Evaluation of a commercial NGS service for detection of bacterial and fungal pathogens in infectious ulcerative keratitis.

OBJECTIVES: To compare results from a commercial next-generation sequencing (NGS) service to corneal cytology and culture for identification of causative organisms in veterinary patients presenting for infectious ulcerative keratitis (IUK). PROCEDURE: Swabs for corneal aerobic and fungal cultures and DNA swabs for NGS were submitted for canine and equine normal controls (n = 11 and n = 4, respectively) and IUK patients (n = 22 and n = 8, respectively) for which microbrush cytology specimens confirmed the presence of infectious organisms. The sensitivity of the NGS results was compared with bacterial and fungal culture results. Concordance between the NGS and culture results was determined. RESULTS: The NGS results were positive for bacterial and fungal organisms in 5 and 1 normal and 18 and 1 IUK cases, respectively. Bacterial and fungal cultures were positive for 7 and 2 normal and 20 and 5 IUK cases, respectively. Sensitivity of NGS was 82.14% (95% confidence interval (CI), 63.11% to 93.94%) and specificity was 76.47% (95% CI, 50.10% to 93.19%). Concordance (complete and partial) between identified bacterial and fungal organisms was found in 79% and 100% of cases, respectively. NGS identified organisms in 3 culture-negative IUK samples. CONCLUSION: A commercial NGS service may be useful in the identification of causative agents in IUK cases with a sensitivity greater than the sensitivity previously reported for aerobic culture. Further testing is needed to determine the clinical significance of additional organisms isolated by NGS from infected cases, as well as organisms isolated from normal corneas.

Prevalence and predictors of bacteremia in dairy calves with diarrhea.

Gastrointestinal disease is the most common cause of mortality in dairy calves. Septicemia is an important sequela of diarrhea, and the possibility of bacteremia is the primary justification for empirical antimicrobial therapy. Prior reports estimate that approximately one-third of diarrheic calves are bacteremic; however, those estimates may not be representative of routine cases in heifer calves on commercial dairy operations early in the course of disease. We hypothesized that the prevalence of bacteremia in calves with diarrhea and systemic signs of illness is less than prior estimates (∼31%), and that clinical signs or hematological values would be associated with the presence or absence of bacteremia. Female calves less than 21 d of age with and without diarrhea were enrolled from 2 commercial dairy farms over a 10-wk period. Diarrheic calves were enrolled if they were newly diagnosed, had loose to watery stool, had either dehydration (assessed by skin tent and eye position) or depression (assessed by suckle reflex and standing ability), and had no prior antimicrobial treatments. Complete health assessments were conducted at 0, 7, and 14 d following enrollment. An aseptic jugular venous sample was collected and cultured using aerobic and anaerobic methods, and bacterial species were identified using mass spectrometry. Poisson regression models were used to identify associations with bacteremia and compute adjusted prevalence ratios. The prevalence of bacteremia in diarrheic and healthy calves was 9.26% (10/108, 95% confidence interval: 4.5-16%) and 14.8% (4/27, 95% confidence interval: 1.4-28.2%), respectively. Among calves with diarrhea, those with a fever (>39.7°C) or depression were 4.8 and 6.5 times more likely, respectively, to have bacteremia. Only 1 of 47 calves (2%) without signs of depression was bacteremic. The prevalence of bacteremia in diarrheic calves with signs of systemic illness (depression or dehydration) was significantly lower than previous estimates, and bacteremia was rare among calves without observed depression. Antimicrobial therapy targeting bacteremia is not currently justified in routine cases of diarrhea in preweaning calves without signs of depression. These results suggest a substantial opportunity for more targeted antimicrobial therapy to improve antimicrobial stewardship.

Using minimum inhibitory concentration values of common topical antibiotics to investigate emerging antibiotic resistance: A retrospective study of 134 dogs and 20 horses with ulcerative keratitis.

OBJECTIVES: To identify the minimum inhibitory concentration (MIC) distribution for commonly used topical antibiotics from isolates of dogs and horses with ulcerative bacterial keratitis, and to investigate changes in MIC values over time and following treatment with topical fluoroquinolones. ANIMALS STUDIED: One hundred thirty-four client-owned dogs and 20 client-owned horses with bacterial ulcerative keratitis. PROCEDURE: Minimum inhibitory concentration values for 14 topical antibiotics were reported for canine and equine cases of bacterial ulcerative keratitis between 2013 and 2018. Changes in MIC values over time and after treatment with topical fluoroquinolones were reported. RESULTS: The three most common bacterial genera isolated were Staphylococcus, Streptococcus, and Pseudomonas. Together, these represented 79.4% of canine cases and 77.4% of equine cases. Overall, isolates from horses tended to have lower MIC values, as did Pseudomonas isolates from both dogs and horses, compared to other bacterial genera, especially Staphylococcus spp. The MIC values of erythromycin and trimethoprim sulfa for Staphylococcus spp., and the MIC value of moxifloxacin for Pseudomonas significantly increased over time. Previous topical fluoroquinolone use was associated with a significant increase in the MIC value of ofloxacin in canine and equine Staphylococcus isolates and current topical fluoroquinolone use was associated with significant increases in the MIC values of ciprofloxacin, moxifloxacin, and ofloxacin in canine Staphylococcus isolates. CONCLUSION: Patients previously or currently treated with topical fluoroquinolones, particularly in Staphylococcus infections, may require alternative antibiotics or additional antibiotic classes other than fluoroquinolones. Bacterial culture with MIC susceptibility testing should be highly recommended when a Staphylococcal infection is suspected.

Population Structure and Antimicrobial Resistance of Canine Uropathogenic Escherichia coli.

Escherichia coli is the most common cause of human and canine urinary tract infection (UTI). Clonal groups, often with high levels of antimicrobial resistance, are a major component of the E. coli population that causes human UTI. While little is known about the population structure of E. coli that causes UTI in dogs, there is evidence that dogs and humans can share fecal strains of E. coli and that human-associated strains can cause disease in dogs. In order to better characterize the E. coli strains that cause canine UTI, we analyzed 295 E. coli isolates obtained from canine urine samples from five veterinary diagnostic laboratories and analyzed their multilocus sequence types, phenotypic and genotypic antimicrobial resistance profiles, and virulence-associated gene repertoires. Sequence type 372 (ST372), an infrequent human pathogen, was the predominant sequence type in dogs at all locations. Extended-spectrum ß-lactamase-producing isolates with blaCTX-M genes were uncommon in canine isolates but when present were often associated with sequence types that have been described in human infections. This provides support for occasional cross-host-species sharing of strains that cause extraintestinal disease and highlights the importance of understanding the role of companion animals in the overall transmission patterns of extraintestinal pathogenic E. coli.

Multilaboratory Survey To Evaluate Salmonella Prevalence in Diarrheic and Nondiarrheic Dogs and Cats in the United States between 2012 and 2014.

Eleven laboratories collaborated to determine the periodic prevalence of Salmonella in a population of dogs and cats in the United States visiting veterinary clinics. Fecal samples (2,965) solicited from 11 geographically dispersed veterinary testing laboratories were collected in 36 states between January 2012 and April 2014 and tested using a harmonized method. The overall study prevalence of Salmonella in cats (3 of 542) was <1%. The prevalence in dogs (60 of 2,422) was 2.5%. Diarrhea was present in only 55% of positive dogs; however, 3.8% of the all diarrheic dogs were positive, compared with 1.8% of the nondiarrheic dogs. Salmonella-positive dogs were significantly more likely to have consumed raw food (P = 0.01), to have consumed probiotics (P = 0.002), or to have been given antibiotics (P = 0.01). Rural dogs were also more likely to be Salmonella positive than urban (P = 0.002) or suburban (P = 0.001) dogs. In the 67 isolates, 27 unique serovars were identified, with three dogs having two serovars present. Antimicrobial susceptibility testing of 66 isolates revealed that only four of the isolates were resistant to one or more antibiotics. Additional characterization of the 66 isolates was done using pulsed-field gel electrophoresis and whole-genome sequencing (WGS). Sequence data compared well to resistance phenotypic data and were submitted to the National Center for Biotechnology Information (NCBI). This study suggests an overall decline in prevalence of Salmonella-positive dogs and cats over the last decades and identifies consumption of raw food as a major risk factor for Salmonella infection. Of note is that almost half of the Salmonella-positive animals were clinically nondiarrheic.

Multicenter molecular investigation of recurrent Escherichia coli bacteriuria in dogs.

Escherichia coli is the most common cause of recurrent urinary tract infection (UTI) in dogs. UTI recurrence comprises of persistent, unresolved E. coli infection or reinfection with a different strain of E. coli. Differentiating between these processes is clinically important but is often impossible with routine diagnostics. We tested the hypothesis that most recurrent canine E. coli bacteriuria is due to recurrence of the same E. coli strain involved in the initial infection. Molecular typing was performed on 98 urinary E. coli isolated from dogs with recurrent bacteriuria from five veterinary diagnostic laboratories in the United States. Of the 42 dogs in this study with multiple E. coli bacteriuria observations, a single strain of E. coli caused recurrent bacteriuria in 26 (62 %) dogs, in some cases on multiple occasions for prolonged periods of time (up to eight months). A single E. coli strain was detected during both subclinical bacteriuria and clinically-apparent UTI in three dogs. Isolates with the P-fimbrial adhesin genes papA and papC were associated with recurrence by the same strain of E. coli. Multiple isolations of a single strain of E. coli associated with recurrent bacteriuria suggests that E. coli may be maintained within the urinary tract of some dogs for prolonged periods of time. In some patients, the same strain can cause both clinical UTI and subclinical bacteriuria. This indicates that in dogs, the urinary bladder may serve as a subclinical, long-term reservoir of E. coli that may cause clinical UTI in the future.

Current state and future directions for veterinary antimicrobial resistance research.

Antimicrobial resistance (AMR) is a critical One Health concern with implications for human, animal, plant, and environmental health. Antimicrobial susceptibility testing (AST), antimicrobial resistance testing (ART), and surveillance practices must be harmonized across One Health sectors to ensure consistent detection and reporting practices. Veterinary diagnostic laboratory stewardship, clinical outcomes studies, and training for current and future generations of veterinarians and laboratorians are necessary to minimize the spread of AMR and move veterinary medicine forward into an age of better antimicrobial use practices. The purpose of this article is to describe current knowledge gaps present in the literature surrounding ART, AST, and clinical or surveillance applications of these methods and to suggest areas where AMR research can fill these knowledge gaps. The related Currents in One Health by Maddock et al, JAVMA, March 2024, addresses current limitations to the use of genotypic ART methods in clinical veterinary practice.

A One Health perspective on the use of genotypic methods for antimicrobial resistance prediction.

Antimicrobial resistance is a global One Health concern with critical implications for the health of humans, animals, and the environment. Phenotypic methods of bacterial culture and antimicrobial susceptibility testing remain the gold standards for the detection of antimicrobial resistance and appropriate patient care; however, genotypic-based methods, such as PCR, whole genome sequencing, and metagenomic sequencing, for detection of genes conferring antimicrobial resistance are increasingly available without inclusion of appropriate standards for quality or interpretation. Misleading test results may lead to inappropriate antimicrobial treatment and, in turn, poor patient outcomes and the potential for increased incidence of antimicrobial resistance. This article explores the current landscape of clinical and methodological aspects of antimicrobial susceptibility testing and genotypic antimicrobial resistance test methods. Additionally, it describes the limitations associated with employing genotypic-based test methods in the management of veterinary patients from a One Health perspective. The companion Currents in One Health by Maddock et al, AJVR, March 2024, addresses current and future needs for veterinary antimicrobial resistance research.

Previous Antibiotic Exposure Reshapes the Population Structure of Infecting Uropathogenic Escherichia coli Strains by Selecting for Antibiotic Resistance over Urovirulence.

Antibiotic therapy is the standard of care for urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC). However, previous antibiotic therapy may impart a selective pressure that influences the population structure and pathogenic potential of infecting UPEC strains. Here, we conducted a 3-year study using whole-genome-sequencing analysis and retrospective medical record review to characterize how antibiotic exposure influenced the phenotypic antibiotic resistance, acquired resistome, virulome, and population structure of 88 UTI-causing E. coli strains from dogs. A majority of UTI-associated E. coli strains were from phylogroup B2 and clustered within sequence type 372. Previous antibiotic exposure was associated with a population shift toward UPEC from phylogroups other than the typical urovirulent phylogroup B2. The specific virulence profiles within the accessory virulome that were associated with antibiotic use were elicited by the effect of antibiotics on UPEC phylogenetic structure. Among phylogroup B2, antibiotic exposure increased the quantity of genes within the resistome and the odds of developing reduced susceptibility to at least one antibiotic. Non-B2 UPEC strains harbored a more diverse and greater resistome that conferred reduced susceptibility to multiple antibiotic classes following antibiotic exposure. Collectively, these data suggest that previous antibiotic exposure establishes an environment that provides a selective edge to non-B2 UPEC strains through their diverse and abundant antibiotic resistance genes, despite their lack of urovirulence genes. Our findings highlight the necessity for judicious use of antibiotics as we uncover another mechanism by which antibiotic exposure and resistance can influence the dynamics of bacterial infectious disease. IMPORTANCE Urinary tract infections (UTIs) are one of the most common infections of dogs and humans. While antibiotic therapy is the standard of care for UTIs and other infections, antibiotic exposure may influence the pathogenic profile of subsequent infections. We used whole-genome sequencing and retrospective medical record review to characterize the effect of systemic antibiotic therapy on the resistance, virulence, and population structure of 88 UTI-causing UPEC strains isolated from dogs. Our results indicate that antibiotic exposure alters the population structure of infecting UPEC strains, providing a selective edge for non-B2 phylogroups that harbor diverse and abundant resistance gene catalogues but fewer urovirulence genes. These findings highlight how antibiotic resistance can influence pathogen infection dynamics and have clinical implications for the judicious use of antibiotics for bacterial infections.

Ingesta-associated choledocholithiasis in horses: 2 cases and literature review.

Equine ingesta-associated choledocholithiasis is a rare cause of morbidity and mortality. We describe here the clinical, gross, histologic, and microbiologic features of this condition in 2 horses and compare the features to 2 previous cases. Case 1 was a 4-y-old Thoroughbred mare with colic. Case 2 was an 18-y-old American Paint Horse mare with colic, chronic weight loss, and inappropriate mentation. Both had elevated biochemical markers of hepatocellular injury and cholestasis and were euthanized given a poor prognosis. Case 1 had a well-formed 5-cm choledocholith surrounding a piece of hay, and had chronic neutrophilic cholangiohepatitis, bridging fibrosis, and extrahepatic obstruction. Case 2 had an ill-formed choledocholith with occasional hay fragments, wood stick, and twigs, and had regionally extensive hepatocellular necrosis with mild neutrophilic cholangiohepatitis and bridging fibrosis. Enterococcus casseliflavus and Escherichia coli were isolated in both cases; Clostridium spp. were also isolated from case 2. All 4 reported cases had increased activity of cholestatic enzymes, hyperbilirubinemia, portal inflammation, and bridging fibrosis. Colic, pyrexia, leukocytosis with neutrophilia, and elevated hepatocellular enzyme activity were documented in 3 cases. Foreign material in all 4 cases was plant origin (choledochophytolithiasis), including hay (n = 2), sticks/twigs (n = 2), and grass awns (n = 1). Ingesta-associated choledocholithiasis may be considered as a cause of colic, pyrexia, and elevated cholestatic biomarkers in horses.

Lack of SARS-CoV-2 Viral RNA Detection among a Convenience Sampling of Ohio Wildlife, Companion, and Agricultural Animals, 2020-2021.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in humans in late 2019 and spread rapidly, becoming a global pandemic. A zoonotic spillover event from animal to human was identified as the presumed origin. Subsequently, reports began emerging regarding spillback events resulting in SARS-CoV-2 infections in multiple animal species. These events highlighted critical links between animal and human health while also raising concerns about the development of new reservoir hosts and potential viral mutations that could alter the virulence and transmission or evade immune responses. Characterizing susceptibility, prevalence, and transmission between animal species became a priority to help protect animal and human health. In this study, we coalesced a large team of investigators and community partners to surveil for SARS-CoV-2 in domestic and free-ranging animals around Ohio between May 2020 and August 2021. We focused on species with known or predicted susceptibility to SARS-CoV-2 infection, highly congregated or medically compromised animals (e.g., shelters, barns, veterinary hospitals), and animals that had frequent contact with humans (e.g., pets, agricultural animals, zoo animals, or animals in wildlife hospitals). This included free-ranging deer (n = 76 individuals), free-ranging mink (n = 57), multiple species of bats (n = 59), and other wildlife in addition to domestic cats (n = 275) and pigs (n = 184). In total, we tested 792 individual animals (34 species) via rRT-PCR for SARS-CoV-2 RNA. SARS-CoV-2 viral RNA was not detected in any of the tested animals despite a major peak in human SARS-CoV-2 cases that occurred in Ohio subsequent to the peak of animal samplings. Importantly, we did not test for SARS-CoV-2 antibodies in this study, which limited our ability to assess exposure. While the results of this study were negative, the surveillance effort was critical and remains key to understanding, predicting, and preventing the re-emergence of SARS-CoV-2 in humans or animals.

Pathogenomics and clinical recurrence influence biofilm capacity of Escherichia coli isolated from canine urinary tract infections.

Biofilm formation enhances bacteria's ability to colonize unique niches while protecting themselves from environmental stressors. Escherichia coli that colonize the urinary tract can protect themselves from the harsh bladder environment by forming biofilms. These biofilms promote persistence that can lead to chronic and recurrent urinary tract infections (UTI). While biofilm formation is frequently studied among urinary E. coli, its association with other pathogenic mechanisms and adaptations in certain host populations remains poorly understood. Here we utilized whole genome sequencing and retrospective medical record analysis to investigate associations between the population structure, phenotypic resistance, resistome, virulome, and patient demographic and clinical findings of 104 unique urinary E. coli and their capacity to form biofilms. We show that population structure including multilocus sequence typing and Clermont phylogrouping had no association with biofilm capacity. Among clinical factors, exposure to multiple antibiotics within that past 30 days and a clinical history of recurrent UTIs were positively associated with biofilm formation. In contrast, phenotypic antimicrobial reduced susceptibility and corresponding acquired resistance genes were negatively associated with biofilm formation. While biofilm formation was associated with increased virulence genes within the cumulative virulome, individual virulence genes did not influence biofilm capacity. We identified unique virulotypes among different strata of biofilm formation and associated the presence of the tosA/R-ibeA gene combination with moderate to strong biofilm formation. Our findings suggest that E. coli causing UTI in dogs utilize a heterogenous mixture of virulence genes to reach a biofilm phenotype, some of which may promote robust biofilm capacity. Antimicrobial use may select for two populations, non-biofilm formers that maintain an arsenal of antimicrobial resistance genes to nullify treatment and a second that forms durable biofilms to avoid therapeutic insults.

Escherichia coli pathotype contamination in raw canine diets.

OBJECTIVE: To investigate the prevalence of Escherichia coli contamination and E coli virulence gene signatures consistent with known E coli pathotypes in commercially available conventional diets and raw-meat-based diets (RMBDs). SAMPLE: 40 diets in total (19 conventionally cooked kibble or canned diets and 21 RMBDs) obtained from retail stores or online distributors. PROCEDURES: Each diet was cultured for E coli contamination in 3 separate container locations using standard microbiological techniques. Further characterization of E coli isolates was performed by polymerase chain reaction-based pathotype and virulence gene analysis. RESULTS: Conventional diets were negative in all culture based testing. In RMBDs, bacterial contamination was similar to previous reports in the veterinary literature, with 66% (14/21) of the RMBDs having positive cultures for E coli. Among the 191 confirmed E coli isolates from these diets, 31.9% (61/191) were positive for virulence genes. Categorized by pathotype, isolates presumptively belonging to the neonatal meningitis E coli pathotype (15.7% [30/191]) were the most common, followed by enterohemorrhagic E coli (10.5% [20/191]), enteropathogenic E coli (5.8% [11/191]), uropathogenic E coli (2.1% [4/191]), and diffusely adherent E coli (1.6% [3/191]). CLINICAL RELEVANCE: The results of this study reaffirmed the bacteriologic risks previously associated with RMBDs. Furthermore, potential zoonotic concerns associated with identified pathotypes in these diets may have significant consequences for owners in the animals' home environment. Potential risk associated with bacterial contamination should be addressed in animals fed RMBDs.

Escherichia coli probiotic exhibits in vitro growth-limiting effects on clinical feline uropathogenic E coli isolates.

OBJECTIVE: To characterize uropathogenic Escherichia coli (UPEC) in cases of clinical feline urinary tract infection (UTI) and subclinical bacteriuria and investigate the in vitro effects of E coli strain Nissle 1917 on isolate growth. ANIMALS: 40 cats with positive E coli culture results for urine collected during routine evaluation. PROCEDURES: Characterization of UPEC isolates was performed by PCR-based phylotype analysis and serotyping. Nissle 1917 effects on growth inhibition and competitive overgrowth against UPEC isolates were evaluated in vitro using a plate-based competition assay. RESULTS: Feline phylogroups were similar to previous human and feline UPEC studies, with most of the isolates belonging to phylogroup A (42.5%), B2 (37.5%), and D (15.0%). Fifty-two percent of isolates were found to be resistant to antimicrobials, with 19% of these being multidrug resistant (MDR). Nissle 1917 adversely affected the growth of 82.5% of all isolates and 100% of MDR isolates in vitro. The median zone of inhibition was 3.33 mm (range, 1.67 to 10.67 mm). Thirteen isolates were affected via competitive overgrowth and 20 via growth inhibition. CLINICAL RELEVANCE: UPEC isolates from cats were similar in phylogroup analysis to human and dog isolates. The in vitro effects of Nissle 1917 on UPEC warrant additional studies to determine if similar results can be duplicated in vivo.

Implementation of an antimicrobial stewardship program in a veterinary medical teaching institution.

Widespread use of antimicrobials in human and veterinary medicine drives the emergence and dissemination of resistant bacteria in human, animal, and environmental reservoirs. The AVMA and FDA Center for Veterinary Medicine have both taken public positions emphasizing the importance of incorporating antimicrobial stewardship in veterinary clinical settings; however, a model for implementing a comprehensive antimicrobial stewardship program in veterinary practice is not readily available. In 2015, The Ohio State University College of Veterinary Medicine began developing a veterinary antimicrobial stewardship program modeled on existing programs in human health-care institutions and the 7 core elements of a successful hospital antimicrobial stewardship program, as defined by the CDC. The program includes comprehensive antimicrobial use guidelines, active environmental surveillance, and enhanced infection control procedures in The Ohio State University Veterinary Medical Center, along with routine monitoring and reporting of antimicrobial prescribing practices and antimicrobial susceptibility patterns of common pathogens isolated from patients and the hospital environment. Finally, programs have been developed to educate clinicians, staff, and students on antimicrobial resistance and appropriate antimicrobial prescribing practices. The antimicrobial stewardship program has been designed to help clinicians and students confidently make judicious antimicrobial use decisions and provide them with actionable steps that can help them act as strong stewards while providing the best care for their patients. This report describes our program and the process involved in developing it, with the intent that the program could serve as a potential model for other veterinary medical institutions.

Comparison of Two Bacterial Transport Media for Culture of Tonsilar Swabs from Bighorn Sheep ( Ovis canadensis ) and Mountain Goats ( Oreamnos americanus ).

Duplicate tonsilar swabs were collected from 77 bighorn sheep ( Ovis canadensis ) and 19 mountain goats ( Oreamnos americanus ) in Utah. Swabs were refrigerated in bacterial transport medium or frozen in cryopreservation medium prior to bacteriologic culture. The cryopreservation medium yielded comparable or superior bacterial growth while permitting more flexibility in specimen shipment to the laboratory.