Use of a chromogenic medium with and without selective enrichment to screen for carbapenemase-producing Enterobacterales (CPE) from canine and feline fecal specimens during an outbreak of NDM-5-producing Escherichia coli.

Carbapenemase-producing Enterobacterales (CPE) are one of the most urgent threats to human healthcare globally. Descriptions of CPE outbreaks in veterinary hospitals suggest the need for screening strategies for CPE from companion animals. Our aim was to optimize a chromogenic agar method with and without selective enrichment to isolate CPE from companion animal feces in an ongoing outbreak of New Delhi metallo-ß-lactamse-5 Escherichia coli. A limit of detection (LOD) assay for spiked canine and feline feces was performed for both methods using a carbapenamase-producing E. coli (24213-18); the LOD (1.5 × 103 cfu/g of feces) was equivalent to that reported for human fecal specimens. We screened 1,247 companion animal fecal specimens for carriage of CPE by 1) direct plating to chromogenic agar and 2) plating to chromogenic agar following selective enrichment. Twenty-one specimens were positive for CPE by both direct culture and enrichment culture. No specimens were positive with selective enrichment and negative by direct culture. A selective enrichment step did not result in any increased recovery of CPE from companion animals, which suggests that enrichment broth may not be necessary for outbreak surveillance testing. It is important to continue to validate methods for the detection of CPE in companion animals as outbreaks become more common in veterinary facilities.

Detection of Viable Streptococcus equi equi Using Propidium Monoazide Polymerase Chain Reaction.

There is debate around the clinical significance of Streptococcus equi subsp. equi detection in low numbers using quantitative real-time PCR (qPCR). Propidium monoazide (PMA) qPCR has been used to differentiate DNA from viable and nonviable bacterial cells. The aim of this study was to evaluate the ability of PMA eqbE SEQ2190 triplex qPCR to differentiate DNA from viable and nonviable S. equi in positive and suspect positive clinical specimens. Fifty-seven stored (frozen and refrigerated) positive (36) or suspect positive (21) clinical specimens (determined via SeeI qPCR as the gold standard) were tested using eqbE SEQ2190 triplex qPCR with (+) and without (-) PMA pretreatment. Cycle thresholds were higher when using PMA indicating a mixture of heat killed and viable cells. Number of S. equi positive specimens were as follows: 6/57 eqbE + PMA, 13/57 eqbE -PMA (Chi- squared 3.1, p = .079); 10/57 SEQ2190 +PMA, 53/57 SEQ2190 -PMA (Chi- squared 65.6, p < .0001). The mean cycle thresholds were as follows: 23.88 eqbE -PMA, 29.89 eqbE + PMA (p = .04); 24.9 SEQ2190 -PMA, 31.9 SEQ2190 +PMA (p < .0001). PMA qPCR can be used to determine S. equi viability, but testing should be performed on fresh specimens.

Benefits and challenges of creating veterinary antibiograms for empiric antimicrobial selection in support of antimicrobial stewardship and advancement of one-health goals.

Antibiograms are important tools for antimicrobial stewardship that are often underutilized in veterinary medicine. Antibiograms summarize cumulative antimicrobial susceptibility testing (AST) data for specific pathogens over a defined time period; in veterinary medicine, they are often stratified by host species and site of infection. They can aid practitioners with empiric therapy choices and assessment of antimicrobial resistance trends within a population in support of one-health goals for antimicrobial stewardship. For optimal application, consideration must be given to the number of isolates used, the timeframe of sample collection, laboratory analytical methodology, and the patient population contributing to the data (eg, treatment history, geographic region, and production type). There are several limitations to veterinary antibiograms, including a lack of breakpoint availability for bacterial species, a lack of standardization of laboratory methodology and technology for culture and AST, and a lack of funding to staff veterinary diagnostic laboratories at a level that supports antibiogram development and education. It is vital that veterinarians who use antibiograms understand how to apply them in practice and receive relevant information pertaining to the data to utilize the most appropriate antibiogram for their patients. This paper explores the benefits and challenges of developing and using veterinary antibiograms and proposes strategies to enhance their applicability and accuracy. Further detail regarding the application of veterinary antibiograms by privately practicing clinicians is addressed in the companion Currents in One Health article by Lorenz et al (JAVMA, September 2023).

Risk factors for meticillin-resistant Staphylococcus aureus (MRSA) carriage in MRSA-exposed household pets.

BACKGROUND: Household pets can carry meticillin-resistant Staphylococcus aureus (MRSA) introduced to the home by their human companions. Specific factors promoting pet carriage of this pathogen have not been fully elucidated. OBJECTIVE: This study evaluated MRSA cultured from pets and the home environment in households where a human infected with MRSA had been identified, and aimed to determine potential risk factors for pet MRSA carriage. MATERIALS AND METHODS: Humans diagnosed with community-associated MRSA (CA-MRSA) skin or soft-tissue infection (SSTI) in the mid-Atlantic United States were identified. One hundred forty-two dogs and cats from 57 affected households were identified of which 134 (94.4%) pets and the household environment were sampled for bacterial culture, PCR confirmation and spa-typing for MRSA strain determination. Samples were obtained 3 months later from 86 pets. RESULTS: At baseline, 12 (9.0%) pets carried MRSA. Potential risk factors associated with carriage included pet bed (environmental) MRSA contamination, flea infestation and prior antimicrobial use in the pet. Pets tended to carry human-adapted MRSA strains and spa-types of MRSA isolates cultured from pets were concordant with strains cultured from the home environment in seven of eight homes (87.5%) at baseline. CONCLUSIONS AND CLINICAL RELEVANCE: Results may inform risk-based veterinary clinical recommendations and provide evidence for selective pet testing as a possible alternative to early removal of pets from the homes of humans infected with MRSA. MRSA contamination of the home environment is likely an important risk factor for pet MRSA carriage, and household interventions should be considered to reduce risk of MRSA carriage in exposed pets.

In vitro efficacy of 0.2% and 0.4% sodium oxychlorosene against meticillin-resistant Staphylococcus pseudintermedius.

BACKGROUND: There is a need for alternative topical therapies as a consequence of the increased prevalence of meticillin-resistant Staphylococcus pseudintermedius (MRSP) skin infections in dogs. Sodium oxychlorosene has been used as a topical antibacterial agent in human medicine since 1955. OBJECTIVES: To determine whether 0.2% and 0.4% sodium oxychlorosene solutions have a bactericidal effect (>3-log reduction) on MRSP strains isolated from canine skin infections. METHODS AND MATERIALS: A genetically heterogeneous collection of MRSP isolates from dogs was assembled from laboratories across the United States. Time-kill assays were performed with 0.2% and 0.4% sodium oxychlorosene on a 0.5 McFarland standard [approximately 108 colony-forming units (cfu/ml)] suspension of each strain. The average bacterial counts (cfu/ml) of each MRSP strain then were determined at 5, 10, 20 and 60 s after exposure to sodium oxychlorosene; cfu/ml data were converted to log10 scale to calculate microbial reduction. RESULTS: The average bacterial counts following exposure to the 0.2% solution at 5, 10, 20 and 60 s were 6.94 × 104 , 5.63 × 103 , 2.96 × 102 and 1.48 × 102  cfu/ml, respectively. For the 0.4% solution, the average bacterial count at 5 s was 2.12 × 103  cfu/ml. No bacterial growth was observed for any MRSP strain by 10 s. The greatest reduction in cfu/ml occurred within 5 s following exposure to each solution 3.4-log and 4.9-log reduction for 0.2% and 0.4%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: 0.2% and 0.4% sodium oxychlorosene solutions have a bactericidal effect (>99.9% reduction) against MRSP in vitro. Further in vivo studies are necessary to determine whether it is an appropriate alternative therapy for canine pyoderma.

Risk factors for antimicrobial resistance among Staphylococcus isolated from pets living with a patient diagnosed with methicillin-resistant Staphylococcus aureus infection.

It has been suggested that pets play a critical role in the maintenance of methicillin-resistant (MR) and multidrug-resistant (MDR) Staphylococcus spp. in the household. We examined risk factors for carriage of antimicrobial-resistant coagulase-positive staphylococci, with particular attention to Staphylococcus aureus and Staphylococcus pseudintermedius isolated from pets living in households of people diagnosed with methicillin-resistant S. aureus (MRSA) skin or soft-tissue infection. We analyzed data collected cross-sectionally from a study conducted in 2012 that evaluated the transmission of MRSA and other staphylococci from humans, their pets and the environment (Pets and Environmental Transmission of Staphylococci [PETS] study). We used unadjusted and adjusted stratified logistic regression analyses with household-clustered standard errors to evaluate the association between demographic, healthcare-related, contact-related and environmental risk factors and MDR Staphylococcus spp. isolated from dogs and cats. Staphylococcal isolates obtained from dogs (n = 63) and cats (n = 47) were included in these analyses. The use of oral or injectable antimicrobials by the pets during the prior year was the main risk factor of interest. Based on our results, 50% (12/24) of S. aureus, 3.3% (1/30) of S. pseudintermedius and 25% (14/56) of other coagulase-positive staphylococci (CPS) were determined to be MDR. S. aureus isolates were more likely to be MDR compared with S. pseudintermedius. We did not find a significant statistical association between the use of oral or injectable antimicrobials in the prior year and the presence of MDR bacteria. The results suggest that drivers of antimicrobial resistance in household staphylococci may vary by bacterial species, which could have implications for one health intervention strategies for staphylococci and inform the investigation of other reverse zoonoses, such as COVID-19.

Characterization of 2 Klebsiella pneumoniae carbapenemase-producing Enterobacterales isolated from canine rectal swabs.

Globally, carbapenemase-producing Enterobacterales (CPE) cause life-threatening, hospital-acquired infections in people, and have been reported recently among veterinary patients. Organisms that produce a Klebsiella pneumoniae carbapenemase (KPC) are one of the most common CPE isolated from people but have been reported only rarely in animals. We characterized 2 KPC-producing Enterobacterales isolated from companion animal rectal swabs during the response to an outbreak caused by a strain of blaNDM-5 Escherichia coli. Both isolates were characterized by whole-genome sequencing (WGS) and analysis. The first isolate (case A) was from an immunosuppressed 6-y-old Yorkshire Terrier and was identified as E. coli (ST372) with a blaKPC-18 gene and an IncFII plasmid. The second isolate (case B) was from a 3-y-old Labrador Retriever with acute diarrhea and was identified as Citrobacter koseri with a blaKPC-2 gene, multiple plasmids (ColRNAI, pKPC-CAV1193), and a putative enterotoxin gene (senB). Further research is needed to determine what role animals might play in the epidemiology of CPE in communities. It is imperative that all CPE isolated from companion animals be fully characterized by WGS and the associated case examined. All veterinary isolates should be sequenced and shared for surveillance, monitoring, and investigation purposes.

PennDemic Simulation Framework: An Innovative Approach to Increase Student Interest and Confidence in Disasters Preparedness/Response and Interdisciplinary Teamwork.

An interdisciplinary group from two higher-education institutions in Philadelphia developed a novel framework for interprofessional education. This framework was applied to two different scenarios disease outbreak and natural disaster, which were used in simulations in 2018 and 2020. By design, these simulations included students from a broad range of disciplines, beyond the typical healthcare fields. Students were first grouped by discipline and were then placed in interdisciplinary teams for the rest of the scenario. Students were administered four surveys throughout which included 10 point-Likert scale and free response items. A statistically significant post-simulation increase in student interest and confidence was found. Survey analysis also revealed higher scores of positive group behaviors among interdisciplinary teams when compared to discipline groups. Importantly, students realized the importance of broad representation of disciplines for disaster preparedness. The PennDemic framework may be helpful for teams looking to develop simulations to build interest and confidence in disaster preparedness/response and interdisciplinary teamwork.

How companion animal veterinarians in the United States perceive financial constraints on antibiotic decision-making.

BACKGROUND: The misuse of antibiotics is a persistent problem in both human and veterinary medicine. While complex social and behavioural factors drive inappropriate use in human medicine, less is known about factors that impact antibiotic use in companion animal medicine. OBJECTIVE: To identify the perceptions that veterinarians practicing companion animal medicine hold about the influence of financial considerations on antibiotic use. METHODS: Semi-structured qualitative interviews were conducted with veterinarians practicing companion animal medicine in a major metropolitan area in the Eastern United States. Respondents were sampled purposefully, and data were analysed using a thematic analysis approach. RESULTS: Interviews were conducted with 36 veterinarians from 19 practices. Veterinarians believed that their clients' willingness to pay for diagnostic testing or treatment interfered with their ability to make appropriate decisions about antibiotic use. Concerns over antibiotic expiration and subsequent financial losses limited which antibiotics veterinarians stocked. Some veterinarians feared that restricting antibiotic use to appropriate uses could harm their business and lead to economic euthanasia of their patients. CONCLUSIONS: Veterinarians perceive that financial factors can impede their ability to appropriately prescribe antibiotics. Interventions that address the financial aspects of prescribing have the potential to improve antibiotic decision-making in veterinary medicine.

Risk Factors for the Acquisition of a blaNDM-5 Carbapenem-Resistant Escherichia coli in a Veterinary Hospital.

Carbapenem-resistant Enterobacteriaceae (CRE) are an urgent antibiotic resistant threat. Only sporadic reports of CRE in companion animals have been described. Our objective was to identify risk factors associated with the acquisition of a blaNDM-5 CR-Escherichia coli strain as part of an outbreak investigation at a tertiary veterinary hospital in the United States. A matched case-control study was conducted among companion animals admitted during July 1, 2018, through June 30, 2019. The 15 identified blaNDM-5 CR-E coli cases were matched 1:2 with controls (culture negative for blaNDM-5 CR-E coli) based on species and number of days of hospitalization before bacterial culture sample collection. The association between exposure to various procedures and hospital services and the acquisition of blaNDM-5 CR-E. coli was assessed through conditional logistic regression. Case patients had significantly higher odds of exposure to the anesthesia service (odds ratio [OR] = 12.8, P = .017), the surgical service (OR = 4.0, P = .046), and to endotracheal intubation (OR = 10.0, P = .03). Veterinary hospitals should be aware of the potential for transmission of CRE via anesthetic and surgical procedures, especially those that require the placement of endotracheal tubes.

Diagnostic clinical microbiology.

Technological advancements have changed the way clinical microbiology laboratories are detecting and identifying bacterial, viral, parasitic, and yeast/fungal pathogens. Such advancements have improved sensitivity and specificity and reduce turnaround time to reporting of clinically important results. This article discusses and reviews some traditional methodologies along with some of the technological innovations introduced into diagnostic microbiology laboratories. Some insight to what might be available in the coming years is also discussed.

Detection of Streptococcus equi subsp. equi in guttural pouch lavage samples using a loop-mediated isothermal nucleic acid amplification microfluidic device.

BACKGROUND: Rapid point-of-care (POC) detection of Streptococcus equi subsp. equi (S. equi) would theoretically reduce the spread of strangles by identifying index and carrier horses. HYPOTHESIS: That the eqbE isothermal amplification (LAMP) assay, and the same eqbE LAMP assay tested in a microfluidic device format, are comparable to a triplex real-time quantitative polymerase chain reaction (qPCR) assay that is commonly used in diagnostic labs. SAMPLES: Sixty-eight guttural pouch lavage (GPL) specimens from horses recovering from strangles. METHODS: Guttural pouch lavage specimens were tested for S. equi retrospectively using the benchtop eqbE LAMP, the eqbE LAMP microfluidic device, and compared to the triplex qPCR, that detects 2 S. equi-specific genes, eqbE and SEQ2190, as the reference standard using the receiver operating characteristic area under the curve (ROC). RESULTS: The 27/68 specimens were positive by benchtop eqbE LAMP, 31/64 by eqbE LAMP microfluidic device, and 12/67 by triplex qPCR. Using the triplex PCR as the reference, the benchtop eqbE LAMP showed excellent discrimination (ROC Area = 0.813, 95% confidence interval [CI] = 0.711-0.915) as did the LAMP microfluidic device (ROC Area = 0.811, 95% CI = 0.529-0.782). There was no significant difference between the benchtop LAMP and LAMP microfluidic device (ROC Area 0.813 ± 0.055 vs 0.811 ± 0.034, P = .97). CONCLUSIONS: The eqbE LAMP microfluidic device detected S. equi in GPL specimens from convalescent horses. This assay shows potential for development as a POC device for rapid, sensitive, accurate, and cost-efficient detection of S. equi.

SODAPOP: A Metacognitive Mnemonic Framework to Teach Antimicrobial Selection.

Mnemonics are used widely throughout medical education to help manage large amounts of information and to promote a systematic approach to complex problems. SODAPOP is a metacognitive mnemonic that offers learners a framework for veterinary clinical decision making to support optimal antimicrobial selection. SODAPOP has students consider the source and organism before they decide to treat; then they consider the antimicrobials to which the organism is susceptible with regard to contraindications in the patient; and, ultimately, the options are weighed and a plan is formulated. A preliminary study showed that students' perception of SODAPOP was favorable and that exposure to SODAPOP improved student confidence levels. Further research is needed to determine whether SODAPOP improves students' optimal antimicrobial selection. SODAPOP could be a potentially helpful teaching tool because it can be mapped to the Association of American Veterinary Medical Colleges competency-based veterinary education framework under subcompetencies 1.3 and 4.2. A mnemonic such as SODAPOP could be integrated throughout the veterinary curriculum both in basic science courses (microbiology) and with real cases during clinical rotations.

Modification of empirical antimicrobial regimens in large animal medicine.

BACKGROUND: Empirical antimicrobial regimens can be modified following new diagnostic information or when empirical treatment fails. Little is known about the frequency or clinical context in which these modifications occur. We characterised these modifications in a large animal hospital to identify when antimicrobial use could be optimised. METHODS: Chart reviews were performed for all inpatients and outpatients administered antimicrobials at a large animal veterinary referral and teaching hospital in 2017-2018 (n=1163 visits) to determine when and why empirical regimens were modified. Multinomial logistic regression was performed to identify factors associated with reasons for modification. RESULTS: Empirical antimicrobial regimens were modified in 17.3 per cent of visits. The main reasons were parenteral-oral conversions in horses and failure of disease prevention or treatment in ruminants. Empirical therapy for disease prevention was more likely to be modified because of complications in ruminants and in animals on the emergency/critical care service. Empirical therapy for disease treatment was more often modified for reasons other than de-escalation in ruminants and in animals with longer lengths of stay. CONCLUSIONS: Empirical antimicrobial regimens were modified infrequently and mostly for purposes of parenteral-oral conversion in horses and lack of response in ruminants. De-escalation of antimicrobials administered for disease treatment, when guided by diagnostics, is a major tenet of judicious antimicrobial use. However, more research is needed to determine when and how antimicrobial regimens administered for disease prevention should be modified.

New Delhi Metallo-ß-Lactamase-5-Producing Escherichia coli in Companion Animals, United States.

We report isolation of a New Delhi metallo-ß-lactamase-5-producing carbapenem-resistant Escherichia coli sequence type 167 from companion animals in the United States. Reports of carbapenem-resistant Enterobacteriaceae in companion animals are rare. We describe a unique cluster of blaNDM-5-producing E. coli in a veterinary hospital.

The otic microbiota and mycobiota in a referral population of dogs in eastern USA with otitis externa.

BACKGROUND: Canine otitis externa (OE) is a common inflammatory disease that is frequently complicated by secondary bacterial and/or yeast infections. The otic microbial population is more complex than appreciated by cytological methods and aerobic culture alone. HYPOTHESIS/OBJECTIVES: Differences in bacterial and fungal populations of the external ear canal will correlate with specific cytological and culture-based definitions of bacterial and Malassezia otitis. ANIMALS: Forty client-owned dogs; 30 with OE and 10 with healthy ears. METHODS AND MATERIALS: Prospective study comparing cytological samples, aerobic bacterial cultures and culture-independent sequencing-based analyses of the external ear canal. Subjects with OE included 10 dogs with only cocci [≥25/high power field (HPF)] on cytological evaluation and culture of Staphylococcus spp.; 10 dogs with rods (≥25/HPF) and exclusive culture of Pseudomonas aeruginosa; 10 dogs with only yeast on cytological results morphologically compatible with Malassezia spp. (≥5/HPF). RESULTS: Staphylococcus was the most abundant taxa across all groups. Ears cytologically positive for cocci had decreased diversity, and all types of OE were associated with decreased fungal diversity compared to controls. CONCLUSIONS AND CLINICAL IMPORTANCE: Cytological and culture-based assessment of the ear canal is not predictive of the diverse microbiota of the ear canal in cases of Pseudomonas or Malassezia otitis. Less abundant bacterial taxa in cases of staphylococcal OE are worth scrutiny for future biological therapy.

Global Burden of Colistin-Resistant Bacteria: Mobilized Colistin Resistance Genes Study (1980-2018).

Colistin is considered to be an antimicrobial of last-resort for the treatment of multidrug-resistant Gram-negative bacterial infections. The recent global dissemination of mobilized colistin resistance (mcr) genes is an urgent public health threat. An accurate estimate of the global prevalence of mcr genes, their reservoirs and the potential pathways for human transmission are required to implement control and prevention strategies, yet such data are lacking. Publications from four English (PubMed, Scopus, the Cochrane Database of Systematic Reviews and Web of Science) and two Chinese (CNKI and WANFANG) databases published between 18 November 2015 and 30 December 2018 were identified. In this systematic review and meta-analysis, the prevalence of mcr genes in bacteria isolated from humans, animals, the environment and food products were investigated. A total of 974 publications were identified. 202 observational studies were included in the systematic review and 71 in the meta-analysis. mcr genes were reported from 47 countries across six continents and the overall average prevalence was 4.7% (0.1-9.3%). China reported the highest number of mcr-positive strains. Pathogenic Escherichia coli (54%), isolated from animals (52%) and harboring an IncI2 plasmid (34%) were the bacteria with highest prevalence of mcr genes. The estimated prevalence of mcr-1 pathogenic E. coli was higher in food-animals than in humans and food products, which suggests a role for foodborne transmission. This study provides a comprehensive assessment of prevalence of the mcr gene by source, organism, genotype and type of plasmid.

Complete Genome Sequence of a Carbapenem-Resistant Escherichia coli Isolate with bla NDM-5 from a Dog in the United States.

The carbapenem resistance gene bla NDM-5 was identified in an Escherichia coli strain isolated from a dog. We report here the complete genome sequence of this E. coli strain; the bla NDM-5 gene was present on a large IncFII multidrug-resistant plasmid. This is the first bla NDM-5-carrying E. coli strain from an animal in the United States.

Enhancing the one health initiative by using whole genome sequencing to monitor antimicrobial resistance of animal pathogens: Vet-LIRN collaborative project with veterinary diagnostic laboratories in United States and Canada.

BACKGROUND: Antimicrobial resistance (AMR) of bacterial pathogens is an emerging public health threat. This threat extends to pets as it also compromises our ability to treat their infections. Surveillance programs in the United States have traditionally focused on collecting data from food animals, foods, and people. The Veterinary Laboratory Investigation and Response Network (Vet-LIRN), a national network of 45 veterinary diagnostic laboratories, tested the antimicrobial susceptibility of clinically relevant bacterial isolates from animals, with companion animal species represented for the first time in a monitoring program. During 2017, we systematically collected and tested 1968 isolates. To identify genetic determinants associated with AMR and the potential genetic relatedness of animal and human strains, whole genome sequencing (WGS) was performed on 192 isolates: 69 Salmonella enterica (all animal sources), 63 Escherichia coli (dogs), and 60 Staphylococcus pseudintermedius (dogs). RESULTS: We found that most Salmonella isolates (46/69, 67%) had no known resistance genes. Several isolates from both food and companion animals, however, showed genetic relatedness to isolates from humans. For pathogenic E. coli, no resistance genes were identified in 60% (38/63) of the isolates. Diverse resistance patterns were observed, and one of the isolates had predicted resistance to fluoroquinolones and cephalosporins, important antibiotics in human and veterinary medicine. For S. pseudintermedius, we observed a bimodal distribution of resistance genes, with some isolates having a diverse array of resistance mechanisms, including the mecA gene (19/60, 32%). CONCLUSION: The findings from this study highlight the critical importance of veterinary diagnostic laboratory data as part of any national antimicrobial resistance surveillance program. The finding of some highly resistant bacteria from companion animals, and the observation of isolates related to those isolated from humans demonstrates the public health significance of incorporating companion animal data into surveillance systems. Vet-LIRN will continue to build the infrastructure to collect the data necessary to perform surveillance of resistant bacteria as part of fulfilling its mission to advance human and animal health. A One Health approach to AMR surveillance programs is crucial and must include data from humans, animals, and environmental sources to be effective.