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

Figure 1.

Study design, survival curve, and clinical assessment of ESKAPEE pathogens in BALB/c mice. (a) Animals were immunosuppressed with 150 mg/kg cyclophosphamide (CP) at Day−4 and 100 mg/kg at Day−1 and then inoculated with one of the ESKAPEE pathogens or sterile saline (Naïve). Clinical observations were made every 4 hours post-inoculation (HPI) throughout the assessment period. Animals were removed from the study if they were found dead in cage (FDIC). Surviving animals were euthanized at Day 7. (b) Percent survival by group. P-values represent the significance between mice inoculated with one of the ESKAPEE pathogens vs. the naïve group. For E. faecium, P = 0.0428. For the remaining pathogens, P < 0.0001. (c) Cumulative clinical scoring demonstrates disease progression over time. The dotted line represents euthanasia of naïve mice.

Figure 2.

Assessment of weight loss vs. hours post-inoculation (HPI) with ESKAPEE pathogens in BALB/c mice. Each animal was weighed every 12 hours at noon and midnight. The starting weight for each mouse was calculated as the average of the 3 weights taken on Days−4, −1, and Day 0. Weight loss percentage was calculated against that average starting weight and is represented as a change from 100% of the original weight. Mixed-effects analysis with Dunnett’s multiple comparisons test was used to determine statistical significance. Compared against naïve mice, weight loss was statistically significant in all inoculated groups. (a) Naïve mice. (b) Mice inoculated with E. faecium (P < 0.0001). (c) Mice inoculated with S. aureus (P < 0.0001). (d) Mice inoculated with K. pneumoniae (P < 0.0001). (e) Mice inoculated with A. baumannii (P < 0.0001). (f) Mice inoculated with P. aeruginosa (P = 0.0021). (g) Mice inoculated with E. cloacae (P < 0.0001). (h) Mice inoculated with E. coli (P < 0.0001).

Figure 3.

Assessment of internal temperature vs. hours post-inoculation (HPI) with ESKAPEE pathogens in BALB/c mice. ESKAPEE pathogen inoculated mice are presented in two groups: survivors and FDIC. A total of 19–20 mice (n ≥ 19) were used for each bacterial species (Table 2). Mixed-effects analysis was used to determine statistical significance. P-values represent the significance between mice that were survivors vs. FDIC for each ESKAPEE pathogen. The median temperature of each group and naïve animals is plotted over time. A black circle represents internal temperature for naïve mice. A white circle represents internal temperature for mice that ultimately survived. An X represents internal temperature for mice that were ultimately FDIC. Mice inoculated with (a) E. faecium, (b) S. aureus, (d) K. pneumoniae, (e) A. baumannii, (e) P. aeruginosa, (f) E. cloacae, or (g) E. coli. The dotted line represents euthanasia of naïve mice.

Figure 4.

Assessment of external temperature vs. hours post-inoculation (HPI) with ESKAPEE pathogens in BALB/c mice. ESKAPEE pathogen inoculated mice are presented in two groups: survivors and FDIC. A total of 19–20 mice (n ≥ 19) were used for each bacterial species (Table 2). Mixed-effects analysis was used to determine statistical significance. P-values represent the significance between mice that were survivors vs. FDIC for each ESKAPEE pathogen. The median temperature of each group and naïve animals is plotted over time. A black circle represents external temperature for naïve mice. A white circle represents external temperature for mice that ultimately survived. An X represents external temperature for mice that were ultimately FDIC. Mice inoculated with (a) E. faecium, (b) S. aureus, (d) K. pneumoniae, (e) A. baumannii, (e) P. aeruginosa, (f) E. cloacae, or (g) E. coli. The dotted line represents euthanasia of naïve mice.

Figure 5.

Histopathological analysis of pulmonary tissue post-inoculation with ESKAPEE pathogens in BALB/c mice. Hematoxylin and eosin (H&E) stained lung at 40× magnification. (a) Naïve, no abnormal findings. (b) E. faecium, alveolar septal necrosis, mild. (c) S. aureus, multifocal septal thickening, increased interstitial macrophages, increased alveolar macrophages. (d) K. pneumoniae, peribronchiolar and alveolar bacteria, septal necrosis, with septal and intrahistiocytic bacteria. (e) A. baumannii, septal necrosis, intrahistiocytic bacteria, extracellular bacteria in septa and alveoli. (f) P. aeruginosa, intracellular and extracellular bacteria, septal necrosis. (g) E. cloacae, alveolar septal necrosis, increased interstitial histiocytes, and intrahistiocytic bacteria. (h) E. coli, intrabronchiolar and alveolar extracellular bacteria, intrahistiocytic bacteria, septal necrosis, alveolar haemorrhage. Scale bars set at 100 µm.