Biosurfactants as Agents to Reduce Adhesion of Pathogenic Bacteria ...

Curr Microbiol (2010) 61:554–559 DOI 10.1007/s00284-010-9652-z

Biosurfactants as Agents to Reduce Adhesion of Pathogenic Bacteria to Polystyrene Surfaces: Effect of Temperature and Hydrophobicity Ana Eliza Zeraik • Marcia Nitschke

Received: 25 November 2009 / Accepted: 11 April 2010 / Published online: 27 April 2010 Ó Springer Science+Business Media, LLC 2010

Abstract Polystyrene surfaces were conditioned with surfactin and rhamnolipid biosurfactants and then assessed regarding the attachment of Staphylococcus aureus, Listeria monocytogenes, and Micrococcus luteus. The effect of different temperatures (35, 25, and 4°C) on the anti-adhesive activity was also studied. Microbial adhesion to solvents and contact angle measurements were performed to characterize bacteria and material surfaces. The results showed that surfactin was able to inhibit bacterial adhesion in all the conditions analyzed, giving a 63–66% adhesion reduction in the bacterial strains at 4°C. Rhamnolipid promoted a slight decrease in the attachment of S. aureus. The anti-adhesive activity of surfactin increased with the decrease in temperature, showing that this is an important parameter to be considered in surface conditioning tests. Surfactin showed good potential as an anti-adhesive compound that can be explored to protect surfaces from microbial contamination.

Introduction Biofilms are a matter of concern to many industries such as medical and food, since bacteria can colonize medical devices and food processing surfaces, altering their properties. Furthermore, it may represent an important source of contamination, releasing pathogenic bacteria. Biofilms are more resistant to antimicrobial and sanitizing agents,

A. E. Zeraik M. Nitschke (&) Chemistry Institute of Saõ Carlos, University of Saõ Paulo, Avenida Trabalhador Saõ-Carlense, 400, PO Box 780, Saõ Carlos, SP CEP 13560-970, Brazil e-mail: [email protected]

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impairing the control of this form of microbial organization, when compared to free cells [4, 5, 14]. Bacterial adhesion to surfaces is one of the initial steps that leads to biofilm formation [4]. Consequently, procedures that inhibit or reduce bacterial attachment may reduce biofilm formation. The presence of polymers, protein, and other molecules in the medium can affect bacterial attachment, because these molecules can accumulate on surfaces, hence altering their properties. Factors that affect this include hydrophobicity, electrostatic charges, and surface free energy [7, 15]. Much effort has been directed to avoid bacterial attachment, such as studies on changing the superficial properties of contact surfaces by conditioning biosurfactants (BS) [8, 13, 17]. BS, surface active products of microbial origin, have several advantages over synthetic surfactants. BS have low toxicity, are biodegradable, present chemical diversity, are effective under extreme environmental conditions—such as temperature, pH, and ionic strength—show strong surface activity, emulsifying ability, and have antimicrobial and anti-adhesive properties [6, 10, 18]. In addition, BS can be obtained from renewable substrates, reducing the costs and enlarging the possibility for commercial production [12]. Despite the large number of studies on BS, only a small part is dedicated to the anti-adhesive properties. Moreover, the mechanisms that lead to the anti-adhesive effects of BS are not completely understood. Therefore, more studies are needed in order to consider BS in the design of strategies for reducing bacterial adhesion. The aim of this study was to evaluate the anti-adhesive activity of surfactin and rhamnolipid BS against Listeria monocytogenes, Staphylococcus aureus, and Micrococcus luteus attachments to polystyrene surfaces, as well as the influence of temperature and physicochemical properties of cells on the anti-adhesive behavior.

A. E. Zeraik, M. Nitschke: Biosurfactant Reduces Adhesion

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Materials and Methods

Physicochemical Characterization of Cell Surfaces

Bacterial Strains and Inoculum Preparation

To evaluate the Lewis acid–base properties and the hydrophilic/hydrophobic nature of bacterial surfaces, a MATS test (microbial adhesion to solvents) was performed according to the methodology developed by BellonFontaine et al. [1]. The solvents used were: Chloroform (an acidic solvent) and hexadecane (apolar), diethyl ether (a basic solvent) and hexane (apolar). A bacterial suspension containing approximately 108 CFU ml-1 in 2.4 ml of NaCl 0.15 mol l-1 and 0.4 ml of solvent was shaken by vortexing for 2 min to form an emulsion. The mixture was allowed to stand for 15 min to ensure the complete separation of both phases. The optical density of the aqueous phase was measured at 400 nm. The adhesion percentage to each solvent was calculated by the equation: % Adh = (1 - A/A0) 9 100, where A0 is the absorbance of the bacterial suspension before mixing and A is the absorbance after mixing [8].

Listeria monocytogenes ATCC 19112, Staphylococcus aureus ATCC 25923 and Micrococcus luteus ATCC 4698 were obtained from Fundaçaõ Oswaldo Cruz Culture Collection (Rio de Janeiro- Brazil) and stored at -20°C in tryptic soy broth supplemented with 20% glycerol (v/v). The bacterial strains were cultivated in tryptic soy agar supplemented with 6 g l-1 of yeast extract and incubated at 35°C for 24 h. The cell mass was removed from the medium using an inoculating loop, suspended in 10 ml of NaCl 0.15 mol l-1 and adjusted to a concentration of approximately 1010 CFU ml-1. Biosurfactants Rhamnolipid from Pseudomonas aeruginosa LBI was obtained as previously described by Nitschke et al. [12] and presented a surface tension of 30.96 mN m-1, critical micelle concentration (CMC) of 43.21 mg l-1 and purity of 65%. Surfactin from Bacillus subtilis LB5a showed a surface tension of 26.6 mN m-1, CMC of 33 mg l-1, purity of 78% and was obtained in accordance with Nitschke and Pastore [11]. Surface Conditioning Ninety-six-well polystyrene microtiter plates were filled with 200 ll of biosurfactant solutions. The concentrations used were 0.1% (w/v) for surfactin and 0.4% (w/v) for rhamnolipid [13]. The plates were incubated for 24 h at three different temperatures (35, 25, and 4°C) and then washed three times with sterile water. The control wells were maintained without treatment. Anti-Adhesive Assay After conditioning with BS, 200 ll of bacterial suspension (2 9 109 CFU/well) was added to polystyrene microtiter plates and incubated at 35, 25, and 4°C for 4 h or at different time intervals (kinetics studies). Unattached bacteria were removed by washing the wells three times with water. The adherent microorganisms were fixed for 15 min with 200 ll of methanol. The wells were then stained with 200 ll of crystal violet (1% aqueous solution w/v) for 15 min, rinsed under running tap water, and dried. The bound dye was released with 200 ll of glacial acetic acid (33% v/v). The quantitative analysis of bacterial adhesion was performed by reading the optical density of the wells in a microtiter plate reader (Thermoplate) at 630 nm.

Contact Angle Measurements Contact angle was determined using 2 cm2 polystyrene coupons. The conditioning of the surfaces with BS was performed as described above at 25°C. Afterwards the surfaces were rinsed with 10 ml of distilled water and left to dry at room temperature. The surface characteristics of the material with and without adsorbed biosurfactant were assessed by water contact angle measurements using the sessile drop technique at 20°C and OCA 15 (Optical Contact Angle) equipment (Dataphysics). The results are an average of at least 20 measurements taken from three independent samples. Scanning Electron Microscopy 1 cm2 polystyrene coupons were immersed in ethanol, subjected to ultrasonic cleaning for 10 min, washed three times with distilled water and left to dry at room temperature. The conditioning procedure was performed with surfactin 0.1% (w/v) for 24 h at 4°C. After rinsing with 10 ml of distilled water, the samples were immersed in a suspension of Staphylococcus aureus (approximately 1010 CFU ml-1) for 4 h at 4°C and rinsed again with 10 ml of distilled water. The dehydration procedure was carried out by transferring the samples to ethanol/water solutions of increasing concentrations (10, 25, 40, 50, 70, 80, 90, and 95%) for 15 min in each solution. The samples were maintained desiccated until gold sputtering and visualized by scanning electron microscope (LEO) operating at 20 kV.

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Statistical Analysis Results are the mean values and standard deviations (SD) of at least three independent experiments. A one-way analysis of variance (ANOVA) was performed using Origin Software 7.5 (Origin Lab Corporation). To locate significant differences, Tukey’s test (P \ 0.05) was used.

Results BS Anti-Adhesive Assay Kinetic studies on bacterial adhesion were performed to verify the adhesion profiles and the effect of biosurfactant conditioning with respect to time. Figure 1 illustrates the adhesion kinetics of S. aureus, L. monocytogenes, and M. luteus on polystyrene surfaces treated at 25°C with or without BS. With this assay, the adhesion period of 4 h was selected for further studies. To evaluate the effect of

Fig. 1 Adhesion kinetic of S. aureus ATCC 25923 (a), L. monocytogenes ATCC 19112 (b), and M. luteus ATCC 4698 (c) on polystyrene surfaces conditioned with surfactin (open square) or

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temperature on the anti-adhesive effect of BS, the conditioning procedure and the adhesion assay were performed at 35, 25, and 4°C, in order to find the best anti-adhesive conditions. Table 1 shows the data obtained from these experiments. Surfactin treatment showed a significant decrease in adhesion of all the bacteria studied and in all conditions evaluated. The reduction in adhesion promoted by surfactin treatment increased with the decrease of temperature. The most significant result was obtained at 4°C, reaching 63–66% of inhibition on bacterial attachment. The conditioning of polystyrene surfaces with rhamnolipid significantly decreased S. aureus attachment at all temperatures studied; although to a lower degree than surfactin. L. monocytogens showed a reduction in the adhesive behavior when rhamnolipid was adsorbed on the surface at 35°C. M. luteus showed a significant increase in adhesion when the surface was treated with rhamnolipid at the three temperatures, represented by the negative values in Table 1.

rhamnolipid (open triangle); control (filled square). Conditioning and adhesion test were performed at 25°C

A. E. Zeraik, M. Nitschke: Biosurfactant Reduces Adhesion Table 1 Effect of temperature on bacterial adhesion to polystyrene surfaces conditioned with biosurfactants

Microorganisms

557

Temperature (°C)

L. monocytogenes ATCC 19112

35

S. aureus ATCC 25923

0.00a

36.83b

21.25c

a

b

21.18c

b

-8.15c

0.00

b

46.44

6.50a

0.00a

55.99b

21.72c

a

b

-10.07c

b

5.79a

b

16.62c

b

-22.67c

0.00

a

25

M. luteus ATCC 4698 L. monocytogenes ATCC 19112

Surfactin

a


Within each line, values with the same letters are not significantly different (P \ 0.05)

Control

0.00

M. luteus ATCC 4698 L. monocytogenes ATCC 19112

Bacterial adhesion inhibition (%)

0.00

a

4

0.00

a


0.00

a

M. luteus ATCC 4698

0.00

42.02 33.92

46.47 65.83 66.02 62.92

Rhamnolipid

Table 2 Percentage affinity of bacterial cells to the solvents used in MATS test Microorganism

% Adhesion (mean ± SD) Chloroform

Hexadecane

Diethyl ether

83.75 ± 1.67

76.21 ± 4.82

19.25 ± 1.50

67.26 ± 2.78d


96.98 ± 0.87a

94.94 ± 1.65b

44.46 ± 6.30c

94.58 ± 3.53b

a

b

c

91.87 ± 0.79

b

Hexane

L. monocytogenes ATCC 19112 M. luteus ATCC 4698

a

89.69 ± 2.00

c

47.79 ± 6.83

74.20 ± 3.2d

Within each line, values with the same letters are not significantly different (P \ 0.05)

Physicochemical Characterization of Cell Surfaces by MATS Test Table 2 shows that the bacterial strains studied have higher affinity with chloroform than with hexadecane and with hexane when compared with diethyl ether, indicating a predominance of basic properties and weak acid properties on its cell surfaces. Regarding the hydrophobicity of the cell surfaces, similar characteristics were also found; all of the bacteria studied were classified as strongly hydrophobic, since the adhesion to hexadecane was higher than 55% [2]. S. aureus showed the highest hydrophobic characteristic, reaching almost 95% of adhesion to hexadecane. Contact Angle Measurements The surface conditioning with BS caused alterations on the water contact angle, when surfactin was used, a decrease from 82° to around 76° was observed, indicating that the surface became less hydrophobic. When the treatment was performed with rhamnolipid, there was a slightly increase from 82° to 84°, suggesting some increase in the surface hydrophobicity (The values for the contact angles were significantly different, P \ 0.05). Scanning Electron Microscopy Analysis The reduction in adhesion promoted by conditioning the surface with surfactin was visualized by scanning electron

microscopy (Fig. 2). The surface colonization by S. aureus noticeably decreased on the treated polystyrene surfaces when compared to the untreated ones.

Discussion The mechanisms involved in anti-adhesive activity are not completely elucidated; however, the inhibitory effect of BS seems to be dependent on the type of biosurfactant, microorganism, and surface properties [19]. Rodrigues et al. [17] postulated that BS reduce hydrophobic interactions and consequently microbial adhesion. Hydrophobic surfaces have shown to be particularly colonized by microorganisms, probably because these surfaces facilitate the close approach between microorganism and solid substratum, favoring the elimination of interfacial water present in the interacting surfaces [17]. Consequently, when a surface is conditioned with biosurfactant, it becomes more hydrophilic, with an expected decrease of microbial attachment. Our data indicated a decrease in hydrophobicity on surfaces treated with surfactin and also a substantial decrease of bacterial attachment, in agreement with the aforementioned explanation. Nevertheless, the slight decrease in the contact angle values observed is not enough to explain the inhibition observed (reaching 66%), suggesting that other factors may contribute to this effect. Surface charge of microorganism and substratum, the presence of fimbria, flagella, surface proteins, and the

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Fig. 2 SEM micrographs of S. aureus adhesion to polystyrene surfaces untreated (a) and treated (b) with surfactin for 24 h at 4°C

production of EPS by bacteria [2, 7] are features involved in the adhesion process. The MATS test showed a similar pattern for all bacteria studied. They were all strongly hydrophobic, and it may explain the great affinity with the hydrophobic polystyrene surface. The bacteria also presented a predominance of basic or electron donating properties. The basic characteristic may result from the presence of chemical groups such as COO- and HSO3- on the bacterial cell surface [16]. Considering that surfactin is an anionic surfactant, the anti-adhesive effect observed can be in part due to the electrostatic repulsion between bacteria and the molecules of surfactin adsorbed onto the polystyrene surface. However, rhamnolipid is also an anionic biosurfactant, but the reduction in adhesion was observed mainly for S. aureus and L. monocytogenes at 35°C. These differences could be attributed to the chemical composition of both BS (one is a lipopeptide and the other a glycolipid), their molecular orientation on surfaces, as well as their surface properties. Di Bonaventura et al. [3] showed that the temperature is an important feature in the adhesion of Listeria monocytogenes to abiotic surfaces. The biofilm formation by L. monocytogenes Lm86 on polystyrene was directly correlated with the increase in temperature, a fact also observed in our work for all the strains evaluated (data not shown). Regarding the temperature effect on the antiadhesive behavior of surfactin, we have noted that although a significant reduction on bacterial attachment was observed in all temperatures, the best results were obtained at 4°C (Table 1). This anti-adhesive response under low temperatures may be associated to an increase in the number of biosurfactant molecules adsorbed to the surface since an increase in the temperature of an adsorbing system will usually decrease the adsorption of ionic surfactants [9]. Moreover, under low temperatures, the hydrophobicity, motility and other attachment factors (EPS, surface proteins) of bacterial cells can be modified, hence favoring the anti-adhesive activity of surfactin. In summary, our results demonstrate that bacterial attachment to polystyrene can be reduced by using

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surfactin and that its effect can be increased at low temperatures, showing the importance of this parameter when studying surfactants such as anti-adhesive agents. Acknowledgments The authors would like to thank the financial support provided by Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnolo´gico (CNPq) and Coordenaçaõ de Aperfeiçoamento de Pessoal de Nı´vel Superior (CAPES).

References 1. Bellon-Fontaine MN, Rault J, van Oss CJ (1996) Microbial adhesion to solvents: a novel method to determine the electrondonor/electron-acceptor or Lewis acid–base properties of microbial cells. Colloids Surf B 7:47–53 2. Chae MS, Schraft H, Hansen LT et al (2006) Effects of physicochemical surface characteristics of Listeria monocytogenes strains on attachment to glass. Food Microbiol 23:250–259 3. Di Bonaventura G, Piccolomini R, Paludi D et al (2008) Influence of temperature on biofilm formation by Listeria monocytogenes on various food-contact surfaces: relationship with motility and cell surface hydrophobicity. J Appl Microbiol 104:1552–1561 4. Donlan RM, Costeron JW (2002) Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 15:167–193 5. Dunne MW (2002) See any good biofilm lately? Clin Microbiol Rev 15:155–166 6. Gautam KK, Tyagi VK (2006) Microbial surfactants: a review. J Oleo Sci 55:155–166 7. Goulter RM, Gentle IR, Dykes GA (2009) Issues in determining factors influencing bacterial attachment: a review using the attachment of Escherichia coli to abiotic surfaces as an example. Lett Appl Microbiol 49:1–7 8. Meylheuc T, van Oss CJ, Bellon-Fontaine MN (2001) Adsorption of biosurfactant on solid surfaces and consequences regarding the bioadhesion of Listeria monocytogenes LO28. J Appl Microbiol 91:822–832 9. Myers D (2006) Surfactant science and technology, 3rd edn. Wiley, Hoboken, pp 337–349 10. Nitschke M, Costa SGVAO (2007) Biosurfactants in food industry. Trends Food Sci Technol 18:252–259 11. Nitschke M, Pastore GM (2004) Biosurfactant production by Bacillus subtilis using cassava processing effluent. Appl Biochem Biotechnol 112:163–172 12. Nitschke M, Costa SGVAO, Haddad R et al (2005) Oil wastes as unconventional substrates for rhamnolipid biosurfactant production by Pseudomonas aeruginosa LBI. Biotechnol Prog 21:1562–1566

A. E. Zeraik, M. Nitschke: Biosurfactant Reduces Adhesion 13. Nitschke M, Arau´jo LV, Costa SGVAO et al (2009) Surfactin reduces the adhesion of food-borne pathogenic bacteria to solid surfaces. Lett Appl Microbiol 49:241–247 14. Palmer RJ, White DC (1997) Developmental biology of biofilms: implications for treatment and control. Trends Microbiol 5:435–440 15. Palmer J, Flint S, Brooks J (2007) Bacterial cell attachment, the beginning of a biofilm. J Ind Microbiol Biotechnol 34:577–588 16. Pelletier C, Bouley C, Cayuela C et al (1997) Cell surface characteristics of Lactobacillus casei subsp. casei, Lactobacillus paracasei subsp. paracasei, and Lactobacillus rhamnosus strains. Appl Environ Microbiol 63:1725–1731

559 17. Rodrigues LR, Banat MI, van der Mei HC et al (2006) Interference in adhesion of bacteria and yeasts isolated from explanted voice prostheses to silicone rubber by rhamnolipid biosurfactants. J Appl Microbiol 100:470–480 18. Singh P, Cameotra SS (2004) Potential applications of microbial surfactants in biomedical sciences. Trends Biotechnol 22:142–146 19. Walencka E, Ro´zalska S, Sadowska B et al (2008) The influence of Lactobacillus acidophilus-derived surfactants on staphylococcal adhesion and biofilm formation. Folia Microbiol 53:61–66

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