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.

LISTERIA MONOCYTOGENES INFECTION IN FREE-RANGING RED FOXES (VULPES VULPES) AND EURASIAN LYNX (LYNX LYNX) IN SWITZERLAND.

Listeria monocytogenes is an ubiquitous environmental saprophytic bacterium causing listeriosis in domestic animals, humans, and occasionally wildlife. In animals, this foodborne zoonotic disease mainly occurs in ruminants and it is rare in carnivores. Seven red foxes (Vulpes vulpes) and one Eurasian lynx (Lynx lynx) were diagnosed with listeriosis between 2010 and 2021 at the Institute for Fish and Wildlife Health, Bern, Switzerland. Necropsy and histopathology revealed meningitis (six of seven red foxes), hepatitis (six of seven red foxes), pneumonia (five of seven red foxes), splenitis (two of seven red foxes) and splenomegaly (the Eurasian lynx, two of seven red foxes). Listeria monocytogenes was isolated from either lung, spleen, liver, or kidney of all animals. Serotyping detected L. monocytogenes serotype 1/2a in five red foxes and the Eurasian lynx and serotype 4b in two red foxes. Six red foxes were positive for canine distemper virus (CDV) by polymerase chain reaction, whereas the Eurasian lynx and one red fox were negative. One red fox that was positive for CDV and listeriosis was also diagnosed with salmonellosis. The identified L. monocytogenes serotypes are among the three most frequently isolated serotypes (1/2a, 1/2b, and 4b) from food or the food production environment and those that cause most listeriosis cases in humans and animals. Coinfection with CDV in six red foxes questions the role of CDV as potential predisposing factor for septicemic listeriosis. The detection of listeriosis in the regionally endangered Eurasian lynx and in carnivores highly abundant in urban settings, such as red foxes, reinforces the importance of wildlife health surveillance in a One Health context and adds the Eurasian lynx to the list of carnivores susceptible to the disease. Further investigations are required to assess the prevalence and epidemiology of L. monocytogenes in free-ranging carnivores and its interaction with CDV.

Emergence of OXA-48-producing Enterobacter hormaechei in a Swiss companion animal clinic and their genetic relationship to clinical human isolates.

BACKGROUND: Enterobacter hormaechei producing the carbapenemase OXA-48 was identified repeatedly in infections in companion animals hospitalized at a Swiss veterinary clinic where OXA-48-producing Klebsiella pneumoniae was previously reported. OBJECTIVES: To determine the genetic relatedness of animal and human E. hormaechei strains collected in Switzerland during 2017-22 and their mobile genetic elements. METHODS: Hybrid assemblies for phylogenetic and comparative analysis of animal (n = 9) and human (n = 25) isolates were obtained by sequencing with Illumina, PacBio and Oxford Nanopore Technologies. Antimicrobial susceptibility was tested by broth microdilution. RESULTS: The animal strains were identified as E. hormaechei subsp. xiangfangensis ST114 (n = 6) and ST418 (n = 2), and E. hormaechei subsp. hoffmannii ST78 (n = 1). Human E. hormaechei belonged to subspecies steigerwaltii (n = 10), xiangfangensis (n = 13), hoffmannii (n = 1) and hormaechei (n = 1), with a heterogeneous ST distribution differing from the animal strains, except for two ST114. Core-gene SNP analysis confirmed the clonality of the animal ST114 and ST418 isolates (0 to 10 SNPs), and close relatedness of animal and human ST114 strains (80-120 SNPs). The strains harboured the blaOXA-48 gene on ca. 63 kb IncL-type plasmids (n = 27); on ca. 72 kb IncL plasmids co-harbouring blaCTX-M-14 (n = 2); and on ca. 150-180 kb IncFIB (n = 4) or hybrid IncFIB/IncL (n = 1) plasmids. The blaOXA-48-harbouring plasmids and the blaDHA-1-carrying ISCR1 element in one animal ST114 and both ST418 clones were likely acquired from previously spreading K. pneumoniae strains. CONCLUSIONS: Common ecological niches favour the spread of plasmid-borne carbapenemases among Enterobacterales and the emergence of MDR E. hormaechei clones.

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.

Two high-risk clones of carbapenemase-producing Klebsiella pneumoniae that cause infections in pets and are present in the environment of a veterinary referral hospital.

OBJECTIVES: Infections with carbapenem-resistant Enterobacterales (CRE) are an emerging problem in pets and a major threat to public health. We determined the genetic relationships among carbapenemase-producing Klebsiella pneumoniae (CPKp) strains causing infections in hospitalized pets in a veterinary clinic and those found in the environment. METHODS: WGS was performed with both the Illumina and Nanopore platforms. Searches of genetic features were performed using several databases and bioinformatics tools, and phylogeny was assessed by whole-genome MLST (wgMLST) using SeqSphere and SNP calling with Snippy. RESULTS: WGS analysis of the CPKp strains identified all environmental and almost all animal strains as the high-risk clone ST11, with the exception of two strains that belonged to ST307. All CPKp belonged to novel complex types (CTs) and carried a conjugative 63 kb IncL plasmid encoding the carbapenemase gene blaOXA-48, yersiniabactin and other virulence factors. Although all CPKp ST11 strains carried additional similar IncR plasmids harbouring multiple antimicrobial resistance genes (ARGs), such as the plasmid-mediated blaDHA-1 AmpC gene, some structural variations were observed. The two ST307 strains carried identical 156 kb MDR IncFIB(K) plasmids with several ARGs, including the blaCTX-M-15 ESBL gene. Both wgMLST and cgSNP analysis confirmed that CPKp strains of the same ST were genetically highly related independent of the source of isolation. CONCLUSIONS: This study demonstrated that the clinical CPKp strains were highly related to those contaminating the clinical environment. These findings confirmed nosocomial spread and highlight veterinary hospitals as a source of CPKp, which may further spread to animals, the environment and humans.

Trueperella pecoris sp. nov. isolated from bovine and porcine specimens.

A novel Gram-stain-positive bacterium was isolated from a purulent bovine milk sample, the bovine placenta from an abortion, the udder secretion of a heifer and the lung of a pig that had succumbed from suppurative bronchopneumonia in Switzerland from 2015 to 2019. The strains grew best under aerobic conditions with 5 % CO2 and colonies were non-haemolytic and greyish-white. They were non-motile and negative for catalase and oxidase. The genomes of the four strains 19M2397T, 15A0121, 15IMD0307 and 19OD0592 were obtained by sequencing. The results of phylogenetic analyses based on the 16S rRNA gene grouped them within the genus Trueperella in the family Arcanobacteriaceae. The genomes had DNA G+C contents of 61.2-62.2 mol% and showed digital DNA-DNA hybridization (dDDH) values of 21.4-22.8 % and average nucleotide identity (ANI) values of approximately 77 % to their closest relatives Trueperella pyogenes and Trueperella bernardiae. With respect to the presence in different livestock species we propose the name Trueperella pecoris sp. nov. The type strain is 19M2397T (=CCOS 1952T=DSM 111392T), isolated from the udder secretion of a heifer diagnosed with summer mastitis in 2019.

Actinomycosis in a gray four-eyed opossum (Philander opossum) caused by a novel species of Schaalia.

BACKGROUND: Infective lesions of the jaws and adjacent tissues (lumpy jaw disease, LJD) have been recognized as one major cause of death of captive macropods. Fusobacterium necrophorum and Actinomyces species serve as the main source of LJD in kangaroos and wallabies. Currently, little is reported about LJD or similar diseases in opossums. CASE PRESENTATION: Here we report a case of actinomycosis resembling the entity lumpy jaw disease in a gray four-eyed opossum, caused by a novel species of Schaalia. A 2.8 year old male Philander opossum was presented with unilateral swelling of the right mandible. After an initial treatment with marbofloxacin, the opossum was found dead the following day and the carcass was submitted for necropsy. Postmortem examination revealed severe mandibular skin and underlying soft tissue infection with subsequent septicemia as the cause of death. Histological examination demonstrated Splendore-Hoeppli phenomenon, typically seen in classical cases of actinomycosis. Bacteriology of liver and mandibular mass yielded a previously undescribed species of Schaalia, whose 16 S rRNA gene sequence was 97.0 % identical to Schaalia canis. Whole genome sequencing of the opossum isolate and calculation of average nucleotide identity confirmed a novel species of Schaalia, for which no whole genome sequence is yet available. CONCLUSIONS: The herewith reported Schaalia infection in the gray four-eyed opossum resembling classical actinomycosis gives a novel insight into new exotic animal bacterial diseases. Schaalia species may belong to the normal oral microbiome, as in macropods, and may serve as a contributor to opportunistic infections. Due to the lack of current literature, more insights and improved knowledge about Schaalia spp. and their pathogenicity will be useful to choose appropriate therapy regimens and improve the treatment success rate and outcome in exotic and endangered species.

Clostridium perfringens-Associated Necrotic Enteritis-Like Disease in Coconut Lorikeets (Trichoglossus haematodus).

Several outbreaks of necrotic enteritis-like disease in lorikeets, from which Clostridium perfringens was consistently isolated, are described. All lorikeets had acute, segmental, or multifocal fibrinonecrotizing inflammatory lesions in the small and/or the large intestine, with intralesional gram-positive rods. The gene encoding C. perfringens alpha toxin was detected by PCR (polymerase chain reaction) on formalin-fixed, paraffin-embedded (FFPE) tissues in 20 out of 24 affected lorikeets (83%), but it was not amplified from samples of any of 10 control lorikeets (P < .0001). The second most prevalent C. perfringens toxin gene detected was the beta toxin gene, which was found in FFPE from 7 out of 24 affected lorikeets (29%). The other toxin genes were detected inconsistently and in a relatively low number of samples. These cases seem to be associated with C. perfringens, although the specific type involved could not be determined.

Methicillin-Resistant Staphylococcus aureus Strains in Swiss Pigs and Their Relation to Isolates from Farmers and Veterinarians.

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged over the last few decades as a One Health problem with an increasing prevalence in various animal species. The most notable animals are pigs, as asymptomatic carriers, and horses, where there is often an association with infections. The current study looked at the course of MRSA prevalence in Swiss livestock since 2009, with a special focus on pigs, followed by screening of veterinarians and farmers. Livestock isolates were obtained from the Swiss monitoring program and then characterized by spa typing. Concentrating on the year 2017, we analyzed the prevalence of MRSA in Swiss veterinarians and farmers, followed by whole-genome sequencing of selected human and animal strains. The phylogeny was assessed by applying core-genome multilocus sequence typing (MLST) and single-nucleotide polymorphism (SNP) analyses, followed by screening for resistance genes and virulence factors. The prevalence of MRSA in Swiss pigs showed a dramatic increase from 2% in 2009 to 44% in 2017. Isolates typically belonged to clonal complex 398 (CC398), split between spa t011 and t034. The higher prevalence was mainly due to an increase in t011. spa t034 strains from farmers were found to be closely associated with porcine t034 strains. The same could be shown for spa t011 strains from horses and veterinarians. spa t034 strains had a high number of additional resistance genes, and two strains had acquired the immune evasion cluster. However, all but one of the pig spa t011 strains clustered in a separate group. Thus, the increase in pig spa t011 strains does not directly translate to humans.IMPORTANCE MRSA is an important human pathogen; thus, its increasing prevalence in livestock over the last decade has a potentially large impact on public health. Farmers and veterinarians are especially at risk due to their close contact with animals. Our work demonstrates a dramatic increase in MRSA prevalence in Swiss pigs, from 2% in 2009 to 44% in 2017. Whole-genome sequencing allowed us to show a close association between farmer and pig strains as well as veterinarian and horse strains, indicating that the respective animals are a likely source of human colonization. Furthermore, we could demonstrate that pig spa t011 strains cluster separately and are probably less likely to colonize humans than are pig spa t034 strains. This research may provide a basis for a more substantiated risk assessment and preventive measures.

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.

Typing of mecD Islands in Genetically Diverse Methicillin-Resistant Macrococcus caseolyticus Strains from Cattle.

Macrococcus caseolyticus belongs to the normal bacterial flora of dairy cows and does not usually cause disease. However, methicillin-resistant M. caseolyticus strains were isolated from bovine mastitis milk. These bacteria had acquired a chromosomal island (McRI mecD -1 or McRI mecD -2) carrying the methicillin resistance gene mecD To gain insight into the distribution of McRI mecD types in M. caseolyticus from cattle, 33 mecD-containing strains from Switzerland were characterized using molecular techniques, including multilocus sequence typing, antibiotic resistance gene identification, and PCR-based McRI mecD typing. In addition, the same genetic features were analyzed in 27 mecD-containing M. caseolyticus strains isolated from bovine bulk milk in England/Wales using publicly available whole-genome sequences. The 60 strains belonged to 24 different sequence types (STs), with strains belonging to ST5, ST6, ST21, and ST26 observed in both Switzerland and England/Wales. McRI mecD -1 was found in different STs from Switzerland (n = 19) and England/Wales (n = 4). McRI mecD -2 was only found in 7 strains from Switzerland, all of which belonged to ST6. A novel island, McRI mecD -3, which contains a complete mecD operon (mecD-mecR1m-mecIm [where the subscript m indicates Macrococcus]) combined with the left part of McRI mecD -2 and the right part of McRI mecD -1, was found in heterogeneous STs from both collections (Switzerland, n = 7; England/Wales, n = 21). Two strains from England/Wales carried a truncated McRI mecD -3. Phylogenetic analyses revealed no clustering of strains according to geographical origin or carriage of McRI mecD -1 and McRI mecD -3. Circular excisions were also detected for McRI mecD -1 and McRI mecD -3 by PCR. The analyses indicate that these islands are mobile and may spread by horizontal gene transfer between genetically diverse M. caseolyticus strains.IMPORTANCE Since its first description in 2017, the methicillin resistance gene mecD has been detected in M. caseolyticus strains from different cattle sources and countries. Our study provides new insights into the molecular diversity of mecD-carrying M. caseolyticus strains by using two approaches to characterize mecD elements: (i) multiplex PCR for molecular typing of McRI mecD and (ii) read mapping against reference sequences to identify McRI mecD types in silico In combination with multilocus sequence typing, this approach can be used for molecular characterization and surveillance of M. caseolyticus carrying mecD.

An unusual case of bovine anthrax in the canton of Jura, Switzerland in 2017.

BACKGROUND: Anthrax caused by Bacillus anthracis is a zoonotic disease mainly affecting herbivores. The last Swiss outbreak was over 20 years ago. We describe a recent anthrax outbreak involving two cows from the same herd. One cow was designated as a peracute clinical case with sudden death and typical lung lesions, while the other cow presented with protracted fever and abortion. CASE PRESENTATION: On April 29th 2017, a 3.5-year-old Montbéliard dairy cow was found dead while out at pasture with haemorrhage from the nose. The veterinarian suspected pneumonia and performed a necropsy on site. Subsequently, a lung and liver sample were sent to the laboratory. Unexpectedly, Bacillus anthracis was isolated, a pathogen not found in Switzerland for decades. Several days later, a second cow from the same farm showed signs of abortion after protracted fever. Since these symptoms are not typical for anthrax, and the bacteria could not be demonstrated in blood samples from this animal, a necropsy was performed under appropriate biosafety measures. Subsequently, Bacillus anthracis could be isolated from the placenta and the sublumbal lymph nodes but not from the blood, liver, spleen and kidney. The outbreak strain (17OD930) was shown to belong to the lineage B.Br.CNEVA, the same as Swiss strains from previous outbreaks in the region. We speculate that the disease came from a temporarily opened cave system that is connected to an old carcass burial site and was flushed by heavy rainfall preceding the outbreak. CONCLUSION: Even in countries like Switzerland, where anthrax is very rare, new cases can occur after unusual weather conditions or ground disturbance. It is important for public officials to be aware of this risk to avoid possible spread.

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.

Whole genome-based antimicrobial resistance, virulence, and phylogenetic characteristics of Trueperella pyogenes clinical isolates from humans and animals.

Trueperella pyogenes is an opportunistic zoonotic bacterial pathogen, whose antimicrobial resistance, virulence, and genetic relatedness between strains from animals and humans are barely studied. These characteristics were therefore analyzed for clinical T. pyogenes strains from 31 animals of 11 different species and 8 humans determining their complete circular genome sequence and antimicrobial susceptibility. The MICs of 19 antimicrobials including 3 antiseptics correlated to the resistance genes identified in silico within the genomes revealing a predominance of resistance to streptomycin (aadA9), sulfamethoxazole (sul1), and tetracycline (tet(33), tet(W/N/W)) among strains from humans and cattle. Additional resistance genes (erm(X), erm(56), cmx, drfA1, aadA1, aph(3'')-Ib (strA), aph(6)-Id (strB), aac(3)-IVa, aph(4)-Ia) were found only sporadically. The resistance genes were localized on genetic elements integrated into the chromosome. A cgMLST-based phylogenetic analysis revealed two major clusters each containing genetically diverse strains. The human strains showed the closest relatedness to strains from cattle. Virulence genes coding for fimbriae (fimA, fimC), neuroamidase (nanP, nanH), pyolysin (plo), and collagen binding protein (cbpA) were identified in strains from different hosts, but no correlation was observed between virulence factors and strain origin. The existence of resistance genes typically found in Gram-negative bacteria within the Gram-positive T. pyogenes indicates a wider capacity to adapt to antimicrobial selective pressure. Moreover, the presence of similar antimicrobial resistance profiles found in cattle and human strains as well as their closest relatedness suggests common zoonotic features and cattle as the potential source for human infections.

Zoonotic bacterial and parasitic intestinal pathogens in foxes, raccoons and other predators from eastern Germany.

In this study, we investigated faecal specimens from legally hunted and road-killed red foxes, raccoons, raccoon dogs, badgers and martens in Germany for parasites and selected zoonotic bacteria. We found that Baylisascaris procyonis, a zoonotic parasite of raccoons, had spread to northeastern Germany, an area previously presumed to be free of this parasite. We detected various pathogenic bacterial species from the genera Listeria, Clostridium (including baratii), Yersinia and Salmonella, which were analysed using whole-genome sequencing. One isolate of Yersinia enterocolitica contained a virulence plasmid. The Salmonella Cholerasuis isolate encoded an aminoglycoside resistance gene and a parC point mutation, conferring resistance to ciprofloxacin. We also found tetracycline resistance genes in Paeniclostridium sordellii and Clostridium baratii. Phylogenetic analyses revealed that the isolates were polyclonal, indicating the absence of specific wildlife-adapted clones. Predators, which scavenge from various sources including human settlements, acquire and spread zoonotic pathogens. Therefore, their role should not be overlooked in the One Health context.

Comparison of genotypes and antibiotic resistances of Campylobacter jejuni and Campylobacter coli on chicken retail meat and at slaughter.

Multilocus sequence typing (MLST) and antibiotic resistance patterns of Campylobacter jejuni and Campylobacter coli from retail chicken meat showed high overlap with isolates collected at slaughterhouses, indicating little selection along the production chain. They also showed significant common sequence types with human clinical isolates, revealing chicken meat as a likely source for human infection.

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.

Fatal gastritis and enterocolitis due to concurrent Helicobacter pylori and Campylobacter jejuni infection in a captive cheetah (Acinonyxjubatus).

A 3.5-year-old female cheetah (Acinonyx jubatus) died after a 10-day history of anorexia, regurgitation and diarrhoea despite symptomatic therapy. At gross post-mortem examination, the stomach was blood-filled with mucosal thickening and multifocal ulcerations. The intestinal mucosa was thickened and reddened, and the intestinal lumen was filled with dark red to black pasty content. Gastric histological lesions were compatible with gastritis due to Helicobacter infection, which was confirmed by polymerase chain reaction. Histology of the intestines revealed a severe necrotizing neutrophilic enterocolitis with abundant intralesional curved to spiral bacteria, corresponding to Campylobacter jejuni, which were subsequently isolated from both small and large intestinal contents. No other intestinal pathogens were detected despite thorough investigations. These findings suggest that C. jejuni may have played an aetiological role in the enterocolitis. Such an association has not been previously reported in non-domestic felids.

Quality of MALDI-TOF mass spectra in routine diagnostics: results from an international external quality assessment including 36 laboratories from 12 countries using 47 challenging bacterial strains.

OBJECTIVES: Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is a widely used method for bacterial species identification. Incomplete databases and mass spectral quality (MSQ) still represent major challenges. Important proxies for MSQ are the number of detected marker masses, reproducibility, and measurement precision. We aimed to assess MSQs across diagnostic laboratories and the potential of simple workflow adaptations to improve it. METHODS: For baseline MSQ assessment, 47 diverse bacterial strains, which are challenging to identify by MALDI-TOF MS, were routinely measured in 36 laboratories from 12 countries, and well-defined MSQ features were used. After an intervention consisting of detailed reported feedback and instructions on how to acquire MALDI-TOF mass spectra, measurements were repeated and MSQs were compared. RESULTS: At baseline, we observed heterogeneous MSQ between the devices, considering the median number of marker masses detected (range = [2-25]), reproducibility between technical replicates (range = [55%-86%]), and measurement error (range = [147 parts per million (ppm)-588 ppm]). As a general trend, the spectral quality was improved after the intervention for devices, which yielded low MSQs in the baseline assessment as follows: for four out of five devices with a high measurement error, the measurement precision was improved (p-values <0.001, paired Wilcoxon test); for six out of ten devices, which detected a low number of marker masses, the number of detected marker masses increased (p-values <0.001, paired Wilcoxon test). DISCUSSION: We have identified simple workflow adaptations, which, to some extent, improve MSQ of poorly performing devices and should be considered by laboratories yielding a low MSQ. Improving MALDI-TOF MSQ in routine diagnostics is essential for increasing the resolution of bacterial identification by MALDI-TOF MS, which is dependent on the reproducible detection of marker masses. The heterogeneity identified in this external quality assessment (EQA) requires further study.

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).