Diphtheria is still endemic in many countries and, as exemplified by the FSU outbreak, is a potentially resurgent disease. Furthermore, given the low levels of protection within adult populations (in particular seniors), accurate and reproducible typing methods are required to monitor and characterize C. diphtheriae
. Although numerous typing techniques for C. diphtheriae
have been described, their use is often hindered by limited reproducibility and subjective analysis. MLST is able to circumvent these limitations by directly analyzing nucleotide information within selectively neutral housekeeping genes. The data produced are objective and, due to their portability, amenable to international collaborations. The C. diphtheriae
MLST database can be accessed at http://pubmlst.org/cdiphtheriae
This is the first use of MLST to characterize isolates of C. diphtheriae. MLST effectively typed 150 diverse C. diphtheriae isolates and confirmed findings of previous studies indicating that there is significant intraspecies genetic diversity. The data presented demonstrate that recombination has played a role in the evolution of C. diphtheriae. This was made evident by splits decomposition analysis and in the significant discordance observed between all MLST gene trees. However, since the congruence was deemed to show no greater similarity than that to trees of random topology within only two MLST loci, genetic exchange does not obscure all phylogenetic signals. Analysis of the complete genome sequence of C. diphtheriae reveals recent acquisition of pathogenicity factors. The observed recombination in this study highlights an obvious opportunity for these and other determinants to move across the population of C. diphtheriae.
To validate the accuracy and discriminatory power of the MLST scheme, the data were compared to an available subset of strains typed by the current gold standard, ribotyping. The MLST data were generally in concordance with the ribotyping findings. However, MLST provided greater strain resolution in two instances and ribotyping in one. MLST identified an SLV within three Sankt-Peterburg ribotype isolates and was able to distinguish between two ribotype Lyon isolates, which De Zoysa et al. had previously distinguished (5
). As with AFLP, PFGE, and RAPD studies (3
), MLST was unable to differentiate between the two predominant ribotypes associated with the FSU outbreak: Sankt-Peterburg and Rossija. Spoligotyping studies of this epidemic clonal group showed a clear divergence between these two ribotypes and suggested that the Rossija ribotype may have originated from one particular subpopulation of ribotype Sankt-Peterburg (21
). Likewise, it is clear by MLST that the Vladimir and Lyon ribotypes are clonally derived (eBURST group 5), as are the Cluj and Gatchina ribotypes (eBURST group 11).
MLST also identified a clonal complex (eBURST group 9) associated with an epidemiologically linked diphtheria outbreak in Haiti and the Dominican Republic, which both share the island of Hispaniola. A suspected diphtheria outbreak is believed to have originated in the Fond des Blancs region of Haiti in 2004 (CDC, personal communication). From 2001 to 2003, 13 diphtheria cases in Haiti and 120 in the Dominican Republic were reported (www.who.int/immunization_monitoring/data/en/
). This increased significantly, to 253 cases in Haiti and 177 in the Dominican Republic, from 2004 to 2006 (see above URL). Of 28 isolates obtained during the outbreak, 93% belonged to a clonal complex comprising ST-31 and ST-4 isolates (eBURST group 9). Since two further isolates, collected in 2000 and 1995, were ST-31 and ST-4, respectively, it is evident that the outbreak strains were circulating in the preepidemic period and may belong to the regions of endemicity for a C. diphtheriae
While members of some clonal complexes were globally distributed, other groups were associated with specific locations. Notably, members of eBURST group 8 were obtained from geographically disparate countries (Thailand, Russia, and Guatemala), whereas exclusively Russian isolates belonged to eBURST group 10. However, these findings may be the result of sampling limitations and require a wider sample analysis.
It was not possible to identify a definitive association between ST and toxigenicity for biotypes mitis, intermedius, and gravis. Likewise, there was not always a clear association between biotype and ST, indicating that biotypes are not necessarily stable epidemiological markers, which is wholly consistent with C. diphtheriae being identified in this work as having only a weakly clonal structure. However, isolates identified by biotype and nontoxigenic status as typical belfanti strains clustered together within lineage II.
MLST provides a valuable tool for monitoring and characterizing endemic and epidemic C. diphtheriae strains. The data produced are portable, reproducible, and unambiguous. Strain discrimination was in accordance with ribotyping data, and clonal complexes associated with disease outbreaks were identified.