João A. Carriço

Illustration of a Common Framework for Relating Multiple Typing Methods by Application to Macrolide-Resistant Streptococcus pyogenes

ABSTRACT The studies that correlate the results obtained by different typing methodologies rely solely on qualitative comparisons of the groups defined by each methodology. We propose a framework of measures for the quantitative assessment of correspondences between different typing methods as a first step to the global mapping of type equivalences. A collection of 325 macrolide-resistant Streptococcus pyogenes isolates associated with pharyngitis cases in Portugal was used to benchmark the proposed measures. All isolates were characterized by macrolide resistance phenotyping, T serotyping, emm sequence typing, and pulsed-field gel electrophoresis (PFGE), using SmaI or Cfr9I and SfiI. A subset of 41 isolates, representing each PFGE cluster, was also characterized by multilocus sequence typing (MLST). The application of Adjusted Rand and Wallace indices allowed the evaluation of the strength and the directionality of the correspondences between the various typing methods and showed that if PFGE or MLST data are available one can confidently predict the emm type (Wallace coefficients of 0.952 for both methods). In contrast, emm typing was a poor predictor of PFGE cluster or MLST sequence type (Wallace coefficients of 0.803 and 0.655, respectively). This was confirmed by the analysis of the larger data set available from http://spyogenes.mlst.net and underscores the necessity of performing PFGE or MLST to unambiguously define clones in S. pyogenes.

Global optimal eBURST analysis of multilocus typing data using a graphic matroid approach

BackgroundMultilocus Sequence Typing (MLST) is a frequently used typing method for the analysis of the clonal relationships among strains of several clinically relevant microbial species. MLST is based on the sequence of housekeeping genes that result in each strain having a distinct numerical allelic profile, which is abbreviated to a unique identifier: the sequence type (ST). The relatedness between two strains can then be inferred by the differences between allelic profiles. For a more comprehensive analysis of the possible patterns of evolutionary descent, a set of rules were proposed and implemented in the eBURST algorithm. These rules allow the division of a data set into several clusters of related strains, dubbed clonal complexes, by implementing a simple model of clonal expansion and diversification. Within each clonal complex, the rules identify which links between STs correspond to the most probable pattern of descent. However, the eBURST algorithm is not globally optimized, which can result in links, within the clonal complexes, that violate the rules proposed.ResultsHere, we present a globally optimized implementation of the eBURST algorithm – goeBURST. The search for a global optimal solution led to the formalization of the problem as a graphic matroid, for which greedy algorithms that provide an optimal solution exist. Several public data sets of MLST data were tested and differences between the two implementations were found and are discussed for five bacterial species: Enterococcus faecium, Streptococcus pneumoniae, Burkholderia pseudomallei, Campylobacter jejuni and Neisseria spp.. A novel feature implemented in goeBURST is the representation of the level of tiebreak rule reached before deciding if a link should be drawn, which can used to visually evaluate the reliability of the represented hypothetical pattern of descent.ConclusiongoeBURST is a globally optimized implementation of the eBURST algorithm, that identifies alternative patterns of descent for several bacterial species. Furthermore, the algorithm can be applied to any multilocus typing data based on the number of differences between numeric profiles. A software implementation is available at http://goeBURST.phyloviz.net.

Analysis of genomic sequences by Chaos Game Representation

MOTIVATION Chaos Game Representation (CGR) is an iterative mapping technique that processes sequences of units, such as nucleotides in a DNA sequence or amino acids in a protein, in order to find the coordinates for their position in a continuous space. This distribution of positions has two properties: it is unique, and the source sequence can be recovered from the coordinates such that distance between positions measures similarity between the corresponding sequences. The possibility of using the latter property to identify succession schemes have been entirely overlooked in previous studies which raises the possibility that CGR may be upgraded from a mere representation technique to a sequence modeling tool. RESULTS The distribution of positions in the CGR plane were shown to be a generalization of Markov chain probability tables that accommodates non-integer orders. Therefore, Markov models are particular cases of CGR models rather than the reverse, as currently accepted. In addition, the CGR generalization has both practical (computational efficiency) and fundamental (scale independence) advantages. These results are illustrated by using Escherichia coli K-12 as a test data-set, in particular, the genes thrA, thrB and thrC of the threonine operon.

Analysis of Typing Methods for Epidemiological Surveillance of both Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Strains

ABSTRACT Sequence-based methods for typing Staphylococcus aureus, such as multilocus sequence typing (MLST) and spa typing, have increased interlaboratory reproducibility, portability, and speed in obtaining results, but pulsed-field gel electrophoresis (PFGE), remains the method of choice in many laboratories due to the extensive experience with this methodology and the large body of data accumulated using the technique. Comparisons between typing methods have been overwhelmingly based on a qualitative assessment of the overall agreement of results and the relative discriminatory indexes. In this study, we quantitatively assess the congruence of the major typing methods for S. aureus, using a diverse collection of 198 S. aureus strains previously characterized by PFGE, spa typing, MLST, and, in the case of methicillin-resistant S. aureus (MRSA), SCCmec typing in order to establish the quantitative congruence between the typing methods. The results of most typing methods agree in that MRSA and methicillin-susceptible S. aureus (MSSA) differ in terms of diversity of genetic backgrounds, with MSSA being more diverse. Our results show that spa typing has a very good predictive power over the clonal relationships defined by eBURST, while PFGE is less accurate for that purpose but nevertheless provides better typeability and discriminatory power. The combination of PFGE and spa typing provided even better results. Based on these observations, we suggest the use of the conjugation of spa typing and PFGE typing for epidemiological surveillance studies, since this combination provides the ability to infer long-term relationships while maintaining the discriminatory power and typeability needed in short-term studies.

Adjusted Wallace Coefficient as a Measure of Congruence between Typing Methods

ABSTRACT We propose a new coefficient, the adjusted Wallace coefficient (AW), and corresponding confidence intervals (CI) as quantitative measures of congruence between typing methods. The performance of the derived CI was evaluated using simulated data. Published microbial typing data were used to demonstrate the advantages of AW over the Wallace coefficient.

High Rates of Transmission of and Colonization by Streptococcus pneumoniae and Haemophilus influenzae within a Day Care Center Revealed in a Longitudinal Study

ABSTRACT Day care centers (DCCs) are unique settings where young children are at increased risk for colonization by pneumococci and Haemophilus influenzae. Although point prevalence studies in DCCs are frequent, only a few longitudinal studies on the dynamics of colonization have been published. We conducted a 1-year longitudinal study with 11 sampling periods on nasopharyngeal carriage of pneumococci and H. influenzae among 47 children who attended a single DCC. All isolates were antibiotyped and genotyped by pulsed-field gel electrophoresis. Pneumococci were also serotyped. Of the 414 samples obtained, 61.4% contained pneumococci, and 87% contained H. influenzae. Only 8.3% of the samples were negative for both species. Twenty-one pneumococcal clones and 47 H. influenzae clones were identified. Introduction of clones occurred during all year. Ninety-eight percent and 96% of all pneumococcal and H. influenzae isolates, respectively, belonged to clones shared by more than one child. Children were sequentially colonized with up to six pneumococcal clones (mean, 3.6) and five serotypes and nine H. influenzae clones (mean, 7.1). Clones with increased capacity for transmission and/or prolonged colonization were identified in both species. These two fitness properties appeared to be independent. In conclusion, among DCC attendees, a high rate of acquisition and turnover of strains was observed, and all children were overwhelmingly colonized by clones shared with others. DCCs are units where permanent introduction of new clones occurs, and attendees, as a whole, provide a pool of hosts where the fittest clones find privileged opportunities to persist and expand.

Assessment of Band-Based Similarity Coefficients for Automatic Type and Subtype Classification of Microbial Isolates Analyzed by Pulsed-Field Gel Electrophoresis

ABSTRACT Pulsed-field gel electrophoresis (PFGE) has been the typing method of choice for strain identification in epidemiological studies of several bacterial species of medical importance. The usual procedure for the comparison of strains and assignment of strain type and subtype relies on visual assessment of band difference number, followed by an incremental assignment to the group hosting the most similar type previously seen. Band-based similarity coefficients, such as the Dice or the Jaccard coefficient, are then used for dendrogram construction, which provides a quantitative assessment of strain similarity. PFGE type assignment is based on the definition of a threshold linkage value, below which strains are assigned to the same group. This is typically performed empirically by inspecting the hierarchical cluster analysis dendrogram containing the strains of interest. This approach has the problem that the threshold value selected is dependent on the linkage method used for dendrogram construction. Furthermore, the use of a linkage method skews the original similarity values between strains. In this paper we assess the goodness of classification of several band-based similarity coefficients by comparing it with the band difference number for PFGE type and subtype classification using receiver operating characteristic curves. The procedure described was applied to a collection of PFGE results for 1,798 isolates of Streptococcus pneumoniae, which documented 96 types and 396 subtypes. The band-based similarity coefficients were found to perform equally well for type classification, but with different proportions of false-positive and false-negative classifications in their minimal false discovery rate when they were used for subtype classification.

Comparison of Molecular Typing Methods for Characterization of Staphylococcus epidermidis: Proposal for Clone Definition

ABSTRACT In the present study we give some direction on the selection of the most appropriate typing method(s) to be used for the characterization of Staphylococcus epidermidis, in view of the most recent findings on the evolution, population structure, and epidemiology of this species. In order to achieve this aim, quantitative assessment of the correlation of the results of three typing methods—pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and staphylococcal chromosomal cassette mec (SCCmec) typing, which target different regions of the chromosome that evolve at different rates—was performed. In order to evaluate the discriminatory ability and the strength and direction of the correlation of the different typing methods, Simpsons index of diversity (SID), the adjusted Rand coefficient (AR), and the Wallace coefficient (W) were calculated. PFGE was the most discriminatory method (SID = 99%), followed by MLST (SID = 90%) and SCCmec typing (SID = 75%). The values of AR and W (0.10 < AR < 0.30; 0.50 < W < 0.75) indicated that the partition of the same isolate collection by PFGE, MLST, and SCCmec typing provided results that had only a poor correlation with each other. However, the information provided by the combination of PFGE and SCCmec enabled the prediction of the results obtained by MLST at the level of the clonal complex with a high degree of precision (W > 0.90). We propose that clones of S. epidermidis be defined by the combination of the PFGE type followed by the SCCmec type, which provides reliable information on the short-term epidemiology and the ability to predict with consistency long-term clonal evolution.

Molecular Characterization of Methicillin-Resistant Staphylococcus epidermidis Clones: Evidence of Geographic Dissemination

ABSTRACT Denmark and Iceland are countries where the frequency of methicillin-resistant Staphylococcus aureus is very low due to strict infection control and restrictive antibiotic use policies. In contrast, methicillin-resistant S. epidermidis (MRSE) continues to be isolated as a nosocomial pathogen. The molecular typing by pulsed-field gel electrophoresis (PFGE) of 136 MRSE isolates from five hospitals in Denmark and 94 MRSE isolates from one hospital in Iceland collected in 1997 and 1998 defined 40 different patterns. Closely related PFGE types were found in isolates recovered in Iceland, Denmark, Mexico, Uruguay, Greece, and Cape Verde, evidencing for the first time the geographic clonal dissemination of MRSE strains. The large majority (87.4%) of the MRSE isolates studied were multiresistant.

Analysis of Invasiveness of Pneumococcal Serotypes and Clones Circulating in Portugal before Widespread Use of Conjugate Vaccines Reveals Heterogeneous Behavior of Clones Expressing the Same Serotype

ABSTRACT To estimate the invasive disease potential of serotypes and clones circulating in Portugal before extensive use of the seven-valent pneumococcal conjugate vaccine, we analyzed 475 invasive isolates recovered from children and adults and 769 carriage isolates recovered from children between 2001 and 2003. Isolates were serotyped and genotyped by pulsed-field gel electrophoresis, and a selection of isolates were also characterized by multilocus sequence typing. We found that the diversities of serotypes and genotypes of pneumococci responsible for invasive infections and carriage were identical and that most carried clones could also be detected as causes of invasive disease. Their ability to do so, however, varied substantially. Serotypes 1, 3, 4, 5, 7F, 8, 9N, 9L, 12B, 14, 18C, and 20 were found to have an enhanced propensity to cause invasive disease, while serotypes 6A, 6B, 11A, 15B/C, 16F, 19F, 23F, 34, 35F, and 37 were associated with carriage. In addition, significant differences in invasive disease potential between clones sharing the same serotype were found among several serotypes, namely, 3, 6A, 6B, 11A, 14, 19A, 19F, 22F, 23F, 34, and NT. This heterogeneous behavior of the clones was found irrespective of the serotypes overall invasive disease potential. Our results highlight the importance of the genetic background when analyzing the invasive disease potential of certain serotypes and provide an important baseline for its monitoring following conjugate vaccine use. Continuous surveillance should be maintained, and current research should focus on uncovering the genetic determinants that contribute to the heterogeneity of invasive disease potential of clones sharing the same serotype.