CHARACTERIZATION OF BIOSURFACTANTS PRODUCED BY …€¦ · 1.1.1. Characteristics The genus Candida...

UNIVERSITEIT GENT

FACULTEIT FARMACEUTISCHE WETENSCHAPPEN

Vakgroep Farmaceutische Analyse

Laboratorium voor Farmaceutische Microbiologie

Academiejaar 2008-2009

CHARACTERIZATION OF BIOSURFACTANTS

PRODUCED BY LACTIC ACID BACTERIA

AND THEIR INHIBITORY EFFECT ON

CANDIDA ALBICANS BIOFILM FORMATION

Experimenteel onderzoek

Università degli studi del Piemonte Orientale (Novara, Italia)

Prof. Dr. M.G. Martinotti

Ilse VANLERBERGHE

Eerste Master in de Farmaceutisch zorg

Promotor

Prof. Dr. H. Nelis

Commissarissen

Prof. Dr. T. Coenye

Prof. Dr. S. De Smedt

AUTEURSRECHT

“De auteur en de promotor geven de toelating deze masterproef voor consultatie

beschikbaar te stellen en delen ervan te kopiëren voor persoonlijk gebruik. Elk ander

gebruik valt onder de beperkingen van het auteursrecht, in het bijzonder met betrekking

tot de verplichting uitdrukkelijk de bron te vermelden bij het aanhalen van de resultaten

uit deze masterproef.”

2 juni 2009 Promotor Auteur Prof. Dr. H. Nelis Ilse Vanlerberghe

THANKS TO

Thank you, professor Nelis and professor Martinotti, for giving me the great chance to study abroad. I’m really grateful for all your help with the correction of

this thesis and for letting me taste of the fascinating world of microbiology.

Dank u, Sofie Thys,om mijn reisgezel te zijn. Samen hebben we veel nieuwe dingen ontdekt, veel geduld gehad, maar vooral veel genoten. Ik kon me geen betere Erasmuspartner voorstellen.

Ringrazio Rosa Montella per essere stata la miglior partner di laboratorio al mondo.

Grazie per avermi fatto sorridere quando invece avevo voglia di piangere. Ti auguro buona fortuna per la tesi. Ti voglio bene.

Dank u, Koen De Vos, om mij op Erasmus te

laten gaan en mij te steunen en te helpen. Ik heb genoten van je bezoekjes, je mailtjes en onze babbels.

Samen komen we er wel.

Thank you, friends from Pakistan, for making Vercelli like a home for us. Thank you for the trips, the spicy foods, the movies, the serious and sometimes ridiculous conversations…We will never forget

the things we promised never to forget. Ci vediamo!

Dank u, mama, papa en Veerle, om mij opnieuw een buitenlandse ervaring te gunnen. Jullie onvoorwaardelijke steun, de vele lieve emailtjes, het leuke bezoek en

het nalezen van deze thesis maakten mijn Italiaanse droom nog aangenamer.

INDEX

1. INTRODUCTION 1

1.1. CANDIDA ALBICANS 1

1.1.1. Characteristics 1

1.1.2. Biofilms 2

1.2. LACTIC ACID BACTERIA 5

1.3. BIOSURFACTANTS 8

2. OBJECTIVES 12

3. EXPERIMENTAL 13

3.1. MATERIALS 13

3.1.1. Strains 13

3.1.1.1. Candida albicans 13

3.1.1.2. Lactic acid bacteria 13

3.1.2. Media 13

3.1.3. Other materials 16

3.2. METHODS 18

3.2.1. General characteristics of the three isolates 18

3.2.1.1. Catalase test, Gram staining, oil spreading test and 18

lactic acid production test

3.2.1.2. Growth curves of the CV8LAC, INS9T7I and ME11T7I isolates 18

3.2.1.3. Biofilm formation by the CV8LAC, INS9T7I and ME11T7I 18

isolates in MRS and ROG medium.

3.2.2. Biofilm formation by Candida albicans 20

3.2.3. Biosurfactants produced by the CV8LAC, INS9T7I 21

and ME11T7I isolates

3.2.3.1. Biosurfactants activity 21

3.2.3.2. Extraction of biosurfactants 22

3.2.3.3. Characterization of the extracted biosurfactants 22

3.2.3.4. Biosurfactants tested on Candida albicans biofilm 23

4. RESULTS AND DISCUSSION 27

4.1. GENERAL CHARACTERISTICS OF THE THREE ISOLATES 27

4.1.1. Catalase test, Gram staining, oil spreading test and lactic acid 27

production test

4.1.2. Growth curves of the CV8LAC, INS9T7I and ME11T7I isolates 27

4.1.3. Biofilm formation by the CV8LAC, INS9T7I and ME11T7I isolates 29

4.2. BIOFILM FORMATION BY CANDIDA ALBICANS 29

4.3. BIOSURFACTANTS PRODUCED BY THE CV8LAC, INS9T7I 31

AND ME11T7I ISOLATES

4.3.1. Biosurfactants activity 31

4.3.2. Extraction of biosurfactants 32

4.3.3. Characterization of the extracted biosurfactants 33

4.3.4. Biosurfactants tested on Candida albicans biofilm 35

4.3.4.1. Precoating experiments 35

4.3.4.2. Coincubation experiments 37

5. CONCLUSIONS 39

6. BIBLIOGRAPHY 40

LIST OF ABBREVIATIONS

CFU Colony Forming Unit

CMC Critical Micelle Concentration

DNA Deoxyribonucleic acid

EPS Exo-Polymeric Substance

HLB Hydrophylic Lipophylic Balance

LAB Lactic Acid Bacteria

LB Luria Bertani

MRS Man Rogosa Sharpe

OD Optical Density

PBS Phosphate Buffered Saline

PS Physiological Saline

ROG Rogosa

SDB Sabouraud Dextrose Broth

UMY Universal Medium for Yeast

YNB Yeast Nitrogen Base

Introduction

1

1. INTRODUCTION

1.1 CANDIDA ALBICANS

1.1.1. Characteristics

The genus Candida belongs to the kingdom of Fungi, the phylum of

Ascomycota, the class of Hemiascomycetes, the order of Saccharomycetales and the

family of Candidaceae. It comprises about a quarter of all yeast species (Calderone,

2002). Candida albicans is a dimorphic fungus. It mainly occurs as oval budding

yeast on mucosal surfaces as a component of the normal flora, but forms hyphae

(filamentous forms) or pseudohyphae when invading (figure.1.1) (Mims et al., 2004).

FIGURE 1.1: DIFFERENT FORMS OF CANDIDA ALBICANS.

(http://overcomingcandida.com/images/candida_gallery/candida_albicans_stages.jpg)

Candida albicans is a commensal of the skin, the vagina and the respiratory and

gastrointestinal tracts. It induces infections of different kinds, classified as superficial,

locally invasive and systemic infections. Superficial infections are the most common

ones, mainly occurring on the skin and on the mucous membranes of the oral cavity,

the vagina and the respiratory tract. Locally invasive candidiasis is seen in stressed,

suppressed and antibiotic-treated individuals. Hence, Candida albicans behaves as a

Introduction

2

conditional to an opportunistic pathogen, yielding as ulcerations of intestinal,

respiratory or genital-urinary tracts. Systemic candidiasis is the most severe type. It

causes invasive infections of the parenchyma of visceral organs, such as heart,

kidneys, liver, spleen, lungs and brains (Rose et al., 1983).

When Candida albicans grows on a surface, it triggers mechanisms like biofilm

formation and invasion (Kumamoto and Vinces, 2005).

Pathogenic fungi in the genus Candida are recognized as one of the major

causes of hospital-acquired infections. The infectious activity lies in the formation of

biofilms on implanted devices such as shunts, voice prostheses, urinary or

intravascular catheters, prosthetic heart valves, endotracheal tubes, cardiac

pacemakers and joint replacements. Recently the ‘US National Nosocomial Infections

Surveillance System’ ranked Candida species as the fourth most common cause of

sepsis. Candida species also play a role in nosocomial pneumonias and urinary tract

infections. Despite antifungal therapy, mortality of patients with invasive candidiasis

is about 40%. In addition to the diseases caused by Candida, biofilm formation can

also cause device failure (Douglas,2003; Ramage et al., 2006; Chandra et al., 2001).

1.1.2. Biofilms

“Biofilms are structured microbial communities attached to a surface.

Individual microorganisms in biofilms are embedded within a matrix of –often slimy-

extracellular polymers, and characteristically display a phenotype that is markedly

different from that of planktonic cells.” (Douglas, 2003)

In their natural habitats, the majority of micro-organisms are found in biofilms

attached to surfaces and not as planktonic (free-living) organisms. In that way they are

protected from stress factors and can survive in non-optimal conditions (Nikolaev &

Plakunov, 2007).

Another characteristic of biofilms is that their susceptibility to antimicrobial

agents is lower than that of planktonic forms. Clinically important antifungal agents

like amphotericin B, fluconazole , flucytosine, itraconazole and ketoconazole appear

to be less active against Candida albicans biofilms than against planktonic cells.

Introduction

3

Suggested mechanisms of drug resistance are: (i) restricted penetration of drugs

through the biofilm matrix, (ii) limitation of nutrients and decreased growth rate, (iii)

presence of ‘persister’ cells and (iv) expression of resistance genes, like those

encoding efflux pumps (Mukherjee & Chandra, 2004).

Candida albicans biofilms consist of a mixture of morphological forms.

Examination of biofilm development, using scanning electron microscopy, shows that

biofilm formation occurs in 4 phases ( figure 1.2.). It starts with the attachment and

colonization of yeast cells (=blastospores) to a surface. After 3-6 hours, division,

growth and proliferation of yeast cells allow the formation of a basal layer of

anchoring cells. After 48 hours, fully mature biofilms are produced and they consist of

a dense network of yeasts, hyphae, pseudohyphae and extracellular matrix. In the last

phase there is further dissemination of the organism because newly formed yeast cells

on the surface of the biofilm are released from the biofilm (Nobile & Mitchell, 2006;

Ramage et al., 2006; Seneviratne et al., 2008; Kruppa, 2008).

FIGURE 1.2. STAGES IN THE FORMATION OF CANDIDA ALBICANS BIOFILM

ON A POLYVINYL CHLORIDE (PVC) CATHETER SURFACE. (Douglas, 2003)

a Catheter surface with an adsorbed conditioning film of host proteins (black dots) such as fibronectin, fibrinogen and salivary factors b Initial adhering of yeasts ( in red) to the surface c Division, growth and proliferation of yeast cells allow the formation of basal layers of anchoring cells. d Maturation of the biofilm: formation of hyphae (green) , pseudohyphae and extracellular matrix (orange). Mature biofilms contain numerous microcolonies with interspersed water channels allowing circulation of nutrients.

Introduction

4

The extracellular matrix, also known as the exopolymeric substance (EPS) is

made by cells within the biofilm and consists predominantly of carbohydrates,

proteins and DNA. The amount of extracellular material increases with the incubation

time, until Candida albicans colonies are completely enclosed (Chandra et al., 2001;

Kruppa, 2008; Muhkerjee & Chandra, 2004). Research by Nobile and Mitchel (2006)

and by Douglas (2003), shows that more EPS is produced in biofilms growing under

high-flow conditions than in biofilms growing under low-flow conditions. It is

expected that one of the goals of the matrix is to strengthen the biofilm structure under

rough environmental conditions. However, very little is known about the role of the

matrix.

Mature Candida biofilms consist of a complex three-dimensional structure.

Water channels between hyphal cells allow the influx of nutrients from the

environment through the biomass to the bottom layers and facilitate the disposal of

waste products (Ramage et al., 2006; Douglas, 2003; Seneviratne et al., 2008).

The fact that Candida spp. are normal commensals of humans facilitates the

contact with implanted biomaterials. These biomedical devices are usually surrounded

by body fluids, so their surfaces are often covered by a glycoproteinaceous

conditioning film after implantation. This conditioning film facilitates the attachment

of planktonic cells. The initial attachment of Candida cells to biomaterials is mediated

by nonspecific interactions between cells and the substratum, such as hydrophobic

and electrostatic forces. In addition, specific adhesins on the fungal surface recognize

ligands in the conditioning films such as serum proteins (fibrinogen and fibronectin)

and salivary factors. Besides, Candida cells can coaggregate and bind with bacteria

already colonizing these devices (Ramage et al., 2006).

Another biochemical facilitator for biofilm formation described in the literature

is cell to cell communication. Neighboring Candida albicans cells sense each other’s

presence and communicate through signaling molecules, a process called quorum

sensing. Two quorum-sensing molecules have been characterized in Candida

albicans: farnesol and tyrosol. It is generally accepted that farnesol inhibits biofilm

formation through inhibition of the morphological transition from yeasts to hyphae.

Tyrosol on the other hand, accelerates the morphological transition and probably

Introduction

5

protects Candida from phagocytic killing by neutrophils (Nobile and Mitchell, 2006;

Alem et al., 2006; Kruppa, 2008).

Different materials and growth media support the growth of biofilms in the

laboratory. The structure of the resulting biofilms is influenced by parameters such as

medium composition, temperature and the nature of the substratum. Furthermore,

different strains of the same Candida species can differ in their ability to form

biofilms. There are ‘strong’ and ‘weak’ biofilm forming strains within each Candida

species (Kumamoto and Vinces, 2005; Seneviratne et al., 2008).

Candida biofilms are very difficult to eradicate because of their high antifungal

resistance. Hence, when a Candida biofilm proliferates in vivo, the only possible

solution to eliminate the infection is to remove the substrate that supports the biofilm

growth. However, substrate removal is often difficult or impossible due to the

patient’s condition, the anatomic location or the underlying disease. These restrictions

make that Candida biofilms, with increasing frequency and severity, have a huge

impact on the health of patients, resulting in a growing economic problem. Concerns

about the alarming effects of in vivo Candida proliferation on human health and the

treating cost, have led researchers to focus on prevention and management of biofilms

(Ramage et al., 2006). Dusane et al. (2008) suggest surface-active agents of chemical

and biological origin as potential biofilm disruptors.

1.2 LACTIC ACID BACTERIA

Lactic acid bacteria (LAB) are Gram-positive, non-sporulating, low-GC (< 55

mol% Guanine Cytosine base pairs in DNA), non-respiring, fastidious, acid-tolerant

and catalase-negative cocci, coccobacilli or rods. The lack of a respiratory chain

causes them to exhibit a fermentative metabolism, some species are aerotolerant,

others are strictly anaerobic. Depending on how they ferment hexoses, we distinguish

between homo- or hetero- fermentative lactic acid bacteria. The homo-fermentative

LAB use the glycolysis (Embden-Meyerhof-Parnas) pathway, resulting in lactic acid

as the main end product. Hetero-fermentative LAB use the 6-phosphogluconate

/phosphoketolase pathway resulting in lactic acid, carbon dioxide and ethanol (or

acetic acid) as the major end products. Lactic acid can be produced as the D-(-)-

Introduction

6

isomer, as the L-(+)- isomer, or as a racemic mixture (Stiles and Holzapfel,1997;

Wessels et al., 2004; Jehanno et al. 1992; Ström 2005).

LAB play a role in the digestive system of humans and animals. Some live as

commensals in the human oral cavity, the intestinal tract and the vagina, having

beneficial influence on these human ecosystems. As such, they are potential

candidates for application as probiotics. In addition, they are economically important

in the food industry. The natural microflora of many fermented foods such as milk,

meats, vegetables and cereal products is predominated by LAB which serve as

preservatives by lowering the pH to 4 (due to lactic acid formation) and thereby

inhibiting the growth of most other microorganisms. The lowered pH can also change

the food-texture by precipitating some proteins. However, the fermentation and the

growth capacity of LAB is self-limited due to their sensitivity to acidic pH

environments (Stiles and Holzapfel, 1997; www.waksmanfoundation.org).

Apart from lactic acid, other compounds produced by LAB used in the food-

industry include: aroma compounds, exo-polysaccharides and bacteriocins. LAB are

also used in food processing because they lower the carbohydrate content of the foods

they ferment. The most important LAB genera found in foods are Lactobacillus,

Leuconostoc, Streptococcus and Pediococcus (Stiles and Holzapfel, 1997; Wessels et

al., 2004).

Lactobacilli are used for the production of cheese, yoghurt, fermented plant

foods, fermented meats, wine, beer and sourdough bread (Stiles and Holzapfel, 1997).

In addition, they are commensals of the urogenital tract of healthy pre-menopausal

women. When the Lactobacilli of the urinary tract are overwhelmed by uropathogens,

it results in an urinary tract infection. Lactobacilli can interfere with pathogens

through several mechanisms: (i) their cells (viable and non-viable) and cell wall

fragments can competitively inhibit uro-pathogen-adherence to uro-epithelial cells,

polymers and catheter surfaces, (ii) Lactobacilli can coaggregate with uro-pathogens

to eliminate them or (iii) they may produce a variety of metabolic by-products with

antimicrobial activity, like hydrogen peroxide, lactic acid, bacteriocins and

biosurfactants (Velraeds et al., 1996).

Introduction

7

In general, Lactobacilli can be divided into three groups based on their

phenotypic characteristics. Group 1 includes the obligatory homo-fermentative

Lactobacilli that ferment glucose exclusively to lactic acid. The best known species of

this group is Lb. acidophilus, extensively studied for its typical ‘probiotic’ properties.

Group 2 includes facultative hetero-fermentative Lactobacilli that ferment hexoses to

lactic acid. Also pentoses can be fermented into lactic and acetic acids by an inducible

phosphoketolase. Important food-associated species in this group include Lb. casei

and Lb. plantarum. Lb. casei occurs in dairy products, the human mouth and the

human intestine. Group 3 includes the obligatory hetero-fermentative Lactobacilli that

ferment hexoses to lactic acid, acetic acid, ethanol and carbon dioxide (Stiles and

Holzapfel,1997).

The genus Leuconostoc consists of hetero-fermentative cocci, typically living

on plants. They produce D-(-)-lactate from glucose which distinguishes them from L-

(+)-lactate-producing Lactococci and DL-lactate-producing hetero-fermentative

Lactobacilli (Stiles and Holzapfel,1997).

The genus Pediococcus is homo-fermentative and is associated with the

spoilage of beer. It consists of cocci forming tetrads or pairs. Most species produce

DL-lactate from glucose. They are part of the normal flora of the oral cavity and the

gastrointestinal tract and are used as probiotics in the food industry (Stiles and

Holzapfel, 1997; Carr et al., 2002).

Streptococci cause a large number of human and animal diseases. Based on

16S rRNA sequence similarities streptococci are divided into three groups, namely

Streptococcus sensu strictu, Enterococcus and Lactococcus. Other species in the

genus Streptococcus belong to the viridans group. Streptococcus thermophilus is used

as a starter organism for yogurt and cheese production (Stiles and Holzapfel,1997).

Introduction

8

1.3 BIOSURFACTANTS

Biosurfactants are amphipathic molecules mostly excreted by micro-organisms

outside the cells, and in some cases attached to the cells, predominantly during growth

on water-immiscible substrates. They contain both hydrophilic and hydrophobic parts.

Hydrophilic parts can consist of amino acids or peptides, phosphate, alcohol and

mono- di- or poly-saccharides. Hydrophobic parts consist of unsaturated or saturated

fatty acids (Desai & Banat, 1997).

Biosurfactants prefer to proliferate at the interface between fluid phases with

different polarity. In that way they are able to reduce surface and interfacial tension.

In view of these characteristics biosurfactants are used as emulsifiers, de-emulsifiers,

wetting and foaming agents and detergents (Desai & Banat, 1997). Biosurfactants

belong to a wide range of chemical classes: glycolipids, lipopeptides, polysaccharide-

protein complexes, phospholipids, fatty acids and neutral lipids. It is considered that

different groups of biosurfactants can have diverse properties and physiological

functions (Rodrigues et al., 2006a).

Some biosurfactants play an essential role in the survival of biosurfactant-

producing microorganisms. They facilitate nutrient transport, act as biocides and

promote the interaction between the microbe and the host. In addition, biosurfactants

increase the surface area and the bioavailability of hydrophobic substrates and are

able to modify the microbial surface hydrophobicity, affecting microbial adhesion and

detachment to/from solid surfaces. Furthermore, they can bind heavy metals and they

play a role in bacterial pathogenesis, quorum sensing and biofilm formation

(Rodrigues et al., 2006a; Rivardo et al., 2009; Ron & Rosenberg, 2001).

Microbial surfactants have a lower toxicity and a higher biodegradability than

synthetic surfactants. They are also effective at extreme temperatures and extreme pH

values. Microbial surfactants are produced through fermentation for use in

environmental protection, oil recovery and food-processing industries. Besides,

biosurfactants have their use in the medical world as antibacterial, antifungal and

antiviral agents (Rodrigues et al., 2006a; Desai & Banat, 1997).

Introduction

9

The activity of biosurfactants is determined by measurement of the critical

micelle concentration (CMC), the stabilization or destabilization of emulsions and the

hydrophilic-lipophilic balance (HLB) (Desai & Banat, 1997).

To define the critical micelle concentration of a surfactant, the surface tension

at an air/water interface for different concentrations of surfactant is measured with a

tensiometer. When surfactant concentrations increase, a reduction of surface tension is

observed. Surfactant molecules are initially enriched at the surface until the surface is

saturated. At that point, further increase in surfactant concentration will no longer

decrease the surface tension. This point is known as the CMC (Figure 1.3). Above the

CMC, the surfactants start to form aggregates inside the liquid known as micelles.

Micelles can be spherical, ellipsoid, cylindrical or bilayer structures depending on the

molecular geometry of the surfactants and the solution properties. In aqueous

solutions, micelles are aggregates having the hydrophilic regions of the surfactants at

their surfaces, while the hydrophobic regions accumulate in their centers. Contrarily,

water- in- oil- micelles have the hydrophilic groups in their centers and the

hydrophobic groups at their surfaces. The surface tension of pure distilled water is

about 72 mN/m and addition of a surfactant lowers this value to approximately 30

mN/m (Desai & Banat, 1997; Rivardo et al., 2009; http://www.kruss.de).

FIG.1.3. EXPLANATION OF THE CRITICAL MICELLE CONCENTRATION.

(http://www.kruss.de/en/theory/measurement/surface-tension/cmc-measurement.html)

Introduction

10

Biosurfactants may stabilize or destabilize emulsions. In an emulsion one

liquid phase is dispersed as microscopic droplets in another liquid phase. The

emulsifying activity of surfactants is determined by their ability to generate turbidity

when hydrocarbons are added to an aqueous system. The de-emulsifying activity of

surfactants is assayed by adding the surfactants to a standard emulsion made by

synthetic surfactants (Desai and Banat, 1997).

The HLB is a relative value, indicating whether a surfactant will promote

water-in-oil or oil-in-water emulsion and is determined by comparing it with

surfactants with known HLB values and properties (Desai and Banat, 1997).

Excreted biosurfactants can form a conditioning film on an interface, thereby

changing the hydrophobicity of the interface (Ron & Rosenberg, 2001).

Biosurfactants may affect both cell-to-cell and cell-to-surface interactions depending

on the initial microbial hydrophobicity, the biosurfactant structure and its

concentration. As a result, the interface is made less supportive for microbial

deposition, influencing the adhesion, desorption and ability to co-aggregate of

microorganisms (Rivardo et al. 2009; Walencka et al. 2008).

It is generally recognized that biosurfactants inhibit the adhesion of pathogenic

organisms to solid surfaces and infection sites. Rodrigues et al. (2006a) described

specific anti-adhesive-activities of rhamnolipids produced by Pseudomonas

aeruginosa, glycolipids produced by Streptococcus thermophilus and surfactants

produced by Lactococcus lactis against several bacterial and yeast strains isolated

from voice prostheses. Surfactants produced by Streptococcus mitis have an anti-

adhesive activity against Streptococcus mutans and surfactin derived from Bacillus

subtilis decreases the amount of biofilm formed by Salmonella Typhimurium,

Salmonella enterica, Escherichia coli and Proteus mirabilis in polyvinyl urethral

catheters. The biosurfactants secreted by Lactobacillus fermentum RC-14 reduce

infections caused by Staphylococcus aureus associated with surgical implants and

inhibit the adhesion of uropathogenic bacteria, including Enterococcus faecalis.

Biosurfactants produced by Lactobacillus acidophilus inhibit biofilm formation by

uropathogens and yeasts on silicone rubber. From this, we may assume that

Introduction

11

biosurfactants develop an anti-adhesive biological coating. In the future, the latter can

be useful for the protection of indwelling medical devices.

Although biosurfactants are recognized to have a huge potential, they are still

not common in today’s medical and anti-infective practice. Reasons for this are the

high production and extraction cost and the lack of information on toxicity in human

systems. Further investigation is needed to validate the use of biosurfactants for

biomedical and health-related applications (Rodrigues et al., 2006a).

Objectives

12

2. OBJECTIVES

The goals of this research are:

(1) characterization of the lactic acid bacteria, primarily isolated in the laboratory,

by performing a catalase test, Gram staining and a lactic acid production test,

by constructing growth curves, by examining biosurfactant production through

the oil spreading test and by determining their ability to form biofilms using

the MBECTM device.

(2) characterization of biosurfactants, extracted from the lactic acid bacteria, by

determining the surface tension reduction activity and the Critical Micelle

Concentration.

(3) investigation of the anti-biofilm activities of the extracted biosurfactants on

Candida albicans CA11225 and CA2894 biofilms, using the crystal violet

assay. Experiments are performed by: (i) adding yeast culture to biosurfactant

precoated flatbottemed 96 well polystyrene microtiter plates or by (ii)

incubating biosurfactants together with yeast culture in flatbottemed 96 well

polystyrene microtiter plates.

Experimental

13

3. EXPERIMENTAL

3.1. MATERIALS

3.1.1. Strains

3.1.1.1 Candida albicans

Experiments are conducted by using two different strains of Candida albicans.

CA2894, isolated from the human tongue, is provided by the Belgian Co-ordinated

Collections of Microorganisms (BCCM). CA11225, isolated from blood, is provided

by the German Collection of Microorganisms and Cell Cultures (DSMZ).

3.1.1.2 Lactic acid bacteria

The lactic acid bacteria are isolated from fruits and vegetables in the laboratory

of professor Martinotti. They grow on both MRS-agar and Rogosa agar. MRS-agar is

designed to enhance selective growth of Lactobacilli, although growth of Leuconostoc

spp. and Pediococci can also occur. Rogosa agar is a selective medium for the

isolation of Lactobacilli. It suppresses the growth of many strains of other lactic acid

bacteria, having a high acetate concentration and a low pH

(http://www.oxoid.com/UK/blue/prod_detail/prod_detail).

The experiments described below are conducted with isolates CV8LAC,

isolated from cabbage, INS9T7I, isolated from lettuce and ME11T7I, isolated from

apples.

3.1.2. Media

Luria-Bertani broth (LB-broth) is made by adding 5 g of Peptone (Fluka

Chemika), 10 g of Tryptone (Fluka Chemika) and 10 g of Sodium chloride

(Fluka Chemika) to 1 L of distilled water. The mixture is sterilized by

autoclaving at 121°C for 15 minutes.

Man Rogosa and Sharpe (MRS) is composed as illustrated in table 3.1.

It is provided by Oxoid. Fifty two grams are suspended in 1 L of distilled

water at 60°C and are mixed until completely dissolved. The mixture is

dispensed into the final containers and sterilized by autoclaving at 121 °C for

15 minutes.

Experimental

14

TABLE 3.1 COMPOSITION OF MRS (OXOID)

Component Concentration

(g/L)


(g/L)

Tryptone 10.0

Dipotassium hydrogen phosphate 2.0

Meat extract 8.0 Tween® 80 1.0

Yeast extract 4.0 Magnesium sulfate 2.0

D-(+)-glucose 20.0 Manganese sulfate 0.04

Rogosa-broth (ROG) is provided by Oxoid. The composition is represented in

table 3.2. Eighty two grams are suspended in 1 L of distilled water and are

brought to boiling till complete dissolution. One thousand thirty two

microliters of glacial acetic acid are added and the solution is mixed

thoroughly. The mixture is heated to 90-100°C for 2-3 minutes with frequent

agitation and is distributed into the final containers. Rogosa broth must not be

autoclaved.

TABLE 3.2 COMPOSITION OF ROGOSA BROTH (OXOID)


(g/L)


(g/L)

Tryptone 10.0 Potassium dihydrogen

phosphate

6.0

Ammonium

citrate

2.0 Tween® 80 1.0

Yeast extract 5.0 Magnesium sulfate 0.575

D-(+)-glucose 20.0 Manganese sulfate 0.12

Sodium acetate 15.0 Iron (II) sulfate 0.034

Yeast Nitrogen Base (YNB) is provided by Fluka Chemika. Its composition is

given in table 3.3. The medium is prepared using 6.7 g of YNB in 100 mL of

distilled water, supplemented with 100 mM glucose (Sifin) and filter-

sterilized.

Experimental

15

TABLE 3.3. COMPOSITION OF YNB (FLUKA)


(µg/L)


(g/L)

P-amino-benzoic

acid

200.0 Ammonium

sulfate

5.0

Biotin 2.0 Calcium chloride 0.1

Boric acid 500.0 Magnesium

sulfate

0.575

Calcium

pantothenate

400.0 DL-methionine 0.020

Manganese sulfate 400.0 Iron (II) sulfate 0.034

Niacin 400.0 Potassium

phospate

1.0

Potassium iodide 100.0 L-histidine HCL 0.010

Pyridoxine HCL 400.0 Inositol 0.002

Copper sulfate 40.0 Magnesium

sulfate

0.5

Ferric chloride 200.0 Sodium chloride 0.1

Folic acid 2.0 DL-tryptophan 0.020

Riboflavin 200.0

Sodium molybdate 200.0

Thiamine HCl 400.0

Zinc sulfate 400.0

Universal Medium for Yeast (UMY) is made by mixing 3.0 g/L of yeast

extract (Microbiol® Diagnostics), 3.0 g/L of malt extract (Biolife Italiana

S.r.l), 5.0 g/L of peptone (Oxoid), 10.0 g/L of glucose (Sifin) and 15 g/L of

agar (Oxoid). The mixture is dispensed into the final containers and sterilized

by autoclaving at 121 °C for 15 minutes.

Sabouraud Dextrose Broth pH 5.6 (SDB) by Sigma Biochemika consists of 10

g/L of Mycological Peptone and 20 g/L of Dextrose. Thirty grams of

Sabouraud Dextrose Broth are suspended in 1 L of distilled water. The

Experimental

16

suspension is boiled to dissolve the medium completely and is sterilized by

autoclaving at 121°C for 15 minutes

3.1.3. Other materials

Sodium chloride, necessary for the physiological saline (PS) is provided by

Fluka Chemika.

Hydrogen peroxide-solution (3%) for the catalase test is a solution of

Hydrogen peroxide (30%, Carlo ERBA Reagenti) in sterile distilled water.

McFarland 1.0 standard is prepared by mixing specific amounts of barium

chloride and sulfuric acid to form a barium sulphate precipitate, which causes

turbidity of the solution. The barium sulphate precipitate (provided by Fluka

Chemika) used in the experiments has an optical density of 0.25 at a

wavelength of 550 nm, corresponding to a concentration of 3X108 CFU/mL in

case of lactic acid bacteria and to a concentration of 1X107 CFU/mL in case of

Candida albicans.

Tween 20® (= poly-oxy-ethylene-sorbitan mono-laurate) is provided by Sigma

Biochemikal. The concentration needed in the LB-Tween 20®- mixture is 1%.

Phosphate buffered saline pH 7 (PBS), used for the exctraction of

biosurfactants, following Velraeds et al. (1996) is made by first adding 0.544 g

KH2PO4 (Sigma) to 4 mL of distilled water (solution 1). Then 1.22 g of

K2HPO4 (Riedel-de Haën) is added to 7 mL of distilled water (solution 2).

Next, 3.85 mL of solution 1 and 6.15 mL of solution 2 are mixed with 90 mL

of distilled water and 1.75 g of NaCl (Fluka Chemika) are added. Finally,

distilled water is added to obtain an amount of 200 mL.

Crystal Violet (2%) is a mixture of 2 solutions, A and B. Solution A is

composed of 2.0 g of crystal violet (Fluka Chemika) and 20 mL of ethyl

alcohol (95%, Fluka Chemika). Solution B is composed of 0.8 g of ammonium

oxalate (Fluka Chemika) and 80.0 mL of distilled water. The solution should

be stored for 24 hours before use. The stain is stable.

Experimental

17

Methanol is provided by Fluka Chemika.

Ethyl acetate is provided by Fluka Chemika.

Anaerobic conditions are created by putting the plates in plastic pouches,

containing an AnaeroGenTM Compact-back (OXOID) and an anaerobic

indicator (OXOID). The pouches are closed with sealing clips.

The Lambda 35 spectrophotometer from Perkin Elmer® One sourceTM

Laboratory Services is used to make the growth curves.

The tensiometer is the Sigma 703D from KSV instruments (Finland).

The pH-meter is provided by Hanna Instruments.

The model of spectrophotometer to measure growth in the microtiter plates is

an Ultramark, Bio-Rad Microplate Imaging System.

The sonicator is an Aquasonic 250HT from VWR International,

(Mississauga,Canada).

Three types of 96-well microtiter plates are used in the experiments. The

standard polystyrene flat bottomed 96 well microtiter plates provided by

Bioster, the microtiter plates used in the sonicator by Nunc A/S and the

MBECTM plates from Innovotech (Edmonton, AB, Canada).

The centrifuge is a Sorvall RC 5B plus.

Experimental

18

3.2. METHODS

3.2.1 General characteristics of the three isolates

3.2.1.1 Catalase test, Gram staining, oil spreading test and lactic acid production test

The isolates were subjected to the catalase test in order to detect if they possess

the catalase enzyme, using 3% hydrogen peroxide. Catalase positive bacteria produce

oxygen bubbles when added to hydrogen peroxide.

Gram staining was performed to distinguish whether the isolates were Gram

positive or negative and to examine their different morphologies. To find out if the

isolates produced biosurfactants, an oil spreading test was performed. This test

measured the diameter of clear zones appearing when 20 µL of a biosurfactant

containing solution is placed on an oil-water surface (Rodrigues et al., 2006b). An oil-

water surface is made by filling a Petri dish (9 cm diameter) with tap water, followed

by the addition of 20 µL of motor oil 10W-40 (Selenia). The oil forms a hydrophobic

film on the surface.

The lactic acid production of the CV8LAC, INS9T7I and ME11T7I isolates

was examined using Biolog microtiter test plates.

3.2.1.2 Growth curves of the CV8LAC, INS9T7I and ME11T7I isolates

Growth curves of the isolates were constructed according to Velraeds et al.

(1996). Each time 400 ml of MRS broth was incubated with 10ml of an overnight

subculture of every isolate. To monitor growth, at regular time intervals during 26

hours, (i) the optical density was measured at 550 nm and (ii) several dilutions (1:10)

were plated on MRS-agar to measure the number of CFU/ mL. In parallel, the

production of biosurfactants in the supernatant was determined, using the oil

spreading test (cf. 3.2.1.1).

3.2.1.3 Biofilm formation by the CV8LAC, INS9T7I and ME11T7I isolates in MRS

and ROG medium

Based on Harrison et al. (2006), we used the Calgary Biofilm device (MBECTM,

Innovotech, Edmonton, AB, Canada) for biofilm production. The MBECTM consists

of a polystyrene lid with 96 pegs, which have a surface area of 108,9 mm² each. This

pegs fit inside a standard 96-well microtiter plate. Starting from a cryogenic stock (-

Experimental

19

80°C) of the 3 lactic acid bacteria, a first culture was made on an MRS agar plate and

then incubated in anaerobiosis at 28 °C for 24 hours. The subculture was checked for

purity and from this first subculture a second subculture was made in a similar way.

The colonies on the latter were used to inoculate approximately 10 mL of PS (0.9 %

NaCl) with a sterile swab. For each isolate, a suspension was made by adding bacteria

until the turbidity was comparable to that of a McFarland 1.0 standard. From this

suspension, a 30-fold dilution - the inoculum - was made in MRS broth or ROG broth,

yielding a concentration of about 1.0x107 CFU/mL.

For biofilm formation, each well of the MBECTM device was filled with 200 µL

of the inoculum. Some wells were filled with pure MRS medium or pure ROG

medium, serving as sterility controls. The microtiter plate was incubated on a rotary

shaker (130 rpm) at 37°C for 24 hours. The division of the microtiter plate is depicted

in figure 3.1.

1 2 3 4 5 6 7 8 9 10 11 12 A 200µl

ROG 200 µl ROG

200 µl ROG

200 µl ROG

200 µl ROG

200 µl ROG

200 µl ROG

200 µl ROG

200 µl ROG

200 µl ROG

200 µl ROG

200µl ROG

B 200µl ROG

200 µl ROG

200 µl ROG

200 µl ROG

200 µl ROG

200 µl ROG

200 µl ROG

200 µl ROG

200 µl ROG

200 µl ROG

200 µl ROG

200µl ROG

C 200µl ROG

CV8LAC in ROG

CV8LAC in ROG

CV8LAC in ROG

ME11T7I in ROG

ME11T7I in ROG

ME11T7I in ROG

ME11T7I in ROG

INS9T7I in ROG

INS9T7I in ROG

INS9T7I in ROG

200µl ROG

D 200µl ROG

CV8LAC in ROG

CV8LAC in ROG

CV8LAC in ROG

ME11T7I in ROG

ME11T7I in ROG

ME11T7I in ROG

ME11T7I in ROG

INS9T7I in ROG

INS9T7I in ROG

INS9T7I in ROG

200µl ROG

E 200µl MRS

CV8LAC in MRS

CV8LAC in MRS

CV8LAC in MRS

ME11T7I in MRS

ME11T7I in MRS

ME11T7I in MRS

ME11T7I in MRS

INS9T7I in MRS

INS9T7I in MRS

I NS9T7I in MRS

200µl MRS

F 200µl MRS

CV8LAC in MRS

CV8LAC in MRS

CV8LAC in MRS

ME11T7I in MRS

ME11T7I in MRS

ME11T7I in MRS

ME11T7I in MRS

INS9T7I in MRS

INS9T7I in MRS

INS9T7I in MRS

200µl MRS

G 200µl MRS

200µl MRS

200µl MRS

200µl MRS

200µl MRS

200µl MRS

200µl MRS

200µl MRS

200µl MRS

200µl MRS

200µl MRS

200µl MRS

H 200µl MRS

200µl MRS

200µl MRS

200µl MRS

200µl MRS

200µl MRS

200µl MRS

200µl MRS

200µl MRS

200µl MRS

200µl MRS

200µl MRS

FIGURE 3.1.: DIVISION OF THE MBECTM DEVICE USED FOR BIOFILM

FORMATION WITH THE CV8LAC, ME11T7I AND INS9T7I ISOLATES.

In parallel, a serial dilution (10-fold) was carried out in a second microtiter plate

and 20 µL of the dilutions were streaked on MRS agar plates, which were incubated

overnight at 28 °C in anaerobiosis. To control the inoculum’s cell number, the

colonies were counted after 24 hours and the average number of colony forming units

(CFU) per milliliter was calculated.

Experimental

20

To remove the planktonic cells, after a 24 hour incubation, the lid of the

MBECTM with the pegs on which biofilm adhered, was washed by placing it for one

minute in successively two microtiter plates filled with 180 mL of PS. Then, the

biofilm was detached by putting the lid in a NUNC microtiter plate containing 200 µL

of Luria Bertani (LB) + Tween20® per well and by placing this plate in a sonicator at

60 Hz for 15 minutes.

To calculate the average number of CFU’s per peg, the LB + TWEEN20®

solution, containing the biofilm cells, was serially diluted (1:10) in microtiter plates

and 20 µL of each dilution was streaked on MRS agar. The plates were incubated

overnight in anaerobic conditions at 28 °C and the colonies were counted the next

day.

3.2.2 Biofilm formation by Candida albicans

Based on the methods described by Jin et al. (2003) and Peeters et al. (2007)

to form and quantify Candida albicans biofilms, a protocol was developed to perform

experiments using polystyrene 96 well microtiter plates.

Starting from an overnight broth culture of Candida albicans in Yeast

Nitrogen Base (YNB), a suspension was made containing approximately 107 CFU

/mL. This concentration is reached by inoculating YNB medium with Candida

albicans colonies until the turbidity is similar to the McFarland 1.0 Standard. Six rows

of a 96 well microtiter plate were inoculated with 150 µL of this suspension, while the

seventh row served for sterility control, only containing pure media. The last row

functioned as a blank control and remained empty.

After 3 hours of adhesion at 37°C on a rotary shaker at 75 rpm, the supernatant

(containing non-adhered cells) was removed, the wells were rinsed with 100 µL of PS

and 150 µL of new medium was added to each well. Then the plate was incubated at

37°C on a rotary shaker at 75 rpm for a minimum of 24 hours.

Quantification of the biofilm was done using the crystal violet assay (Peeters

et al, 2007; Christensen et al., 1985). In a first step, the supernatant was removed and

the residue in the wells was rinsed twice with 100 µL of physiological solution. Then,

Experimental

21

the biofilm was fixed for 15 minutes by adding 100 µL of 99% methanol to each well

(except the blank). Again the supernatant was removed and the plate was air dried,

after which 100 µl of a solution of crystal violet (2%) was added and left in contact

for 20 minutes. The excess crystal violet was removed with a pipette and the residue

in the wells was washed with tap water and air dried.

The biofilm biomass was quantified with a spectrophotometer measuring the

light absorbance at a wavelength of 595 nm. Absorbance values higher than 3 times

the standard deviation of the sterility control (± 0.120) imply production of mature

biofilm. Values below three times the standard deviation of the sterility control (±

0.120) mean that no biofilm formation has occurred.

Different culture media (YNB, SDB, UMY) and different incubation times

(24h, 48h, 72h) were tested to get an indication of the optimal conditions for biofilm

growth.

3.2.3. Biosurfactants produced by the CV8 LAC, INS9T7I and ME11T7I isolates

3.2.3.1. Biosurfactants activity

To test whether biosurfactant molecules were attached to the bacterial cell wall

or excreted in the supernatant, the oil spreading test was used, as described in 3.2.1.1.

First, each isolate was plated on MRS agar and incubated for 72 hours in anaerobiosis

at 28°C. Then one or two colonies of each isolate were inoculated in 3 tubes

containing 20 mL of MRS broth and incubated for 24 hours in anaerobiosis.

Thereafter, the culture broths were first tested by the oil spreading test (3.2.1.1) and

then centrifuged for 15 minutes at 4000 rpm to separate the cells from the supernatant.

The cells were washed twice in 5 mL of PS, resuspended in 500 µL PS and subjected

to the oil spreading test. The oil spreading test was also performed on the supernatant.

Production of biosurfactants was examined after 5 and 20 hours of bacterial

growth by carrying out the oil spreading test on the isolates cultured in MRS broth.

Experimental

22

3.2.3.2. Extraction of biosurfactants

Assuming that the biosurfactants were excreted in the supernatant, an organic

solvent extraction was done. Twenty milliliter of an overnight culture of INS9T7I,

ME11T7I or CV8LAC in MRS broth were added to 1 L of MRS broth and incubated

for 5 hours at 28°C, followed by 10 minutes centrifugation at 5000 rpm. The

supernatant was acidified with hydrochloric acid (6M) to pH 2. At this pH, the iso-

electric point of the biosurfactants was reached resulting in a decrease of their

solubility. After one night at 4°C, the precipitated biosurfactants were extracted from

the broth using a mixture of ethyl acetate and methanol (4:1 v/v). The biosurfactants

move from the hydrophilic phase (MRS broth) into the organic, hydrophobic phase.

After removal of the remaining water with anhydrous sodium sulphate, the extract

was brought in an evaporation flask, which was put on a rotavapor until a thin film of

biosurfactants remained. The biosurfactants were recovered by adding sufficient

acetone and transferred into another flask. Then the solvent was evaporated. The dry

extract was collected, weighed and stored at room temperature.

To exclude that biosurfactant molecules were attached to the bacterial cell

wall, an extraction was done using the method described by Velraeds et al. (1996).

First, 15 mL of an overnight culture of INS9T7I, ME11T7I or CV8LAC in MRS

broth were added to 600 mL of MRS broth and incubated for 5 hours or 20 hours at

28°C. Then, cells were separated from the supernatant by centrifugation (10000g, 5

minutes, 10°C), washed twice in demineralized water and re-suspended in 100 mL of

PBS. To release biosurfactants, the lactic acid bacteria in PBS were stored for 2 hours

at room temperature with gentle stirring. Finally, cells were again separated from the

supernatant by centrifugation and the supernatant, potentially containing the

biosurfactants, was filtered through a 0.22 µm pore size filter (Millipore). The filtrate

and the broth supernatant were subjected to the oil spreading test (3.2.1.1.).

3.2.3.3. Characterization of the extracted biosurfactants

To measure the surface tension of the biosurfactants, 25 mL of a biosurfactant

solution (minimally 500 µg/mL) was prepared in demineralized water and adjusted to

pH 8 by adding hydrochloric acid or sodium hydroxide. Measurements were carried

out at room temperature using a Sigma 703D tensiometer equipped with a du Noüy

platinum iridium ring. The critical micelle concentration was determined by diluting

Experimental

23

the biosurfactants, replacing 5 mL of the solution with 5 mL of distilled water after

each measurement. Surfactants exhibit a specific surface tension curve as a function

of their concentration and from this, the CMC can be calculated. Initially the

surfactant molecules are enriched at the water surface. During this phase the surface

tension decreases linearly with the surfactant concentration, resulting in a steep linear

plot. When the surface is saturated with biosurfactants, a further increase in surfactant

concentration no longer decreases the surface tension, resulting in a plot with a lower

slope. The CMC is obtained by calculating the intersection of the trendlines for the

concentration dependent and the concentration independent sections.

3.2.3.4. Biosurfactants tested on Candida albicans biofilm

To test if the extracted biosurfactants, derived from the CV8LAC, INS9T7I

and ME11T7I isolates, could inhibit the biofilm formation by Candida albicans

CA11225 and CA2894, the protocol, followed by the crystal violet quantification, as

described in 3.2.2, was slightly modified.

Precoating experiments

According to Rivardo et al. (2009), a series of experiments was conducted in

which the 96 well microtiter plates were initially coated with different concentrations

of biosurfactants. Based on the different CMC values, we used an initial concentration

of 1600 µg/mL in the experiments conducted with biosurfactants produced by

CV8LAC, and a concentration of 800 µg/mL in the experiments conducted with

biosurfactants produced by INS9T7I and ME11T7I.

After dissolving 8000 µg or 16000 µg of biosurfactants in 10 mL of

physiological solution, the solution was filter sterilized and 200 µL was put in each

well of the B-row of a 96-well microtiter plate. In each of the rows C to G, the

solution was diluted by adding 100 µL of PS to 100 µL of biosurfactant solution of

the row above. The A-row remained empty to serve later (when Candida is added) as

control group for biofilm formation without biosurfactants. The last row was filled

with pure YNB and functioned as sterility control. The division of the microtiter plate

is shown in figure 3.2. The microtiter plate was incubated on a rotary shaker at 75 rpm

for 24 hours at 37°C, then the coating solution was removed and 100 µL of the

Candida albicans inoculum (107 CFU/mL), prepared as described in 3.2.2, were

Experimental

24

added to each well. The microtiter plate was again incubated on a rotary shaker at 75

rpm for 48 hours at 37°C and finally quantification was done by using the crystal

violet assay (3.2.2).

1 2 3 4 5 6 7 8 9 10 11 12

A (a) (a) (a) (a) (a) (a) (a) (a) (a) (a) (a) (a) B (b) (b) (b) (b) (b) (b) (b) (b) (b) (b) (b) (b) C (c) (c) (c) (c) (c) (c) (c) (c) (c) (c) (c) (c) D (d) (d) (d) (d) (d) (d) (d) (d) (d) (d) (d) (d) E (e) (e) (e) (e) (e) (e) (e) (e) (e) (e) (e) (e) F (f) (f) (f) (f) (f) (f) (f) (f) (f) (f) (f) (f) G (g) (g) (g) (g) (g) (g) (g) (g) (g) (g) (g) (g) H (h) (h) (h) (h) (h) (h) (h) (h) (h) (h) (h) (h) (a) row A remains empty (b) add 200µL of biosurfactants with a concentration of X µg ml-1 (c) add 100µL of PS & 100 µL of row B surfactant- concentration= X/2 µg mL-1 (d) add 100µL of PS & 100 µL of row C surfactant- concentration= X/4 µg mL-1 (e) add 100µL of PS & 100 µL of row D surfactant- concentration= X/8 µg mL-1 (f) add 100µL of PS & 100 µL of row E surfactant- concentration= X/16 µg mL-1

(g) add 100µL of PS & 100 µL of row F surfactant- concentration= X/32 µg mL-1 (h) sterility control: add 150 µL of pure YNB-medium

FIGURE 3.2. DIVISION OF A 96 WELL MICROTITER PLATE IN THE FIRST

PHASE OF THE COATING EXPERIMENT.

Coincubation experiments

In another set of experiments, Candida albicans inocula were distributed in

each well together with different biosurfactant dilutions (Rivardo et al., 2009).

Similarly to the coating experiment, different initial concentrations for the three

different biosurfactants were used, based on their CMC values.

For biosurfactants produced by CV8LAC, we started with 3200 µg/mL in

order to obtain concentrations ranging from 1067 µg/mL to 50 µg/mL after addition

of the Candida albicans inocula (107 CFU/mL, prepared as described in 3.2.2), as

explained in figure 3.3. For biosurfactants produced by INS9T7I and ME11T7I, we

started with 1600 µg/mL in order to obtain concentrations ranging from 534 µg/mL to

Experimental

25

25 µg/mL after addition of the Candida albicans inocula (107 CFU/mL, prepared as

described in 3.2.2) as explained in figure 3.4.

The microtiter plates were incubated on a rotary shaker at 75 rpm for 48 hours

at 37°C and finally quantification was performed using the crystal violet assay (3.2.2).

1 2 3 4 5 6 7 8 9 10 11 12

A (a) (a) (a) (a) (a) (a) (a) (a) (a) (a) (a) (a) B (b) (b) (b) (b) (b) (b) (b) (b) (b) (b) (b) (b) C (c) (c) (c) (c) (c) (c) (c) (c) (c) (c) (c) (c) D (d) (d) (d) (d) (d) (d) (d) (d) (d) (d) (d) (d) E (e) (e) (e) (e) (e) (e) (e) (e) (e) (e) (e) (e) F (f) (f) (f) (f) (f) (f) (f) (f) (f) (f) (f) (f) G (g) (g) (g) (g) (g) (g) (g) (g) (g) (g) (g) (g) H (h) (h) (h) (h) (h) (h) (h) (h) (h) (h) (h) (h) First step: (a) row A remains empty (b)add 100 µL of a biosurfactant solution with a concentration of 3200 µg mL-1 (c)add 50 µL of YNB and add 50 µL of row B surfactant concentration= 1600 µg mL-1 (d)add 50 µL of YNB and add 50 µL of row C surfactant concentration= 800 µg mL-1 (e)add 50 µL of YNB and add 50 µL of row D surfactant concentration= 400 µg mL-1 (f)add 50 µL of YNB and add 50 µL of row E surfactant concentration= 200 µg mL-1 (g)add 50 µL of YNB and add 50 µL of row F surfactant concentration= 100 µg mL-1 (h) blank control Second step: (a) add 100 µL of Candida albicans inoculum (107 CFU/mL) (b) add 100 µL of Candida albicans inoculum (107 CFU/mL) surfactant concentration= 1067µg mL-1 (c) add 100 µL of Candida albicans inoculum (107 CFU/mL) surfactant concentration= 534µg mL-1

(d) add 100 µL of Candida albicans inoculum (107 CFU/mL) surfactant concentration= 267µg mL-1 (e) add 100 µL of Candida albicans inoculum (107 CFU/mL) surfactant concentration= 133µg mL-1 (f) add 100 µL of Candida albicans inoculum (107 CFU/mL) surfactant concentration= 67 µg mL-1 (g) add 100 µL of Candida albicans inoculum (107 CFU/mL) surfactant concentration= 50µg mL-1

(h) blank control

FIGURE 3.3. ORGANISATION OF THE MICROTITER PLATE IN THE

COINCUBATION EXPERIMENT WITH BIOSURFACTANTS PRODUCED BY

CV8LAC.

Experimental

26

1 2 3 4 5 6 7 8 9 10 11 12

A (a) (a) (a) (a) (a) (a) (a) (a) (a) (a) (a) (a) B (b) (b) (b) (b) (b) (b) (b) (b) (b) (b) (b) (b) C (c) (c) (c) (c) (c) (c) (c) (c) (c) (c) (c) (c) D (d) (d) (d) (d) (d) (d) (d) (d) (d) (d) (d) (d) E (e) (e) (e) (e) (e) (e) (e) (e) (e) (e) (e) (e) F (f) (f) (f) (f) (f) (f) (f) (f) (f) (f) (f) (f) G (g) (g) (g) (g) (g) (g) (g) (g) (g) (g) (g) (g) H (h) (h) (h) (h) (h) (h) (h) (h) (h) (h) (h) (h) First step: (a) row A remains empty (b)add 100 µl of a biosurfactant solution with a concentration of 1600 µg mL-1 (c)add 50µL of YNB and add 50µL of row B surfactant concentration= 800 µg mL-1 (d)add 50µL of YNB and add 50µL of row C surfactant concentration= 400 µg mL-1 (e)add 50µL of YNB and add 50µL of row D surfactant concentration= 200 µg mL-1 (f)add 50µL of YNB and add 50 µL of row E surfactant concentration= 100 µg mL-1 (g)add 50µL of YNB and add 50 µL of row F surfactant concentration= 50 µg mL-1 (h) blank control Second step: (a) add 100 µL of Candida albicans inoculum (107 CFU/mL) (b) add 100 µL of Candida albicans inoculum (107 CFU/mL) surfactant concentration= 534 µg mL-1 (c) add 100 µL of Candida albicans inoculum (107 CFU/mL) surfactant concentration= 267 µg mL-1

(d) add 100 µL of Candida albicans inoculum (107 CFU/mL) surfactant concentration= 133 µg mL-1 (e) add 100 µL of Candida albicans inoculum (107 CFU/mL) surfactant concentration= 67 µg mL-1 (f) add 100 µL of Candida albicans inoculum (107 CFU/mL) surfactant concentration= 33 µg mL-1 (g) add 100 µL of Candida albicans inoculum (107 CFU/mL) surfactant concentration= 25 µg mL-1

(h) blank control

FIGURE 3.4. ORGANISATION OF THE MICROTITER PLATE IN THE

COINCUBATION EXPERIMENT WITH BIOSURFACTANTS PRODUCED BY

INS9T7I AND ME11T7I.

Results and discussion

27

4. RESULTS AND DISCUSSSION

4.1 GENERAL CHARACTERISTICS OF THE THREE ISOLATES

4.1.1 Catalase test, Gram staining, oil spreading test and lactic acid production

test

The catalase test shows that the isolates are catalase negative, while Gram

staining reveals that all the isolates are Gram positive, occurring in various

morphologies: cocci, rods and coccobacilli. The results from the oil spreading test are

shown in table 4.1. As the diameter of the clear zone for CV8LAC, ME11T7I and

INS9T7I is significantly higher than for the pure MRS broth, we can conclude that the

three strains produce biosurfactants. The three isolates test positive for production of

both DL-lactic acid and L-lactic acid using the Biolog microtiter test plate.

TABLE 4.1: RESULTS OF THE OIL SPREADING TEST

Name Isolate Diameter of the clear zone (cm) a

INS9T7I 2.1 ME11T7I 1.5 CV8LAC 2.8

PURE MRS-broth 1.0

a A Petri dish (9 cm diameter) was filled with tap water and a droplet of motor oil was added. The oil forms a hydrophobic film on the surface. Twenty microliters of broth culture of each isolate was deposited on this surface. The diameter of the appearing clear zones was measured .

4.1.2. Growth curves of the CV8LAC, INS9T7I and ME11T7I isolates.

Growth curves are constructed for the three lactic acid bacteria in order to

determine a relation between cell growth and biosurfactant production. All three

isolates show similar growth curves and a similar capability to produce biosurfactants.

Biosurfactant production was determined by carrying out the oil spreading test on the

supernatant. For all isolates, the biosurfactant production mainly occurs after 5-6

hours in the mid-exponential growth phase. The stationary growth phase is reached

after approximately 12 hours. However, all isolates keep producing biosurfactants

during the 26 hours but in a lesser amount. Results are shown in figures 4.2, 4.3 and

4.4.


28

a A Petri dish (9 cm diameter) was filled with tap water and a droplet of motor oil was added. The

oil forms a hydrophobic film on the surface. Twenty microliter of supernatant was deposited on this surface. The diameter of the appearing clear zones was measured.

FIGURE 4.2. GROWTH CURVE OF CV8LAC AND RESULTS OF THE OIL

SPREADING TEST.

a see figure 4.2.

FIGURE 4.3. GROWTH CURVE OF INS9T7I AND RESULTS OF THE OIL

SPREADING TEST.

a See figure 4.2.

FIGURE 4.4. GROWTH CURVE OF ME11T7I AND RESULTS OF THE OIL

SPREADING TEST.


29

4.1.3 Biofilm formation CV8LAC, ME11T7I, INS 9T7I

The protocol to study biofilm formation by using the Calgary Biofilm

device (3.2.1.3) was applied to the three lactic acid bacteria. Figure 4.5 shows the

results.

FIGURE 4.5 VIABLE CELLS IN THE BIOFILM, OBTAINED FROM ONE PEG

OF THE MBEC DEVICE, PRODUCED BY THE CV8LAC, INS9T7I OR ME11T7I

ISOLATES.

The highest mean viable cell count per peg (see 3.2.3.1) on both media is

obtained for CV8LAC, with slightly higher results in ROG medium than in MRS

medium. Biofilm formation for INS9T7I and ME11T7I is similar and there are no

differences between MRS or ROG medium. Looking back to the growth curves (see

4.1.2), CV8LAC also shows the highest oil spreading values, from which we conclude

that CV8LAC produces the highest amount of biosurfactants. Because of this we can

suggest that there is a possible link between biofilm formation and biosurfactant

production, although to confirm this further research is necessary.

4.2 BIOFILM FORMATION BY CANDIDA ALBICANS

To determine the optimal conditions for biofilm production of the Candida

albicans strains, experiments were conducted using three different media: YNB,

UMY and SDB. The amount of biofilm biomass was quantified using the crystal

violet (2%) assay after an incubation of 24, 48 and 72 hours. Crystal violet is a basic

dye that binds to negatively charged surface molecules and polysaccharides in the

extracellular matrix. (Peeters et al., 2007) Results are shown in figures 4.6 and 4.7.


30

FIGURE 4.6: EVOLUTION IN BIOFILM BIOMASS GROWTH OF CA11225 AS A

FUNCTION OF TIME, QUANTIFIED USING THE CRYSTAL VIOLET ASSAY.

FIGURE 4.7: EVOLUTION IN BIOFILM BIOMASS GROWTH OF CA2894 AS A

FUNCTION OF TIME, QUANTIFIED USING THE CRYSTAL VIOLET ASSAY.

The highest optical densities are reached using YNB medium. Biofilm formation

increases as a function of time, with a big increase between 24 and 48 hours of

incubation and a smaller increase between 48 and 72 hours of incubation. Given these

results, we use YNB in all the following experiments for testing the influence of

biosurfactants on Candida albicans biofilm and we let the biofilms grow for 48 hours.

YNB shows the best results. A possible explanation for this is the fact that YNB is a

rich medium, containing growth factors. All three media contain glucose.


31

The type of substrate and the nature of the media used to grow Candida albicans

biofilms can influence the morphology (blastospores, pseudohyphae or hyphae) and

growth of Candida albicans cells. Biofilms grown using YNB contained mostly

blastospores (Chandra & Ghannoun 2004).

Since crystal violet staining measures the total biomass formed, it can also be

interesting to quantify the number of active cells, e.g. using the reduction of a

tetrazolium salt (XTT).

4.3 BIOSURFACTANTS PRODUCED BY THE CV8LAC, ME11T7I AND INS9T7I

ISOLATES.

4.3.1. Biosurfactants activity

To verify whether biosurfactants are attached to the bacterial cells or excreted in

the supernatant, the oil spreading test is performed on both the cells and the

supernatant. The results summarized in table 4.2 demonstrate that there is no visible

clear zone when carrying out the oil spreading test on the cells, while large clear

zones occur when the test is applied to the supernatants. Hence, we can conclude that

the biosurfactants are excreted in the supernatant.

To determine the optimal incubation time for extraction of significant amounts

of biosurfactants, another oil spreading test was performed on the broth supernatants

after 5 and 20 hours of bacterial growth. Figure 4.8 confirms the results obtained

previously (4.1.2). We conclude that a higher amount of biosurfactants is produced

after 5 hours (mid-exponential growth phase), than after 20 hours (stationary growth

phase) of growth. Considering this, we will perform an organic solvent extraction

after 5 hours of incubation to obtain biosurfactants for testing the influence of

biosurfactants on Candida albicans biofilm formation.


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TABLE 4.2: RESULTS OF THE OIL SPREADING TEST, APPLIED TO THE

BACTERIAL CELLS AND THE SUPERNATANTS FOR THE THREE

ISOLATES.

Diameter of the clear zone (cm) a

Isolate cells + supernatant supernatant cells

CV8LAC 2.30 2.40 0.00

ME11T7I 1.90 2.00 0.00

INS9T7I 2.10 2.00 0.00

a A Petri dish (9 cm diameter) was filled with tap water and a droplet of motor oil was added. The oil forms a hydrophobic film on the surface. Twenty microliter of the solution that need to be tested, was deposited on this surface. The diameter of the appearing clear zones was measured.

a see table 4.2.

FIGURE 4.8: PRODUCTION OF BIOSURFACTANTS ( DIAMETER OF ZONES

IN THE OIL SPREADING TEST) AFTER 5 AND 20 HOURS OF GROWTH OF

THE THREE ISOLATES

4.3.2 Extraction of biosurfactants

The amounts of biosurfactants obtained via an organic solvent extraction of 600

ml of supernatants from the three isolates are displayed in table 4.3.


33

TABLE 4.3: AMOUNTS OF BIOSURFACTANTS EXTRACTED IN AN

ORGANIC SOLVENT a.

Amount of biosurfactants extracted (mg)

CV8LAC 951.9

INS9T7I 848.4

ME11T7I 714.0 a ethyl acetate : methanol (4:1, v/v)

To confirm the hypothesis that biosurfactants were not attached to the bacterial

cell, an extraction is performed, according to Velraeds et al. (1996). None of the

filtrates, resulting from the extraction obtained after 5 and 20 hours of bacterial

growth, produce a clear zone in the oil spreading test. On the other hand, broth

supernatant produces positive clear zones. Again we can conclude that the

biosurfactants are excreted in the supernatant.

4.3.3.Characterization of the extracted biosurfactants

Tensiometry is used to determine the CMC of each extracted biosurfactant. The

surface tension for pure distilled water is 70.92 mN/m. The biosurfactants lower this

value to more or less 40 mN/m. Figure 4.9, 4.10 and 4.11 illustrate the results. CMC-

values were calculated as explained in material and methods ( see 3.2.3.3.). The CMC

of the biosurfactants produced by CV8LAC is approximately 106 µg/mL, the CMC of

the surfactants produced by INS9T7I is about 40.6 µg/mL and the CMC of the

ME11T7I surfactants is approximately 35.4 µg/mL.


34

FIGURE 4.9: SURFACE TENSION AS A FUNCTION OF CONCENTRATION OF

BIOSURFACTANTS PRODUCED BY CV8LAC

FIGURE 4.10: SURFACE TENSION AS A FUNCTION OF CONCENTRATION

OF BIOSURFACTANTS PRODUCED BY INS9T7I.

FIGURE 4.11: SURFACE TENSION AS A FUNCTION OF CONCENTRATION

OF BIOSURFACTANTS PRODUCED BY ME11T7I.


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4.3.4 Biosurfactants tested on Candida albicans biofilm

Biosurfactants were tested at different concentrations for their effect on the

growth of Candida albicans biofilm. The results of the growth control (without

biosurfactants) are compared with the results of the growth when biosurfactants were

added.

4.3.4.1 Precoating experiments

Precoating of the microtiter wells with biosurfactants affected biofilm formation

by both Candida albicans strains. Results are shown in figures 4.12, 4.13 and 4.14.

With Candida albicans CA11225 exposed to CV8LAC biosurfactants, there was

a decrease in OD595 approximately from 0.225 to 0.05, using surfactant concentrations

ranging from 50 to 1600 µg/mL. There was a decrease from 0.450 to 0.09 in the

experiments with Candida albicans CA2894 exposed to CV8LAC biosurfactants at

the same concentrations. Hence, we can conclude that precoating with CV8LAC

surfactants inhibits the formation of Candida albicans biofilm on polystyrene flat

bottomed 96 well microtiter plates. (figure 4.12)

FIGURE 4.12. GROWTH OF CANDIDA ALBICANS CA11225 AND CA2894

BIOFILMS IN THE PRESENCE OF BIOSURFACTANTS (PRECOATED ON THE

WELLS OF A MICROTITER PLATE) PRODUCED BY CV8LAC

INS9T7I biosurfactants tested on Candida albicans CA11225 biofilm, gave a

decrease in OD595 from 0.200 to 0.05, using surfactant concentrations ranging from 25

to 800 µg/ mL. With Candida albicans CA2894 biofilm exposed to INS9T7I


36

biosurfactants in the same concentrations, there was a decrease from 0.450 to 0.100.

In conclusion precoating with INS9T7I surfactant seems to inhibit the formation of

Candida albicans biofilms on polystyrene flat bottomed 96 well microtiter plates

(figure 4.13).



WELLS OF A MICROTITER PLATE) PRODUCED BY INS9T7I.

ME11T7I biosurfactants in concentrations ranging from 25 to 800 µg/ mL tested

on Candida albicans CA11225 biofilm, gave a decrease in OD595 from 0.200 to 0.075.

The effect of using the same ME11T7I biosurfactant concentrations with Candida

albicans CA2894 biofilm, resulted in a OD595 decrease from 0.450 to 0.100 (fig 4.14).

This shows that precoating with ME11T7I surfactant inhibits the formation of

Candida albicans biofilms on polystyrene flat bottomed 96 well microtiter plates.


37



WELLS OF A MICROTITER PLATE) PRODUCED BY ME11T7I.

4.3.4.2 Coincubation experiments

CV8LAC biosurfactants at concentrations ranging from 50 µg/mL to 1067

µg/mL reduce the OD595 of Candida albicans CA11225 from about 0.2 to

approximately 0.05. The OD595 of Candida albicans CA2894 is reduced from 0.4 to

0.1. These results are displayed in figure 4.15.


BIOFILMS COINCUBATED WITH CV8LAC BIOSURFACTANTS.

The biosurfactants produced by INS9T7I at concentrations ranging from 33

µg/mL to 534 µg/mL reduce the OD595 of Candida albicans CA11225 biofilm from

approximately 0.280 to approximately 0.075. A concentration of 25 µg/mL seems less


38

active. The OD595 of Candida albicans CA2894 biofilm is reduced from

approximately 0.4 to approximately 0.75. These results are displayed in figure 4.16.


BIOFILMS COINCUBATED WITH INS9T7I BIOSURFACTANTS.

ME11T7I biosurfactants at concentrations ranging from 25 µg/mL to 534 µg/mL

reduce the OD595 of Candida albicans CA11225 biofilm from approximately 0.280 to

0.075. A concentration of 25 µg/mL was less active. The OD595 of Candida albicans

CA2894 biofilm could be reduced from approximately 0.4 to about 0.125. In the latter

experiment a concentration of 25 µg/mL seems to be effective. The results are

displayed in figure 4.17.


BIOFILMS COINCUBATED WITH ME11T7I BIOSURFACTANTS.

Conclusions

39

5. CONCLUSIONS

1. The isolates from the fresh fruits and vegetables, growing on both MRS and ROG

agar, were catalase negative and Gram positive. This confirms the fact that they

belong to the lactic acid bacteria. Experiments using the MBEC device showed that

they were able to form biofilms. In addition, they excreted active biosurfactants in

their supernatants.

2. The biosurfactants, extracted from the CV8LAC, ME11T7I and INS9T7I isolates,

were characterized by determining their CMC’s. The biosurfactants produced by

CV8LAC have a CMC of 106 µg/mL, the biosurfactants derived from ME11T7I have

a CMC of 35.4 µg/mL and the ones derived from INS9T7I have a CMC of 40.6

µg/mL. They all lowered the surface tension of pure distilled water from 70.92 mN/m

to approximately 40.00 mN/m.

3. The anti-biofilm activity of the extracted biosurfactants, produced by lactic acid

bacteria, was examined on Candida albicans strains CA11225 and CA2894.

• The biosurfactants derived from the CV8LAC, INS9T7I and ME11T7I

isolates all showed an inhibitory activity at different concentration on the

biofilm formation by both Candida albicans CA11225 and CA2894.

• The results showed that precoating of the wells prior to inoculation was as

effective as coincubation of both surfactants and the yeast cultures.

Since all biosurfactants proved inhibitory, there is a potential of lactic acid

bacteria, producing biosurfactants, to prevent biofilm formation of Candida albicans

on medical devices and hence, reduce the number of nosocomial infections involving

these biofilms.

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