Complete genome sequence of Arcanobacterium wilhelmae strain DSM 102162 isolated from the genital tract of a Rhinoceros unicornis.

Previous case reports indicate Arcanobacterium's opportunistic pathogenic potential. However, the true diversity of the genus remains understudied. Here, we present the complete genome of Arcanobacterium wilhelmae isolated from a diseased rhinoceros, suspected to play a role in its condition. These genomic data may enable future advancements in understanding Arcanobacterium pathogenicity.

Corynebacterium ulcerans Infections in Eurasian Beavers (Castor fiber).

The Eurasian beaver (Castor fiber) has been reintroduced successfully in Germany since the 1990s. Since wildlife is an important source of zoonotic infectious diseases, monitoring of invasive and reintroduced species is crucial with respect to the One Health approach. Three Eurasian beavers were found dead in the German federal states of Bavaria, North Rhine-Westphalia and Baden-Wuerttemberg in 2015, 2021 and 2022, respectively. During post-mortem examinations, Corynebacterium (C.) ulcerans could be isolated from the abscesses of two beavers and from the lungs of one of the animals. Identification of the bacterial isolates at the species level was carried out by spectroscopic analysis using MALDI-TOF MS, FT-IR and biochemical profiles and were verified by molecular analysis based on 16-23S internal transcribed spacer (ITS) region sequencing. Molecular characterization of the C. ulcerans isolates using whole-genome sequencing (WGS) revealed a genome size of about 2.5 Mbp and a GC content of 53.4%. Multilocus sequence typing (MLST) analysis classified all three isolates as the sequence type ST-332. A minimum spanning tree (MST) based on cgMLST allelic profiles, including 1211 core genes of the sequenced C. ulcerans isolates, showed that the beaver-derived isolates clearly group on the branch of C. ulcerans with the closest relationship to each other, in close similarity to an isolate from a dog. Antibiotic susceptibility testing revealed resistance to clindamycin and, in one strain, to erythromycin according to EUCAST, while all isolates were susceptible to the other antimicrobials tested.

Production of Satratoxin G and H Is Tightly Linked to Sporulation in Stachybotrys chartarum.

Stachybotrys chartarum is a toxigenic fungus that is frequently isolated from damp building materials or improperly stored forage. Macrocyclic trichothecenes and in particular satratoxins are the most potent mycotoxins known to be produced by this fungus. Exposure of humans or animals to these secondary metabolites can be associated with severe health problems. To assess the pathogenic potential of S. chartarum isolates, it is essential to cultivate them under conditions that reliably promote toxin production. Potato dextrose agar (PDA) was reported to be the optimal nutrition medium for satratoxin production. In this study, the growth of S. chartarum genotype S strains on PDA from two manufacturers led to divergent results, namely, well-grown and sporulating cultures with high satratoxin concentrations (20.8 ± 0.4 µg/cm2) versus cultures with sparse sporulation and low satratoxin production (0.3 ± 0.1 µg/cm2). This finding is important for any attempt to identify toxigenic S. chartarum isolates. Further experiments performed with the two media provided strong evidence for a link between satratoxin production and sporulation. A comparison of three-point and one-point cultures grown on the two types of PDA, furthermore, demonstrated an inter-colony communication that influences both sporulation and mycotoxin production of S. chartarum genotype S strains.

Erysipelothrix anatis sp. nov., Erysipelothrix aquatica sp. nov. and Erysipelothrix urinaevulpis sp. nov., three novel species of the genus, and emended description of Erysipelothrix.

Seven genotypically distinct strains assigned to the genus Erysipelothrix were isolated in different laboratories from several animal sources. Strain D17_0559-3-2-1T and three further strains were isolated from samples of duck, pig and goose. The strains had >99 % 16S rRNA gene sequence similarity to each other and to strain VA92-K48T and two further strains isolated from samples of medical leech and a turtle. The closest related type strains to the seven strains were those of Erysipelothrix inopinata (96.74 %) and Erysipelothrix rhusiopathiae (95.93 %). Average nucleotide identity, amino acid identity and in silico DNA-DNA hybridization results showed that the strains represented two separate novel species. One further phylogenetically distinct strain (165301687T) was isolated from fox urine. The strain had highest 16S rRNA gene sequence similarity to the type strains of Erysipelothrix tonsillarum (95.67 %), followed by Erysipelothrix piscisicarius (95.58 %) and Erysipelothrix larvae (94.22 %) and represented a further novel species. Chemotaxonomic and physiological data of the novel strains were assessed, but failed to unequivocally differentiate the novel species from existing members of the genus. MALDI-TOF MS data proved the discrimination of at least strain 165301687T from all currently described species. Based on the presented phylogenomic and physiological data, we propose three novel species, Erysipelothrix anatis sp. nov. with strain D17_0559-3-2-1T (=DSM 111258T= CIP 111884T=CCM 9044T) as type strain, Erysipelothrix aquatica sp. nov. with strain VA92-K48T (=DSM 106012T=LMG 30351T=CIP 111492T) as type strain and Erysipelothrix urinaevulpis sp. nov. with strain 165301687T (=DSM 106013T= LMG 30352T= CIP 111494T) as type strain.

Corynebacterium pseudotuberculosis Infections in Alpacas (Vicugna pacos).

Alpacas are the major camelid species in Europe held for hobbies, animal-aided therapy, and commercial reasons. As a result, health-related issues associated with alpacas are of growing significance. This especially holds true for one of the most serious infectious diseases, caseous lymphadenitis, which is caused by the bacterial pathogen Corynebacterium (C.) pseudotuberculosis. Our study focuses on post-mortem examinations, the laboratory diagnostic tool ELISA, and the immunoblot technique for the detection of specific antibodies against C. pseudotuberculosis and detection of the causative pathogen in alpaca herds. We examined a total of 232 alpacas living in three herds. Four of these alpacas were submitted for post-mortem examination, revealing abscesses, apostematous and fibrinous inflammation in inner organs, pleura, and peritoneum. Serological investigation using a commercial ELISA based on phospholipase D (PLD) as antigen and an in-lab ELISA based on whole cell antigens (WCA) revealed an overall seroprevalence of 56% and 61.2%, respectively. A total of 247 alpaca sera originating from 232 animals were tested comparatively using the in-lab and the commercial ELISA and showed a substantial degree of agreement, of 89.5% (Cohen's kappa coefficient of 0.784), for both tests. Further comparative serological studies using the two ELISAs and the immunoblot technique were carried out on selected sera originating from 12 breeding stallions and six breeding mares for which epidemiological data and partial C. pseudotuberculosis isolates were available. The results showed the immunoblot to have a sensitivity that was superior to both ELISAs. In this context, it should be emphasized that evaluation of these investigations and the epidemiological data suggest an incubation period of one to two months. Antibiotic susceptibility testing of 13 C. pseudotuberculosis isolates based on the determination of minimal inhibitory concentrations using the broth microdilution method revealed uniform susceptibility to aminopenicillins, cephalosporines, macrolides, enrofloxacin, florfenicol, tetracycline, sulfonamid/trimethoprime, tiamulin, gentamicin, neomycin, spectinomycin, and vancomycin, but resistance to colistin, nitrofurantoin, and oxacillin.

Fatal Infection in an Alpaca (Vicugna pacos) Caused by Pathogenic Rhodococcus equi.

Rhodococcus (R.) equi is a pathogen primarily known for infections in equine foals, but is also present in numerous livestock species including New World camelids. Moreover, R. equi is considered an emerging zoonotic pathogen. In this report, we describe in detail a fatal rhodococcal infection in an alpaca (Vicugna pacos), to our best knowledge, for the first time. The alpaca died due to a septicemic course of an R. equi infection resulting in emaciation and severe lesions including pyogranulomas in the lungs and pericardial effusion. The onset of the infection was presumably caused by aspiration pneumonia. R. equi could be isolated from the pyogranulomas in the lung and unequivocally identified by MALDI-TOF MS analysis and partial sequencing of the 16S rRNA gene, the 16S-23S internal transcribed spacer (ITS) region and the rpoB gene. The isolate proved to possess the vapA gene in accordance with tested isolates originating from the lungs of infected horses. The R. equi isolates revealed low minimal inhibitory concentrations (MIC values) for doxycycline, erythromycin, gentamycin, neomycin, rifampicin, trimethoprim/sulfamethoxazole, tetracycline and vancomycin in antibiotic susceptibility testing. Investigations on the cause of bacterial, especially fatal, septicemic infections in alpacas are essential for adequately addressing the requirements for health and welfare issues of this New World camelid species. Furthermore, the zoonotic potential of R. equi has to be considered with regard to the One Health approach.

First isolation of Arcanobacterium pinnipediorum from a grey seal pup (Halichoerus grypus) in the UK.

In the present study, a single Arcanobacterium (A.) pinnipediorum strain isolated from discharge of a jaw swelling of a grey seal pup (Halichoerus grypus) in England, UK, was identified. This strain was further characterized by phenotypical investigations, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), by Fourier transform infrared spectroscopy (FT-IR), and genotypically by sequencing the 16S rRNA gene and the genes gap encoding glyceraldehyde 3-phosphate dehydrogenase, tuf encoding elongation factor tu, and rpoB encoding the ß subunit of bacterial RNA polymerase. The present study gives a first detailed characterization of the species A. pinnipediorum from a grey seal in the UK. However, the route of infection of the grey seal with the bacterial pathogen remains unclear.

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.

Genome-Based Analysis of Klebsiella spp. Isolates from Animals and Food Products in Germany, 2013-2017.

The increase in infections with multidrug-resistant and virulent Klebsiella pneumoniae (K. pneumoniae) strains poses a serious threat to public health. However, environmental reservoirs and routes of transmission for Klebsiella spp. that cause infections in humans and in livestock animals are not well understood. In this study, we aimed to analyze the distribution of antibiotic resistance genes and important virulence determinants (ybt, clb, iro, iuc, rmpA/A2) among 94 Klebsiella spp. isolates from different animal and food sources isolated between 2013 and 2017 in Germany. Antibiotic susceptibility testing was performed, and the genomes were sequenced by Illumina and Nanopore technology. Genetic relationships were assessed by conducting core genome multilocus sequence typing (cgMLST). Kleborate was used to predict resistance and virulence genes; Kaptive was used to derive the capsule types. The results revealed that 72 isolates (76.6%) belonged to the K. pneumoniae sensu lato complex. Within this complex, 44 known sequence types (STs), 18 new STs, and 38 capsule types were identified. Extended-spectrum beta-lactamase (ESBL) genes were detected in 16 isolates (17.0%) and colistin resistance in one (1.1%) K. pneumoniae isolate. Virulence genes were found in 22 K. pneumoniae isolates. Overall, nine (9.6%) and 18 (19.1%) isolates possessed the genes ybt and iuc, respectively. Notably, aerobactin (iuc lineage 3) was only detected in K. pneumoniae isolates from domestic pigs and wild boars. This study provides a snapshot of the genetic diversity of Klebsiella spp. in animals and food products in Germany. The siderophore aerobactin was found to be more prevalent in K. pneumoniae strains isolated from pigs than other sources. Further investigations are needed to evaluate if pigs constitute a reservoir for iuc lineage 3.

Streptococcus catagoni sp. nov., isolated from the respiratory tract of diseased Chacoan peccaries (Catagonus wagneri).

Novel catalase-negative, Gram-stain-positive, beta-haemolytic, coccus-shaped organisms were isolated from Chacoan peccaries that died from respiratory disease. The initial API 20 Strep profiles suggested Streptococcus agalactiae with acceptable identification scores, but the 16S rRNA gene similarity (1548 bp) to available sequences of streptococci was below 98 %. Next taxa of the genus Streptococcus, displaying highest similarities to the strains from this study, were S. bovimastitidis NZ1587T (97.5 %), S. iniae ATCC 29178T (97.5 %), S. hongkongensis HKU30T (97.4 %), S. parauberis DSM 6631T (97.1 %), S. penaeicida CAIM 1838T (97.1 %), S. pseudoporcinus DSM 18513T (97.0 %), S. didelphis DSM 15616T (96.6 %), S. ictaluri 707-05T (96.6 %), S. uberis JCM 5709T (96.5 %) and S. porcinus NCTC 10999T (96.4 %). All other Streptococcus species had sequence similarities of below 96.4 %. A sodA gene as well as whole genome-based core genome phylogeny of three representative strains and 145 available Streptococcus genomes confirmed the unique taxonomic position. Interstrain average nucleotide identity (ANI) and amino acid identity (AAI) values were high (ANI >96 %; AAI 100%), but for other streptococci clearly below the proposed species boundary of 95-96 % (ANI <75 %; AAI <83 %). Results were confirmed by genome-to-genome distance calculations. Pairwise digital DNA-DNA hybridization estimates were high (>90 %) between the novel strains, but well below the species boundary of 70 % for closely related Streptococcus type strains (23.5-19.7 %). Phenotypic properties as obtained from extended biochemical profiles and MALDI-TOF mass spectrometry supported the outstanding rank. Based on the presented molecular and physiological data of the six strains, we propose a novel taxon for which we suggest the name Streptococcus catagoni sp. nov. with the type strain 99-1/2017T (=DSM 110457T=CCUG 74072T) and five reference strains.

Streptobacillus felis, a member of the oropharynx microbiota of the Felidae, isolated from a tropical rusty-spotted cat.

Streptobacillus felis is a fastidious microorganism and a novel member of the potentially zoonotic bacteria causing rat bite fever. Since its description, this is the second isolation of S. felis in a diseased member of the Felidae. Interestingly, the strain from this study was isolated from a zoo held, rusty-spotted cat (Prionailurus rubiginosus), with pneumonia, thereby indicating a possible broader host range in feline species. A recent preliminary sampling of domestic cats (Felis silvestris forma catus) revealed that this microorganism is common in the oropharynx, suggesting that S. felis is a member of their normal microbiota. Due to unawareness, fastidiousness, antibiotic sensitivity and lack of diagnostics the role of S. felis as a cat and human pathogen might be under-reported as with other Streptobacillus infections. More studies are necessary to elucidate the role of S. felis in domestic cats and other Felidae in order to better estimate its zoonotic potential.

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).

Epidemiological analysis of Arcanobacterium phocae isolated from cases of mink dermatitis of a single farm.

The present study was designed to identify nine Arcanobacterium phocae strains isolated from cases of mink dermatitis of a single farm in Finland and characterize the strains for epidemiological relationships. All nine strains and previously described A. phocae used for comparative purposes were identified and further characterized phenotypically, by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), by Fourier Transform Infrared Spectroscopy (FT-IR) and genotypically by detection of phocaelysin encoding gene phl with a previously developed loop-mediated isothermal amplification (LAMP) assay and by sequencing 16S rRNA gene and gene phl, the elongation factor tu encoding gene tuf and the ß subunit of bacterial RNA polymerase encoding gene rpoB. Genetic relatedness among isolates was determined using whole-genome single nucleotide polymorphism (wgSNP) analysis. The wgSNP results, partly the MALDI-TOF MS and FT-IR analyses and sequencing of the genes, revealed that the nine A. phocae strains recovered from a single farm showed close sequence similarities among each other and differed from previously investigated A. phocae strains isolated from other farms and animals in Finland and from the A. phocae type strain. This indicated a close epidemiological relationship of the A. phocae strains isolated from a single farm and that the nine A. phocae strains of the present study might have developed from a common ancestor.

Reliable differentiation of a non-toxigenic tox gene-bearing Corynebacterium ulcerans variant frequently isolated from game animals using MALDI-TOF MS.

Corynebacterium (C.) ulcerans is a zoonotic member of the C. diphtheriae group and is known to cause abscesses in humans and several animal species. Toxigenic strains, expressing the tox gene encoding diphtheria toxin, are also able to cause diphtheria in humans. In recent years, a non-toxigenic but tox gene-bearing (NTTB) variant of C. ulcerans has been identified that was frequently isolated from clinically healthy as well as from diseased wildlife animals, especially wild boars (Sus scrofa scrofa) in Germany and Austria. The described clinical cases showed similar signs of disease and the isolated corynebacteria displayed common genetic features as well as similar spectroscopic characteristics, therefore being assigned to a so called wild boar cluster (WBC). This study describes the establishment and validation of a method using MALDI-TOF mass spectrometry for a reliable differentiation between various members of the C. diphtheriae group at species level as well as a reliable sub-level identification of C. ulcerans isolates of the WBC variant. For this study 93 C. ulcerans isolates from wildlife animals, 41 C. ulcerans isolates from other animals and humans, and 53 isolates from further representatives of the C. diphtheriae group, as well as 26 non-diphtheriae group Corynebacteria collected via the MALDI user platform from seven MALDI users were used. By assigning 86 C. ulcerans isolates to the WBC the extensive geographical distribution of this previously less noticed variant in two Central European countries could be shown.

Artificial neural network-assisted Fourier transform infrared spectroscopy for differentiation of Salmonella serogroups and its application on epidemiological tracing of Salmonella Bovismorbificans outbreak isolates from fresh sprouts.

Salmonellae represent one of the most common bacterial infection reagents in both humans and animals. For detection and epidemiological elucidation of Salmonella infections, determination of Salmonella serotypes and differentiation between different Salmonella isolates is crucial. In the first part of this study, Artificial Neural Network (ANN)-assisted Fourier transform infrared (FTIR) spectroscopy was used to establish a method for subtyping Salmonella isolates according to their serogroups. For this, 290 Salmonella strains from 35 different serogroups were used to establish an ANN for differentiation between infrared spectra of 10 different Salmonella serogroups (B, C1, C2-C3, D1/D2, E1, E4, F, G, H, O:55) vs. the remaining serogroups. In the final ANN, sensitivity values ranged between 90 and 100% for most of the 10 serogroups under investigation. In the second part of this study, ANN-assisted FTIR spectroscopy was applied for epidemiological distinction of Salmonella Bovismorbificans outbreak isolates from fresh sprouts vs. isolates from other sources. Four Salmonella Bovismorbificans isolates from human and food origin in the context of a Southern German outbreak were successfully discriminated from other S. Bovismorbificans isolates from various sources. ANN-assisted FTIR spectroscopy is thus an effective tool for discrimination of Salmonella isolates at or even below serogroup level.

Streptococcus castoreus, an uncommon group A Streptococcus in beavers.

Streptococcus castoreus is a rarely encountered beta-haemolytic group A Streptococcus with high tropism for the beaver as host. Based on 27 field isolates under study, evidence strongly suggests that S. castoreus behaves as an opportunistic pathogen in beavers. Although it belongs to the resident mucosal microbiota, this Streptococcus species is associated with purulent lesions in diseased animals. With few exceptions, isolates proved to be highly similar in a panel of phenotypic (including biochemistry, resistance pattern, MALDI-TOF mass spectrometry and Fourier transform-infrared spectroscopy) and classic molecular (16S rRNA and sodA gene) analyses, and thus did not show any specific pattern according to host species or spatio-temporal origin. Conversely, S. castoreus isolates were differentiated into a multitude of pulsed-field gel electrophoresis 'pulsotypes' that did not seem to reflect true epidemiologic lineages. In contrast, single reactions of genomic fingerprinting using BOX-, (GTG)5- and RAPD-PCRs revealed at least subclusters with respect to host species, geographic origin or year, and confirmed the co-colonization of individuals with more than one isolate. In addition to isolates from free-ranging Eurasian beavers (Castor fiber), this study includes S. castoreus from captive North American beavers (Castor canadensis) for the first time.

Duplex PCR-ELONA for the detection of pork adulteration in meat products.

In this work, a duplex PCR-Enzyme Linked Oligonucleotide Assay (ELONA) is reported for the sensitive and reliable detection of pork adulteration in beef and chicken products, two of the most widely consumed meat types in the world. The strategy relies on the use of species-specific tailed primers for duplex amplification and simple dilution of the PCR reactions for direct colorimetric detection via hybridization, eliminating the need for any other post-amplification steps. A high sensitivity was achieved, with as low as 71-188 pg of genomic DNA able to be detected using mixtures of control DNA from each species. The strategy was validated using DNA add-mixtures as well as DNA extracted from raw meat mixtures and 0.5-1% w/w pork could be easily detected when mixed with beef or chicken. The proposed approach is simple, sensitive and cost-effective compared to equivalent commercial kits suitable for detecting adulterant pork levels in meat products.

Tox-positive Corynebacterium ulcerans in hedgehogs, Germany.

Toxigenic Corynebacterium ulcerans may cause both respiratory and cutaneous diphtheria in humans. As a zoonotic emerging pathogen it has been isolated from a wide variety of animals living in captivity, such as livestock, pet, zoo and research animals and additionally in a large number of different wild animals. Here we report the isolation of tox-positive C. ulcerans in four hedgehogs with cutaneous diphtheria and pneumonia, respectively.