Candida albicans-Staphylococcus aureus polymicrobial peritonitis modulates host innate immunity

Abstract

Despite advances in medical device fabrication and antimicrobial treatment therapies, fungal-bacterial polymicrobial peritonitis remains a serious complication for surgery patients, those on peritoneal dialysis, and the critically ill. Using a murine model of peritonitis, we have demonstrated that monomicrobial infection with Candida albicans or Staphylococcus aureus is nonlethal. However, coinfection with these same doses leads to a 40% mortality rate and increased microbial burden in the spleen and kidney by day 1 postinfection. Using a multiplex enzyme-linked immunosorbent assay, we have also identified a unique subset of innate proinflammatory cytokines (interleukin-6, granulocyte colony-stimulating factor, keratinocyte chemoattractant, monocyte chemoattractant protein-1, and macrophage inflammatory protein-1α) that are significantly increased during polymicrobial versus monomicrobial peritonitis, leading to increased inflammatory infiltrate into the peritoneum and target organs. Treatment of coinfected mice with the cyclooxygenase (COX) inhibitor indomethacin reduces the infectious burden, proinflammatory cytokine production, and inflammatory infiltrate while simultaneously preventing any mortality. Further experiments demonstrated that the immunomodulatory eicosanoid prostaglandin E2 (PGE2) is synergistically increased during coinfection compared to monomicrobial infection; indomethacin treatment also decreased elevated PGE2 levels. Furthermore, addition of exogenous PGE2 into the peritoneal cavity during infection overrode the protection provided by indomethacin and restored the increased mortality and microbial burden. Importantly, these studies highlight the ability of fungal-bacterial coinfection to modulate innate inflammatory events with devastating consequences to the host.

Fig 1

C. albicans-S. aureus coinfection results in mortality by day 1 postinoculation. Mice were injected intraperitoneally with 7 × 10⁶ CFU of C. albicans (light gray bar), 8 × 10⁷ CFU of S. aureus (black bar), or the same doses of each pathogen simultaneously (8.7 × 10⁷ CFU total; striped bar). Mice injected with sterile saline only served as controls (white bar). Mice infected with 8.7 × 10⁷ CFU of C. albicans alone (dark gray bar) or S. aureus alone (hashed bar) served as monomicrobial controls. Overall mortality in the coinfected group was found to be statistically significant using Fisher's exact test (P < 0.05; n = 10).

Fig 2

Microbial burden is increased in the target organs during coinfection. Mice were inoculated and sacrificed 1 day later. (A and B) Quantitative counts of both C. albicans (CA) and S. aureus (SA) in kidneys (A) and spleens (B). Mono- and polyinfection groups were compared by the Mann-Whitney U test. *, P < 0.05; horizontal lines, medians. (C) Small staphylococcal abscesses were found growing among a dense network of C. albicans hyphae colonizing the spleen (H&E stain, arrows). Surface colonization of the kidney during polymicrobial infection demonstrates biofilm-like structures with S. aureus attached to a scaffolding of C. albicans hyphae (arrows). The images on the bottom row are magnifications of the boxed areas in the images above.

Fig 3

Coinfection with C. albicans and S. aureus modulates host innate immunity. Mice were inoculated and sacrificed 1 day later. The differential expression of 23 cytokines and chemokines in the (A) kidneys and (B) spleens of mice infected with C. albicans alone, S. aureus alone, or both pathogens (striped bar) is shown. Control mice were injected with sterile saline. Significant increases were found for IL-6, G-CSF, KC, MCP-1, and MIP-1α during polymicrobial versus monomicrobial infection. Values were plotted as the median and compared by the Mann-Whitney U test. *, P < 0.05 for C. albicans versus C. albicans plus S. aureus coinfection; °, P < 0.05 for S. aureus versus C. albicans plus S. aureus coinfection. Error bars represent interquartile ranges.

Fig 4

Coinfection results in both mature and immature neutrophil influx into the peritoneal cavity. At 1 day postinfection with C. albicans alone, S. aureus alone, or both pathogens, mice underwent a peritoneal lavage procedure. (A) Cells were stained with Diff-Quick reagent to identify cells containing granules and multilobed nuclei. White arrows, mature and immature (band-like) neutrophils recovered from the peritoneal cavity. (B) Cells were also immunostained with an FITC-conjugated Gr-1/Ly6G primary antibody and counterstained with DAPI to determine polymorphic nuclei. The numbers of Gr-1-positive cells per field were counted and calculated as the total percentage of cells enumerated. Values were plotted as the median. Infection groups were compared by the Mann-Whitney U test. *, P < 0.05 for C. albicans versus C. albicans plus S. aureus coinfection; °, P < 0.05 for S. aureus versus C. albicans plus S. aureus coinfection. Error bars represent interquartile ranges.

Fig 5

Microbial burden and cytokine production early during infection. (A) Microbial burden between monomicrobial and polymicrobial infections for both organisms in the spleen at 8 h; (B) levels of the 5 cytokines previously identified were assessed in the spleen at 8 h postinoculation. Control mice were injected with sterile saline. Values were plotted as the median and compared by the Mann-Whitney U test. *, P < 0.05 for C. albicans versus C. albicans plus S. aureus coinfection; °, P < 0.05 for S. aureus versus C. albicans plus S. aureus coinfection. Error bars represent interquartile ranges.

Fig 6

Effect of indomethacin (Indo) on survival and microbial growth. (A) Mice received either vehicle or indomethacin at a dose of 5 mg/kg by i.p. injection 4 h prior to and 8 h after polymicrobial infection. Mortality was assessed for up to 5 days postinfection. Overall mortality was significant using Fisher's exact test (P < 0.05; n = 10). (B) Growth of C. albicans and S. aureus was monitored for up to 8 h in medium supplemented with vehicle alone or indomethacin at physiologically relevant concentrations. OD, optical density.

Fig 7

Microbial burden and cytokine levels in the target organs during coinfection are decreased by indomethacin administration. Mice were singly or dually infected; control mice were injected with sterile saline only. All mice received indomethacin at a dose of 5 mg/kg or vehicle 4 h prior to and 8 h after infection and sacrificed at 1 day postinoculation. (A and B) Quantitative counts of C. albicans and S. aureus in kidneys (A) and spleens (B) during infection; (C and D) cytokine levels in the kidneys (C) and spleens (D) of coinfected mice treated with indomethacin or vehicle. Infection groups were compared by the Mann-Whitney U test. *, P < 0.05. Horizontal bars and values were plotted as the medians; error bars represent interquartile ranges.

Fig 8

Indomethacin treatment reduces neutrophil influx into the peritoneal cavity and target organs. Mice were dually infected with C. albicans and S. aureus. All mice received either indomethacin at a dose of 5 mg/kg or vehicle 4 h prior to and 8 h after infection and underwent peritoneal lavage immediately following sacrifice at 1 day postinoculation. (A) The numbers of Gr-1-positive polymorphonuclear cells per field were counted and calculated as the total percentage of cells enumerated. Infection groups were compared by the Mann-Whitney U test. *, P < 0.05. Error bars represent interquartile ranges. (B) Periodic acid-Schiff-stained spleen tissue obtained from coinfected mice receiving vehicle (left) or indomethacin (right). Yellow arrows, neutrophils; black arrows, microbial burden.

Fig 9

Indomethacin abrogates coinfection-mediated synergistic increases in PGE₂ synthesis in the peritoneal cavity. Mice were infected with C. albicans alone, S. aureus alone, or a combination of both pathogens. Control mice were injected with sterile saline only. For experiments testing the efficacy of cyclooxygenase inhibition, coinfected mice received indomethacin at a dose of 5 mg/kg or vehicle alone at 4 h prior to and 8 h after infection. At 1 day after infection, mice were euthanized and immediately underwent peritoneal lavage. Values are plotted as the median. *, P < 0.05 for C. albicans versus C. albicans plus S. aureus coinfection; °, P < 0.05 for S. aureus versus C. albicans plus S. aureus coinfection. Error bars represent interquartile ranges. PGEM, prostaglandin E₂ metabolite.

Fig 10

Addition of PGE₂ in the peritoneal cavity increases mortality and microbial burden. All mice were administered indomethacin 4 h before or 8 h after polymicrobial infection. At 1 h prior to and 9 h after infection, mice were administered 1 μg of PGE₂ or vehicle by i.p. injection. Uninfected mice receiving both indomethacin and PGE₂ served as controls. (A) Survival of mice was significantly decreased in the group receiving PGE₂ compared to the group receiving vehicle alone (Fisher's exact test, P < 0.05; n = 10). (B) PGE₂ levels were measured by competitive EIA at 1 day postinoculation; groups were compared by the Mann-Whitney U test. *, P < 0.05. Values are plotted as medians. Error bars represent interquartile ranges. (C and D) Microbial burdens of both C. albicans and S. aureus in the kidney (C) and spleen (D) of mice receiving PGE2 (Indo+PGE2) or vehicle (Indo) at 1 day postinoculation. The poly- and monoinfection groups were compared by the Mann-Whitney U test. *, P < 0.05; horizontal lines, medians.