Multilocus sequence typing of Corynebacterium ulcerans provides evidence for zoonotic transmission and for increased prevalence of certain sequence types among toxigenic strains

Abstract

Human-to-human-transmitted Corynebacterium diphtheriae was historically the main pathogen causing diphtheria and has therefore been studied extensively in the past. More recently, diphtheria caused by toxigenic Corynebacterium ulcerans is an emerging disease in several industrial countries, including the United Kingdom, the United States, France, and Germany. However, toxigenic C. ulcerans has so far been almost neglected in the development of epidemiologic tools. One of the most important tools in modern epidemiology to understand transmission pathways is sequence typing of pathogens. Here, we provide a protocol for multilocus sequence typing (MLST) to type C. ulcerans strains rapidly and relatively cost-effectively. Applying MLST to C. ulcerans for the first time, we show that related sequence types (STs) might be associated with the presence of the diphtheria toxin gene, which encodes diphtheria toxin (DT), the most important diphtheria-causing virulence factor. Interestingly, we found only two very closely related STs in the isolates derived from six dogs. Additionally, our data show that all STs derived from animals which were at least twice present in our analysis were found in humans as well. This finding is congruent with zoonotic transmission of C. ulcerans.

FIG 1

goeBURST diagram for the MLST data set of 44 C. ulcerans isolates. (A) An eBURST diagram was calculated using PHYLOViZ with the goeBURST algorithm. STs were grouped according to their allelic profiles. Solid lines indicate single-locus variants, except for the two lines marked with arrows, which indicate double-locus variants. The dotted lines reflect triple-locus variants and therefore more distantly related isolates. Each circle represents one ST, and the size represents the number of isolates in each ST. Exact numbers of isolates per ST are given in Table 2. Isolates which tested positive for the tox gene by PCR are colored red; negative isolates are colored blue. In the right panel, a pie chart depicts the fractions of tox-positive and -negative strains in the analysis. (B) eBURST diagram tree as in panel A. The isolates are color coded according to their host, as given in the key at right. The pie chart depicts the isolates from each host as fractions of the total number of isolates.

FIG 2

Neighbor-joining tree based on concatenated MLST sequencing data for 44 C. ulcerans isolates. The NJ tree was calculated using MEGA 6.0 (22). The strain identifiers are given at the ends of the branches. The numbers give the values for the bootstrapping test of the tree with 100 repetitions. The sequence type is given under “ST,” and the results of the tox-PCR are given in the rightmost panel. The lineage of STs 326, 330, 331, and 332 is highlighted in light gray. All isolates in this cluster were tox positive.