Master thesis performed at:

GHENT UNIVERSITY UNIVERSITY OF EASTERN PIEDMONT

FACULTY OF PHARMACEUTICAL SCIENCES 'AMEDEO AVOGADRO'

Department of Pharmaceutical Analysis Department of Pharmaceutical Sciences

Laboratory of Pharmaceutical Microbiology Laboratory of Microbiology

Academic year 2011-2012

Anti-adhesive effect of biosurfactant AC7 on Candica albicans and characterization of the

biosurfactant producing strain Bacillus subtilis AC7.

Liesbeth MISSIAEN

First Master of Pharmaceutical Care

Promotor

Prof. Dr. H. Nelis

Commissioners

Prof. Dr. T. Coenye

Dr. Apr. G. Brackman

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COPYRIGHT "The author and the promoters give the authorization to consult and to copy parts of this

thesis for personal use only. Any other use is limited by the laws of copyright, especially

concerning the obligation to refer to the source whenever results from this thesis are cited."

June 1, 2012 Promoter Author Prof. dr. H. Nelis Liesbeth Missiaen

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SUMMARY

Resistance to antibiotics is a pivotal and precarious problem in the microbiological and medical

world. Biofilm formation is a frequently the cause of chronic infections on a lot of medical devices. As

antibiotics are highly resistant to biofilms, alternatives are searched. Biosurfactants produced by

several bacteria are known to possess antimicrobial and anti-adhesive activity against pathogens.

In this thesis, biochemical tests were carried out to confirm the identity of the biosurfactant AC7

producing strain, previously genotypically identified as Bacillus subtilis. All results of the biochemical

tests performed to characterize Bacillus subtilis AC7 were in agreement with the biochemical

properties of Bacillus subtilis described by Koneman (1995).

Subsequently, the critical micelle concentration of biosurfactant AC7 was determined, measuring

the surface tension using different concentrations of biosurfactant AC7. As all results were consistent

with the presence of a potent low molecular weight lipopeptidic biosurfactant, i.e. surfactin, B.

subtilis AC7 is able to produce a potent biosurfactant.

The anti-adhesive activity on Candida albicans 40 of biosurfactant AC7 produced by B. subtilis

was evaluated on silicone disks at several conditions. The method used to coat silicone disks with

biosurfactant AC7 is a modification of the method described by Rivardo et al. (2009). The evaluation

of the C. albicans biofilm formation was carried out according to Chandra et al. (2009). Biosurfactant

A7 has a good anti-adhesive activity against Candida albicans 40 on silicone elastomeric disks.

Different conditions of pre-coating and incubation gave similar results. The obtained results

demonstrate that the adherence of C. albicans to silicone elastomeric disks can be reduced by using

biosurfactant AC7 and that its effect can be increased at low temperatures, showing the importance

of this parameter when studying surfactants as anti-adhesive agents. The anti-adhesive activity of

biosurfactant AC7 seems to be only effective in early phase of adhesion.

Finally, positive results were obtained for a first screening of biosurfactant AC7. Optimization

of the method by simulating the real condition in human body could be a promising future

perspective.

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SAMENVATTING

Resistentie van antibiotica is een groot, zorgwekkend wereld in de microbiologische en

medische wereld. Vorming van biofilms is een vaak voorkomend probleem bij medische implantaten.

Aangezien biofilms erg resistent zijn tegen antibiotica, wordt er naar C. albicans alternatieven

gezocht. Meerdere bacteriën produceren biosurfactantia die antimicrobiële en anti-adhesieve

activiteit vertonen tegenover pathogenen.

Om de identiteit van de biosurfactant producerende strain AC7 te bevestigen, die vooraf

genotypisch bepaald werd als zijnde Bacillus subtilis, werden in deze thesis een aantal biochemische

testen uitgevoerd. Alle resultaten van de biochemische testen die uitgevoerd werden om B. subtilis

AC7 te karakteriseren, waren in overeenstemming met de biochemische eigenschappen van B.

subtilis zoals beschreven door Koneman (1995).

Vervolgens werd de kritische micellaire concentratie van biosurfactant AC7 bepaald door de

oppervlaktespanning van verschillende concentraties biosurfactant AC7 te meten. Aangezien de

resultaten bevestigen dat er een potent biosurfactant met laag moleculair gewicht aanwezig was,

kan men zeggen dat B. subtilis AC7 capabel is om een goed werkzaam biosurfactant te produceren.

Het anti-adhesief effect van biosurfactant AC7, geproduceerd door B. subtilis, op Candida

albicans 40 werd geëvalueerd op silicone disks onder verschillende condities. De methode die

gebruikt werd om de disks te coaten met biosurfactant AC7 is een aanpassing van de methode

beschreven door Rivardo et al. (2009). De C. albicans biofilm vorming werd geëvalueerd volgens

Chandra et al. (2009). Biosurfactant AC7 vertoonde een goede anti-adhesieve activiteit tegenover 40

op elastomere silicone disks. De verschillende condities qua pre-coating en incubatie gaven

gelijkaardige resultaten. Deze toonden aan dat de aanhechting van C. albicans aan elastomere

silicone disks verlaagd kan worden door applicatie van biosurfactant AC7 en dat het effect verbeterd

kan worden door te werken bij lagere temperaturen. Hieruit kan men afleiden dat temperatuur een

belangrijke parameter is wanneer surfactantia als anti-adhesieve agentia bestudeerd worden. Deze

anti-adhesieve activiteit bleek echter enkel effectief tijdens de eerste fase van de adhesie.

Uiteindelijk werden positieve resultaten verkregen voor de eerste screening van biosurfactant AC7.

Optimalisatie van de methode door een reële conditie, gelijkaardig aan het menselijke lichaam, te

creëren kan een belovend vooruitzicht zijn.

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Thanks to…

Als eerste wil ik graag Prof. Dr. H. Nelis bedanken voor het grondig en kritisch nalezen van

mijn werk. Verder wil ik hem bedanken om mij de kans te hebben geboden om via mijn

thesis een buitenlandse ervaring te beleven.

I also would like to thank Prof. L. Fracchia and Dr. M. Cavallo, for their support during my

labwork and help with my thesis report. Thanks a lot to all the collaborators for the good

atmosphere at the lab.

Vervolgens wil ik mijn ouders, broer, vriend, familie en vrienden bedanken voor de continue

steun die ik van hen kreeg tijdens mijn verblijf in het buitenland.

Subsequently, I would like to thank all the people of the residence in Vercelli. I spend a great

time with them during my stay in Italy.

Ten laatste, maar zeker niet in het minst, wil ik graag mijn reisgenoot Elien bedanken voor de

steun in het labo en voor de supermomenten die wij samen beleefd hebben.

Aan allen een welgemeende dank.

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LIST OF ABBREVIATIONS

ABC ATP-binding cassette

B. subtilis Bacillus subtilis

BS Biosurfactant

C. albicans Candida albicans

CFU Colony Forming Units

CMC Critical Micelle Concentration

CMC Carboxymethylcellulose

CSLM Confocal scanning laser microscopy

CV Crystal violet

CVC-RI Central venous catheter-related infections

EPS Extracellular polymeric substance

ECM Extracellular matrix

FBS Fetal Bovine Serum

HIV Human immunodeficiency virus

LB Luria Bertani

M Molair = 1 mol per Liter

MAPK Mitogenactivated protein kinase

MELs Mannosylerythritol lipids

MFS Major facilitator superfamily

MIC Minimal inhibitory concentration

MRSA Methicillin-resistant Staphylococcus aureus

OD Optical density

PBS Phosphate Buffered Saline

RI Refractive Index

rpm rotations per minute

SAC Surface-active compounds

SDA Sabouraud Dextrose Agar

SEM Scanning electron microscopy

T Transmittance

YNB Yeast Nitrogen Base

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1. INTRODUCTION .............................................................................................................. 1

1.1. BIOFILM .......................................................................................................................... 1

1.1.1. Definition ............................................................................................................................. 1

1.2. CANDIDA ALBICANS ........................................................................................................ 2

1.2.1. Candida albicans: cell structure and virulence .................................................................... 2

1.2.2. Candida albicans biofilm ..................................................................................................... 4

1.2.3. Candida albicans biofilm resistance mechanisms ............................................................... 5

1.2.3.1. Physiology .................................................................................................................... 6

1.2.3.2. Density ......................................................................................................................... 6

1.2.3.3. Mutations .................................................................................................................... 7

1.2.3.4. Overexpressed targets ................................................................................................ 7

1.2.3.5. Efflux ............................................................................................................................ 7

1.2.3.6. Extracellular matrix ..................................................................................................... 7

1.2.3.7. Persisters ..................................................................................................................... 8

1.3. BIOSURFACTANTS ........................................................................................................... 8

1.3.1. Definition ............................................................................................................................. 8

1.3.2. Classification ........................................................................................................................ 9

1.3.2.1. Low molecular weight compounds ............................................................................. 9

1.3.2.2. High molecular weight compounds ........................................................................... 10

1.3.3. Biosurfactans versus synthetic surfactants ....................................................................... 10

1.3.4. Characteristics and functions ............................................................................................ 10

1.3.4.1. Antimicrobial activity of biosurfactants .................................................................... 11

1.3.4.2. Anti-adhesive activity of biosurfactants .................................................................... 12

1.4. INFECTIONS ON MEDICAL DEVICES .............................................................................. 13

1.4.1. Infections on central venous catheters ............................................................................. 15

2. OBJECTIVES ................................................................................................................... 17

3. MATERIALS AND METHODS ......................................................................................... 18

3.1. MATERIALS ................................................................................................................... 18

3.1.1. Instruments ....................................................................................................................... 18

3.1.2. Strains ................................................................................................................................ 18

3.1.2.1. Bacillus sp. AC7 .......................................................................................................... 18

3.1.2.2. Candida albicans 40. .................................................................................................. 18

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3.1.3. Media ................................................................................................................................. 19

3.1.4. Reagents ............................................................................................................................ 19

3.2. BIOSURFACTANT AC7 PRODUCTION ............................................................................ 20

3.3. PREPARATION OF BIOSURFACTANT AC7 STOCK SOLUTION ........................................ 20

3.4. CRITICAL MICELLE CONCENTRATION ........................................................................... 20

3.5. CHARACTERIZATION BACILLUS SUBTILIS AC7 STRAIN : BIOCHEMICAL TESTS ............. 21

3.5.1. Oxidase test ....................................................................................................................... 21

3.5.2. Catalase test ...................................................................................................................... 21

3.5.3. Tests on nitrate reduction, indole production, urease activity, esculin hydrolysis and

gelatin hydrolysis. .............................................................................................................................. 22

3.5.4. Motility test ....................................................................................................................... 22

3.5.5. Growth in 6% NaCl LB-broth .............................................................................................. 22

3.5.6. Protease test...................................................................................................................... 23

3.5.7. Cellulase test ..................................................................................................................... 23

TABLE 3.2. : COMPOUNDS PRESENT IN THE MEDIUM FOR THE CELLULASE TEST. ........................... 23

3.6. INHIBITION OF C. ALBICANS-ADHESION ON SILICONE USING PRE-COATING WITH

BIOSURFACTANT AC7 ............................................................................................................... 24

3.6.1. Treatment of the silicone elastomeric disks ..................................................................... 24

3.6.2. Pre-coating method ........................................................................................................... 25

3.6.2.1. Pre-coating phase ...................................................................................................... 25

3.6.2.2. Adhesion phase ......................................................................................................... 26

3.6.3. Quantification of Candida albicans 40 adhesion to silicone disks by crystal violet staining

method 27

3.6.4. Quantification by plate counting ....................................................................................... 28

3.6.5. Calculation of C. albicans 40 inhibition of adhesion to silicone disks ............................... 28

3.6.6. Statistical analysis .............................................................................................................. 29

4. RESULTS ........................................................................................................................ 30

4.1. CRITICAL MICELLE CONCENTRATION OF BIOSURFACTANT AC7 ................................. 30

4.2. BIOCHEMICAL TESTS FOR THE CONFIRMATION OF THE IDENTITY OF THE

BIOSURFACTANT AC7 PRODUCING STRAIN ............................................................................ 31

4.2.1. Catalase test, oxidase test, motility test and growth at 6% NaCl ..................................... 31

4.2.2. Tests on nitrate reduction, indole production, urease, esculin hydrolysis and gelatin

hydrolysis. .......................................................................................................................................... 31

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4.2.3. Protease test...................................................................................................................... 32

4.2.4. Cellulase ............................................................................................................................. 32

4.3. ANTI-ADHESIVE ACTIVITY OF BIOSURFACTANT AC7 ON SILICONE ELASTOMERIC DISKS:

CRYSTAL VIOLET STAINING ....................................................................................................... 33

4.3.1. Negative control ................................................................................................................ 33

4.3.2. Pre-coating of disks at 37°C and incubation at 37°C for 90 minutes ................................ 34

4.3.3. Pre-coating of disks 37°C and Incubation for at 37°C 24 hours........................................ 36

4.3.4. Pre-coating of disks at 4°C and incubation at 4°C for 90 minutes .................................... 36

4.3.5. Pre-coating at 4°C and incubation at 37°C for 90 minutes ............................................... 38

4.3.6. Pre-coating at 37°C and incubation at 4°C for 90 minutes................................................ 39

4.3.7. Pre-coating at 25°C and incubation at 37°C for 90 minutes.............................................. 41

4.3.8. Pre-coating at 25°C and incubation at 37°C for 4 hours ................................................... 42

4.4. ANTI-ADHESIVE ACTIVITY OF BIOSURFACTANT AC7 ON SILICONE ELASTOMERIC DISKS:

PLATE COUNTING METHOD ..................................................................................................... 43

4.4.1. Pre-coating at 25°C and incubation at 37°C for 90 minutes.............................................. 43

4.4.2. Pre-coating at 25°C and incubation at 37°C for 4 hours ................................................... 44

5. DISCUSSION .................................................................................................................. 45

5.1. CRITICAL MICELLE CONCENTRATION ........................................................................... 45

5.2. CHARACTERISATION OF BACILLUS SUBTILIS AC7: BIOCHEMICAL TESTS ...................... 45

5.3. ANTI-ADHESION ACTIVITY OF BIOSURFACTANT AC7 ON SILICONE ELASTOMERIC DISKS

46

5.4. FUTURE PERSPECTIVE ................................................................................................... 49

6. CONCLUSION ................................................................................................................ 50

7. BIBLIOGRAPHY .............................................................................................................. 51

1

1. INTRODUCTION

1.1. BIOFILM

1.1.1. Definition

“The microbial biofilm, composed by a single or by multiple species, is defined as a

sessile community of microbial cells irreversibly attached to a substratum or an interface and

also among them, embedded in a matrix of extracellular polymeric substances as their own

products, exhibiting a modified phenotype concerning the rate of growth and gene

transcription (Lazar and Chifiriuc, 2010).” Bacteria and fungi are able to change between

planktonic growth and sessile multicellular communities, biofilms. Up to 80% of all

microorganisms in the environment live in biofilm communities (Donlan, 2002).

FIGURE 1.1. CYCLE OF BIOFILM FORMATION. (1) PLANKTONIC CELLS ATTACH TO THE SURFACE. (2)

PRODUCTION OF EXTRACELLULAR POLYMERIC SUBSTANCE (EPS) MAKES THE ATTACHMENT IRREVERSIBLE. (3)

AND (4) GROWTH, DEVELOPMENT AND MATURATION OF THE BIOFILM. (5) FREE PLANKTONIC CELLS ARE

RELEASED. (http://www.advancedhealing.com/blog/2009/09/25/dr-ettingers-biofilm-

protocol-for-lyme-and-gut-pathogens/)

As illustrated in figure 1.1., the cycle of biofilm formation consists of several steps. First

planktonic cells, cells which are free living, adhere to the surface. Then, due to the

production of extracellular polymeric substance (EPS) the adherence becomes irreversible,

so the cells are fully attached to the surface. Subsequently, the biofilm develops further and

matures. The last step is the release of free planktonic cells, a process called “biofilm

dispersal” and so the cycle of biofilm formation is complete (Shunmugaperumal, 2010).

2

Morphology and architecture of biofilms are examined using fluorescence

microscopy, scanning electron microscopy (SEM) an confocal scanning laser microscopy

(CSLM) SEM is able to show more details of the surface morphology than fluorescence

microscopy. Moreover, CSLM allows a visualization of the three-dimensional (3D)

architecture at different depths of the biofilms and can also measure the biofilm thickness

without destroying it (Chandra et al., 2001).

1.2. CANDIDA ALBICANS

1.2.1. Candida albicans: cell structure and virulence

Candida albicans (C. albicans) is a normal fungal human commensal, living in the oral

cavity, the gastrointestinal tract, skin and vagina (Ramage et al., 2011). It is an opportunistic

pathogen (Ruiz-Herrera et al., 2005), which means that when a patient is

immunosuppressed, like in the case of HIV (Human immunodeficiency virus) infection, cancer

chemotherapy, immunosuppressive drugs, diabetes and old age, the microorganism

becomes pathogenic (Odds, 1987; Sandven, 2000).

FIGURE 1.2. : SCHEMATIC VISUALIZATION OF CANDIDA ALBICANS AS A COMMENSAL ORGANISM AND ITS

PATHOGENIC STATUS IN AN IMMUNOCOMPROMISED HOST (Ruiz-Herrera et al., 2005).

In those cases, C. albicans becomes pathogen and can cause serious diseases,

commonly designated as candidiasis (Ruiz-Herrera et al., 2005; Kojic and Darouiche, 2004).

This process is illustrated in figure 1.2..

3

The outer surface of C. albicans, as shown in figure 1.3., is composed of some typical

compounds: lipids, chitin, glucans and proteins (Ruiz-Herrera, 2005). Phospholipomannan is

a lipid at the outer surface of C. albicans and assumed to have a function in adhesion,

protection and signaling. Mutation in a gene necessary for the synthesis of

phospholipomannan reduced virulence in a mouse model (Mille et al., 2004). Chitin is a

polysaccharide of N-acetylglucosamine units linked by β-1-4-bonds (Ruiz-Herrera et al.,

2005). As chitin is not present in the human host, it would be a good target for antifungals.

Two inhibitors of chitin synthesis, polyoxins an nikkomycins, had high activity against

Candida albicans growth in vitro but not in vivo. So their chemical structure has to be

modified to obtain the in vivo antifungal activity (Ruiz-Herrera and San-Blas, 2003). C.

albicans only possesses β-glucans in its cell wall, which is the main polysaccharide in fungal

cells. This polymer of glucose contains both β-1-3 and β-1-6 glycosidic linkages (Ruiz-Herrera

et al., 2005). Wall proteins play several roles in C. albicans, they are crucial for its life. Cell

wall proteins include enzymes and structural proteins. They further play a role in

pathogenicity and the immune response (Ruiz-Herrera et al., 2005).

FIGURE 1.3. : VISUALISATION OF THE COMPOSITION OF THE CELL WALL AND CYTOPLASMATIC MEMBRANE OF

CANDIDA ALBICANS (Kartsonis et al., 2003).

The cell wall of C. albicans plays a crucial role in the pathogenesis because: (1) it is the

contact point with the host cells, (2) it possesses important antigens on its surface, (3) it

mediates adherence and (4) it is responsible for the cross-talk with the host, as it possesses

the “glycan code” (Ruiz-Herrera et al., 2005). The glycan code is determined by the nature of

4

the sugar, the anomer type of linkage and branching, and the length of the oligosaccharide

chains (Poulain and Jouault, 2004). C. albicans is able to regulate its glycan code to interact

with the host (Poulain and Jouault, 2004).

C. albicans has several virulence factors : adhesins, filament formation, proteolytic and

lipolytic enzymes and probably phenotypic switching. Adherence to host cells of C. albicans

is mediated by adhesins, which is a first virulence factor (Calderone and Fonzi, 2001). As C.

albicans is a dimorphic fungus, it is able to switch from the yeast to the hyphal filamentous

formation during tissue invasion. This morphological change is a second important virulence

factor. Further, hydrolytic enzymes destroy the cytoplasmatic membrane of cells when

Candida invades the body. Proteolytic and lipolytic enzymes cleave bonds in proteins and

lipids, respectively. They degrade host surface molecules and they can also attack molecules

of the host defense system (Schaller et al., 2005). Finally, phenotypic switching makes C.

albicans able to adapt to changes in the environment when it invades a host (Soll, 2002).

1.2.2. Candida albicans biofilm

Candida albicans biofilm formation has three phases of development: (1) early phase:

adherence of yeast cells to the surface, (2) intermediate phase: formation of a matrix with

switching from yeast to hyphal and pseudohyphal forms, (3) maturation phase: increase of

the matrix and formation of a three-dimensional architecture (Chandra et al, 2001; Hawser

et al.,1994).

FIGURE 1.4. : GROWTH FORMS OF C. ALBICANS.

(http://overcomingcandida.com/images/

candida_gallery/candida_albicans_stages.jpg)

5

Different morphological forms, which are shown in figure 1.4., are important in

biofilm formation. For example, the wild-type of C. albicans produced a two-layer biofilm,

whereas one mutant, incapable of yeast growth, produced only the basal layer and another

mutant, incapable of hyphal growth, only the outer layer (Kojic and Darouiche, 2004). So

dimorphism appears to be needed for the full biofilm architecture and structure and is a

crucial element for the pathogenicity of C. albicans (Baillie and Douglas, 1999).

Light microscopy has demonstrated that at first (2h-4h) only budding yeast cells are

present in early biofilm formation. Later (4h) the budding yeast cells start to filament

forming pseudo-hyphae and real hyphae.

Confocal scanning laser microscopy (CSLM) has demonstrated that C. albicans

biofilms possess a complex three dimensional architecture, with structural heterogeneity

and channels, which allow the influx of nutrients and the efflux of waste products and the

organization of micro-niches throughout the biofilm (Ramage et al., 2001).

Candida biofilms have been studied by in vitro and in vivo models (Tournu and Van

Dijck, 2011). In vivo models are more important because they simulate the real situation in

an animal. In vitro methods are useful at first because of their lower cost.

Microbial biofilms are notoriously resistant many antimicrobial agents, including

antibiotics, antiseptics and industrial biocides. Bacterial cells in biofilms are 10-1000 times

more resistant to antibiotics than planktonic cells (Donlan and Costerton, 2002).

Only caspofungin has been shown to be fully effective against C. albicans biofilms (Ramage

et al, 2002).

1.2.3. Candida albicans biofilm resistance mechanisms

According to Ramage et al. (2011) there are a lot of elements which enhance fungal

biofilm resistance, so the resistance is multifaceted. The following section lists some crucial

elements of fungal biofilm resistance (figure 1.5.).

6

FIGURE 1.5. : OVERALL VIEW OF C. ALBICANS BIOFILM RESISTANCE MECHANISMS (Ramage et al., 2011).

1.2.3.1. Physiology

The physiological state of the C. albicans cells play a minor role in the resistance of biofilms

(Ramage et al., 2011). Using of the XTT-assay, it has been confirmed that biofilm cells are

doing mitochondrial respiration during their development (Chandra et al, 2001). C. albicans

biofilms, growing under conditions of glucose or iron limitation (Baillie and Douglas, 1998) or

in an anaerobic environment were all highly resistant to amphotericin B, a polyene

antifungal drug (Dumitru et al., 2004).

1.2.3.2. Density

The more dense the structure of a biofilm is, the more resistance to it becomes to

antibiotics. Perumal et al. (2007) showed that planktonic and resuspended biofilm cells were

susceptible to azoles at a cell concentration of 10³ cells/mL, but became highly resistant

when the cell concentration was increased tenfold. In a biofilm, there is quorum sensing,

defined as: “The ability of microorganisms to communicate and coordinate their behavior via

the secretion of signaling molecules in a population-dependent manner (Mukherjee et al.,

2003)”. They become “social” and they work together. So cell density is a second important

factor influencing fungal biofilm resistance. (Ramage et al., 2011).

7

1.2.3.3. Mutations

Mutations, such as point mutations in genes, can lead to a modified target with lower

or no affinity for a drug. For example, the target of azoles is the 14α-demethylase enzyme

encoded by ERG11. 14α-demethylase is an important enzyme in the ergosterol biosynthesis.

Azoles block this enzyme and hence the ergosterol biosynthesis, which leads to

accumulation of toxic sterol intermediates and inhibits growth (Akins, 2005; Cannon et al.,

2007). When a mutation in this gene occurs, the azole can no longer bind to its target, so the

activity of the antifungal agent is lost (reviewed by Ramage et al., 2011).

1.2.3.4. Overexpressed targets

When C. albicans biofilms are highly exposed to antimycotica such as fluconazole,

genes encoding the target for antimycotics, e.g. the 14α-demethylase in the ergosterol

biosynthetic pathway, may be upregulated (increased transcription) so that there is an

overexpression of the target. As a result, the target can no longer be saturated by the

antimycotic (e.g. fluconazole) and its activity is lost (reviewed by Ramage et al., 2011).

1.2.3.5. Efflux

Another kind of resistance is due to the increased expression of efflux pumps. The

main two types of efflux are mediated by the ATP-binding cassette (ABC) and the major

facilitator superfamily (MFS) transporters respectively (Albertson et al., 1996; Lopez-Ribot et

al., 1999; Sanglard et al. 1997). Efflux accounts for the insensitivity of C. albicans to azoles

and triazoles, e.g. (Williams et al., 2011; White, 1997). Echinocandins are not susceptible to

the action of efflux pumps (Miller and Bassler, 2001).

1.2.3.6. Extracellular matrix

The extracellular matrix (ECM) protects the cells in the biofilm from host immunity

and antifungal compounds (Ramage et al., 2011). The ECM is composed of proteins,

hexosamine, phosphate, uronic acid, and carbohydrates (Al-Fattani and Douglas; 2006). One

of the most important carbohydrates in the ECM of Candida albicans is beta- 1,3-glucan,

8

because beta-1,3-glucanase enhances the detachment of biofilms from their substrate (Al-

Fattani and Douglas; 2006). Moreover, beta-1-3-glucanase enhances the activity of

fluconazole and amphotericin B against Candida albicans biofilm (Nett et al., 2007). The

synthesis of ECM by biofilm cells is less in static than in dynamic conditions (Al-Fattani and

Douglas; 2006). So biofilm formation is stimulated on mucosal and abiotic sites where there

is a fluid flow, such as on the oral mucosa, the urethra, or central venous catheters.

1.2.3.7. Persisters

Persister cells defined as “dormant variants of regular cells that form stochastically in

microbial populations and are highly tolerant to antibiotics (Lewis, 2010)”. They are not

mutants but phenotypic variants and occur as a small fraction in the biofilm. Persisters play

an important role in chronic infections (Fauvart et al., 2011). When antibiotics/antimycotics

are given, the majority of cells are killed but persisters survive the treatment. After the

antibiotic therapy is finished, the persister cells repopulate and the infection relapses (Lewis,

2001, 2010).

1.3. BIOSURFACTANTS

1.3.1. Definition

Biosurfactants are amphiphilic molecules, produced extracellularly or part of the cell

membrane by microorganisms (Mulligan, 2005). Many organisms, such as bacteria, yeasts

and fungi, are able to produce a wide range of amphipathic compounds, with both an

hydrophilic and hydrophobic part present in the same molecule. They are called

biosurfactants or bioemulsifiers. They reduce surface and interfacial tensions at liquid-solid

interfaces and the interfacial tension between immiscible liquids or at the solid-liquid

interfaces,respectively (Smyth et al., 2010a, 2010b). An interfacial film is formed by

biosurfactants or surface-active compounds (SAC) in heterogeneous systems. This interfacial

film influences properties of the surface such as surface energy and wettability (Fracchia et

al., 2011). Biosurfactants are considered to play a major role as the survival of the producing

9

organisms because they improve the transport of nutrients, interaction with a host and they

can act as biocidal agents (Rodrigues et al., 2006a).

1.3.2. Classification

Surface-active compounds are classified (SAC) according to their structural

properties, the producing organism and their molecular mass. Their hydrophilic part

generally consists of an acid, peptide cations, or anions, mono-, di- or polysaccharides, while

the hydrophobic part contains a unsaturated or saturated hydrocarbon or fatty acid chain

(Chen et al., 2010a, 2010b).

1.3.2.1. Low molecular weight compounds

Low molecular weight biosurfactants include lipopeptides and glycopeptides.

Many Bacillus species produce lipopeptides. The latter vary in their fatty acid and

their peptide chains (Jacques, 2010; Thavasi et al., 2011). Surfactin, produced by Bacillus

subtilis (B. subtilis) and discovered by Arima and collaborators (1968), is considered as the

most active biosurfactant known so far (Ron and Rosenberg, 2001). Surfactin can lower the

surface tension of water, from 72 mN m-1 to 27 mN m-1 at a concentration of 500 µg/mL

(Arima et al., 1968) . This cyclic lipopeptide is composed of a seven amino-acid ring structure

coupled to a fatty-acid chain by a lactone linkage (Jacques, 2010). Due to the presence of

two negative charges in the surfactin molecule, as shown in figure 1.6., surfactin can bind to

heavy metals, such as zinc and copper, and remove them from contaminated soil and

sediments (Thimon et al., 1992, Mulligan, 2005).

FIGURE 1.6. : MAIN LOW MOLECULAR WEIGHT BIOSURFACTANT: SURFACTIN (Mulligan, 2005).

Another compound of which the chemical structure and physio-chemical properties

are similar to those of surfactin is lichenysin. It is produced by Bacillus licheniformis

(Horowitz et al., 1990). Other important biosurfactants belonging to the lipopeptides are

10

those of the iturin and the fengycins families (Jacques, 2010). Serrawettins are also

lipopeptides. These are nonionic cyclodepsipeptide biosurfactants produced by Serratia

marcescens (Matsuyama et al., 2010) and have anti-tumor and anti-nematode activities.

Glycolipids are mostly mono- or disaccharides acylated with long chain fatty acids or

hydroxyl fatty acids. Best studied subclasses are rhamnolipids, mannosylerythritol lipds

(MELs), sophorolipids and thehalolipids (reviewed by Fracchia et al., 2011).

1.3.2.2. High molecular weight compounds

High molecular weight compounds are polymeric biosurfactants. They are composed

of lipoproteins, proteins, polysaccharides, lipopolysaccharides or complexes containing

several of these structures. The best studied one is emulsan, which is a lipopolysaccharide

isolated from Acinetobacter calcoacetius RAG-1 31012. It is a complex of an anionic

heteropolysaccharide and a protein (Ron and Rosenberg, 2001; Rosenberg and Ron, 1997,

1999).

Alasan, a complex of an anionic polysaccharide and a protein, is another high

molecular weight biosurfactant isolated from Acinetobacter radioresistens (Smyth, 2010b).

Because of their high emulsifying activities, high molecular weight biosurfactants are also

called bioemulsifiers (Smyth et al., 2010b).

1.3.3. Biosurfactans versus synthetic surfactants

Biosurfactants have several advantages over synthetic surfactants. Due to their

biological origin they are more biodegradable (Zajic et al., 1977) and more compatible with

the environment. Besides they exhibit a lower toxicity, high selectivity and a specific activity

at extreme temperature, pH and salinity (Banat et al. 2000; Banat, 1995a, 1995b; Wei et al.,

2003). Because of their biological origin, biosurfactants are generally considered safer than

synthetic surfactants.

1.3.4. Characteristics and functions

Because of its amphiphilic nature, the main characteristic of a biosurfactant is their

ability to lower the water surface tension. Drop collapse (Bodour and Miller-Maier, 1998)

11

and oil spreading test (Morikawa et al., 2000) are two easy, fast and often used methods to

measure the surface activity of biosurfactants. The surface or interfacial tension will

decrease until the biosurfactant reaches the critical micelle concentration (CMC). When the

CMC is reached, biosurfactant monomers start to associate and form several typical

structures such as micelles, vesicles and bilayers. Those structures are not a consequence of

formation of covalent bounds. Only hydrophobic, van der Waals and hydrogen bounds are

present (Maier, 2003; Raza et al,2010). The CMC is defined as the minimum concentration

necessary to initiate micelle formation (Becher, 1965). The orientations of the hydrophobic

and hydrophilic parts depend on the polarity of the solution. In highly polar solutions, such

as aqueous solutions, the hydrophilic head group will be directed to the fluid, while the

hydrophobic tails will be oriented to each other to protect themselves from the polar

solution. In oil, a highly nonpolar solution, the hydrophilic groups will be directed to each

other while the hydrophobic tails have affinity for the nonpolar solvent and so they will be

oriented towards the oil (Soberon-Chavez and Maier, 2010). The surfaces of microorganisms

are generally negatively charged, so the ionic conditions and pH influence the interaction

between the biosurfactant and the microbial surface (Craig et al., 1993). The CMC is also

dependent on the temperature (Mulligan, 2005).

1.3.4.1. Antimicrobial activity of biosurfactants

During the last decades, few antibiotics have been explored. Some biosurfactants

have antimicrobial activity, they have been evaluated as potential alternatives for antibiotics

(Hancock and Chapelle, 1999). Lipopeptides possess the highest antimicrobial activity

because they are able to self-associate and form pore-bearing channels or micellar

aggregates inside a lipid membrane (Carillo et al., 2003; Deleu et al., 2008). Surfactin has

antimicrobial activity. It can influence the ordering of the fatty acid chains because it can

disrupt the membrane due to hydrophobic interactions (Bonmatin et al., 2003). A

biosurfactant produced by Bacillus circulans showed antimicrobial activity against several

bacteria including methicillin-resistant Staphylococcus aureus (MRSA). So this biosurfactant

could possibly be used as antimicrobial agent (Das et al., 2008). Another example is the

antimicrobial lipopeptide, daptomycin, which is produced by Streptomyces roseosporus. It is

12

highly active against multi-resistant bacteria such as MRSA and has been commercialized as

Cubicin® (reviewed by Fracchia, 2011).

1.3.4.2. Anti-adhesive activity of biosurfactants

Biosurfactants have been demonstrated to inhibit the adhesion of pathogenic

organisms to surfaces or infection sites (Rodrigues et al, 2006a). As adherence is the first

step of biofilm formation, it can decrease colonization and so avoid infections

(Shunmugaperumal, 2010; Singh and Cameotra, 2004). Moreover, antibiotics often

becoming highly resistant, so the anti-adhesion activity of biosurfactants might be an

effective therapeutic alternative (Fracchia et al., 2011).

Surfactin is often studied because of its anti-adhesion activity. It was able to avoid

biofilm formation of Salmonella Thyphimurium, Salmonella enterica, Escherichia coli and

Proteus mirabilis in polyvinyl chloride wells and vinyl urethral catheters (Mireles et al., 2001).

Rivardo and collaborators (2009) studied the two lipopeptide biosurfactants, produced by

Bacillus subtilis V9T14 and Bacillus licheniformis V19T21. Both biosurfactants inhibited the

formation of biofilms by pathogenic strains of Staphylococcus aureus and Escherichia coli on

polystyrene. Anti-adhesive activity has been studied in both co-incubation and pre-coating

experiments, where respectively the biosurfactant is added in the inoculum and

biosurfactant is pre-coated on the surface before incubation, respectively (Rivardo et al,

2009). Chemical analysis elucidated the chemical composition of lipopeptide V9T14. It

consists of 77% surfactin and 23% fengycin (Pecci et al., 2010).

Zeraik and Nitschke (2010) evaluated the effect of different temperatures (35°C, 25°C,

4°C) on the anti-adhesion of Staphylococcus aureus, Listeria monocytogenes and

Micrococcus luteus on polystyrene surfaces treated with surfactin biosurfactant. Pre-coating

for 24 hours and incubation for 4 hours and other time intervals was done at 35°C, 25°C and

4°C. The results are shown in table 1.1. The highest reduction of adhesion, 63 – 66%, was

obtained after pre-coating and incubation, both at 4°C. So, for surfactin, decreasing the

precoating temperature and incubation temperature, increases the reduction of adhesion

(Zeraik and Nitschke, 2010). According to Myers (2006), the high anti-adhesive reduction at

4°C may be due to the higher adsorption of a higher amount of biosurfactant molecules that

13

are adsorbed to the polystyrene surface at lower temperature. Moreover, at low

temperatures, the hydrophobicity, motility and other attachment factors (adhesins) of the

cells can be different, positively favoring the anti-adhesive activity of the biosurfactant

(Zeraik and Nitschke, 2010).

TABLE 1.1. : BACTERIAL INHIBITION OF ADHESION TO POLYSTYRENE SURFACES OF L. MONOCYTOGENES, S.

AUREUS AND M. LUTEUS BY SURFACTIN AT DIFFERENT PRECOATING AND INCUBATION TEMPERATURES

(Zeraik and Nitschke, 2010).

Hence, because of their antimicrobial and anti-adhesive activities the importance of

biosurfactants has increased. They are used for a variety of biomedical applications.

Moreover, no new or effective chemical antibiotics have been explored during the last

decades (Hancock and Chapelle, 1999). So biosurfactants might be appropriate alternatives

for synthetic drugs and antimicrobial compounds (Banat et al., 2000).

1.4. INFECTIONS ON MEDICAL DEVICES

Many nosocomial infections are associated with medical devices a lot of them are

caused by Candida species, especially those devices involving the bloodstream and the

urinary tract (Jarvis, 1995). These infections are resistant to immune defense mechanisms

and hard to treat with antimicrobial agents due to biofilm formation. (Habash and Reid,

1999). Microorganisms which grow in biofilms are less susceptible to antibiotics than

planktonic, free living cells of the same microorganism (Bryers, 2008; Costerton et al., 1999).

Microbial infections, highly resistant to antibiotics because of biofilm formation, have been

observed on a lot of medical devices: central venous catheters, urinary catheters, voice

14

prostheses, hip prostheses, contact lenses, etc. For this reason, medical devices, such as

central venous catheters (Sousa et al., 2011), silicone rubber voice prostheses (Rodrigues et

al., 2006), urinary catheters, heart valves, pacemakers (Kojic and Darouiche, 2004) and

contact lenses (Rodrigues et al., 2004b), are often coated with antimicrobial and anti-

adhesive agents to suppress biofilm formation. (Basak et al., 2009; von Eiff et al., 2005).

Rodrigues (2011) evaluated the effect of biosurfactants on refractive index (RI) and

transmittance (T) of contact lenses. As more biosurfactant was used, RI increased and T

decreased. As a result, the use of biosurfactants as coating agents on contact lenses is

limited.

After implantation of a medical device, there is a competition between integration of

the medical device in the tissue and colonization by bacteria on the surface of the implant.

For a good implant, the integration must be effective before the adhesion of bacteria

(Gristina, 1987). So, the most critical period is the 6 hours after implantation of the device

because in this time the implant is most susceptible to colonization of microorganisms on

the surface (Castelli et al., 2006; Poelstra et al., 2002). Catheter-related infections represent

a great risk for morbidity and mortality (Sousa et al., 2011). The adherence of bacteria to the

surfaces of medical devices is said to be the principal pathogenic mechanism of implant

infections (von Eiff et al. 2005).

Medical device-related infections can be prevented by prophylactic use of antibiotics

and biocides. They can reduce the biofilm formation on the devices. The use of these

antibiotics is controversial because of the increasing antimicrobial resistance, especially in

high-risk patient groups (Lynch and Robertson, 2008). As biofilms usually become highly

resistant to antibiotics and antimicrobial agents, the approach has been adopted in order to

limit the colonization by the microorganisms. So the anti-adhesive function of biosurfactant

gained importance (Fracchia et al., 2011).

Rodrigues and collaborators used biosurfactants produced by several Lactobacilli to

inhibit the adhesion of the pathogenic microorganisms to silicone rubber (Rodrigues et al.,

2004, 2006b, c, 2007).

15

Luna et al. (2011) and Gudina et al. (2010) determined respectively the anti-adhesive

and antimicrobial effect of biosurfactants produced by Candida sphaerica UCP and

Lactobacillus paracasei ssp. A20 on C. albicans and other microorganisms with success.

1.4.1. Infections on central venous catheters

Catheter-related bloodstream infections frequently occur in the intensive care unit.

They increase the duration of stay in the hospital, the costs and their morbidity is high

(McGee and Gould, 2003; Frasca et al., 2010). C. albicans is an important cause of central

venous catheter related infections (Donlan, 2008). A bloodstream infection caused by C.

albicans is called candidemia (Raad et al., 2003). Candidemia has been defined in Beck-

Sague and Jarvis (1993) as: “The isolation of Candida species from ≤ 1 blood culture specimen

associated with clinical manifestations of bloodstream infections, such as fever, chills, or

hypertension.”

Various proteins such as fibrin, fibrinogen, fibronectin, collagen, elastin, laminin,

vitronectin, thrombospondin or Willebrand’s factor form a film on the surface of the

catheter after insertion. They enhance microbial adhesion and biofilm formation (Casey et

al., 2008). A few days after insertion, a biofilm embedded in its extracellular matrix is

irreversibly attached to the catheter (Sekhar et al., 2010).

The nature of the material used for the catheter influences the biofilm formations by

C. albicans. On latex or silicone elastomer, biofilm formation was enhanced compared with

polyvinylchloride, whereas it was lower on polyurethane or 100% silicone (Hawser et

al,1994).

Central venous catheter-related infections (CVC-RI) are commonly treated with

antimicrobial agents, according to antimicrobial susceptibility test results. Treatments are

frequently unsuccessful because those agents were tested on planktonic cells, whereas

biofilms are much more resistant (Nadell et al., 2009). Removal of the catheter is often

required (Curtin et al., 2003).

Many strategies have been proposed to avoid biofilm formation on central venous

catheters. Some of them have been reviewed by Sousa et al. (2011): coating with substances

having antimicrobial or antiseptic properties, antimicrobial locks (ALT) and catheter surface

16

modification. In ALT a high concentration, 100- to 1000-fold higher than the minimal

inhibitory concentration, of antimicrobial agent is placed in a catheter in a volume adequate

to fill the lumen. The catheter is then “locked” into place for a long period to prevent its

colonization and thereby the risk of infection (Berrington and Gould, 2001; Lynch and

Robertson, 2008).

In conclusion, a lot of research is necessary to minimize or avoid biofilm formation on

central venous catheters in order to decrease CVC-RI (Sousa et al., 2011).

17

2. OBJECTIVES

The general aim of this thesis was to evaluate the ability of a biosurfactant produced by an

endophytic Bacillus subtilis strain (named AC7) to inhibit the adhesion of a Candida albicans

strain of nosocomial origin, isolated from a central venous catheter, to medial-grade silicone

disks. Specifically, the following aspects were studied.

1. The ability of biosurfactant AC7 to lower the surface tension in aqueous solution and

determination of the critical micelle concentration of biosurfactant AC7.

2. Confirmation of the identity of biosurfactant producing strain AC7, previously

genotypically identified as Bacillus subtilis. strain by various biochemical tests.

3. Anti-adhesive effect of biosurfactant AC7 on Candida albicans 40 on the adhesion to

silicone elastomeric disks

Influence of temperature on biosurfactant pre-coating. Effect of temperature on

C. albicans 40 adhesion to silicone.

Comparison of two different methods for the evaluation of the inhibition of C.

albicans 40 adhesion.

18

3. MATERIALS AND METHODS

3.1. MATERIALS

3.1.1. Instruments

PH-meter: HANNA instruments pH 11 microprocessor pH-meter

Autoclave: pbi international FEDEGARI AUTOCLAVI SPA

Magnetic stirrer: ARED heating magnetic stirrer high power VELP scientific

Centrifuge A: RC5B plus centrifuge, Sorvall, Haverhill, USA

Centrifuge B: Thermo Scientific, CL10 centrifuge

Sonicator: Elma S30H Elmasonic

Tensiometer: KSV Sigma 703D

Balance A: KERN 440-35N (sensitivity: 0.01g)

Balance B: Sartonius CP324S-OCE (sensitivity: 0.0001g)

Camera: Nikon coolpix 995

3.1.2. Strains

3.1.2.1. Bacillus sp. AC7

The biosurfactant (BS) producer strain of Bacillus subtilis AC7 is a bacterial

endophyte isolated from a Robinia pseudoacacia tree in Novara. These trees are native to

southeastern United States, but have been cultivated in Europe. Cultures of B. subtilis AC7

were stored at -80 °C in a Luria Bertani broth (LB) supplemented with glycerol (25%V/V).

When needed, the strain was thawed and grown at 28 °C on a LB-agar.

3.1.2.2. Candida albicans 40.

C. albicans 40 was isolated from a central venous catheter (courtesy of Hospital

“Maggiore della Carità”, Novara). Pure cultures of C. albicans 40 were stored at -80 °C in

Sabouraud dextrose broth. Glycerol (25%V/V) was added to prevent formation of water

crystals in the cells. When needed, the strain was thawed and cultured at 37°C on Sabouraud

dextrose agar.

19

3.1.3. Media

Sabouraud 4% Dextrose Agar (SDA) is a complex medium which promotes the growth

of fungi. SDA consists of 30 g SD broth (Fluka), 20 g glucose (Biolife), 15 g agar (Fluka),

dissolved in 1L deionized water (final pH 5.6).

Luria Bertani (LB) broth (Fluka) is a rich complex grow medium used for the growth of

many bacteria. One liter LB broth contains 10 g tryptone (Fluka), 5 g yeast extract (Sigma-

Aldrich) and 10 g NaCl (Sigma-Aldrich) (final pH 7).

The above mentioned media are sterilized by autoclaving for 15 minutes at 121 °C. The

pH was controlled by a pH-meter.

Yeast nitrogen base is a complex medium containing a variety of different minerals,

vitamins, amino acids and buffering compounds, which are essential for the growth of

yeasts. Ammonium sulfate is the source of nitrogen. 50 mM of glucose is added as carbon

source. YNB is termolabile and has to be sterilized by filtration. It is obvious to prepare first a

stock solution was prepared by dissolving 6.7 g of YNB (Fluka) and 9.01 g glucose (Biolife) in

100 mL of distilled water and sterilized the medium using a 0.22 µm filter. This stock solution

was stored at 4 °C. The work solution was prepared aseptically by diluting the stock solution

10 times with sterile distilled water. The final pH of the solution was 7.

3.1.4. Reagents

Phosphate buffered saline (PBS) is composed of 8 g NaCl (Sigma-Aldrich), 0.2 g KCl (Sigma-

Aldrich), 1.44 g Na₂HPO₄ (Sigma-Aldrich) and 0.42 g KH₂PO₄ (Sigma-Aldrich). These salts were

dissolved in 1L distilled water. The pH was adjusted to 7.4. The solution was mixed on a

magnetic stirrer and sterilized by autoclaving for 15 minutes at 121°C.

20

3.2. BIOSURFACTANT AC7 PRODUCTION

For the biosurfactant production a colony of Bacillus subtilis AC7 culture was

transferred into 20 mL LB broth. This suspension was incubated for 4 hours at 28°C. Two mL

of the pre-culture was transferred into 500 mL LB broth. This was done in quadruplicate.

The erlenmeyer flasks were incubated at 28°C for 24 hours at 140 rotations per minute

(rpm). Solutions were poured into centrifuge tubes and centrifuged for 20 minutes at

approximately 5000 rpm (Centrifuge A). The biosurfactant was extracted from the bacterial

supernatant with ethylacetate/methanol (4:1) (Sigma-Aldrich) according to the method

described by Rivardo et al. (2009). The remaining water in the organic phase was removed

by anhydrous sodium sulfate (Sigma-Aldrich). The organic phase was evaporated to dryness

under vacuum using a rotary evaporator RV10 (IKA). Then the biosurfactant was

resuspended with acetone (Sigma-Aldrich) and transferred into 1.5 mL tubes to dry

(Eppendorf). The biosurfactant was weighed after drying in the 1.5 mL tubes. In this way Lot

17/02/2012 AC7LB24h was produced.

3.3. PREPARATION OF BIOSURFACTANT AC7 STOCK SOLUTION

A solution of biosurfactant 2000 µg/mL (2 mg/mL) was made for use.

Hundred milligrams of biosurfactant AC7 were dissolved in 50 mL PBS by adding a few drops

NaOH 10 M. Finally, the solution was brought to pH 7 using 1 M HCl. This solution was

sterilized in a sterile flask by membrane filtration (0.22 µm) and stored at 4°C.

3.4. CRITICAL MICELLE CONCENTRATION

A biosurfactant solution of 2000 µL/mL in alkaline demineralized water (pH=8,3) was

prepared. The surface tension was measured by a tensiometer (KSV Sigma 703D). A platinum

ring was emerged each time in 20 mL of the solution. The force necessary to move the ring

from the liquid to the air phase was measured by placing the ring underneath the surface of

the solutions and moving it slowly, with a constant velocity, out of the liquid into the air.

21

MilliQ water (Millipore, Italy) was used for calibration. Critical micelle concentration (CMC)

was determined on serially diluted (1:2) biosurfactant solutions in MilliQ water. Dilutions

were made with alkaline demineralized water (pH= 8,3). The surface tension of each dilution

was measured in quadruplicate. A curve was plotted with the surface tension (mN/m) as a

function of the concentrations of biosurfactant AC7 (µg/mL). The curve consisted of a

concentration dependent and a concentration independent section. The CMC was calculated

from the intercept of the regression lines in the concentration dependent and concentration

independent region of the curve. The corresponding concentration of biosurfactant was the

CMC. This experiment was done in duplicate.

3.5. CHARACTERIZATION BACILLUS SUBTILIS AC7 STRAIN : BIOCHEMICAL TESTS

In order to better characterize the biosurfactant AC7 producing strain, genotypically

identified as Bacillus subtilis by DSMZ in German, some tests were chosen among those

indicated by Koneman et al. (1995) for the identification of Bacilli. Avicelase,

carboxymethylcellulase and protease tests were also carried out in order to evaluate some

enzymatic activities of this strain that could be useful for biotechnological applications.

3.5.1. Oxidase test

B. subtilis AC7 was plated on a LB agar and incubated for 24 hours at 28°C. A loopfull of

B. subtilis AC7 was spread on an oxidase detection strip (Microgen products) with a plastic

sterile loop. The appearance of a purple color on the strip indicated that the cytochrome

oxidase enzyme is present. Pseudomonas fluorescens, a typically oxidase-positive species,

was used as a positive control.

3.5.2. Catalase test

B. subtilis AC7 was plated on a LB agar and incubated for 24 hours at 28°C. One colony

of B. subitilis was dissolved in a drop of 3% H₂O₂ deposited on a glass microscope slide. The

formation of bubbles indicates the presence of the catalase enzyme.

22

3.5.3. Tests on nitrate reduction, indole production, urease activity, esculin

hydrolysis and gelatin hydrolysis.

Five tests of the API NE kit (Biomérieux) were used in order to evaluate nitrate

reduction, indole production, urease activity, esculin hydrolysis and gelatin hydrolysis.

Instructions of the API 20 NE test were applied. Distilled water was distributed into the

bottom of the tray to create a humid atmosphere. Some colonies of B. subtilis AC 7 were

diluted in 2 mL of API NaCl 0.85% medium. Tests were inoculated by distributing the saline

suspension into the tubes very slowly to avoid the formation of bubbles. Paraffin oil was

added to the cupule of the urease test until a convex meniscus was formed. Reading and

interpretation were done according to the API 20 NE instruction sheet.

3.5.4. Motility test

The motility was carried out using the method described by Koneman et al. The growth

medium used is outlined in table 3.1.. The low agar concentration allows the strain to move

easily through it. Six milliliters of the medium were added into sterile tubes. Bacillus subtilis

AC7 strain was inoculated in the medium with a sterile plastic needle. Diffusion of the B.

subtilis strain besides the inoculation point indicates mobility (Koneman et al., 1995).

Compound quantity

Meat extract (Microbiol) 3 g

Peptone (Fluka) 5 g

Agar (Fluka) 4 g

Distilled water 1000 mL

Final pH 7.3

TABLE 3.1. : GROWTH MEDIUM FOR MOTILITY TEST

3.5.5. Growth in 6% NaCl LB-broth

Twenty milliliters of 6% LB-broth were poured into a 100 mL Erlenmeyer flask and

inoculated with one colony of B. subtilis AC7. Growth was observed after incubation at 28 °C

for 24 hours at 140 rpm (Koneman et al., 1995).

23

3.5.6. Protease test

The ability of B. subtilis to hydrolyze casein (a protease activity) was tested

qualitatively on skim milk-agar. The medium was composed of 20% skim milk powder

(Oxoid) dissolved in 100 mL distilled water and a medium composed of 2% agar (Fluka)

dissolved in 100 mL distilled water, was autoclaved separately, at 121°C for 15 minutes,

cooled to 55 °C and mixed with the first one. This test was preformed with both a fresh B.

subtilis culture and with a liquid culture. For the solid culture, one B. subtilis colony was

inoculated in the middle of the plate. For the liquid culture, a hole was made in the middle of

the skim milk agar plates by a sterile punch and 50 µL of the bacterial supernatant was

poured into the hole. Both tests were done in triplicate. The plates were incubated at 45°C

for 12 hours. Protease activity was indicated by a clear halo around the bacterial

colony/supernatant (Dean and Ward, 1991; Koneman et al., 1995).

3.5.7. Cellulase test

The ability of B. subtilis AC7 to degrade carboxymethylcellulose and crystalline

cellulose was assessed. 1 % carboxymethylcellulose (CMC) (Fluka) and 1 % crystalline

cellulose (Avicel – Sigma) were added respectively into the 2 different bottles which

contained the medium described in table 3.2..

Compound quantity

Ferric citrate (Sigma-Aldrich) 2.5 mg

Ammonium tartrate (Sigma-Aldrich) 125 mg

KH₂PO₄ (Sigma-Aldrich) 200 mg

MgSO₄.7H₂O (Fluka) 250 mg

CaCl₂.2H₂O (Fluka) 2.5 mg

MnSo₄.H₂O (Fluka) 1.9 mg

ZnSO₄.7H₂O ( Sigma-Aldrich) 2.5 mg

Yeast extract (Sigma-Aldrich) 25 mg

Agar (Fluka) 7.5 g

Distilled water 500 mL

TABLE 3.2. : COMPOUNDS PRESENT IN THE MEDIUM FOR THE CELLULASE TEST.

24

A loopfull of B. subtilis AC7 was spread on the cellulose media to form of a

quadrangle in the middle of the plate. The experiments were carried out in duplicate. A hole

was made in the middle of the agar by a punch to contain 50 µL of the supernatant of B.

subtilis AC 7 which has been incubated overnight in LB broth. Plates had been incubated for

5 days at 28°C. A solution of 1 mg/mL Congo red (composition shown in table 3.3.) was

added to the plates for 15 minutes. Congo red was then, poured off and plates were treated

with 4 mL 1 M NaCl for 15 minutes. Appearance of transparent areas around the bacterial

growth indicates a positive reaction (Teather and Wood, 1982).

Solution A Solution B

NaCl

(Sigma-Aldrich) 2 g

Congo red powder

(Sigma-Aldrich) 0.5 g

Distilled water 20 mL

Ethanol (Sigma-

Adlrich) 80 mL

TABLE 3.3. : COMPOSITION OF THE CONGO RED SOLUTION. SOLUTIONS A AND B

WERE MIXED TOGETHER TO OBTAIN THE WORKING SOLUTION.

3.6. INHIBITION OF C. ALBICANS-ADHESION ON SILICONE USING PRE-COATING WITH

BIOSURFACTANT AC7

3.6.1. Treatment of the silicone elastomeric disks

The disks were treated according to the method of Busscher et al. (1997). First the

disks were submerged into a 2% solution of RBS 35, which is an industrial detergent, and

sonicated for 5 minutes (Elma S30H Elmasonic) to detach the fatty acids from the surface.

This step is followed by two washes in 1 L ultrapure water to remove the RBS 35. Secondly

the disks were immersed in methanol and sonicated again for 5 minutes. The methanol was

then removed by two washes in 1L ultrapure water. Disks were transferred into a glass petri

dish (diameter of 12 cm) and deposited on a water absorbing paper in order to prevent the

attachment of the disks to the surface. Finally, the plate was sterilized by autoclaving for 15

minutes at 121 °C.

25

3.6.2. Pre-coating method

The method used to coat silicone disks with biosurfactant AC7 is a modification of the

method described by Rivardo et al. (2009). The evaluation of the C. albicans biofilm

formation was carried out according to Chandra et al. (2009).

3.6.2.1. Pre-coating phase

The experiment is carried out in 12 well plates (Greiner bio-one). The 12 well plates

are sterile, free of detectable DNase, RNase, human DNA and pyrogens. The material is non-

cytotoxic. Sterile disks were transferred with sterile tweezers into the twelve well plate. Two

milliliters of biosurfactant AC7 2000 µL/mL were added to six wells and 2 mL PBS were

added to the remaining six wells, as positive control. At first there were one negative

control and 5 positive controls. After knowing that the negative control is almost the same

every time, the negative control was replaced by another positive control. So then 6 samples

containing biosurfactant AC7 2000 µg/mL and 6 positive controls containing PBS were

prepared (see figure 3.4.). Subsequently, the plates were incubated at a certain temperature

(see table 3.5.) in static condition. After 24 hours, the supernatant was removed from the

wells, the disks were allowed to dry and transferred into a new plate.

FIGURE 3.4. : SCHEMATIC VISUALIZATION OF A 12 WELL PLATE

26

3.6.2.2. Adhesion phase

Two colonies of C. albicans 40 (diameter 1 mm) were inoculated into 25 mL YNB and

glucose 50 mM pH 7.0 in a 100 mL Erlenmeyer flask and incubated at 37°C on a rotary shaker

at 140 rpm. After 18 hours incubation, the culture was centrifuged for 10 minutes at 3500

rpm (Centrifuge B), washed twice with PBS and resuspended in 25 mL PBS with 10% fetal

bovine serum (FBS). This suspension was diluted to an optical density (OD) of 1.0, measured

using a spectrophotometer (Biophotometer Eppendorf, Hamburg, Germany) at 600 nm. This

absorbance corresponds approximately to 10⁷ CFU/mL. In order to confirm the correct

concentration of C. albicans OD1 suspension, serial dilutions (10⁻¹ till 10⁻⁵) were prepared

and 25 µL of dilutions 10⁻³ till 10⁻⁵ were plated in triplicate onto SDA plates. Plates were

incubated at 37°C for 24 hours and C. albicans colonies were counted using a

stereomicroscope (Nikon SMZ200).

Two milliliters of C. albicans OD1 suspension were added to each well except in the

negative control where 2 mL of PBS with 10% FBS (Fetal Bovine Serum) was added. Then, the

plates were incubated for 90 minutes in static position at a certain temperature (see table

3.5.). Subsequently, the disks were gently washed three times with sterile PBS to remove the

Candida cells which are not attached to the surface of the disks.

In one experiment, the disks were transferred to another plate containing 2 mL YNB

with 10% FBS in each well and incubated at 37 °C for 24 hours.

TABLE 3.5. : DIFFERENT CONDITIONS FOR PRE-COATING PLATES AND INCUBATION.

Figure (results section) Pre-coating (°C) Incubation

Temperature (°C) Time

4.3. 37 37 90 minutes

4.4. 37 37 24 hours

4.6. 4 4 90 minutes

4.8. 4 37 90 minutes

4.10. 37 4 90 minutes

4.13. and 4.18. 25 37 90 minutes

4.15. and 4.19 25 37 4 hours

27

3.6.3. Quantification of Candida albicans 40 adhesion to silicone disks by crystal

violet staining method

According to Gudina et al. (2010) and Luna (2011), crystal violet staining was used to

quantify the amount of adhesion of C. albicans 40 present on silicone disks to evaluate the

anti-adhesion effect of biosurfactant AC7. The solution for crystal violet (CV) staining was

prepared according to the procedure outlined in table 3.6.. Solutions A and B were mixed

and stored for 24 hours before use. Then the resulting stain is stable.

TABLE 3.6. : PROCEDURE FOR THE PREPARATION OF CRYSTAL VIOLET SOLUTION (= HUCKER’S STAIN)

The disks derived from the experiment above described were transferred into another

12 well plate to quantify C. albicans 40 adhesion using the crystal violet staining. Candida

cells were fixed on the elastomeric disks surface by dipping in methanol (99%) (Sigma-

Aldrich) for 15 minutes. After drying, the disks were submerged in 2 mL crystal violet

solution for 5 minutes. After approximately 8 washes with distilled water (until the distilled

water becomes clear), the disks were incubated overnight at 28 °C. Disks were then

decolorized by adding 4 mL of acetic acid (33%) (Sigma-Aldrich) to each well and incubated

at 180 rpm for 15 minutes. Subsequently, four aliquots of 200 µL of each crystal violet

solution were transferred into in a 96 well plate (Bioster) and the absorbance was measured

at 570 nm with a spectrophotometer (Ultramark microplate imaging system). For each

condition, two independent experiments were conducted, with the exception of the

experiments carried out at 24 hours and 4 hours. Experiments with pre-coating at 25 °C and

37 °C and incubation at 37 °C for 90 minutes were done in triplicate.

Solution A Solution B

crystal violet certified (Fluka) 2.0 g

ammonium oxalate

(Fluka) 0.8 g

ethyl alcohol 95% (Sigma-Aldrich) 20 mL distilled water 80.0 mL

28

3.6.4. Quantification by plate counting

In order to assess cell adhesion of C. albicans 40 to silicone disks, the plate counting

method was also used in the experiments with pre-coating at 25 °C and incubation at 37 °C

for 90 minutes and 4 hours. After the adhesion phase, the disks were transferred into tubes

containing 10mL PBS. The tubes were vortexed and sonicated (Elmasonic S30H, Elma) four

times for 30 seconds in order to efficiently detach cells from the disks. The cell suspension

was then serially diluted in PBS (10⁻¹ to 10⁻³) and 25 µL of the three dilutions were plated out

in triplicate on Sabouraud dextrose agar. Plates were incubated for 24 hours at 37°C. After

incubation, the colonies were counted using a stereomicroscope and the number of cells

was calculated according to the following formula (2) and expressed as mean log₁₀ CFU/disk.

N= colony forming units/ml in primary dilution =∑

( ) (1)

∑ C = Sum of colonies on the plates considered.

V1 = Volume of inoculum plated, expressed in mL.

n1= number of plates considered for the first dilution.

n2= number of plates considered for the second dilution.

d=factor of dilution corresponding with the first dilution

CFU /disk = N * V2 (2)

N= colony forming units/mL in the first dilution (1)

V2 = Volume of the first dilution.

3.6.5. Calculation of C. albicans 40 inhibition of adhesion to silicone disks

The percentage of adhesion inhibition was calculated according to following formula:

[1 – (Ac/A₀)] x 100

Where Ac is the absorbance or CFU/disk of the pre-coated samples and A₀ the absorbance or

CFU/disk of the positive controls.

29

3.6.6. Statistical analysis

In all experiments, the standard deviation was calculated en visualized by error bars in the

graphs. The Mann-Whitney U-test for unmatched samples was performed when the aim was

to investigate whether the difference between the means of 2 groups of experimental values

obtained under different conditions could be considered as statistically significant. “The

Mann-Whitney U-test is a non-parametric technique for comparing the medians of two

unmatched samples. It may be used with as few as four observations in each sample because

the values of observations are converted to their ranks (Fowler et al., 1998).”

30

4. RESULTS

4.1. CRITICAL MICELLE CONCENTRATION OF BIOSURFACTANT AC7

A solution of biosurfactant AC7 with a concentration 2000 µg/mL reduced water surface

tension from 73.56 mN/m to 33.07 mN/m. The plot shown in figure 4.1. visualizes the

surface tension as a function of the concentration of biosurfactant AC7. Serial dilutions

starting from 2000 µg/mL of biosurfactant AC7 demonstrated an approximately constant

value of 33 mN/m up to the concentration of 500 µg/mL and then the surface tension

increased slowly to 38.52 mN/m at a concentration of 250 µg/mL. Then the surface tension

increased faster for concentrations lower than 250 µg/mL. The CMC was calculated as the

intercept of two straight lines extrapolated from the concentration dependent and

independent sections of the curve. The CMC value for biosurfactant AC7 was calculated as

129.5 µg/mL.

FIGURE 4.1.: PLOT OF SURFACE TENSION AS A FUNCTION OF THE CONCENTRATION OF BIOSURFACTANT AC7.

y = -0.0029x + 38.31 R² = 0.864

y = -0.2096x + 65.071 R² = 0.7455

25.00

35.00

45.00

55.00

65.00

75.00

85.00

0 250 500 750 1000 1250 1500 1750 2000

Surf

ace

te

nsi

on

(m

N/m

)

Biosurfactant AC7 concentration (µg/mL)

concentration independentsection

concentration dependentsection

31

4.2. BIOCHEMICAL TESTS FOR THE CONFIRMATION OF THE IDENTITY OF THE

BIOSURFACTANT AC7 PRODUCING STRAIN

4.2.1. Catalase test, oxidase test, motility test and growth at 6% NaCl

Catalase test : A drop of H₂O₂ was poured on the microscopic glass slide. Some colonies of B.

subtilis AC7 were dissolved in the fluid. A formation of bubbles was visible so the B. subtilis

AC7 strain is catalase positive.

Oxidase test : The oxidase strip became purple when a colony of Pseudomonas fluorescens

was added. The paper didn’t color when a colony of the AC7 strain was added. So the

positive control, Pseudomonas fluorescens confirms that the strips work. The AC7 strain is

oxidase negative.

Motility test : The Bacillus AC7 strain diffused beyond the inoculation point. So the AC7

strain is a motile strain.

Growth in 6% NaCl LB-broth : Growth was observed after incubation for 24 hours at 28°C. So

the AC7 strain has the ability to grow in a LB-agar medium with 6% NaCl.

4.2.2. Tests on nitrate reduction, indole production, urease, esculin hydrolysis and

gelatin hydrolysis.

These tests were carried out because they respresent important properties to characterize

Bacillus sp. As demonstrated in figure 4.2., the tests gave the following results:

Test Result

- NO₃ = Nitrate reduction: pink coloration Positive

- TRP = Indole production: remains transparent Negative

- URE = Urease: yellow coloration Negative

- ESC = Esculin hydrolysis: grey coloration Positive

- GEL = Gelatin hydrolysis: black coloration Positive

32

FIGURE 4.2. : RESULTS OF NITRATE REDUCTION, INDOLE PRODUCTION,

UREASE, ESCULIN HYDROLYSIS AND GELATIN HYDROLYSIS (TESTS API NE)

4.2.3. Protease test

The protease activity, degradation of casein, of AC7 strain was semi-quantitative

determined by measuring the diameters of the transparent zone. Diameters, as illustrated in

table 4.1., were observed after incubation at 28°C for 24 hours. So the AC7 strain has a

protease activity.

Incubated on skim milk

agar

Mean diameter (cm)

after 24 hours

supernatant B. subtilis AC7 2.8 ± 0.28

colonies of B. subtilis AC7 3.7 ± 0.71

TABLE 4.1. : RESULTS OF THE PROTEASE ACTIVITY OF B. SUBTILIS AC7 ON SKIM MILK AGAR.

4.2.4. Cellulase

Results of cellulose degradation are presented in table 4.2.. The AC7 strain possess an

enzyme to degrade cellulose on the CMC plates. Figures 4.3. and 4.4. respectively show the

degradation of cellulose by the AC7 strain and its supernatant. On the avicel plates,

degradation was also visible but diameters were impossible to measure because the

degradation crossed the border of the petri dishes (data not shown). It was confirmed that

also on avicel medium the AC7 strain also shows a protease activity.

Incubated on CMC

Mean diameter (cm)

after 5 days

supernatant B. subtilis AC7 4.6 ± 0.07

colonies of B. subtilis AC7 5.0 ± 0.28

TABLE 4.2. : RESULTS OF THE CELLULOSE ACTIVITY OF BACILLUS AC7 ON CMC.

33

FIGURE 4.3. : CELLULASE ACTIVITY B. SUBTILIS AC7. FIGURE 4.4.: CELLULASE ACTIVITY SUPERNATANT

OF B. SUBTILIS AC7

4.2.5. Conclusion of the biochemical tests

The results of the above biochemical tests are consistent with the properties of a Bacillus

subtilis strain as previously genotypically identified by DSMZ in Germany.

4.3. ANTI-ADHESIVE ACTIVITY OF BIOSURFACTANT AC7 ON SILICONE ELASTOMERIC DISKS:

CRYSTAL VIOLET STAINING

The ability of biosurfactant AC produced by Bacillus subtilis AC7 to decrease the adhesion

of Candida albicans 40 to elastomeric silicone disks was tested under different conditions.

The elastomeric disks were pre-coated with biosurfactant AC7.

4.3.1. Negative control

At first, one negative control was included in each test. The negative controls without

biosurfactant AC 7 consisted of silicone disks pre-coated for 24 hours with PBS and were

incubated with PBS and 10% FBS for 90 minutes. The negative controls with biosurfactant

AC7 consisted of silicone disks pre-coated with biosurfactant AC7 2000 µg/mL and incubated

with PBS and 10% FBS for 90 minutes. Both negative controls were prepared in order to

determine the background color from the crystal violet staining. As shown in figure 4.5., the

34

mean absorbance at 570 nm of the negative controls was 0.065 and its standard deviation

was 0.012. Negative controls with biosurfactant AC 7 are visualized in figure 4.6.. The mean

absorbance was 0.075 and its standard deviation was 0.008. So subtraction of the

absorbance of negative control from the absorbances of the positive controls and of the

samples pre-coated with biosurfactant AC7 was not necessary and discarded in following

experiments.

FIGURE 4.5. : NEGATIVE CONTROL WITHOUT BIOSURFACTANT AC7.

FIGURE 4.6. : NEGATIVE CONTROL WITH BIOSURFACTANT AC7 2000 µg/mL.

4.3.2. Pre-coating of disks at 37°C and incubation at 37°C for 90 minutes

In this experiment, silicone disks were pre-coated with biosurfactant AC7 at a

concentration of 2000 µg/mL for 24 hours at 37°C and incubated at 37°C with C. albicans

suspension OD1 for 90 minutes. Positive controls were pre-coated with PBS for 24 hours at

37°C and incubated with C. albicans suspension OD1 for 90 minutes at 37°C. As shown in

figure 4.7. there was a reduction of adhesion of 42.3% to the silicone disks treated with

biosurfactant AC7 2000 µg/mL in comparison with the positive control disks. The Mann-

Whitney value was 0.00432, so the reduction is statistically significant. The OD1 suspension

of C. albicans contained 7.2 x 10⁶ CFU/mL.

0

0.02

0.04

0.06

0.08

0.1

Ab

sorb

ance

57

0 n

m

NegativecontrolwithoutbiosurfactantAC7

0

0.02

0.04

0.06

0.08

0.1

Ab

sorb

ance

57

0 n

m

Negativecontrol withbiosurfactantAC7 2000µg/mL

35

FIGURE 4.7. : RESULT OF CRYSTAL VIOLET STAINING OF SILICONE DISKS PRECOATED WITH BIOSURFACTANT AC7

AT 37°C FOR 24 HOURS AND INCUBATION AT 37°C FOR 90 MINUTES. * INDICATES A SIGNIFICANT DIFFERENCE

WITH THE POSITIVE CONTROL (P<0.05).

Figure 4.8. is shows a picture of the silicone disks after crystal violet staining. For the

positive controls the of the stained C. albicans cells are distributed all over the disks and

more densely in comparison with the treated disks. Microscopic pictures of the disks are

shown in figure 4.9. and figure 4.10..

FIGURE 4.8. : SILICONE DISKS AFTER CRYSTAL VIOLET STAINING (CONDITIONS: PRE-COATING

AT 37°C FOR 24 HOURS AND INCUBATION AT 37°C FOR 90 MINUTES).

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

Ab

sorb

ance

57

0 n

m

positive control

BS 2000µg/mL*

36

FIGURE 4.9. : MICROSCOPIC PICTURE (10-FOLD ZOOMED) OF A POSITIVE CONTROL DISK, (BS AC7 0

µG/ML)(NIKON ECLIPSE E400).

FIGURE 4.10. : MICROSCOPIC PICTURE (10-FOLD ZOOMED) SAMPLE, (BS AC7 2000 µG/ML). (NIKON ECLIPSE

E400).

CONDITIONS OF BOTH FIGURES: PRECOATING AT 37°C FOR 24 HOURS AND INCUBATION AT 37°C FOR 90

MINUTES.

4.3.3. Pre-coating of disks 37°C and Incubation for at 37°C 24 hours.

In this experiment, silicone disks were pre-coated with biosurfactant AC7 2000 µg/mL

for 24 hours at 37°C and incubated at 37°C with C. albicans suspension OD1 for 90 minutes.

Positive control disks were pre-coated with PBS for 24 hours at 37°C and incubated with C.

albicans suspension OD1 for 90 minutes at 37°C. Subsequently, all disks were incubated with

C. albicans 40 in YNB medium for 24 hours. Only a reduction of adherence of 1.7% was

obtained for the pre-coated silicone disks. This reduction was not statistically significant

because the value of the Mann-Whitney value is 0.08225. The OD1 suspension of C.

albicans contained 7.2 x 10⁶ CFU/mL.

4.3.4. Pre-coating of disks at 4°C and incubation at 4°C for 90 minutes

In a next experiment, silicone disks were pre-coated with biosurfactant AC7 2000

µg/mL for 24 hours at 4°C and incubated at 4°C with C. albicans suspension OD1 for 90

minutes. Positive controls were pre-coated with PBS for 24 hours at 4°C and incubated with

C. albicans suspension OD1 for 90 minutes at 4°C. Figure 4.12. illustrates that there was a

reduction of adhesion of 50.2% on the silicone disks treated with biosurfactant AC7 2000

37

µg/mL in comparison with the positive control disks. The Mann-Whitney value is 0.004329,

so the reduction is statistically significant. The OD1 suspension of C. albicans contained 6.2 x

10⁶ CFU/mL.

FIGURE 4.12. : RESULT OF CRYSTAL VIOLET STAINING OF SILICONE DISKS PRECOATED WITH BIOSURFACTANT

AC7 AT 4°C FOR 24 HOURS AND INCUBATION AT 4°C FOR 90 MINUTES. * INDICATES A SIGNIFICANT DIFFERENCE

WITH THE POSITIVE CONTROL (P<0.05).

Figure 4.13. shows a picture of the silicone disks after crystal violet staining. The

negative control is blank. On the positive control disks, a strange shape is present in the

middle of the disk. On the surface of the disks treated with biosurfactant AC7, only small

violet spots are visible.

FIGURE 4.13. : SILICONE DISKS AFTER CRYSTAL VIOLET STAINING (CONDITIONS: PRE-COATING

AT 4°C FOR 24 HOURS AND INCUBATION AT 4°C FOR 90 MINUTES).

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45A

bso

rban

ce 5

70

nm

positive control

BS 2000µg/mL

*

38

4.3.5. Pre-coating at 4°C and incubation at 37°C for 90 minutes

In this experiment, silicone disks were pre-coated with biosurfactant AC7 2000 µg/mL

for 24 hours at 4°C and incubated at 37°C with C. albicans suspension OD1 for 90 minutes.

Positive controls were pre-coated with PBS for 24 hours at 4°C and incubated with C.

albicans suspesion OD1 for 90 minutes at 37°C. Under this condition, a reduction of adhesion

of 31.9% was obtained for the silicone disks treated with biosurfactant AC7 2000 µg/mL in

comparison with the positive control disks, which were not treated with biosurfactant AC7

(figure 4.14.). The value of the Mann-Whitney test is 0.01515, so the reduction is statistically

significant. The OD1 suspension of C. albicans contained 6.1 x 10⁶ CFU/mL.

FIGURE 4.14. : RESULT OF CRYSTAL VIOLET STAINING OF SILICONE DISKS PRECOATED WITH BIOSURFACTANT

AC7 AT 4°C FOR 24 HOURS AND INCUBATION AT 37°C FOR 90 MINUTES. * INDICATES A SIGNIFICANT

DIFFERENCE WITH THE POSITIVE CONTROL (P<0.05).

The color of the silicone with crystal violet staining is illustrated in figure 4.15.. Again

a distribution of the violet colored Candida cells all over the silicone disks is observed for the

positive control. A lower number Candida cells is visible on the silicone disks treated with

biosurfactant AC7, indicating a reduction of adhesion in this case.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

Ab

sorb

ance

57

0 n

m positive control

BS 2000 µg/mL*

39

FIGURE 4.15. : SILICONE DISKS AFTER CRYSTAL VIOLET STAINING (CONDITIONS: PRE-COATING

AT 4°C FOR 24 HOURS AND INCUBATION AT 37°C FOR 90 MINUTES).

4.3.6. Pre-coating at 37°C and incubation at 4°C for 90 minutes

In this experiment, silicone disks were precoated with biosurfactant AC7 2000 µg/mL

for 24 hours at 37°C and incubated at 4°C with C. albicans suspension OD1 for 90 minutes.

Positive controls were pre-coated with PBS for 24 hours at 37°C and incubated with C.

albicans suspension OD1 for 90 minutes at 4°C. A substantial decrease of adhesion,

illustrated in figure 4.16., was obtained for this condition. The reduction of adhesion was up

to 59.5% for the silicone disks treated with biosurfactant in comparison with the untreated

disks. This result is statistically significant because the result of the Mann-Whitney test was

0.004329. The OD1 suspension of C. albicans contained 6.2 x 10⁶ CFU/mL.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

Ab

sorb

ance

57

0 n

m

positive control

BS 2000µg/mL*

40

FIGURE 4.16. : RESULT OF CRYSTAL VIOLET STAINING OF SILICONE DISKS PRECOATED WITH BIOSURFACTANT

AC7 AT 37°C FOR 24 HOURS AND INCUBATION AT 4°C FOR 90 MINUTES. * INDICATES A SIGNIFICANT

DIFFERENCE WITH THE POSITIVE CONTROL (P<0.05).

A homogeneous distribution of colored C. albicans cells on the disks was obtained for

the positive control after crystal violet staining (figure 4.13.). The biosurfactant treated disks

displayed only purple speckles due to the attachment of less C. albicans cells. Microscopic

pictures are shown in figure 4.14. and figure 4.15..

FIGURE 4.13. : SILICONE DISKS AFTER CRYSTAL VIOLET STAINING (CONDITIONS: PRE-COATING

AT 37°C FOR 24 HOURS AND INCUBATION AT 4°C FOR 90 MINUTES).

FIGURE 4.14. : MICROSCOPIC PICTURE (10-FOLD ZOOMED) POSITIVE CONTROL DISK (BS AC7 0 µG/ML) (NIKON

ECLIPSE E400). FIGURE 4.15. : MICROSCOPIC PICTURE (10-FOLD ZOOMED) SAMPLE OF A PRE-COATED DISK (BS

AC7 2000 µG/ML) (NIKON ECLIPSE E400). CONDITIONS OF BOTH FIGURES: PRECOATING AT 4°C FOR 24 HOURS

AND INCUBATION AT 4°C FOR 90 MINUTES.

41

4.3.7. Pre-coating at 25°C and incubation at 37°C for 90 minutes

In this experiment, silicone disks were pre-coated with biosurfactant AC7 2000 µg/mL

for 24 hours at 25°C and incubated at 37°C with C. albicans suspension OD1 for 90 minutes.

Positive controls were pre-coated with PBS for 24 hours at 25°C and incubated with C.

albicans suspension OD1 for 90 minutes at 37°C. In this case, as shown in figure 4.16., the

adhesion of the C. albicans cells on disks pre-coated with biosurfacant AC7 decreased by

31.7%. A higher number of C. albicans cells adhere to the surface of the untreated disks in

comparison with the disks which were pre-coated with biosurfactant AC7. The Mann-

Whitney test gave a statistically significant value of 0.002165. The C. albicans suspension

OD1 contained 6.3 x 10⁶ CFU/mL.

FIGURE 4.16.: RESULT OF CRYSTAL VIOLET STAINING OF SILICONE DISKS PRECOATED WITH BIOSURFACTANT

AC7 AT 25°C FOR 24 HOURS AND INCUBATION AT 37°C FOR 90 MINUTES. * INDICATES A SIGNIFICANT

DIFFERENCE WITH THE POSITIVE CONTROL (P<0.05).

Figure 4.17. shows a picture of the silicone disks after crystal violet staining. Almost a

uniform distribution of colored C. albicans cells was visible on the positive control disks. On

the surface of the disks pre-coated with biosurfactant AC7 the coloration was not

homogeneous neither dense.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

Ab

sorb

ance

57

0 n

m positive control

BS 2000 µg/mL*

42

FIGURE 4.17. : SILICONE DISKS AFTER CRYSTAL VIOLET STAINING (CONDITIONS: PRE-COATING

AT 25°C FOR 24 HOURS AND INCUBATION AT 37°C FOR 90 MINUTES).

4.3.8. Pre-coating at 25°C and incubation at 37°C for 4 hours

In this experiment, silicone disks were pre-coated with biosurfactant AC7 2000 µg/mL

for 24 hours at 25°C and incubated at 37°C with C. albicans suspension OD1 for 4 hours.

Positive controls were pre-coated with PBS for 24 hours at 25°C and incubated with C.

albicans suspesion OD1 for 4 hours at 37°C. Figure 4.18. shows the reduction of adhesion

present for biosurfactant pre-coated silicone disks in comparison with the untreated disks. A

reduction of adhesion of 18.6% was obtained. This result is significant due to the Mann-

Whitney value of 0.008658. The concentration of C. albicans suspension had a concentration

of 6.2 x 10⁶ CFU/mL.

FIGURE 4.18. : RESULT OF CRYSTAL VIOLET STAINING OF SILICONE DISKS PRECOATED WITH BIOSURFACTANT

AC7 AT 25°C FOR 24 HOURS AND INCUBATION AT 37°C FOR 4 HOURS. * INDICATES A SIGNIFICANT DIFFERENCE

WITH THE POSITIVE CONTROL (P<0.05).

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

Ab

sorb

ance

57

0 n

m

positivecontrol

BS2000µg/mL

*

43

As shown is figure 4.19. the density of the C. albicans cells on the disks is high in all

cases. A more homogeneous distribution of purple colored C. albicans cells can be observed

on the positive controls. In contrast, there are more transparent spots on the disks treated

with biosurfactant AC7.

FIGURE 4.19. : : SILICONE DISKS AFTER CRYSTAL VIOLET STAINING (CONDITIONS: PRE-COATING

AT 25°C FOR 24 HOURS AND INCUBATION AT 4°C FOR 4 HOURS).

4.4. ANTI-ADHESIVE ACTIVITY OF BIOSURFACTANT AC7 ON SILICONE ELASTOMERIC

DISKS: PLATE COUNTING METHOD

4.4.1. Pre-coating at 25°C and incubation at 37°C for 90 minutes

In this experiment, silicone disks were pre-coated with biosurfactant AC7 2000 µg/mL

for 24 hours at 25°C and incubated at 37°C with C. albicans suspension OD1 for 90 minutes.

Positive controls were pre-coated with PBS for 24 hours at 25°C and incubated with C.

albicans suspension OD1 for 90 minutes at 37°C. A reduction of 73.1%, as visualized in figure

4.20., was obtained by plate counting. This result was statistically significant as the Mann-

Whitney test gave of value of 0.004329. The C. albicans suspension used, contained 6.5 x 10⁶

CFU/mL.

44

FIGURE 4.20. : RESULT OF ADHESION OF C. ALBICANS TO THE SILICONE ELASTOMERIC SURFACE COATED WITH

BIOSURFACTANT 2000µG/ML, AS DETERMINED BY PLATE COUNTING. CONDITIONS: PRE-COATING AT 25°C,

INCUBATION AT 37°C FOR 90 MINUTES. * INDICATES A SIGNIFICANT DIFFERENCE WITH THE POSITIVE CONTROL

(P<0.05).

4.4.2. Pre-coating at 25°C and incubation at 37°C for 4 hours

In this experiment, silicone disks were pre-coated with biosurfactant AC7 2000 µg/mL

for 24 hours at 25°C and incubated at 37°C with C. albicans suspension OD1 for 4 hours.

Positive controls were pre-coated with PBS for 24 hours at 25°C and incubated with C.

albicans suspension OD1 for 4 hours at 37°C. A reduction of adhesion of 26.5% was obtained

for this condition and showed in figure 4.21.. This result was not statistically significant as

the Mann-Whitney value was 0.3095. The C. albicans suspension OD1 contained 6.5 x 10⁶

CFU/mL.

FIGURE 4.21. : RESULT OF ADHESION OF C. ALBICANS TO THE SILICONE ELASTOMERIC SURFACE COATED WITH

BIOSURFACTANT 2000µG/ML, AS DETERMINED BY PLATE COUNTING. CONDITIONS: PRE-COATING AT 25°C,

INCUBATION AT 37°C FOR 4 HOURS.

4.5

4.7

4.9

5.1

5.3

5.5

5.7

5.9

6.1

log₁

₀ C

FU/d

isk positive

control

BS 2000µg/mL

6

6.1

6.2

6.3

6.4

6.5

6.6

log

₁₀ c

fu/d

isk positive

control

BS 2000µg/ml

*

45

5. DISCUSSION

The aim of this thesis was to evaluate the ability of a biosurfactant, produced by an

endophytic AC7 strain previously identified as B. subtilis by DSMZ in Germany, to inhibit the

adhesion of a C. albicans strain of nosocomial origin, isolated from a central venous catheter,

to medical-grade silicone disks. Different conditions of pre-coating and incubation were used

in order to assess the influence of temperature. Two different methods for evaluation of the

inhibition of C. albicans adhesion were compared. Experiments to determine the ability of

biosurfactant AC7 to lower the surface tension in aqueous solution and to estimate the

critical micelle concentration were also conducted. Chemical characterization of BS AC7

revealed that it is composed of 94% surfactin and 6% fengycin. The quantitative analysis

indicates that surfactin accounts for about 65% of the crude extract. The chemical analyses

(LC-MS/MS) on BS AC7 were carried out by the Laboratory of Analytical Chemistry (Prof.

Gianna Allegrone) of the Department of Pharmaceutical Sciences in Novara.

5.1. CRITICAL MICELLE CONCENTRATION

Bacillus sp. AC7 is able to produce a potent biosurfactant. The surface tension of culture

supernatant was decreased down to 33.07 mN/m and the CMC calculated value was 129.5

µg/ml. These results are consistent with the presence of a potent low molecular weight

lipopeptidic biosurfactant, i.e. surfactin. This molecule is known to reduce surface tension to

27 mN/m and its CMC lies between 20 and 160 µg/mL (Desai and Banat, 1997; Rivardo et al.,

2009).

5.2. CHARACTERISATION OF BACILLUS SUBTILIS AC7: BIOCHEMICAL TESTS

The strain of Bacillus AC 7 was previously genotypically identified as B. subtilis.

Biochemical tests were performed to confirm its identity by determining some of its

biochemical properties. All results of the biochemical tests are in agreement with to the

biochemical properties of B. subtilis, as described by Koneman et al. (1995). Moreover, the

strain was able to degrade crystalline cellulose and carboxymethylcellulose as well as milk

proteins.

46

5.3. ANTI-ADHESION ACTIVITY OF BIOSURFACTANT AC7 ON SILICONE ELASTOMERIC DISKS

According to Chandra et al. (2008) two methods are mainly used for biofilm

quantification. Firstly, XTT and MTT are colorimetric assays that determine the metabolic

activity of the cells. Secondly dry weight determination, in which the total biomass of the

biofilm is measured (Chandra et al., 2008). Metabolic activity of cells is proportional to the

color intensity of the formazan product, which is measured by a spectrophotometer

(Chandra et al., 2001). The crystal violet staining method has been applied for the

quantification of the adhesion of microorganisms on plastic, such as C. albicans, E. coli,

Lactobacillus casei, Streptococcus spp., etc. because it is an easy, cheap and fast method

(Gudina et al., 2010; Luna et al., 2011). Another valid method is the viable cell counting

method but it is not commonly used since it is time consuming and labor intensive (Rivardo

et al., 2009).

In this thesis, the effect of biosurfactant on C. albicans adhesion was studied on

elastomeric silicone disks. The method used to coat the silicone disks with biosurfactant AC7

was derived from the method of Rivardo et al. (2009). The evaluation of the C. albicans

biofilm formation was carried out according to Chandra et al. (2008). Initially, the

experiments involved by crystal violet staining since it is an easy, cheap and quick method.

Subsequently, two experiments were done using plate counting method and compared with

previously obtained CV results.

Several factors influence Candida biofilm formation. For example, cell surface

hydrophobicity correlates positively with Candida biofilm formation (Li, Yan and Xu, 2003)

and gentle shaking also improves biofilm formation (Hawser et al., 1998). Although it is more

realistic to work in flow-based systems, biofilms are often studied in static conditions for

some economic reasons such as lower cost, sample throughput and technical considerations

(Busscher and van der Mei, 2006). The experiments in this thesis were done in static

conditions.

The C. albicans cells suspension in PBS was supplemented with 10% fetal bovine serum

because this condition simulates the physiological condition in the body. Blood serum

consists of several proteins. Serum stimulates germ tube production, which enhances biofilm

formation (Krom and Darouiche, 2009). Ramage and collaborators (2001) studied the

47

influence of serum and saliva conditioning films on C. albicans adhesion and biofilm

formation and observed that initially, after 30 minutes, the adhesion increased with both

conditions but especially with serum. On the contrary, after 4 hours and 24 hours incubation

there was no significant difference between the disks treated with saliva and serum and

untreated disks.

Data obtained previously in the Laboratory of Microbiology in Novara demonstrated that

biosurfactant AC7 has no antimicrobial activity. The minimal inhibitory concentration (MIC)

test demonstrated that planktonic Candida cells were not killed by different concentrations

of biosurfactant AC7. So the decrease of C. albicans cells on silicone disks observed in the

experiments of this thesis is only due to inhibition of adhesion.

Luna and collaborators (2011) determined the anti-adhesive and antimicrobial effects of

biosurfactant produced by Candida sphaerica UCP in 96-well flat-bottomed plastic tissue

culture plates. For the anti-adhesive assay, a range of concentrations of the biosurfactant

(0.625 to 10 mg/mL) were brought in contact with the surfaces 18 hours at 4°C. This was

called the pre-coating phase. Then suspensions of several microorganisms, including Candida

albicans, were added to the wells and incubated for 4 hours at 4°C. Subsequently, plates

were stained with by crystal violet and the fixed CV solubilized with 33% (v/v) glacial acetic

acid, the absorbance was measured at 595 nm and the percentage of microbial inhibition

was calculated. The antimicrobial activity of a biosurfactant, isolated from C. sphaerica, at a

concentration of 10 mg/mL was 57% against C. albicans, whereas the anti-adhesive effect at

the highest concentration of biosurfactant was 100% . The same conditions of pre-coating

and incubation were used by Gudina et al. (2010) with a biosurfactant isolated from

Lactobacillus paracasei ssp. A20. The results of antimicrobial activity against C. albicans were

up to 100 ± 0.2 % for the highest concentration of biosurfactant (50mg/mL), whereas the

anti-adhesive effect on C. albicans was only 29.5 ± 1.6 %. So this biosurfactant demonstrated

excellent antimicrobial activity against C. albicans, in contrast with a minimal anti-adhesive

effect.

In this thesis, the highest anti-adhesion effect, 59.5%, using the crystal violet method was

obtained under the condition of pre-coating at 37°C for 24 hours and incubation at 4°C. The

experiment with pre-coating at 4°C for 24 hours and incubation at 4°C for 90 minutes,

48

yielded a reduction of 50.2%. Zeraik and Nitschke (2010) described experiments carried out

at different pre-coating and incubation temperatures (4°C, 25°C and 35°C). In one

experiment, pre-coating and incubation were carried out at same temperature. The highest

reduction of Staphylococcus aureus, Listeria monocytogenes and Micrococcus luteus

adhesion was observed with pre-coating using the biosurfactant produced by Bacillus subtilis

LB5a and incubation at 4°C for 4 hours. According to Myers (2006), the strong reduction at

4°C might be due to an higher amount of biosurfactant adsorbed to the polystyrene surface

at a lower temperature (Myers, 2006). Moreover, at low temperatures, the hydrophobicity,

motility and other attachment factors (adhesins) of the cells can be different, positively

favoring the anti-adhesive activity of the biosurfactant (Zeraik and Nitschke, 2010).

In order to simulate the physiological conditions in the body, experiments with incubation

at 37°C were carried out. The experiment with pre-coating at 37°C for 24 hours and

incubation 37°C for 90 minutes showed 42.3% reduction of adhesion, whereas pre-coating

at 4 °C for 24 hours and incubation 37°C for 90 minutes demonstrated 31.9% reduction of

adhesion. Similarly to what was observed previously, pre-coating at 4 °C did not seem to

improve anti adhesive activity compared to pre-coating at 37°.

In summary, the obtained results demonstrate that the attachment of C. albicans to

silicone elastomeric disks can be reduced by using biosurfactant AC7 and that its effect can

be increased at low temperatures, showing the importance of this parameter when studying

surfactants as anti-adhesive agents.

Subsequently, a pre-coating experiment at 25°C was conducted, in order to reduce the

cost of the process because no cooling or heating system is required to pre-coat the silicone

material. Pre-coating at 25°C for 24 hours and incubation at 37°C for 90 minutes were

evaluated by crystal violet staining and by plate counting. The reductions in adhesion were

31.7% and 73.1% respectively.

Finally, an experiment with pre-coating at 25°C and incubation at 37°C for 4 hours

was carried out in combination with crystal violet staining and plate counting. The reduction

in adhesion (18,6%, crystal violet and 26.5%, plate counting) may be interesting because,

according to Poelstra et al. (2002) the most critical period for the adhesion is in the first six

hours after the implantation of a medical device. This is explained by the fact that there is

49

competition between the immune system and the microorganisms which try to cause an

infection. In the experiment carried out in this thesis, four hours of incubation time were

chosen.

The reduction of adhesion determined by plate counting was higher than that

obtained after crystal violet staining. In the experiment with pre-coating at 25°C for 24 hours

and incubation at 37°C for 90 minutes and 4 hours, 73.1% and 26.5% reduction of adhesion

was obtained respectively, using plate counting.

According to the obtained results, the anti-adhesion activity of biosurfactant AC7

seems to be only effective in the first phase of adhesion. In a 4 hours incubation experiment,

reduction was lower and with incubation at 37°C for 24 hours no statistical difference was

found between treated and untreated disks.

Higher reductions of adhesion were observed using plate counting compared to CV

staining. Possible explanations were for this observation were considered. As observed by

SEM analyses carried out previously at the University of Trento, the surface of silicone disks

is not homogeneous. Due to this rough surface crystal violet can be withheld in the grooves

of the silicone disks, resulting in an overestimation of the number of cells. Moreover, small

granules of crystal violet might be retained by the cells so that a relatively darker solution is

obtained after decolorization, again resulting in an overestimation of C. albicans cells.

5.4. FUTURE PERSPECTIVE

In this thesis, only a first screening of the anti-adhesive activity of the biosurfactant AC7

was accomplished. In the future, the method has to be optimized to create conditions more

similar to human body, those with e.g. using flow conditions. Also, the contact angle has to

be determined to study the distribution of the biosurfactant on the surface of the disks. The

pre-coating process of biosurfactant, also, has to be improved, for example by fixing the

biosurfactant to the surface of the elastomeric silicone disk by plasma treatment, in order to

create a more uniform and stable treatment (Hegemann et al., 2003). As biosurfactant AC7

seems to be effective during the first phase of adhesion, antimycotics such as fluconazole

can be combined with biosurfactant AC7 biosurfactant in order to act in an additive or

synergistic way with the latter’s anti-adhesive activity.

50

6. CONCLUSION

Bacillus sp. AC7 is able to produce a potent biosurfactant. The surface tension of culture

supernatant was decreased towards 33.07 mN/m and the CMC value was of 129.5 µg/mL.

These results are consistent with the presence of a potent low molecular weight lipopeptidic

biosurfactant, i.e. surfactin.

All the results of the biochemical tests performed to characterize B. subtilis AC7 were in

agreement with the biochemical properties of B. subtilis described by Koneman et al. (1995).

Consequently, the biosurfactant producing strain, previously genotypically identified B.

subtilis strain, is confirmed to have all tested properties of B. subtilis.

Biosurfactant A7 produced by B. subtilis AC7 has a good anti-adhesive activity of against

Candida albicans 40 on silicone elastomeric disks.

Different conditions of pre-coating and incubation gave similar results. The obtained

results demonstrate that the attachment to silicone elastomeric disks can be reduced by

using biosurfactant AC7 and that its effect can be increased at low temperatures, showing

the importance of this parameter when studying surfactants as anti-adhesive agents. This

anti-adhesion activity of biosurfactant AC7 seems to be only effective in early phase of

adhesion.

Higher reductions of adhesion were observed using plate counting compared to CV

staining. Possible explanations were for this observation were considered. As observed by

SEM analyses carried out previously at the University of Trento, the surface of silicone disks

is not homogeneous. Due to this rough surface crystal violet can be withheld in the grooves

of the silicone disks, resulting in an overestimation of the number of cells. Moreover, small

granules of crystal violet might be retained by the cells so that a relatively darker solution is

obtained after decolorization, again resulting in an overestimation of C. albicans cells.

In conclusion, first screening of biosurfactant AC7 produced by B. subtilis AC7 to prevent

adhesion of C. albicans 40 on silicone disks gave good results. Optimization of the pre-

coating and incubation method could be a future promising perspective.

51

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