Morphogenesis is not required for Candida albicans-Staphylococcus aureus intra-abdominal infection-mediated dissemination and lethal sepsis

Abstract

Intra-abdominal polymicrobial infections cause significant morbidity and mortality. An established experimental mouse model of Staphylococcus aureus-Candida albicans intra-abdominal infection results in ∼60% mortality within 48 h postinoculation, concomitant with amplified local inflammatory responses, while monomicrobial infections are avirulent. The purpose of this study was to characterize early local and systemic innate responses during coinfection and determine the role of C. albicans morphogenesis in lethality, a trait involved in virulence and physical interaction with S. aureus. Local and systemic proinflammatory cytokines were significantly elevated during coinfection at early time points (4 to 12 h) compared to those in monoinfection. In contrast, microbial burdens in the organs and peritoneal lavage fluid were similar between mono- and coinfected animals through 24 h, as was peritoneal neutrophil infiltration. After optimizing the model for 100% mortality within 48 h, using 3.5 × 10(7) C. albicans (5× increase), coinfection with C. albicans yeast-locked or hypha-locked mutants showed similar mortality, dissemination, and local and systemic inflammation to the isogenic control. However, coinfection with the yeast-locked C. albicans mutant given intravenously (i.v.) and S. aureus given intraperitoneally (i.p.) failed to induce mortality. These results suggest a unique intra-abdominal interaction between the host and C. albicans-S. aureus that results in strong inflammatory responses, dissemination, and lethal sepsis, independent of C. albicans morphogenesis.

FIG 1

Microbial burden in monomicrobial and polymicrobial peritoneal infection with C. albicans and S. aureus. Mice (n = 5 mice/group) were injected i.p. with 0.2 ml of 3.5 × 10⁷ CFU/ml C. albicans alone (7 × 10⁶ CFU), 0.2 ml of 4 × 10⁸ CFU/ml S. aureus alone (8 × 10⁷ CFU), or 0.2 ml containing 3.5 × 10⁷ CFU/ml C. albicans and 4 × 10⁸ CFU/ml S. aureus (8.7 × 10⁷ total organisms). At various time points postinoculation, mice were sacrificed to quantitate C. albicans (mono-CA [open circles] versus poly-CA [closed circles]) and S. aureus (mono-SA [open squares] versus poly-SA [closed squares]) in peritoneal lavage fluid (A) and spleen (B). Results are expressed as the median CFU ± interquartile range. Shown are cumulative data from two repeat experiments. Mono- and polyinfection groups were analyzed using the Mann-Whitney U test. *, P < 0.05 for C. albicans versus C. albicans-S. aureus; °, P < 0.05 for S. aureus versus C. albicans-S. aureus.

FIG 2

Microbial burden in dead versus surviving coinfected mice. Mice were inoculated i.p. with C. albicans (CA) and S. aureus (SA) as described in the legend to Fig. 1. Mice (n = 5 to 10 mice/group) were sacrificed when moribund or after 5 days postinoculation (p.i.) to assess fungal and bacterial CFU in peritoneal lavage fluid (A) and spleen (B) (C. albicans, open circles; S. aureus, closed circles). Results are expressed as the median CFU. Shown are cumulative data from three repeat experiments.

FIG 3

Local proinflammatory cytokines during polymicrobial peritoneal infection with C. albicans and S. aureus. Mice (n = 5 mice/group) were inoculated i.p. with C. albicans (CA) and S. aureus (SA) as described in the legend to Fig. 1. At various time points postinoculation, mice were sacrificed, and the peritoneal cavity was lavaged. IL-6 (with inset showing 24 h p.i.) (A), TNF-α (B), and IL-1β (C) concentrations were quantified in peritoneal lavage fluid by ELISA from C. albicans-infected mice (white bars), S. aureus-infected mice (black bars), and coinfected mice (diagonally striped bars). Results are expressed as the mean cytokine level ± standard error of the mean (SEM). Shown are cumulative data from two repeat experiments. Data were analyzed using the unpaired Student's t test. *, P < 0.05 for C. albicans versus C. albicans-S. aureus; °, P < 0.05 for S. aureus versus C. albicans-S. aureus.

FIG 4

PMN infiltration in monomicrobial and polymicrobial peritoneal infection with C. albicans and S. aureus. Mice (n = 5 mice/group) were inoculated i.p. with C. albicans (CA) and S. aureus (SA) as described in the legend to Fig. 1. At various time points postinoculation, peritoneal lavage fluid from mice infected with C. albicans alone (open circles), S. aureus alone (closed squares), or both pathogens (closed triangles) was cytospun and stained with H&E. PMNs and total cells from each infection group were counted in 5 nonadjacent fields, and the percentage of PMNs was calculated. Results are expressed as the mean percentage of PMNs ± SEM. Shown are cumulative data from two repeat experiments. Data were analyzed using the unpaired Student's t test. *, P < 0.05 for CA versus C. albicans-S. aureus; °, P < 0.05 for SA versus C. albicans-S. aureus.

FIG 5

Systemic proinflammatory cytokines in polymicrobial peritoneal infection with C. albicans and S. aureus. Mice (n = 5 mice/group) were inoculated i.p. with C. albicans (CA) and S. aureus (SA) as described in the legend to Fig. 1. At various time points postinoculation, mice were sacrificed, and serum was analyzed for IL-6 (A) and TNF-α (B) by ELISA. Cytokine concentrations were analyzed in mice infected with C. albicans alone (white bars), S. aureus alone (black bars), and both pathogens combined (striped bars). Results are expressed as the mean cytokine level ± SEM. Shown are cumulative data from two repeat experiments. Data were analyzed using the unpaired Student's t test. *, P < 0.05 for C. albicans versus C. albicans-S. aureus; °, P < 0.05 for S. aureus versus C. albicans-S. aureus.

FIG 6

Dissemination of microbial burdens in monomicrobial and polymicrobial peritoneal infections with C. albicans and S. aureus. Mice (n = 5 mice/group) were inoculated i.p. with C. albicans (CA) and S. aureus (SA) as described in the legend to Fig. 1. At various time points postinoculation, mice were sacrificed, and the microbial burden in the brain was assessed in mice with monomicrobial and polymicrobial infections (C. albicans, open circles; S. aureus, closed circles). Results are expressed as the median CFU. Shown are cumulative data from two repeat experiments. Data were analyzed using the Mann-Whitney U test. °, P < 0.05 for S. aureus versus C. albicans-S. aureus.

FIG 7

Effects of increased C. albicans inocula on mortality during coinfection. Mice (n = 5 mice/group) were inoculated i.p. with the standard inocula of 7 × 10⁶ C. albicans and 8 × 10⁷ S. aureus cells (1× CA/SA [closed triangles]), 3.5 × 10⁷ C. albicans and 8 × 10⁷ S. aureus cells (5× CA/1× SA [closed circles]), or with C. albicans alone (5× CA [open circles]). Mice were monitored for morbidity/mortality through 5 days p.i. Results are from one experiment with all groups inclusive.

FIG 8

C. albicans morphogenesis is not required for dissemination or synergistic mortality. Mice (n = 4 or 5 mice/group) were inoculated with S. aureus (SA) and either C. albicans (CA) strain TNRG1, TUME6, or the isogenic control strain TT21 using 5× inocula as described in the legend to Fig. 7. (A) Survival of mice coinfected with S. aureus and TNRG1 using a 5× inoculum (dark gray line), S. aureus and TUME6 using a 5× inoculum (light gray line), or S. aureus and the isogenic control using a 5× inoculum (white line). Analysis was performed using the Kaplan-Meier test (P < 0.05). (B) At 24 h postinoculation, brains from coinfected mice were assessed for fungal and bacterial burden (C. albicans, open circles; S. aureus, closed circles). Results are expressed as the median CFU. Data shown are cumulative data from two repeat experiments, except for the TUME6 and TUME6-S. aureus inoculation, which was performed once. Data were analyzed using the Mann-Whitney U test.

FIG 9

C. albicans morphogenesis is not required for induction of local and systemic inflammation. Mice (n = 5 mice/group) were inoculated with S. aureus (SA) and either C. albicans (CA) strain TNRG1, TUME6, or isogenic control strain TT21 at the 5× inoculum as described in the legend to Fig. 7. At 24 h p.i., peritoneal lavage fluid (A to C) and serum (D to F) were collected and analyzed for IL-6 (A and D), TNF-α (B and E), and IL-1β (C and F). Results are expressed as the mean cytokine level ± SEM. Shown are cumulative data from two repeat experiments, except TUME6 and TUME6-S. aureus inoculation, which was performed once. Data were analyzed using the unpaired Student's t test. *, P < 0.05.