Science against microbial pathogens: communicating current research and technological advances ______________________________________________________________________________ A. Mndez-Vilas (Ed.

Antimicrobial resistance to disinfectants in biofilms

P.Arajo, M.Lemos, F.Mergulho, L. Melo and M.Simes*
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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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 Nutrients Antimicrobials Dispersing agents Biological contaminants Contaminants in raw material Biofilm detachment Enzymes Pathogenic or harmful organisms Environmental conditions pH Temperature Retention time 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

Antimicrobial action

Interaction Mechanisms

Examples

Chemical reactions Thiol containing cytoplasmic Metabolic inhibition and membrane bound enzymes e.g. dehydrogenases Oxidation of thiol (predominantly) groups Isothiazolinone Organomercury Salts of heavy metals Hypochlorite Glutaraldehyde Formaldehyde Chloroacetamide

Biomolecules (e.g. proteins, RNA, DNA) with amino, imino, amide, carboxyl and thiol groups (nucleophilic)

Inhibition of cellular metabolism and replication. Possible cell wall damage Metabolic inhibition; lysis Metabolic inhibition

General alkylation reactions

Amino groups in proteins

Halogenation

Hypochlorite Chlorine-releasing agents Hydrogen peroxide Peracetic acid EDTA Oxine

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

Free radical oxidation (e.g. hydroxyl radicals)

Release of cellular Chelation of metal ions contents; High susceptibility to stress; metabolic inhibition.

Damage in replication

Intercalation

Aminoacridines

Ionic interactions Cytoplasmic membrane integrity; membrane-bound enzyme environment and function Leakage; respiratory Electrostatic interaction with inhibition; intracellular phospholipids coagulation Quaternary ammonium compounds Clorhexidine Polyhexamethilene Biguanides

Physical interactions Transmembrane pH gradient; membrane integrity Leakage; disruption of transport, respiratory and energy coupling processes Penetration/partition into phospholipid bilayer; possible displacement of phospholipid molecules; intra membrane molecular cycling Phenols Weak acids Parabens Tetrachlorosalisylanilide Phenoxyethanol 2-phenylethanol Aliphatic alcohols Anionic surfactants

Membrane integrity Cytoplasmic membrane integrity; membrane-bound enzyme environment and function

Leakage Leakage, uncoupling of energy processes; lysis

Solution of phospholipids Membrane-protein solubilization

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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 (crossresistance)[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]. Mretr[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/EBBEBI/105085/2008.

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