Classical Diphtheria Caused by Corynebacterium ulcerans in Germany: Amino Acid Sequence Differences between Diphtheria Toxins from Corynebacterium diphtheriae and C. ulcerans

Sir—Corynebacterium ulcerans is increasingly recognized as an emerging pathogen in various countries, including the United States [1], the United Kingdom [2], Japan [3], The Netherlands [4], Switzerland [5], and Italy [6]. In some of these countries, C. ulcerans is now the leading cause of classical pharyngeal diphtheria [2]. The ability of C. ulcerans infection to mimic diphtheria is explained by the fact that C. ulcerans—similar to Corynebacterium diphtheriae and Corynebacterium pseudotuberculosis—carries lysogenic β-corynephages coding for the diphtheria toxin (DT). We previously described 2 C. ulcerans isolates that were associated with cases of extrapharyngeal diphtheria (one was isolated from a cutaneous infection and the other caused lethal necrotizing sinusitis); sequencing of the respective tox gene of both strains revealed differences in nucleotide and amino acid sequences between C. diphtheriae and C. ulcerans DTs [7]. Here, we report a case of classical diphtheria caused by C. ulcerans in a 53-year-old woman who developed severe pharyngeal swelling and a whitish pseudomembrane. Her family practitioner obtained a swab specimen from the lesion, which grew C. ulcerans (API code 0111326), as detected by biochemical methods (API Coryne; bioMérieux) and 16S rRNA gene sequencing. The presence of DT was tested both by tox PCR and an Elek test, as described elsewhere [7]. Because of the severity of the swelling, the patient was referred to an ear, nose, and throat specialist who treated the infection with 2 injections of penicillin (1.2 million units). The patient recovered completely. She had received a complete course of primary immunizations during childhood; however, booster immunizations were never administered. Because no sequence data are available for the tox gene of C. ulcerans associated with classical pharyngeal diphtheria, we sequenced the complete tox gene of this strain (X959; GenBank accession number AY703827) and compared the amino acid sequence with those of a C. diphtheriae strain (GenBank accession number V01536) and 2 C. ulcerans strains, A2911 and A6361, that cause extrapharyngeal disease (GenBank accession numbers AY141013 and AY141014, respectively) [7]. The C. diphtheriae strain and the 3 C. ulcerans strains differed from each other in 26 amino acids (homology, 95.4%), 13 of which belonged to different amino acid classes (table 1). Most differences were located in the B fragment of the DT. In 3 positions (aa14, aa31, and aa262), the DT from C. ulcerans X959 was unique, harboring an amino acid that was not present in the other DTs. Interestingly, the DTs from both the C. diphtheriae and the C. ulcerans X959 strains have a serine in position 22 of the A fragment and an alanine in position 183 of the A fragment, whereas the DTs of the C. ulcerans A2911 and A6361 strains have a leucine in position 22 of the A fragment and a glutamine position 183 of the A fragment. Because these are the only differences between the DTs of the C. diphtheriae and C. ulcerans X959 strains, on the one hand, and the DTs of the 2 extrapharyngeal C. ulcerans strains, on the other hand, it might be speculated that these amino acids could be involved in pseudomembrane formation; however, loss of activity in mutated C. diphtheriae DT has not yet been linked to aa22 or aa183. Amino acid sequence differences in diphtheria toxin of Corynebacterium diphtheriae, Corynebacterium ulcerans X959, C. ulcerans A2911, and C. ulcerans A6361. The difference between C. diphtheriae and C. ulcerans tox genes was further corroborated by C. ulcerans tox—specific PCR using primers DT1 with 1467R or 1586R, respectively [7], yielding a positive PCR result only for the 3 C. ulcerans strains, and not for the C. diphtheriae strain. To our knowledge, this is the first report of classical diphtheria caused by C. ulcerans in Germany since the description of this species in 1995 [8]. Moreover, the sequence data on the isolated strain that caused classical diphtheria in the patient we describe confirm the previous results that we obtained for 2 isolates of C. ulcerans that caused extrapharyngeal infections, suggesting that DTs from C.diphtheriae and C. ulcerans are different, independent from an association with pharyngeal or extrapharyngeal disease. In addition, this first sequenced DT from a C. ulcerans isolate associated with classical diphtheria indicates that C. ulcerans DT amino acid sequences are less well conserved than their C. diphtheriae counterparts [9]. Potential conflicts of interest. All authors: no conflicts.