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Keywords:

  • Candida ;
  • bacteria;
  • biofilms;
  • artificial urine;
  • catheters

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Scanning electron microscopy for MSBs and DSBs of C. albicans with P. aeruginosa/E. coli
  6. The effect of glucose supplemented artificial urine on DSB growth of C. albicans and P. aeruginosa
  7. The effect of glucose supplemented artificial urine on DSB growth of C. albicans with E. coli
  8. MIC of bacterial LPS on C. albicans biofilms
  9. The relative efficacy of live bacteria (P. aeruginosa, E. coli) vs their commercially prepared LPS on the inhibition of C. albicans biofilm growth
  10. Discussion
  11. Conclusions
  12. References
  13. Supporting Information

Mixed Candida-bacterial biofilms in urinary catheters are common in hospitalized patients. (i) The aims of this study were to evaluate, quantitatively and qualitatively, the in vitro development of mono- and dual-species biofilms (MSBs and DSBs) of Candida albicans and two enteric gram-negative bacilli (EGNB; Pseudomonas aeruginosa or Escherichia coli) on Foley catheter (FC) discs, (ii) to determine the biofilm growth in tryptic soy broth or glucose supplemented artificial urine (AU) and (iii) to assess the inhibitory effects of EGNB and their lipopolysaccharides (LPS) on Candida biofilm growth. The growth of MSBs and DSBs on FC discs was monitored by cell counts and SEM. The metabolic activity of LPS-treated Candida biofilms was determined by the XTT reduction assay. Candida albicans and EGNB demonstrated significant inter- and intra-species differences in biofilm growth on FC discs (p < 0.01). Pseudomonas aeruginosa suppressed Candida albicans significantly (p < 0.001) in DSBs. Compared with MSBs, DSB of EGNB in glucose supplemented AU demonstrated robust growth. Escherichia coli and its LPS, significantly suppressed Candida biofilm growth, compared with Pseudomonas aeruginosa and its LPS (p < 0.001). Candida albicans and EGNB colonization in FC is significantly increased in AU with glucose, and variably modified by Escherichia coli, Pseudomonas aeruginosa and their corresponding LPS.

Urinary catheters are essential devices used in the management of critically ill patients. However, secondary infections associated with such catheter-related organisms are thought to precipitate systemic disease that account for an estimated 25–40% of nosocomial infections [1]. Some of the predominant pathogens which invade these devices and develop biofilms include enteric gram-negative bacilli such as Pseudomonas aeruginosa and Escherichia coli, and Klebsiella species, as well as the yeast species particularly belonging to the genus Candida [1, 2].

The pathogenesis of catheter-related urinary tract infections (UTIs) has been characterized to some extent [3, 4]. Once implanted, urinary catheters rapidly acquire a biofilm comprising one or more opportunistic uropathogens embedded in an extracellular matrix, either derived from their own metabolic products and/or from body fluids [4, 5]. In the case of the opportunistic yeast, Candida albicans, they attach to the catheter surface and grow into a multi-layered mass encapsulated in an exopolymer matrix [6], frequently co-infested with enteric gram-negative bacilli [7]. The cohabitation of C. albicans, P. aeruginosa, and E. coli in many biological fluids of human origin is well documented [8]. It is also thought that the sequestrated polymicrobial biofilm on urinary catheters provides a protective environment for the constituent microorganisms to evade the activity of antimicrobial agents [9].

There is clear evidence that bacterial species cohabit in intraluminal biofilms of urinary catheters and trigger and perpetuate urinary tract infections [10]. It has also been reported that such increased propensity for catheter colonization may be promoted by glucosuria, such as in patients with uncontrolled diabetes mellitus [2, 11, 12]. Geerlings et al. for instance have shown increased growth of uropathogenic bacteria in urine samples of patients with diabetes mellitus [11]. This finding combined with the fact that sugars such as sucrose and glucose promote candidal adherence to abiotic surfaces [13, 14], imply that the latter may promote and modulate biofilm formation on urinary catheter surfaces as well. However, there is little data on the colonization profiles of enteric gram-negative bacilli and/or Candida on urinary catheter surfaces particularly in a milieu laced with either urine or artificial urine (AU). The effect of glucose in such settings is also not known.

Furthermore, catheter-related urinary tract infections are notoriously resistant to antimicrobial therapy. Hence, some have suggested alternative approaches such as microbial antagonism as a solution to this problem [15]. We have previously demonstrated, for instance, the growth suppression of Candida biofilms on polystyrene surfaces by lipopolysaccharides (LPS) of a number of gram-negative bacteria [16, 17]. Yet, similar investigations with either LPS or dietary sugars have not been conducted to evaluate candidal biofilm growth on urinary catheter material. In particular, the dose-response effect of P. aeruginosa, E. coli, or their LPS on the in vitro biofilm growth of C. albicans on such catheter material is not known. Hence, we evaluated the antagonistic interactions of cohabiting Candida and enteric bacilli, and their LPS, on Foley Catheter (FC) biofilms in a milieu of AU. Clinical extrapolation of our findings may, in the longer term, assist the management of patients with catheter-related UTIs.

The specific objectives of this study, therefore, were (i) to quantitatively evaluate the mixed biofilm growth of C. albicans, and either P. aeruginosa or E. coli on FC discs in comparison with their mono-species counterparts, (ii) to evaluate the resultant biofilm architecture using scanning electron microscopy, (iii) to compare the effect of AU with/without glucose on mixed biofilm growth of C. albicans with either P. aeruginosa or E. coli, on FC discs, (iv) to assess the minimum inhibitory concentration (MIC) of P. aeruginosa or E. coli LPS on Candida biofilm viability and (v) to compare the susceptibility of C. albicans biofilms to live P. aeruginosa or E. coli or their corresponding commercially prepared LPS counterparts.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Scanning electron microscopy for MSBs and DSBs of C. albicans with P. aeruginosa/E. coli
  6. The effect of glucose supplemented artificial urine on DSB growth of C. albicans and P. aeruginosa
  7. The effect of glucose supplemented artificial urine on DSB growth of C. albicans with E. coli
  8. MIC of bacterial LPS on C. albicans biofilms
  9. The relative efficacy of live bacteria (P. aeruginosa, E. coli) vs their commercially prepared LPS on the inhibition of C. albicans biofilm growth
  10. Discussion
  11. Conclusions
  12. References
  13. Supporting Information

Microorganisms

Ten clinical isolates each of P. aeruginosa, E. coli and C. albicans were selected from the stock cultures in the laboratories of Oral Bio-sciences, Prince Phillip Dental Hospital, University of Hong Kong. The origins of these microorganisms are provided as supplementary material (supplementary Table 1). The identity of each organism was confirmed with API 32 C (for Candida) and API 20 E (for P. aeruginosa and E. coli) identification systems (bioMérieux, Marcy I'Etoile, France). All fungal and bacterial isolates were stored at 4°C on Sabouraud's dextrose agar and Blood agar (SDA and BA; Oxoid Ltd., Hampshire, UK) plates respectively. At the time of the study, isolates were subcultured onto either fresh SDA or BA plates and were checked for purity. The best biofilm-producing isolate from each species was selected for further experiments as described below.

Growth media

Yeast nitrogen base (YNB; Difco, Sparks, MD, USA) solution supplemented with 100 mM glucose was used for culturing Candida species while, BA, MacConkey agar (MCA; Oxoid Ltd., Hampshire, UK) and Tryptic soy broth (TSB; Difco Laboratories, Detroit, MI, USA) were utilized for P. aeruginosa and E. coli culture.

Preparation of the standard microbial inocula

Candida albicans, P. aeruginosa and E. coli were subcultured on SDA and BA, respectively, for 18 h at 37°C. A loopful of the overnight Candida growth was inoculated into YNB medium, P. aeruginosa and E. coli inoculated into TSB medium and each incubated for 18 h in an orbital shaker (75 rpm) at 37°C. The resulting cells were harvested, washed twice in phosphate-buffered saline (PBS, pH 7.2, Ca2+ and Mg2+ free; Sigma; St. Lois, MQ, USA), and resuspended in PBS. Concentrations of C. albicans, P. aeruginosa and E. coli were adjusted to 107 cells/mL by spectrophotometry and confirmed by haemocytometric counting.

Preparation and sterilization of catheter discs

A Foley catheter (FC; 100% Silicone Foley Catheter; Mansfield, Mexico) was used to obtain FC discs of 0.5 cm diameter that was then sterilized by autoclaving at 121°C/15 psi for 45 min.

Preparation of artificial urine (AU)

Artificial urine was prepared as described by Chutipongtanate and Thongboonkerd [18].

Experimental design

Mono-species biofilm (MSB) development on FC discs

MSBs of P. aeruginosa, E. coli and C. albcians were developed on FC discs as described by Bandara et al. [16] with slight modifications. As has been described earlier, 200 μLs of standard cell suspensions of C. albicans, P. aeruginosa, or E. coli (107 cells/mL) were transferred into selected wells of a flat-bottomed 96-well microtitre plate (IWAKI, Tokyo, Japan). FC discs were immersed in the microbial suspension and incubated for 90 min at 37°C in an orbital shaker at 75 rpm to promote microbial adherence and thereafter washed twice with PBS, and re-incubated in TSB medium at 37°C for 48 h. The FC discs with biofilms were then washed with PBS, transferred to 1 mL of PBS in eppendorf tubes and vortexed at 150 rpm for 30 s to disperse the biofilm cells. The fungal and bacterial growth was evaluated by the spiral plater method on SDA and MCA agar plates at 90 min, 24 h, and 48 h, and qualitatively with SEM.

DSBs of C. albicans with either P. aeruginosa or E. coli on FC discs

DSBs of C. albicans with either P. aeruginosa or E. coli were developed on FC discs as described in earlier studies [16]. The MSB and DSB growth at 90 min, 24 h, and 48 h was quantified by colony forming units (cfus) on SDA (for yeast) and on TSB supplemented with 1 mg/mL chloramphenicol (for bacteria) by the spiral plater method.

The MSB and DSB development was also assessed qualitatively with SEM at the three different time points.

MSB and DSB development of C. albicans with either P. aeruginosa or E. coli on FC discs with glucose supplemented artificial urine

The effect of glucose supplemented artificial urine (AU) on MSB and DSB development of C. albicans with either P. aeruginosa or E. coli was investigated using a protocol similar to the above described method. However, the organisms were cultured in AU supplemented with varying concentrations of glucose; that is, 50 mM, 100 mM, 150 mM and 200 mM. The control AU suspensions were devoid of glucose. After the adhesion phase, AU suspensions were aspirated and MSBs of C. albicans, P. aeruginosa, E. coli, and their respective DSB's, were developed in fresh samples of AU supplemented with glucose and the appropriate controls as described above.

Preparation of LPS

LPS purified from P. aeruginosa and E. coli (1 mg/mL, lyophilized powder, Sigma; Aldrich St. Louis, MQ, USA), and YNB supplemented with 100 mM glucose were mixed to yield a final concentration of 100 μg/mL LPS in the medium of the test sample. LPS was replaced with sterile PBS in the control.

Determination of minimum inhibitory concentration (MIC) of bacterial LPS

MICs of commercially prepared LPS obtained from E. coli and P. aeruginosa against the clinical C. albicans isolate used in the above studies were determined by a broth microdilution assay in accordance with the CLSI guidelines [19]. Briefly, fungal cell suspensions (103 cells/mL) were incubated with LPS (1 μL) and RPMI 1640 (99 μL) supplemented with 0.165 M 3-(N-morpholino) propanesulphonic acid. Lipopolysaccharide was tested within a concentration range of 50–300 μg/mL for both E. coli and P. aeruginosa isolates. After 24 h of incubation at 35°C in a moist, dark, orbital shaker, fungal growth was measured using spectrophotometry [19].

Comparative effect of live bacteria (P. aeruginosa and E. coli) and the respective commercially prepared LPS on the inhibition of C. albicans biofilm growth on Foley catheter discs

Biofilms of C. albcans and bacteria were developed on FC discs immersed in TSB medium as described above with the following conditions: (i) control; 200 μLs of C. albicans (107 cells/mL), (ii) test 1; 100 μLs of each of C. albicans and P. aeruginosa (107 cells/mL, 1:1 ratio), (iii) test 2; 200 μLs of C. albicans supplemented with 200 μg/mL P. aeruginosa LPS (P. aeruginosa LPS, MIC = 200 μg/mL), (iv) test 3; 100 μLs of each of C. albicans and E. coli (107 cells/mL, 1:1 ratio), (v) test 4; 200 μLs of C. albicans supplemented with 150 μg/mL, E. coli LPS (E. coli LPS, MIC = 150 μg/mL).

FC discs immersed in C. albicans suspensions with live bacteria or their respective LPS were incubated at 37°C in an orbital shaker at 75 rpm for 90 min. After the adhesion phase, each FC disc was washed twice with PBS and incubated in TSB with standard bacterial suspensions, of P. aeruginosa LPS (200 μg/mL) or E. coli LPS (150 μg/mL), respectively, for a 7 h period. All the foregoing assays were performed on three different occasions using triplicate samples.

Quantitative analyses

Spiral plating and colony forming units assay

The resulting colony forming units of microbial growth in MSB and DSB on each FC disc was vortexed, serially diluted, and inoculated onto SDA supplemented with chloramphenicol and MCA agar plates by the spiral plater method and quantified after 48 h incubation at 37°C.

All the foregoing assays were performed on three different occasions using triplicate samples.

Scanning electron microscopy

MSBs of C. albicans, P. aeruginosa, E. coli, and their DSBs, on FC discs were prepared as described above. The morphotypes and phenotypes of C. albicans, P. aeruginosa and E. coli biofilms at various stages, and under varying growth conditions, were visualized with a scanning electron microscope (Philip XL30CP) in high-vacuum mode at 10 kV, and the images processed as documented by Bandara et al. [16].

Statistical analysis

Statistical analysis was performed using SPSS software (version 16.0). Mann–Whitney U test was performed to compare the significant differences between control and each test sample of the bacterial/candidal biofilm. Data obtained from C. albicans and the two bacterial species at different time points were pooled, and evaluated using Wilcoxon matched-pairs test. A p-value of <0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Scanning electron microscopy for MSBs and DSBs of C. albicans with P. aeruginosa/E. coli
  6. The effect of glucose supplemented artificial urine on DSB growth of C. albicans and P. aeruginosa
  7. The effect of glucose supplemented artificial urine on DSB growth of C. albicans with E. coli
  8. MIC of bacterial LPS on C. albicans biofilms
  9. The relative efficacy of live bacteria (P. aeruginosa, E. coli) vs their commercially prepared LPS on the inhibition of C. albicans biofilm growth
  10. Discussion
  11. Conclusions
  12. References
  13. Supporting Information

MSB growth of C. albicans, P. aeruginosa, and E. coli on FC discs

Intra-strain differences in MSB growth on FC discs were observed for all 10 bacterial isolates of P. aeruginosa (p < 0.001), E. coli (p < 0.02), and for the 10 C. albicans isolates (p < 0.001) (Fig. 1). A similar, significant intra-species difference in biofilm growth was also observed between P. aeruginosa and E. coli (p < 0.001).

image

Figure 1. Mono-species biofilms (MSB) growth of Candida albicans, Pseudomonas aeruginosa and Escherichia coli on foley catheter FC discs immersed in tryptic soy broth (TSB) medium (each dot represents one clinical isolate).

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DSB growth of C. albicans and P. aeruginosa/E. coli on FC discs

DSBs of C. albicans/P. aeruginosa on FC discs

In DSBs of Ca/Pa it was noted that Candida biofilms were significantly (p < 0.001) inhibited by P. aeruginosa. The mean% reduction in Candida biofilm growth was 95.8% at the 90 min adhesion phase (Table 1). The co-culture of the two microbial species clearly demonstrated the antagonistic effect of P. aeruginosa on candidal biofilm growth, which almost completely eradicated (99.9%) of the yeast community in the 48 h mixed biofilm (Table 1; Fig. 2). We also noted a concomitant significant reduction in bacterial growth at 90 min (p < 0.001; 52.3%) and at 48 h (p < 0.001; 38.85), whereas no such significant decline in bacterial cell viability was observed at 24 h (Table 1).

Table 1. The viable cfu's ± SD of mono-species biofilms (MSBs) and dual-species biofilms (DSBs) of Candida albicans and Pseudomonas aeruginosa formed in tryptic soy broth (TSB) medium on foley catheter (FC) discs at (i) 90 min, (ii) 24 h, and (iii) 48 h
  C. albicans P. aeruginosa  C. albicans and P. aeruginosa
MeanSDMeanSDMeanSDp-value
(i)90 min2.20 × 1053.36 × 1041.91 × 1066.86 × 105 C. albicans 9.20 × 1032.20 × 103<0.0001
P. aeruginosa 9.11 × 1052.47 × 1050.001
(ii)24 h1.39 × 1062.64 × 1056.58 × 1071.19 × 107 C. albicans 7.07 × 1032.98 × 103<0.0001
P. aeruginosa 5.89 × 1079.17 × 1060.188
(iii)48 h4.76 × 1069.89 × 1058.44 × 1071.67 × 107 C. albicans 4.24 × 1031.01 × 103<0.0001
P. aeruginosa 5.17 × 1071.92 × 1070.001
image

Figure 2. SEM images of Candida albicans SC5314 and Pseudomonas aeruginosa biofilms formed in tryptic soy broth (TSB) medium on foley catheter (FC) discs, (A) Mono-species biofilms (MSB) of C. albicans at 90 min, 24 h and 48 h, (B) Dual-species biofilms (DSBs) of C. albicans and Pseudomonas aeruginosa at 90 min, 24 h, and 48 h, (C) Mono-species biofilms (MSB) of P. aeruginosa at 90 min, 24 h, and 48 h.

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DSBs of C. albicans/E. coli on FC discs

In general, Candida biofilms were significantly suppressed in the Ca/Ec dual-species biofilms (p < 0.001). The mean% reduction in Candida biofilm growth was 95.0, 96.4, and 97.7% at 90 min, 24 h and 48 h, respectively, compared with that of control Candida biofilms (Table 2). In contrast, E. coli growth was noted to increase significantly at all three time points; at 24 h (p < 0.001) and particularly at 48 h (p < 0.0001) compared with that of the single species E. coli control counterpart. Bacterial biofilm mass increased after 48 h of continuous growth compared with the yeast growth. These results imply that the E. coli were antagonistic to C. albicans biofilm growth, akin to that of P. aeruginosa. The degree of growth suppression of C. albicans by P. aeruginosa was higher at 90 min, compared with that of E. coli (52.3% vs 38.8%), whereas the percentage reduction of growth was lower for P. aeruginosa when compared with that of E. coli at 24 h (10.5% vs 59.2%) and 48 h (38.8% vs 44.0%) time points.

Table 2. The viable cfu's ± SD of mono-species biofilms (MSBs) and dual-species biofilms (DSBs) of Candida albicans and Escherichia coli formed in tryptic soy broth (TSB) medium on foley catheter (FC) discs at (i) 90 min, (ii) 24 h, and (iii) 48 h
  C. albicans E. coli  C. albicans and E. coli
MeanSDMeanSDMeanSDp-value
(i)90 min2.20 × 1053.36 × 1041.78 × 1065.95 × 105 C. albicans 1.10 × 1042.27 × 103<0.0001
E. coli 1.09 × 1064.91 × 1050.016
(ii)24 h1.39 × 1062.64 × 1053.38 × 1071.41 × 107 C. albicans 4.95 × 1042.50 × 104<0.0001
E. coli 1.38 × 1073.67 × 1060.001
(iii)48 h4.76 × 1069.89 × 1054.29 × 1079.39 × 106 C. albicans 1.10 × 1052.02 × 104<0.0001
E. coli 2.40 × 1077.68 × 106<0.0001

Scanning electron microscopy for MSBs and DSBs of C. albicans with P. aeruginosa/E. coli

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Scanning electron microscopy for MSBs and DSBs of C. albicans with P. aeruginosa/E. coli
  6. The effect of glucose supplemented artificial urine on DSB growth of C. albicans and P. aeruginosa
  7. The effect of glucose supplemented artificial urine on DSB growth of C. albicans with E. coli
  8. MIC of bacterial LPS on C. albicans biofilms
  9. The relative efficacy of live bacteria (P. aeruginosa, E. coli) vs their commercially prepared LPS on the inhibition of C. albicans biofilm growth
  10. Discussion
  11. Conclusions
  12. References
  13. Supporting Information

C. albicans/P. aeruginosa

MSBs of C. albicans initially formed small, discrete, and scanty microcolonies on the FC disc during the (90 min) adhesive phase and later developed into a thin base film at the end of 24 h (Fig. 2A) as opposed to a robust biofilm growth of P. aeruginosa (Fig. 2C). Both Candida and P. aeruginosa subsequently grew in thickness and fully formed MSBs were observed at 48 h (Figs 2A and 2C). The growth suppressive effect of P. aeruginosa on Candida throughout the adhesion and biofilm phase of growth resulted in a skeletal layer of blastospores on the catheter surface at 48 h (Fig. 2B), whereas P. aeruginosa showed a thick and a confluent biofilm at this stage. Most interestingly, this result was consistent across several other combinations of C. albicans and P. aeruginosa (Ca/Pa) biofilms we tested in our laboratories (results not shown).

C. albicans/E. coli

Unlike P. aeruginosa, E. coli showed extremely scanty adhesion on FC discs at 90 min (Fig. 3C). In mixed biofilms at the initial adhesion stage, Candida blastospores appeared to develop germ tubes rather than hyphae, which were not observed in the Ca/Pa biofilms described above. In DSBs, E. coli adhered to both C. albicans filaments as well as the blastospores, some of which appeared to be dead or dying with deformed and crenated cell surfaces even at 24 h (Fig. 3B). In the control MSBs, both Candida blastospore and hyphal growth was seen on the FC disc at 24 h and at 48 h time points (Fig. 3A). In particular, E. coli MSBs showed good growth at 24 h and relatively robust growth at 48 h (Fig. 3C).

image

Figure 3. SEM images of Candida albicans SC5314 and Escherichia coli biofilms formed in tryptic soy broth (TSB) medium on foley catheter (FC) discs, (A) Mono-species biofilms (MSB) of C. albicans at 90 min, 24 h and 48 h, (B) Dual-species biofilms (DSBs) of C. albicans and E. coli at 90 min, 24 h and 48 h, (C) MSB of E. coli at 90 min, 24 h, and 48 h.

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The effect of glucose supplemented artificial urine on DSB growth of C. albicans and P. aeruginosa

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Scanning electron microscopy for MSBs and DSBs of C. albicans with P. aeruginosa/E. coli
  6. The effect of glucose supplemented artificial urine on DSB growth of C. albicans and P. aeruginosa
  7. The effect of glucose supplemented artificial urine on DSB growth of C. albicans with E. coli
  8. MIC of bacterial LPS on C. albicans biofilms
  9. The relative efficacy of live bacteria (P. aeruginosa, E. coli) vs their commercially prepared LPS on the inhibition of C. albicans biofilm growth
  10. Discussion
  11. Conclusions
  12. References
  13. Supporting Information

In MSBs, there was no significant difference in the adherence of C. albicans to FC discs between glucose supplemented AU and control during the initial 90 min (Table 3). However, a significantly high (p < 0.001) biofilm mass was observed for C. albicans at both 24 h and 48 h in glucose supplemented AU compared to control AU samples. In contrast, in the absence of glucose, MSBs of P. aeruginosa demonstrated a significantly robust biofilm growth both at 90 min and at 48 h (p < 0.001), but not at 24 h where the test and control samples showed similar growth.

Table 3. The viable cfu's ± SD of mono-species biofilms (MSBs) and dual-species biofilms (DSBs) of Candida albicans and Pseudomonas aeruginosa formed in artificial urine (AU) supplemented with 6.7 mM glucose on foley catheter (FC) discs at (i) 90 min, (ii) 24 h, and (iii) 48 h
  C. albicans P. aeruginosa  C. albicans and P. aeruginosa
MeanSDMeanSDMeanSDp-value
(i)90 minAU without glu1.46 × 1043.13 × 1031.34 × 1062.23 × 105 C. albicans 1.48 × 1041.52 × 1030.821
AU with 16.7 mM glu1.62 × 1041.63 × 1037.56 × 1051.69 × 1051.11 × 1042.22 × 103<0.0001
      P. aeruginosa 1.78 × 1061.14 × 1060.268
     1.28 × 1062.25 × 105<0.0001
(ii)24 hAU without glu5.56 × 1031.24 × 1031.38 × 1071.90 × 106 C. albicans 4.58 × 1029.19 × 101<0.0001
AU with 16.7 mM glu1.56 × 1042.86 × 1031.39 × 1071.64 × 1061.65 × 1032.90 × 103<0.0001
      P. aeruginosa 1.19 × 1073.28 × 1060.160
     6.87 × 1065.36 × 105<0.0001
(iii)48 hAU without glu5.84 × 1039.79 × 1021.64 × 1072.96 × 106 C. albicans 1.17 × 1032.74 × 102<0.0001
AU with 16.7 mM glu1.05 × 1041.70 × 1033.78 × 1061.20 × 1064.87 × 1031.32 × 103<0.0001
      P. aeruginosa 1.82 × 1077.45 × 1060.515
     1.33 × 1074.24 × 106<0.0001

In the presence of glucose, the growth of C. albicans in DSBs on FC discs showed a significant difference (p < 0.001) in yeast growth at all three time points between the control and the test sample. However, C. albicans biofilm growth in DSBs in glucose supplemented AU was lower than its MSB counterpart at all three time points, (90 min, 24 h, and 48 h). In contrast, P. aeruginosa growth in DSBs in sugar supplemented AU was much more profuse during the three time points, than its MSB counterpart. However, compared to AU devoid of glucose, a significant difference in biofilm growth (p < 0.001), was only noted at 24 h, and not at the end of either 90 min (adhesion phase) or at 48 h (Table 3). These results clearly indicate that C. albicans biofilm growth is suppressed in the presence of P. aeruginosa.

The effect of glucose supplemented artificial urine on DSB growth of C. albicans with E. coli

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Scanning electron microscopy for MSBs and DSBs of C. albicans with P. aeruginosa/E. coli
  6. The effect of glucose supplemented artificial urine on DSB growth of C. albicans and P. aeruginosa
  7. The effect of glucose supplemented artificial urine on DSB growth of C. albicans with E. coli
  8. MIC of bacterial LPS on C. albicans biofilms
  9. The relative efficacy of live bacteria (P. aeruginosa, E. coli) vs their commercially prepared LPS on the inhibition of C. albicans biofilm growth
  10. Discussion
  11. Conclusions
  12. References
  13. Supporting Information

As with MSBs of C. albicans described above, E. coli did not demonstrate a significant difference in adherence to FC catheter surfaces when the control and glucose-laced samples were compared at 90 min (Table 4).

Table 4. The viable cfu's ± SD of mono-species biofilms (MSBs) and dual-species biofilms (DSBs) of Candida albicans and Escherichia coli formed in artificial urine (AU) supplemented with 6.7 mM glucose on foley catheter (FC) discs at (i) 90 min, (ii) 24 h and (iii) 48 h
  C. albicans E. coli  C. albicans and E. coli
MeanSDMeanSDMeanSDp-value
(i)90 minAU without glu1.46 × 1043.13 × 1039.22 × 1051.63 × 105 C. albicans 6.62 × 1038.57 × 102<0.0001
AU with 16.7 mM glu1.62 × 1041.63 × 1039.62 × 1051.62 × 1054.82 × 1031.39 × 103<0.0001
      E. coli 5.64 × 1051.55 × 105<0.0001
     6.58 × 1051.28 × 105<0.0001
(ii)24 hAU without glu5.56 × 1031.24 × 1031.09 × 1063.18 × 105 C. albicans 3.67 × 1028.06 × 101<0.0001
AU with 16.7 mM glu1.56 × 1042.86 × 1034.60 × 1065.83 × 1054.40 × 1031.42 × 103<0.0001
      E. coli 3.56 × 1051.33 × 105<0.0001
     4.87 × 1061.44 × 1060.613
(iii)48 hAU without glu5.84 × 1039.79 × 1022.17 × 1067.81 × 105 C. albicans 7.93 × 1021.68 × 102<0.0001
AU with 16.7 mM glu1.05 × 1041.70 × 1032.87 × 1074.80 × 1065.09 × 1041.72 × 104<0.0001
      E. coli 2.33 × 1067.07 × 1050.641
     6.00 × 1061.73 × 106<0.0001

In DSBs, C. albicans demonstrated no significant biofilm growth between the control and glucose supplemented AU during the adhesion phase of 90 min (Table 4). Similarly, in E. coli and Candida DSBs, the latter did not show a significant difference in adhesion when the control and glucose supplemented AU samples were compared. Remarkably, in DSBs, C. albicans biofilm growth was significantly less than its MSB counterpart (p < 0.005), whereas E. coli biofilm growth was more profuse in the presence of glucose at both 24 h and 48 h (p < 0.001) (Fig. 4). These results imply that E. coli has an inhibitory effect on C. albicans biofilm growth on FC discs either in the presence or absence of glucose (Table 4).

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Figure 4. SEM images of Candida albicans SC5314 and Escherichia coli biofilms on FC discs in artificial urine (AU), (A) Mono-species biofilms (MSB) of E. coli at 90 min, 24 h and 48 h, (B) Dual-species biofilms (DSBs) of C. albicans and E. coli at 90 min, 24 h and 48 h, (C) Mono-species biofilms (MSB) of E. coli in AU with 16.7 mM glucose at 90 min, 24 h and 48 h, (D) Dual-species biofilms (DSBs) of E. coli and C. albicans in AU with 16.7 mM glucose at 90 min, 24 h, and 48 h.

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MIC of bacterial LPS on C. albicans biofilms

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Scanning electron microscopy for MSBs and DSBs of C. albicans with P. aeruginosa/E. coli
  6. The effect of glucose supplemented artificial urine on DSB growth of C. albicans and P. aeruginosa
  7. The effect of glucose supplemented artificial urine on DSB growth of C. albicans with E. coli
  8. MIC of bacterial LPS on C. albicans biofilms
  9. The relative efficacy of live bacteria (P. aeruginosa, E. coli) vs their commercially prepared LPS on the inhibition of C. albicans biofilm growth
  10. Discussion
  11. Conclusions
  12. References
  13. Supporting Information

The MIC assay of P. aeruginosa and E. coli LPS against Candida biofilms revealed that LPS caused a dose-dependent loss of yeast cell viability. The minimum inhibitory concentrations (MICs) of E. coli and P. aeruginosa LPS that inhibited 48 h C. albicans biofilms were 150 μg/mL and 200 μg/mL respectively (Fig. 5).

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Figure 5. The minimum inhibitory concentrations of Escherichia coli and Pseudomonas aeruginosa lipopolysaccharides (LPS) that inhibit 48 h Candida albicans biofilms on foley catheter (FC) discs.

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The relative efficacy of live bacteria (P. aeruginosa, E. coli) vs their commercially prepared LPS on the inhibition of C. albicans biofilm growth

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Scanning electron microscopy for MSBs and DSBs of C. albicans with P. aeruginosa/E. coli
  6. The effect of glucose supplemented artificial urine on DSB growth of C. albicans and P. aeruginosa
  7. The effect of glucose supplemented artificial urine on DSB growth of C. albicans with E. coli
  8. MIC of bacterial LPS on C. albicans biofilms
  9. The relative efficacy of live bacteria (P. aeruginosa, E. coli) vs their commercially prepared LPS on the inhibition of C. albicans biofilm growth
  10. Discussion
  11. Conclusions
  12. References
  13. Supporting Information

A significant reduction in C. albicans biofilm growth on the FC disc was observed in the presence of either 107 cells/mL P. aeruginosa (p < 0.005) or E. coli (p < 0.001) after 7 h of incubation. However, no significant difference (p < 0.05) in the reduction in biofilm growth was seen for these two bacterial species. A concentration of 200 μg/mL of P. aeruginosa LPS (MIC of P. aeruginosa) was also significantly (p < 0.001) effective in impeding Candida biofilm growth compared to 150 μg/mL E.coli LPS (MIC of E. coli), which did not suppress C. albicans biofilm growth. These results indicate that live E. coli were significantly more effective in inhibiting C. albicans biofilms during a 7 h incubation period compared to its LPS (Fig. 6).

image

Figure 6. The effect of live bacteria (Pseudomonas aeruginosa and Escherichia coli) and their corresponding lipopolysaccharides (LPS) on the inhibition of a 7 h Candida albicans biofilm on foley catheter (FC) discs.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Scanning electron microscopy for MSBs and DSBs of C. albicans with P. aeruginosa/E. coli
  6. The effect of glucose supplemented artificial urine on DSB growth of C. albicans and P. aeruginosa
  7. The effect of glucose supplemented artificial urine on DSB growth of C. albicans with E. coli
  8. MIC of bacterial LPS on C. albicans biofilms
  9. The relative efficacy of live bacteria (P. aeruginosa, E. coli) vs their commercially prepared LPS on the inhibition of C. albicans biofilm growth
  10. Discussion
  11. Conclusions
  12. References
  13. Supporting Information

A majority of infections in patients using short-term indwelling urinary catheters are caused by single organisms while infections in patients requiring long-term catheters are frequently polymicrobial in nature and enteric gram-negative bacilli such as E. coli, P. aeruginosa, and Candida species are commonly isolated from such infections [20, 21]. Recent studies have shown that both in vivo and in vitro suppression of C. albicans growth by P. aeruginosa, E.coli and other bacteria [1, 15, 22, 23]. Thein et al. have reported C. albicans inhibition by P. aeruginosa [22]. The current results are similar to those of Bandara et al. [23] who showed that P. aeruginosa totally inhibited 48 h C. albicans biofilm growth on polystyrene and also concur with the findings of Trautner et al. [15], who noted that E. coli 83972 significantly inhibited C. albicans growth on catheter surfaces.

The role of glucose in urine and its effect on candidal colonization process on Foley catheter surfaces and the consequent biofilm growth is poorly understood. However, none of the previous work has determined the effect of AU and glucose on candidal biofilm development on FC discs and the inhibitory effects of live bacteria or their LPS on the formation of C. albicans biofilms. Thus, we are the first to demonstrate that the increased sugar content in AU (such as in uncontrolled diabetics) may promote candidal colonization on FC surfaces via biofilm formation.

Scanning electron microscopy

SEM of C. albicans and E. coli biofilms on FC discs in TSB medium showed a similar composition and architecture for both MSBs and DSBs and in general appeared to develop only fairly basal, yeast blastospore layers devoid of extracellular matrix and hyphal elements. We propose this to be due to the static conditions found in vitro. Indeed, a dynamic liquid flow over the surface of Candida biofilm, a condition found in vivo, has been shown to favour increased biofilm growth [6]. MSBs of C. albicans and the two bacterial strains displayed multiple layers of blastospores, pseudohyphae, and hyphal cells on AU-coated FC discs consistent with those formed by standard methods [24] that was evident in the SEMs obtained for this study.

Influence of dietary carbohydrates on biofilm growth on FC discs

There is documented evidence that dietary sugars promote increased candidal adherence and biofilm formation on different substrates including latex urinary Foley catheter material in vitro [25, 26]. In this study, we noted a significantly increased biofilm mass on FC discs at both 24 h and 48 h for Candida in glucose supplemented AU. In addition, it is also known that dietary glucose promotes other virulence characteristics of the yeast such as carboxylic acids [27], extracellular proteinases [28], and phospholipase production [29] that further enhance the virulence of the fungus.

The results of this study clearly imply that glucose in the ambient environment significantly promotes both MSB and DSB development of uropathogenic bacteria, P. aeruginosa and E. coli at both 24 h and 48 h. The foregoing observations also imply that an indwelling urinary catheter, bathed in glucose-rich urine, typical in patients with uncontrolled diabetes mellitus, may be an excellent niche for polymicrobial biofilm growth.

Bacterial lipopolysaccharides

It is known that lipopolysaccharide, an intrinsic component of the gram-negative bacterial cell wall [30], variably modulates in vitro biofilm formation of Candida species on polystyrene material in a species-specific and temporal manner [16, 17]. Therefore, biofilm growth of C. albicans was assessed following exposure of FC discs to either live P. aeruginosa, E. coli, or their LPS after a 7 h incubation period. However, there was no significant difference in biofilm growth between the live cells and its LPS. This implies that LPS per se is a powerful suppressant of Candida biofilm growth.

We also noted that in the presence of P. aeruginosa LPS (150 μg/mL), C. albicans can only proliferate as unicellular budding yeasts, but with increasing concentrations of LPS it undergoes severe morphological changes, leading to elongated growth forms, constricted hyphal elements, and dead cells. A similar result was seen with E. coli LPS although not to the same extent as P. aeruginosa. Indeed, a recently published article using E. coli 83972 strain describes the efficacy of bacterial interference as an effective method of significantly impeding colonization of urinary catheters by a variety of uropathogens including C. albicans [15, 31]. Because of the highly conducive milieu offered by FC for mixed biofilm development it is quite a challenge to suppress such biofilm formation. Therefore, the growth suppression of C. albicans due to live E. coli and commercially prepared P. aeruginosa LPS observed here might provide insights into new strategies that can be explored for the control of catheter associated urinary tract infection.

Conclusions

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Scanning electron microscopy for MSBs and DSBs of C. albicans with P. aeruginosa/E. coli
  6. The effect of glucose supplemented artificial urine on DSB growth of C. albicans and P. aeruginosa
  7. The effect of glucose supplemented artificial urine on DSB growth of C. albicans with E. coli
  8. MIC of bacterial LPS on C. albicans biofilms
  9. The relative efficacy of live bacteria (P. aeruginosa, E. coli) vs their commercially prepared LPS on the inhibition of C. albicans biofilm growth
  10. Discussion
  11. Conclusions
  12. References
  13. Supporting Information

To conclude, our data indicate that the candidal/bacterial strain type is important in the initial colonization profile and subsequent ultrastructure of mixed species biofilms on catheter surfaces. In DSBs, P. aeruginosa seems to be far superior in killing the filamentous/hyphal form of C. albicans, compared to E. coli. The inhibitory effect of live bacterial cells was replicated to a great extent by LPS of P. aeruginosa. It should be noted that the results of this study need to be extrapolated with caution, given the small number of strains investigated. Despite this, it provides interesting basic information on the behaviour of polymicrobial biofilms on Foley catheter surfaces.

The authors are very grateful for the Research Grants Council of the University of Hong Kong for supporting this study (HKU Project code: 201109176041).

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Scanning electron microscopy for MSBs and DSBs of C. albicans with P. aeruginosa/E. coli
  6. The effect of glucose supplemented artificial urine on DSB growth of C. albicans and P. aeruginosa
  7. The effect of glucose supplemented artificial urine on DSB growth of C. albicans with E. coli
  8. MIC of bacterial LPS on C. albicans biofilms
  9. The relative efficacy of live bacteria (P. aeruginosa, E. coli) vs their commercially prepared LPS on the inhibition of C. albicans biofilm growth
  10. Discussion
  11. Conclusions
  12. References
  13. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Scanning electron microscopy for MSBs and DSBs of C. albicans with P. aeruginosa/E. coli
  6. The effect of glucose supplemented artificial urine on DSB growth of C. albicans and P. aeruginosa
  7. The effect of glucose supplemented artificial urine on DSB growth of C. albicans with E. coli
  8. MIC of bacterial LPS on C. albicans biofilms
  9. The relative efficacy of live bacteria (P. aeruginosa, E. coli) vs their commercially prepared LPS on the inhibition of C. albicans biofilm growth
  10. Discussion
  11. Conclusions
  12. References
  13. Supporting Information
FilenameFormatSizeDescription
apm12098-sup-0001-TableS1.docxWord document12KTable S1. The origin of the microorganisms used in the study. Ca, Candida albicans; Pa, Pseudomonas aeruginosa; Ec, Escherichia coli; NPC, nasopharyngeal carcinoma.

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