Science against microbial pathogens: communicating current research and technological advances

______________________________________________________________________________
A. Méndez-Vilas (Ed.)

Antimicrobial resistance to disinfectants in biofilms

P.Araújo, M.Lemos, F.Mergulhão, L. Melo and M.Simões*
LEPAE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, s/n,
4200-465 Porto, Portugal
*mvs@fe.up.pt

Microorganisms have a natural capacity to attach to surfaces, to multiply and to embed themselves in a slimy matrix,
forming biofilms. These allow complex interactions among different species and surfaces. There is significant evidence
that bacterial growth in attached communities constituted the first form of life on the planet, and it is estimated that the
majority of microbial activity on earth is resultant from biofilms, rather than microbes growing isolated in the form of
planktonic cells. Industrial units, particularly food processing plants, provide good environmental conditions for biofilm
proliferation, such as an abundant source of nutrients, which can create a significant problem mainly because most of these
microorganisms can be pathogens. Therefore, cleaning and disinfection in food processing plants are important issues that
deserve full consideration. However, the phenotype of biofilms provides intrinsic resistance to cleaning and disinfection.
Nowadays, there are no biofilm control strategies that inactivate, remove or prevent their regrowth after antimicrobial
treatment.This study provides information on the problems of biofilm formation and on the main strategies used for their
control. Information on the antimicrobial mode of action of biocides and biofilm resistance mechanisms is also provided.
Keywords: antimicrobialagents; biofilms; disinfectant; antimicrobial mode of action; resistance

1. Introduction
The survival of the fittest is a biological principle applicable to all living beings, and although different organisms have
developed their own survival mechanisms, all have one common factor that relates survival with the ability to adapt to
constant changes in the environment. Microorganisms are particularly adaptable to changes in the environment because
of their high reproduction rates, which allows them to transfer survival characteristics to future generations in short
periods of time[1].Surfaces seem to play a major role in the survival of microbial cells. In fact, there are factors that
contribute to the adhesion of microorganisms, near the surface: the high concentration of nutrients, an optimum pH, and
the low hydrodynamic force that is exerted on the adhered cells. Adhesion of microorganisms to surfaces in biofilms
represents an ecological advantage and is a prevalent form of survival in hostile environments [2]. Genetic load and
regulation of microorganisms are determining factors for biofilm formation, which still depends on the properties of the
adhesion surface, type of bacterial cells, hydrodynamic conditions and surrounding environmental factors [3].
Biofilm formation is a complex process that involves several steps. Initially, it is necessary that the surface of
adhesion becomes preconditioned, either intentionally or by adhesion of macromolecules present in the circulating
fluid. These favorable characteristics attract microorganisms that are adsorbed to the surfaces, either in reversible or
irreversible way. The persistent microorganisms that remain on the surface after irreversible adsorption, produce
signaling molecules as well as extracellular polymeric substances (EPS) [4]. EPS are an intricate network formed
essentially by polysaccharides, proteins, phospholipids, teichoic acids and even nucleic acids. It is also possible to find
mineral crystals, silt particles, milk residues as calcium phosphate [5] and sometimes blood components or dirt in the
EPS composition due to the conditions under which the biofilms were formed [3, 6]. The convective and diffusional
transport of oxygen and nutrients to the biofilm takes place through existing channels in the biofilm.
In the biofilm formation process there is also proliferation and cell growth, accompanied by secretion of matrix
exopolymers and transport of products to the exterior of the biofilm [5]. At the same time that cells and nutrients are
transported and accumulated on the surface, some portions of the biofilm are removed and enter the surrounding
streams[7]. The development of matured biofilms is also determined by the balance of growth and detachment
(sloughing and erosion) events.
Biofilms are difficult to eradicate, so prevention of its occurrence would be the optimal strategy for their control.
Although biofilm removal can occur naturally by intrinsic processes, mechanical removal by human action is a common
strategy in food industry, though very expensive because of the need to open the process machinery. However,
currently, there is no known technique to control or prevent biofilm formation without undesirable side effects [5].
Biofilms have a diversity of defense mechanisms. When compared to their planktonic equivalents, biofilm cells are
10-1000 times more resistant to antimicrobials [8]. The way how microorganisms develop resistance is not well
understood. Some hypothesis mentioned the function of EPS as a polymer that could interact with antimicrobials,
quenching their activity, before they could reach the cells embedded in the matrix. EPS can either bond to the
antimicrobials, delaying their diffusion, or chemically react with them, causing their inactivation [9]. A deeper
understanding of biofilm resistance mechanisms is necessary in order to develop new and more effective biofilm control
strategies.

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A. Méndez-Vilas (Ed.)

2. Biofilms in the industry

In all industries, especially in the food industry, the proliferation of microorganisms is very common even when the
manufacturers take all the “by-the-book” contingency plans. Therefore, contamination is normally caused by biofilm
formation due to ineffective or complete lack of cleaning. Organic molecules are able to deposit themselves in all types
of surfaces, and the water used for manufacturing provides good conditions for microbial growth. Also, the physical
characteristics of the substratum influence initial attachment. Moreover, industrial plants have intricate process lines
with critical points where built up of fouling is expected. Gaskets, dead ends, joints, valves, corners, cracks, or crevices
are parts of those critical points [5, 10].
Biofilms appear in all kinds of industries resulting in serious operation and maintenance costs by reducing
operational efficacy in heat exchangers, increasing operational pressure in desalination plants, causing blockage of
tubes in water distribution systems, increasing energy consumption and accelerating corrosion of metal surfaces
requiring the replacement of damaged parts [3, 11]. Also, biofilms may be a public health concern as the biological
deposits can be a reservoir of spoilage and/or pathogenic bacteria. Salmonella spp., Listeria monocytogenes, Yersinia
enterocolitica, Campylobacter jejuni and Escherichia coli are frequently associated with biofilms from food processing
plants [3].

Chemical contaminants Biological contaminants Environmental conditions

Contaminants in raw material pH

Nutrients Biofilm detachment Temperature
Antimicrobials Enzymes Retention time
Dispersing agents Pathogenic or harmful organisms Processing time

Microbial growth

Biofilm formation
Blockage of process lines
Product contamination
Product spoilage

Fig. 1 Microbial growth in food industry (adapted from[12]).

Biofilms usually differ according to the environmental conditions under which they were formed, i.e. temperature,
pH, type of nutrients available, and type of bacteria. For instance, dairy industries commonly have biofilms composed
by Pseudomonas fluorescens, E. coli, Shigellaspp., Staphylococcus aureus and Bacillus cereus. Shrimp factories
normally have P. fluorescens and P. putida as biofilm colonizers; in fish factories is common to find biofilms composed
by Enterobacteriaceae and Serratialiquefaciens. In a caviar plant, biofilms of Neisseriaceaespp., Pseudomonas spp.,
Vibrio spp. and Listeria spp.were reported[13, 14].

3. Cleaning and disinfection

The aim of microbial control is theelimination, or reduction of microorganisms and their activity to an acceptable level,
as well as the prevention and control of the formation of biological deposits on process equipment [12]. Therefore,
programs are established to control microbial proliferation, as Good Manufacturing Practice (GMP) and Hazard
Analysis and Critical Control Points (HACCP) plans [14]. Biofilm control in dairy plants normally includes a process
called Clean-in-Place (CIP), which includes the cleaning of the plant without dismantling or opening the equipment.
CIP consists on running alternated cycles of detergent and disinfectant solutions, with water rinses with increased
hydrodynamics (high turbulence and flow velocities) through the plant [10]. This method typically uses caustic acids,
surfactants, biocides and sometimes includes enzymes [3, 10, 15,16].
An ineffective cleaning plan leads to operation and maintenance costs caused by the downtime of the production and
by contaminated or spoiled products. The choice of the material for surfaces also plays an important role [5]. For
instance, polyvinylchloride increases the risk of contamination because of its deterioration over time [12]. Stainless

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steel may be a better option because it is more resistant to mechanical stresses like grinding, brushing, lapping, and
electrolytical or mechanical cleaning[5, 16]. For that reason, in a cleaning and disinfection plan, it is of major
importance to gather the maximum information about the system together with flow diagrams (information about
volume, residence time, cycle time, half-life time, etc.) [5].
The risk of biofilm formation is increased if events such as intermittent operation, unattended risk areas (i.e. filters),
inconsistent raw water composition, lack of cleaning after failures, and poor access to surfaces exist in the plant. The
risk can be lessened by the exclusion of light, use of short piping, inert materials and smooth materials; good air
circulation; working at low temperatures, in dry conditions, and general quality control [17].
Cleaning and disinfection should be thorough and systematic, being of utmost importance that the nature and age of
the fouling layer are known information. Knowing which microorganisms require elimination is essential to define the
right biocide or combinations of biocides, point for biocide injection, required concentration, temperature levels and
exposure time, according to the hydrodynamics of the system. Type and nature of contaminating residues are also key
factors. All the parameters mentioned above must be taken into account when designing a disinfection plan. The
disinfectant should be kept at a concentration equal or superior than the minimum inhibitory concentration for the
period of time defined ideal for disinfection [5, 12,18]. In order to achieve long lasting stable results, follow up actions
are required, such as monitoring the presence of microorganisms and the formation of deposits on surfaces.

4. Biocides
The European Standard of 24 April 1998 (CE/8/98), defines biocidal products as active substances, or preparations that
contain one or more active substances, that are presented to the user in their final form, and whose function is to
destroy, stop the growth, make harmless, avoid or control by any mean the action of a pathogenic organism by a
biological or chemical process.
The use of biocides in biofilm control is well accepted and very common. Although biocides are used for the
reduction of number of microorganisms, their simple use does not necessarily reduce the biofilm formation rate. It is
essential to use the correct amount of the correct biocide, with the correct frequency. The incorrect application is
expensive and leads to unwanted results [19]. Current methods of disinfection include application of chemical
compounds like alcohols, aldehydes, anilides, biguanides, bis-phenols, diamidines, halogen-releasing agents,
halophenols, peraceticacid, heavy metal derivatives, peroxygens, phenols and cresols, quaternary ammonium
compounds (QACs), chlorine-releasing agents and ozone[5, 20].
Each bacterial strain reacts differently to each chemical compound, either by its phenotypic characteristics (e.g.
properties of the cell wall) or due to resistance mechanisms (coded by its genotype or induced). Thus, it is fundamental
that when selecting one or more biocides, an evaluation on the efficacy on the eradication of dominant microorganisms
present on that system is performed. Only after having information about the nature of the microbial population to treat
it is possible to determine the relation between the minimum inhibitory concentration and the contact period of a
biocide to a given contaminant [21]. Table 1 provides information on the mechanisms of action, typical targets,
resulting effects and examples of biocides.
Within biocide mechanisms of action four major categories can be found: the oxidants, the electrophilic agents, the
cationic membrane biocides and the weak acids. Oxidants act via radical-mediated reaction oxidizing organic material;
electrophilic agents react covalently with cellular nucleophiles to inactivate enzymes; cationic membrane active
biocides destabilize membranes leading to rapid cell lysis; and finally the weak acids interfere with the ability of the cell
membrane to maintain a proper pH balance, resulting in acidification of the cell interior and widespread disruption of
metabolism[22].

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Table 1Mechanisms of interaction of several biocides according to their cellular targets and antimicrobial actions (adapted
from [18]).
Cellular targets
Thiol containing cytoplasmic Metabolic inhibition
and membrane bound enzymes
e.g. dehydrogenases
Biomolecules (e.g. proteins,
RNA, DNA) with amino,
imino, amide, carboxyl and
thiol groups (nucleophilic)
Amino groups in proteins
Enzyme and protein thiol
groups
Divalent cation-mediated outer
membrane integrity, principal
target region Gram negative
cell wall. Metal ion-requiring
enzyme processes
Intercalation between DNA
base pairs
Cytoplasmic membrane
integrity; membrane-bound
enzyme environment and
function
Transmembrane pH gradient;
membrane integrity
Membrane integrity
Cytoplasmic membrane
integrity; membrane-bound
enzyme environment and
function
Science against microbial pathogens: communicating current research and technological advances
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A. Méndez-Vilas (Ed.)
Antimicrobial action Interaction Mechanisms Examples
Chemical reactions
Oxidation of thiol groups Isothiazolinone
(predominantly) Organomercury
Salts of heavy metals
Hypochlorite
Inhibition of cellular General alkylation reactions Glutaraldehyde
metabolism and Formaldehyde
replication. Chloroacetamide
Possible cell wall
damage
Metabolic inhibition; Halogenation Hypochlorite
lysis Chlorine-releasing agents
Metabolic inhibition Free radical oxidation (e.g. Hydrogen peroxide
hydroxyl radicals) Peracetic acid
Release of cellular Chelation of metal ions EDTA
contents; High Oxine
susceptibility to stress;
metabolic inhibition.
Damage in replication Intercalation Aminoacridines
Ionic interactions
Leakage; respiratory Electrostatic interaction with Quaternary ammonium
inhibition; intracellular phospholipids compounds
coagulation Clorhexidine
Polyhexamethilene
Biguanides
Physical interactions
Leakage; disruption of Penetration/partition into Phenols
transport, respiratory phospholipid bilayer; possible Weak acids
and energy coupling displacement of phospholipid Parabens
processes molecules; intra membrane Tetrachlorosalisylanilide
molecular cycling Phenoxyethanol
2-phenylethanol
Leakage Solution of phospholipids Aliphatic alcohols
Leakage, uncoupling Membrane-protein Anionic surfactants
of energy processes; solubilization
lysis
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A. Méndez-Vilas (Ed.)

5. Mechanisms of antimicrobial action

A classical approach which is used to determine the mechanism of action of a biocide establishes a correlation between
the minimum inhibitory concentration and the resulting biochemical and physiological changes in the organism[18]. An
antimicrobial effect can be defined as an interaction between an active substance and specific targets in the microbial
cell. In target approach the active ingredients contact with a variety of cellular structures (cell wall, cytoplasmic
membrane, membrane enzymes, cytoplasm, and genetic material). Experiments conducted comparing different strains
revealed that Gram-negative bacteria, which have the supplementary protection of the cell wall, are more resistant to the
bactericidal effects than Gram-positive bacteria [23-25]. The biocides pass through the cell wall by
pores(porin).Thispenetration,accordingtoPaulus[26]isdependentonthesize,chargeandlipophilicpropertiesofmolecules.Ifas
ubstanceissolubleinwateranditsmolecularweightisaround600Da,there is a great probability of passing through the
channel formed by the porin. It is also possible that the biocide penetrates the cell wall after causing its destabilization
and disintegration. Finally, thebiocidereachesthecytoplasmicmembraneastheprimarysiteofaction.Dependingontheaction
spectrum, these substances could be designated as biostatics (if they only inhibit the microorganism growth or
multiplication) or as biocides (if they are able to kill the microorganisms).
Theprocessoftransportingthebiocidetothecellsurface,adsorption,diffusion,penetrationandinteractionwiththetargetcellc
omponentisnotinstantaneous, and the duration of this process can be different accordingly to the biocide. The
differences depend on the action mode, as well as the chemical composition and physico-chemical properties of the
biocidal agent.
Biocidal compounds come from a variety of chemical classes. Fig. 2 shows the antimicrobial mode of action of
biocide on diverse types of microorganisms.

Fig. 2 - Antimicrobial mode of action of biocides (adapted from [19]). CRAs – Chlorine removal agents; QACs – Quaternary
ammonium compounds.

Biocides may cause a series of self-destructive events in microorganisms, resulting in sub-lethal damage to cell
death. Typical damage caused by biocidal compounds involves the disruption of the transmembranar proton motive

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force leading to an uncoupling of oxidative phosphorylation and inhibition of active transport across the membrane;
inhibition of respiration or catabolic/anabolic reactions; disruption of replication; loss of membrane integrity resulting
in leakage of essential intracellular constituents such as potassium cation, inorganic phosphate, pentoses, nucleotides
and nucleosides, and proteins; lysis and coagulation of intracellular material [18].

6. Factors affecting biocide action

Cleaning is often not efficient in the removal of biofilms. Bactericidal activity is influenced by the surrounding media
but a correct cleaning plan is also very important. The main environmental factors that could influence the activity of a
biocide are pH, water hardness, presence of additives and temperature [19]. Biocide concentration, exposure time,
presence of organic compounds and type of microorganisms are also key factors in the antimicrobial action. Many
biocides have an optimum pH range of activity. For example, glutaraldehyde and cationic biocides such as
chlorhexidineand QACs are most active at alkaline pH, whereas hypochlorites and phenolics are more potent at acid
pH. Additives as corrosion inhibitors or conditioning agents may also influence and even reduce or inactivate activity.
The activity of biocides against Gram-negative organisms may be enhanced by permeabilisers, which increase the cell
permeability. Russell[27] reported that ethylenediaminetetraacetic acid (EDTA), chelates divalent cations from the
outer membrane, especially on P.aeruginosa. Also, polylysine and polyethyleneimine act by cation displacement.
Activity can also be increased by a combination of biocides, or addition of an efflux inhibitor to an antibacterial
compound. Elevated temperatures raise the activity of biocides, but this method finds now little practical use[28, 29]. In
general, the efficacy of disinfectants increases with temperature. When disinfection occurs at low temperatures, the use
of higher concentrations of biocides or elongation of the contact time may increase effectiveness[30]. The antibacterial
activity of biocides is determined by their chemical reactivity to certain organic groups. Biocides do not react
independently with fixed groups or groups of the cell border. Oxidizing biocides react with any oxidizing organic
group, not only with living cells. In food industries, deficient cleaning may not eliminate contaminating substances,
such as carbohydrates, fat, proteins, calcium phosphate, blood residues or dirt. [5]. These contaminants may have a high
impact on the cleaning and disinfection steps. This happens because most of the antimicrobial activity of chemical
compounds may be reduced in the presence of organic material, which can react with oxidative disinfectants and may
also neutralize tenside-based disinfectants and non-ionic surfactants[8, 31].
The effect of disinfectants is concentration dependent. Generally, an user-concentration is given by the manufacturer
based on simple laboratory tests measuring efficacy in suspension and without additives, which may not be efficient to
kill attached microorganisms[31].In a practical disinfecting setting, the disinfectant may be diluted due to residual water
left after the cleaning process. In order to avoid dilution, the equipment design should prevent and facilitate running of
water off the surfaces instead of accumulation. Furthermore, surfaces should be allowed to dry up reasonably before
disinfection[30].Biocides such as phenolics or alcohols typically loose activity with dilution, whereas QACs,
chlorhexidine, glutaraldehyde, ortho-pthalaldehyde retain much of their activity with dilution [27].
It is of utmost importance that the nature and age [32]of biofouling is known, as well as characteristics like the
location, and the type of microorganism (bacteria, spores, yeasts and moulds, protozoa) or biological entities (prions,
viruses) [33]. The characteristics of the surface to be cleaned are also relevant, because of the side-effects of cleaning on
equipment materials (i.e. some cleaning agents can be corrosive) [5].The total number of target cells should be taken
into account since high initial numbers of bacteria may result in some persistant cells, which promotes biofilm regrowth
[27, 30]. In cases where the concentration of the disinfectant is limited, the bactericidal effect may be reduced in the
presence of high numbers of bacteria. Also, the growth phase of bacteria will influence their susceptibility to
disinfectants. It is known that bacteria in exponential phase of growth are more sensitive to disinfectants than stationary
phase bacteria[30]. The biofilm cells are more resistant to biocides as a result of their physiological heterogeneity
(including the presence of dormant cells) and the presence of EPS, which hinders the diffusion of biocides into the cells
[34].

7. Microbial resistance to biocides

When exposed to a harmful stress environment, bacteria will do all in their power to survive [27]. External stress, like
environmental conditions, has different effects on different organisms, leading to natural responses like inhibition
and/or inactivation of the cells. Any deviation from the normal environmental conditions might result in reduced growth
rates. When bacteria are exposed to sub-lethal levels of biocides, only minor cell damage is caused. The consequences
of that may include changes in their phenotype and induction of gene expression, giving rise to a more resistant
population. Resistance mechanisms are the means that living organisms have to respond to continuously changing
environment in order to survive. Resistance is the description of the relative insusceptibility, viability or multiplication
of a microorganism, to a certain chemical treatment under certain conditions. It may be temporary or permanent and
relates either to the first generation organisms as to the next [28].Thus, there are three documented types of resistance:
inherent resistance, also termed natural or intrinsic; acquired resistance, due to the occurrence of a mutation, and usually

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mediated by plasmids; and finally, resistance by adaptation which occurs when a community of bacteria acquires
resistance to an antimicrobial, it may also acquire resistance to other antimicrobials of the same type (cross-
resistance)[35, 36].
Many studies have been performed in order to access microbial resistance to biocides [5, 21, 27, 29, 30, 35, 37-54].
Common defense mechanisms against biocides were reported in literature such as the ability to produce enzymes that
could destroy the biocides, changes in the permeability of the cytoplasmic membrane to prevent the entry of the biocide
and also changes in the composition of the cell wall. Spontaneous mutations, by exposure to sub-lethal concentrations
of antimicrobials, may occur at chromosomal or plasmid levels. There is evidence of a regulated adapted response in
growing E. coli exposed to hydrogen peroxide, with the cells becoming resistant to normally lethal doses of peroxide
and the synthesis of around 40 new proteins [27].Efflux is another resistance mechanism. Poole [45, 46] documented
multidrug efflux systems, that are encoded by chromosomes and customary of Gram-negative bacteria. Finally, changes
at the phenotypic level, i.e. the ability to form biofilms, are an adaptive form of resistance.
Adhered cells have a phenotype that confers increased resistance to antibiotics and biocides, when compared with
suspended cells. The physiological state of biofilm cells is different from that their planktonic counterparts[55]. There
are several characteristics that underlie the increased resistance of bacterial films. The resistance mechanism is more
evident in biofilms due to the presence of EPS. The biofilm structure dictates its susceptibility to antimicrobial agents, it
is a mechanism that can be either physical or chemical. EPSare electrically charged and may be responsible for binding
the antimicrobial agents before they have the opportunity to reach specific targets in the cell, hindering the diffusion of
biocides [8, 9,56]. The biocide could also react and be neutralized by components of the biofilms [5]. A model for the
reduced susceptibility of thick biofilms to chlorine antimicrobials is described under the reaction-diffusion kinetics. The
organic components of the biofilm consume the chlorine [57].Phenotypic tolerance to oxidizing biocides can also result
from the biofilm mode of growth, which is often invoked as the underlying factor in failure of biocide activity in the
water treatment and pulp industries. [36]
When bacteria are starved for nutrients, they shift from exponential growth to slow or no growth. This setback is
usually accompanied by an increase in antibiotic resistance [8, 58]. Mahand O`Toole [8] reported that different cells
subjected to slightly different environments within the same biofilm, resulted in different growth rates. This fact has
influence in the resistance to antimicrobials because it was proven that bacteria have different degrees of resistance
according to their state: resistance increased as both planktonic and biofilm cultures approached stationary phase,
biofilm cells were 15-times more resistant than the planktonic cells [59].

8. Future remarks
Biofilms are a problem in all industries where water is involved in the process. The chemical control of biofilms is an
important issue because of its complexity, and also due to the fact that resistance to biocides is unavoidable. The
solution is to continually find new strategies and new compounds for biofilm eradication. Likewise, the development of
improved cleaning regimes and improved product quality, plant performance, and economic returns should be sought
[10].Industrial processes need biocides that retain their activity under “dirty” conditions, work in low volumes, have
low costs and avoid corrosion. Particularly in the food industry, consumers and governmental agencies agents demand
chemical agents that are less toxic and less susceptible to microbial resistance. Natural products have been introduced
into the market, such as grape fruit seed extract, [30], or phytochemicals [60]. In general, their effects are limited
compared to conventional disinfectants[30]. Møretrø[30] refers anti-biofilm specific compounds as alternative drugs
with the function of selectively block virulence, quorum sensing, and/or biofilm formation and not affecting planktonic
growth of the bacteria. The addition of enzymes to the biofilms to degrade the EPS may also potentiate the action of
antimicrobials [34, 61].
A better understanding of biofilm formation process is required from the early stages to the maturation, by a
combined perspective of their physical, chemical and biological phenomena. The investigation of mechanisms of action
of antimicrobial sheads towards the study of interactions of these with different cellular targets and their killing effects,
allowing the development of strategies to enhance the biocidal effect. Among these effects are the increase of the initial
interaction of the biocide or its accumulation in the target cell by intracellular releasing, optimization of synergistic
combinations of several compounds and triggering autocidal effects [18].
The process will also involve the source, i.e. the food industry, which must progressively improve the production
processes, always taking into account health and sanitation quality standards (ISO 22000; BRC-IOP; etc.).HACCP
plans should predict measures to control the possible contaminants present that could reduce biocide efficacy,
monitoring of biofilms in processing lines and the plant layout, in order to guarantee an efficient cleaning and
disinfection program[5].

Acknowledgements: The authors acknowledge the financial support provided by the Operational Programme for Competitiveness
Factors –COMPETE and the Portuguese Foundation for Science and Technology -FCT, through Project Bioresist – PTDC/EBB-
EBI/105085/2008.

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