Development and characterization of a fecal-induced peritonitis model of murine sepsis: results from a multi-laboratory study and iterative modification of experimental conditions

Abstract

Background: Preclinical sepsis models have been criticized for their inability to recapitulate human sepsis and suffer from methodological shortcomings that limit external validity and reproducibility. The National Preclinical Sepsis Platform (NPSP) is a consortium of basic science researchers, veterinarians, and stakeholders in Canada undertaking standardized multi-laboratory sepsis research to increase the efficacy and efficiency of bench-to-bedside translation. In this study, we aimed to develop and characterize a 72-h fecal-induced peritonitis (FIP) model of murine sepsis conducted in two independent laboratories. The experimental protocol was optimized by sequentially modifying dose of fecal slurry and timing of antibiotics in an iterative fashion, and then repeating the experimental series at site 1 and site 2.

Results: Escalating doses of fecal slurry (0.5-2.5 mg/g) resulted in increased disease severity, as assessed by the modified Murine Sepsis Score (MSS). However, the MSS was poorly associated with progression to death during the experiments, and mice were found dead without elevated MSS scores. Administration of early antibiotics within 4 h of inoculation rescued the animals from sepsis compared with late administration of antibiotics after 12 h, as evidenced by 100% survival and reduced bacterial load in peritoneum and blood in the early antibiotic group. Site 1 and site 2 had statistically significant differences in mortality (60% vs 88%; p < 0.05) for the same dose of fecal slurry (0.75 mg/g) and marked differences in body temperature between groups.

Conclusions: We demonstrate a systematic approach to optimizing a 72-h FIP model of murine sepsis for use in multi-laboratory studies. Alterations to experimental conditions, such as dose of fecal slurry and timing of antibiotics, have clear impact on outcomes. Differences in mortality between sites despite rigorous standardization warrants further investigations to better understand inter-laboratory variation and methodological design in preclinical studies.

Keywords: Animal models of sepsis; Fecal-induced peritonitis; Multi-laboratory; National Preclinical Sepsis Platform; Preclinical reproducibility; Preclinical sepsis; Sepsis.

Fig. 1

Dose titration of fecal slurry shows a threshold disease severity effect at site 1. Kaplan–Meier survival curves (A), temperature (B), MSS (C), and percent weight change (D) over time in FIP-treated and sham-treated mice at site 1. See series 1 for experimental details (Table 1). Data are presented as median ± interquartile range for sham-treated mice (n = 8; 4 male, 4 female), 0.5 mg/g fecal slurry (n = 6; 3 male, 3 female), 0.75 mg/g fecal slurry (n = 6; 3 male, 3 female), 1.0 mg/g fecal slurry (n = 6; 3 male, 3 female), 1.5 mg/g fecal slurry (n = 9; 5 male, 4 female), and 2.5 mg/g fecal slurry (n = 6; 3 male, 3 female)

Fig. 2

Dose titration of fecal slurry shows a threshold disease severity effect at site 2. Kaplan–Meier survival curves (A), temperature (B), MSS (C), percent weight change (D), and Hill equation (E) over time in FIP-treated and sham-treated mice at site 2. See series 3 for experimental details (Table 1). Data are presented as median ± interquartile range from sham-treated mice (n = 6; 3 male, 3 female), 0.5 mg/g fecal slurry (n = 8; 4 male, 4 female), 0.625 mg/g fecal slurry (n = 8; 4 male, 4 female), and 0.75 mg/g fecal slurry (n = 8; 4 male, 4 female)

Fig. 3

Reproducibility of fecal-induced peritonitis model between sites. Kaplan–Meier survival curves (A), temperature (B), MSS (C), and percent weight change (D) over time in FIP-treated and sham-treated mice. Data are presented as median ± interquartile range from FIP mice with 0.75 mg/g at site 1 (n = 20; 10 male and 10 female) and FIP mice with 0.75 mg/g at site 2 (n = 8; 4 male and 4 female); sham animals were pooled between both sites (n = 6; 3 males and 3 females). See series 3 at site 1 and site 2 for experimental details (Table 1). Survival curves between site 1 and site 2 were analyzed using a Log-rank (Mantel–Cox) test; p-values < 0.05 were considered significant

Fig. 4

The Murine Sepsis Score (MSS) is not reliably associated with death. MSS for mice that either reached humane endpoint (n = 8), were found dead (n = 13), or died during handling (n = 5). Animals received FIP with 0.75 mg/g; see series 3 for experimental details at site 1 and site 2 (Table 1). Data are presented as a violin plot. MSS for animals found dead was within acceptable range for ongoing experimentation

Fig. 5

Timing of antibiotic administration shows effect on disease severity. Kaplan–Meier survival curves (A), temperature (B), MSS (C), and percent weight change (D) over time in FIP-treated and sham-treated mice. Animals received FIP with 0.75 mg/g; see series 2 (early) and series 3 (late) at site 1 for experimental details (Table 1). Data are presented as median ± interquartile range from sham-treated mice (n = 8; 4 male, 4 female), FIP-treated mice with early intervention (n = 10; 5 male, 5 female), and FIP-treated mice with late intervention (n = 20; 10 male, 10 female). Bacterial loads for PCF (E), and bacterial loads for blood (F) were recorded for each mouse. Data are presented as violin plots. Confluent bacterial loads were omitted as the colonies were uncountable

Fig. 6

Disease severity in FIP model is dependent on fecal slurry batch. Kaplan–Meier survival curves (A), temperature (B), MSS (C), and percent weight change (D) over time in FIP-treated and sham-treated mice. Animals received FIP with 0.75 mg/g; see series 3 (2021) and series 4 (2020) at site 2 for experimental details (Table 1). Data are presented as median ± interquartile range from sham-treated mice (n = 4; 2 male, 2 female), FIP-treated mice with 2020 batch (n = 8; 4 male, 4 female), and FIP-treated mice with 2021 batch (n = 8; 4 male, 4 female)

Fig. 7

Differential septic response to inoculum between fecal slurry batches. The microbiota composition of the fecal slurry batches from 2020 (n = 3) and 2021 (n = 3) was determined using 16S rRNA amplicon sequencing. Bar plots depict the relative abundance of the top 25 bacterial genera from the 2020 batch and the 2021 batch (A). Principal coordinate analysis (PCoA) of the fecal slurry microbiota was calculated from the Bray–Curtis dissimilarity distance of CLR transformed ASVs (B). Statistical significance was determined using a permutational ANOVA (PERMANOVA). p values as shown. Taxonomic diversity as represented by C Shannon diversity and D Chao1 was calculated for individual fecal slurry samples at the ASV level and differences between the 2020 batch and 2021 batch were determined using a Wilcoxon test, p values as shown. Differential abundance analysis was performed using linear discriminant analysis effect size (LEfSe) and analysis of composition of microbiomes (ANCOM) to determine differentially abundant bacterial genera between the 2020 batch and the 2021 batch (E). Bacterial genera identified by both tools to be differentially abundant are shown here. A cladogram depiction of the bacterial taxa identified as differentially abundant between the 2020 fecal slurry batch and 2021 fecal slurry batch by LEfSe (F)

Fig. 8

Septic non-survivor mice exhibit elevated levels of coagulation and inflammation. Plasma levels of TAT (A) and IL-6 (B) were quantified and compared to levels observed in septic non-survivors, septic survivors, and sham mice. Data are presented as violin plots representing sham mice (n = 4), septic non-survivors (n = 7), and septic survivors (n = 6–7). Statistical significance was determined using a one-way ANOVA. p-values < 0.05 were considered significant