Corynebacterium oculi-related bacterium may act as a pathogen and carrier of antimicrobial resistance genes in dogs: a case report.

BACKGROUND: The genus Corynebacterium comprises well-known animal and human pathogens as well as commensals of skin and mucous membranes. Species formerly regarded as contaminants are increasingly being recognized as opportunistic pathogens. Corynebacterium oculi has recently been described as a human ocular pathogen but has so far not been reported in dogs. CASE PRESENTATION: Here we present two cases of infection with a novel Corynebacterium sp., a corneal ulcer and a case of bacteriuria. The two bacterial isolates could not be identified by MALDI-TOF MS. While 16 S rRNA gene (99.3% similarity) and rpoB (96.6% identity) sequencing led to the preliminary identification of the isolates as Corynebacterium (C.) oculi, whole genome sequencing revealed the strains to be closely related to, but in a separate cluster from C. oculi. Antimicrobial susceptibility testing showed high minimal inhibitory concentrations of lincosamides, macrolides, tetracycline, and fluoroquinolones for one of the isolates, which also contained an erm(X) and tet-carrying plasmid as well as a nonsynonymous mutation leading to an S84I substitution in the quinolone resistance determining region of GyrA. CONCLUSIONS: While the clinical signs of both dogs were alleviated by antimicrobial treatment, the clinical significance of these isolates remains to be proven. However, considering its close relation with C. oculi, a known pathogen in humans, pathogenic potential of this species is not unlikely. Furthermore, these bacteria may act as reservoir for antimicrobial resistance genes also in a One Health context since one strain carried a multidrug resistance plasmid related to pNG3 of C. diphtheriae.

Wielerella bovis gen. nov., sp. nov. a member of the family Neisseriaceae associated with bovine endocarditis.

Seven bacterial strains isolated from bovine endocarditis in six animals from different geographic regions were investigated in a polyphasic taxonomic approach. Phylogenetic analysis based on 16S rRNA gene sequences placed all seven isolates on a distinct, monophyletic cluster in the family Neisseriaceae with closest similarity to type strains of Alysiella filiformis (97.06 %) and Kingella kingae (96.34 %). Whole genome sequence analysis of isolates confirmed their species status, with an average nucleotide identity >96 % between isolates and <80 % to other type species of genera of Neisseriaceae while digital DNA-DNA hybridization values were >80 % and<18 %, respectively. The DNA G+C content was 42.5-43.0 mol%. Whole genome sequence based phylogeny showed the isolates being monophyletic and separated from established genera, thereby forming a new genus within the family Neisseriaceae. Similarly, analysis of MALDI-TOF MS reference spectra clustered the isolates close together and clearly separated from other genera, making this the method of choice for identification. Biochemical markers based on classical as well as commercial identification schemes allowed separation from closely related Neisseriaceae genera, even though the new taxon is biochemically not very active. Major fatty acids are C12 : 0, C14 : 0 and C16 : 0. The major quinone is ubiquinone Q-8. In the polar lipid profile, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phospholipid were predominant. We propose the novel genus Wielerella with the type species Wielerella bovis gen. nov., sp. nov. The type strain is CCUG 44465T (=DSM 113289T=JF 2483T) isolated post mortem from a cow with endocarditis in Switzerland.

Detection of treponemes in digital dermatitis lesions of captive European bison (Bison bonasus).

A newly-discovered foot disease of unknown origin in captive European Bison (Bison bonasus) was recently detected at Berne Animal Park. Dermatitis of the interdigital cleft of varying degrees of severity was diagnosed in all animals (n = 10). The aim of this study was to describe the gross and histological lesions of the interdigital cleft found in 10 captive European bison and to identify involved potential pathogens in affected feet using molecular-based methods for Treponema spp., Dichelobacter nodosus and Fusobacterium necrophorum. Lesions were scored according to the degree of gross pathology at limb level. In a single animal, the gross lesions were restricted to focal lesions on the dorsal aspect of the digital skin of each foot (score 1), whereas all other animals showed at least one foot with extended lesions including the interdigital cleft (score 2). The presence of viable spirochaetes was observed in all animals using dark field microscopy. Applying fluorescence in situ hybridisation (FISH) on biopsies, Treponema spp. were identified, infiltrating the skin lesions in varying numbers in nine animals. Nested PCRs for Treponema medium, Treponema phagedenis and Treponema pedis of swab samples showed three positive animals out of ten for the latter two, whereas pooled biopsy samples were positive in all ten animals for at least T. phagedenis (9/10) and/or T. pedis (7/10), while all samples were negative for T. medium. However, none of these Treponema species could be isolated and sequence analysis of the amplified products showed 100% match of 365 base pairs (bp) to Treponema phylotype PT3 and almost full match (530 of 532 bp, 99.6%) to Treponema phylotype PT13. The presence of T. phagedenis, PT3 and PT13 phylotypes was confirmed by FISH analyses. The phylotypes of T. phagedenis were present in all hybridized positive biopsies of Treponema spp., and PT13 and PT3 were less abundant. Neither D. nodosus nor F. necrophorum were detected. The histological Treponema score was mostly mild. Digital dermatitis in captive European Bison is contagious and differs from bovine digital dermatitis, concerning associated pathogens as well as gross appearance.

A filter-assisted culture method for isolation of Treponema spp. from bovine digital dermatitis and their identification by MALDI-TOF MS.

Digital dermatitis (DD) is a major infectious foot disease of cattle worldwide. Some DD stages are associated with lameness, and the disease has significant economic and animal welfare consequences. The pathogenesis of the disease is not yet fully understood, but Treponema spp. have been associated consistently with clinical cases. Isolation of these fastidious bacteria is difficult and cumbersome. We describe an improved method enabling the culturing of the 3 Treponema spp. (T. pedis, T. phagedenis, and T. medium) from bovine foot specimens derived from DD lesions, using a combination of membrane filtering and subsequent growth on selective agar media. The entire procedure from sampling to verification of individual Treponema spp. takes up to 24 d. In addition, we established a MALDI-TOF MS-based identification method to be applied for confirmation of the different Treponema spp. This scheme provides an unambiguous, simple, and straightforward identification procedure for DD-associated Treponema spp.

Mannheimia pernigra sp. nov., isolated from bovine respiratory tract.

Over a period of 1 year, 270 isolates identified as Taxon 39 of Bisgaard were obtained from the nasopharynx of veal calves at 11 epidemiologically independent Swiss fattening farms. Two isolates from each farm and the Australian Taxon 39 reference strain BNO311 were further characterized by genetic and phenotypic methods. Phylogenetic analysis of 16S rRNA and recN gene sequences placed the isolates in a single, distinct cluster within the genus Mannheimia. As to the rpoB gene, most isolates clustered together, but four strains formed a separate cluster close to Mannheimia varigena. Genome sequence analysis of isolates from both rpoB clusters confirmed their species status, with an average nucleotide identity (ANI) >98.9 % between isolates and <84 % to the closest species, M. varigena. Based upon whole genome sequences, the G+C content was determined as 39.1 mol%. Similarly, analysis of MALDI-TOF MS reference spectra clustered the isolates clearly separated from the other Mannheimia species, making this the method of choice for identification. In addition, numerous biochemical markers based on classical as well as commercial identification schemes were determined, allowing separation from other Mannheimia species and identification of the new taxon. Major fatty acids for strain 17CN0883T are C14 : 0, C16 : 0, C16 : 1 ω7c and C18 : 1 ω7c. Major respiratory quinones are ubiquinone-7 and ubiquinone-8. We propose the name Mannheimia pernigra sp. nov. for former Taxon 39 of Bisgaard. The type strain is 17CN0883T (=CCUG 74657T=DSM 111153T) isolated from a veal calf in Switzerland.

Treponema phagedenis (ex Noguchi 1912) Brumpt 1922 sp. nov., nom. rev., isolated from bovine digital dermatitis.

'Treponema phagedenis' was originally described in 1912 by Noguchi but the name was not validly published and no type strain was designated. The taxon was not included in the Approved Lists of Bacterial Names and hence has no standing in nomenclature. Six Treponema strains positive in a 'T. phagedenis' phylogroup-specific PCR test were isolated from digital dermatitis (DD) lesions of cattle and further characterized and compared with the human strain 'T. phagedenis' ATCC 27087. Results of phenotypic and genotypic analyses including API ZYM, VITEK2, MALDI-TOF and electron microscopy, as well as whole genome sequence data, respectively, showed that they form a cluster of species identity. Moreover, this species identity was shared with 'T. phagedenis'-like strains reported in the literature to be regularly isolated from bovine DD. High average nucleotide identity values between the genomes of bovine and human 'T. phagedenis' were observed. Slight genomic as well as phenotypic variations allowed us to differentiate bovine from human isolates, indicating host adaptation. Based on the fact that this species is regularly isolated from bovine DD and that the name is well dispersed in the literature, we propose the species Treponema phagedenis sp. nov., nom. rev. The species can phenotypically and genetically be identified and is clearly separated from other Treponema species. The valid species designation will allow to further explore its role in bovine DD. The type strain for Treponema phagedenis sp. nov., nom. rev. is B43.1T (=DSM 110455T=NCTC 14362T) isolated from a bovine DD lesion in Switzerland.

Otitis in a cat associated with Corynebacterium provencense.

BACKGROUND: The role of corynebacteria in canine and feline otitis has not been investigated in detail; however, members of this genus are increasingly recognized as pathogens of otitis in both human and veterinary medicine. CASE PRESENTATION: Here we report the first case of feline otitis associated with the recently described species Corynebacterium provencense. A seven-month old cat presented with a head tilt and ataxia was diagnosed with peripheral vestibular syndrome associated with an otitis media/interna. This took place 6 weeks after resection of a polyp, having initially shown a full recovery with topical ofloxacin and glucocorticoid treatment. Bacteriology of an ear swab yielded a pure culture of corynebacteria, which could not be identified at the species level using routine methods. However, the 16S rRNA gene sequence was 100% identical to the recently published novel corynebacterium species, Corynebacterium provencense. Whole genome sequencing of the cat isolate and calculation of average nucleotide identity (99.1%) confirmed this finding. The cat isolate was found to contain additional presumptive iron acquisition genes that are likely to encode virulence factors. Furthermore, the strain tested resistant to clindamycin, penicillin and ciprofloxacin. The cat was subsequently treated with chloramphenicol, which lead to clinical improvement. CONCLUSION: Corynebacteria from otitis cases are not routinely identified at the species level and not tested for antimicrobial susceptibility in veterinary laboratories, as they are not considered major pathogens. This may lead to underreporting of this genus or animals being treated with inappropriate antimicrobials since corynebacteria are often resistant to multiple drugs.

Uruburuella testudinis sp. nov., isolated from tortoise (Testudo).

A polyphasic taxonomic analysis was carried out on 11 uncommon Gram-stain-negative, non-motile, catalase- and oxidase-positive, but indole-negative, bacterial strains isolated from tortoises. Phenotypically and genetically they represented a homogeneous group of organisms most closely related to, but distinct from, Uruburuella suis. In a reconstructed 16S rRNA gene tree they clustered on a monophyletic branch next to U. suis with gene similarities between strains of 99.5-100%, and of up to 98.2% with U. suis . DNA-DNA hybridization indicated the organisms represented a novel species with only 40% DNA-DNA similarity with U. suis . Partial sequencing of rpoB resulted in two subclusters confirming the 16S rRNA gene phylogeny; both genes allowed clear separation and identification of the novel species. Furthermore, they could be unambiguously identified by matrix-assisted laser desorption ionization time-of-flight MS, where, again, they formed a highly homogeneous cluster separate from U. suis and other members of the family Neisseriaceae . The major fatty acids were C(16 : 0) and summed feature C(16 : 1)ω7c/iso-C(15 : 0) 2-OH. The DNA G+C content was 54.4 mol%. Based on phenotypic and genetic data we propose classifying these organisms as representatives of a novel species named Uruburuella testudinis sp. nov. The type strain is 07_OD624(T) ( = DSM 26510(T) = CCUG 63373(T)).

Routine phenotypic identification of bacterial species of the family Pasteurellaceae isolated from animals.

Pasteurellaceae are bacteria with an important role as primary or opportunistic, mainly respiratory, pathogens in domestic and wild animals. Some species of Pasteurellaceae cause severe diseases with high economic losses in commercial animal husbandry and are of great diagnostic concern. Because of new data on the phylogeny of Pasteurellaceae, their taxonomy has recently been revised profoundly, thus requiring an improved phenotypic differentiation procedure to identify the individual species of this family. A new and simplified procedure to identify species of Actinobacillus, Avibacterium, Gallibacterium, Haemophilus, Mannheimia, Nicoletella, and Pasteurella, which are most commonly isolated from clinical samples of diseased animals in veterinary diagnostic laboratories, is presented in the current study. The identification procedure was evaluated with 40 type and reference strains and with 267 strains from routine diagnostic analysis of various animal species, including 28 different bacterial species. Type, reference, and field strains were analyzed by 16S ribosomal RNA (rrs) and rpoB gene sequencing for unambiguous species determination as a basis to evaluate the phenotypic differentiation schema. Primary phenotypic differentiation is based on beta-nicotinamide adenine dinucleotide (beta-NAD) dependence and hemolysis, which are readily determined on the isolation medium. The procedure divides the 28 species into 4 groups for which particular biochemical reactions were chosen to identify the bacterial species. The phenotypic identification procedure allowed researchers to determine the species of 240 out of 267 field strains. The procedure is an easy and cost-effective system for the rapid identification of species of the Pasteurellaceae family isolated from clinical specimens of animals.