Corynebacterium uberis sp. nov. frequently isolated from bovine mastitis.

Several strains belonging to the genus Corynebacterium, but not to any described species of the genus were isolated from bovine mastitic milk samples over the past five years in the diagnostic unit of the University of Bern. Six of these strains (18M0132T, 17M2518, 18M0913, 19M0083, 20M1046 and 20M1090) that were phenotypically similar were further characterized genotypically. Gram-positive coryneform rods were catalase positive, facultative anaerobe and CAMP-test negative. Whole genome sequencing and subsequent phylogenetic analysis revealed their genome size to be 2.53 Mb and their G + C content to be between 65.4 and 65.5 mol%. Digital DNA-DNA hybridisation (dDDH) showed the highest similarity of only less than 20% with Corynebacterium mastitidis and Corynebacterium frankenforstense, which indicated that the isolates belong to an undescribed Corynebacterium species. This was confirmed by studying the average nucleotide identity (ANI) where the accepted species boundary is around 95% and which ranged between 70.3% and 74.9% with the most closely related species C. mastitidis. We established MALDI-TOF fingerprints of the species, which allows a clear separation from related species and can be used by other laboratories for diagnostic purposes. Based on our analyses we conclude that the selected strains belong to a previously undescribed species and propose the name Corynebacterium uberis sp. nov. The proposed type strain is 18M0132T (=DSM 111922T, = CCOS 1972T)

Field validation of an antibiotic-free hoof spray to effectively treat ovine footrot by eliminating virulent Dichelobacter nodosus.

Ovine footrot caused by Dichelobacter nodosus is a highly contagious hoof disease negatively impacting animal welfare and causing major economic losses to the sheep industry. Bactericidal footbaths have shown to be an efficient treatment option and will be used in the national footrot control program in Switzerland. However, the application of footbaths is laborious and economically not sound for small flock holders. We therefore tested in a field study the Intra Repiderma spray for its applicability and efficacy to treat ovine footrot. Ten independent flocks fulfilling defined parameters (e.g. clinical signs, positive for D. nodosus, flock size) could be identified and were included in the study. Farms were visited weekly to fortnightly and clinical scores and swabs for D. nodosus real-time (rt)PCR were taken. Treatment with the Intra Repiderma spray was started after initial claw trimming at the very first visit and was carried out three times within a week. Clearly visible clinical improvement was evident after one week of treatment. Virulent D. nodosus amounts on feet declined constantly during treatment which was continued until all sheep of a flock tested rtPCR-negative (1-10 weeks). Results indicate that a highly effective improvement of clinical signs and complete elimination of virulent D. nodosus can be achieved with the spray treatment. Therefore, it is a valuable alternative to cumbersome footbaths especially for small flocks. A sustainable control of footrot and its pathogen in a successfully treated flock can be maintained by strict biosecurity measures and continued treatment as far as necessary

IS711-based real-time PCR assay as a tool for detection of Brucella spp. in wild boars and comparison with bacterial isolation and serology

<p>Abstract</p> <p>Background</p> <p>Control of brucellosis in livestock, wildlife and humans depends on the reliability of the methods used for detection and identification of bacteria. In the present study, we describe the evaluation of the recently established real-time PCR assay based on the <it>Brucella</it>-specific insertion sequence IS<it>711 </it>with blood samples from 199 wild boars (first group of animals) and tissue samples from 53 wild boars (second group of animals) collected in Switzerland. Results from IS<it>711 </it>real-time PCR were compared to those obtained by bacterial isolation, Rose Bengal Test (RBT), competitive ELISA (c-ELISA) and indirect ELISA (i-ELISA).</p> <p>Results</p> <p>In the first group of animals, IS<it>711 </it>real-time PCR detected infection in 11.1% (16/144) of wild boars that were serologically negative. Serological tests showed different sensitivities [RBT 15.6%, c-ELISA 7.5% and i-ELISA 5.5%] and only 2% of blood samples were positive with all three tests, which makes interpretation of the serological results very difficult. Regarding the second group of animals, the IS<it>711 </it>real-time PCR detected infection in 26% of animals, while <it>Brucella </it>spp. could be isolated from tissues of only 9.4% of the animals.</p> <p>Conclusion</p> <p>The results presented here indicate that IS<it>711 </it>real-time PCR assay is a specific and sensitive tool for detection of <it>Brucella </it>spp. infections in wild boars. For this reason, we propose the employment of IS<it>711 </it>real-time PCR as a complementary tool in brucellosis screening programs and for confirmation of diagnosis in doubtful cases.</p

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

First European report of Francisella tularensis subsp. holarctica isolation from a domestic cat

Francisella tularensis subsp. holarctica is a select agent causing life-threatening tularemia. It has been isolated from humans and animals, mainly lagomorphs and rodents, rarely other wild carnivore species. Increasing numbers of human tularemia cases have been reported during the last 5 years in Switzerland. Here we report the first isolation of Francisella tularensis subsp. holarctica from a domestic cat in Europe and compare its genome sequence with other Swiss isolates. The cat isolate shows a close phylogenetic relationship with a contemporary hare isolate from close geographic proximity, indicating a possible epidemiological link

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

Clinical and epidemiological analysis of Campylobacter fetus subsp. fetus infections in humans and comparative genetic analysis with strains isolated from cattle.

BACKGROUND Campylobacter fetus subspecies fetus (CFF) is an important pathogen for both cattle and humans. We performed a systematic epidemiological and clinical study of patients and evaluated the genetic relatedness of 17 human and 17 bovine CFF isolates by using different genotyping methods. In addition, the serotype, the dissemination of the genomic island containing a type IV secretion system (T4SS) and resistance determinants for tetracycline and streptomycin were also evaluated. METHODS The isolates from patients diagnosed with CFF infection as well as those from faecal samples of healthy calves were genotyped using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), as well as single locus sequence typing (SLST) targeting cmp1 and cmp2 genes encoding two major outer membrane proteins in CFF. The presence of the genomic island and identification of serotype was determined by PCRs targeting genes of the T4SS and the sap locus, respectively. Tetracycline and streptomycin resistance phenotypes were determined by minimal inhibitory concentration. Clinical data obtained from medical records and laboratory data were supplemented by data obtained via telephone interviews with the patients and treating physicians. RESULTS PFGE analysis defined two major clusters; cluster A containing 16 bovine (80 %) isolates and cluster B containing 13 human (92 %) isolates, suggesting a host preference. Further genotypic analysis using MLST, SLST as well as sap and T4SS PCR showed the presence of genotypically identical isolates in cattle and humans. The low diversity observed within the cmp alleles of CFF corroborates the clonal nature of this pathogen. The genomic island containing the tetracycline and streptomycin resistance determinants was found in 55 % of the isolates in cluster A and correlated with phenotypic antibiotic resistance. CONCLUSIONS Most human and bovine isolates were separated on two phylogenetic clusters. However, several human and bovine isolates were identical by diverse genotyping methods, indicating a possible link between strains from these two hosts

Identification and Molecular Characterization of a Novel Hordeivirus Associated With Yellow Mosaic Disease of Privet (Ligustrum vulgare) in Europe.

Wild plants serve as a large reservoir of known and yet-unknown viruses and as a source of viral pathogens of cultivated plants. Yellow mosaic disease of forest shrub Ligustrum vulgare (privet) was recurrently observed in Europe for more than 100 years. Using a universal virus identification approach based on deep sequencing and de novo assembly of viral small interfering (si)RNAs we identified a causative agent of this disease in Switzerland and reconstructed its complete 3-segmented RNA genome. Notably, a short 3'-terminal common region (CR) attached to each segment via a ∼53-71 nucleotide poly(A) tract, as determined by RT-PCR sequencing, was initially identified as an orphan siRNA contig with conserved tRNA-like secondary structure. Phylogenomic analysis classified this virus as a novel member in the genus Hordeivirus of family Virgaviridae, which we named ligustrum mosaic virus (LigMV). Similar to other hordeiviruses, LigMV formed rod-shape virions (visualized by electron microscopy), was transmitted through seeds and could also be mechanically transmitted to herbaceous hosts Chenopodium quinoa and Nicotiana benthamiana. Blot hybridization analysis identified genomic and subgenomic RNAs, sharing the 3'-CR and likely serving as monocistronic mRNAs for seven evolutionarily-conserved viral proteins including two subunits of viral RNA-dependent RNA polymerase, coat protein, triple gene block proteins mediating viral movement and cysteine-rich suppressor of RNA silencing. Analysis of size, polarity, and hotspot profiles of viral siRNAs suggested that they are produced by the plant antiviral Dicer-like (DCL) proteins DCL2 and DCL4 processing double-stranded intermediates of genomic RNA replication. Whole genome sequencing of French and Austrian isolates of LigMV revealed its genetic stability over a wide geographic range (>99% nucleotide identity to Swiss isolates and each other), suggesting its persistence and spread in Europe via seed dispersal

Genomic Characterization and Antimicrobial Susceptibility of Dromedary-Associated Staphylococcaceae from the Horn of Africa.

Members of the Staphylococcaceae family, particularly those of the genus Staphylococcus, encompass important human and animal pathogens. We collected and characterized Staphylococcaceae strains from apparently healthy and diseased camels (n = 84) and cattle (n = 7) in Somalia and Kenya. We phenotypically characterized the strains, including their antimicrobial inhibitory concentrations. Then, we sequenced their genomes using long-read sequencing, closed their genomes, and subsequently compared and mapped their virulence- and resistance-associated gene pools. Genome-based phylogenetics revealed 13 known Staphylococcaceae and at least two novel species. East African strains of different species encompassed novel sequence types and phylogenetically distant clades. About one-third of the strains had non-wild-type MICs. They were resistant to at least one of the following antimicrobials: tetracycline, benzylpenicillin, oxacillin, erythromycin, clindamycin, trimethoprim, gentamicin, or streptomycin, encoded by tet(K), blaZ/blaARL, mecA/mecA1, msrA/mphC, salA, dfrG, aacA-aphD, and str, respectively. We identified the first methicillin- and multidrug-resistant camel S. epidermidis strain of sequence type (ST) 1136 in East Africa. The pool of virulence-encoding genes was largest in the S. aureus strains, as expected, although other rather commensal strains contained distinct virulence-encoding genes. We identified toxin-antitoxin (TA) systems such as the hicA/hicB and abiEii/abiEi families, reported here for the first time for certain species of Staphylococcaceae. All strains contained at least one intact prophage sequence, mainly belonging to the Siphoviridae family. We pinpointed potential horizontal gene transfers between camel and cattle strains and also across distinct Staphylococcaceae clades and species. IMPORTANCE Camels are a high value and crucial livestock species in arid and semiarid regions of Africa and gain importance giving the impact of climate change on traditional livestock species. Our current knowledge with respect to Staphylococcaceae infecting camels is very limited compared to that for other livestock species. Better knowledge will foster the development of specific diagnostic assays, guide promising antimicrobial treatment options, and inform about potential zoonotic risks. We characterized 84 Staphylococcaceae strains isolated from camels with respect to their antimicrobial resistance and virulence traits. We detected potentially novel Staphylococcus species, resistances to different classes of antimicrobials, and the first camel multidrug-resistant S. epidermidis strain of sequence type 1136

Novel tetracycline resistance gene tet(65) located on a multi-resistance Corynebacterium plasmid.

BACKGROUND Corynebacterium (C.) sp. 22KM0430 related to C. oculi and isolated from a dog exhibited resistance to tetracycline, and its WGS analysis revealed a putative resistance gene on a 35 562-bp plasmid also harbouring the MLSB resistance gene erm(X). OBJECTIVES To characterize the novel tetracycline resistance gene tet(65) and demonstrate its functionality by expression in C. glutamicum and Escherichia coli and plasmid curing of the host strain. METHODS tet(65) was cloned with and without its repressor tetR(65) and expressed in C. glutamicum DSM20300 and E. coli DH5α. Plasmid was cured by non-selective passages. Minimal inhibitory concentrations (MICs) of tetracyclines were determined according to CLSI guidelines. Association of tet(65) with efflux was shown by the addition of reserpine to MIC assays. Phylogenetic position and transmembrane structure of Tet(65) were analysed using MEGA11 and DeepTMHMM. RESULTS Tet(65) shows 73% amino acid identity with the closest related Tet(Z), contains 12 transmembrane domains and is structurally related to the Major Facilitator Superfamily. The tetracycline MICs decreased in the plasmid-cured strain and increased when tet(65) was expressed in C. glutamicum and in E. coli. The MICs of tetracycline decreased in the presence of reserpine indicating that tet(65) functions as an efflux pump. A GenBank search also identified tet(65) in C. diphtheriae and Brevibacterium (B.) casei and B. luteolum. CONCLUSIONS A novel tetracycline efflux gene tet(65) was identified in a C. oculi related species and was also present in the human pathogen C. diphtheriae and in Brevibacterium species indicating broader potential for dissemination