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Abstract Background Bacterial infection of bile is a common cause of hepatobiliary disease in cats. Whether bile harbors a core microbiota in health or in cases of suspected hepatobiliary disease in cats is unknown. Objectives Establish if gallbladder bile in apparently healthy cats harbors a core microbiota composed of bacterial taxa common to many individuals. Compare results of bile cytology, bile culture, and 16S rRNA gene amplicon sequencing in apparently healthy cats and cats with suspected hepatobiliary disease. Animals Forty‐three client‐owned cats with suspected hepatobiliary disease and 17 control cats. Methods Bile was collected by ultrasound guided cholecystocentesis (cats with suspected hepatobiliary disease) or laparotomy after euthanasia (controls). Bile samples underwent cytologic examination, aerobic and anaerobic culture, and DNA was extracted for 16S rRNA gene amplification and sequencing. Results Microbiome sequencing did not identify a core microbiota in control cats or cats having bile sampled because of clinical suspicion for hepatobiliary disease. Microbiome profiles from control cats were indistinguishable from profiles obtained from sampling instruments and reagents that were not exposed to bile (technical controls). Bacterial taxa that could not be explained by contamination or off‐target amplification were identified only in samples from cats with bactibilia and positive bile culture results for Escherichia coli. In several E. coli positive samples, microbiome sequencing also identified a small number of potentially co‐infecting bacterial genera not identified by culture. Conclusions and Clinical Importance Cat bile does not harbor a core microbiota. Uncultured bacteria may contribute to pathogenesis of hepatobiliary disease in cats with bile E. coli infection.

Abstract Background Clinicians face several dilemmas regarding tracheal washes (TWs) for the diagnosis of respiratory disease, including method and prediction of bacterial growth from cytology results. Objective To compare cytology and culture of endotracheal and transtracheal washes and identify factors associated with discordancy and bacterial growth. Animals Two hundred forty‐five dogs with respiratory disease. Methods Retrospective study. Tracheal wash submissions were included if cellularity was sufficient for cytologic interpretation and aerobic cultures were performed. Collection technique, cytology, bacterial growth, and antibiotic history were analyzed. Results Fewer transtracheal specimens (9/144, 6.3%) were excluded for hypocellularity than endotracheal (28/174, 16.1%); otherwise, results were similar and were combined. Of 281 specimens with cellularity sufficient for interpretation, 97 (34.5%) had bacteria on cytology and 191 (68.0%) had bacterial growth. Cytology positive/culture negative discordancy was uncommon (8/97, 8%). Cytology negative/culture positive discordancy was frequent (102/184, 55.4%), but occurred less often (28/184, 14.2%) when only 1+ growth or greater was considered positive. Oropharyngeal contamination was associated with bacterial growth, but not discordancy. No association was found between antibiotic administration and bacterial growth. Conclusions and Clinical Importance Endotracheal wash fluid, in particular, should be screened for gross mucus or turbidity to maximize the likelihood of an adequate specimen. Otherwise, endotracheal and transtracheal specimens were similar. Presence of bacteria on cytology was a good predictor of any growth, while their absence was a good predictor of the absence of growth of 1+ or more. Recent antibiotic usage should not discourage TW culture if there is compelling reason to avoid delay.

The leading cause of treatment failure in Staphylococcus aureus infections is the development of biofilms. Biofilms are highly tolerant to conventional antibiotics which were developed against planktonic cells. Consequently, there is a lack of antibiofilm agents in the antibiotic development pipeline. To address this problem, we developed a platelet-rich plasma (PRP)-derived biologic, termed BIO-PLY (for the BIOactive fraction of Platelet-rich plasma LYsate) which has potent in vitro bactericidal activity against S. aureus synovial fluid free-floating biofilm aggregates. Additional in vitro studies using equine synoviocytes and chondrocytes showed that BIO-PLY protected these cells of the joint from inflammation. The goal of this study was to test BIO-PLY for in vivo efficacy using an equine model of infectious arthritis. We found that horses experimentally infected with S. aureus and subsequently treated with BIO-PLY combined with the antibiotic amikacin (AMK) had decreased bacterial concentrations within both synovial fluid and synovial tissue and exhibited lower systemic and local inflammatory scores compared to horses treated with AMK alone. Most importantly, AMK+BIO-PLY treatment reduced the loss of infection-associated cartilage proteoglycan content in articular cartilage and decreased synovial tissue fibrosis and inflammation. Our results demonstrate the in vivo efficacy of AMK+BIO-PLY and represents a new approach to restore and potentiate antimicrobial activity against synovial fluid biofilms.

Biochemical testing is an important clinical tool in evaluating the physiology of reptiles and amphibians. Suitable point of care analyzers can allow for rapid delivery of results, but small patient size can inhibit sufficient sample collection. This study evaluated the utility of sample dilution with sterile distilled water as a means of biochemical evaluation when sample volume is limited. Blood was collected from 12 eastern box turtles (Terrapene carolina carolina) and 12 marine toads (Rhinella marinus) and analyzed via i-STAT CHEM8+ cartridges. Two undiluted samples and two samples diluted 1 : 1 with sterile water were evaluated immediately following collection for each animal in the study. Analytes reported in the diluted samples were limited to glucose, ionized calcium, and total carbon dioxide. The expected dilution ratio value of diluted to undiluted samples was 0.5, of which glucose in both turtles and toads was nearest. Dilution ratio values for ionized calcium, however, were higher than expected in both turtles and toads. Sample dilution is not recommended for most analytes included on the CHEM8+ cartridge due to values occurring outside the limits of detection for the analyzer. Glucose and ionized calcium values obtained on diluted samples should be interpreted with caution but may provide clinical utility in reptile and amphibian patients where sample volume is limited.

To date studies have not investigated the culture-independent microbiome of bile from dogs, a species where aseptic collection of bile under ultrasound guidance is somewhat routine. Despite frequent collection of bile for culture-based diagnosis of bacterial cholecystitis, it is unknown whether bile from healthy dogs harbors uncultivable bacteria or a core microbiota. The answer to this question is critical to understanding the pathogenesis of biliary infection and as a baseline to exploration of other biliary diseases in dogs where uncultivable bacteria could play a pathogenic role. A pressing example of such a disease would be gallbladder mucocele formation in dogs. This prevalent and deadly condition is characterized by excessive secretion of abnormal mucus by the gallbladder epithelium that can eventually lead to rupture of the gallbladder or obstruction of bile flow. The cause of mucocele formation is unknown as is whether uncultivable, and therefore unrecognized, bacteria play any systematic role in pathogenesis. In this study we applied next-generation 16S rRNA gene sequencing to identify the culture-negative bacterial community of gallbladder bile from healthy dogs and gallbladder mucus from dogs with mucocele formation. Integral to our study was the use of 2 separate DNA isolations on each sample using different extraction methods and sequencing of negative control samples enabling recognition and curation of contaminating sequences. Microbiota findings were validated by simultaneous culture-based identification, cytological examination of bile, and fluorescence in-situ hybridization (FISH) performed on gallbladder mucosa. Using culture-dependent, cytological, FISH, and 16S rRNA sequencing approaches, results of our study do not support existence of a core microbiome in the bile of healthy dogs or gallbladder mucus from dogs with mucocele formation. Our findings further document how contaminating sequences can significantly contribute to the results of sequencing analysis when performed on samples with low bacterial biomass.