Antimicrobial Resistance Characteristics of Fecal Escherichia coli and Enterococcus Species in U.S. Goats: 2019 National Animal Health Monitoring System Enteric Study.

Escherichia coli and Enterococcus species are normal bacteria of the gastrointestinal tract and serve as indicator organisms for the epidemiology and emergence of antimicrobial resistance in their hosts and the environment. Some E. coli serovars, including E. coli O157:H7, are important human pathogens, although reservoir species such as goats remain asymptomatic. We describe the prevalence and antimicrobial resistance of generic E. coli, E. coli O157:H7, and Enterococcus species collected from a national surveillance study of goat feces as part of the National Animal Health Monitoring System (NAHMS) Goat 2019 study. Fecal samples were collected from 4918 goats on 332 operations across the United States. Expectedly, a high prevalence of E. coli (98.7%, 4850/4915) and Enterococcus species (94.8%, 4662/4918) was found. E. coli O157:H7 prevalence was low (0.2%; 10/4918). E. coli isolates, up to three per operation, were evaluated for antimicrobial susceptibility and 84.7% (571/674) were pansusceptible. Multidrug resistance (MDR; ≥3 classes) was uncommon among E. coli, occurring in 8.2% of isolates (55/674). Resistance toward seven antimicrobial classes was observed in a single isolate. Resistance to tetracycline alone (13.6%, 92/674) or to tetracycline, streptomycin, and sulfisoxazole (7.0% 47/674) was the most common pattern. All E. coli O157:H7 isolates were pansusceptible. Enterococcus isolates, up to four per operation, were prioritized by public health importance, including Enterococcus faecium and Enterococcus faecalis and evaluated. Resistance to lincomycin (93.8%, 1232/1313) was most common, with MDR detected in 29.5% (388/1313) of isolates. The combination of ciprofloxacin, lincomycin, and quinupristin resistance (27.1%, 105/388) was the most common pattern detected. Distribution and characteristics of antimicrobial resistance in E. coli and Enterococcus in the U.S. goat population from this study can inform stewardship considerations and public health efforts surrounding goats and their products.

Prevalence, Antimicrobial Resistance, and Diversity of Campylobacter Isolated from U.S. Goat Feces: 2019 NAHMS Survey.

Goats are often asymptomatic carriers of Campylobacter, including the foodborne pathogen Campylobacter jejuni. Infections can have significant and economically detrimental health outcomes in both humans and animals. The primary objective of this study was to estimate the prevalence of Campylobacter in U.S. goat herds. Campylobacter species were isolated from 106 of 3,959 individual animals and from 42 of 277 goat operations that participated in fecal sample collection as part of the National Animal Health Monitoring System Goat 2019 study. Weighted animal-level prevalence was 2.3% (SE = 0.5%) and operation prevalence was 13.0% (SE = 3.2%). Animal-level prevalence ranged widely from 0 to 70.0%, however, 52.4% of positive operations (22/42) had only a single isolate. C. jejuni was the most frequently isolated species (68.9%; 73/106), followed by C. coli (29.3%, 31/106). A total of 46.2% (36/78) of viable isolates were pan-susceptible to 8 antimicrobials. Resistance to tetracycline (TET) was observed in 44.9% (35/78) of isolates, while 12.8% (10/78) were resistant to ciprofloxacin (CIP) and nalidixic acid (NAL). Among all isolates, a single resistance profile CIP-NAL-TET was observed in 3.8% (3/78) of isolates. A total of 35 unique sequence types (STs) were identified, 11 of which are potentially new. Multiple C. jejuni STs were observed in 48.1% (13/27) of positive operations. Goats with access to surface water, operations reporting antibiotics in the feed or water (excluding ionophores and coccidiostats), and operations reporting abortions and without postabortion management tasks had significantly greater odds of being Campylobacter positive. This snapshot of the U.S. goat population enriches the limited pool of knowledge on Campylobacter species presence in U.S. goats.

Comparative Genomics of Atypical Enteropathogenic Escherichia coli from Kittens and Children Identifies Bacterial Factors Associated with Virulence in Kittens.

Typical enteropathogenic Escherichia coli (tEPEC) is a leading cause of diarrhea and associated death in children worldwide. Atypical EPEC (aEPEC) lacks the plasmid encoding bundle-forming pili and is considered less virulent, but the molecular mechanism of virulence is poorly understood. We recently identified kittens as a host for aEPEC where intestinal epithelial colonization was associated with diarrheal disease and death. The purposes of this study were to (i) determine the genomic similarity between kitten aEPEC and human aEPEC isolates and (ii) identify genotypic or phenotypic traits associated with virulence in kitten aEPEC. We observed no differences between kitten and human aEPEC in core genome content or gene cluster sequence identities, and no distinguishing genomic content was observed between aEPEC isolates from kittens with nonclinical colonization (NC) versus those with lethal infection (LI). Variation in adherence patterns and ability to aggregate actin in cultured cells mirrored descriptions of human aEPEC. The aEPEC isolated from kittens with LI were significantly more motile than isolates from kittens with NC. Kittens may serve as a reservoir for aEPEC that is indistinguishable from human aEPEC isolates and may provide a needed comparative animal model for the study of aEPEC pathogenesis. Motility seems to be an important factor in pathogenesis of LI associated with aEPEC in kittens.

Optimizing a Screening Protocol for Potential Extended-Spectrum ß-Lactamase Escherichia coli on MacConkey Agar for Use in a Global Surveillance Program.

The increasing prevalence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli is worrisome. Coordinated efforts to better understand global prevalence and risk factors are needed. Developing lower- and middle-income countries need reliable, readily available, and cost-effective solutions for detecting ESBL E. coli to contribute to global surveillance. We evaluated MacConkey agar supplemented with ceftriaxone or cefotaxime as a screening method for accurately detecting and quantifying potential ESBL E. coli MacConkey agar from eight manufacturers, representing seven countries, was prepared with 2 or 4 µg/ml ceftriaxone or cefotaxime. Four E. coli strains (NC11, ATCC 25922, CM-13457, and CM-10455) and one Klebsiella pneumoniae strain (CM-11073) were grown overnight, serially diluted, and plated in triplicate for enumeration on all medium combinations. After recovery was assessed, US-1 MacConkey agar with cefotaxime was used to further evaluate the reproducibility and detection of potential ESBL E. coli from poultry cecal (n = 30) and water (n = 30) samples. Results indicated the recovery of E. coli 13457 from four MacConkey agar manufacturers was reduced by up to 4 log CFU/ml, and phenotypic differences in colony size and color were apparent for each manufacturer for control E. coli strains. A true ESBL, NC11, was not reduced with 4 µg/ml cefotaxime. From ceca and water, potential ESBL E. coli isolates were only confirmed from MacConkey agar with 4 µg/ml cefotaxime, where 45% and 16.6% of E. coli isolates phenotypically expressed ESBL production. The quality and reproducibility of MacConkey agar varied by manufacturer, which suggests that a single manufacturer and medium type should be selected for global monitoring efforts so that training and interpretation can be standardized.

Sterility and concentration of liposomal bupivacaine single-use vial when used in a multiple-dose manner.

OBJECTIVE: To evaluate the sterility of bupivacaine liposome injectable suspension (Nocita®) used in a multiple-dose fashion for 5 days. STUDY DESIGN: Triplicate liposomal bupivacaine vials were stored under two conditions, (1) room temperature (24°C) and (2) refrigerated temperature (5°C). A 3-mL aliquot was withdrawn from each vial daily. Samples were inoculated in tryptic soy broth in triplicate and then incubated for 24 hours at 37°C and subcultured every 48 hours onto blood agar and Sabouraud dextrose agar, respectively. Separate 1.5-mL aliquots of liposomal bupivacaine were centrifuged at 3500 g to separate liposome-encapsulated bupivacaine from the solution. Concentration of unencapsulated bupivacaine was analyzed via high-pressure liquid chromatography. Data were analyzed by using mixed effects procedure with multiple comparisons. SAMPLE POPULATION: Ten 20-mL vials of bupivacaine liposome injectable suspension stored under two conditions, (1) room temperature (24°C) and (2) refrigerated temperature (5°C). RESULTS: Five days of repeated withdrawal from the single-use vials yielded no bacterial growth. One control vial, which was opened and punctured once on the last day of the experiment, yielded fungal growth of an Aspergillus spp, likely an environmental contaminant. The concentration of free bupivacaine did not significantly differ until the fifth day of sampling. CONCLUSION: When aseptic technique was used, liposomal bupivacaine remained sterile for 5 days. Concentrations of free bupivacaine were unchanged from baseline for 4 days in both refrigerated and room temperature conditions. CLINICAL SIGNIFICANCE: Single-use liposomal bupivacaine vials can be used extralabel in a multiple-dose fashion for up to 4 days when stored either refrigerated or room temperature when sterile technique is used.

Efficacy of vaporized hydrogen peroxide for repeated sterilization of a single-use single-incision laparoscopic surgery port.

OBJECTIVE: To determine the ability of vaporized hydrogen peroxide (VHP) to sterilize a single-use single-incision laparoscopic surgery port and its associated components after repeated simulated uses. STUDY DESIGN: Prospective in vitro experimental study. SAMPLE POPULATION: Six single-use single-incision laparoscopic surgery ports with associated cannulas and insufflation tubing. METHODS: Ports, cannulas, and tubing were subjected to 10 cycles of simulated use, bacterial inoculation with Staphylococcus pseudintermedius and Escherichia coli, decontamination and sterilization, and testing via culture based on their treatment group designation. Bacteriological scores were compared among the negative control, positive control, and 4 treated ports and components. RESULTS: There was no difference in bacteriological scores between treated ports, cannulas, and insufflation tubing and the negative control port and components. Bacteriological scores of ports and components undergoing 6-10 cycles were not significantly different from scores of ports and components undergoing 5 or fewer sterilization cycles. No difference was found in detection of bacteria from treated ports by biopsy of the foam versus sampling via wash. CONCLUSION: This study suggests that a single-use single-incision laparoscopic port and its associated components can be effectively sterilized after multiple simulated uses by using VHP. CLINICAL SIGNIFICANCE: Reuse of a single-use single-incision laparoscopic port is a safe and effective method of cost reduction in veterinary patients.

Agreement between Parallel Canine Blood and Urine Cultures: Is Urine Culture the Poor Man's Blood Culture?

Bloodstream infections are a significant cause of morbidity and mortality in critically ill dogs, but due to cost and difficulties in sample acquisition, blood cultures are infrequently obtained. In ill dogs, urine cultures may be recommended as surrogates for blood cultures. In order to determine the outcome agreement between parallel urine and blood cultures, we retrospectively analyzed parallel blood and urine specimens submitted for culture from dogs at the NC State Veterinary Hospital between 2011 and 2016. Positive cultures were reported from 15% of the submitted blood specimens and 23% of the submitted urine specimens. A total of 295 urine and blood samples were submitted in parallel, with positive growth demonstrated in 14 concordant and five discordant pairs. A kappa statistic comparing blood and urine culture outcomes was 0.266 (fair) when all parallel growth was included, including concordant and discordant results, and 0.170 (poor) when restricted to parallel concordant growth. The sensitivity of urine to reflect concordant bloodstream bacterial organisms was 30%, with a specificity of 87%. The positive and negative predictive values were 30% and 88%, respectively. Of dogs with both specimens positive on bacterial culture, 7 of 7 (100%) with suspected urogenital infection sources were concordant. All dogs with discordant bloodstream and urinary infections were immunosuppressed. Urinary coagulase-positive Staphylococcus isolates were most likely to be concordant with bloodstream infections. In conclusion, we found that urine culture is neither a substitute nor a screen for blood culture. Blood cultures should be performed in any potentially septic animal, especially those that are considered immunosuppressed.

Susceptibility of Escherichia coli O157:H7 to Disinfectants In Vitro and in Simulated Footbaths Amended with Manure.

Escherichia coli O157:H7 is a human pathogen associated with gastrointestinal disease and hemolytic uremic syndrome. Direct contact with ruminants and their feces at agritourism or livestock interaction events is a known source of transmission. Footbath use is a pathogen reduction strategy that may decrease the transmission of E. coli O157:H7 at these interactions. The efficacy of chemical disinfectants in footbaths is not well reported. Our objective was to determine the susceptibility of E. coli O157:H7 toward commonly used disinfectants in vitro and within contaminated footbaths. The minimum inhibitory concentration and the minimum bactericidal concentration (MIC and MBC) and the time-to-kill were determined in vitro for seven E. coli O157:H7 strains using five disinfectant compounds (didecyldimethylammonium chloride [DDAC], glutaraldehyde, hydrogen peroxide, phenol, and sodium hypochlorite). Time-kill assays were performed within simulated footbaths at 22°C, 37°C, and 42°C with and without organic contamination using three commercial disinfectants with similar active ingredients (0.26% Clorox® Bleach, 0.034% Virex® II 256, and 1% Virkon™ S). The MBCs of disinfectants toward E. coli O157:H7 were 3.2, 625, 40, 5000, and 320 ppm of DDAC, glutaraldehyde, hydrogen peroxide, phenol, and sodium hypochlorite, respectively. At 2 × MIC, E. coli O157:H7 reached a 3 log10(colony-forming unit [CFU]/mL) reduction on contact with glutaraldehyde, by 20 min with DDAC and sodium hypochlorite, and by 4 h with phenol and hydrogen peroxide. In simulated footbaths, the commercial disinfectants reduced concentrations by 3 log10(CFU/mL) on contact in the absence of organic contamination, but viable E. coli O157:H7 was recovered from organically contaminated Clorox Bleach and Virex II 256 footbaths. No E. coli O157:H7 was recovered from the Virkon S footbaths after 10 min. This study highlights the ability for organic contamination to compromise the efficacy of disinfectants in footbaths and the importance of choosing an appropriate footbath disinfectant to retain the efficacy.

Association of Atypical Enteropathogenic Escherichia coli with Diarrhea and Related Mortality in Kittens.

Diarrhea is responsible for the death of approximately 900,000 children per year worldwide. In children, typical enteropathogenic Escherichia coli (EPEC) is a common cause of diarrhea and is associated with a higher hazard of death. Typical EPEC infection is rare in animals and poorly reproduced in experimental animal models. In contrast, atypical EPEC (aEPEC) infection is common in both children and animals, but its role in diarrhea is uncertain. Mortality in kittens is often attributed to diarrhea, and we previously identified enteroadherent EPEC in the intestines of deceased kittens. The purpose of this study was to determine the prevalence and type of EPEC in kittens and whether infection was associated with diarrhea, diarrhea-related mortality, gastrointestinal pathology, or other risk factors. Kittens with and without diarrhea were obtained from two shelter facilities and determined to shed atypical EPEC at a culture-based prevalence of 18%. In contrast, quantitative PCR detected the presence of the gene for intimin (eae) in feces from 42% of kittens. aEPEC was isolated from kittens with and without diarrhea. However, kittens with diarrhea harbored significantly larger quantities of aEPEC than kittens without diarrhea. Kittens with aEPEC had a significantly greater severity of small intestinal and colonic lesions and were significantly more likely to have required subcutaneous fluid administration. These findings identify aEPEC to be prevalent in kittens and a significant primary or contributing cause of intestinal inflammation, diarrhea, dehydration, and associated mortality in kittens.

Efficacy of an Escherichia coli O157:H7 SRP Vaccine in Orally Challenged Goats and Strain Persistence Over Time.

Small ruminants have been implicated in outbreaks of Escherichia coli O157:H7 at livestock exhibitions throughout the United States. Additionally, goat meat or milk may serve as a reservoir for foodborne transmission of the organism. These associations highlight the public health importance of an effective strategy to reduce E. coli O157:H7 shedding in goats. We examined the efficacy of the SRP® vaccine in goats orally challenged with E. coli O157:H7. Mixed-breed goats (n = 14) were randomly allocated into vaccinated and unvaccinated treatments (n = 7 per treatment). Goats were housed with a vaccinated and unvaccinated animal in each pen. Feces were collected for 3 weeks, then at necropsy, gastrointestinal contents were collected to determine the concentration of E. coli O157:H7. Three isolates per positive sample were saved and evaluated by pulsed-field gel electrophoresis (PFGE) to assess strain persistence over time. The mean concentration of E. coli O157:H7 in the feces of goats was numerically reduced in the vaccinated treatment; however, it was not statistically significant. In addition, the total number of days goats were fecal positive for E. coli O157:H7 were not different between vaccinated and unvaccinated treatments. Pulsotypes of isolates revealed that goats initially shed two of the four challenge strains of E. coli O157:H7, after which there was a distinct shift to two different strains. Further work is needed to evaluate cost-effective intervention strategies that reliably reduce E. coli O157:H7 shedding in goats, particularly those that may reduce the risk of transmission at public events, including petting zoos and fairs.

Effect of high-pressure processing of bovine colostrum on immunoglobulin G concentration, pathogens, viscosity, and transfer of passive immunity to calves.

This study aimed to determine the effects of high-pressure processing on the immunoglobulin concentration, microbial load, viscosity, and transfer of passive immunity to calves when applied to bovine colostrum as an alternative to thermal pasteurization. A pilot study using Staphylococcus aureus was conducted to determine which pressure-time treatments are most appropriate for use with bovine colostrum, with the goals of maximizing bacterial inactivation while minimizing IgG content and viscosity changes. Following the pilot study, an inoculation study was conducted in which first-milking colostrum samples from Holstein-Friesian cows were inoculated with known concentrations of various bacteria or viruses and pressure processed at either 300 MPa for up to 60min or at 400MPa for up to 30min. The recovery of total native aerobic bacteria, Escherichia coli, Salmonella enterica ssp. enterica serovar Dublin, Mycobacterium avium ssp. paratuberculosis, bovine herpesvirus type 1, and feline calicivirus were determined after processing. Colostrum IgG content was measured before and after pressure processing. Shear stress and viscosity for each treatment was determined over shear rates encompassing those found during calf feeding and at normal bovine body temperature (37.8°C). Following a calf trial, serum IgG concentration was measured in 14 calves fed 4 L of colostrum pressure processed at 400MPa for 15min. In the pilot study, S. aureus was effectively reduced with pressure treatment at 300 and 400MPa (0, 5, 10, 15, 30, and 45min), with 2 treatments at 400MPa (30, 45min) determined to be inappropriate for use with bovine colostrum due to viscosity and IgG changes. High-pressure processing at 300MPa (30, 45, and 60min) and 400MPa (10, 15, and 20min) was shown to effectively reduce total native aerobic bacteria, E. coli, Salmonella Dublin, bovine herpesvirus type 1, and feline calicivirus populations in bovine colostrum, but no decrease occurred in Mycobacterium avium ssp. paratuberculosis. All inoculation study pressure treatments insignificantly decreased IgG content of colostrum. Treatment of colostrum at 400MPa for 15min during the calf trial decreased IgG content of colostrum. Treatment at 400MPa for 15min increased colostrum viscosity, with 2 of 14 samples requiring dilution with water for calf feeding. Calves fed pressure-processed colostrum had similar serum IgG but lower efficiency of absorption than calves fed heat-treated colostrum. The results of this study suggest that high-pressure processing of bovine colostrum maintains an acceptable IgG level while decreasing bacterial and viral counts. Changes in viscosity sometimes made calf feeding more difficult, but still feasible. Additional research to optimize this technology for on-farm use is necessary.

Molecular confirmation of shampoo as the putative source of Pseudomonas aeruginosa-induced postgrooming furunculosis in a dog.

BACKGROUND: An acute onset furunculosis due to Pseudomonas aeruginosa following grooming is a well recognized entity. Although contaminated shampoos have been suspected to be the source of the infection, a molecular confirmation of this association has been missing. OBJECTIVE: This case report describes a dog with postgrooming furunculosis in which Pseudomonas aeruginosa with an identical genetic fingerprint was isolated from the skin lesions as well as from the shampoo used prior to the disease onset. RESULTS: The dog presented for lethargy, anorexia, pain and rapidly progressing skin lesions consistent with haemorrhagic papules, pustules, coalescing ulcers and crusts localized to the dorsal and lateral aspects of the thorax and gluteal region, which developed within 24 h after a bath. Cytology demonstrated suppurative inflammation with occasional intracellular rod-shaped bacteria. Bacterial culture from skin lesions and the shampoo bottle yielded Pseudomonas aeruginosa with an identical pulsed-field gel electrophoresis pattern. Treatment with oral ciprofloxacin and topical antimicrobial shampoo resulted in a complete resolution of skin lesions within eight weeks. CONCLUSION AND CLINICAL IMPORTANCE: Our clinical investigation suggests a link between Pseudomonas-contaminated shampoo and development of postgrooming furunculosis, and underscores the need for hygienic management of shampoos to help limit this disease.

Diagnostic utility of the total nucleated cell count for differentiation of septic and sterile peritoneal effusions in dogs.

BACKGROUND: Rapid and accurate diagnosis of septic peritonitis is critical for initiating appropriate medical and surgical management. OBJECTIVES: The aim of this study was to determine the diagnostic utility of the total nucleated cell count (TNCC), absolute neutrophil count, neutrophil percentage, and total protein (TP) to distinguish septic versus non-septic peritoneal effusions in dogs. METHODS: Electronic medical records were retrospectively searched for peritoneal fluid samples from 2008 to 2018 and classified as septic or non-septic based on bacterial culture and/or cytology results. Receiver operator characteristic curves (ROCs) were used to describe the overall diagnostic utility of each test, with optimal cutpoints analyzed to dichotomize continuous variables. Positive and negative likelihood ratios were calculated at these cutpoints. RESULTS: A total of 166 unique samples, including 87 septic and 79 non-septic peritoneal effusions, were included. There were no significant differences in dog sex, age, or days hospitalized between groups. Septic effusions had significantly higher TP, TNCC, absolute neutrophil count, and neutrophil percentage compared with non-septic effusions. The area under the curve of the ROC curves was TNCC (0.80), absolute neutrophil count (0.80), neutrophil percentage (0.64), and TP (0.63). For TNCC and absolute neutrophil count, optimal cutoffs were 17.13 × 103 cells/µL and 19.88 × 103 cells/µL, resulting in positive and negative likelihood ratios of 2.39 and 0.28 and 2.85 and 0.28, respectively. CONCLUSIONS: Total nucleated cell counts and absolute neutrophil counts aid in the differentiation of septic and non-septic peritoneal effusions with similar diagnostic utility but are not sufficiently sensitive or specific to use without concurrent microscopic evaluation.

Presence of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli in Food-Producing and Companion Animals and Wildlife on Small-Holder Farms of Floreana Island, Galápagos Islands.

Background: Antimicrobial resistance (AR) has led to increasing human and animal morbidity and mortality and negative consequences for the environment. AR among Escherichia coli (EC) is on the rise, with serious concerns about extended-spectrum ß-lactamase-producing E. coli (ESBL-EC). In the Galápagos Islands, where antimicrobials are available without a prescription, growing demands for food production can drive antimicrobial use. Food producing animals are at the interface of wildlife and environmental health on the smallest human-inhabited Galápagos Island, Floreana. We sought to determine if ESBL-EC were present in Floreana Island farm animal species and nearby wildlife and the relatedness of ESBL-EC isolates identified. Materials and Methods: During July 4-5, 2022, we visited 8 multispecies farms, representing 75% of food-producing animal production on Floreana, and collected 227 fecal samples from farm animals and wildlife. Each sample was plated on MacConkey agar supplemented with cefotaxime (4 µg/mL). Results: ESBL-EC was isolated from 20 (9%) fecal samples collected from pigs (N = 10), chickens (N = 6), wildlife (N = 3), and dog (N = 1). All ESBL-EC isolates were from samples taken at three (38%) of the eight farms. Fifteen (75%) of the ESBL-EC isolates were from a single farm. All ESBL-EC isolates were multidrug resistant. The most prevalent ESBL genes belonged to the blaCTX-M group. Among the typeable isolates from the farm with the largest proportion of ESBL-EC isolates (N = 14), we observed nine unique pulsed-field gel electrophoresis (PFGE) patterns, with identical patterns present across pig and chicken isolates. PFGE patterns in the three farms with ESBL-EC isolates were different. Conclusions: These results lend support for future routine AR monitoring activities at the livestock-wildlife interface in Galápagos to characterize potential interspecies transmission of AR bacteria and AR genes in this unique protected ecosystem, and the related human, animal, and environmental health impacts, and to formulate interventions to reduce AR spread in this setting.

Temperature has a greater effect than salinity on microbial survival in saltwater from a single equine hydrotherapy unit.

OBJECTIVE: To determine the ability of bacteria commonly isolated from equine limb wounds to survive in saltwater obtained from an equine hydrotherapy unit at different salinity concentrations and temperatures. METHODS: Saltwater samples were obtained over a 2-week period (January 22, 2024 to February 2, 2024) from an equine hydrotherapy unit used for clinical patients, kept at either full salinity per manufacturer recommendations or diluted to half salinity to mimic the dilution that likely occurs in the clinical setting between cases when holding tanks are replenished with tap water only. Samples were then autoclaved to eliminate preexisting bacterial contamination before individual inoculation with Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus zooepidemicus. Each inoculated sample was maintained at 2, 22, or 44 °C to represent typical holding tank conditions. The bacterial concentration was determined at each condition every 24 hours up to and including 96 hours. The lower limit of detection was set at 1 CFU/mL. RESULTS: Salinity did not affect bacterial survival. Bacterial concentrations generally decreased with increasing temperature over time. Escherichia coli, S aureus, and S zooepidemicus concentrations decreased to the lower limit of detection at 44 °C by 24 to 48 hours, while P aeruginosa concentrations significantly decreased over 24 hours but remained well above the lower limit of detection. CONCLUSIONS: Common bacterial isolates of equine limb wounds can survive in typical saltwater hydrotherapy conditions. CLINICAL RELEVANCE: Further investigation is warranted to determine the clinical relevance of these findings including protocols for hydrotherapy unit disinfection, wastage of treatment water, and case inclusion/exclusion criteria.

Continuous sampling of healthy and mastitic quarters of lactating cattle by ultrafiltration after intramammary ceftiofur hydrochloride administration.

BACKGROUND: Pharmacological activity of intramammary drugs depends on adequate drug concentrations within the cistern, but sampling is often limited. Insight into the active drug concentration within the mammary cistern may assist in determining effective and appropriate therapeutic decisions for cows being treated for mastitis. OBJECTIVE: Evaluate the disposition of ceftiofur hydrochloride administered intramammary in diseased and nondiseased quarters. Whole milk and ultrafiltrate sampling techniques were compared. ANIMALS: Ten mature, late lactation Holstein (n = 9) and Jersey (n = 1) dairy cows (422-670 kg) with naturally occurring clinical mastitis, producing between 1.4 and 15.9 kg/day of milk. METHODS: Ultrafiltration probes were placed in both mastitic and healthy quarters. Each quarter was treated with 2 doses of 125 mg ceftiofur hydrochloride suspension, and whole milk and milk ultrafiltrate samples were collected. Ceftiofur concentrations in composite whole milk and milk ultrafiltrate were analyzed. RESULTS: The maximum concentration of ceftiofur was higher in ultrafiltrate samples, but no differences were identified in healthy or mastitic quarters. The use of ultrafiltration probes provides a novel technique for free drug concentrations within the mastitic and healthy bovine mammary gland. CONCLUSIONS AND CLINICAL IMPORTANCE: Significant inter- and intracow variability and lower daily milk weights may overestimate ceftiofur concentrations available within the cistern. The pharmacokinetic (PK) parameters reported in milk ultrafiltrate will help establish a link between the PK and the corresponding drug effect, potentially providing a meaningful rationale for the selection of a safe and effective dose in cows with mastitis.

Prevalence and molecular epidemiology of carbapenemase-producing Enterobacterales isolated from dog and cat faeces submitted to veterinary laboratories in the USA.

AIMS: To estimate the prevalence of carbapenemase-producing Enterobacterales (CPE) carriage among pets using faecal specimens submitted to veterinary diagnostic laboratories throughout the US. A secondary aim was to employ whole-genome sequencing (WGS) to characterize isolates of CPE from companion animals and compare them to publicly available CPE genomes. METHODS AND RESULTS: To estimate the prevalence of CPE in companion animals in the USA, a multicenter surveillance study including 8 different veterinary diagnostic laboratories from across the USA was conducted. Briefly, remnant faecal specimens from dogs and cats were screened using two selective agar plates (CHROMID Carba and MacConkey with 1 mg/L cefotaxime and 0.125 mg/L meropenem) and presumptive CPE isolates screened by the modified carbapenemase inactivation method for carbapenemase production. A total of 2393 specimens were screened and yielded 196 isolates for carbapenemase screening. A total of 5 isolates from 4 dogs and 1 cat at 3 different veterinary diagnostic laboratories were confirmed to produce a carbapenemase (0.21%). Whole-genome sequencing (WGS) revealed two E. coli (ST167) isolates that both produced an NDM-5 carbapenemase, two Enterobacter hormaechei (ST171) isolates that produced an NDM-5 carbapenemase and a KPC-4 carbapenemase respectively and one Klebsiella oxytoca (ST199) that produced an Oxa-48-type carbapenemase. Both E. coli isolates were found to be within at least 22 SNPs of previously characterized canine and human CPE isolates. CONCLUSIONS: This study demonstrates that the prevalence of CPE among companion animals is relatively low (0.21%) but that given the genetic relatedness of animal isolates to human isolates, additional surveillance is needed.

Relating Antimicrobial Resistance and Virulence in Surface-Water E. coli.

The role of the environment in the emergence and spread of antimicrobial resistance (AMR) is being increasingly recognized, raising questions about the public health risks associated with environmental AMR. Yet, little is known about pathogenicity among resistant bacteria in environmental systems. Existing studies on the association between AMR and virulence are contradictory, as fitness costs and genetic co-occurrence can be opposing influences. Using Escherichia coli isolated from surface waters in eastern North Carolina, we compared virulence gene prevalence between isolates resistant and susceptible to antibiotics. We also compared the prevalence of isolates from sub-watersheds with or without commercial hog operations (CHOs). Isolates that had previously been evaluated for phenotypic AMR were paired by matching isolates resistant to any tested antibiotic with fully susceptible isolates from the same sample date and site, forming 87 pairs. These 174 isolates were evaluated by conventional PCR for seven virulence genes (bfp, fimH, cnf-1, STa (estA), EAST-1 (astA), eae, and hlyA). One gene, fimH, was found in 93.1% of isolates. Excluding fimH, at least one virulence gene was detected in 24.7% of isolates. Significant negative associations were found between resistance to at least one antibiotic and presence of at least one virulence gene, tetracycline resistance and presence of a virulence gene, resistance and STa presence, and tetracycline resistance and STa presence. No significant associations were found between CHO presence and virulence, though some sub-significant associations merit further study. This work builds our understanding of factors controlling AMR dissemination through the environment and potential health risks.

Microbiome analysis of bile from apparently healthy cats and cats with suspected hepatobiliary disease.

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.

Gallbladder microbiota in healthy dogs and dogs with mucocele formation.

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.