Fungal β-1,3-glucan increases ofloxacin tolerance of Escherichia coli in a polymicrobial E. coli/Candida albicans biofilm

Abstract

In the past, biofilm-related research has focused mainly on axenic biofilms. However, in nature, biofilms are often composed of multiple species, and the resulting polymicrobial interactions influence industrially and clinically relevant outcomes such as performance and drug resistance. In this study, we show that Escherichia coli does not affect Candida albicans tolerance to amphotericin or caspofungin in an E. coli/C. albicans biofilm. In contrast, ofloxacin tolerance of E. coli is significantly increased in a polymicrobial E. coli/C. albicans biofilm compared to its tolerance in an axenic E. coli biofilm. The increased ofloxacin tolerance of E. coli is mainly biofilm specific, as ofloxacin tolerance of E. coli is less pronounced in polymicrobial E. coli/C. albicans planktonic cultures. Moreover, we found that ofloxacin tolerance of E. coli decreased significantly when E. coli/C. albicans biofilms were treated with matrix-degrading enzymes such as the β-1,3-glucan-degrading enzyme lyticase. In line with a role for β-1,3-glucan in mediating ofloxacin tolerance of E. coli in a biofilm, we found that ofloxacin tolerance of E. coli increased even more in E. coli/C. albicans biofilms consisting of a high-β-1,3-glucan-producing C. albicans mutant. In addition, exogenous addition of laminarin, a polysaccharide composed mainly of poly-β-1,3-glucan, to an E. coli biofilm also resulted in increased ofloxacin tolerance. All these data indicate that β-1,3-glucan from C. albicans increases ofloxacin tolerance of E. coli in an E. coli/C. albicans biofilm.

FIG 1

Interaction of C. albicans and E. coli in E. coli/C. albicans biofilms. E. coli and C. albicans were grown for 24 h at 37°C using titanium disks as the substrate. After dehydration, samples were visualized using SEM.

FIG 2

Increased ofloxacin tolerance of E. coli in an E. coli/C. albicans biofilm. E. coli (gray bars) and E. coli/C. albicans (black bars) biofilms were treated with different concentrations of ofloxacin (0.39 to 3.13 μM). Afterwards, survival of E. coli was quantified using selective plating. *, P < 0.05; ***, P < 0.001.

FIG 3

Increased ofloxacin tolerance of E. coli in the presence of C. albicans is mainly biofilm specific. Survival of E. coli in planktonic conditions upon ofloxacin treatment (0.09 to 0.75 μM) was quantified in the absence (gray bars) or presence (black bars) of C. albicans. *, P < 0.05.

FIG 4

The extracellular matrix contributes to the observed increased ofloxacin tolerance of E. coli in an E. coli/C. albicans biofilm. E. coli/C. albicans and E. coli biofilms were treated with 0.78 μM ofloxacin with or without matrix-degrading enzymes (50 μg/ml). Afterwards, survival of E. coli was quantified using selective plating. *, P < 0.05; ***, P < 0.001.

FIG 5

Exogenously added laminarin increases ofloxacin tolerance of E. coli in an E. coli biofilm. An E. coli biofilm was treated with different concentrations of ofloxacin in the presence or absence of different concentrations of laminarin (0 to 0.5 mg/ml). Biomass was quantified using crystal violet. **, P < 0.01; ***, P < 0.001.

FIG 6

The presence of C. albicans zap1Δ/zap1Δ increases ofloxacin tolerance of E. coli to a greater extent than does the presence of C. albicans wild type. E. coli/C. albicans biofilms consisting of E. coli wild type and C. albicans wild type (black bars) or zap1Δ/zap1Δ deletion mutant (gray bars) were treated with 0.78 μM ofloxacin. Afterwards, survival of E. coli was quantified using selective plating. Statistical analysis was performed using an unpaired t test. ***, P < 0.001.