Vancomycin Treatment Alters Humoral Immunity and Intestinal Microbiota in an Aged Mouse Model of Clostridium difficile Infection

Abstract

Background: The elderly host is highly susceptible to severe disease and treatment failure in Clostridium difficile infection (CDI). We investigated how treatment with vancomycin in the aged host influences systemic and intestinal humoral responses and select intestinal microbiota.

Methods: Young (age, 2 months) and aged (age, 18 months) C57BL/6 mice were infected with VPI 10463 after exposure to broad-spectrum antibiotics. Vancomycin was given 24 hours after infection, and treatment was continued for 5 days. At select time points, specimens of serum and intestinal tissue and contents were collected for histopathologic analysis, to measure antibody levels and the pathogen burden, and to determine the presence and levels of select intestinal microbiota and C. difficile toxin.

Results: Levels of disease severity, relapse, and mortality were increased, and recovery from infection was slower in aged mice compared to young mice. Serum levels of immunoglobulin M, immunoglobulin A, and immunoglobulin G against C. difficile toxin A were depressed in aged mice, and vancomycin treatment reduced antibody responses in both age groups. While baseline levels of total bacterial load, Bacteroidetes, Firmicutes, and Enterobacteriaceae were mostly similar, aged mice had a significant change in the Firmicutes to Bacteroidetes ratio with vancomycin treatment.

Conclusions: Vancomycin treatment decreases the systemic humoral response to CDI. Increased mortality from and recurrence of CDI in the aged host are associated with an impaired humoral response and a greater susceptibility to vancomycin-induced alteration of intestinal microbiota.

Keywords: Clostridium difficile infection; antibiotic-associated colitis; diarrhea; elderly.

Figure 1.

Effect of Clostridium difficile (VPI10463, 10⁵) infection in young C57BL/6 mice (age, 2 months) and aged C57BL/6 mice (age, 18 months). There were 4 mice per uninfected group and 13 mice per infected groups. *P < .05 between infected young mice and infected aged mice by 2-way analysis of variance with the Bonferroni correction or by the log-rank test. A, Weight change from baseline, prior to infection, up to the day of death. B, Disease severity scores among surviving mice. C, Survival curve (by the Mantel–Cox test).

Figure 2.

Histopathologic analysis of cecal and colonic tissue specimens obtained from Clostridium difficile–infected and control young and aged mice. A, Histopathology scores. *P < .001 between infected young mice and infected aged mice. B–E, Representative cecal tissue specimens (at 100× original magnification) from young uninfected mice (B), aged uninfected mice (C), young infected mice (D), and aged infected mice (E).

Figure 3.

Effect of treatment with vancomycin (50 mg/kg/day) for 5 days (days 1–5 after infection) in Clostridium difficile–infected young mice (age, 2 months) and infected aged mice (age, 18 months). Data are for 8 mice per group. A, Weight change from baseline, prior to infection, up to the day of death. *P < .05 by 2-way analysis of variance (ANOVA) with the Bonferroni correction. B, Disease severity scores of surviving mice. *P < .001 by 2-way ANOVA with the Bonferroni correction. C, Survival curve. P < .0001 by the log-rank (Mantel–Cox) test.

Figure 4.

Clostridium difficile bacterial and toxin burden in 8 infected young mice (age, 2 months) and 8 infected aged mice (age, 18 months) that were able to provide stool specimens for testing. Statistical analysis not performed on days 3 and 14 for aged mice because of an insufficient quantity of stool specimens. *P < .05 and **P < .001 by the unpaired Student t test. TcdA/B, C. difficile toxins A and B.

Figure 5.

Clostridium difficile toxin A (TcdA) antibody levels in infected young mice and infected aged mice. Data are for 2–4 mice in uninfected groups and 4–8 mice in infected groups. Each sample was evaluated in duplicate. A, TcdA immunoglobulin M (IgM) level in sera at day 6. *P < .01 between young infected mice vs young uninfected mice and aged infected mice. B, TcdA immunoglobulin G (IgG) level in sera at day 14. *P < .001 between young infected mice vs young uninfected mice and aged infected mice. C, TcdA immunoglobulin A (IgA) level in sera at day 14. *P < .01 between young infected mice vs young uninfected mice. D, TcdA IgA level in cecal contents at day 14. Statistical analyses were performed by 1-way analysis of variance with the Bonferroni correction.

Figure 6.

Effect of vancomycin treatment on Clostridium difficile toxin A (TcdA) antibody levels in sera of infected young mice and infected aged mice at day 14 after infection. There were 2–4 mice in uninfected groups and 2–8 mice in infected groups. Each sample was evaluated in duplicate. The changes in sera antibody levels in aged mice after infection, with or without treatment, were not statistically significant. *P < .01, **P < .001, and ***P < .0001 by 1-way analysis of variance with the Bonferroni correction. Abbreviations: IgA, immunoglobulin A; IgG, immunoglobulin G.

Figure 7.

Effect of vancomycin treatment on total bacterial content and select intestinal microbiota in infected young mice and infected aged mice. There were 6–8 mice per group. Statistical analysis not performed on days 3 and 14 for aged mice because of an insufficient quantity of stool specimens. *P < .01, **P < .001, and ***P < .0001 by 1-way analysis of variance with the Bonferroni correction; ^#by the unpaired Student t test.