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Binary interactions between the yeast Candida albicans and two gut-associated Bacteroides species
Microbial Pathogenesis 135 (2019) 103619
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Binary interactions between the yeast Candida albicans and two gutassociated Bacteroides species
T
Marisa Valentinea, Eliska Benadéa, Marnel Moutonb, Wesaal Khana, Alfred Bothaa,* a b
Department of Microbiology, Stellenbosch University, Stellenbosch, South Africa Department of Botany and Zoology, Stellenbosch University, Stellenbosch, South Africa
ABSTRACT
The yeast Candida albicans forms part of the natural gut microbiota of healthy human individuals and its interactions with other microbial symbionts can impact host well-being. We therefore studied binary interactions between potentially pathogenic representatives of the gut-associated bacterial genus Bacteroides and C. albicans using anaerobic bacteria/yeast co-cultures prepared with a quarter-strength brain heart infusion (¼ BHI; 9.25 g/l) broth. We found that, except for minor changes observed in the cell numbers of one out of four C. albicans strains tested, yeast growth was largely unaffected by the presence of the bacteria. In contrast, growth of Bacteroides fragilis NCTC 9343 and Bacteroides vulgatus ATCC 8482 was significantly enhanced in the presence of C. albicans. Supplementation of Bacteroides monocultures with dead Candida albicans CAB 392 cells, containing intact outer cell wall mannan layers, resulted in increased bacterial concentrations. Subsequent culturing of the Bacteroides strains in a liquid minimal medium supplemented with candidal mannan demonstrated that B. vulgatus ATCC 8482, unlike B. fragilis NCTC 9343, utilized the mannan. Furthermore, by reducing the initial oxygen levels in monocultures prepared with ¼ BHI broth, bacterial numbers were significantly enhanced compared to in monocultures prepared with ¼ BHI broth not supplemented with the reducing agent L-cysteine hydrochloride. This suggests that C. albicans can stimulate Bacteroides growth via aerobic respiration and/or antioxidant production. The cell-free supernatant of 24-h-old C. albicans CAB 392 monocultures was also found to increase Bacteroides growth and chloramphenicol sensitivity.
1. Introduction Ascomycetous yeasts belonging to the genus Candida are the causative agents of oral candidiasis, the most common opportunistic infection among HIV/AIDS-infected patients [1]. Alarmingly, such mucosal infections can develop into invasive candidiasis when candidal cells penetrate the epithelial barrier, enter the bloodstream and disseminate to various organs. Both superficial and invasive infections of individuals, with compromised host immune systems (e.g. HIV/AIDS) or microbiomes, are predominantly caused by Candida albicans. However, in 30–60% of healthy individuals C. albicans exists as a harmless commensal of the skin and mucosal surfaces, including gastrointestinal, oral and vaginal surfaces. Within the various anatomical sites of the human body C. albicans can potentially interact with the indigenous or transient host microbiota. For example, it was demonstrated that vaginal colonizing lactobacilli can prevent adherence of C. albicans to epithelial cells via competition and biosurfactant production [2]. These lactobacilli were also found to produce lactic and fatty acids, lowering vaginal pH and inhibiting yeast growth. On the other hand, oral streptococci such as
*
Streptococcus gordonii enhanced biofilm and hyphal formation of C. albicans, while the yeast increased Streptococcus mutans virulence [3,4]. In addition to studies on the interactions between C. albicans and members of the oral or vaginal microbiota, interactions between this yeast and the gut microbiota were recently explored. It was shown that the natural gut microbiota in mice can prevent C. albicans-induced gastritis [5]. Furthermore, it was observed that the gut pathogen Salmonella enterica serovar Typhimurium inhibited biofilm formation and filamentation of C. albicans [6]. Similar negative effects on C. albicans were reported for a bacteriocin produced by the opportunistic gut pathogen Enterococcus faecalis [7]. However, in the cecum of the gastrointestinal tract of cefoperazone treated mice, C. albicans promoted E. faecalis colonization while preventing colonization by Lactobacillus species [8]. It is of interest to note that these studies of gut microbiota predominantly focus on interactions between C. albicans and Firmicutes or Proteobacteria. Firmicutes together with Bacteroidetes are the dominant bacterial phyla in the gastrointestinal tract constituting ca. 90% of species in this habitat [9]. Within the Bacteroidetes, the genus Bacteroides represents approximately 25% of the anaerobic bacteria in the colon with
Corresponding author. Department of Microbiology, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa. E-mail address: [email protected] (A. Botha).
https://doi.org/10.1016/j.micpath.2019.103619 Received 31 January 2019; Received in revised form 11 May 2019; Accepted 6 July 2019 Available online 07 July 2019 0882-4010/ © 2019 Elsevier Ltd. All rights reserved.
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Department of Microbiology at the University of Stellenbosch, South Africa. The bacterial strains were maintained anaerobically at 37 °C on brain heart infusion (BHI; Biolab Diagnostics, Merck, Darmstadt, Germany) agar plates. Anaerobic growth conditions were created in anaerobic chambers with Anaerocult A sets (Merck) and verified using Anaerotest indicator strips (Merck) as recommended by the manufacturer. The same method was used to achieve anaerobiosis throughout the study. Yeast strains were maintained aerobically at 26 °C on yeast extract-malt extract (YM) agar plates [15].
representatives of Bacteroides fragilis regarded the most virulent and frequent clinical isolates [10]. Bacteroides fragilis together with Bacteroides vulgatus are responsible for a variety of conditions such as skin and soft tissue infections together with Crohn's disease, respectively. The nature of the interaction between these Bacteroides species and C. albicans in the anaerobic environment of the gut is still unknown. Nevertheless, both B. fragilis and B. vulgatus are known to express enzymes capable of degrading the outer mannan layers of ascomycetous yeast cell walls, namely α-mannanases and/or α-mannosidases [11,12]. Furthermore, it was shown that Bacteroides species can utilize yeast αmannan, including that of C. albicans, as a carbon source [13]. In addition, the anaerobic environment of C. albicans biofilms was found to enhance the survival of B. fragilis [14]. It was also demonstrated that following cefoperazone treatment of mice, C. albicans promoted the recovery of Bacteroidetes [8]. Although these compositional changes were reflected in the increased relative abundance of the family Porphyromonadaceae, it remains to be discovered whether complex interactions in the gut or binary interactions between C. albicans and Bacteroides species contributed to the enhancement of Bacteroidetes growth. The aim of this study was therefore to determine if C. albicans exerts a positive effect on Bacteroides growth under anaerobic conditions. Thus, the first objective was to compare growth of B. fragilis, B. vulgatus and C. albicans in monocultures to growth in Bacteroides/C. albicans cocultures. The second objective was to explore the potential role of yeast α-mannan during this interaction by determining the viability of Bacteroides in monocultures, prepared in a minimal medium, supplemented with mannan extracted from C. albicans cell walls. The third objective was to investigate whether yeast metabolism could influence Bacteroides growth. This was done by determining bacterial growth in monocultures that were supplemented with metabolically inactive (dead) C. albicans cells. The role of yeast respiration was then explored by determining Bacteroides growth in monocultures prepared with a medium containing reduced oxygen levels. In addition, the effect of C. albicans-secreted metabolite(s) on Bacteroides growth was determined by assessing bacterial growth in monocultures prepared with the cellfree supernatant of C. albicans monocultures. The effect of C. albicanssecreted metabolites on Bacteroides antibiotic susceptibility was also evaluated by determining bacterial survival, in the presence and absence of chloramphenicol, in monocultures prepared with the cell-free C. albicans supernatant.
2.2. Preparation of starter inoculums A loopful of growth from each C. albicans strain was inoculated into 5 ml BHI broth contained in a test tube, while bacterial strains were cultured in test tubes containing 10 ml BHI broth. Inoculated BHI test tubes were incubated anaerobically at 37 °C. To obtain the starter inoculums, cells were harvested after 48 h by centrifugation (12 000×g, 5 min) and washed thrice with physiological saline solution (PSS; 8.9 g/ l NaCl). The concentrations of bacterial and yeast cell suspensions were determined using a Petroff-Hausser counting chamber (Neubauer Improved, Marienfeld Superior, Lauda-Königshofen, Germany) and hemocytometer (Neubauer Improved, Marienfeld Superior), respectively. Cell concentrations were adjusted and test tubes containing liquid media were inoculated resulting in a final concentration of log 6 bacterial cells/ml and/or log 6 yeast cells/ml. In all experimentation, unless specified differently, starter inoculums were prepared as described here. 2.3. Growth in mono- and co-cultures A nutrient limited medium was used to increase the potential for cooperative and other types of interactions within bacteria/yeast cocultures [17]. Mono- and co-cultures were subsequently prepared in test tubes each containing 5 ml quarter-strength BHI (¼ BHI; 9.25 g/l) broth using the starter inoculums obtained as described in section 2.2. Co-cultures were prepared for all eight combinations of the Bacteroides and C. albicans strains, i.e. Bacteroides fragilis NCTC 9343 and Bacteroides vulgatus ATCC 8482 were individually co-cultured with each of the following yeast strains: Candida albicans CAB 201, Candida albicans CAB 392, Candida albicans CAB 397 and Candida albicans CAB 1085. Both mono- and co-cultures were incubated anaerobically at 37 °C, while microbial numbers within the cultures were determined as colonyforming units (CFU)/ml on dilution plates after 24, 48 and 72 h. Bacterial cells were enumerated on BHI agar plates that were incubated anaerobically for 96 h at 37 °C. Yeast cells were enumerated on YM agar plates supplemented with 0.2 g/l chloramphenicol (Sigma-Aldrich, St. Louis, USA) after 48 h of aerobic incubation at 30 °C. All monocultures were grown as described here unless indicated otherwise. Also, for all experimentation, bacterial numbers were determined by plating serial
2. Materials and methods 2.1. Microbial strains and culture conditions The Bacteroides and C. albicans strains used in the experimentation (Table 1) were revived from 30 to 20% (v/v) glycerol stocks, respectively, that were stored at −80 °C in the culture collection of the
Table 1 The origins and GenBank accession numbers of the bacterial and yeast strains used in this study. Species Bacterial Bacteroides vulgatus Bacteroides fragilis Yeast Candida albicans Candida albicans Candida albicans Candida albicans
Strain
Origin
GenBank accession noa
ATCC 8482 NCTC 9343
Clinical isolate, American Type Culture Collection Clinical isolate, American Type Culture Collection
AJ867050.1 NR_074784.2
CAB CAB CAB CAB
Veterinary isolate, Columba livia domestica, South Africa Clinical isolate, Tygerberg Hospital, South Africa Clinical isolate, Tygerberg Hospital, South Africa Environmental isolate, Plankenburg River, South Africa
MK248726 KJ534504 KJ534505 KJ534503
201 392b 397b 1085c
a
GenBank accession numbers for taxonomic informative gene sequences representing either the bacterial 16S or yeast D1/D2 region of the ribosomal RNA gene. C. albicans CAB 392 and CAB 397 were previously stored as MRC 8908 and MRC 8912, respectively, in the Program on Mycotoxins and Experimental Carcinogenesis (PROMEC) Unit culture collection of the South African Medical Research Council. c Isolate obtained from a river that is known to be polluted with sewage [16]. b
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dilutions on BHI agar plates as described in this section.
contained in a microcentrifuge tube. The resulting suspension was harvested via centrifugation (12 000×g, 5 min) and the pellet (ca. 100 mg wet weight) suspended in 2 ml AmB solution. The suspension was incubated for 4 h at 22 °C, centrifuged and the pellet was once more treated with 2 ml AmB solution. The dead yeast cells were washed twice by suspension in DMSO, followed by a third wash step with dH2O. To confirm that the mannan layers of the AmB killed cells were intact, both dead and live C. albicans CAB 392 cells that were used as inoculums were stained with concanavalin A, fluorescein conjugate (Life Technologies, CA, USA). Images of the fluorescently stained yeast cells were captured using a Nikon Eclipse E400 epifluorescence microscope (Nikon, Tokyo, Japan) equipped with a fluorescein isothiocyanate (FITC; excitation/emission 465–495/515–555 nm) filter set, Nikon DS-Fi2 camera and Nikon Digital Sight DS-U3 camera controller. The relative fluorescence intensity of 50 AmB killed and live C. albicans CAB 392 cells, respectively, were measured with ImageJ software (National Institute of Health, Maryland, USA). The integrated density of the cells was measured, corrected by subtracting the mean background fluorescence and normalized per cell to be expressed as corrected total cell fluorescence (CTCF). Finally, the dead cells of C. albicans CAB 392 were washed with PSS and added at a final concentration of log 6 cells/ml to B. fragilis NCTC 9343 and B. vulgatus ATCC 8482 monocultures prepared in ¼ BHI broth. The resulting cultures were incubated anaerobically at 37 °C and Bacteroides numbers were determined after 24, 48 and 72 h as described in section 2.3.
2.4. Effect of C. albicans mannan on Bacteroides growth in liquid minimal medium To confirm whether B. fragilis NCTC 9343 and B. vulgatus ATCC 8482 can utilize the outer cell wall mannan layer of C. albicans [12,13], the bacteria were inoculated into a liquid minimal medium containing yeast mannan as a carbon source. The latter was obtained from the cell wall of the clinical strain C. albicans CAB 392 using a modified alkali extraction method [18,19]. For this purpose, C. albicans cells were grown aerobically on YM agar plates that were incubated for 48 h at 30 °C. The yeast cells were transferred from the plates to PSS and washed thrice by centrifugation (10 000×g, 15 min). The resulting biomass was treated twice with 6% (w/v) NaOH for 90 min at 70 °C and centrifuged at 10 000×g for 15 min. The yeast mannan was subsequently precipitated from the alkali-extracted supernatant by adding Fehling's reagent. After centrifugation (10 000×g, 15 min), the resulting pellet was dissolved in cold 3 M HCl and the mannan precipitated with methanol. Residual copper was removed by dialyzing the extracted mannan against citrate buffer [pH 6; 0.0115 M citric acid monohydrate (Saarchem, Univar, Merck) and 0.0885 M trisodium citrate dihydrate (Saarchem)] using a Spectra/Por membrane (molecular weight cut-off 6000–8000) for six days at 8 °C. During a final 48 h dialysis step, the citrate buffer was replaced with distilled water (dH2O) and the removal of copper was confirmed with inductively coupled plasma mass spectrometry (ICP-MS) performed by the Central Analytical Facility at Stellenbosch University. The extracted C. albicans CAB 392 mannan was freeze-dried and stored in a desiccator at 4 °C until use. The extracted yeast mannan [0.25 or 0.5% (w/v)] was added to a liquid minimal medium containing the mannan monomer mannose [0.1% (w/v)]. The minimal medium was prepared according to Martens et al. [20]. Controls containing mannose [0.1% (w/v)] or glucose [0.5% (w/v)] were included in the experimentation. Each Bacteroides strain was inoculated into a test tube with 5 ml minimal medium resulting in a final concentration of log 6 bacterial cells/ml. The inoculated test tubes were incubated anaerobically at 37 °C and Bacteroides numbers were determined after 24 h as described in section 2.3. For glucose controls, Bacteroides numbers were determined after 24, 48 and 72 h.
2.5.2. Growth of Bacteroides in broth medium with reduced oxygen levels The potential effect of oxygen depletion as a result of the metabolic activity of C. albicans on Bacteroides growth was evaluated in ¼ BHI broth supplemented with the reducing agent L-cysteine hydrochloride (0.5 g/l; Sigma-Aldrich) and colorimetric redox indicator resazurin (1 mg/l; BDH Chemicals, Poole, UK) [23]. Test tubes containing 5 ml of the pre-reduced media were inoculated with the B. fragilis NCTC 9343 or B. vulgatus ATCC 8482 starter inoculum. Additionally, Bacteroides monocultures were supplemented with log 6 dead C. albicans CAB 392 cells/ml (prepared as described in section 2.5.1) to test if yeast cells may serve as an additional nutrient source for bacterial cells. All test tubes were incubated anaerobically at 37 °C and Bacteroides numbers were determined after 24, 48 and 72 h as described in section 2.3. Negative controls containing no reducing agent were also included in all experimentation.
2.5. Interactions between Bacteroides and yeast metabolism Similar to section 2.4, C. albicans CAB 392 was used in all subsequent experimentation to study the potential effect of a clinical yeast strain on Bacteroides growth.
2.5.3. Effect of C. albicans metabolites on Bacteroides growth and antibiotic susceptibility The influence of secreted C. albicans metabolites on growth of B. fragilis NCTC 9343 and B. vulgatus ATCC 8482 was determined by culturing the bacteria in the cell-free supernatant of C. albicans CAB 392 monocultures. Thus, following incubation of C. albicans CAB 392 in ¼ BHI broth for 24 h, the supernatant was removed, filter-sterilized (0.20 μm; GVS Filter Technology, Lasec, Ndabeni, RSA; designated as spent medium) and 5 ml added to a sterile empty test tube. The spent medium containing test tube was inoculated to obtain a final concentration of log 6 bacterial cells/ml. Following anaerobic incubation at 37 °C, Bacteroides numbers were determined after 24, 48 and 72 h as described in section 2.3. The influence of C. albicans-secreted metabolites on the antibiotic susceptibility of Bacteroides was also studied, since it was previously shown that metabolites secreted by C. albicans can act synergistically with antibiotics. For example, Jabra-Rizk et al. [24] illustrated synergy
2.5.1. Bacteroides cultures supplemented with metabolically inactive C. albicans cells Metabolically inactive yeasts (dead yeast cells), without compromised outer cell wall mannan layers, were obtained by treating C. albicans CAB 392 cells with amphotericin B (AmB; Merck). This fungicidal compound is known to compromise the ergosterol component of yeast cell membranes [21]. To obtain the AmB dosage for the treatment of yeast cells, the macrobroth dilution method [22] was used to determine the minimum inhibitory concentration of AmB for C. albicans CAB 392, which was found to be 0.06 μg/ml. Subsequently, C. albicans CAB 392 was treated with a 6 mg/ml solution of AmB dissolved in dimethyl sulfoxide (DMSO). Yeast growth was transferred from a 48-h-old anaerobic YM agar plate culture incubated at 37 °C to 1 ml PSS
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between farnesol, a quorum-sensing molecule secreted by C. albicans, and the antibiotic gentamicin. We therefore conducted time-kill assays as described by Petersen et al. [25], with modifications for B. fragilis NCTC 9343 and B. vulgatus ATCC 8482, using the antibiotic chloramphenicol and cell-free supernatant of C. albicans. Reaction mixtures were prepared in 1.5 ml microcentrifuge tubes to contain 899 μl ¼ BHI broth or spent medium of 24-h-old C. albicans CAB 392 monocultures, to which 100 μl chloramphenicol solution (320 or 5120 μg/ml for B. vulgatus and B. fragilis, respectively) was added. Each reaction mixture was inoculated with 1 μl Bacteroides starter inoculum resulting in a final concentration of log 6 cells/ml. Controls, containing either ¼ BHI broth or spent medium, in which the chloramphenicol was replaced with 100 μl dH2O were included in the experimentation. As described by Petersen et al. [25], we determined bacterial numbers within the first 24 h following treatment with the antibiotic. Subsequently, after 5, 10 and 24 h of anaerobic incubation at 37 °C, aliquots were removed and Bacteroides numbers determined as described in section 2.3.
data was determined using the non-parametric Mann-Whitney U and Kruskal-Wallis tests for single and multiple comparisons, respectively. For multiple comparisons, a Fisher's LSD post-host test was performed. All statistical analyses were performed using Statistica version 13.3 (Dell, Round Rock, USA) with the significance level set at p < 0.05. 3. Results 3.1. Growth in mono- and co-cultures Generally, cell concentrations of C. albicans in monocultures did not differ significantly from concentrations observed in co-cultures with B. fragilis NCTC 9343 or B. vulgatus ATCC 8482 over the 72-h period (Fig. 1). After 24 h, however, the concentration of C. albicans CAB 397 decreased in co-culture with B. vulgatus ATCC 8482 while increasing following 48 h of co-cultivation compared to the yeast concentration in monoculture (Fig. 1C). Also, the cell concentration of C. albicans CAB 201 was higher in co-culture with B. vulgatus ATCC 8482 than when in the presence of B. fragilis NCTC 9343 after 24 and 72 h (Fig. 1A). After 24 h of incubation, an initial decline in Bacteroides numbers was observed in monocultures (Fig. 2). Nevertheless, following cocultivation, all the C. albicans strains were found to enhance growth of B. fragilis NCTC 9343 (Fig. 2A). Similarly, B. vulgatus ATCC 8482 growth was promoted by co-culturing with C. albicans for 24 and 48 h compared to in monoculture (Fig. 2B). After 24 h of incubation, bacterial concentrations were higher in B. vulgatus ATCC 8482/C. albicans CAB 201 co-cultures than in co-cultures prepared with this bacterium and C. albicans CAB 392 or C. albicans CAB 1085. Bacteroides vulgatus ATCC 8482 concentrations were not significantly influenced by Candida after 72 h of co-cultivation, except for when cultured with C. albicans CAB 201 for which the highest bacterial concentrations were obtained
2.6. Statistical analyses All data are presented as mean ± 1 standard error of the mean. Data for each time point was analyzed separately. To determine if a treatment affected Bacteroides or C. albicans growth, the treatment was directly compared to the control. For differences between treatments, all data, except the control, were compared. All data, including the control, were simultaneously compared to determine the effect of mannan on Bacteroides growth. Normally distributed data with equal variances were analyzed using one-way ANOVA followed by a Fisher's LSD post-hoc test for multiple comparisons. Normally distributed data with unequal variances were analyzed using t-tests for independent samples by groups. Statistical significance of not normally distributed
Fig. 1. Anaerobic growth at 37 °C determined for different time intervals of (A) C. albicans CAB 201, (B) C. albicans CAB 392, (C) C. albicans CAB 397 and (D) C. albicans CAB 1085 in mono- and co-cultures with Bacteroides in ¼ BHI broth. Mono, yeast concentrations in monoculture; + BF, yeast concentrations in co-culture with B. fragilis NCTC 9343; + BV, yeast concentrations in co-culture with B. vulgatus ATCC 8482. Bars represent the mean of four repeats and whiskers indicate standard error. Dashed lines depict the initial cell concentration of log 6 cells/ml. Statistically significant differences between bars are illustrated by horizontal lines and asterisks (*p < 0.05) as determined using one-way ANOVA and t-test analyses.
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Fig. 2. Anaerobic growth at 37 °C determined for different time intervals of (A) B. fragilis NCTC 9343 and (B) B. vulgatus ATCC 8482 in mono- and co-cultures with C. albicans in ¼ BHI broth. Mono, bacterial concentrations in monoculture; + CAB 201, bacterial concentrations in co-culture with C. albicans CAB 201; + CAB 392, bacterial concentrations in co-culture with C. albicans CAB 392; + CAB 397, bacterial concentrations in co-culture with C. albicans CAB 397; + CAB 1085, bacterial concentrations in co-culture with C. albicans CAB 1085. Bars represent the mean of four repeats and whiskers indicate standard error. Dashed lines depict the initial cell concentration of log 6 cells/ml. Relevant statistically significant differences between bars are illustrated by horizontal lines and asterisks (*p < 0.05, **p < 0.005) as determined using one-way ANOVA, t-test and Fisher's LSD analyses.
at that time point.
mannan [0.25 or 0.5% (w/v)] than in mannose-containing medium only (Fig. 3A). Bacteroides was, however, capable of growing in the liquid minimal medium when glucose was provided as a carbon source (Fig. A.1 ). Bacteroides vulgatus ATCC 8482 responded differently to mannan supplementation of the minimal medium. Cell concentrations of B. vulgatus ATCC 8482 were significantly higher in the minimal medium containing 0.1% (w/v) mannose to which mannan was added compared to in the presence of mannose alone (Fig. 3B). Although there
3.2. Effect of C. albicans mannan on Bacteroides growth in liquid minimal medium Enumeration of bacteria in the liquid minimal medium after 24 h revealed that B. fragilis NCTC 9343 numbers were lower in minimal medium containing 0.1% (w/v) mannose when supplemented with
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Fig. 3. Anaerobic growth at 37 °C determined after 24 h of (A) B. fragilis NCTC 9343 and (B) B. vulgatus ATCC 8482 in liquid minimal medium with mannose as a carbon source with and without mannan. Bars represent the mean of three repeats and whiskers indicate standard error. Dashed lines depict the initial cell concentration of log 6 cells/ml. Statistically significant differences between bars are illustrated by horizontal lines and asterisks (*p < 0.05, **p < 0.005) as determined using Fisher's LSD analyses.
were indications that mannan concentration had an effect on Bacteroides survival, the difference between B. vulgatus ATCC 8482 concentrations in minimal medium with 0.25 or 0.5% (w/v) mannan was not statistically significant (p = 0.11).
albicans CAB 392 cells (Fig. 4A) tended to be slightly more intensely stained than the live yeast cells (Fig. 4B). However, the relative fluorescence intensity (Fig. 4C) did not differ between the live and dead cells that were used as inoculums. This indicated that the outer mannan layers of the yeast cells were not degraded during the killing process. Supplementation of B. fragilis NCTC 9343 monocultures with these dead yeast cells led to increased bacterial numbers after 24 and 48 h, compared to numbers in monocultures prepared with ¼ BHI broth alone (Fig. 5A). Nevertheless, B. fragilis NCTC 9343 growth remained highest
3.3. Bacteroides cultures supplemented with metabolically inactive C. albicans cells Metabolically inactive (dead) concanavalin A, fluorescein-stained C.
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Fig. 4. Micrographs of (A) dead and (B) live C. albicans CAB 392 cells stained with concanavalin A, fluorescein conjugate, showing the intact outer mannan layers surrounding the cells. Low yeast concentrations necessitated the preparation of composite images of cells originating from a single sample (scale bars indicate 10 μm). (C) The relative fluorescence intensity of the cells, presented as corrected total cell fluorescence (CTCF), showed no statistical difference between the dead and live cells. Dead cells, dead C. albicans CAB 392 cells; Live cells, live C. albicans CAB 392 cells.
in co-cultures with live C. albicans CAB 392 cells. Similarly, 24 and 48 h B. vulgatus ATCC 8482 growth in the presence of live C. albicans CAB 392 cells were more than in bacterial monocultures with and without supplemented dead Candida cells (Fig. 5B). At all of the incubation time points, supplementation of B. vulgatus ATCC 8482 monocultures with dead C. albicans CAB 392 cells resulted in higher bacterial concentrations. At 72 h, equal B. vulgatus ATCC 8482 growth was observed in co-cultures with dead and live C. albicans CAB 392 cells.
3.4. Growth of Bacteroides in broth medium with reduced oxygen levels Growth of B. fragilis NCTC 9343 and B. vulgatus ATCC 8482 significantly increased when bacterial monocultures were prepared with ¼ BHI broth containing the reducing agent L-cysteine hydrochloride compared to monocultures prepared with ¼ BHI broth without the reducing agent (Fig. 6). Growth of B. fragilis NCTC 9343 in the prereduced medium was mostly unaffected by supplementation with dead C. albicans CAB 392 cells, except after 48 h of incubation during which
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Fig. 5. Anaerobic growth at 37 °C determined for different time intervals of (A) B. fragilis NCTC 9343 and (B) B. vulgatus ATCC 8482 monocultures in ¼ BHI broth with and without metabolically inactive (dead) C. albicans CAB 392 cells compared to in co-culture with live Candida cells. Monoculture, bacterial concentrations in monoculture; + Dead CAB 392, bacterial concentrations in monocultures supplemented with dead C. albicans CAB 392 cells; + Live CAB 392, bacterial concentrations in co-cultures with live C. albicans CAB 392 cells. Bars represent the mean of four repeats and whiskers indicate standard error. Dashed lines depict the initial cell concentration of log 6 cells/ml. Relevant statistically significant differences between bars are illustrated by horizontal lines and asterisks (*p < 0.05, **p < 0.005) as determined using one-way ANOVA and ttest analyses.
it was observed that dead yeast cells resulted in greater bacterial numbers (Fig. 6A). Supplementation of the pre-reduced medium with dead C. albicans CAB 392 cells did not affect B. vulgatus ATCC 8482 growth (Fig. 6B). Bacterial growth in B. fragilis NCTC 9343/C. albicans CAB 392 cocultures, with live yeast cells, was significantly more than in the prereduced media regardless of the presence of dead yeast cells (Fig. 6A). In contrast, B. vulgatus ATCC 8482 growth was more pronounced in the pre-reduced media than in co-culture with live C. albicans CAB 392 cells (Fig. 6B).
BHI broth (Fig. 7A). Likewise, increased B. vulgatus ATCC 8482 growth was observed after 24, 48 and 72 h of culture in the Candida spent medium (Fig. 7B). Growth of this bacterial species was not as pronounced in co-cultures with live C. albicans CAB 392 cells. In contrast, growth of B. fragilis NCTC 9343 in co-cultures was significantly more than in the spent medium of Candida cells (Fig. 7A). The addition of 512 μg/ml chloramphenicol to the spent medium of C. albicans CAB 392 or freshly prepared ¼ BHI broth generally had no notable effect on the survival of B. fragilis NCTC 9343 (Fig. 8A). Nevertheless, the addition of chloramphenicol to Candida spent medium resulted in a significant reduction in the survival of B. fragilis NCTC 9343 after 5 h, compared to bacterial survival in ¼ BHI broth. Similar results were obtained for B. vulgatus ATCC 8482 treated with 32 μg/ml chloramphenicol after 24 h (Fig. 8B).
3.5. Effect of C. albicans metabolites on Bacteroides growth and antibiotic susceptibility Culturing B. fragilis NCTC 9343 for 48 or 72 h in the spent medium of C. albicans CAB 392 monocultures resulted in significantly more growth compared to bacterial growth observed in freshly prepared ¼
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Fig. 6. Anaerobic growth at 37 °C determined for different time intervals of (A) B. fragilis NCTC 9343 and (B) B. vulgatus ATCC 8482 monocultures in ¼ BHI broth as well as in pre-reduced ¼ BHI broth with and without dead C. albicans cells, compared to in the presence of live C. albicans CAB 392 cells. Bars represent the mean of four repeats and whiskers indicate standard error. Dashed lines depict the initial cell concentration of log 6 cells/ml. Relevant statistically significant differences between bars are illustrated by horizontal lines and asterisks (*p < 0.05, **p < 0.005) as determined using one-way ANOVA, t-test, Mann-Whitney U and Fisher's LSD analyses.
4. Discussion
interactions between microorganisms strongly depends on the microbial strains [26,27]. Similar to our findings, others reported that C. albicans promoted growth of Escherichia coli and Klebsiella pneumoniae [28–30]. To explore the enhancement of Bacteroides growth by C. albicans, Bacteroides monocultures were prepared in liquid minimal media supplemented with extracted Candida mannan. Bacteroides fragilis NCTC 9343 and B. vulgatus ATCC 8482 are known to express enzymes capable of degrading the outer cell wall mannan layers of yeast cells [11,12]. Yeast mannan-degrading enzymes
The current study demonstrated that the yeast C. albicans exerted a significant positive effect on Bacteroides growth under anaerobic conditions, while B. fragilis NCTC 9343 had no notable effect on C. albicans concentrations in co-cultures. Bacteroides vulgatus ATCC 8482, however, affected yeast concentrations in a strain-specific manner and differences were noted in the growth of this bacterial species in the presence of different C. albicans strains. Based on previous studies, the nature of
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Fig. 7. Anaerobic growth at 37 °C determined for different time intervals of (A) B. fragilis NCTC 9343 and (B) B. vulgatus ATCC 8482 monocultures in ¼ BHI broth and spent medium from 24-h-old C. albicans CAB 392 monocultures compared to in co-culture with C. albicans CAB 392. Bars represent the mean of four repeats and whiskers indicate standard error. Dashed lines depict the initial cell concentration of log 6 cells/ml. Relevant statistically significant differences between bars are illustrated by horizontal lines and asterisks (*p < 0.05, **p < 0.005) as determined using one-way ANOVA and t-test analyses.
belonging to the glycoside hydrolases (GH) families GH38, GH76 and GH92 which include α-1,6-mannanase and α-mannosidases were discovered in B. fragilis NCTC 9343. While an α-1,2-mannosidase of the GH92 family is associated with B. vulgatus ATCC 8482. Also, it was shown that representatives of Bacteroides utilized mannan originating from Saccharomyces cerevisiae and C. albicans cells [13]. During our study, supplementation of Bacteroides monocultures in ¼ BHI broth with dead C. albicans CAB 392 cells, containing intact outer cell wall mannan layers as determined using fluorescence microscopy, stimulated bacterial growth. The finding supported the contention that yeast cells may serve as an additional nutrient source for the bacteria. It was
therefore hypothesized that the representatives of Bacteroides used in our study may utilize Candida mannan as a nutrient source. Upon testing this hypothesis, we found that B. fragilis NCTC 9343 survived better in the liquid minimal medium with mannose as a sole carbon source, compared to in the medium supplemented with both mannose and mannan. This phenomenon could have been the result of complexation between non-utilized mannan and essential ions needed for growth of this particular strain, since it is known that metal ions can adhere to the yeast cell wall (including the mannan layer) [31]. The addition of mannan to the mannose-containing minimal medium, however, enhanced survival of B. vulgatus ATCC 8482 under the
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Fig. 8. Percentage survival of (A) B. fragilis NCTC 9343 and (B) B. vulgatus ATCC 8482 in ¼ BHI broth compared to in C. albicans CAB 392 spent medium when treated with 512 or 32 μg/ml chloramphenicol, respectively. Bars represent the mean of three repeats and whiskers indicate standard error. Statistically significant differences between bars are illustrated by horizontal lines and asterisks (*p < 0.05) as determined using one-way ANOVA analyses.
conditions tested in this study. Our findings were similar to that of Cuskin et al. [13], who showed that B. vulgatus, not B. fragilis, was capable of utilizing mannan from S. cerevisiae cells as a carbon source. It seemed that while yeast mannan may have contributed to higher cell concentrations of B. vulgatus ATCC 8482 in co-culture with live and media supplemented with dead C. albicans CAB 392 cells, this complex polymer did not contribute to the enhanced growth of B. fragilis NCTC 9343. Further investigation revealed that the metabolism of C. albicans also played a role during this binary interaction. It was found that during co-cultivation of C. albicans and the obligate anaerobe, Clostridium difficile, aerobic respiration or antioxidant
production by the yeast may decrease oxygen to a level that can allow bacterial proliferation [32]. During our study, supplementation of the ¼ BHI broth with the reducing agent L-cysteine hydrochloride resulted in enhanced growth of Bacteroides in monocultures after 24 h, compared to bacterial growth in monocultures without the reducing agent. The initial decline in Bacteroides numbers in monocultures, prepared with the latter medium, may thus have been the result of available oxygen. On the other hand, growth of the anaerobic Bacteroides species in coculture with live C. albicans cells may have been enhanced in ¼ BHI broth of which the oxygen levels were lowered by yeast metabolism. Interestingly, a decrease in the cell concentrations of B. fragilis NCTC
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9343 was observed after 48 h within the pre-reduced ¼ BHI broth, but supplementation of the medium with dead C. albicans CAB 392 cells resulted in more bacterial growth after the same incubation period. Providing further evidence that C. albicans cells may serve as an additional nutrient source for Bacteroides. The presence of live C. albicans CAB 392 cells in the medium led to more B. fragilis NCTC 9343 growth, compared to growth of this bacterium either in pre-reduced ¼ BHI broth or pre-reduced ¼ BHI broth with dead yeast cells. This indicated that direct contact or multiple factors are simultaneously needed for the stimulatory effect exerted by Candida on bacterial growth. Researchers suggested that interactions between microorganisms can be contact-dependent [33,34]. In contrast, B. vulgatus ATCC 8482 grew better in ¼ BHI broth with reduced oxygen levels than in the presence of live C. albicans CAB 392 cells. This suggested that anaerobic conditions are essential for growth of B. vulgatus ATCC 8482 and that the metabolism of C. albicans is unable to remove sufficient quantities of oxygen from the medium in order to support anaerobic bacterial growth. Additionally, this bacterium showed more growth in the spent medium of C. albicans CAB 392 than in freshly prepared ¼ BHI broth. Consequently, it seemed that although C. albicans may overall positively influence B. vulgatus, this effect may be counteracted by contact-dependent negative factors that were absent in the spent medium. The spent medium of Candida monocultures significantly promoted Bacteroides growth. The metabolite(s) involved, which could either be produced intracellularly and exported to the extracellular environment or a product of membrane-associated enzymes, need to be identified in future studies. Other authors demonstrated that certain molecules secreted by C. albicans affect bacterial growth. For example, farnesol secreted by C. albicans suppressed Acinetobacter baumannii growth [34]. We also found that the spent medium of C. albicans CAB 392 increased the susceptibility of Bacteroides to chloramphenicol. Secreted metabolites of Candida may be acting synergistically with the antibiotic. On the other hand, active growth and concomitant protein synthesis by Bacteroides cells in the spent medium may increase the susceptibility of the
bacterial cells to chloramphenicol, which is a known inhibitor of protein synthesis [35]. In conclusion, cell concentrations of C. albicans (specifically C. albicans CAB 201, C. albicans CAB 392 and C. albicans CAB 1085) were unaffected by the presence of B. fragilis NCTC 9343 or B. vulgatus ATCC 8482, while Bacteroides growth was significantly enhanced in co-culture with C. albicans. The interaction between C. albicans and Bacteroides species are complex with multiple factors contributing to the increased growth of Bacteroides in the presence of C. albicans. Strain differences were not apparent in co-cultures with B. fragilis NCTC 9343 and C. albicans. In contrast, strain-specific interactions were noted during cocultivation of B. vulgatus ATCC 8482 and C. albicans. The interaction between B. fragilis NCTC 9343 and C. albicans seemed to be contactdependent, while the interaction between B. vulgatus ATCC 8482 and C. albicans is likely governed by non-contact-dependent mechanisms. Furthermore, the nature of these interactions may be dictated by environmental conditions. For example, in the hypoxic regions of the gastrointestinal tract [36], aerobic respiration and/or antioxidant production by C. albicans may largely contribute to greater Bacteroides growth. However, the cells of C. albicans may serve as an additional nutrient source for the bacteria in anaerobic regions of the gut. It was also found that Bacteroides growth and susceptibility to the antibiotic chloramphenicol increased in the presence of C. albicans supernatant. It is clear that an interaction between Bacteroides species and C. albicans may exist in the human body. Therefore, it is imperative to study Bacteroides and C. albicans within dual-species biofilms and the effect of these microorganisms on each other's virulence. In future, the implications thereof on human health need to be investigated in vivo. Declarations of interest None.
Fig. A.1. Anaerobic growth of Bacteroides at 37 °C determined for different time intervals in liquid minimal medium with glucose [0.5% (w/v)] as a carbon source. BF, B. fragilis NCTC 9343 cell concentrations; BV, B. vulgatus ATCC 8482 cell concentrations. Bars represent the mean of four repeats and whiskers indicate standard error. The dashed line depicts the initial cell concentration of log 6 cells/ml.
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Acknowledgements Funding: This work was supported by the National Research Foundation (NRF) of South Africa.
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Binary interactions between the yeast Candida albicans and two gutassociated Bacteroides species
T
Marisa Valentinea, Eliska Benadéa, Marnel Moutonb, Wesaal Khana, Alfred Bothaa,* a b
Department of Microbiology, Stellenbosch University, Stellenbosch, South Africa Department of Botany and Zoology, Stellenbosch University, Stellenbosch, South Africa
ABSTRACT
The yeast Candida albicans forms part of the natural gut microbiota of healthy human individuals and its interactions with other microbial symbionts can impact host well-being. We therefore studied binary interactions between potentially pathogenic representatives of the gut-associated bacterial genus Bacteroides and C. albicans using anaerobic bacteria/yeast co-cultures prepared with a quarter-strength brain heart infusion (¼ BHI; 9.25 g/l) broth. We found that, except for minor changes observed in the cell numbers of one out of four C. albicans strains tested, yeast growth was largely unaffected by the presence of the bacteria. In contrast, growth of Bacteroides fragilis NCTC 9343 and Bacteroides vulgatus ATCC 8482 was significantly enhanced in the presence of C. albicans. Supplementation of Bacteroides monocultures with dead Candida albicans CAB 392 cells, containing intact outer cell wall mannan layers, resulted in increased bacterial concentrations. Subsequent culturing of the Bacteroides strains in a liquid minimal medium supplemented with candidal mannan demonstrated that B. vulgatus ATCC 8482, unlike B. fragilis NCTC 9343, utilized the mannan. Furthermore, by reducing the initial oxygen levels in monocultures prepared with ¼ BHI broth, bacterial numbers were significantly enhanced compared to in monocultures prepared with ¼ BHI broth not supplemented with the reducing agent L-cysteine hydrochloride. This suggests that C. albicans can stimulate Bacteroides growth via aerobic respiration and/or antioxidant production. The cell-free supernatant of 24-h-old C. albicans CAB 392 monocultures was also found to increase Bacteroides growth and chloramphenicol sensitivity.
1. Introduction Ascomycetous yeasts belonging to the genus Candida are the causative agents of oral candidiasis, the most common opportunistic infection among HIV/AIDS-infected patients [1]. Alarmingly, such mucosal infections can develop into invasive candidiasis when candidal cells penetrate the epithelial barrier, enter the bloodstream and disseminate to various organs. Both superficial and invasive infections of individuals, with compromised host immune systems (e.g. HIV/AIDS) or microbiomes, are predominantly caused by Candida albicans. However, in 30–60% of healthy individuals C. albicans exists as a harmless commensal of the skin and mucosal surfaces, including gastrointestinal, oral and vaginal surfaces. Within the various anatomical sites of the human body C. albicans can potentially interact with the indigenous or transient host microbiota. For example, it was demonstrated that vaginal colonizing lactobacilli can prevent adherence of C. albicans to epithelial cells via competition and biosurfactant production [2]. These lactobacilli were also found to produce lactic and fatty acids, lowering vaginal pH and inhibiting yeast growth. On the other hand, oral streptococci such as
*
Streptococcus gordonii enhanced biofilm and hyphal formation of C. albicans, while the yeast increased Streptococcus mutans virulence [3,4]. In addition to studies on the interactions between C. albicans and members of the oral or vaginal microbiota, interactions between this yeast and the gut microbiota were recently explored. It was shown that the natural gut microbiota in mice can prevent C. albicans-induced gastritis [5]. Furthermore, it was observed that the gut pathogen Salmonella enterica serovar Typhimurium inhibited biofilm formation and filamentation of C. albicans [6]. Similar negative effects on C. albicans were reported for a bacteriocin produced by the opportunistic gut pathogen Enterococcus faecalis [7]. However, in the cecum of the gastrointestinal tract of cefoperazone treated mice, C. albicans promoted E. faecalis colonization while preventing colonization by Lactobacillus species [8]. It is of interest to note that these studies of gut microbiota predominantly focus on interactions between C. albicans and Firmicutes or Proteobacteria. Firmicutes together with Bacteroidetes are the dominant bacterial phyla in the gastrointestinal tract constituting ca. 90% of species in this habitat [9]. Within the Bacteroidetes, the genus Bacteroides represents approximately 25% of the anaerobic bacteria in the colon with
Corresponding author. Department of Microbiology, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa. E-mail address: [email protected] (A. Botha).
https://doi.org/10.1016/j.micpath.2019.103619 Received 31 January 2019; Received in revised form 11 May 2019; Accepted 6 July 2019 Available online 07 July 2019 0882-4010/ © 2019 Elsevier Ltd. All rights reserved.
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Department of Microbiology at the University of Stellenbosch, South Africa. The bacterial strains were maintained anaerobically at 37 °C on brain heart infusion (BHI; Biolab Diagnostics, Merck, Darmstadt, Germany) agar plates. Anaerobic growth conditions were created in anaerobic chambers with Anaerocult A sets (Merck) and verified using Anaerotest indicator strips (Merck) as recommended by the manufacturer. The same method was used to achieve anaerobiosis throughout the study. Yeast strains were maintained aerobically at 26 °C on yeast extract-malt extract (YM) agar plates [15].
representatives of Bacteroides fragilis regarded the most virulent and frequent clinical isolates [10]. Bacteroides fragilis together with Bacteroides vulgatus are responsible for a variety of conditions such as skin and soft tissue infections together with Crohn's disease, respectively. The nature of the interaction between these Bacteroides species and C. albicans in the anaerobic environment of the gut is still unknown. Nevertheless, both B. fragilis and B. vulgatus are known to express enzymes capable of degrading the outer mannan layers of ascomycetous yeast cell walls, namely α-mannanases and/or α-mannosidases [11,12]. Furthermore, it was shown that Bacteroides species can utilize yeast αmannan, including that of C. albicans, as a carbon source [13]. In addition, the anaerobic environment of C. albicans biofilms was found to enhance the survival of B. fragilis [14]. It was also demonstrated that following cefoperazone treatment of mice, C. albicans promoted the recovery of Bacteroidetes [8]. Although these compositional changes were reflected in the increased relative abundance of the family Porphyromonadaceae, it remains to be discovered whether complex interactions in the gut or binary interactions between C. albicans and Bacteroides species contributed to the enhancement of Bacteroidetes growth. The aim of this study was therefore to determine if C. albicans exerts a positive effect on Bacteroides growth under anaerobic conditions. Thus, the first objective was to compare growth of B. fragilis, B. vulgatus and C. albicans in monocultures to growth in Bacteroides/C. albicans cocultures. The second objective was to explore the potential role of yeast α-mannan during this interaction by determining the viability of Bacteroides in monocultures, prepared in a minimal medium, supplemented with mannan extracted from C. albicans cell walls. The third objective was to investigate whether yeast metabolism could influence Bacteroides growth. This was done by determining bacterial growth in monocultures that were supplemented with metabolically inactive (dead) C. albicans cells. The role of yeast respiration was then explored by determining Bacteroides growth in monocultures prepared with a medium containing reduced oxygen levels. In addition, the effect of C. albicans-secreted metabolite(s) on Bacteroides growth was determined by assessing bacterial growth in monocultures prepared with the cellfree supernatant of C. albicans monocultures. The effect of C. albicanssecreted metabolites on Bacteroides antibiotic susceptibility was also evaluated by determining bacterial survival, in the presence and absence of chloramphenicol, in monocultures prepared with the cell-free C. albicans supernatant.
2.2. Preparation of starter inoculums A loopful of growth from each C. albicans strain was inoculated into 5 ml BHI broth contained in a test tube, while bacterial strains were cultured in test tubes containing 10 ml BHI broth. Inoculated BHI test tubes were incubated anaerobically at 37 °C. To obtain the starter inoculums, cells were harvested after 48 h by centrifugation (12 000×g, 5 min) and washed thrice with physiological saline solution (PSS; 8.9 g/ l NaCl). The concentrations of bacterial and yeast cell suspensions were determined using a Petroff-Hausser counting chamber (Neubauer Improved, Marienfeld Superior, Lauda-Königshofen, Germany) and hemocytometer (Neubauer Improved, Marienfeld Superior), respectively. Cell concentrations were adjusted and test tubes containing liquid media were inoculated resulting in a final concentration of log 6 bacterial cells/ml and/or log 6 yeast cells/ml. In all experimentation, unless specified differently, starter inoculums were prepared as described here. 2.3. Growth in mono- and co-cultures A nutrient limited medium was used to increase the potential for cooperative and other types of interactions within bacteria/yeast cocultures [17]. Mono- and co-cultures were subsequently prepared in test tubes each containing 5 ml quarter-strength BHI (¼ BHI; 9.25 g/l) broth using the starter inoculums obtained as described in section 2.2. Co-cultures were prepared for all eight combinations of the Bacteroides and C. albicans strains, i.e. Bacteroides fragilis NCTC 9343 and Bacteroides vulgatus ATCC 8482 were individually co-cultured with each of the following yeast strains: Candida albicans CAB 201, Candida albicans CAB 392, Candida albicans CAB 397 and Candida albicans CAB 1085. Both mono- and co-cultures were incubated anaerobically at 37 °C, while microbial numbers within the cultures were determined as colonyforming units (CFU)/ml on dilution plates after 24, 48 and 72 h. Bacterial cells were enumerated on BHI agar plates that were incubated anaerobically for 96 h at 37 °C. Yeast cells were enumerated on YM agar plates supplemented with 0.2 g/l chloramphenicol (Sigma-Aldrich, St. Louis, USA) after 48 h of aerobic incubation at 30 °C. All monocultures were grown as described here unless indicated otherwise. Also, for all experimentation, bacterial numbers were determined by plating serial
2. Materials and methods 2.1. Microbial strains and culture conditions The Bacteroides and C. albicans strains used in the experimentation (Table 1) were revived from 30 to 20% (v/v) glycerol stocks, respectively, that were stored at −80 °C in the culture collection of the
Table 1 The origins and GenBank accession numbers of the bacterial and yeast strains used in this study. Species Bacterial Bacteroides vulgatus Bacteroides fragilis Yeast Candida albicans Candida albicans Candida albicans Candida albicans
Strain
Origin
GenBank accession noa
ATCC 8482 NCTC 9343
Clinical isolate, American Type Culture Collection Clinical isolate, American Type Culture Collection
AJ867050.1 NR_074784.2
CAB CAB CAB CAB
Veterinary isolate, Columba livia domestica, South Africa Clinical isolate, Tygerberg Hospital, South Africa Clinical isolate, Tygerberg Hospital, South Africa Environmental isolate, Plankenburg River, South Africa
MK248726 KJ534504 KJ534505 KJ534503
201 392b 397b 1085c
a
GenBank accession numbers for taxonomic informative gene sequences representing either the bacterial 16S or yeast D1/D2 region of the ribosomal RNA gene. C. albicans CAB 392 and CAB 397 were previously stored as MRC 8908 and MRC 8912, respectively, in the Program on Mycotoxins and Experimental Carcinogenesis (PROMEC) Unit culture collection of the South African Medical Research Council. c Isolate obtained from a river that is known to be polluted with sewage [16]. b
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dilutions on BHI agar plates as described in this section.
contained in a microcentrifuge tube. The resulting suspension was harvested via centrifugation (12 000×g, 5 min) and the pellet (ca. 100 mg wet weight) suspended in 2 ml AmB solution. The suspension was incubated for 4 h at 22 °C, centrifuged and the pellet was once more treated with 2 ml AmB solution. The dead yeast cells were washed twice by suspension in DMSO, followed by a third wash step with dH2O. To confirm that the mannan layers of the AmB killed cells were intact, both dead and live C. albicans CAB 392 cells that were used as inoculums were stained with concanavalin A, fluorescein conjugate (Life Technologies, CA, USA). Images of the fluorescently stained yeast cells were captured using a Nikon Eclipse E400 epifluorescence microscope (Nikon, Tokyo, Japan) equipped with a fluorescein isothiocyanate (FITC; excitation/emission 465–495/515–555 nm) filter set, Nikon DS-Fi2 camera and Nikon Digital Sight DS-U3 camera controller. The relative fluorescence intensity of 50 AmB killed and live C. albicans CAB 392 cells, respectively, were measured with ImageJ software (National Institute of Health, Maryland, USA). The integrated density of the cells was measured, corrected by subtracting the mean background fluorescence and normalized per cell to be expressed as corrected total cell fluorescence (CTCF). Finally, the dead cells of C. albicans CAB 392 were washed with PSS and added at a final concentration of log 6 cells/ml to B. fragilis NCTC 9343 and B. vulgatus ATCC 8482 monocultures prepared in ¼ BHI broth. The resulting cultures were incubated anaerobically at 37 °C and Bacteroides numbers were determined after 24, 48 and 72 h as described in section 2.3.
2.4. Effect of C. albicans mannan on Bacteroides growth in liquid minimal medium To confirm whether B. fragilis NCTC 9343 and B. vulgatus ATCC 8482 can utilize the outer cell wall mannan layer of C. albicans [12,13], the bacteria were inoculated into a liquid minimal medium containing yeast mannan as a carbon source. The latter was obtained from the cell wall of the clinical strain C. albicans CAB 392 using a modified alkali extraction method [18,19]. For this purpose, C. albicans cells were grown aerobically on YM agar plates that were incubated for 48 h at 30 °C. The yeast cells were transferred from the plates to PSS and washed thrice by centrifugation (10 000×g, 15 min). The resulting biomass was treated twice with 6% (w/v) NaOH for 90 min at 70 °C and centrifuged at 10 000×g for 15 min. The yeast mannan was subsequently precipitated from the alkali-extracted supernatant by adding Fehling's reagent. After centrifugation (10 000×g, 15 min), the resulting pellet was dissolved in cold 3 M HCl and the mannan precipitated with methanol. Residual copper was removed by dialyzing the extracted mannan against citrate buffer [pH 6; 0.0115 M citric acid monohydrate (Saarchem, Univar, Merck) and 0.0885 M trisodium citrate dihydrate (Saarchem)] using a Spectra/Por membrane (molecular weight cut-off 6000–8000) for six days at 8 °C. During a final 48 h dialysis step, the citrate buffer was replaced with distilled water (dH2O) and the removal of copper was confirmed with inductively coupled plasma mass spectrometry (ICP-MS) performed by the Central Analytical Facility at Stellenbosch University. The extracted C. albicans CAB 392 mannan was freeze-dried and stored in a desiccator at 4 °C until use. The extracted yeast mannan [0.25 or 0.5% (w/v)] was added to a liquid minimal medium containing the mannan monomer mannose [0.1% (w/v)]. The minimal medium was prepared according to Martens et al. [20]. Controls containing mannose [0.1% (w/v)] or glucose [0.5% (w/v)] were included in the experimentation. Each Bacteroides strain was inoculated into a test tube with 5 ml minimal medium resulting in a final concentration of log 6 bacterial cells/ml. The inoculated test tubes were incubated anaerobically at 37 °C and Bacteroides numbers were determined after 24 h as described in section 2.3. For glucose controls, Bacteroides numbers were determined after 24, 48 and 72 h.
2.5.2. Growth of Bacteroides in broth medium with reduced oxygen levels The potential effect of oxygen depletion as a result of the metabolic activity of C. albicans on Bacteroides growth was evaluated in ¼ BHI broth supplemented with the reducing agent L-cysteine hydrochloride (0.5 g/l; Sigma-Aldrich) and colorimetric redox indicator resazurin (1 mg/l; BDH Chemicals, Poole, UK) [23]. Test tubes containing 5 ml of the pre-reduced media were inoculated with the B. fragilis NCTC 9343 or B. vulgatus ATCC 8482 starter inoculum. Additionally, Bacteroides monocultures were supplemented with log 6 dead C. albicans CAB 392 cells/ml (prepared as described in section 2.5.1) to test if yeast cells may serve as an additional nutrient source for bacterial cells. All test tubes were incubated anaerobically at 37 °C and Bacteroides numbers were determined after 24, 48 and 72 h as described in section 2.3. Negative controls containing no reducing agent were also included in all experimentation.
2.5. Interactions between Bacteroides and yeast metabolism Similar to section 2.4, C. albicans CAB 392 was used in all subsequent experimentation to study the potential effect of a clinical yeast strain on Bacteroides growth.
2.5.3. Effect of C. albicans metabolites on Bacteroides growth and antibiotic susceptibility The influence of secreted C. albicans metabolites on growth of B. fragilis NCTC 9343 and B. vulgatus ATCC 8482 was determined by culturing the bacteria in the cell-free supernatant of C. albicans CAB 392 monocultures. Thus, following incubation of C. albicans CAB 392 in ¼ BHI broth for 24 h, the supernatant was removed, filter-sterilized (0.20 μm; GVS Filter Technology, Lasec, Ndabeni, RSA; designated as spent medium) and 5 ml added to a sterile empty test tube. The spent medium containing test tube was inoculated to obtain a final concentration of log 6 bacterial cells/ml. Following anaerobic incubation at 37 °C, Bacteroides numbers were determined after 24, 48 and 72 h as described in section 2.3. The influence of C. albicans-secreted metabolites on the antibiotic susceptibility of Bacteroides was also studied, since it was previously shown that metabolites secreted by C. albicans can act synergistically with antibiotics. For example, Jabra-Rizk et al. [24] illustrated synergy
2.5.1. Bacteroides cultures supplemented with metabolically inactive C. albicans cells Metabolically inactive yeasts (dead yeast cells), without compromised outer cell wall mannan layers, were obtained by treating C. albicans CAB 392 cells with amphotericin B (AmB; Merck). This fungicidal compound is known to compromise the ergosterol component of yeast cell membranes [21]. To obtain the AmB dosage for the treatment of yeast cells, the macrobroth dilution method [22] was used to determine the minimum inhibitory concentration of AmB for C. albicans CAB 392, which was found to be 0.06 μg/ml. Subsequently, C. albicans CAB 392 was treated with a 6 mg/ml solution of AmB dissolved in dimethyl sulfoxide (DMSO). Yeast growth was transferred from a 48-h-old anaerobic YM agar plate culture incubated at 37 °C to 1 ml PSS
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between farnesol, a quorum-sensing molecule secreted by C. albicans, and the antibiotic gentamicin. We therefore conducted time-kill assays as described by Petersen et al. [25], with modifications for B. fragilis NCTC 9343 and B. vulgatus ATCC 8482, using the antibiotic chloramphenicol and cell-free supernatant of C. albicans. Reaction mixtures were prepared in 1.5 ml microcentrifuge tubes to contain 899 μl ¼ BHI broth or spent medium of 24-h-old C. albicans CAB 392 monocultures, to which 100 μl chloramphenicol solution (320 or 5120 μg/ml for B. vulgatus and B. fragilis, respectively) was added. Each reaction mixture was inoculated with 1 μl Bacteroides starter inoculum resulting in a final concentration of log 6 cells/ml. Controls, containing either ¼ BHI broth or spent medium, in which the chloramphenicol was replaced with 100 μl dH2O were included in the experimentation. As described by Petersen et al. [25], we determined bacterial numbers within the first 24 h following treatment with the antibiotic. Subsequently, after 5, 10 and 24 h of anaerobic incubation at 37 °C, aliquots were removed and Bacteroides numbers determined as described in section 2.3.
data was determined using the non-parametric Mann-Whitney U and Kruskal-Wallis tests for single and multiple comparisons, respectively. For multiple comparisons, a Fisher's LSD post-host test was performed. All statistical analyses were performed using Statistica version 13.3 (Dell, Round Rock, USA) with the significance level set at p < 0.05. 3. Results 3.1. Growth in mono- and co-cultures Generally, cell concentrations of C. albicans in monocultures did not differ significantly from concentrations observed in co-cultures with B. fragilis NCTC 9343 or B. vulgatus ATCC 8482 over the 72-h period (Fig. 1). After 24 h, however, the concentration of C. albicans CAB 397 decreased in co-culture with B. vulgatus ATCC 8482 while increasing following 48 h of co-cultivation compared to the yeast concentration in monoculture (Fig. 1C). Also, the cell concentration of C. albicans CAB 201 was higher in co-culture with B. vulgatus ATCC 8482 than when in the presence of B. fragilis NCTC 9343 after 24 and 72 h (Fig. 1A). After 24 h of incubation, an initial decline in Bacteroides numbers was observed in monocultures (Fig. 2). Nevertheless, following cocultivation, all the C. albicans strains were found to enhance growth of B. fragilis NCTC 9343 (Fig. 2A). Similarly, B. vulgatus ATCC 8482 growth was promoted by co-culturing with C. albicans for 24 and 48 h compared to in monoculture (Fig. 2B). After 24 h of incubation, bacterial concentrations were higher in B. vulgatus ATCC 8482/C. albicans CAB 201 co-cultures than in co-cultures prepared with this bacterium and C. albicans CAB 392 or C. albicans CAB 1085. Bacteroides vulgatus ATCC 8482 concentrations were not significantly influenced by Candida after 72 h of co-cultivation, except for when cultured with C. albicans CAB 201 for which the highest bacterial concentrations were obtained
2.6. Statistical analyses All data are presented as mean ± 1 standard error of the mean. Data for each time point was analyzed separately. To determine if a treatment affected Bacteroides or C. albicans growth, the treatment was directly compared to the control. For differences between treatments, all data, except the control, were compared. All data, including the control, were simultaneously compared to determine the effect of mannan on Bacteroides growth. Normally distributed data with equal variances were analyzed using one-way ANOVA followed by a Fisher's LSD post-hoc test for multiple comparisons. Normally distributed data with unequal variances were analyzed using t-tests for independent samples by groups. Statistical significance of not normally distributed
Fig. 1. Anaerobic growth at 37 °C determined for different time intervals of (A) C. albicans CAB 201, (B) C. albicans CAB 392, (C) C. albicans CAB 397 and (D) C. albicans CAB 1085 in mono- and co-cultures with Bacteroides in ¼ BHI broth. Mono, yeast concentrations in monoculture; + BF, yeast concentrations in co-culture with B. fragilis NCTC 9343; + BV, yeast concentrations in co-culture with B. vulgatus ATCC 8482. Bars represent the mean of four repeats and whiskers indicate standard error. Dashed lines depict the initial cell concentration of log 6 cells/ml. Statistically significant differences between bars are illustrated by horizontal lines and asterisks (*p < 0.05) as determined using one-way ANOVA and t-test analyses.
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Fig. 2. Anaerobic growth at 37 °C determined for different time intervals of (A) B. fragilis NCTC 9343 and (B) B. vulgatus ATCC 8482 in mono- and co-cultures with C. albicans in ¼ BHI broth. Mono, bacterial concentrations in monoculture; + CAB 201, bacterial concentrations in co-culture with C. albicans CAB 201; + CAB 392, bacterial concentrations in co-culture with C. albicans CAB 392; + CAB 397, bacterial concentrations in co-culture with C. albicans CAB 397; + CAB 1085, bacterial concentrations in co-culture with C. albicans CAB 1085. Bars represent the mean of four repeats and whiskers indicate standard error. Dashed lines depict the initial cell concentration of log 6 cells/ml. Relevant statistically significant differences between bars are illustrated by horizontal lines and asterisks (*p < 0.05, **p < 0.005) as determined using one-way ANOVA, t-test and Fisher's LSD analyses.
at that time point.
mannan [0.25 or 0.5% (w/v)] than in mannose-containing medium only (Fig. 3A). Bacteroides was, however, capable of growing in the liquid minimal medium when glucose was provided as a carbon source (Fig. A.1 ). Bacteroides vulgatus ATCC 8482 responded differently to mannan supplementation of the minimal medium. Cell concentrations of B. vulgatus ATCC 8482 were significantly higher in the minimal medium containing 0.1% (w/v) mannose to which mannan was added compared to in the presence of mannose alone (Fig. 3B). Although there
3.2. Effect of C. albicans mannan on Bacteroides growth in liquid minimal medium Enumeration of bacteria in the liquid minimal medium after 24 h revealed that B. fragilis NCTC 9343 numbers were lower in minimal medium containing 0.1% (w/v) mannose when supplemented with
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Fig. 3. Anaerobic growth at 37 °C determined after 24 h of (A) B. fragilis NCTC 9343 and (B) B. vulgatus ATCC 8482 in liquid minimal medium with mannose as a carbon source with and without mannan. Bars represent the mean of three repeats and whiskers indicate standard error. Dashed lines depict the initial cell concentration of log 6 cells/ml. Statistically significant differences between bars are illustrated by horizontal lines and asterisks (*p < 0.05, **p < 0.005) as determined using Fisher's LSD analyses.
were indications that mannan concentration had an effect on Bacteroides survival, the difference between B. vulgatus ATCC 8482 concentrations in minimal medium with 0.25 or 0.5% (w/v) mannan was not statistically significant (p = 0.11).
albicans CAB 392 cells (Fig. 4A) tended to be slightly more intensely stained than the live yeast cells (Fig. 4B). However, the relative fluorescence intensity (Fig. 4C) did not differ between the live and dead cells that were used as inoculums. This indicated that the outer mannan layers of the yeast cells were not degraded during the killing process. Supplementation of B. fragilis NCTC 9343 monocultures with these dead yeast cells led to increased bacterial numbers after 24 and 48 h, compared to numbers in monocultures prepared with ¼ BHI broth alone (Fig. 5A). Nevertheless, B. fragilis NCTC 9343 growth remained highest
3.3. Bacteroides cultures supplemented with metabolically inactive C. albicans cells Metabolically inactive (dead) concanavalin A, fluorescein-stained C.
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Fig. 4. Micrographs of (A) dead and (B) live C. albicans CAB 392 cells stained with concanavalin A, fluorescein conjugate, showing the intact outer mannan layers surrounding the cells. Low yeast concentrations necessitated the preparation of composite images of cells originating from a single sample (scale bars indicate 10 μm). (C) The relative fluorescence intensity of the cells, presented as corrected total cell fluorescence (CTCF), showed no statistical difference between the dead and live cells. Dead cells, dead C. albicans CAB 392 cells; Live cells, live C. albicans CAB 392 cells.
in co-cultures with live C. albicans CAB 392 cells. Similarly, 24 and 48 h B. vulgatus ATCC 8482 growth in the presence of live C. albicans CAB 392 cells were more than in bacterial monocultures with and without supplemented dead Candida cells (Fig. 5B). At all of the incubation time points, supplementation of B. vulgatus ATCC 8482 monocultures with dead C. albicans CAB 392 cells resulted in higher bacterial concentrations. At 72 h, equal B. vulgatus ATCC 8482 growth was observed in co-cultures with dead and live C. albicans CAB 392 cells.
3.4. Growth of Bacteroides in broth medium with reduced oxygen levels Growth of B. fragilis NCTC 9343 and B. vulgatus ATCC 8482 significantly increased when bacterial monocultures were prepared with ¼ BHI broth containing the reducing agent L-cysteine hydrochloride compared to monocultures prepared with ¼ BHI broth without the reducing agent (Fig. 6). Growth of B. fragilis NCTC 9343 in the prereduced medium was mostly unaffected by supplementation with dead C. albicans CAB 392 cells, except after 48 h of incubation during which
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Fig. 5. Anaerobic growth at 37 °C determined for different time intervals of (A) B. fragilis NCTC 9343 and (B) B. vulgatus ATCC 8482 monocultures in ¼ BHI broth with and without metabolically inactive (dead) C. albicans CAB 392 cells compared to in co-culture with live Candida cells. Monoculture, bacterial concentrations in monoculture; + Dead CAB 392, bacterial concentrations in monocultures supplemented with dead C. albicans CAB 392 cells; + Live CAB 392, bacterial concentrations in co-cultures with live C. albicans CAB 392 cells. Bars represent the mean of four repeats and whiskers indicate standard error. Dashed lines depict the initial cell concentration of log 6 cells/ml. Relevant statistically significant differences between bars are illustrated by horizontal lines and asterisks (*p < 0.05, **p < 0.005) as determined using one-way ANOVA and ttest analyses.
it was observed that dead yeast cells resulted in greater bacterial numbers (Fig. 6A). Supplementation of the pre-reduced medium with dead C. albicans CAB 392 cells did not affect B. vulgatus ATCC 8482 growth (Fig. 6B). Bacterial growth in B. fragilis NCTC 9343/C. albicans CAB 392 cocultures, with live yeast cells, was significantly more than in the prereduced media regardless of the presence of dead yeast cells (Fig. 6A). In contrast, B. vulgatus ATCC 8482 growth was more pronounced in the pre-reduced media than in co-culture with live C. albicans CAB 392 cells (Fig. 6B).
BHI broth (Fig. 7A). Likewise, increased B. vulgatus ATCC 8482 growth was observed after 24, 48 and 72 h of culture in the Candida spent medium (Fig. 7B). Growth of this bacterial species was not as pronounced in co-cultures with live C. albicans CAB 392 cells. In contrast, growth of B. fragilis NCTC 9343 in co-cultures was significantly more than in the spent medium of Candida cells (Fig. 7A). The addition of 512 μg/ml chloramphenicol to the spent medium of C. albicans CAB 392 or freshly prepared ¼ BHI broth generally had no notable effect on the survival of B. fragilis NCTC 9343 (Fig. 8A). Nevertheless, the addition of chloramphenicol to Candida spent medium resulted in a significant reduction in the survival of B. fragilis NCTC 9343 after 5 h, compared to bacterial survival in ¼ BHI broth. Similar results were obtained for B. vulgatus ATCC 8482 treated with 32 μg/ml chloramphenicol after 24 h (Fig. 8B).
3.5. Effect of C. albicans metabolites on Bacteroides growth and antibiotic susceptibility Culturing B. fragilis NCTC 9343 for 48 or 72 h in the spent medium of C. albicans CAB 392 monocultures resulted in significantly more growth compared to bacterial growth observed in freshly prepared ¼
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Fig. 6. Anaerobic growth at 37 °C determined for different time intervals of (A) B. fragilis NCTC 9343 and (B) B. vulgatus ATCC 8482 monocultures in ¼ BHI broth as well as in pre-reduced ¼ BHI broth with and without dead C. albicans cells, compared to in the presence of live C. albicans CAB 392 cells. Bars represent the mean of four repeats and whiskers indicate standard error. Dashed lines depict the initial cell concentration of log 6 cells/ml. Relevant statistically significant differences between bars are illustrated by horizontal lines and asterisks (*p < 0.05, **p < 0.005) as determined using one-way ANOVA, t-test, Mann-Whitney U and Fisher's LSD analyses.
4. Discussion
interactions between microorganisms strongly depends on the microbial strains [26,27]. Similar to our findings, others reported that C. albicans promoted growth of Escherichia coli and Klebsiella pneumoniae [28–30]. To explore the enhancement of Bacteroides growth by C. albicans, Bacteroides monocultures were prepared in liquid minimal media supplemented with extracted Candida mannan. Bacteroides fragilis NCTC 9343 and B. vulgatus ATCC 8482 are known to express enzymes capable of degrading the outer cell wall mannan layers of yeast cells [11,12]. Yeast mannan-degrading enzymes
The current study demonstrated that the yeast C. albicans exerted a significant positive effect on Bacteroides growth under anaerobic conditions, while B. fragilis NCTC 9343 had no notable effect on C. albicans concentrations in co-cultures. Bacteroides vulgatus ATCC 8482, however, affected yeast concentrations in a strain-specific manner and differences were noted in the growth of this bacterial species in the presence of different C. albicans strains. Based on previous studies, the nature of
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Fig. 7. Anaerobic growth at 37 °C determined for different time intervals of (A) B. fragilis NCTC 9343 and (B) B. vulgatus ATCC 8482 monocultures in ¼ BHI broth and spent medium from 24-h-old C. albicans CAB 392 monocultures compared to in co-culture with C. albicans CAB 392. Bars represent the mean of four repeats and whiskers indicate standard error. Dashed lines depict the initial cell concentration of log 6 cells/ml. Relevant statistically significant differences between bars are illustrated by horizontal lines and asterisks (*p < 0.05, **p < 0.005) as determined using one-way ANOVA and t-test analyses.
belonging to the glycoside hydrolases (GH) families GH38, GH76 and GH92 which include α-1,6-mannanase and α-mannosidases were discovered in B. fragilis NCTC 9343. While an α-1,2-mannosidase of the GH92 family is associated with B. vulgatus ATCC 8482. Also, it was shown that representatives of Bacteroides utilized mannan originating from Saccharomyces cerevisiae and C. albicans cells [13]. During our study, supplementation of Bacteroides monocultures in ¼ BHI broth with dead C. albicans CAB 392 cells, containing intact outer cell wall mannan layers as determined using fluorescence microscopy, stimulated bacterial growth. The finding supported the contention that yeast cells may serve as an additional nutrient source for the bacteria. It was
therefore hypothesized that the representatives of Bacteroides used in our study may utilize Candida mannan as a nutrient source. Upon testing this hypothesis, we found that B. fragilis NCTC 9343 survived better in the liquid minimal medium with mannose as a sole carbon source, compared to in the medium supplemented with both mannose and mannan. This phenomenon could have been the result of complexation between non-utilized mannan and essential ions needed for growth of this particular strain, since it is known that metal ions can adhere to the yeast cell wall (including the mannan layer) [31]. The addition of mannan to the mannose-containing minimal medium, however, enhanced survival of B. vulgatus ATCC 8482 under the
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Fig. 8. Percentage survival of (A) B. fragilis NCTC 9343 and (B) B. vulgatus ATCC 8482 in ¼ BHI broth compared to in C. albicans CAB 392 spent medium when treated with 512 or 32 μg/ml chloramphenicol, respectively. Bars represent the mean of three repeats and whiskers indicate standard error. Statistically significant differences between bars are illustrated by horizontal lines and asterisks (*p < 0.05) as determined using one-way ANOVA analyses.
conditions tested in this study. Our findings were similar to that of Cuskin et al. [13], who showed that B. vulgatus, not B. fragilis, was capable of utilizing mannan from S. cerevisiae cells as a carbon source. It seemed that while yeast mannan may have contributed to higher cell concentrations of B. vulgatus ATCC 8482 in co-culture with live and media supplemented with dead C. albicans CAB 392 cells, this complex polymer did not contribute to the enhanced growth of B. fragilis NCTC 9343. Further investigation revealed that the metabolism of C. albicans also played a role during this binary interaction. It was found that during co-cultivation of C. albicans and the obligate anaerobe, Clostridium difficile, aerobic respiration or antioxidant
production by the yeast may decrease oxygen to a level that can allow bacterial proliferation [32]. During our study, supplementation of the ¼ BHI broth with the reducing agent L-cysteine hydrochloride resulted in enhanced growth of Bacteroides in monocultures after 24 h, compared to bacterial growth in monocultures without the reducing agent. The initial decline in Bacteroides numbers in monocultures, prepared with the latter medium, may thus have been the result of available oxygen. On the other hand, growth of the anaerobic Bacteroides species in coculture with live C. albicans cells may have been enhanced in ¼ BHI broth of which the oxygen levels were lowered by yeast metabolism. Interestingly, a decrease in the cell concentrations of B. fragilis NCTC
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9343 was observed after 48 h within the pre-reduced ¼ BHI broth, but supplementation of the medium with dead C. albicans CAB 392 cells resulted in more bacterial growth after the same incubation period. Providing further evidence that C. albicans cells may serve as an additional nutrient source for Bacteroides. The presence of live C. albicans CAB 392 cells in the medium led to more B. fragilis NCTC 9343 growth, compared to growth of this bacterium either in pre-reduced ¼ BHI broth or pre-reduced ¼ BHI broth with dead yeast cells. This indicated that direct contact or multiple factors are simultaneously needed for the stimulatory effect exerted by Candida on bacterial growth. Researchers suggested that interactions between microorganisms can be contact-dependent [33,34]. In contrast, B. vulgatus ATCC 8482 grew better in ¼ BHI broth with reduced oxygen levels than in the presence of live C. albicans CAB 392 cells. This suggested that anaerobic conditions are essential for growth of B. vulgatus ATCC 8482 and that the metabolism of C. albicans is unable to remove sufficient quantities of oxygen from the medium in order to support anaerobic bacterial growth. Additionally, this bacterium showed more growth in the spent medium of C. albicans CAB 392 than in freshly prepared ¼ BHI broth. Consequently, it seemed that although C. albicans may overall positively influence B. vulgatus, this effect may be counteracted by contact-dependent negative factors that were absent in the spent medium. The spent medium of Candida monocultures significantly promoted Bacteroides growth. The metabolite(s) involved, which could either be produced intracellularly and exported to the extracellular environment or a product of membrane-associated enzymes, need to be identified in future studies. Other authors demonstrated that certain molecules secreted by C. albicans affect bacterial growth. For example, farnesol secreted by C. albicans suppressed Acinetobacter baumannii growth [34]. We also found that the spent medium of C. albicans CAB 392 increased the susceptibility of Bacteroides to chloramphenicol. Secreted metabolites of Candida may be acting synergistically with the antibiotic. On the other hand, active growth and concomitant protein synthesis by Bacteroides cells in the spent medium may increase the susceptibility of the
bacterial cells to chloramphenicol, which is a known inhibitor of protein synthesis [35]. In conclusion, cell concentrations of C. albicans (specifically C. albicans CAB 201, C. albicans CAB 392 and C. albicans CAB 1085) were unaffected by the presence of B. fragilis NCTC 9343 or B. vulgatus ATCC 8482, while Bacteroides growth was significantly enhanced in co-culture with C. albicans. The interaction between C. albicans and Bacteroides species are complex with multiple factors contributing to the increased growth of Bacteroides in the presence of C. albicans. Strain differences were not apparent in co-cultures with B. fragilis NCTC 9343 and C. albicans. In contrast, strain-specific interactions were noted during cocultivation of B. vulgatus ATCC 8482 and C. albicans. The interaction between B. fragilis NCTC 9343 and C. albicans seemed to be contactdependent, while the interaction between B. vulgatus ATCC 8482 and C. albicans is likely governed by non-contact-dependent mechanisms. Furthermore, the nature of these interactions may be dictated by environmental conditions. For example, in the hypoxic regions of the gastrointestinal tract [36], aerobic respiration and/or antioxidant production by C. albicans may largely contribute to greater Bacteroides growth. However, the cells of C. albicans may serve as an additional nutrient source for the bacteria in anaerobic regions of the gut. It was also found that Bacteroides growth and susceptibility to the antibiotic chloramphenicol increased in the presence of C. albicans supernatant. It is clear that an interaction between Bacteroides species and C. albicans may exist in the human body. Therefore, it is imperative to study Bacteroides and C. albicans within dual-species biofilms and the effect of these microorganisms on each other's virulence. In future, the implications thereof on human health need to be investigated in vivo. Declarations of interest None.
Fig. A.1. Anaerobic growth of Bacteroides at 37 °C determined for different time intervals in liquid minimal medium with glucose [0.5% (w/v)] as a carbon source. BF, B. fragilis NCTC 9343 cell concentrations; BV, B. vulgatus ATCC 8482 cell concentrations. Bars represent the mean of four repeats and whiskers indicate standard error. The dashed line depicts the initial cell concentration of log 6 cells/ml.
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Acknowledgements Funding: This work was supported by the National Research Foundation (NRF) of South Africa.
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