Antimicrobial resistance and genetic diversity of Klebsiella pneumoniae strains from different clinical sources in horses.

Introduction: Klebsiella pneumoniae is a major cause of infections and reproductive disorders among horses, ranked in recent French studies as the sixth most frequently isolated bacterial pathogen in equine clinical samples. The proportion of multidrug-resistant (MDR) K. pneumoniae is therefore significant in a context where MDR K. pneumoniae strains are considered a major global concern by the World Health Organization. Methods: In this study, we used a genomic approach to characterize a population of 119 equine K. pneumoniae strains collected by two laboratories specialized in animal health in Normandy (France). We describe the main antibiotic resistance profiles and acquired resistance genes, and specify the proportion of virulence-encoding genes carried by these strains. The originality of our panel of strains lies in the broad collection period covered, ranging from 1996 to 2020, and the variety of sample sources: necropsies, suspected bacterial infections (e.g., genital, wound, allantochorion, and umbilical artery samples), and contagious equine metritis analyses. Results: Our results reveal a remarkable level of genomic diversity among the strains studied and we report the presence of 39% MDR and 9% hypervirulent strains (including 5% that are both MDR and hypervirulent). Discussion: These findings clearly emphasize the importance of improving the surveillance of K. pneumoniae in routine equine diagnostic tests to detect high-risk MDR-hypervirulent Klebsiella pneumoniae strains. The circulation of these worrisome strains reveals that they are not being detected by the simple K1, K2, and K5 serotype approach currently implemented in the French horse-breeding sector.

Relationship between rifampicin resistance and RpoB substitutions of Rhodococcus equi strains isolated in France.

OBJECTIVES: Study of the rifampicin resistance of Rhodococcus equi strains isolated from French horses over a 20-year period. METHODS: Rifampicin susceptibility was tested by disk diffusion (DD) and broth macrodilution methods, and rpoB gene sequencing and MLST were performed on 40 R. equi strains, 50.0% of which were non-susceptible to rifampicin. RESULTS: Consistency of results was observed between rifampicin susceptibility testing and rpoB sequencing. Strains non-susceptible to rifampicin by DD had a substitution at one of the sites (Asp516, His526 and Ser531) frequently encountered and conferring rifampicin resistance. High-level resistance was correlated with His526Asp or Ser531Leu substitutions; low-level resistance was correlated with Asp516Tyr substitution, a novel substitution for R. equi. Strains susceptible to rifampicin by DD showed no substitution in the three sites, except for two strains carrying, respectively, the His526Asn and Asp516Val substitutions (previously correlated with low-level rifampicin resistance). Both strains were isolated from an animal from which ten other strains were also isolated and found to be rifampicin-non-susceptible by DD. MLST showed the presence of 10 STs (including the novel ST43), but no association was observed with rifampicin resistance. CONCLUSIONS: This study confirms that certain substitutions in RpoB are more likely to confer high- or low-level rifampicin resistance, describes a new substitution conferring rifampicin resistance in R. equi and suggests non-clonal dissemination of rifampicin-resistant strains in France. Standard DD may miss strains with a low-level rifampicin-resistant substitution; further studies are needed to remedy the absence of R. equi-specific clinical breakpoints.

Resumeq: A Novel Way of Monitoring Equine Diseases Through the Centralization of Necropsy Data.

The French surveillance network for causes of equine mortality (Resumeq) was created in 2015 for the qualitative surveillance of equine mortality through the centralization in a national database of necropsy data and their subsequent epidemiological analysis. It was designed to identify the causes of equine mortality, monitor their evolution over time and space, and detect emerging diseases as early as possible. Resumeq is an event-based surveillance system involving various players and structures. It is organized around a steering body, a scientific and technical support committee and a coordination unit. Different tools have been developed specifically for Resumeq. These include standardized necropsy protocols, a thesaurus for the anatomopathological terms and the causes of equine death, and an interactive web application so that network contributors can display data analysis results. The four French veterinary schools, seventeen veterinary laboratories, and ten veterinary clinics already contribute to the production and centralization of standardized data. To date, the data from around 1,000 equine necropsies have been centralized. While most deaths were located in western France, the geographic coverage is gradually improving. Data analysis allows the main causes of death to be ranked and major threats identified on a local, regional or national level. Initial results demonstrate the feasibility and benefits of this national surveillance tool. Moreover, in the future, this surveillance could take an international dimension if several countries decided to jointly capitalize on their necropsy data.

Identification of pathogenic Leptospira strains in tissues of a premature foal by use of polymerase chain reaction analysis.

Studies were carried out to determine the cause of death in a prematurely born Thoroughbred foal that died 24 hours after birth. Necropsy revealed gross lesions suggestive of septicemia. A commercial Leptospira polymerase chain reaction (PCR) assay designed to specifically amplify the hemolysis-associated protein 1 (hap1) gene present only in pathogenic Leptospira strains detected the presence of Leptospira DNA in various tissues of the foal. Histologic examination of lung, liver, kidney, and myocardium revealed numerous spirochetes in Warthin-Starry-stained tissue sections. Results of PCR analysis and histologic examination suggested a leptospiral infection in the newborn foal. At the moment of death, the infection coexisted with a streptococcal-associated aspiration bronchopneumonia and postpartum septicemia. These findings indicate that the PCR assay based on the amplification of the hap1 gene represents a useful tool for specific detection of pathogenic leptospira in field samples taken from horses.

Investigation of Neospora sp. antibodies in aborted mares from Normandy, France.

Neospora caninum, an apicomplexan protozoan parasite, is recognized as a major cause of abortion in cattle while limited information is presently available on association between equine Neospora infections and abortions. The aim of the present study was to document prevalence of antibodies against Neospora sp. in aborted mares as a clue to the role of N. caninum in mare reproductive failure in Normandy, France. Using an agglutination test, the number of animals with elevated (>80) anti-Neospora sp. antibody titer was higher in a group of 54 aborted mares than in randomly chosen groups of 45 mares and 76 horses sampled for equine arteritis virus and Fasciola hepatica antibodies, respectively (P<0.001). N. caninum DNA was found in 3/91 fetal brains, 2/77 fetal hearts, and 1/1 placenta, and present in both brains and hearts of two fetuses. In 13 cases for which both mare serum and fetus were available, no fetal N. caninum amplification product was present while a large variation of maternal antibody titers was found. Data prompt at additional surveys of association between equine reproductive failure and Neospora sp. infection.

Magnetic resonance imaging for the diagnosis of stress fractures in a horse.

In humans, magnetic resonance (MR) imaging is the method of choice for the diagnosis of stress fractures. In this paper, bilateral stress fracture of the lateral condyle of the third metacarpal bone in a French trotter is described. Results of the radiographic, MR imaging, and histologic examinations are presented, with a focus on the MR signal abnormalities found. Based on this patient, the potential use of MR imaging for the diagnosis of stress fractures in horses is discussed.