Disruption of fungal and bacterial biofilms by lauroyl glucose

Abstract

Aim:  The ability of enzymatically synthesized lauroyl glucose to disrupt fungal (Candida albicans, Candida lipolytica) and bacterial (Pseudomonas aeruginosa PAO1, Pseudomonas aureofaciens) biofilms was investigated.

Methods and Results:  Preformed biofilms of C. albicans and C. lipolytica in polystyrene microtitre plates were disrupted upto 45% and 65%, respectively, while P. aeruginosa and P. aureofaciens biofilms were disrupted by 51% and 57%. Precoating of the microtitre wells with lauroyl glucose affected cell attachment and biofilm growth of all the cultures to a lesser extent. With C. albicans and C. lipolytica, there was 11% and 32% decrease in the development of biofilms, respectively. With P. aeruginosa and P. aureofaciens, the reduction was 21% and 12% after 48 h. Lauroyl glucose effectively inhibited the formation of biofilms on glass slide surfaces when added along with the inoculum. Analysis by confocal laser scanning microscopy showed that the growth of the biofilms was lesser as compared with the control experiments. Lauroyl glucose displayed minimum inhibitory concentration values >500 μg ml⁻¹ for the test cultures and was comparable to that obtained with acetyl salicylate.

Conclusion:  Lauroyl glucose reduces biofilm growth of all the four test cultures on polystyrene and glass surfaces.

Significance and Impact of the Study:  This report is a novel application of the enzymatically synthesized, environmental-friendly nonionic surfactant.

Introduction

Single-celled organisms often exist in the free-floating planktonic forms and as sessile cells that adhere to surfaces. Surface adherent cells form well-developed complexes called biofilms (Costerton et al. 1995). They exhibit structures that are necessary for attachment, show genetic heterogeneity and display diverse community interactions. Biofilm-forming fungi and bacteria are of significance particularly in the medical settings. Among the fungi, Candida species such as Candida albicans, Candida dubliniensis and Candida glabrata are important biofilm formers (Ramage et al. 2001, 2002; Verstrepen and Klis 2006). Listeria monocytogenes, Staphylococcus epidermidis, Salmonella enteritica and Pseudomonas aeruginosa are some of the important bacteria that form biofilms (Farber and Wolff 1992; Mireles et al. 2001; Djordjevic et al. 2002; Werner et al. 2004).

Biofilms tend to form on medical devices such as catheters and prosthetic surfaces. Such biofilms are often difficult to eradicate (d’Enfert 2006). The use of antimicrobial agents (disinfectants and antibiotics) has resulted in target micro-organisms becoming resistant towards such compounds (Stewart 2001; d’Enfert 2006). Finding nonconventional antimicrobial agents such as natural products or substances to which organisms will not develop resistance is of prime importance now. In this respect, surface-active agents of chemical and biological origin have been proposed as potential biofilm disruptors (Velraeds et al. 1996; Mireles et al. 2001; Irie et al. 2005). The use of enzymatically synthesized sugar fatty acid esters (nonionic surfactants) as biofilm disruptors has not been investigated earlier.

Sugar fatty acids are biodegradable amphipathic molecules with polar (sugar) and nonpolar (fatty acid) moieties. Such compounds are synthesized either chemically or enzymatically and display bioactive properties in being anti-tumoural and insecticidal (Nshikawa et al. 1976; Chortyc et al. 1996). From the point of view of being potential biofilm disruptors, sugar esters have antimicrobial properties (Ferrer et al. 2005) and display excellent emulsification and surfactant properties (Nakamura 1997; Watanabe 1999; Kelkar et al. 2007). We therefore hypothesized the use of a representative ester, lauroyl glucose, in disrupting test fungal and bacterial biofilms. The present paper reports biofilm disruption of four test cultures under three sets of conditions: (i) disruption of preformed biofilms in polystyrene microtitre plates; (ii) the effect of precoating polystyrene plates with lauroyl glucose on biofilm formation; and (iii) the effect of co-incubation of lauroyl glucose in culture medium on the formation of biofilms on glass surfaces. The results suggest a possible use of such agents in the disruption of the representative fungal and bacterial biofilm-forming cultures.

Materials and methods

Organisms, growth conditions and enzymatic synthesis of lauroyl glucose

Candida albicans NCIM 3471 and C. lipolytica NCIM 3589 were the fungal isolates used. The bacterial cultures tested were P. aeruginosa PAO1 and P. aureofaciens 30-84. The fungi were grown in yeast nitrogen base (0·7% w/v) with 1% glucose and the bacteria in Luria–Bertani medium with shaking. The cells were harvested, washed twice with phosphate-buffered saline (PBS). Thereafter, a standard inoculum containing 1·0 × 10⁶ cells ml⁻¹ was used for subsequent experiments. The enzymatic production of lauroyl glucose and the subsequent purification were carried out as described earlier by us (Kelkar et al. 2007).

Biofilm formation in microtitre plates

Biofilms were formed in flat-bottomed, 96-well microtitre plates. Standardized suspensions (100 μl) were added into the wells and the cells were allowed to adhere for 1 h. The wells were emptied, washed with PBS and 200 μl of growth medium was added. The plates were incubated for 24 or 48 h. After incubation, the planktonic cells were discarded and weakly adherent cells were removed by washing with PBS. Quantification of the biofilms was performed by the crystal violet method (Djordjevic et al. 2002). All the biofilm formation experiments were carried out in two independent sets with six replicates and the results are expressed as mean values of crystal violet absorbance ± SD from the mean.

Disruption of preformed biofilms in microtitre plates by lauroyl glucose

The biofilms were individually formed in microtitre plates for 24 h. The residual medium was aspirated and the wells were washed with PBS. Lauroyl glucose (100 μg) was added to each well from a stock solution containing 1·0 mg ml⁻¹, in accordance with an earlier report on the use of surfactants as biofilm disruptors (Mireles et al. 2001). Such wells along with the culture medium were incubated further for 24 and 48 h as described previously (Irie et al. 2005) and the residual biofilm was assessed. In control wells, lauroyl glucose was omitted and biofilm growth was monitored in an uninterrupted manner.

Effect of initial precoating with lauroyl glucose on cell attachment and biofilm growth

Wells were coated with 100 μl of 1·0 mg ml⁻¹ solution of lauroyl glucose (100 μg per well) for 24 h to check the effect of the surfactant on initial cell adhesion. The wells were emptied and the unadsorbed lauroyl glucose was transferred to test tubes. The quantity of unadsorbed lauroyl glucose was monitored by performing an emulsification assay as described earlier (Kelkar et al. 2007). Such precoated wells were inoculated with standardized cell suspensions. Respective growth media were added and the plates were incubated for 24 or 48 h and biofilm formation was estimated. The control experiments were carried out in wells that were not precoated with lauroyl glucose. The precoating experiments were also carried out in two independent sets with six replicates and the results are expressed as mean values of crystal violet absorbance ± SD from the mean.

Biofilm disruption on glass surface and confocal scanning laser microscopy (CSLM)

The effect of lauroyl glucose on biofilm formation was studied on glass slide surfaces. The test cultures were grown for 24 h and inoculated into petri plates containing 20 ml of YNB or LB medium, respectively. Lauroyl glucose at a final concentration of 50 or 100 μg ml⁻¹ of the medium was added to individual plates. The plates without lauroyl glucose served as controls. Presterilized microscopic glass slides were immersed into the medium and the plates were incubated for 4 and 24 h. The biofilms were monitored under a CSLM after washing with PBS and staining with 0·01% acridine orange. A CSLM (Model TCS SP2 AOBS) equipped with DM IRE 2-inverted microscope (Leica Microsystems, Germany) was used to image biofilms using a 63 × 1·2 NA water immersion objective. The 488 nm Ar laser and a 500–640-nm band pass emission filter were used to excite and detect the stained cells. Images (20) were collected from the 4- and 24-h-old biofilms measuring an area of 238·1 × 238·1 μm. The area of colonization with and without lauroyl glucose was determined using the digital image analysis freeware, imagej, downloadable from the site http://rsb.info.nih.gov/ij.

Antimicrobial activity of lauroyl glucose (minimum inhibitory concentration, MIC)

The MIC of lauroyl glucose with the test cultures was estimated by the broth microdilution susceptibility test. Cells (1 × 10⁶ per well) were added to microplates containing the diluted antimicrobial agents. These plates were then incubated for 48 h and the MIC values were determined. In addition, the MIC of acetyl salicylate, a known biofilm disruptor was also determined.

Results

When added to preformed biofilms, lauroyl glucose disrupted biofilms of all the four cultures (Fig. 1). Lauroyl glucose dislodged C. albicans biofilms by 45% and C. lipolytica biofilms by 65% when compared with the control experiments after 48 h of incubation. Disruption of 51% was observed in the case of P. aeruginosa and with P. aureofaciens, such disruption was 57% after 48 h of incubation.

Figure 1.  Disruption of preformed biofilms by lauroyl glucose in microtitre plates after 24 h (, control; , test) and 48 h (, control; , test).

In the present study, there was effective coating of lauroyl glucose (80% of the added lauroyl glucose was adsorbed after 24 h when monitored by the emulsification assay). Precoating of the wells resulted in the formation of sub-optimal biofilms when compared with the control experiments (Table 1). This table shows the mean values of crystal violet absorbed at 595 nm ± SD from the mean. A 29% reduction of C. albicans biofilms and 40% decrease for C. lipolytica was observed after 24 h. The effect was less pronounced after further incubation (48 h) and 11% and 32% reduction was observed for C. albicans and C. lipolytica biofilms, respectively. The biofilm growth was affected to a lesser extent in case of P. aeruginosa (27% and 21%) and P. aureofaciens (18 and 12%), respectively, after 24 and 48 h of incubation.

Table 1.   Effect of precoating of lauroyl glucose on biofilm formation

Culture	Crystal violet absorbance (A595)
	24 h		48 h
	Control	Test	Control	Test
Candida albicans	0·27 ± 0·01	0·19 ± 0·01	0·54 ± 0·03	0·48 ± 0·03
Candida lipolytica	0·26 ± 0·01	0·16 ± 0·01	0·55 ± 0·02	0·37 ± 0·01
Pseudomonas aeruginosa	0·33 ± 0·03	0·24 ± 0·01	0·51 ± 0·02	0·40 ± 0·01
Pseudomonas aureofaciens	0·45 ± 0·02	0·37 ± 0·03	0·55 ± 0·03	0·48 ± 0·02

The effect of co-incubation of the test cultures with lauroyl glucose in culture media was studied by using CSLM. In all the cases, control biofilms showed a higher surface coverage (Fig. 2a,c,e,g). The effect of 100 μg ml⁻¹ of lauroyl glucose on biofilms of C. albicans, C. lipolytica, P. aeruginosa and P. aureofaciens is shown in Fig. 2b,d,f and h, respectively. Image analysis and subsequent quantitation of biofilm using imagej software is shown in Table 2. In the presence of lauroyl glucose (50 and 100 μg ml⁻¹), there was a graded decrease in the biofilm formation and a greater reduction in the area that was colonized.

Figure 2.  Representative confocal scanning laser microscopy images of biofilm disruption with 100 μg ml⁻¹ of lauroyl glucose (test) on glass slides after 24 h. (a, b) Candida albicans; (c, d) Candida lipolytica; (e, f) Pseudomonas aeruginosa; (g, h) Pseudomonas aureofaciens. Control: a, c, e, g; Test: b, d, f, h. Bar represents 50-μm scale.

Table 2.   Quantitative data on biofilm disruption obtained from confocal laser scanning microscopic image analysis

Lauroyl glucose concentration	Per cent reduction in area colonized onto glass surface after 24 h
	Candida albicans	Candida lipolytica	Pseudomonas aeruginosa	Pseudomonas aureofaciens
50 μg ml^−1	63·6	41·8	85·6	73·6
100 μg ml^−1	91·2	80·2	92·1	94·9

The MIC of lauroyl glucose was determined and compared with that obtained with acetyl salicylate. For both the disruptors, and for all the four test cultures, the MIC was >500 μg ml⁻¹ (Fig. S1).

Discussion

As stated earlier, sugar esters display important antimicrobial properties; therefore, the effect of a representative sugar ester, lauroyl glucose, on the four test biofilm formers was investigated. Lauroyl glucose was able to dislodge preformed biofilms of all the test cultures (Fig. 1). There are reports on different chemicals individually disrupting preformed fungal and bacterial biofilms. For example, there is a report on the inhibition of C. albicans biofilms by farnesol, a quorum-sensing molecule (Ramage et al. 2002). Similarly, bacterial biofilms of Staph. epidermidis have been disrupted by sodium salicylate (Farber and Wolff 1992). In the present study, a surfactant was able to disrupt representative fungal and bacterial preformed biofilms.

There are problems encountered in eradicating biofilms that have been already been established. Coating biomaterials with antimicrobial agents has been proposed as an alternative method to the application of such agents for disrupting preformed biofilms. Precoating of wells with lauroyl glucose was less effective in preventing biofilm formation (Table 1). Precoating has been effective with some surfactants. For example, surfactin (a surfactant produced by Bacillus subtilis) was effective in controlling Sal. enterica biofilms by the precoating technique (Mireles et al. 2001). Precoating with enzymes has been another novel approach in preventing the formation of bacterial biofilms (Kaplan et al. 2004). The authors have successfully disrupted biofilms of Staph. epidermidis by coating surfaces with the enzyme ‘dispersin’ obtained from Actinobacillus actinomycetemcomitans. Although there are reports on the effectiveness of precoating surfaces with different surfactants and enzymes, in the present study, the precoating technique was not found to be very effective.

Direct microscopic observations of biofilms after exposure to agents are known to provide valuable information on the effect of antimicrobial agents and CSLM analysis was therefore carried out. Lauroyl glucose was able to inhibit growth and biofilm formation of the all the test cultures (Fig. 2a–h and Table 2). Similar types of microscopic observations have been made earlier on the dispersal of Bordetella bronchiseptica biofilms by the spent medium of P. aeruginosa containing rhamnolipid biosurfactant (Irie et al. 2005). The inhibition of Enterococcus faecalis biofilms on glass surfaces using surfactants produced by Lactobacillus spp. has been reported (Velraeds et al. 1996). There was a decrease (77%) in adhesion of E. faecalis cells after 4 h of incubation with the surfactants produced by L. acidophilus RC14 or Lactobacillus fermentum B54. A major advantage observed with lauroyl glucose was the effectiveness of the agent in disrupting all the test biofilm-forming cultures.

The antimicrobial activity (MIC) of lauroyl glucose against the representative fungal and bacterial biofilm-forming cultures was also determined and was comparable with that observed with acetyl salicylate. Although the MIC was higher, disruption experiments were carried out at concentrations reported earlier for other surfactants (Mireles et al. 2001). Sugar esters are earlier reported to display antimicrobial activities (Ferrer et al. 2005). According to the authors, two sugar esters (lauroyl sucrose and lauroyl maltose) inhibited the growth of Bacillus sp. and Lactobacillus plantarum. Lauroyl glucose however did not exhibit antibacterial activity against the cultures that were tested. The authors have also reported that none of the tested carbohydrate esters significantly inhibited the growth of the yeasts, Zygosaccharomyces rouxii and Pichia jadinii, that were tested. The present work reports a novel finding on the antimicrobial activities of lauroyl glucose against the four biofilm-forming test cultures.

In conclusion, lauroyl glucose decreased biofilm formation of representative fungal and bacterial cultures under all the three conditions that were tested. In polystyrene microtitre plates, preformed biofilms were disrupted by lauroyl glucose. Precoating of microtitre plates was not effective in reducing the biofilm formation. Co-incubation of the test cultures with lauroyl glucose in culture media decreased biofilm formation on glass surfaces. The surfactant activity of lauroyl glucose possibly reduced the initial adhesion of cells. Lauroyl glucose inhibited the growth of all the four test cultures. These observations indicate that the antimicrobial and surfactant properties of lauroyl glucose may play a role in inhibiting the overall growth and disrupting biofilms of the test cultures. These results suggest a novel application of lauroyl glucose as a biofilm-disrupting agent.

Acknowledgements

The authors thank Prof. Robert J.C. McLean for the P. aureofaciens 30-84 culture.