Longitudinal temperature measurement can determine humane endpoints in BALB/c mouse models of ESKAPEE infection

ABSTRACT

Antimicrobial resistance (AMR) is a worldwide problem, which is driving more preclinical research to find new treatments and countermeasures for drug-resistant bacteria. However, translational models in the preclinical space have remained static for years. To improve animal use ethical considerations, we assessed novel methods to evaluate survival after lethal infection with ESKAPEE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter cloacae, and Escherichia coli) in pulmonary models of infection. Consistent with published lung infection models often used for novel antimicrobial development, BALB/c mice were immunosuppressed with cyclophosphamide and inoculated intranasally with individual ESKAPEE pathogens or sterile saline. Observations were recorded at frequent intervals to determine predictive thresholds for humane endpoint decision-making. Internal temperature was measured via implanted IPTT300 microchips, and external temperature was measured using a non-contact, infrared thermometer. Additionally, clinical scores were evaluated based on animal appearance, behaviour, hydration status, respiration, and body weight. Internal temperature differences between survivors and non-survivors were statistically significant for E. faecium, S. aureus, K. pneumoniae, A. baumannii, E. cloacae, and E. coli, and external temperature differences were statistically significant for S. aureus, K. pneumoniae, E. cloacae, and E. coli. Internal temperature more precisely predicted mortality compared to external temperature, indicating that a threshold of 85ºF (29.4ºC) was 86.0% predictive of mortality and 98.7% predictive of survival. Based on our findings, we recommend future studies involving BALB/c mice ESKAPEE pathogen infection use temperature monitoring as a humane endpoint threshold.

KEYWORDS:

• Temperature
• ESKAPEE
• murine pulmonary infection model
• BALB/c
• humane endpoints
• replacement, reduction, refinement (3Rs)

Acknowledgements

RD, YA, and DZ were involved in protocol development and study design; ME, WWS, and TF prepared bacterial pathogens; RD, YA, RA, and CC were involved in performing animal observations; RD and TW were responsible for data analysis and figure generation; RD, TW, ME, and DZ were responsible for the writing of the manuscript; all authors were involved in editing and review of the manuscript. We thank Sridhar Samineni and Yoann LeBreton for assistance with animal use protocol development; Tesfaye Mekennon, and Kifle Workagegnehu for performing animal observations; Joe Anderson and Dan Finnegan for histopathological tissue analysis; and all members of the WRAIR/NMRC animal care and husbandry teams, especially Alphonso Evans, for their support of our animals throughout the entire study. This work was supported by the Military Infectious Diseases Research Program under grant W0383_20_WR.

Material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the authors, and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense. Research was conducted under an IACUC-approved animal protocol in an AAALAC International-accredited facility in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, NRC Publication, 2011 edition.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The authors confirm that the study data is available within the article and supplemental materials (openly available in figshare at https://doi.org/10.6084/m9.figshare.22257727.v1).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21505594.2023.2186331

Additional information

Funding

The work was supported by the Military Infectious Diseases Research Program [W0383_20_WR].