Oral Candida biofilm model and Candida Staph interactions

Transcription

1 Oral Candida biofilm model and Candida Staph interactions Mark Shirtliff, PhD Associate Professor Department of Microbial Pathogenesis, School of Dentistry Department of Microbiology and Immunology, School of Medicine University of Maryland Baltimore

2 Background C. albicans Candida albicans is a polymorphic fungus Yeast Pseudohyphae Hyphae (ph, temperature, serum, N) Strains w/ multiple drug resistance Due to increase in immunocompromised population Capable of forming biofilms Commensal >50% humans Common infection in: Hospitalized Immunocompromised Elderly Critically ill Local and systemic disease Mucosal/skin infection Fungemia 40% mortality VAP Wound infections Trends Microbiol Jul;12(7):317-24

3 Background S. aureus Staphylococcus aureus G+ coccus with low G+C content non-spore forming, non-motile facultative anaerobe normal resident of nares (30-70%) Re-emerging human pathogen Rapid development of antimicrobial resistance increased use of indwelling medical devices. Capable of forming biofilms Causes a variety of diseases Skin and soft tissue infections Keratitis Endocarditis Toxic Shock Syndrome Food poisoning Indwelling medical device infections Bacteremia Staphylococcus aureus

4 Candida albicans and Staphylococcus aureus Both capable of forming biofilms and currently rated among the top 3 bloodstream pathogens (CDC). Implications for interaction: Co-isolated from the oral, vaginal, gastrointestinal mucosa. Blood cultures from candidemic patients are associated (11%) with S. aureus co-infection. (Klotz, et. al. 2007) Commonly associated in otitis externa, VAP, diabetic foot wound infections, keratitis, and on catheter lines and denture surfaces Polymicrobial biofilm on catheter disc material.

5 C. albicans enhances S. aureus pathogenicity CA+SA % mortality CA SA Infect Immun. 38(3): Infect Immun. 50(3): Carlson, et al.

6 Retrospective Epidemiological Study Patient records from were screened for various criteria: HIV+ (AIDS - CD4+ T cell count < 200) Documented S. aureus bacteremia 85% of these traced to i.v. needle use, CVC lines 15% of unknown etiologies 25% had current candidiasis 50% had previous documented cases of candidiasis 3.75% 3.75% 85.00% 7.50% Mary Claire Roghmann, MD, MS. Univ. of MD Baltimore, Dept. of Epidemiology

7 Co-localization in the oral cavity Prevalence of Staphylococcus spp and Candida spp in the oral cavity and periodontal pockets of periodontal disease patients. Acta Odontol Latinoam. 2010; 23(1):20-6. Candida albicans, Staphylococcus aureus and Streptococcus mutans colonization in patients wearing dental prosthesis. Med Oral Patol Oral Cir Bucal. 2005; 10 Suppl 1:E Increase in detectable opportunistic bacteria in the oral cavity of orthodontic patients. Int J Dent Hyg. 2009; 7(2): Prevalence of potential respiratory pathogens in the mouths of elderly patients and effects of professional oral care. Arch Gerontol Geriat. 2001; 32:45 55.

8 In vivo dual-species infection model Amp. Prednisolone CD-1 5x10 6 CFU C. albicans 529L S. aureus C. albicans + S. aureus?

9 In vitro interaction of C. albicans and S. aureus in biofilms A B Images of biofilm probed with (A) Tamra-labeled Universal Yeast probe and FlTC-labeled S. aureus probe and (B) Tamra-labeled S. aureus probe and FITC-labeled C. albicans probe.

10 Physical interaction of C. albicans and S. aureus in biofilms p< 0.05 Peters, et al. FEMS Immunol Med Microbiol Aug;59(3):

11 Bacterial adherence to C. albicans hyphae Percent of cells attached to Candida albicans hyphae attached cells per field (100x), % 100.0% 80.0% 60.0% 57.4% 40.0% 24.6% 24.7% 15.1% 20.0% 2.4% 5.6% 0.0% S. aureus S. pyogenes Avg of 10 n=3 S. epidermidis P. aeruginosa B. subtilis E. coli (DH5-a) 10 fields per slide, 100X (#attached cells / #total cells)

12 in vitro co-infection HaCaT human keratinized epithelial cell line Infect with 5x106 CFU C. albicans/s. aureus 20x 20x S. aureus (green) C. albicans (red) 100x

13 in vitro co-infection Dual-species in vitro infection using HaCaT keratinized epithelium

14 Hypothetical Dual-species Model of Infection

15 Adherence ELISA assay * OD, 450 nm C. albicans strain tested

16 Atomic force microscopy (AFM) Bastiann Krom, et al., UMC Groningen, Netherlands.

17 Atomic force microscopy Bastiann Krom, et al., UMC Groningen, Netherlands.

18 ex vivo model of co-infection Tongue Tongue CD-1 S. aureus C. albicans S. aureus CD-1 1h, wash,rpmi, 12h, wash One half homogenize One half fix Culture on selective media Embed in paraffin, section, stain

19 Conclusions Over half of all S. aureus bacteremias have no known portal of entry (del Rio, et al. 2007) C. albicans is a risk factor for S. aureus bacteremia but not vice versa (Klotz, et al. 2007)