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.

Evolution of In Vitro Antimicrobial Susceptibility of Equine Clinical Isolates in France between 2016 and 2019.

The present study described the evolution of antimicrobial resistance in equine pathogens isolated from 2016 to 2019. A collection of 7806 bacterial isolates were analysed for their in vitro antimicrobial susceptibility using the disk diffusion method. The most frequently isolated pathogens were group C Streptococci (27.0%), Escherichia coli (18.0%), Staphylococcus aureus (6.2%), Pseudomonas aeruginosa (3.4%), Klebsiella pneumoniae (2.3%) and Enterobacter spp. (2.1%). The majority of these pathogens were isolated from the genital tract (45.1%, n = 3522). With the implementation of two French national plans (named ECOANTIBIO 1 and 2) in 2012-2016 and 2017-2021, respectively, and a reduction in animal exposure to veterinary antibiotics, our study showed decreases in the resistance of group C Streptococci, Klebsiella pneumoniae and Escherichia coli against five classes, four classes and one class of antimicrobials tested, respectively. However, Staphylococcus aureus, Escherichia coli and Enterobacter spp. presented an increased resistance against all the tested classes, excepted for two fifths of E. coli. Moreover, the percentages of multi-drug resistant strains of Staphylococcus aureus and Enterobacter spp. also increased from 24.5% to 37.4% and from 26.3% to 51.7%, respectively. The data reported here are relevant to equine practitioners and will help to improve knowledge related to antimicrobial resistance in common equine pathogens.

In vitro antimicrobial susceptibility of equine clinical isolates from France, 2006-2016.

OBJECTIVES: This study aimed to analyse antimicrobial susceptibility evolution of equine pathogens isolated from clinical samples from 2006-2016. METHODS: A collection of 25 813 bacterial isolates was studied, clustered according to their origins (respiratory tract, cutaneous, genital and other), and analysed for their antimicrobial susceptibility using the disk diffusion method. RESULTS: The most frequently isolated pathogens were group C Streptococci (27.6%), Escherichia coli (20.0%), Staphylococcus aureus (7.8%), Pseudomonas aeruginosa (4.0%), Enterobacter spp. (3.4%), Klebsiella pneumoniae (2.4%), and Rhodococcus equi (1.8%). Of the isolates, 9512 were from respiratory samples (36.8%), 7689 from genital origin (29.8%), and 4083 from cutaneous samples (15.8%). Over the 11-year period, the frequency of multidrug-resistant (MDR) strains fluctuated between 6.4-20.4% for group C Streptococci and 17-37.7% for Klebsiella pneumoniae. From 2006-2009, 24.5-43.0% of Staphylococcus aureus isolates were MDR; after 2009 the level did not exceeded 27.6%. For Escherichia coli and Enterobacter spp., these levels were mostly >30.0% until 2012, but significantly decreased thereafter (22.5-26.3%). CONCLUSIONS: This study is the first large-scale analysis of equine pathogens, by the number of samples and duration of study. The results showed high levels of MDR strains and the need to support veterinary antimicrobial stewardship to encourage proper use of antibiotics.

Are animals a source of Stenotrophomonas maltophilia in human infections? Contributions of a nationwide molecular study.

Stenotrophomonas maltophilia (Sm) is an archetypal environmental opportunistic bacterium responsible for health care-associated infections. The role of animals in human Sm infections is unknown. This study aims to reveal the genetic and phylogenetic relationships between pathogenic strains of Sm, both animal and human, and identify a putative role for animals as a reservoir in human infection. We phenotypically and genotypically characterized 61 Sm strains responsible for animal infections (mainly respiratory tract infections in horses) from a French nationwide veterinary laboratory network. We tested antimicrobial susceptibility and performed MLST and genogrouping using the concatenation of the seven housekeeping genes from the original MLST scheme. Excluding the eight untypeable strains owing to the lack of gene amplification, only 10 out of the 53 strains yielded a known ST (ST5, ST39, ST162, ST8, ST27, ST126, ST131). The genogroup distribution highlighted not only genogroups (genogroups 5 and 9) comprised exclusively of animal strains but also genogroups shared by human and animal strains. Interestingly, these shared genogroups were primarily groups 2 and 6, which have previously been identified as the two most frequent genogroups among human-pathogenic Sm strains, especially among respiratory pathogens. The antimicrobial susceptibility testing underlined the presence of acquired resistance: 18.8 and 7.5% of the tested isolates were resistant to the sulfonamide-trimethoprim combination and ciprofloxacin, respectively. Animal strains of Sm shared phylogenetic traits with some of the most successful human strains. The exact relationships between the human and animal strains, and the genetic support of these common traits, need to be determined.

Multiplex PCR targeting tpi (triose phosphate isomerase), tcdA (Toxin A), and tcdB (Toxin B) genes for toxigenic culture of Clostridium difficile.

A multiplex PCR toxigenic culture approach was designed for simultaneous identification and toxigenic type characterization of Clostridium difficile isolates. Three pairs of primers were designed for the amplification of (i) a species-specific internal fragment of the tpi (triose phosphate isomerase) gene, (ii) an internal fragment of the tcdB (toxin B) gene, and (iii) an internal fragment of the tcdA (toxin A) gene allowing distinction between toxin A-positive, toxin B-positive (A+B+) strains and toxin A-negative, toxin B-positive (A-B+) variant strains. The reliability of the multiplex PCR was established by using a panel of 72 C. difficile strains including A+B+, A-B-, and A-B+ toxigenic types and 11 other Clostridium species type strains. The multiplex PCR assay was then included in a toxigenic culture approach for the detection, identification, and toxigenic type characterization of C. difficile in 1,343 consecutive human and animal stool samples. Overall, 111 (15.4%) of 721 human samples were positive for C. difficile; 67 (60.4%) of these samples contained A+B+ toxigenic isolates, and none of them contained A-B+ variant strains. Fifty (8%) of 622 animal samples contained C. difficile strains, which were toxigenic in 27 (54%) cases, including 1 A-B+ variant isolate. Eighty of the 721 human stool samples (37 positive and 43 negative for C. difficile culture) were comparatively tested by Premier Toxins A&B (Meridian Bioscience) and Triage C. difficile Panel (Biosite) immunoassays, the results of which were found concordant with toxigenic culture for 82.5 and 92.5% of the samples, respectively. The multiplex PCR toxigenic culture scheme described here allows combined diagnosis and toxigenic type characterization for human and animal C. difficile intestinal infections.

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.

Kaposi's sarcoma-associated herpesvirus-infected primary effusion lymphoma has a plasma cell gene expression profile.

Kaposi's sarcoma-associated herpesvirus is associated with three human tumors: Kaposi's sarcoma, and the B cell lymphomas, plasmablastic lymphoma associated with multicentric Castleman's disease, and primary effusion lymphoma (PEL). Epstein-Barr virus, the closest human relative of Kaposi's sarcoma-associated herpesvirus, mimics host B cell signaling pathways to direct B cell development toward a memory B cell phenotype. Epstein-Barr virus-associated B cell tumors are presumed to arise as a consequence of this virus-mediated B cell activation. The stage of B cell development represented by PEL, how this stage relates to tumor pathology, and how this information may be used to treat the disease are largely unknown. In this study we used gene expression profiling to order a range of B cell tumors by stage of development. PEL gene expression closely resembles that of malignant plasma cells, including the low expression of mature B cell genes. The unfolded protein response is partially activated in PEL, but is fully activated in plasma cell tumors, linking endoplasmic reticulum stress to plasma cell development through XBP-1. PEL cells can be defined by the overexpression of genes involved in inflammation, cell adhesion, and invasion, which may be responsible for their presentation in body cavities. Similar to malignant plasma cells, all PEL samples tested express the vitamin D receptor and are sensitive to the vitamin D analogue drug EB 1089 (Seocalcitol).

Identification of amplified and expressed genes in breast cancer by comparative hybridization onto microarrays of randomly selected cDNA clones.

Microarray analysis using sets of known human genes provides a powerful platform for identifying candidate oncogenes involved in DNA amplification events but suffers from the disadvantage that information can be gained only on genes that have been preselected for inclusion on the array. To address this issue, we have performed comparative genome hybridization (CGH) and expression analyses on microarrays of clones, randomly selected from a cDNA library, prepared from a cancer containing the DNA amplicon under investigation. Application of this approach to the BT474 breast carcinoma cell line, which contains amplicons at 20q13, 17q11-21, and 17q22-23, identified 50 amplified and expressed genes, including genes from these regions previously proposed as candidate oncogenes. When considered together with data from microarray expression profiles and Northern analyses, we were able to propose five genes as new candidate oncogenes where amplification in breast cancer cell lines was consistently associated with higher levels of RNA expression. These included the HB01 histone acetyl transferase gene at 17q22-23 and the TRAP100 gene, which encodes a thyroid hormone receptor-associated protein coactivator, at 17q11-21. The results demonstrate the utility of this microarray-based CGH approach in hunting for candidate oncogenes within DNA amplicons.