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Current Microbiology
ISSN 0343-8651
Curr Microbiol
DOI 10.1007/s00284-013-0412-8
A Comparison of Effects of Broad-
Spectrum Antibiotics and Biosurfactants on
Established Bacterial Biofilms
Gerry A.Quinn, Aaron P.Maloy, Malik
M.Banat & Ibrahim M.Banat
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A Comparison of Effects of Broad-Spectrum Antibiotics
and Biosurfactants on Established Bacterial Biofilms
Gerry A. Quinn
•
Aaron P. Maloy
•
Malik M. Banat
•
Ibrahim M. Banat
Received: 8 March 2013 / Accepted: 26 May 2013
Ó Springer Science+Business Media New York 2013
Abstract Current antibiofilm solutions based on plank-
tonic bacterial physiology have limited efficacy in clinical
and occasionally environmental settings. This has promp-
ted a search for suitable alternatives to conventional ther-
apies. This study compares the inhibitory properties of two
biological surfactants (rhamnolipids and a plant-derived
surfactant) against a selection of broad-spectrum antibiot-
ics (ampicillin, chloramphenicol and kanamycin). Testing
was carried out on a range of bacterial physiologies from
planktonic and mixed bacterial biofilms. Rhamnolipids
(Rhs) have been extensively characterised for their role in
the development of biofilms and inhibition of planktonic
bacteria. However, there are limited direct comparisons
with antimicrobial substances on established biofilms
comprising single or mixed bacterial strains. Baseline
measurements of inhibitory activity using planktonic bac-
terial assays established that broad-spectrum antibiotics
were 500 times more effective at inhibiting bacterial
growth than either Rhs or plant surfactants. Conversely,
Rhs and plant biosurfactants reduced biofilm biomass of
established single bacterial biofilms by 74–88 and
74–98 %, respectively. Only kanamycin showed activity
against biofilms of Bacillus subtilis and Staphylococcus
aureus. Broad-spectrum antibiotics were also ineffective
against a complex biofilm of marine bacteria; however, Rhs
and plant biosurfactants reduced biofilm biomass by 69 and
42 %, respectively. These data suggest that Rhs and plant-
derived surfactants may have an important role in the
inhibition of complex biofilms.
Introduction
Microbial biofilms have been implicated in recalcitrant
healthcare-associated infections [24, 28, 47], the dissemi-
nation of community-acquired diseases [43] and hazardous
concerns in the nutritional and environmental sectors [23].
Biofilms are sessile multicellular bacterial collectives with
distinctly different physiologies from those of independent
free-living bacteria [8]. Many biofilms are resistant to con-
ventional antimicrobial technologies that were developed
using a planktonic model [30]. In an effort to compensate for
enhanced biofilm resistance, therapeutic doses of conven-
tional antibiotics are often increased, accelerating harmful
resistance patterns in bacteria. The detrimental conse-
quences of this in the human population and wider envi-
ronment have prompted a search for alternative solutions.
One such safe and effective alternative to synthetic medi-
cines and antimicrobial agents are biosurfactants [3, 18, 38].
Rhamnolipids (Rhs) are a group of biosurfactants pro-
duced by Pseudomonas aeruginosa. These have one (for
mono-rhamnolipid) or two (for di-rhamnolipids) rhamnose
sugar moieties (hydrophilic moiety) acylated to long-chain
fatty acids or hydroxyl fatty acids (hydrophobic moiety)
Electronic supplementary material The online version of this
article (doi:10.1007/s00284-013-0412-8) contains supplementary
material, which is available to authorized users.
G. A. Quinn (&) A. P. Maloy
Centre of Applied Marine Biotechnology (CAMBio),
Letterkenny Institute of Technology (LYIT), Port Road,
Letterkenny, County Donegal, Ireland
e-mail: gerryquinn@gmail.com
M. M. Banat
University Hospital North Staffordshire (UHNS), Medical
Division City General Site, Newcastle Road, Stoke-on-Trent
ST4 6QG, UK
I. M. Banat
Biomedical Sciences Research Institute, University of Ulster,
Coleraine BT52 1SA, Northern Ireland, UK
123
Curr Microbiol
DOI 10.1007/s00284-013-0412-8
Author's personal copy
through one or two b-hydroxy fatty acid chains [32]. Their
characteristic antimicrobial activity (mostly against
planktonic bacteria) and biomedical applications have been
extensively investigated [3, 4, 7, 13, 37]. However, there is
surprisingly little information available on the extent to
which Rhs and plant biosurfactants (PBs) inhibit mixed-
species pre-existing bacterial biofilms. The inhibitory
activity of several different Rh mixtures has been docu-
mented in relation to pre-existing single-species biofilms of
Salmonella typhimurium [27], Bordetella bronchiseptica
[16], microflora on vocal prosthesis [39] and Bacillus
pumilus [10]. However, these studies have not examined
this inhibitory action in direct comparison to existing
antimicrobial solutions. Mixed bacterial biofilms are also
important in inhibitory tests because many biofilms exist as
complex polymicrobial colonisations [20, 41, 42, 46]. This
complexity can add another dimension to their persistence
[41]. In order to encompass a wide range of biofilm phy-
siologies, this study compared the action of the biosur-
factants with broad-spectrum antimicrobials on mixed
bacterial biofilms.
Materials and Methods
Chemicals and Reagents
All solvents of analytical grade or other purities were
supplied by VWR (HiPerSolv, Chromanorm Range, VWR
international, Poole, Dorset, UK). Microbiological media
and reagents were supplied by Oxoid Ltd (Basingstoke,
Hampshire, UK) unless otherwise stated. Nutrient broth:
Lab-Lemco Powder (1.0 g), yeast extract (2.0 g), peptone
(5.0 g) and sodium chloride (5.0 g).
Broad-spectrum antibiotics: ampicillin (AMP) and kana-
mycin (KAN) were supplied by Gibco (Paisley, Scotland,
UK) and chloramphenicol (CHL) was supplied by Acros
(Geel, Belgium).
Rhs (at a 4 % w/v concentration, containing mono- and
di-rhamnolipids mixtures) were produced as described previ-
ously [32, 35] and were diluted from a stock of 2 mg/ml
solution.
The PB (SC1000) was supplied by Biobased Europe,
Ayrshire, Scotland (http://www.biobasedeurope.com/).
This is a non-ionic surfactant blend of plant oil extracts,
fatty alcohols and tall oil. This colloid is water soluble and
readily biodegradable and was formulated from a stock
concentration of 2 mg/ml.
Bacterial Strains and Culture Conditions
Bacterial strains included Escherichia coli (ATTC 11775),
Citrobacter freundii (ATTC 8090), Klebsiella pneumoniae
(ATTC 13883), Cronobacter sakazkii (ATTC 29544),
Micrococcus luteus (ATTC 4698), Bacillus subtilis (ATTC
6051) and Staphylococcus aureus (ATTC 12600). Bacteria
for all experiments were removed from frozen stocks (-80 °
C
in glycerol) and thawed when necessary. Bacteria were cul-
tured at 37 °C in nutrient broth (Oxoid Ltd) and enumerated
by transferring an aliquot (10 ll) of growing bacteria to
nutrient agar (NA) and incubating overnight at 37 °C.
Minimum Inhibitory Concentration
Minimum inhibitory concentrations (MICs) for planktonic
bacteria were assessed using flat-bottomed 96-well high-
bind plates (Costar
Ò
, Corning Incorporated, Corning, NY,
USA). To maintain consistent comparisons throughout bio-
film analysis, all assays and dilutions were performed using
nutrient broth. This meant that the analysis did not require
media supplements for different species or adjustments for
test substances such as broad-spectrum antibiotics. The
highest concentration of the biosurfactant (test substance)
was diluted through a series of twofold dilutions in 10 wells
of a 96-well plate. To ensure that the MIC was within the
range of observations, pilot experiments were carried out to
determine the optimal range of concentrations for the test
biosurfactant. The same determinations were also performed
for the broad-spectrum antibiotics AMP, CHL and KAN. For
MIC determinations, a 1-ml aliquot of bacteria that was sub-
cultured (18 h at 37 °C) in sterile nutrient broth was incu-
bated for a further 2–3 h in 50 ml of fresh media. The
inoculum was adjusted to 1 9 10
4
cfu (determined from a
growth curve for each bacterium in comparison to their OD
at (600 nm)) and added to a 96-well plate. Appropriate
controls of test substance only and media only were also
added to wells in the same plate. Cultures along with test
substances were shaken (130 rpm) overnight at 37 °C. The
MIC was determined as the lowest concentration of test
substance that inhibited visual growth of test bacteria after
overnight incubation (16–20 h). Broth was removed from
wells that showed visual inhibition and inoculated onto NA.
The minimum concentration of test substance resulting in no
bacterial growth was referred to as the minimum bactericidal
concentration (MBC).
Radial Diffusion Assay
A modification of the ultrasensitive radial diffusion assay
(RDA) was used to detect inhibitory activity of test sub-
stances on solid growth media [33, 21].
Preparation of Single-Species Biofilms
Single-species (homogenous) biofilms were prepared to
assess the inhibition by biosurfactant in comparison to
G. A. Quinn et al.: Biosurfactants and Biofilms
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broad-spectrum antibiotics [33]. Biofilm biomass was
quantified by the crystal violet adhesion assay, commonly
used in biofilm quantification [18, 25, 27, 33, 34]. The
absorbance values were expressed as a percentage of a
control biofilm for each organism, which contained an
identical concentration of bovine serum albumin (BSA)
(Sigma, St Louis, MO, USA). Most antibiotics and bio-
surfactants are also a potential carbon source for bacteria;
therefore, BSA was used as the control substance due to its
relatively benign nature in terms of bacterial inhibitory
activity [6].
Preparation of Self-Assembling Marine Biofilm
(SAMB)
A self-assembling marine biofilm (SAMB) was used to
compare the ability of broad-spectrum antibiotics and
biosurfactants to disperse a complex mixture of bacteria.
This consisted of an assemblage of marine bacteria that
formed a mixed marine bacterial biofilm (which was sub-
sequently characterised in terms of species composition) on
high-bind 99-well polystyrene plates [33]. Mixed biofilms
were cultivated at a low temperature (10 °C) in a dilute
nutrient medium of 50 % nutrient broth and 50 % seawa-
ter. Test substances were added to the four-day-old SAMB
and subsequently cultivated for 4 days. The supernatant in
the test well, referred to as planktonic bacteria, was
removed from the wells. The biofilm biomass was quanti-
fied using the crystal violet adherence assay.
DNA-Based Characterisation of the SAMB: Cloning
After allowing the SAMB to form for 8 days, DNA was
extracted from attached organisms constituting the biofilm.
The biofilm was rinsed three times with sterile PBS to
remove loosely associated bacteria prior to DNA extraction
using a Power Biofilm kit (MO BIO Laboratories Inc.,
Carlsbad, California, USA). DNA was PCR amplified with
each 50-ll reaction containing PCR buffer at 19,1.5mM
MgCl
2
, 0.8 lM of universal primers U519F and U1068R
[2, 45], 200 lM of each deoxynucleoside triphosphate,
1.25 U HotStar Taq polymerase (Qiagen, Hilden, Germany)
and 1.5 ll of DNA. Reactions was run under the following
conditions: 10 min at 95 °C, followed by 28 cycles of 45-s
denaturing (95 °C), 45-s annealing (55 °C), 1-min elonga-
tion (72 °C) and an additional 10-min elongation at 72 °C.
Four replicate PCRs were pooled, purified (MinElute Kit,
Qiagen, Hilden, Germany) and 20 ng of PCR products were
cloned using a TOPO-TA (Invitrogen) cloning kit. Recom-
binant clones were screened to ensure they were carrying
inserts of the correct size prior to purification (QIAprep
Miniprep kit, Qiagen, Hilden, Germany) and 53
plasmids were commercially sequenced (Beckman-Coulter
Genomics, Essex, UK) using the primer U519F. Sequence
reads were manually edited and aligned using Geneious Pro
software version 5.5.6 (Geneious, Auckland, New Zealand).
Sequences were analysed using the CLASSIFIER tool
available through the Ribosomal Database Project [44].
Neighbouring sequences were used to reconstruct phyloge-
netic associations based on the neighbour-joining method
with Tamura–Nei distances [40].
Data Analysis
Statistical analysis was performed using GraphPad Prism 4
software (Hearne Scientific Software Pty Ltd, Melbourne,
Victoria, Australia). For MIC/MBC, data were given as
mean ± standard error of the mean (SEM). For RDA test,
the radius of inhibition (mm) was annotated as mean ±
SEM for each test bacteria. For the calculations of the
inhibition of single species of microbial biofilm, absorbance
values (at 595 nm) from crystal violet adherence assay of
control wells were subtracted from test wells to give a cor-
rected value. This corrected value was expressed as a per-
centage of the control biofilm. The significance of corrected
values relative to the control was calculated using a two-
tailed students t test (unpaired) and annotated as *P \ 0.05,
**P \ 0.01 and ***P \ 0.001. Outlying absorbance values
that were less than the value of a standard blank well (i.e. a
negative absorbance value in the crystal violet adherence
assay) were adjusted using the value of a blank well, which
was assigned as 0.01. Absorbance values from the test sub-
stances were expressed as a percentage of the control/control
biofilm and used to create a bar graph depicting the
mean ± SEM. This procedure was also followed for the
mixed-species marine biofilm.
Results and Discussion
A comparison of solutions to bacterial colonisation cannot
be adequately assessed through exclusively planktonic
bacterial assays. In this manuscript, the efficacy of a Rh, a
PB, AMP, KAN and CHL was compared over a range of
bacterial physiologies. These included planktonic, agar
(stranded planktonic), single-species biofilms and mixed-
species bacterial biofilms [26].
Comparisons of the Minimum Inhibitory
Concentrations of Antibiotics and Biosurfactants
Although bacteria exploit optimal growth conditions, there
are very few environments in which they receive the optimum
nutrition, agitation and aeration provided by laboratory-based
planktonic culture. Rather, research suggests that many bac-
teria assume a more sessile physiology typified by a biofilm in
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