An In Vitro Model for Oral Mixed Biofilms of Candida albicans and Streptococcus gordonii in Synthetic Saliva.

Daniel Montelongo-Jauregui,Anand K Ramasubramanian,Jose L Lopez-Ribot,Anand Srinivasan

doi:10.3389/fmicb.2016.00686

Daniel Montelongo-Jauregui, Anand K Ramasubramanian + Show 2 more

Open Access

https://doi.org/10.3389/fmicb.2016.00686

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Abstract

As a member of the normal human oral microbiota, the fungus Candida albicans is often found in association with Streptococcus gordonii, a member of dental plaque forming bacteria. Evidence suggests that S. gordonii serves as a facilitator of C. albicans adherence to dental tissues, which represents a clinically relevant problem, particularly for immunocompromised individuals that could subsequently develop fungal infections. In this study we describe the development of a relatively simple and economical in vitro model that allows for the growth of mixed bacterial/fungal biofilms in 96-well microtiter plates. We have applied this method to test and compare the growth characteristics of single and dual species biofilms in traditional microbiological media versus a synthetic saliva medium (basal medium mucin, BMM) that more closely resembles physiological conditions within the oral cavity. Results indicated a synergistic effect for the formation of biofilms when both microorganisms were seeded together under all conditions tested. The structural and architectural features of the resulting biofilms were further characterized using scanning electron microscopy and confocal scanning laser microscopy. We also performed drug susceptibility assays against single and mixed species biofilms using commonly used antifungals and antibacterial antibiotics, both in monotherapy and in combination therapy, for a direct comparison of resistance against antimicrobial treatment. As expected, mixed species biofilms displayed higher levels of resistance to antimicrobial treatment at every dose tested in both traditional media and BMM synthetic saliva, as compared to single-species biofilms.

Highlights

Candida albicans is an opportunistic pathogenic fungus able to colonize and cause infections in a variety of host sites, including the oral cavity
RPMI 1640 media was chosen for biofilm growth as it is widely used for the formation of C. albicans biofilms, and THB + 0.02% yeast extract (YE) media is conventionally used for growth of Streptococci biofilms (Loo et al, 2000; Pierce et al, 2008)
We note that mixed biofilms formed in synthetic saliva displayed metabolic activity similar to those formed in RPMI medium, and demonstrated a synergistic effect as compared to their monospecies counterparts

Summary

Introduction

Candida albicans is an opportunistic pathogenic fungus able to colonize and cause infections in a variety of host sites, including the oral cavity. Streptococci of the mitis group, most notably Streptococcus gordonii, are among the early colonizers and comprise a large proportion of the oral microbiota, and the ability of C. albicans to interact with oral streptococci and form biofilm consortia in multiple oral sites has been documented (Jenkinson et al, 1990; Bamford et al, 2009; Silverman et al, 2010; Falsetta et al, 2014; Jakubovics et al, 2014; Xu et al, 2014; Chukkapalli et al, 2015) These interactions are bidirectional and considered to be mutualistic beneficial, leading to a cooperative relationship that greatly contributes to survival, persistence and pathogenicity of these microorganisms in diverse oral niches (Jenkinson et al, 1990; Waltimo et al, 1997; O’Sullivan et al, 2000; Lana et al, 2001; Jenkinson and Douglas, 2002; Holmes et al, 2006; Shirtliff et al, 2009; Morales and Hogan, 2010)

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