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Appl Microbiol Biotechnol (2009) 83:541–553

DOI 10.1007/s00253-009-1987-7

APPLIED MICROBIAL AND CELL PHYSIOLOGY

Anti-adhesion activity of two biosurfactants produced


by Bacillus spp. prevents biofilm formation of human
bacterial pathogens
F. Rivardo & R. J. Turner & G. Allegrone & H. Ceri &
M. G. Martinotti

Received: 20 January 2009 / Revised: 20 March 2009 / Accepted: 21 March 2009 / Published online: 3 April 2009
# Springer-Verlag 2009

Abstract In this work, two biosurfactant-producing Two fractions from each purified biosurfactant, obtained
strains, Bacillus subtilis and Bacillus licheniformis, have by flash chromatography, fractions (I) and (II), showed
been characterized. Both strains were able to grow at high that fraction (II), belonging to fengycin-like family, was
salinity conditions and produce biosurfactants up to 10% responsible for the anti-adhesion activity against biofilm
NaCl. Both extracted-enriched biosurfactants showed of both strains.
good surface tension reduction of water, from 72 to 26–
30 mN/m, low critical micelle concentration, and high Keywords Bacillus . Biosurfactant . Biofilm .
resistance to pH and salinity. The potential of the two Anti-adhesion . Escherichia coli CFT073 .
lipopeptide biosurfactants at inhibiting biofilm adhesion of Staphylococcus aureus ATCC 29213
pathogenic bacteria was demonstrated by using the MBEC
device. The two biosurfactants showed interesting specific
anti-adhesion activity being able to inhibit selectively Introduction
biofilm formation of two pathogenic strains. In particular,
Escherichia coli CFT073 and Staphylococcus aureus Biosurfactants are unique amphiphilic metabolites, pro-
ATCC 29213 biofilm formation was decreased of 97% duced by a wide variety of microorganisms from
and 90%, respectively. The V9T14 biosurfactant active on various biochemical building blocks, able to reduce the
the Gram-negative strain was ineffective against the surface tension of aqueous media (Bodour et al. 2003).
Gram-positive and the opposite for the V19T21. This Biosurfactants have advantages over their synthetic chem-
activity was observed either by coating the polystyrene ical counterparts because of their biodegradability and
surface or by adding the biosurfactant to the inoculum. reduced toxicity, availability from cheap raw materials,
biocompatibility, and the effectiveness at extreme temper-
F. Rivardo : G. Allegrone : M. G. Martinotti (*) ature, pH, and salinity (Kosaric 2001; Plaza et al. 2006;
Department of Chemical, Food, Singh and Cameotra 2004). These properties allow their
Pharmaceutical and Pharmacological Sciences (DiSCAFF), use and possible replacement of chemically synthesized
Drug and Food Biotechnology Centre,
surfactants (Desai and Banat 1997). They are molecules of
University of Eastern Piedmont, Via Bovio 6,
28100 Novara, Italy increasing interest because of their antimicrobial action
e-mail: martinotti@pharm.unipmn.it (Kosaric 2001). Typical desirable properties include
surface tension reduction and low critical micelle concen-
R. J. Turner : H. Ceri
trations. Biosurfactants are able to modify bacterial
Department of Biological Sciences, University of Calgary,
2500 University Drive N.W., surface hydrophobicity and consequently, microbial adhe-
Calgary, AB T2N 1N4, Canada sion to solid surfaces. Their effect depends on the initial
bacterial hydrophobicity as well on the lipopeptides type
H. Ceri
and concentration (Ahimou et al. 2000). A remarkable
Biofilm Research Group, University of Calgary,
2500 University Drive N.W., property of biosurfactant is the inhibitory activity against
Calgary, AB T2N 1N4, Canada bacterial and fungal colonization of surfaces, including
542 Appl Microbiol Biotechnol (2009) 83:541–553

those of biomedical interest. Control of microbial growth Both the strains were deposited in the publicly accessible
is required in many microbiologically sensitive environ- culture collection DSMZ (Braunschweig, Germany) with
ments where surfaces provide favorable conditions for accession number DSM 21038 for Bacillus licheniformis
proliferation of microorganisms. Bacteria growing as a V9T14 and DSM 22265 for Bacillus subtilis V19T21.
biofilm remain a significant challenge in biomedical
field, growing on abiotic material such as catheters and Surface activity
prosthesis, as they tend to be more tolerant to antimi-
crobial treatments. Frequent replacement of the prosthesis A seed culture was prepared by transferring a loop of the
is uncomfortable, costly, time consuming, and may lead V9T14 or V19T21 strain from a LB agar overnight culture
to damage of the cellular tissue of patients. The biofilm into a 100-ml flask containing 20 ml of LB broth and
formation immediately starts once a biomedical device incubating flask at 28°C for 24 h at 130 rpm. The overnight
has been placed in its body niche. Biofilm infection liquid culture was centrifuged at 8,000×g for 10 min. Surface
can be limited by preventing microbial adhesion to the activity was measured by the oil spreading assay (Morikawa
surfaces of medical devices. Surfactants may affect et al. 2000) by using 20 μl of Motor Oil 10 W-40 (Selenia)
the development of flagella, suggesting changes in the previously deposited onto the surface of 20 ml of distilled
attachment capability of bacteria (Splendiani et al. 2006). water in a Petri dish (90 mm in diameter) to form a thin
Lactococcus lactis and Streptococcus thermophilus membrane. Twenty microliters of bacterial supernatant were
produce biosurfactants able to decrease the amount of gently put onto the centre of the oil membrane. Diameters of
bacteria in a multi-species biofilm on voice prosthesis clearly formed oil displaced circle were measured to
(Rodrigues et al. 2004). Mireles and collaborators (2001) determine the presence of biosurfactants.
observed that surfactin could promote dispersal of
preformed biofilm and inhibits Salmonella enterica adhe- Bacterial halotolerance
sion to PVC without inhibiting the growth. Rhamnolipid-
preconditioned PTFE surface was able to reduce Listeria The two Bacillus strains were inoculated in LB broth with
monocytogenes attachment (Meylheuc et al. 2001). 50 and 100 g/L of NaCl and incubated at 28°C for 48 h.
Surfactin and rhamnolipids are able to prevent Escherichia Bacterial growth was monitored at OD595nm, biosurfactant
coli and Proteus mirabilis biofilm attachment (Mireles et production was estimated by the measurement of the
al. 2001). The aims of the present work were to surface tension assessed with a Sigma 703D tensiometer
characterize two biosurfactant-producing Bacillus spp. (KSV) equipped with a du Nouy platinum ring.
isolated from an organic ammendant already known for
its rich microbial biodiversity (Fracchia et al. 2006; Biosurfactant extraction and enrichment
Martinotti et al. 1999) and to analyze their antimicrobial
potential against different biofilm and planktonic forms of For biosurfactant production, a seed culture was prepared
pathogenic bacterial strains. by transferring either a loop of the V9T14 or V19T21
strain, from a LB agar overnight culture, into 10 ml of LB
broth and incubated at 28°C for 4 h at 200 rpm. Thereafter,
Materials and methods 2 ml were inoculated in 500 ml of LB broth in a 2,000-ml
flask and incubated again at 28°C for 24 h at 120 rpm. The
Bacterial strains and culture conditions overnight liquid culture was centrifuged at 8,000×g for
30 min and the supernatant was collected. Bacterial pellet
Two Bacillus strains (V9T14 and V19T21) were isolated was resuspended in distilled water and centrifuged again at
from the organic ammendant ENZYVEBA® Nucleobase 2 8,000×g for 30 min. Supernatants were pooled, acidified to
(EN2) (MARCOPOLO ENGINEERING S.p.a, Borgo San pH 2 with 6 N HCl, stored at 4°C for 4 h and extracted
Dalmazzo, Cuneo, Italy). Pure culture of the two strains three times with ethyl acetate/methanol (4:1). The organic
were obtained by streaking colonies several times onto LB fraction was evaporated to dryness under vacuum condi-
agar and storing them at −80°C in LB broth (Fluka) tion, acetone was added to recover the raw biosurfactant.
supplemented with 30% glycerol. Morphological character- Acetone was evaporated and biosurfactant was collected
istics were defined by observing their bacterial colonies and weighted.
grown on LB agar and nutrient agar supplemented with
0.03% of MnSO4 (NA+Mn) and performing Gram staining Spectrometric analyses
on an overnight culture on NA+Mn agar. Identification was
performed by using the GP-ROD-SB BIOLOG® assay Fourier transform infrared (FT-IR) absorption spectrometry
(Microlog, USA). was used to define the structure of V9T14 and V19T21
Appl Microbiol Biotechnol (2009) 83:541–553 543

biosurfactants. FT-IR spectra were obtained with a Thermo measure was made in triplicate and the average and
Nicolet Avatar 370 FT-IR spectrometer equipped with a standard deviation was calculated.
diffuse reflectance accessory. One milligram of the enriched
sample was mixed thoroughly with 100 mg of homoge- Bacterial biofilm inhibition
nized porcelain-milled KBr (FT-IR grade). A pellet was
prepared using a press. Afterwards, the pellet was imme- Biosurfactant stock solution and dilutions
diately put into the sample holder and FT-IR spectra were
recorded. Data were collected and processed with Ez The extracted dried V9T14 and V19T21 biosurfactants
Omnic software. FT-IR scanning was conducted in ambient were dissolved in PBS (pH 7.2) at the final concentration
conditions. The resolution was set to 4 cm−1 and the of 5,120 μg ml−1. These solutions were filtered through
operating range was 500 to 4,000 cm−1. Sixty-four spectra 0.2-μm filters and then stored at 4°C. For experiments,
per sample were recorded, averaged for each spectrum and stock solutions of both biosurfactants were then serially
corrected against the spectra of pure KBr and ambient air as diluted 1:1 in PBS.
background.
Spectrometric analyses were carried out to characterize Bacterial biofilm
the chemical structure of the enriched V9T14 and V19T21
biosurfactants. In particular TLC, flash chromatography, Biofilms were grown in the Calgary Biofilm device (CBD,
lactone bond determination, mass spectrometry analysis, Innovotech, Edmonton, AB, Canada) as described by
and liquid chromatography-mass spectrometry analysis Harrison et al. (2006). The CBD consists of a polystyrene
were performed (results not shown). lid with 96 pegs that may be fitted inside a standard 96-well
microtiter plate. Each peg of the CBD has a surface area of
Surface tension and critical micelle concentration approximately 109 mm2. For the experiments, frozen stocks
of four bacterial pathogens were used.
To measure the surface tension between biosurfactant E. coli CFT073 was streaked on LB agar, Staphylococcus
solution and air, an extracted-enriched biosurfactant solu- aureus ATCC 29213 and Pseudomonas aeruginosa PA14 on
tion was prepared in alkaline sterile demineralized water at trypticase soy agar (TSA), and the isolate Staphylococcus
500 μg ml−1. Distilled water was used for calibration. epidermidis on Cathion-Adjusted Mueller-Hinton agar
Twenty milliliters of biosurfactant solution were used for and all were incubated overnight at 37°C. A second
each measurements; the ring was placed just below the fresh subculture of each microbial strain was grown
surface of the solution, subsequently the force to move this overnight at 37°C on the appropriate agar medium.
ring from the liquid phase to the air phase was determined Using a cotton swab, colonies from this fresh secondary
in triplicate. subculture were suspended in the respective broth medium to
Critical micelle concentration (CMC) was determined on match a 1.0 McFarland standard, corresponding to approx-
serially diluted biosurfactant solutions in alkaline distilled imately 3.0×108 cfu ml−1. This suspension was diluted
water. Surface tension of each dilution was determined in again 30-fold in broth to create the inoculum for the CBD,
triplicate. Maximal standard deviation associated with these that approximately was 1.0×107 cfu ml−1. Then, 150 μl of
surface activity measurements was 0.30 mN/m. The CMC each bacterial inoculum were added to each well of
of the two biosurfactants was estimated from the intercept 96-well microtiter plates. The CBD peg lid was then fitted
of two straight lines extrapolated from the concentration- inside of this and the assembled device was placed on a
dependent and concentration-independent sections of a gyrorotatory shaker at 150 rpm in a humidified incubator
curve plotted between biosurfactant concentration and for 24 h. Following this period, biofilms were rinsed
surface tension values. twice by inserting the peg lids into microtiter plates with
200 μl/well of 0.9% saline for 2 min to remove loosely
Stability study of biosurfactants adherent cells.
For coating experiments, the CBD was previously
Solutions of extracted-enriched biosurfactants were coated with biosurfactant by dipping the lid of the CBD
prepared in distilled water at 100 μg/ml (final pH adjusted into 200 μl of the biosurfactant solution starting from
to 7.0 with 1 N NaCl) containing different concentration of 5,120 to 80 μg ml−1 previously put in each well of a
NaCl ranging from 0% to saturation (more than 35%) microtiter plate. The CBD assembled with the microtiter
and the surface tension was measured. The pH of the plate was incubated at 37°C on a rotatory shaker at
biosurfactants water solutions was adjusted to different 125 rpm for 24 h, then removed from the microtiter plate
values, ranging from 2.3 to 10.3, using 3 N NaOH or and upside down dried under the hood for 1 min before
3 N HCl and surface tension was measured. Each adding it to the bacterial inocula.
544 Appl Microbiol Biotechnol (2009) 83:541–553

In another set of experiments, the CBD was used uncoated Mississauga, ON, Canada) set at 60 Hz for 10 min. The
and microbial inocula were distributed in each well together disrupted biofilm cells were serially diluted in 0.9%
with biosurfactant stock dilutions to reach concentration saline, and then plated onto LB agar. Agar plates were
ranges from 1 to 20 μg well−1 (5 to 100 μg ml−1) in the final incubated for 24 h at 37°C and then enumerated. Viable
volume of 200 μl. cell counts for planktonic cultures (i.e. starting inocula
and planktonic forms after incubation) were similarly
Viable cell counting carried out by serial dilution in 0.9% saline and plating
onto agar as described for biofilm cells.
The effect of the two biosurfactants was assessed by
determining the viable cell counts after biofilms had Confocal laser scanning microscopy
been rinsed as described above. The lid of the CBD
was then inserted into 200 μl of LB broth added with Pegs were broken from the lid of the CBD using pliers
1% Tween 20 in the wells of a microtiter plate. (Harrison et al. 2006) and then rinsed once with 0.9% saline
Biofilms were disrupted from the peg surface using an to remove planktonic bacteria. Prior to examination by
Aquasonic 250HT ultrasonic cleaner (VWR International, confocal laser scanning microscopy (CLSM), biofilms were

Table 1 Metabolic characteristics of the V9T14 and V19T21 strains

Substrates V9T14 V19T21 Substrates V9T14 V19T21

2,3-butanediol + ± Gentiobiose + +
2’-deoxy adenosine + ± Glycerol + +
3-methyl glucose + + Glycyil-L-glutamic acid ± −
Acetic acid ± − Glycogen + ±
α-cyclodextrin + ± Inosine + +
Adenosine + + Inulin ± −
Adenosine-5’-monophosphate − − L-alanine ± ±
α-D-glucose + + L-alanyl-glycine ± −
α-ketoglutric acid ± − L-arabinose ± −
α-ketovaleric acid ± − L-asparagine ± +
α-methyl-D-glucoside + + L-glutamic acid + +
Amygdalin ± − L-lactic acid − −
Arbutin + + L-malic acid ± +
β-cyclodextrine + + L-serine ± +
β-methyl-D-glucoside + + Maltose + +
D-alanine ± ± Maltotriose + +
D-cellobiose ± + m-inositol + −
Dextrin + + N-acetyl-ββ-D-mannosamine ± −
D-fructose + + N-acetyl-D-glucosamine + +
D-galactose − ± N-acetyl-L-glutamic acid − −
D-gluconic acid + + Palatinose + +
D-lactic acid methyl ester ± ± Pyruvatic acid methyl ester ± +
D-L-α-glycerol phosphate − − Pyruvic acid + +
D-mannitol − + Salicin + +
D-mannose + + Succinamic acid ± −
D-melezitose + ± Sucrose + +
D-psicose + + Thymidine + +
D-ribose + + Thymidine-5’-monophosphate ± −
D-sorbitol ± + Turanose + +
D-tagatose − − Tween 40 ± ±
D-trehalose + + Uridine + +

Substrate utilization is expressed as positive (+), negative (−), or intermediate (±)


Appl Microbiol Biotechnol (2009) 83:541–553 545

Table 2 Morphological characteristics of the V9T14 and V19T21 GP-ROD-SB identification of the BIOLOG® system, the
strains
strain V9T14 was identified as B. licheniformis with a
V9T14 V19T21 probability of 99%, a similarity of 0.639, and a distance of
5.49 while the strain V19T21 was identified as B. subtilis
LB agar Shape: circular Shape: circular with a probability of 99%, a similarity of 0.797, and a
Edges: entire Edges: undulate distance of 3.05. Metabolic characteristics of B. licheniformis
Elevation: pulvinate Elevation: raised V9T14 and B. subtilis V19T21 are given in Table 1. The two
Color: yellowish, bright Color: pale yellow, mat strains mainly differ for D-mannitol and m-inositol metabo-
Consistency: mucous Consistency: dry lism. Morphological characteristics of the two strains grown
NA+Mn Shape: circular Shape: circular on different agar are shown in Table 2. On LB agar,
Edges: entire Edges: entire B. licheniformis V9T14 colonies were pulvinate, yellowish,
Elevation: raised Elevation: flat bright, and mucous while those of B. subtilis V19T21 were
Color: pale yellow, bright Color: white yellow, mat raised, pale yellow, mat, and dry. Almost similar results were
Consistency: mucous Consistency: dry observed on NA+Mn.

Growth curve and biosurfactant production


fluorescently stained with acridine orange (AO) (Sigma
Chemical, St. Louis, MO, USA). To stain biofilms, pegs B. licheniformis V9T14 and B. subtilis V19T21 showed
were immersed in 0.1% w/v AO in PBS for 5 min at room similar growth curves and similar capability to produce
temperature in the dark. Fluorescently stained biofilms were biosurfactants (Fig. 1). Oil spreading diameter of V19T21
placed in two drops of 0.9% saline on the surface of a glass after 12 h of growth was larger than that of V9T14, but at
coverslip. These pegs were examined using a Leica DM 24 h both values were similar and slightly decreased in the
IRE2 spectral confocal and multiphoton microscope with a next 24 h.
Leica TCS SP2 acoustic optical beam splitter (AOBS) (Leica B. licheniformis V9T14 was able to grow in the presence of
Microsystems, Richmond Hill, ON, Canada). Fluorescent- 5% NaCl and was able to produce biosurfactant in the presence
labelled biofilm was excited at 476 nm. Line averaging (×2) of the halogen, reaching a surface tension of 36 mN/m after
was used to capture images with reduced noise. A ×63 water 24 h. Bacterial growth at 10% NaCl was highly slackened and
immersion objective was used in all imaging experiments. biosurfactant production was inhibited (Fig. 2a–c).
Image capture and two-dimensional projections of z-stacks B. subtilis V19T21 was able to grow in the presence of
were performed using Leica Confocal Software (LCS, Leica 5% NaCl and was able to produce biosurfactant, reaching
Microsystems). a surface tension of 33 mN/m after 24 h. At 10% NaCl,
bacterial growth was delayed and reached the end of the
Interpretation of results

The efficacy of biofilm adhesion inhibition was assessed by


determining the minimal biofilm eradication concentration
(MBEC) after 24 h by viable cell count. Each test was
performed at least three times in triplicate on separate cultures.

Statistical analysis

The Student’s t test was performed when the aim was to


investigate whether the difference in between the experi-
mental values obtained under different conditions could be
considered significant.

Results

Strain identification and characteristics


Fig. 1 Growth and biosurfactant production capability of B.
Gram staining revealed that the isolates were spore-forming, licheniformis V9T14 and B. subtilis V19T21. Values are average
Gram-positive, rods. By using the conventional method for of three growth cultures
546 Appl Microbiol Biotechnol (2009) 83:541–553

Fig. 2 Time-course of bacterial growth, biosurfactant production, and surface tension of B. licheniformis V9T14 (a, b, c) and B. subtilis V19T21
(d, e, f)

log phase at 48 h; biosurfactant production was not small decrease to 30 mN/m from 25% to saturation (more
detectable after 24 h, but surface tension was reduced to than 35%) was observed. CMC of V9T14 biosurfactant
30 mN/m after 48 h of incubation (Fig. 2d–f). showed a small increase at 5% NaCl, from 6.4 μg/ml to
7 μg/ml, then drastically decrease to 0.5 μg/ml at NaCl
Biosurfactants properties saturation (Fig. 4a). Initial surface tension of the V19T21
biosurfactant (35 mN/m) showed a small decrease to
Extracted-enriched V9T14 biosurfactant at 500 μg mL−1, 32 mN/m at 10% NaCl, then increased to a value of
decreased water surface tension from 68.8 to 26.3 mN/m, 38 mN/m at 25% NaCl. The best surface activity was
while the V19T21 biosurfactant, at the same concentration, detected at saturation of NaCl, with a value of 29.5 mN/m.
reduced surface tension to 26.1 mN/m (Fig. 3). Serial CMC of V19T21 biosurfactant increased from 6.0 μg/ml to
dilutions of V9T14 and V19T21 biosurfactants showed a
constant surface tension value of 26 mN/m up to the
concentration of 50 μg ml−1 (Fig. 3). Then, values slowly
increased to ranges between 27 and 30 mN/m up to the
concentration of 10 μg ml−1. The CMC values for the
V9T14 and V19T21 biosurfactants were 6.7 μg ml−1 and
6.0 μg ml−1, respectively.

Effect of NaCl concentration and pH on the surface activity

The biosurfactants V9T14 and V19T21 showed surface


activity up to saturation of NaCl concentration (Fig. 4a).
Initial surface tension of V9T14 biosurfactant solution was
Fig. 3 A plot of surface tension as a function of concentration of
33 mN/m and remains almost stable for concentration up to V9T14 and V19T21 biosurfactants after purification. Standard
25% showing values ranging from 31 to 33 mN/m. Then a deviation was ranging between ±0.3 mN/m
Appl Microbiol Biotechnol (2009) 83:541–553 547

Fig. 4 Surface tension and


CMC of V9T14 and V19T21
biosurfactants as a function of
NaCl concentration (a) and pH
(b). For NaCl dependence (a),
biosurfactants were dissolved in
water and the final pH was
adjusted to 7.0 with 1 N NaOH.
For pH dependence (b), pH was
adjusted with 3 N NaOH or
3 N HCl and surface tension
was measured. Surface tension
of distilled water at pH 7.0 was
71.2 mN/m

10 μg/ml at 5% NaCl then restored to 6–7 μg/ml up to from N–H stretching mode, at 1,655 cm−1 resulting from
NaCl saturation. the stretching mode of the CO–N bond and the 1,535 cm−1
Figure 4b shows the effect of various pH values on resulting from the deformation mode of N–H bond
biosurfactants surface activity. V9T14 biosurfactant has a combined with C–N stretching mode. These results suggest
poor surface activity at pH from 2 to 4, decreasing from that V9T14 and V19T21 biosurfactants contain peptide-like
71 mN/m to about 40 mN/m; while good surface activity moieties. The bands at 2,960 to 2,860 and 1,470 to
was detected starting from pH 5 to 10 (30–33 mN/m), with 1,370 cm−1 resulting from the C–H stretching mode reflect
maximum activity at pH 5 (30 mN/m). the presence of an aliphatic chain (–CH2–, –CH3). The
The V19T21 biosurfactant showed low decrease of absorption region at 1,740–1,680 cm−1 was due to lactone
surface tension up to pH 4 (from 70 to 40 mN/m) . The carbonyl absorption. The spectroscopic analysis indicated
best range of surface activity was found between pH 5 to above confirmed that the two biosurfactants were
10 (30–34 mN/m) and the maximum activity at pH 6 lipopeptides consistent with belonging to fengycin-like
(28 mN/m). and surfactin-like family of structures.

Spectroscopic analysis Influence of V9T14 and V19T21 biosurfactants on biofilm


formation by different bacterial strains
The FT-IR spectrum (Fig. 5) of V9T14 and V19T21
biosurfactants in KBr, showed strong absorption bands, The anti-adhesive effect of the two biosurfactants was
indicating the presence of peptides at 3,300 cm−1 resulting considered positive if, after 24 h of growth, there was a ≥1

Fig. 5 FT-IR spectra of


V9T14 and V19T21 purified
biosurfactants
548 Appl Microbiol Biotechnol (2009) 83:541–553

Fig. 6 The adhesion of E. coli


CFT073 biofilm in the presence
of different concentration of
V9T14 biosurfactant by
precoating the CBD’s pegs (a)
and by adding different amount
of V9T14 to each well of a
96-well plate (b). * indicates a
significant (P<0.01) difference
from no biosurfactant

log10 (90%) difference in the mean of CFU/peg compared Statistical analysis reveals that the results with and without
to the growth control. The four strains tested efficiently V19T21 biosurfactant against S. aureus ATCC 29213 are
formed biofilms on the used media. significantly different.
The increase of V9T14 biosurfactant concentration, There was no apparent effect on planktonic survivability
promoted a decrease in E. coli CFT073 adherent viable (Fig. 8) by the presence of V9T14 (p=0.46) and V19T21
cell count, to reach a maximal inhibition of 97% compared (p=0.38) biosurfactants, at every concentration tested.
to the growth control with a precoating concentration of V9T14 biosurfactant was not able to inhibit the adhesion
2,560 μg/ml (Fig. 6a) or by adding 10 μg/well (Fig. 6b) of of other microorganisms (Fig. 9a), in particular S. aureus
biosurfactant with the inoculum. Statistical analysis of the ATCC 29213, P. aeruginosa PA14, and the isolate S.
biofilm viable cell count in the presence or absence of epidermidis. Viable cell count showed no difference in
V9T14 showed a significant activity of the biosurfactant bacterial population of these microorganisms when V9T14
(p<0.0001). biosurfactant was present on the pegs or in the culture
The V19T21 biosurfactant behavior was diametrically broth, even with high concentration of biosurfactant.
opposed, the increase of its concentration did not meet an Likewise, biosurfactant V19T21 was not able to inhibit
increase of activity (Fig. 7a, b), but biofilm adhesion was microbial adhesion of other strains such as P. aeruginosa
restored at the growth control level. The maximum PA14, E. coli CFT073, and S. epidermidis (Fig. 9b)
inhibition versus S. aureus (reduction of 90% adhesion) Figure 10 presents CLSM images of E. coli CFT073 and
was observed at low concentration of V19T21 biosurfac- S. aureus ATCC 29213 growth control (Fig. 10a and b,
tant, with a precoating solution of 160–320 μg/ml or by respectively) and adhesion in presence of V9T14 and
adding 5–25 μg/ml (1 to 5 μg/well) to the biofilm V19T21 biosurfactants (Fig. 10c and d, respectively).
inoculum, a value just higher than the calculated CMC. E. coli CFT073 showed a dramatic decrease of adhesion

Fig. 7 The adhesion of


S. aureus ATCC 29213 biofilm
in the presence of different
concentration of V19T21
biosurfactant by precoating the
CBD’s pegs (a) and by adding
different amount of V19T21 to
each well of a 96-well plate
(b). * indicates a significant
(P<0.01) difference from no
biosurfactant
Appl Microbiol Biotechnol (2009) 83:541–553 549

observed by using V19T21(I) and V19T21(II) fractions


against both strains (Fig. 12a, b).

Discussion

In this study we have shown that two biosurfactant


producers B. subtilis V19T21 and B. licheniformis V9T14,
isolated and extracted from an organic ammendant, are able
to produce very efficient and effective biosurfactants. Both
strains were able to produce amylase and cellulase even if
B. licheniformis V9T14 showed a higher production of both
enzymes than B. subtilis V19T21 (data not shown). Over
the range evaluated, our study showed that NaCl concen-
Fig. 8 Influence of V9T14 and V19T21 biosurfactants on planktonic trations have little effect on B. subtilis V19T21 biosurfac-
E. coli CFT073 and S. aureus ATCC 29213 growth tant production, but at 10% inhibited the production by B.
licheniformis V9T14. Both B. subtilis and B. licheniformis
when V9T14 biosurfactant was present on the peg or in had been found to produce biosurfactant at high concentra-
culture broth. S. aureus ATCC 29213 biofilm was similarly tion of halogens (Yakimov et al. 1995). Halophyles and
inhibited by V19T21 biosurfactant. halotolerants may have an important role to play as surface-
active releasing agents. Biosurfactants secreted by halo-
Influence of purified fractions obtained by flash phyles are highly stable and may have applications as
chromatography on biofilm formation of E. coli CFT073 mobility controllers and emulsifying agents in the oil
and S. aureus ATCC 29123 industry (Cameotra and Makkar 1998). Moreover, V9T14
and V19T21 biosurfactants had high stability over a wide
Figures 11 and 12 show the influence of four purified range of pH. Similar results have been recently observed by
fractions, named V9T14(I), V9T14(II) and V19T21(I), Ghojavand et al. (2008) on the biosurfactant produced by
V19T21(II), obtained from the two solvent extracted the strain B. subtilis PTCC 1696.
biosurfactants (data not shown). The surface tensions and CMC values observed for both
V9T14 fraction (I) was able to inhibit adhesion of E. coli V9T14 and V19T21 biosurfactants were comparable to those
CFT073 of about 50% (0.4±0.1 Log10) at concentration of observed by others (Cooper et al. 1981; Makkar and
8 μg/ml (Fig. 11a) while it induced an increased adhesion Cameotra 1998; Yakimov et al. 1995). Organoleptic charac-
of S. aureus ATCC 29213 of about 1 Log10 (Fig. 11b). teristics with bad odor of the two enriched biosurfactants
V9T14 fraction (II) inhibited the adhesion of E. coli were also observed by Abdel-Mawgoud et al. (2008). The
CFT073 of about 90–95% (1.4±0.2 Log10) (Fig. 11a) and infrared spectra analysis showed that their structure belongs
of S. aureus ATCC 29213 of about 90% (1 Log10) at to lipopeptides compounds as found for other biosurfactants
concentration of 8 μg/ml (Fig. 11b). Similar trends were (Dehghan-Noude et al. 2005; Joshi et al. 2008).

Fig. 9 Effect of V9T14 (a) and


V19T21 (b) biosurfactants
against microbial adhesion of
different strains
550 Appl Microbiol Biotechnol (2009) 83:541–553

Fig. 10 Confocal laser scanning


microscopy analysis of biofilm
formation growth control (a, b)
and inhibition of V9T14 (c) and
V19T21 (d) biosurfactants on
E. coli CFT073 (left column)
and S. aureus ATCC 29213
(right column) adhesion.
Bacteria were incubated in
the Calgary Biofilm Device
in LB broth with or without
biosurfactants. Concentration
of V9T14 and V19T21
biosurfactants were 10 and
4 μg/well

The need of new antimicrobial agents to overcome bacterial energy saving and several microorganisms use it
bacterial antibiotic tolerance or resistance in biofilms leads to protect their ecological niche. Rhamnolipids produced by P.
to investigations towards novel strategies to fight against aeruginosa were able to disperse a Bordetella bronchiseptica
bacterial chronic infection. Bacteria demonstrate diverse biofilm (Irie et al. 2005) reducing significantly the
strategies to protect themselves from environmental assaults viability of the microorganism. Rodrigues et al. (2006)
and facilitate their own survival. Adhesion is needed for demonstrated that rhamnolipids inhibit bacterial adhesion

Fig. 11 Influence of biosurfac-


tant fractions V9T14(I) and
V9T14(II) on biofilm adhesion
of E. coli CFT073 (a) and
S. aureus ATCC 29213 (b)
Appl Microbiol Biotechnol (2009) 83:541–553 551

Fig. 12 Influence of biosurfac-


tant fractions V19T21(I) and
V19T21(II) on biofilm adhesion
of E. coli CFT073 (a) and S.
aureus ATCC 29213 (b)

of various strains (e.g. S. epidermidis, Streptococcus V9T14(II) and V19T21(II) as well (data not shown). The
salivarius) within a range from 21% to 81%. A bio- different fractions ratio among V9T14 and V19T21 extracts
surfactant produced by Lactobacillus was able to inhibit could explain the observed specificity of action. Fraction
the adhesion of Enterococcus faecalis of 99% (Velraeds et (II) was the major responsible of the activity against both
al. 2000; Velraeds et al. 1996). Harshey et al. (2003) strains. The best activity against S. aureus ATCC 29213
demonstrated that lipopeptides were able to inhibit biofilm was observed at low concentration, while an increase of
formation when used to coat surfaces. S. enterica biofilm concentration showed no anti-adhesion activity. The con-
adhesion on urinary catheters was abolished by precoating centration needed to observe the activity against E. coli
the surface of the device with 5 μg of surfactin and the CFT073 was higher than that against S. aureus. Fraction (I)
same effect was observed for E. coli, while was com- showed anti-adhesion activity against E. coli CFT073 but
pletely ineffective against P. aeruginosa (Mireles et al. enhanced the adhesion of S. aureus ATCC 29213. This
2001). The decreased bacterial adhesion and the reduction enhancement can contrast the anti-adhesion effect of
of biofilm population can be clinically useful in the fraction (II) against S. aureus, giving to this a small range
removal of bacterial colonization from medical devices of activity at low concentration. It has been demonstrated
surfaces and in UTIs. Huang et al. (2008) observed the that blocking Staphylococci quorum sensing enhances
efficacy of a mixture of surfactin and fengycin (1:1) bacterial biofilm formation (Fowler et al. 2008; Otto
against planktonic E. coli. 2001). In fact, synthetic cyclic pentapeptides, analogues of
Our results showed that precoating of the pegs prior to quorum sensing autoinducers, were found to stimulate
inoculation was just as effective as including biosurfactant S. aureus adhesion to surfaces (Fowler et al. 2008). In
in the growth medium. The two treatments were not addition, it has been demonstrated that the Staphylococci
statistically different. A reduction of 97% of E. coli autoinducers, in contrast to the Gram-negative’s, show the
CFT073 for biosurfactant V9T14 and of 90% of S. aureus phenomenon of cross-inhibition: autoinducers of the self
ATCC 29213 biofilms for biosurfactant V19T21 was induce, whereas autoinducers of non-self inhibit the
observed. To explain the observed specificity, further quorum sensing response (Otto 2001, 2004). This inhibition
analyses were carried out to understand the differences in leads to an increase of adherence.
chemical structures between the two compounds. Surpris- Other studies showed that sub-MIC concentration of
ingly, V9T14 and V19T21 analyzed by HPLC evidenced biocides are known to increase Staphylococci biofilm
similar composition, presenting principally two groups of formation, while in Gram-negative bacteria they exert
molecules for each extract, named fraction (I) and fraction anti-adhesive properties (Carsenti-Etesse et al. 1993;
(II). Fractions (I) showed identical elution peaks belonging Houari and Di Martino 2007).
to surfactin-like family in both extracts and fractions (II) as For these reasons, we think that V9T14 biosurfactant,
well, identical elution peaks belonging to fengycin family richer in fraction (II) than V19T21, can be prevalently
(data not shown). Surfactin-like fraction (I) was the most active against E. coli CFT073, while the amount of fraction
abundant in both extracts and formed by a mixture of (II) in V9T14 is probably too high to be effective against S.
isoforms, while fengycin-like fraction (II), also formed by a aureus ATCC 29213. For the same reasons, V19T21,
mixture of isoforms, was more abundant in V9T14 extract poorer in fraction (II) than V9T14, presents a concentration
than in V19T21. Furthermore, the fractions V9T14(I) and of this fraction too low to be effective against E. coli
V19T21(I) differed in homologues and isoforms ratio, CFT073 but sufficient to exert its anti-adhesion activity
552 Appl Microbiol Biotechnol (2009) 83:541–553

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