Candida albicans enhances meropenem tolerance of Pseudomonas aeruginosa in a dual-species biofilm

Abstract

Background: Pseudomonas aeruginosa is an opportunistic bacterium that infects the airways of cystic fibrosis patients, surfaces of surgical and burn wounds, and indwelling medical devices. Patients are prone to secondary fungal infections, with Candida albicans being commonly co-isolated with P. aeruginosa. Both P. aeruginosa and C. albicans are able to form extensive biofilms on the surfaces of mucosa and medical devices.

Objectives: To determine whether the presence of C. albicans enhances antibiotic tolerance of P. aeruginosa in a dual-species biofilm.

Methods: Single- and dual-species biofilms were established in microtitre plates and the survival of each species was measured following treatment with clinically relevant antibiotics. Scanning electron microscopy and confocal microscopy were used to visualize biofilm structure.

Results: C. albicans enhances P. aeruginosa biofilm tolerance to meropenem at the clinically relevant concentration of 5 mg/L. This effect is specific to biofilm cultures and is dependent upon C. albicans extracellular matrix polysaccharides, mannan and glucan, with C. albicans cells deficient in glycosylation structures not enhancing P. aeruginosa tolerance to meropenem.

Conclusions: We propose that fungal mannan and glucan secreted into the extracellular matrix of P. aeruginosa/C. albicans dual-species biofilms play a central role in enhancing P. aeruginosa tolerance to meropenem, which has direct implications for the treatment of coinfected patients.

Figure 1.

C. albicans increases the tolerance of P. aeruginosa to meropenem in a dual-species biofilm. (a) Preformed 24 h biofilms were incubated for 18 h in MHB containing no antibiotic or 5 mg/L meropenem. Data are the mean ± SEM from five biological replicates. Data were analysed using two-way ANOVA and Holm–Sidak’s multiple comparisons test (**P < 0.01). NS, not significant. (b) Scanning electron microscopy analysis of biofilms. Meropenem treatment of mono-species P. aeruginosa biofilms results in death of bacterial cells, whilst the presence of C. albicans in the dual-species biofilm enhances meropenem tolerance; the tight association of P. aeruginosa cells to fungal surfaces is visible. C. albicans alone is unaffected by meropenem. (c) 3D reconstructions of biofilms from confocal z-stacks. Red indicates propidium iodide stain (dead cells), green indicates Syto 9 dye (DNA) and blue indicates calcofluor white stain (chitin).

Figure 2.

Increased tolerance to meropenem is dependent on fungal viability. Preformed 24 h biofilms were incubated for 18 h in MHB containing no antibiotic or 5 mg/L meropenem. Data are the mean ± SEM from five biological replicates. Data were analysed using two-way ANOVA and Holm–Sidak’s multiple comparisons test (**P <0.01). NS, not significant.

Figure 3.

C. dubliniensis also enhances tolerance of P. aeruginosa to meropenem in dual-species biofilms. Preformed 24 h biofilms were incubated for 18 h in MHB containing no antibiotic or 5 mg/L meropenem. Data are the mean ± SEM from three biological replicates. Data were analysed using two-way ANOVA and Holm–Sidak’s multiple comparisons test (*P < 0.05). NS, not significant.

Figure 4.

Mannan and glucan enhance P. aeruginosa biofilm tolerance to meropenem. Fungal polysaccharides were used at a final concentration of 0.25 mg/mL. (a) Mannan and glucan enhance P. aeruginosa biofilm tolerance to 5 mg/L meropenem. Preformed 24 h biofilms were incubated for 18 h in MHB containing no antibiotic or 5 mg/L meropenem. Data are the mean ± SEM from six biological replicates. (b) The effects of mannan and glucan are not additive. (c) Enhanced meropenem tolerance from mannan and glucan is biofilm specific. Planktonic cultures supplemented with exogenous mannan and/or glucan were grown for 3 h and subsequently incubated for 18 h in MHB containing 5 mg/L meropenem. Data are the mean ± SEM from three biological replicates. Data were analysed using two-way ANOVA and Holm–Sidak’s multiple comparisons test (*P < 0.05, **P < 0.01 and ****P < 0.0001). NS, not significant.

Figure 5.

C. albicans cell wall glycosylation is important for protection against meropenem. (a) Schematic diagram representing the structure of N-mannan (including phosphomannan) and O-mannan of C. albicans.^, The points of truncation of the mutants used in this study are indicated by arrows. The pmr1Δ mutant causes loss of a Golgi Ca²⁺/Mn²⁺-ATPase, affecting numerous mannosyltransferases, so the extent of truncation of the α-1,6-mannose backbone is variable.^, (b) N-mannan glycosylation is important for protection against meropenem. Preformed 24 h biofilms were incubated for 18 h in MHB containing no antibiotic or 5 mg/L meropenem. The N-mannan glycosylation mutants (mnn4Δ, pmr1Δ or mnn2Δ) inhibit the ability of C. albicans to protect P. aeruginosa. Tolerance to meropenem is restored in reconstituted control strains. (c) The mnn2–26Δ sextuple mutant, in which only the unsubstituted α-1,6-mannose backbone of N-mannan remains, inhibits the ability of C. albicans to protect P. aeruginosa. (d) O-mannan glycosylation is important for protection against meropenem. The mnt1Δ/mnt2Δ double mutant inhibits the ability of C. albicans to protect P. aeruginosa. Meropenem tolerance is restored when MNT1 is reconstituted. Data are the mean ± SEM from three biological replicates. Data were analysed using two-way ANOVA and Holm–Sidak’s multiple comparisons test (**P < 0.01, ***P < 0.001 and ****P < 0.0001 in panels b, c and d). NS, not significant. (e) Scanning electron microscopy analysis of biofilms. Deletion of genes required for fungal N-mannan biosynthesis (mnn4) or O-mannan biosynthesis (mnt1/mnt2) reduced the ability of C. albicans to protect P. aeruginosa from meropenem, as indicated by the reduction in the number of bacterial cells following meropenem treatment; the majority of surviving bacteria are in close contact with fungal cells. When the genes (MNN4 or MNT1) are reconstituted, the protective effect is restored, as evidenced by the abundance of P. aeruginosa cells coating the fungi in the meropenem-treated samples.

Figure 6.

C. albicans enhances meropenem tolerance of a P. aeruginosa CF isolate. Preformed 24 h biofilms were incubated for 18 h in MHB containing no antibiotic or 5 mg/L meropenem. Data are the mean ± SEM from five biological replicates. Data were analysed using two-way ANOVA and Holm–Sidak’s multiple comparisons test (*P < 0.05). NS, not significant.