Zoonotic transmission of diphtheria toxin‐producing Corynebacterium ulcerans

Diphtheria caused by toxin‐producing Corynebacterium ulcerans is a re‐emerging human disease that can cause local and systemic sequelae. In Australia, toxigenic diphtheria is a rare notifiable communicable disease, due to high‐vaccination coverage. The public health management of cutaneous cases of toxigenic C. ulcerans varies between jurisdictions, as opposed to the more uniform public health response to toxigenic Corynebacterium diphtheriae presenting as respiratory or laryngeal diphtheria.

Humans are the only known reservoir of C. diphtheriae (Centers for Disease Control, Prevention (US); Education, Information, Partnership Branch, 2005).However, toxigenic C. ulcerans and C. pseudotuberculosis have been isolated from wild and domesticated animals and have demonstrated a capacity for zoonotic transmission (Belchior et al., 2009;Othieno et al., 2019).
C. ulcerans in particular has emerged as a cause of illness in humans, having first been reported as a human pathogen in 1920 (Gilbert, 1926).Indeed, C. ulcerans appears to be displacing C. diphtheriae as the dominant cause of respiratory diphtheria in the United Kingdom (UK) and Western Europe (Bonmarin et al., 2009;Gower et al., 2020) and is increasing in incidence in the United States (Otshudiema et al., 2021).Generally, infection is associated with mild respiratory and cutaneous manifestations with more serious manifestations thought to be linked to immunocompromise or inadequate vaccination (Gower et al., 2020).C. ulcerans has been isolated from many domesticated and wild animals (Eisenberg et al., 2015;Seto et al., 2008;Sting et al., 2023;Tejedor et al., 2000;Terriere et al., 2022;Thomas et al., 2022); the earliest reports of human infection were associated with the drinking of unpasteurised bovine products (Hacker et al., 2016), though companion animals such as cats and dogs are more frequently implicated in recent reports (Abbott et al., 2020;Carfora et al., 2018;Museux et al., 2023).
The natural history of diphtheria and human-human transmission is understood to differ between vaccinated and unvaccinated individuals, with unvaccinated cohorts experiencing a greater rate of infection (Truelove et al., 2020).Human-to-human transmission of C. ulcerans has not been established, but case reports suggest that in many cases where asymptomatic human contacts were positive for toxigenic C. ulcerans, animal contact was also present (Konrad et al., 2015;Martini et al., 2019;Othieno et al., 2019;Wagner et al., 2010).Whether humans are a reservoir for C. ulcerans is not definitive, however, in a European carriage study, toxigenic C. ulcerans was not shown to be a human nasopharyngeal commensal, unlike other Corynebacterium species (Teutsch et al., 2017).The zoonotic reservoir and zoonotic-human transmission are established with changes occurring in international public health management guidelines regarding the management of animal contacts (Bundesministerium für Gesundheit Deutschland -Robert Koch Institut, 2018;Hacker et al., 2016; Ministre de la Santé France, Haut Conseil de la santé publique, 2021; Queensland Government Department of Health, 2023; UK Health Security Agency, Department of Health and Social Care, 2023).Currently, in Australia, only one state's guidelines, Queensland, recommends contacting biosecurity "to discuss the practicality of screening companion animals" (Queensland Government Department of Health, 2023); other jurisdictional guidelines are silent on this, highlighting a need for review.
In humans, the diphtheria toxoid vaccine has been in widespread use since the 1940's and is highly effective in reducing symptomatic disease but not transmission (Truelove et al., 2020;Wiedermann, 2020).Vaccination coupled with widespread anti-microbial use is understood to be responsible for the reduction in the number of notified cases and elimination in some countries (Truelove et al., 2020).However, many regions have observing an increasing incidence of disease caused by toxigenic diphtheroid and non-diphtheroid Corynebacterium isolates in the past 10 years (Clarke et al., 2019).Causes may include inadequate vaccination resulting from vaccine hesitancy and waning immunity, lower vaccination coverage among refugees and from health service disruption due to COVID-19 and conflict, increasing globalization and importation of disease from affected areas (Clarke et al., 2019).The role of the animal reservoir in circulating and perpetuating human C. ulcerans disease is not yet described.
We report a case of cutaneous diphtheria due to toxigenic C. ulcerans, with evidence of zoonotic transmission from domestic dog companions.This is the first case of diphtheria from C. ulcerans and only the third diphtheria illness notified in Western Australia since 1990.Furthermore, we provide a summary of the literature base regarding cases of zoonotic transmission of C. ulcerans and propose guidance on the public health management of cases presumed to have been transmitted by animals.Routine bacterial microscopy and culture (on standard selective and non-selective media) were performed on finger swabs from the human case, incubated at 35°C and interrogated at 24 and 48 h for growth.Isolate identification was performed using Matrix-assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF) (Bruker Biotyper; Bruker Daltonik GmbH, Bremen, Germany).Bacterial microscopy and culture were also performed on paired canine nose and throat swabs.The presence of the diphtheria toxin tox gene in human and animal specimens was determined using a published polymerase chain reaction (PCR) assay (Pallen et al., 1994).Elek testing for diphtheria toxin was not available and thus not performed.Comparative analysis of tox gene sequences was performed using ABRIcate which includes interrogation of the Virulence Factor Database (VFDB) (Liu et al., 2022).

| ME THODS
Antimicrobial susceptibility testing was performed using gradient diffusion tests (E-test®; bioMerieux, Marcy-l'Étoile, France) for penicillin, vancomycin, cotrimoxazole, clindamycin, ciprofloxacin and doxycycline according to methods and breakpoints published by the Clinical and Laboratory Standards Institute (CLSI) (Hindler & Richter, 2016).A maximum parsimony tree was generated using Molecular Evolutionary Genetics Analysis (MEGA) X software (version 10.2.0) (Kumar et al., 2018) using a reference-free core genome alignment generated from Split Kmer Analysis (SKA, version 1) (Harris, 2018) built from 113 published C. ulcerans genomes (accession numbers listed in Dataset S1), which enabled determination of pairwise single nucleotide polymorphism (SNP) distances and sequence alignments.
The bootstrap value was set to 1000, with the Subtree-Pruning-Regrafting (SPR) as the maximum parsimony search parameter.

| C A S E SUMMARY
A 55-year-old Western Australian resident, without recent travel, sustained a knife laceration to her right middle finger while preparing raw chicken at home.Within 24 h of injury, the wound was erythematous with localised pain.She presented to an emergency department a week later with increasing pain and ascending cellulitis and was subsequently admitted for management.The case TA B L E 1 Prevalence of Corynebacterium ulcerans infection among different animal species.had an extensive medical history with multiple comorbidities that included end-stage heart failure, type 2 diabetes mellitus, ischaemic heart disease, chronic kidney disease, hypertension, Factor V

Animal
Leiden mutation and clotting and stroke events, and SARS-CoV-2 infection 1 month prior.During admission, an adult diphtheria/ tetanus booster was administered, intravenous (IV) cefazolin 1 g eight-hourly (3 doses plus a 2 g loading dose) was commenced, and after 1 day the patient was discharged on oral flucloxacillin 500 mg six-hourly for 5 days.
Swab specimens from the infected finger of the case were collected and submitted to PathWest Laboratory Medicine, WA (PathWest) for routine bacterial culture (as described above).
Abundant gram-positive bacilli were seen on microscopy, and within 24 h of incubation, there was moderate pure growth of white-grey colonies (Figure 1) which were subsequently identified as C. ulcerans by MALDI-TOF.The presence of the diphtheria tox gene was confirmed by PCR from the clinical isolate.On susceptibility testing, minimum inhibitory concentrations for penicillin and clindamycin were in the intermediate range (0.25 and 1.0 μg/mL respectively), while the isolate was susceptible to the remaining antimicrobials.
Based on these findings, the patient was recalled to the emergency department for further review and management, 12 days after the initial injury.On presentation, the wound was well healed with a good range of movement, minimal pain and only minor erythema.
Features of complicated diphtheria toxin-related disease including cardiac, respiratory and neurological symptoms were absent, and she was discharged home on 7 days of oral doxycycline.
Notification to public health for urgent investigation occurred concurrently.The case was interviewed and provided with information on the risk of transmission and wound hygiene.Primary immunisation in childhood and a subsequent booster of adult diphtheria/ tetanus 11 years prior to the current illness were confirmed.Case isolation was recommended until the course of doxycycline was completed and the wound was healed.Nose and throat swabs were submitted for culture to assess C. ulcerans carriage, which returned negative.
Five household contacts were identified, and urgent nose and throat swabs submitted for these contacts were also negative.
They were advised to monitor for symptoms and seek medical re- evidence of cutaneous or respiratory disease and were asymptomatic.The two canine isolates were referred to PathWest for comparison with the human isolate.All the three isolates were found to have the same antibiotic susceptibility profile.Genetically identical diphtheria toxin genes were confirmed in the three isolates, and matched 100% with a published diphtheria toxin gene found previously in C.
ulcerans (GenBank® accession number: CP011913).There was a maximum of three SNP differences between the isolates suggesting a common source between the canine and human isolates.A maximum parsimony phylogenetic tree was constructed to identify the closest neighbour to isolates in this study, which were from Germany (Figure 2).The phylogenetic tree demonstrates highly related C. ulcerans isolates intertwined between humans and animals, supporting zoonotic transmission theories.

| RE VIE W OF ZOONOTI C TR AN S MISS I ON C A S E S AND MANAG EMENT A PPROACH
Given the global re-emergence of this pathogen, a literature review was completed, which identified 17 cases of reported zoonotic transmission of respiratory and cutaneous C. ulcerans summarized in Table 2.A MEDLINE search was performed with the combination of search terms: 'Corynebacterium ulcerans', 'diphtheria', 'zoonoses' and 'human' for publications in the English language since 1966.In 12 cases, comparative genetic testing (by whole-genome sequencing, multilocus sequence typing, pulsed-field gel electrophoresis or ribotype analysis) demonstrated that the animal isolates were identical or highly similar to the human case isolates, establishing zoonotic transmission as a highly probable source.Comparative genomics was not performed by Corti et al. (2012), and in a further 3 studies, zoonotic transmission was only inferred on clinical and epidemiological grounds as animal contacts were unable to be tested (Hatanaka et al., 2003;Konrad et al., 2015;Moore et al., 2015).Of the 12 cases with comparative genetic testing, six were thought to have acquired infection from pet dogs (Fuursted et al., 2015;Hogg et al., 2009;Lartigue et al., 2005;Meinel et al., 2015;Monaco et al., 2017;Othieno et al., 2019), and six from pet cats (Berger et al., 2019;Hoefer et al., 2022;Vandentorren et al., 2014;Wake et al., 2021;Yasuda et al., 2018;Yoshimura et al., 2014).In one of the case reports, recent diphtheria toxoid vaccination was reported (in this case, 1 year prior) (Vandentorren et al., 2014), with most cases reporting vaccination status as inadequate or unknown.Interestingly, one report identified both asymptomatic carriage in human and canine household contacts.Transmission was surmised to be zoonotic; however, human-to-human could not be excluded (Othieno et al., 2019).A study by Meinel et al. provided  Outside of these aforementioned jurisdictions, no other specific guidelines on the public health management of C. ulcerans were identified in the public domain.

| DISCUSS ION
Our cutaneous diphtheria case reinforces the zoonotic transmission potential of C. ulcerans, particularly for inadequately vaccinated and/or immune-compromised individuals.Both respiratory and cutaneous presentations of toxigenic C. ulcerans resulting from animal contacts have been described, with severe illness occurring in some cases.This growing body of evidence has highlighted the threat of zoonotic transmission in reversing long-term progress toward diphtheria elimination.
Human and animal prevalence studies are lacking in their ability to determine the natural reservoir for C. ulcerans.Questions remain as to the breadth of species able to be colonized and infected; the extent of zoonotic colonization between species; the frequency of toxigenic conversion and symptomatic illness in those colonized; the role of environmental factors on acquisition and transmission; and the animal and human behaviours that confer the greatest risk to humans.With pet ownership increasing globally (HealthforAnimals, 2022), and an ageing population with potentially greater rates of immune compromise, the burden of C.
ulcerans infection may increase.These questions all have implications for optimal public health management of this pathogen.
From our review of global literature and our own experiences in managing this case, we recommend the following measures be considered: Where susceptibility to several agents is demonstrated, consultation with expert veterinarians regarding optimal selection is advised, with consideration of alternatives to penicillin given the anecdotal reports of penicillin non-response particularly in canines.
4. That animal companions who undertake treatment be re-swabbed for persistent carriage if they will continue to be in contact with inadequately vaccinated or immune-compromised humans.
Our investigation has several limitations.First, detection of the diphtheria toxin gene by PCR does not confirm that the diphtheria strain is toxin-producing because strains containing a non-functional Maximum parsimony phylogenetic tree of the core genome of the three Corynebacterium ulcerans isolates from our study (coloured dark purple) and 113 isolates published in the National Centre for Biotechnology Information (NCBI) database.Colours on the tip node represent the country of origin, while host species is represented in the outer ring and separated into four categories; canis lupus familiaris (green), homo sapiens (yellow), other host (red) and unknown host (purple).The regular phylogenetic tree shows in greater resolution 14 isolates, including the three from our study, that cluster closely together in the circular phylogenetic tree.toxin gene have been described (Melnikov et al., 2022).The Elek test is considered the gold standard for detection of diphtheria toxin (Melnikov et al., 2022).While we were unable to demonstrate phenotypic toxin production in this case, the Elek test is time-consuming, labour intensive and not suited to screening large numbers of isolates (Melnikov et al., 2022).In line with this, Elek testing for diphtheria toxin has not been available in Australia since the late 1990's.Second, we were not able to complete a second round of canine clearance swabs after repeat treatment with enrofloxacin, so are not able to confirm that this treatment approach resulted in clearance.

TA B L E 2 Overview of zoonotic transmission of
In these situations, the role of public health authorities, veterinarian authorities, private veterinarian services and pet owners is complex and therefore national and/or jurisdictional authorities would benefit from outlining a coordinated local approach to case management and animal management.These approaches must balance the above recommendations with potential costs relating to the administrative burden of arranging animal testing

Impacts•
This article describes the increasing burden of Corynebacterium ulcerans in humans and highlights the zoonotic transmission potential of this re-emerging pathogen.• Best practices in the public health management of animal contacts of C. ulcerans cases are not yet well described; lessons can be learned from our experience in managing a case of zoonotically acquired C. ulcerans.• Jurisdictions should adopt a coordinated local approach to the management of C. ulcerans cases, inclusive of multidisciplinary stakeholders.part of the urgent public health response to a case of C. ulcerans under the Western Australian Public Health Act 2016.Written consent was obtained from both the case, and the owners of the canine companion animals, who reviewed the final manuscript, figures and tables in entirety.

For
whole genome experiments, DNA was extracted from the pure subculture of the human and canine C. ulcerans isolates using the QIAsymphony DSP DNA Mini from the QIAsymphony DNA extraction system (QIAGEN, The Netherlands).Genomic libraries were prepared using the Illumina Nextera XT DNA Library Preparation Kit (Illumina, United States) as per the manufacturer's instructions.The Illumina MiSeq platform and the MiSeq Reagent Kit v3 (both from Illumina, United States) were used to sequence paired-end reads which generated 23-31 contigs.
Abbott et al. (2020) view and testing if symptoms developed.One contact had acute respiratory symptoms at the time of review and was excluded from occupational activities until negative respiratory virus multiplex PCR and negative swab culture results were returned.Based on antibiotic susceptibility patterns and expert microbiological advice, oral doxycycline was prescribed for the adult household contacts and phenoxymethylpenicillin was prescribed for the paediatric household contact.One contact was confirmed to have received a diphtheria toxoid-containing vaccine within the last 10 years and therefore only the remaining contacts received a diphtheria toxoid booster.Case interviews explored potential sources of infection.The raw chicken being prepared at the time of the initial wound was considered a plausible source, but no samples of this product were available for testing.It was identified that two pet German Shepherd dogs lived in the house and had close direct contact with all household members.As Australian state public health guidelines are not explicit about the management of animal contacts, opinion was sought from relevant animal authorities to ascertain whether canine testing should be performed.The two dogs were screened using nose and throat swabs, from which C. ulcerans was isolated in both animals (moderate growth in both; one also had heavy growth of Staphylococcus pseudintermedius).These isolates were similarly tox gene PCR positive.The dogs were assessed by a veterinarian for F I G U R E 1 The human Corynebacterium ulcerans isolate growing on tellurite (left) and horse blood agar (right).

A
10-day course of oral clearance antibiotics with amoxicillin and clavulanic acid (750/187.5mg twice daily) was provided to each of the dogs.Clearance testing 2 weeks after antibiotic course completion revealed one of the dogs remained an asymptomatic nasal carrier of C. ulcerans (light growth).International expert opinion was sought regarding causes of ineffective carrier clearance despite treatment with an antimicrobial agent selected against the susceptibility profile, with one possibly being the anecdotal reports of penicillin non-response in canines with C. ulcerans in South Africa (Henton, n.d.).A second course of marbofloxacin or enrofloxacin was recommended due to the sensitivity profile, lipophilic properties that promote distribution, and canine pharmacokinetics.Enrofloxacin was preferred over marbofloxacin due to cost.No further clearance specimens were obtained due to resourcing limitations.
1. That, in the absence of a plausible alternative source, animal companions of human cases of C. ulcerans should be examined for signs of infection and be tested for C. ulcerans carriage/ infection.Testing should occur regardless of symptoms/signs in the animal and should include nasal and throat swabs (noting that nasopharyngeal swabs would be preferable to nasal swabs but are often impractical to obtain in animals), as well as swabs of any cutaneous lesions.2. That animal swabs returning a positive result for C. ulcerans should have toxin testing and antimicrobial susceptibility testing completed, the latter with a focus on antimicrobial agents typically used in animals.3.That animal companions who are found to carry C. ulcerans are treated with antimicrobial agents based on susceptibility testing.
and follow-up, and expenses related to logistics and transport, testing and antibiotic treatments.Factors such as potential inconvenience to owners and potential distress to animal companions should also be considered.Where possible, protocols should be inclusive of local roles and responsibilities and funding arrangements.In conclusion, our experience in responding to a zoonotically acquired C. ulcerans case has highlighted a need for greater surveillance and understanding of this re-emerging pathogen, in both animals and humans, and for improved consistency in public health responses.

Human case immune and vaccination status Transmission path and genetic analysis Animal symptom status Type of animal contact
Corynebacterium ulcerans reported in the literature since 1966.Overview of local and international guidelines on public health management of Corynebacterium ulcerans diphtheria which are available open access and updated from 2012.