Nontoxigenic Tox-Bearing Corynebacterium ulcerans Infection among Game Animals, Germany

Tobias Eisenberg

, Peter Kutzer, Martin Peters, Andreas Sing, Matthias Contzen, and Jörg Rau

Author affiliations: Landesbetrieb Hessisches Landeslabor, Gießen, Germany (T. Eisenberg); Landeslabor Berlin-Brandenburg, Frankfurt (Oder), Germany (P. Kutzer); Chemisches und Veterinäruntersuchungsamt Westfalen, Standort Arnsberg, Germany (M. Peters); Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Oberschleißheim, Germany (A. Sing);Chemisches und Veterinäruntersuchungsamt Stuttgart, Fellbach, Germany (M. Contzen, J. Rau)

Abstract

Corynebacterium ulcerans may cause diphtheria in humans and caseous lymphadenitis in animals. We isolated nontoxigenic tox-bearing C. ulcerans from 13 game animals in Germany. Our results indicate a role for game animals as reservoirs for zoonotic C. ulcerans.

The Corynebacterium species C. diphtheriae, C. ulcerans, and C. pseudotuberculosis form the C. diphtheriae group, as shown by 16S rRNA gene sequence analysis (1). Strains of this group carrying lysogenic β-corynephages might produce the tox-encoded diphtheria toxin (DT) (2). Moreover, C. ulcerans and C. pseudotuberculosis may produce phospholipase D, the major virulence factor involved in caseous lymphadenitis, which is a disease that mainly affects sheep, goats, and horses (3).

From a public health perspective, diphtheria is the most critical human disease attributed to coryneform bacteria (3). In recent years, cases of diphtheria caused by C. ulcerans have outnumbered those caused by C. diphtheriae (4). C. diphtheriae carriage is nearly exclusively restricted to humans; C. ulcerans is a zoonotic pathogen and has been found in various animal species that have contact with humans (5). C. ulcerans is most closely related to C. pseudotuberculosis, and distinction between these species is often difficult when using standard bacteriological methods (5). The aim of this study was to comprehensively characterize 13 C. ulcerans strains isolated from game animals in Germany.

The Study

Strains of C. ulcerans were isolated during routine bacteriological investigations in conjunction with necropsies of wild animals that were found dead or that had suspicious lesions during 1997–2013. Isolates of coryneform bacteria were subjected to conventional biochemical tests (3), and were evaluated after prolonged incubation at 37°C for as long as 14 days. For further characterization, commercial tests API Coryne and VITEK2-compact with cards for coryneform bacteria and corynebacteria and anaerobes (bioMérieux, Nürtingen, Germany) were used according to the manufacturer’s instructions.

We conducted the reverse CAMP test by using Staphylococcus aureus American Type Culture Collection (ATCC [Manassas, VA, USA]) 25923 and the CAMP test by using Rhodococcus equiATCC 33701 according to standard procedures on Columbia sheep blood agar (Oxoid, Wesel, Germany) (3). We determined DT production using a modified Elek test (6); we used C. diphtheriae NCTC 10648 and C. diphtheriae NCTC 10356 as positive and negative controls, respectively; and performed a cytotoxicity assay using Vero cells (7). The rpoB and tox genes were partially amplified by using primer pairs C2700F/C3130R and DT1/DT2, respectively, as described (5).

Figure 1

Thumbnail of Cluster analysis of respective spectra obtained by Fourier-transform infrared-spectroscopy by using OPUS Software version 4.2 (BrukerOptics, Ettlingen, Germany). In each case, 2 infrared spectra of isolates from game animals and a selection of several Corynebacterium ulcerans and C. pseudotuberculosis strains were used for calculation by using the Ward algorithm. The dendrogram depicts the arrangement of isolates in groups according to their spectral differences.

Figure 1. . Cluster analysis of respective spectra obtained by Fourier-transform infrared-spectroscopy by using OPUS Software version 4.2 (BrukerOptics, Ettlingen, Germany). In each case, 2 infrared spectra of isolates from game animals and...

PCR products were purified for sequence analysis by using the Double Pure Combi Kit (Bio&SELL, Nürnberg, Germany). Both strands of the rpoB and tox PCR products were sequenced by Microsynth (Balgach, Switzerland) by using the amplification primers. Sequence analysis was performed by using the BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi

) sequence analysis tool. Additionally, coryneform isolates in which C. ulcerans was suspected were analyzed by using Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) and by using Biotyper version 3.3.1.0 (BrukerBiotyper; BrukerDaltonics, Bremen, Germany). The database used (DB 4613) comprised spectra from 71Corynebacterium species including C. diphtheriae, C. ulcerans, and C. pseudotuberculosis. For Fourier-transform infrared (FT-IR) spectroscopy, bacterial isolates were harvested and prepared as described (5). IR spectra were recorded by using an FT-IR spectrometer (Tensor 27 with High Throughput Screening eXTension HTS-XT module) and OPUS software version 4.2 (BrukerOptics, Ettlingen, Germany). IR spectra of isolates from game animals and selected C. ulcerans and C. pseudotuberculosis strains were compared by cluster analysis by using the second derivation of vector normalized spectra (8). The dendrogram obtained depicts the arrangement of isolates in groups according to their spectral differences (Figure 1).