Biofilm control with antimicrobial agents

182 Pages·2014·3.07 MB·English

by  Paula Araújo

BIOFILM CONTROL WITH ANTIMICROBIAL AGENTS:
THE ROLE OF THE EXOPOLYMERIC MATRIX

A Dissertation presented to the
UNIVERSITY OF PORTO
for the degree of Doctor in
Chemical and Biological Engineering
by
Paula Alexandra da Silva Araújo

Supervisor: Professor Manuel Simões
Co-supervisor: Professor Filipe Mergulhão

LEPABE – Laboratory for Process, Environment, Biotechnology and Energy Engineering
Department of Chemical Engineering
Faculty of Engineering, University of Porto
June, 2014
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“We live in an island surrounded by a sea of ignorance.
As our island of knowledge grows, so does the shore of our ignorance.”
John Archibald Wheeler (1992)

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“Ser poeta é ser mais alto, é ser maior
Do que os homens! Morder como quem beija!
É ser mendigo e dar como quem seja
Rei do Reino de Aquém e de Além Dor!
É ter de mil desejos o splendor
E não saber sequer que se deseja!
É ter cá dentro um astro que flameja,
É ter garras e asas de condor!
É ter fome, é ter sede de Infinito!
Por elmo, as manhãs de oiro e de cetim...
É condensar o mundo num só grito!
E é amar-te, assim perdidamente...
É seres alma, e sangue, e vida em mim
E dizê-lo cantando a toda a gente!”

Florbela Espanca (1923)
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ACKNOWLEDGEMENTS
This is the place reserved to make a retrospective on the last 3 years of my career, (life),
and PhD dissertation. This has been a journey where I gained knowledge from different
people who made a difference that mattered.
I wish to express my gratitude to my supervisor Professor Manuel Simões for his
help, patience and support. My co-supervisor Professor Filipe Mergulhão, and Professor
Luís Melo are recognized as well. Without you it would be impossible to complete this
thesis. You were all very helpful throughout the whole process by providing assistance
and guidance.
The Faculty of Engineering of University of Porto and LEPABE are acknowledged.
These were the places where the work was performed. My deepest gratitude in
particular to Joana Moreira, Carla Ferreira, Joana Malheiro, and Margarida Pereira. And
for the others from the old E303 lab and the new E007/008 labs who helped me a lot
going around the place (whether suggesting better methods or even when searching
for a much needed tool). Thanks for all the discussions which were extremely helpful.
The insights of Idalina on the manuscripts are comprehensively appreciated. The help
of Paula Pinheiro and Silvia Faia is fully acknowledged as well. To all of you thanks for
making the labs a pleasant place.
I gratefully acknowledge the financial support provided by the Operational
Programme for Competitiveness Factors (COMPETE), the European Regional
Development Fund (FEDER), and by the Portuguese Foundation for Science and
Technology (FCT) through the Project Bioresist— PTDC/EBB-EBI/105085/2008. The
project Susclean – KBBE.2011.2.3-01/287514 is also acknowledged.
Thank you Hans just for being there, giving me those pep talks, and a vision for
future endeavors.
Jorge Trigo, I thank you for being my teacher, friend and for the important lessons
about the walk of life.
To my friends Lobo, Bony, Zé, João and Rita thank you for your friendship, I cherish
all the moments spent with you, with all the laugh and warm feelings. Thanks for being
here.
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To my family, Mamã, Papá, Sis, João, Beatriz, Luiz, Hugo, Marieta, and Ana thank
you for all the love, tenderness, support and incentive much needed to keep me going
with bravery even when everything seemed to be falling apart. I love you all.
I would like to reinforce my gratitude for Luiz for all the time spent double checking
the manuscripts.
And last but not the least, to my Bubas just for being there doing what you do,
which is mostly a bit of all things acknowledged before and a tad more. I love you!

March 13th 2014

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ABSTRACT
Biofilms, accumulated microorganisms and extracellular compounds on a surface, are
able to thrive in all environments. Biofilm presence in the food industry can cause
negative effects, being associated to lower industrial operational efficiencies, as well as
microbial contamination of the final product. There are many strategies that attempt to
control biofilm proliferation, however, no control strategy is completely effective. Thus,
the development of new and more effective treatments and improving of the
conventional strategies is in demand. In an effort to overcome biofilm resistance new
compounds must be discovered and their antimicrobial properties assessed.
Additionally, the association between different chemical agents could potentiate their
singular antimicrobial efficacy.
The main objective of this study was to develop biofilm control strategies and to
understand the biofilm behavior to these conditions. Therefore a selection of factors
associated with biofilm resistance were studied. Bacillus cereus and Pseudomonas
fluorescens are common contaminants in the food industry and were selected as
microbial models. Several antimicrobial agents were screened using a colony biofilm
test. These consisted as biofilms developed in as colonies in the top of polycarbonate
membranes. The efficacy of selected agents with putative antimicrobial quenching
substances was studied using respirometry. The killing and removal efficacy of
treatments with antimicrobial agents was assessed using 96-well microtiter plates. To
mimic close-to-practice conditions, biofilms were developed in a flow cell system and
characterized. Control strategies potentiating current antimicrobial agents, and new
agents were performed using biofilms developed in the referred bioreactors.
The diffusion of ethanol, isopropanol, sodium hypochlorite, chlorine dioxide,
hydrogen peroxide, bezalkonium chloride (BAC), benzyldimethyldodecylammonium
chloride (BDMDAC), cetyltrimethylammonium bromide (CTAB), ciprofloxacin,
erythromycin, streptomycin and tetracycline was assessed on colony biofilms.
Ciprofloxacin, streptomycin, BAC and CTAB were selected to assess their biofilm control
efficacy. These products had distinct abilities to diffuse through the biofilms (high
diffusion – BAC and ciprofloxacin; low diffusion – CTAB and streptomycin). It was
concluded that the diffusion ability of antimicrobial agents is not directly correlated with
biofilm killing and removal efficacy. BAC and CTAB were selected for the following
studies due use in industrial cleaning and disinfection practices.
Known constituents of the extracellular polymeric matrix of biofilms (alginate and
humic acids), and selected disinfection-interfering agents from the European Standard
EN – 1276 (bovine serum albumin and yeast extract) were used to challenge the
antimicrobial efficacy of the selected quaternary ammonium compounds as soiling
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agents. The minimum bactericidal concentration of the chemicals was assessed. The
interfering agents simulated “clean” soiling conditions. Within the range of
concentrations tested the interfering substances mildly reduced the action of the
antimicrobial agents. Humic acids were able not only of reducing the antimicrobials
efficacy, but also to increase P. fluorescens respiratory activity. It was shown that humic
acids should be considered as a potential interfering agent when developing cleaning
and disinfection solutions, due to its strong interaction with the quaternary ammonium
compounds tested.
The biofilms grown in the flow cell system at varying linear flow velocities (applied
in food industry) showed different characteristics. The biofilms developed at the lowest
linear flow velocity (u = 0.1 m.s-1) differed by being thicker and more hydrated than the
biofilms developed at the two higher linear flow velocities (u = 0.4 m.s-1 and
u = 0.8 m.s-1). These biofilms were more compact, with higher bacterial cell numbers
and more exopolymeric substances (proteins and polysaccharides). In spite of these
differences, the dry biofilm mass per area was similar, as well as the expression of the
major outer membrane proteins from the biofilm cells. The biofilms developed at higher
linear flow velocities were selected for further studies of control strategies, due to
higher resistance characteristics (cells and exopolymeric substances).
Halogen-based products are recognized for their relevant antimicrobial properties.
Thus, selected halogen-based products (CTAB, 3-bromopropionyl chloride -BrCl, 3-
bromopropionic acid -BrOH and sodium hypochlorite -SH) were used in order to
understand their antimicrobial activity against both planktonic and biofilm cells of
P. fluorescens. The mode of action of these products is cell membrane disruption,
causing leakage of essential cellular constituents. The results demonstrate comparable
effects of BrCl and BrOH to those of sodium hypochlorite that makes them a potential
alternative to sodium hypochlorite. However, CTAB was the most efficient agent.
The addition of enzymes as an aid to biofilm control treatments, applied alone or
in combination with BAC and CTAB, had the ability to kill and remove the biofilms
developed in microtiter plates and in the flow cell system. The combination enzyme-
biocides was synergistic on biofilm control. The treatments allowed both long term
effects (additional biofilm removal and colony forming units reduction were observed
in the hours following the treatments), as well as biofilm regrowth.
The presented studies in this thesis clearly underline the importance to study
biofilm control strategies under representative conditions for practice, being stress
conditions determinants of different biofilm responses. Biofilm control should be a
multifactorial approach due to the many features that biofilms have that provides them
an increased protection. It is, therefore, necessary to incessantly find new control
strategies because microorganisms will adapt and find new ways to overcome the
biofilm control treatments.
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