterns with ≤6 band class differences. D; MST with permutation resampling with majority summary based on band matching class, each single node represents a distinct DL pattern, the collapsed nodes represent closely related patterns with ≤2 band class differences.</p

Congruence between typing methods determined by the Adjusted Wallace coefficient [95% CI].

<p>CI, confidence interval.</p><p>MLST-CC, MLST clonal complex.</p><p>MLVA-CC, MLVA clonal complex.</p

Simpson's index of diversity of the different typing methods.

<p>SID, Simpson's index of diversity.</p><p>CI, confidence interval.</p

Graphical representation of 44 isolates belonging to MLST-CC235 analyzed by PFGE, DL, MLST and MLVA.

<p>A; Dendrogram generated by PFGE banding pattern, the clustering was done by UPGMA using the Dice coefficient with a tolerance position of 1%. 19 PFGE types were obtained with a cut-off of 80% of similarity. B; Dendrogram generated by the Pearson cluster analysis of rep-PCR results. PFGE-type, ST, MT and virtual gel are shown for each isolate. STs are depicted in colored boxes. A similarity index cut-off value of 95% was used by the DiversiLab® software to define genetic classification and to highlight the 9 DL-types. C; Minimum Spanning Tree illustrating the clonal complex MLST-CC235, the predominant ST235 is the primary founder surrounded by its SLVs (STs 976, 230, 989 and 227). Each color was assigned to each distinct ST. D; Minimum Spanning Tree of MLST-CC235 isolates typed by MLVA, Each circle represents an MT and 13 different colors were assigned to each distinct MT.</p