Inhibition of Microbial Biofilm on Different Surfaces Treated with Biosurfactant from Lactobacillus spp

Background: The use and potential commercial application of biosurfactants in the medical field has increased during the past decade. Their antibacterial, antifungal and antiviral activities make them relevant molecules for applications in combating many diseases and as therapeutic agents. In addition, their role as antiadhesive agents against several pathogens indicates their utility as suitable anti-adhesive coating agents for medical insertional materials leading to a reduction in a large number of hospital infections. In the present study the ability of the Lactobacillus acidophilus biosurfactant to inhibit the Enterococcus faecalis and Staphylococcus epidermidis biofilm on Foley catheter and microtiter plate wells were investigated. Materials and Methods: The surfaces were coated with biosurfactant solution, subsequently the tested bacteria were inoculated to the surfaces. Quantification of biofilm was performed by a spectrophotometric method (measuring the optical density (OD540)), which measures the total biofilm biomass, including bacterial cells and extracellular matrix. Results: The amounts of biofilm were determined after 24 hr of incubation. biosurfactant layers caused a marked inhibition of Enterococcus faecalis biofilm formation on microtiter plate well (OD= 0.227) and Foley catheter (OD= 0.112) compare with uncoated surfaces (OD= 0.439, 0.297 respectively), similar antiadhesive activity were obtained on Staphylococcus epidermidis biofilm, as the amount of biofilm formation on microtiter plate well (OD=0.118) and Foley catheter (OD=0.099) were reduced markedly when compare with uncoated surface (OD=0.213,0.188 respectively). The biofilm amount formed by Enterococcus faecalis and Staphylococcus epidermidis were reduced by 48.2% and 44.6 % on microtiter plate wells and 62.2% and 47.3% on Foley catheter respectively, after coating those surfaces by biosurfactants. Conclusions: Biosurfactants have the potential to be used as a preventive strategy to delay the onset of pathogenic biofilm growth on catheters and other materials, thus may lowering the large number of hospital infections without the use of synthetic drugs and chemicals. Introduction The origin of the uropathogens in uncomplicated urinary tract infection and bacterial vaginosis is the fecal flora. For more than 30 years, the key factor in pathogenesis has been regarded as the ability of the pathogens to attach to epithelial cells, thereby allowing them a niche in which to establish, multiply, spread, and avoid host defenses (Reid, 2001). Journal of Kirkuk University –Scientific Studies, vol.7, No.1, 2012 25 It has been reported that more than 50 species colonize the healthy vagina (Redondo-Lopez, 1990). Studies have shown that urogenital cells are covered by dense bacterial biofilms whose composition changes constantly but in which lactobacilli predominate, at least until menopause (Reid, 2001). Uropathogenic organisms emerge from the intestine and come into contact with these biofilms on vaginal and urethral cells, yet little is known about what happens thereafter. It is presumed that uropathogens can either bypass the microflora or successfully enter the biofilms, survive, and continue their ascension into the bladder (Wold et al., 1992). Biofilms have been described in the human host for more than 20 years but their role in health has not been studied as well as their association with disease (Costerton et al., 1981). These biofilms are defined as structured communities of microbial species embedded in a biopolymer matrix on either biotic or abiotic substrata. Both abiotic and biotic surfaces such as mineral, metal, animal or plant surfaces, lung and intestine, and all types of medical implants are subject to bacterial colonization and biofilm formation (Fux et al., 2003). Most importantly, the biofilm is characterized by its resistance to biocides, antibiotic chemotherapy, and clearance by humoral or cellular host defense mechanisms (Kreft, 2005). Lactobacilli are Gram-positive rods, primarily facultative or strict anaerobes that generally have a fastidious growth requirement. They prefer an acidic environment and help create one by producing lactic and other acids. In general, lactobacilli have not been associated with disease and for more than 100 years have been regarded as nonpathogenic members of the intestinal and urogenital floras (Reid, 2001). In more recent years, the use of probiotics per se and lactobacilli specifically has received greater attention as an alternative, inexpensive, and natural remedy to restore and maintain health (Shalev et al., 1996 ), namely the ability to adhere to and colonize tissues and the capacity to inhibit the pathogenesis of disease causing organisms (Hamilton-Miller, 1997). Microbial compounds that exhibit pronounced surface and emulsifying activities are classified as biosurfactants. Biosurfactants comprise a wide range of chemical structures, such as glycolipids, lipopeptides, polysaccharide–protein complexes, phospholipids, fatty acids and neutral lipids (Ahimou, et al., 2001). Biosurfactants are amphipathic molecules with both hydrophilic and hydrophobic moieties that partition preferentially at the interface between fluid phases that have different degrees of polarity and hydrogen bonding, such as oil and water or air and water interfaces. Some biosurfactants are suitable alternatives to synthetic Journal of Kirkuk University –Scientific Studies, vol.7, No.1, 2012 26 medicines and antimicrobial agents and may be used as safe and effective therapeutic agents (Singh and Cameotra, 2004 and Rodrigues et al., 2006). The aim of this study was to investigate the potential of Lactobacillus acidophilus biosurfactant as an antiadhesive coating for different solid surfaces. Materials and Methods Bacterial Strains Two uropathogenic bacteria were used in the study: Enterococcus faecalis and Staphylococcus epidermidis and these strains were clinical isolates from urinary tract infections and were identified by using conventional methods. For isolation and identification of Lactobacillus acidophilus, vaginal swabs were taken from lateral vaginal wall of healthy women and were inoculated on MRS medium (de Man, Rogosa and Sharpe) and incubated overnight at 37oC in an atmosphere containing 5% CO2. To identify the strain each colony from MRS agar was subjected to Gram stain test, as well as test for catalase activity, cell shape, motility, growth at various temperatures (15, 45 and 50 oC) and tolerance of different salt levels (MRS broth with 2,4 and 6.5% NaCl). Finally the carbohydrate fermentation characteristics were determined by API 50 CHL system (Biomerieux, France) (Velraeds et al., 1996).


Introduction
The origin of the uropathogens in uncomplicated urinary tract infection and bacterial vaginosis is the fecal flora. For more than 30 years, the key factor in pathogenesis has been regarded as the ability of the pathogens to attach to epithelial cells, thereby allowing them a niche in which to establish, multiply, spread, and avoid host defenses (Reid, 2001).
It has been reported that more than 50 species colonize the healthy vagina (Redondo-Lopez, 1990). Studies have shown that urogenital cells are covered by dense bacterial biofilms whose composition changes constantly but in which lactobacilli predominate, at least until menopause (Reid, 2001). Uropathogenic organisms emerge from the intestine and come into contact with these biofilms on vaginal and urethral cells, yet little is known about what happens thereafter. It is presumed that uropathogens can either bypass the microflora or successfully enter the biofilms, survive, and continue their ascension into the bladder (Wold et al., 1992).
Biofilms have been described in the human host for more than 20 years but their role in health has not been studied as well as their association with disease (Costerton et al., 1981). These biofilms are defined as structured communities of microbial species embedded in a biopolymer matrix on either biotic or abiotic substrata. Both abiotic and biotic surfaces such as mineral, metal, animal or plant surfaces, lung and intestine, and all types of medical implants are subject to bacterial colonization and biofilm formation (Fux et al., 2003). Most importantly, the biofilm is characterized by its resistance to biocides, antibiotic chemotherapy, and clearance by humoral or cellular host defense mechanisms (Kreft, 2005).
Lactobacilli are Gram-positive rods, primarily facultative or strict anaerobes that generally have a fastidious growth requirement. They prefer an acidic environment and help create one by producing lactic and other acids. In general, lactobacilli have not been associated with disease and for more than 100 years have been regarded as nonpathogenic members of the intestinal and urogenital floras (Reid, 2001).
In more recent years, the use of probiotics per se and lactobacilli specifically has received greater attention as an alternative, inexpensive, and natural remedy to restore and maintain health (Shalev et al., 1996 ), namely the ability to adhere to and colonize tissues and the capacity to inhibit the pathogenesis of disease causing organisms (Hamilton-Miller, 1997).
Microbial compounds that exhibit pronounced surface and emulsifying activities are classified as biosurfactants. Biosurfactants comprise a wide range of chemical structures, such as glycolipids, lipopeptides, polysaccharide-protein complexes, phospholipids, fatty acids and neutral lipids (Ahimou, et al., 2001). Biosurfactants are amphipathic molecules with both hydrophilic and hydrophobic moieties that partition preferentially at the interface between fluid phases that have different degrees of polarity and hydrogen bonding, such as oil and water or air and water interfaces. Some biosurfactants are suitable alternatives to synthetic medicines and antimicrobial agents and may be used as safe and effective therapeutic agents (Singh andCameotra, 2004 andRodrigues et al., 2006). The aim of this study was to investigate the potential of Lactobacillus acidophilus biosurfactant as an antiadhesive coating for different solid surfaces.

Bacterial Strains
Two uropathogenic bacteria were used in the study: Enterococcus faecalis and Staphylococcus epidermidis and these strains were clinical isolates from urinary tract infections and were identified by using conventional methods.
For isolation and identification of Lactobacillus acidophilus, vaginal swabs were taken from lateral vaginal wall of healthy women and were inoculated on MRS medium (de Man, Rogosa and Sharpe) and incubated overnight at 37ºC in an atmosphere containing 5% CO2. To identify the strain each colony from MRS agar was subjected to Gram stain test, as well as test for catalase activity, cell shape, motility, growth at various temperatures (15, 45 and 50 ºC) and tolerance of different salt levels (MRS broth with 2,4 and 6.5% NaCl). Finally the carbohydrate fermentation characteristics were determined by API 50 CHL system (Biomerieux, France) (Velraeds et al., 1996).

Adhesion assay 1-Microtiter Plate
The quantification assay of biofilm was performed as previously described (Mireles et al., 2001). Typically, 25µl of an overnight culture of tested bacteria (Staphylococcus epidermidis and Enterococcus faecalis) in Trypticase soy broth (TSB) was used to inoculate at least 20 wells of microtiter plate containing 175 µl of sterile TSB. The covered microtiter dish was sealed with Parafilm during incubation at 35°C. Cultures were removed and the wells were rinsed with distilled water. After drying at room temperature for 15 min, 200 µl of crystal violet (1%) was added to the wells for 20 min. The stained biofilms were rinsed several times with distilled water, allowed to dry at room temperature for 15 min, and extracted with 200 µl of 95% ethanol. The optical densities (OD) were estimated using ELISA reader spectrophotometer (230S Organon Teknika, Austria). At least 10 wells filled with 200 µl of sterile TSB and used as controls.

2-Foley catheters assay
An overnight culture (10µl) of tested bacteria (Enterococcus faecalis and Staphylococcus epidermidis) in TSB was inoculated into 500 µl of sterile TSB and injected into clear urethral catheters. The catheters were capped at both ends and incubated at 35°C overnight. Cultures were removed and the catheters were rinsed with distilled water. After drying at room temperature for 15 min, 700 µl of crystal violet (1%) was added to the catheters for 20 min. The stained biofilms were rinsed several times with distilled water and allowed to dry at room temperature for 15 min, then 700 µl of 95% ethanol were added for distaining, finally 200 µl of them was transferred to a clean MTP for measuring the optical density (Mireles et al., 2001).

Production of Biosurfactant
For a select number of Lactobacillus strains, 600-ml cultures in MRS broth were grown overnight (for 18 h), then the cells were harvested by centrifugation at 10,000 rpm for 5 min, washed twice in demineralized water, and resuspended in 100 ml of PBS. The lactobacilli were left at room temperature for 2 h with gentle stirring for biosurfactant production. Subsequently, the bacteria were removed by centrifugation, and the remaining supernatant liquid was filtered through a 0.22-µm-pore-size filter (Millipore). About 10-ml of the supernatant was used immediately in the adhesion assay (Velraeds et al., 1996).

Inhibition assay
Both MTP wells and catheter were filled with (200 µl and 500 µl respectively) of freshly produced biosurfactant and left at room temperature for overnight adsorption. Subsequently, the two tested bacteria (Enterococcus faecalis and Staphylococcus epidermidis) suspension was added. Bacterial inoculums, media and growth conditions and reading the results were as described above for adhesion assay (Velraeds et al., 1996, Mireles et al., 2001.

Measuring the optical density (OD)
The OD of all assays was measured as following:

Mean OD (control) -Mean OD (test) = OD biofilm
For tested bacteria at least mean of 20 MTP wells were used and for control at least mean of 10 wells were used. Three catheters were used for bacterial biofilm formation and one catheter as control. The optical densities of control wells subtracted from the test wells to estimate only the biofilm binding stain.

Results
Different amount of biofilm (bacterial adhesion) were recorded on different abiotic surfaces by two uropathogenic bacteria. As shown in figure 1, the amount of biofilm formed by Enterococcus faecalis on microtiter plate wells as measured by optical density (0.439) were higher than those formed on Foley catheters (0.297), similar results were observed when Staphylococcus epidermidis were used as the amount of biofilm formed on microtiter plate wells were higher (0.213) than those formed on Foley catheter (0.188), (Figure 2).

Fig.2: The mean optical densities of stained Staphylococcus epidermidis biofilm on different surfaces with and without biosurfactant
The antiadhesive activities of biosurfactant were investigated in the present study, stationary phase Lactobacillus aciophilus were used for biosurfactant production.
The amounts of Enterococcus faecalis biofilm (adherent bacteria) were markedly reduced when the microtiter plate wells (0.227) and Foley catheter (0.122) pre-coated with biosurfactant compared with the uncoated surfaces (Figure 1, 3 and 4). Similar antiadhesive effect of biosurfactant were recorded on Staphylococcus epidermidis adhesions on microtiter plate wells (0.188) and Foley catheters (0.099) compared to uncoated surfaces (figure 2).

Fig.3: Stained Enterococcus faecalis biofilm with and without
biosurfactants after 24hr of incubation.

Fig. 4: Stained Enterococcus faecalis biofilm on Foley catheter with and without biosurfactants after 24hr of incubation
On the basis of the data, it was concluded that the biosurfactants from Lactobacillus acidophilus decreased the amount of biofilm of Enterococcus faecalis and Staphylococcus epidermidis by 62.2% and 47.3% on catheters and by 48.2% and 44.6% on microtiter plate wells respectively (table1).

Biofilm without Biosurfactant Biofilm with Biosurfactant
Discussion Velraeds and coworkers (1996) reported that fifteen isolates of lactobacillus were found to produce biosurfactant.
Many studies found that the production of biosurfactant per bacterial cell was generally greater and more rapid when the bacteria were harvested in the stationary phase than when they were harvested in the midexponential growth phase, the crude substance was analyzed and they were found that crude substance contain proteins and carbohydrates, and the activity was not due to lipoteichoic acid or glycosyldiglycerides or to factors such as acid or bacteriocins, which inhibit bacterial growth (Reid, 2001, Mireles et al., 2001, Velraeds et al.,1997and Velraeds et al., 1998. In our study we used a stationary phase Lactobacillus acidophilus (18 hr) for biosurfactant production. Pre coating Foley catheters and microtiter plate wells with biosurfactant solution before inoculation with uropathogenic bacteria resulted in a decrease in the amount of biofilm formed by Enterococcus faecalis (40.80%, 62.20%) and Staphylococcus epidermidis (37.70%, 48.20%), these results have great potential for practical applications.
Velraeds and his group (1998) reported that the adsorbed biosurfactant (surlactin) layers were particularly effective at preventing adhesion to silicone rubber of Enterococcus faecalis, E. coli and Staphylococcus epidermidis strains. (Ried, 2001) also found that the biosurfactant adsorbed to surfaces and inhibited the initial adhesion of Enterococcus faecalis by 70%. (Heinemann et al. 2000) showed that Lactobacillus fermentum RC-14 releases surface-active components that can inhibit adhesion of uropathogenic bacteria, including Enterococcus faecalis.
Efforts in the development of strategies to prevent the microbial colonization of silicone rubber voice prostheses have been reported by Rodrigues et al. (2004 and2006) The ability of biosurfactants obtained from the probiotic strains, Lactococcus lactis 53 and Streptococcus thermophilus A, to inhibit adhesion of four bacterial and two yeast strains isolated from explanted voice prostheses to pre-coated silicone rubber was evaluated. The results obtained by those researchers showed that the biosurfactants were effective in decreasing the initial deposition rates, as well as the number of bacterial cells adhering after 4 h, for all microorganisms tested. Over 90% reductions in the initial deposition rates were achieved for most of the bacterial strains tested. Mireles and co-workers (2001), reported that Pre-coating vinyl urethral catheters by running the surfactin solution through them before inoculation with media resulted in a decrease in the amount of biofilm formed by Salmonella typhimurium, Salmonella enterica, E. coli and Proteus mirabilis.
Singh et al (2004) concluded that biosurfactants have been found to inhibit the adhesion of pathogenic organisms to solid surfaces or to infection sites; thus, prior adhesion of biosurfactants to solid surfaces might constitute a new and effective means of combating colonization by pathogenic microorganisms.
As a conclusion, biosurfactants produced by Lactobacillus acidophilus have the potential to be used as a preventive strategy to delay the onset of pathogenic biofilm growth on catheters and other medical insertional materials, thus lowering the large number of hospital infections without the use of synthetic drugs and chemicals, especially at a time when drug resistance among causal organisms for many life-threatening diseases is on the rise.