Corynebacterium diphtheriae and Corynebacterium ulcerans: development of EUCAST methods and generation of data on which to determine breakpoints.

BACKGROUND: Evidence-based clinical susceptibility breakpoints have been lacking for antimicrobial agents used for diphtheria. OBJECTIVES: We aimed to evaluate broth microdilution and disc diffusion methods and create a dataset of MIC values and inhibition zone diameters (ZDs) from which breakpoints could be determined. METHODS: We included 400 recent clinical isolates equally distributed by species (Corynebacterium diphtheriae and Corynebacterium ulcerans) and by national surveillance programmes (France and Germany). Non-duplicate toxigenic and non-toxigenic isolates were chosen to enable the inclusion of a diversity of susceptibility levels for the 13 agents tested. Broth microdilution and disc diffusion, using EUCAST methodology for fastidious organisms, were used. RESULTS: The distributions of MIC and ZD values were largely in agreement among methods and countries. Breakpoints to allow categorization of WT isolates as susceptible, i.e. susceptible (S) or susceptible, increased exposure (I) were determined for 12 agents. The data supported a breakpoint for benzylpenicillin and amoxicillin of resistant (R) > 1 mg/L since WT isolates were inhibited by 1 mg/L or less. WT isolates were categorized as I (S ≤ 0.001 mg/L) for benzylpenicillin, emphasizing the need for increased exposure, and S (S ≤ 1 mg/L) for amoxicillin. Erythromycin breakpoints were set at S ≤ 0.06 mg/L and R > 0.06 mg/L. The corresponding ZD breakpoints were determined for all agents except amoxicillin, for which categorization was based on benzylpenicillin results. CONCLUSIONS: This work provided a large set of antimicrobial susceptibility data for C. diphtheriae and C. ulcerans, using a harmonized methodology. The dataset allowed EUCAST and experts in the diphtheria field to develop evidence-based breakpoints in January 2023.

Management and outcome of cutaneous diphtheria in adolescent refugees in Germany, June 2022 - October 2023.

OBJECTIVES: From September 2022 an increase in Corynebacterium diphtheriae (C. diphtheriae) infections was reported in Europe. Our study focuses on 31 adolescent and young adult refugees with cutaneous C. diphtheriae infections detected in Germany. We examined treatment regimens and outcomes to provide targeted insights into the management of this infection. METHODS: We distributed a standardized survey, focused on children and adolescents presenting to paediatric clinics through the German Paediatric Infectious Diseases Society (DGPI) and additional professional contacts in Germany. Data were extracted from routine medical documentation and reported anonymously. RESULTS: A total of 31 individuals with cutaneous C. diphtheriae infection were reported by 9 centres. Two of these showed diphtheria toxin (DT) related systemic symptoms and four exhibited systemic inflammation requiring complex management. The remaining 25 cases, with exclusively cutaneous manifestations, were afebrile. Treatment with topical antiseptics and systemic antibiotics, mainly aminopenicillin/beta-lactamase inhibitors (BLI) (35%) or clindamycin (25%), achieved eradication in all but two cases treated with aminopenicillin/BLI. Treatment duration varied between 5 and 17 days. CONCLUSIONS: In refugees presenting with chronic skin wounds, C. diphtheriae should be included into the differential diagnosis. Fever seems to be a valuable marker to differentiate severe cases with potentially DT-mediated sequelae from exclusively cutaneous diphtheria (CD). For afebrile CD, topical antiseptics and oral antibiotic therapy with clindamycin for 7 days, followed by clinical surveillance appears to be a safe treatment regimen. Patients with CD who present with fever or pharyngitis should be thoroughly investigated including blood and pharyngeal swab cultures.

Detection of diphtheria toxin production by toxigenic corynebacteria using an optimized Elek test.

PURPOSE: Diphtheria, still present in many countries of the world, is caused by toxigenic strains of species of the Corynebacterium diphtheriae complex, mainly Corynebacterium diphtheriae and the emerging zoonotic pathogen C. ulcerans. The immunoprecipitation test according to Elek is the gold standard for detection of the major virulence factor diphtheria toxin (DT) in toxigenic corynebacteria. Due to its sophisticated methodological requirements, the classical Elek test is performed mainly by specialized reference laboratories. It was revealed that the current modification of the Elek test does not detect the toxin in weakly toxigenic isolates. Therefore, a more robust method for detecting free DT is urgently needed, especially for toxigenic C. ulcerans strains which are known to produce often much lower amounts of DT than C. diphtheriae. METHODS: Thirty-one tox-positive C. ulcerans isolates with a negative standard Elek test result previously determined as NTTB (non-toxigenic tox bearing) were re-analyzed in this study using a modified immunoprecipitation method optimized regarding different parameters including type and concentration of antitoxin, medium volume, inoculum distance from the antitoxin disk and position of controls. RESULTS: All 31 C. ulcerans strains tested positive in the optimized Elek test. CONCLUSION: Only with a reliable and easy-to-handle method for detecting the toxigenicity of C. ulcerans, it is possible to assess the etiological role of this emerging zoonotic bacterium in human pathology.

Outbreak of imported diphtheria with Corynebacterium diphtheriae among migrants arriving in Germany, 2022.

From July 2022, cases of imported diphtheria with toxigenic Corynebacterium diphtheriae remarkably increased among migrants arriving in Germany. Up to 30 September 2022, 44 cases have been reported to the national public health institute, all laboratory-confirmed, male, and mainly coming from Syria (n = 21) and Afghanistan (n = 17). Phylogeny and available journey information indicate that most cases (n = 19) were infected along the Balkan route. Active case finding, increased laboratory preparedness and epicentre localisation in countries along this route are important.

Widespread Disease in Hedgehogs (Erinaceus europaeus) Caused by Toxigenic Corynebacterium ulcerans.

Toxin-producing Corynebacterium ulcerans, a causative agent of diphtheria in humans, was isolated from 53 hedgehogs in Belgium during the spring of 2020. Isolates showed low levels of acquired antimicrobial drug resistance. Strain diversity suggests emergence from an endemic situation. These findings stress the need for raising public awareness and improved wildlife disease surveillance.

Corynebacterium rouxii, a recently described member of the C. diphtheriae group isolated from three dogs with ulcerative skin lesions.

Corynebacterium (C.) diphtheriae is one of the two etiological pathogens for human diphtheria with significant morbidity and mortality. Recently, members of its biovar Belfanti have been described as two novel species, C. belfantii and C. rouxii. The most important virulence factor and also the premise to cause diphtheria is the isolate's capacity to encode and express the diphtheria toxin (DT). In contrast to C. ulcerans, which represents a potentially zoonotic pathogen, C. diphtheriae (incl. the novel deduced species) has almost exclusively been found to comprise a human pathogen. We here report three rare cases of C. rouxii isolation from dogs suffering from disseminated poly-bacterial exsudative to purulent dermatitis and a traumatic labial defect, respectively. The isolates were identified as C. diphtheriae based on commercial biochemistry and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS) analysis. However, recently described specific spectral peaks were highly similar to spectra of C. rouxii, which was confirmed by whole genome sequencing. Further investigations of the dog isolates for the presence of DT by tox gene qPCR revealed negative results. The findings from this study point out that skin infections in companion animals can be colonized by uncommon and so believed human specific pathogens, thereby resembling the clinical signs of cutaneous diphtheria.

Toxigenic Corynebacterium diphtheriae-Associated Genital Ulceration.

In October 2016, an adolescent boy sought care for acute genital ulceration in Cologne, Germany. We presumed a sexually transmitted infection, but initial diagnostic procedures yielded negative results. He was hospitalized because swab samples from the lesion grew toxigenic Corynebacterium diphtheriae, leading to the diagnosis of possibly sexually transmitted cutaneous diphtheria.

Antimicrobial susceptibility of Corynebacterium diphtheriae and Corynebacterium ulcerans in Germany 2011-17.

BACKGROUND: Diphtheria is mainly caused by diphtheria-toxin-producing strains of Corynebacterium diphtheriae and Corynebacterium ulcerans. The recommended first-line antibiotic is penicillin or erythromycin, but reliable susceptibility data are scarce. OBJECTIVES: To define WT MIC distributions of 12 antimicrobial agents and provide data for the determination of tentative epidemiological cut-off values (TECOFFs) for potentially toxigenic corynebacteria and to evaluate the potential usefulness of a gradient test (Etest) for susceptibility testing of penicillin, erythromycin and clindamycin. METHODS: For the 421 human or veterinary isolates from the period 2011-17, MICs of 12 antimicrobial agents were determined. Etest performance was evaluated for penicillin, erythromycin and clindamycin. RESULTS: MIC distributions were characterized and TECOFFs could be set for 11 out of 24 antibiotic/species combinations. The current EUCAST clinical breakpoints, predominantly determined for Corynebacterium species other than C. diphtheriae and C. ulcerans, divide the WT MIC distributions of penicillin and clindamycin, thereby making reproducible susceptibility testing of C. diphtheriae and C. ulcerans difficult. For erythromycin, 4% of C. diphtheriae and 2% of C. ulcerans had MICs higher than those for WT isolates. Phenotypically detectable resistance to other antibiotics was rare. Etest underestimated MICs of penicillin and lower concentrations needed to be included for erythromycin, while for clindamycin the Etest was not a good surrogate method. CONCLUSIONS: MIC distributions based on reference broth microdilution for potentially toxigenic Corynebacterium spp. were developed. For five and six agents, TECOFFs were suggested for C. diphtheriae and C. ulcerans, respectively, but for Corynebacterium pseudotuberculosis the number of isolates was too low.

Corynebacterium silvaticum sp. nov., a unique group of NTTB corynebacteria in wild boar and roe deer.

A total of 34 Corynebacterium sp. strains were isolated from caseous lymph node abscesses of wild boar and roe deer in different regions of Germany. They showed slow growth on Columbia sheep blood agar and sparse growth on Hoyle's tellurite agar. Cellular fatty acid analysis allocated them in the C. diphtheriae group of genus Corynebacterium. MALDI-TOF MS using specific database extensions and rpoB sequencing resulted in classification as C. ulcerans. Their quinone system is similar to C. ulcerans, with major menaquinone MK-8(H2). Their complex polar lipid profile includes major lipids phosphatidylinositol, phosphatidylinositol-mannoside, diphosphatidylglycerol, but also unidentified glycolipids, distinguishing them clearly from C. ulcerans. They ferment glucose, ribose and maltose (like C. ulcerans), but do not utilise d-xylose, mannitol, lactose, sucrose and glycogen (like C. pseudotuberculosis). They showed activity of catalase, urease and phospholipase D, but variable results for alkaline phosphatase and alpha-glucosidase. All were non-toxigenic, tox gene bearing and susceptible to clindamycin, penicillin and erythromycin. In 16SrRNA gene and RpoB protein phylogenies the strains formed distinct brancheswith C. ulcerans as nearest relative.Whole genome sequencing revealed the unique sequence type 578, a distinctbranch in pangenomic core genome MLST, average nucleotide identities <91%, enhancedgenome sizes (2.55 Mbp) and G/C content (54.4 mol%) compared to related species.These results suggest that the strains represent a novel species, for which wepropose the name Corynebactriumsilvaticum sp. nov., based on their first isolation from forest-dwellinggame animals. The type strain isKL0182T (= CVUAS 4292T = DSM 109166T = LMG 31313T= CIP 111 672T).

Rapid establishment of laboratory diagnostics for the novel coronavirus SARS-CoV-2 in Bavaria, Germany, February 2020.

The need for timely establishment of diagnostic assays arose when Germany was confronted with the first travel-associated outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Europe. We describe our laboratory experiences during a large contact tracing investigation, comparing previously published real-time RT-PCR assays in different PCR systems and a commercial kit. We found that assay performance using the same primers and probes with different PCR systems varied and the commercial kit performed well.

NGS-based phylogeny of diphtheria-related pathogenicity factors in different Corynebacterium spp. implies species-specific virulence transmission.

BACKGROUND: Diphtheria toxin (DT) is produced by toxigenic strains of the human pathogen Corynebacterium diphtheriae as well as zoonotic C. ulcerans and C. pseudotuberculosis. Toxigenic strains may cause severe respiratory diphtheria, myocarditis, neurological damage or cutaneous diphtheria. The DT encoding tox gene is located in a mobile genomic region and tox variability between C. diphtheriae and C. ulcerans has been postulated based on sequences of a few isolates. In contrast, species-specific sequence analysis of the diphtheria toxin repressor gene (dtxR), occurring both in toxigenic and non-toxigenic Corynebacterium species, has not been done yet. We used whole genome sequencing data from 91 toxigenic and 46 non-toxigenic isolates of different pathogenic Corynebacterium species of animal or human origin to elucidate differences in extracted DT, DtxR and tox-surrounding genetic elements by a phylogenetic analysis in a large sample set. RESULTS: Sequences of both DT and DtxR, extracted from whole genome sequencing data, could be classified in four distinct, nearly species-specific clades, corresponding to C. diphtheriae, C. pseudotuberculosis, C. ulcerans and atypical C. ulcerans from a non-toxigenic toxin gene-bearing wildlife cluster. Average amino acid similarities were above 99% for DT and DtxR within the four groups, but lower between them. For DT, subgroups below species level could be identified, correlating with different tox-comprising mobile genetic elements. In most C. diphtheriae, tox genes were located within known prophages. In contrast, in C. ulcerans diverse tox-including mobile elements could be identified: either prophages differing from C. diphtheriae prophages or an alternative pathogenicity island (PAI) described previously. One isolate showed a different, shorter tox-comprising putative PAI. Beyond the tox-overlapping elements, most isolates harbored a variety of additional prophages. CONCLUSION: Our NGS data from 137 isolates indicate the existence of different genetic backgrounds of DT-mediated pathogenicity in different Corynebacterium species and evolution of once acquired pathogenicity features with the strains. Different groups of pathogenicity-related elements within C. ulcerans imply that tox transmission pathways between isolates may differ in the zoonotic species and contribute to their emerging pathogenic potential.

Genetic diversity and delineation of Salmonella Agona outbreak strains by next generation sequencing, Bavaria, Germany, 1993 to 2018.

BackgroundIn 2017, a food-borne Salmonella Agona outbreak caused by infant milk products from a French supplier occurred in Europe. Simultaneously, S. Agona was detected in animal feed samples in Bavaria.AimUsing next generation sequencing (NGS) and three data analysis methods, this study's objectives were to verify clonality of the Bavarian feed strains, rule out their connection to the outbreak, explore the genetic diversity of Bavarian S. Agona isolates from 1993 to 2018 and compare the analysis approaches employed, for practicality and ability to delineate outbreaks caused by the genetically monomorphic Agona serovar.MethodsIn this observational retrospective study, three 2017 Bavarian feed isolates were compared to a French outbreak isolate and 48 S. Agona isolates from our strain collections. The later included human, food, feed, veterinary and environmental isolates, of which 28 were epidemiologically outbreak related. All isolates were subjected to NGS and analysed by: (i) a publicly available species-specific core genome multilocus sequence typing (cgMLST) scheme, (ii) single nucleotide polymorphism phylogeny and (iii) an in-house serovar-specific cgMLST scheme. Using additional international S. Agona outbreak NGS data, the cluster resolution capacity of the two cgMLST schemes was assessed.ResultsWe could prove clonality of the feed isolates and exclude their relation to the French outbreak. All approaches confirmed former Bavarian epidemiological clusters.ConclusionEven for S. Agona, species-level cgMLST can produce reasonable resolution, being standardisable by public health laboratories. For single samples or homogeneous sample sets, higher resolution by serovar-specific cgMLST or SNP genotyping can facilitate outbreak investigations.

Whole genome sequencing suggests transmission of Corynebacterium diphtheriae-caused cutaneous diphtheria in two siblings, Germany, 2018.

In September 2018, a child who had returned from Somalia to Germany presented with cutaneous diphtheria by toxigenic Corynebacterium diphtheriae biovar mitis. The child's sibling had superinfected insect bites harbouring also toxigenic C. diphtheriae. Next generation sequencing (NGS) revealed the same strain in both patients suggesting very recent human-to-human transmission. Epidemiological and NGS data suggest that the two cutaneous diphtheria cases constitute the first outbreak by toxigenic C. diphtheriae in Germany since the 1980s.

Geographically Diverse Clusters of Nontoxigenic Corynebacterium diphtheriae Infection, Germany, 2016-2017.

From 2016 through the middle of 2017, the German Consiliary Laboratory on Diphtheria noted an increase in nontoxigenic Corynebacterium diphtheriae isolates submitted from cities in northern Germany. Many patients for whom epidemiologic data were available were homeless, alcohol or drug abusers, or both. After performing routine diagnostics and multilocus sequence typing (MLST), we analyzed isolates of sequence type (ST) 8 and previously submitted isolates by whole-genome sequencing. Results were analyzed for phylogenetic relationship by core genome MLST (cg-MLST) and whole-genome single-nucleotide polymorphism profiles. Next-generation sequencing-based cg-MLST revealed several outbreak clusters caused by ST8; the geographic focus was in the metropolitan areas of Hamburg and Berlin. To achieve enhanced analytical depth, we used additional cg-MLST target genes and genome-wide single-nucleotide polymorphisms. We identified patient characteristics and detected transmission events, providing evidence that nontoxigenic C. diphtheriae infection is a potential public health threat in industrialized countries.

Screening for infectious diseases among newly arrived asylum seekers, Bavaria, Germany, 2015.

Background and aimAs a consequence of socioeconomic and political crises in many parts of the world, many European Union/European Economic Area (EU/EEA) countries have faced an increasing number of migrants. In the German federal state of Bavaria, a mandatory health screening approach is implemented, where individuals applying for asylum have to undergo a medical examination that includes serological testing for HIV and hepatitis B, screening for tuberculosis, and until September 2015, stool examination for Salmonella spp. and Shigella spp.. Methods: Data from mandatory screening of all first-time asylum seekers in Bavaria in 2015 was extracted from the mandatory notification and laboratory information system and evaluated. Results: The HIV positivity and hepatitis B surface antigen (HBsAg) positivity rate of tested samples from asylum seekers were 0.3% and 3.3%, respectively, while detection rate of active tuberculosis was between 0.22% and 0.38%. The rates for HIV, hepatitis B, and tuberculosis among asylum seekers were similar to the corresponding prevalence rates in most of their respective countries of birth. Only 47 Salmonella spp. (0.1%) were isolated from stool samples: 45 enteric and two typhoid serovars. Beyond mandatory screening, louse-borne relapsing fever was found in 40 individuals. Conclusions: These results show that mandatory screening during 2015 in Bavaria yielded overall low positivity rates for all tested infectious diseases in asylum seekers. A focus of mandatory screening on specific diseases in asylum seekers originating from countries with higher prevalence of those diseases could facilitate early diagnosis and provision of treatment to affected individuals while saving resources.

Corynebacterium species nasopharyngeal carriage in asymptomatic individuals aged ≥ 65 years in Germany.

PURPOSE: The prevalence of protective anti-diphtheria toxin antibodies decreases with age. Therefore, the elderly might serve as reservoir for potentially toxigenic Corynebacterium (C.) species (C. diphtheriae, C. ulcerans, and C. pseudotuberculosis). This study aimed to examine the colonization rate of the nasopharynx with corynebacteria of individuals aged 65 years and older. METHODS: In the period from October 2012 to June 2013, nasal and throat swabs were taken from 714 asymptomatic subjects aged 65-106 years (average age 77.2) at three regions in Germany and investigated for Corynebacterium species. RESULTS: A total of 402 strains of Corynebacterium species were isolated from 388 out of 714 asymptomatic subjects (carriage rate 54.3%). The carriage rate was significantly higher in study participants living in retirement homes (68.4%) compared to those living autonomously at home (51.1%). Strains were isolated mostly from the nose (99%). Corynebacterium accolens was the most often isolated species (39.8%), followed by C. propinquum (24.1%), C. pseudodiphtheriticum (19.4%), and C. tuberculostearicum (10.2%). No C. diphtheriae, C. ulcerans, and C. pseudotuberculosis strains were isolated. A subsample of 74 subjects was tested serologically for anti-diphtheria antibodies. Protective anti-diphtheria toxin antibodies were found in 29.7% of the subjects; 70.3% showed no protective immunity. CONCLUSIONS: These results suggest that carriage of potentially toxigenic corynebacteria is very rare among people aged 65 and older in Germany. However, the low prevalence of protective anti-diphtheria toxin antibodies might pose a risk for acquiring diphtheria especially for the elderly.

Zoonotic transmission of toxigenic Corynebacterium ulcerans strain, Germany, 2012.

Severe necrotizing fasciitis was diagnosed in a 53-year-old man in Germany in 2012. Toxigenic Corynebacterium ulcerans was grown from a wound swab sample. One of the patient's 2 dogs was found to harbor a toxigenic C. ulcerans strain. Results of next generation sequencing of both isolates supported recent zoonotic transmission of this bacterial pathogen.

Prevalence and correlates of young people's sexual aggression perpetration and victimisation in 10 European countries: a multi-level analysis.

Data are presented on young people's sexual victimisation and perpetration from 10 European countries (Austria, Belgium, Cyprus, Greece, Lithuania, the Netherlands, Poland, Portugal, Slovakia and Spain) using a shared measurement tool (N = 3480 participants, aged between 18 and 27 years). Between 19.7 and 52.2% of female and between 10.1 and 55.8% of male respondents reported having experienced at least one incident of sexual victimisation since the age of consent. In two countries, victimisation rates were significantly higher for men than for women. Between 5.5 and 48.7% of male and 2.6 and 14.8% of female participants reported having engaged in a least one act of sexual aggression perpetration, with higher rates for men than for women in all countries. Victimisation rates correlated negatively with sexual assertiveness and positively with alcohol use in sexual encounters. Perpetration rates correlated positively with attitudes condoning physical dating violence and with alcohol use in men, and negatively with sexual assertiveness in women. At the country level, lower gender equality in economic power and in the work domain was related to higher male perpetration rates. Lower gender equality in political power and higher sexual assertiveness in women relative to men were linked to higher male victimisation rates.