Staphylococcus aureus Synergized with Candida albicans to Increase the Pathogenesis and Drug Resistance in Cutaneous Abscess and Peritonitis Murine Models

Abstract

The mixed species of Staphylococcus aureus and Candida albicans can cause infections on skin, mucosa or bloodstream; however, mechanisms of their cross-kingdom interactions related to pathogenesis and drug resistance are still not clear. Here an increase of S. aureus proliferation and biofilm formation was observed in S. aureus and C. albicans dual-species culture, and the synergistic pathogenic effect was then confirmed in both local (cutaneous abscess) and systemic infection (peritonitis) murine models. According to the transcriptome analysis of the dual-species culture, virulence factors of S. aureus were significantly upregulated. Surprisingly, the beta-lactams and vancomycin-resistant genes in S. aureus as well as azole-resistant genes in C. albicans were also significantly increased. The synergistic effects on drug resistance to both antibacterial and antifungal agents were further proved both in vitro and in cutaneous abscess and peritonitis murine models treated by methicillin, vancomycin and fluconazole. The synergistic interactions between S. aureus and C. albicans on pathogenesis and drug resistance highlight the importance of targeting the microbial interactions in polyspecies-associated infections.

Keywords: Candida albicans; Staphylococcus aureus; drug resistance; synergistic effect; virulence.

Figure 1

C. albicans promoted proliferation and biofilm formation of S. aureus. (a) GFP expressing S. aureus was co-cultured with C. albicans. GFP fluorescence was detected at 24 h (n = 7). (b) Scanning electron micrographs of single-species or dual-species biofilms. (c) Biofilm thickness of single-species or dual-species biofilms determined by crystal violet staining (n = 5). ***: p < 0.001.

Figure 2

C. albicans and S. aureus synergistically increased pathogenicity in local infection murine model and in systemic infection murine model. (a) Images of infected mice skin after 7 day inoculation of normal saline, C. albicans, S. aureus or both organisms. Cutaneous abscess is indicated by red arrows. (b) Abscess area measured at day 0, 5 and 7 (n = 4); *: p < 0.05 compared to the S. aureus + C. albicans group. (c) S. aureus burdens obtained from the infected skin after 7 day inoculation (n = 3). *: p < 0.05. (d) C. albicans burdens obtained from the infected skin after 7 day inoculation (n = 3). *: p < 0.05. (e) Representative histological images the mice skin. Necrosis is indicated by black arrows and subcutaneous nodule is indicated by green arrows. (f) Survival curves of mice intraperitoneally infected with C. albicans, S. aureus or both organisms (n = 5); *: p < 0.05 compared to the S. aureus + C. albicans group.

Figure 3

Staphylococcal genes related to virulence were activated by C. albicans. (a) Heat map of differentially expressed staphylococcal virulence genes (n = 3). (b) Relative expression of virulence factors in S. aureus or co-cultured S. aureus measured by RT-qPCR (n = 3). All gene expression levels were normalized by 16 s rRNA gene expression. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Figure 4

C. albicans augmented beta-lactam and vancomycin-resistant gene expression in S. aureus. (a) Heat map of differentially expressed staphylococcal genes related to drug resistance (n = 3). (b) Relative expression of genes related to antibiotic resistance in S. aureus or co-cultured S. aureus measured by RT-qPCR (n = 3). All gene expression levels were normalized by 16 s rRNA gene expression. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Figure 5

Fungal ergosterol biosynthesis and drug transmembrane transportation were up-regulated by S. aureus. (a) Heat map of differentially expressed fungal ergosterol biosynthesis genes (n = 3). (b) Relative expression of selected ergosterol biosynthesis genes in C. albicans or co-cultured C. albicans measured by RT-qPCR (n = 3). All gene expression levels were normalized by 18 s rRNA gene expression. (c) Heat map of differentially expressed fungal genes related to drug transmembrane transportation (n = 3). (d) Relative expression of drug transmembrane transportation genes in C. albicans or co-cultured C. albicans measured by RT-qPCR (n = 3). All gene expression levels were normalized by 18 s rRNA gene expression. *: p < 0.05; ***: p < 0.001.

Figure 6

C. albicans increased the drug resistance of S. aureus in vitro as well as in cutaneous abscess and peritonitis murine models. (a) Antimicrobial assay of the single- and dual-species biofilms quantified by CFU (n = 3); *: p < 0.05; **: p < 0.01; #: p < 0.05 compared to the control S. aureus group. (b) Images of infected mice skin after 7 day inoculation of microbial cultures and intervention with antibiotics. Cutaneous abscess is indicated by red arrows. (c) Abscess area measured at day 0, 5 and 7 (n = 4); *: p < 0.05 compared to the S. aureus + VAN group; #: p < 0.05 compared to the S. aureus + MEZ group. (d) Staphylcococcal burdens obtained from the infected skin after 7 day inoculation of C. albicans and S. aureus and intervention with antibiotics (n = 3); *: p < 0.05. (e) Representative histological images mice skin. Necrosis is indicated by green arrows. (f) Survival curves of mice intraperitoneally infected with germs and intervention with antibiotics (n = 5); *: p < 0.05 compared to the S. aureus + C. albicans + VAN group; #: p < 0.05 compared to the S. aureus + C. albicans + MEZ group.

Figure 7

S. aureus elevated the drug resistance of C. albicans in vitro as well as in cutaneous abscess and peritonitis murine models. (a) Antimicrobial assay of the single- and dual-species biofilms quantified by CFU (n = 3); *: p < 0.05, #: p < 0.001 compared to the control C. albicans group. (b) Images of infected mice skin after 7 day inoculation of microbial cultures and intervention with antibiotics. Cutaneous abscess is indicated by red arrows. (c) Abscess area measured at day 0, 5 and 7 (n = 4); *: p < 0.05. (d) Fungal burdens obtained from the infected skin after 7 day inoculation of C. albicans and S. aureus and intervention with antibiotics (n = 3); *: p < 0.05. (e) Representative histological images mice skin. Necrosis is indicated by green arrows. (f) Survival curves of mice intraperitoneally infected with germs and intervention with antibiotics (n = 5); *: p < 0.05.