Molecular and Epidemiological Characterization of Toxigenic and Nontoxigenic Corynebacterium diphtheriae, Corynebacterium belfantii, Corynebacterium rouxii, and Corynebacterium ulcerans Isolates Identified in Spain from 2014 to 2019

Abstract

This study examines the microbiological and epidemiological characteristics of toxigenic and nontoxigenic Corynebacterium isolates submitted to the national reference laboratory in Spain, between 2014 and 2019, in order to describe the current situation and improve our knowledge regarding these emerging pathogens. Epidemiological information was extracted from the Spanish Surveillance System. Microbiological and molecular characterization was carried out using phenotypic methods, multilocus sequence typing (MLST), whole-genome sequencing (WGS), and core genome MLST (cgMLST). Thirty-nine isolates were analyzed. Twenty-one isolates were identified as Corynebacterium diphtheriae (6 toxigenic), 14 as C. belfantii, 4 as C. ulcerans (3 toxigenic), and 1 as C. rouxii One C. diphtheriae isolate was identified as nontoxigenic tox gene bearing (NTTB). Ages of patients ranged from 1 to 89 years, with 10% (3/30) of nontoxigenic and 22% (2/9) of toxigenic isolates collected from children less than 15 years. Twenty-five of the patients were males (17/30 in nontoxigenic; 8/9 in toxigenic). MLST identified 28 sequence types (STs), of which 7 were described for the first time in Spain. WGS analysis showed that 10 isolates, including 3 toxigenic isolates, harbored a variety of antibiotic resistance genes in addition to the high prevalence of penicillin resistance phenotypically demonstrated. Phylogenetic analysis revealed one cluster of isolates from family members. Risk information was available for toxigenic isolates (9/39); 3 patients reported recent travels to countries of endemicity and 3 had contact with cats/dogs. One unvaccinated child with respiratory diphtheria had a fatal outcome. Including nontoxigenic Corynebacterium infections in disease surveillance and using WGS could further improve current surveillance.

Keywords: Corynebacterium; Corynebacterium infections; diphtheria; diphtheria toxin; epidemiology; microbiology.

FIG 1

Nontoxigenic and toxigenic C. diphtheriae, C. belfantii, and C. ulcerans isolates in Spain by age and sex groups, 2014 to 2019.

FIG 2

ST profiles of toxigenic and nontoxigenic C. diphtheriae, C. belfantii, and C. ulcerans isolates in Spain, 2014 to 2019. The asterisk (*) indicates the C. ulcerans isolates and underlined STs correspond to C. belfantii isolates. The STs that were identified once are gray, and those that have been identified in more than one specimen share a color.

FIG 3

cgMLST analysis. (A) Minimum spanning tree of C. diphtheriae and C. belfantii isolates generated using a cluster distance threshold of ≤5 alleles. Species and biovars are color coded as follows: Corynebacterium belfantii (turquoise), Mitis (yellow), and Gravis (green); and all toxigenic isolates are marked with a solid red circle. The reference genomes used are white. (B) Neighbor joining tree generated from the C. diphtheriae cgMLST scheme. Reference genomes used were C. belfantii (FRC0043), C. diphtheriae bv. Gravis (NCTC 13129) and Mitis (ISS3319), and C. diphtheriae subsp. lausannense (CHUV2995). Isolates were color-coded according to their subtypes as in A, with red rectangles denoting the toxigenic isolates. (C) Minimum spanning tree of the C. ulcerans isolates generated using a cluster distance threshold of ≤5 alleles. Isolates are color-coded according to their toxigenicity; toxigenic isolates (purple), nontoxigenic isolates (green), and the reference genome used NCTC 017317 (white). (D) Neighbor joining tree of C. ulcerans isolates consisted of 1,209 target loci and 961 more loci for an accessory target scheme. Toxigenic isolates are denoted with a red rectangle.

FIG 4

cgMLST analysis of C. belfantii and C. rouxii. (A) Minimum spanning tree of C. belfantii and C. rouxii isolates generated using a cluster distance threshold of ≤5 alleles. Reference genomes for C. belfantii (FRC0043) and C. rouxii (FRC0190) are white. (B) Neighbor joining tree generated from the cgMLST scheme using the reference genomes for C. belfantii (FRC0043) and C. rouxii (FRC0190) (in white).