Antimicrobial resistance in biofilms - Formatex Research...

Antimicrobial resistance in biofilms

M.G. Paraje1 1 Department of Pharmacy, IMBIV-CONICET, Faculty of Chemical Sciences, National University of Córdoba, Haya de la

Torre y Medina Allende, Ciudad Universitaria (5000), Córdoba, Argentina. [email protected]

The ability of microorganisms to form biofilms is closely related to infectious diseases, and environmental and biotechnological processes. Biofilms constituting a microbial multicellular lifestyle and are defined as organized communities of bacteria, collaborating among themselves and being attached to an inert or living surface contained in a self-produced polymeric matrix made principally of exopolysaccharide. The structural nature of the biofilms and the characteristics of the sessile cells, produce resistance towards the antimicrobial agents, leading to a protected environment against adverse conditions and the host´s defenses. Despite decades of research, very little is known about the molecular mechanisms of antibiotic resistance in biofilms. Although several theories have been proposed, the precise mechanism of how this sensitivity is altered has still not been clarified. Nevertheless, it is possible to separate these mechanisms into intrinsic (or innate) and extrinsic (or induced) resistance factors to biofilms. Nevertheless, because of the heterogeneous nature of biofilms, it is likely that multiple mechanisms of antimicrobial resistance occur. However, additional mechanisms must also exist to be able account for increased biofilm antibiotic resistance. Although methods to test biofilm-growing bacteria have already been developed, their clinical relevance with regard to the prediction of clinically successful therapies still awaits confirmation.

Keywords: biofilms; antimicrobial agents; mechanism of action; intrinsic resistance factors; extrinsic resistance factors.

1. What are biofilms?

Antonie van Leeuwenhoek (1684) was the first to display the "animalcules" (bacteria) found in plaque scraped from his teeth plaque, and described in a report to the Royal Society of London: "The numbers of these animalcules in the scurf of a man’s teeth are so many that I believe they exceed the number of men in a kingdom." In a 1940 H. Heukelekian and A. Heller in an issue of the Journal of Bacteriology wrote a development takes place either as bacterial slime or colonial growth attached to surfaces, and Zobell (1943) noted an "effect" in seawater and described many of the fundamental characteristics of attached microbial communities. Biofilm science and biofilm engineering have been active fields of study since sessile communities were first described and named in 1978. However, the first conceptual term used was “aufwuchs” (growth in German, 1975), and later other term was employed, but deemed inappropriate because it implies “around plants ". The group of Dr. Costerton, in 1978, used the name “biofilms” as a more generic term for microorganisms adhering to wet surfaces in freshwater ecosystems [1]. Donald (2002) [2] made a widely accepted description of biofilms. Microorganisms constitute the most successful form of life on earth, in terms of total numbers, their phylogenetic diversity and the extent of habitats colonized. They impact on human existence and well-being, either directly by influencing human development, health and disease, or indirectly, by carrying out processes in the natural or man-made environments [3]. Bacteria exist in two principal forms, as free-floating planktonic replicating cells and in biofilms. Microbiologists, due to historical reasons, have traditionally focused on the results of empirical research work on free-floating microorganisms growing in suspension in a liquid growth medium. However, it is now generally acknowledged that the majority of microbial cells on earth are living in spatially distinct communities, referred to as biofilms, a form in which they behave very differently. In fact, it is now known that 99 % of all bacteria exist in biofilms, with only 1 percent living in the planktonic state. As it has been estimated that 65% of microbial infections are associated with biofilms [4-7], this is now one of the hottest topics in microbiology [8]. Biofilms constituting a microbial multicellular lifestyle and are defined as organized communities of bacteria, collaborating among themselves and being attached to an inert or living surface contained in a self-produced polymeric matrix made principally of exopolysaccharide [2, 9]. This matrix contains polysaccharides, proteins and DNA originating from microbes, and the bacterial consortium can consist of one or more species living in a sociomicrobiological way. Direct structural examination of biofilms has shown that their component micro-colonies, which are composed of cells embedded in matrix material, are bisected by ramifying the water channels that carry the bulk fluid into the community by convective flow [10, 11]. This allows cells to form long-term relationships, interact with each other and establish metabolic cooperation. Biofilms can be composed of a population developed from a single species, or from a community derived of multiple microbial species. Hypotheses about the ecological advantages of forming biofilms include protection from the environment, nutrient availability, metabolic cooperation, and the acquisition of new genetic traits [9, 12, 13].

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Microbial multicellular communities or biofilms come in a great variety of sizes and shapes, with some of the most common types containing mushroom-like, pillar-like, hilly, or flat multicellular structures, which allow cells to form long-term relationships, interact with each other and establish metabolic cooperation [9, 10]. However, many of the underlying processes are interdependent and require cooperation between various bacterial species with different metabolic capacities. Therefore, the fact that, in biofilms, the participating microbial members are situated in close proximity seems to be advantageous, since metabolites can easily be transferred and metabolized further [9, 12-14]. The association of bacteria with a surface and the development of a biofilm can be viewed as a survival mechanism, with bacteria benefiting by acquiring nutrients and protection from biocides. In cases of adverse conditions such as desiccation, osmotic shock, or exposure to toxic compounds, UV radiation, or predators, the microbial community as a whole can provide protection. Moreover, biofilms are also sites where genetic material is easily exchanged because of the proximity of the cells, thus maintaining a large gene pool. Biofilms thrive wherever there is water, such as in the kitchen, on contact lenses and in the gut linings of animals. When the urban sprawl is extensive, biofilms can be seen with the naked eye, coating the inside of water pipes or dangling in slippery and green forms from plumbing [7-15].

2. What are the stages of biofilm development?

The first step is the adhesion of pioneer bacteria, with some of the planktonic or free-floating bacteria approaching the surface (live or alive) and becoming attached to the “boundary layer”, the quiescent zone at the surface where the flow velocity falls to zero. Some of these cells strike and are adsorbed to the surface for only a finite time, before being deadsorbed, in a process called “reversible adsorption” [16]. This initial attachment is based on electrostatic attraction and physical forces, but due to not any chemical attachments. Some of these reversibly adsorbed cells begin to make preparations for a lengthy stay by forming structures which may then permanently bind then to the surface within the next few hours, the pioneer cells proceed to reproduce and the daughter cells, which form microcolonies on the surface and begin to produce a polymer matrix around the microcolonies [9, 17], in an irreversible steps. Subsequently, in the next stage focal areas of the biofilm dissolve and the liberated bacterial cells are then able to spread to other locations where new biofilms can be formed, and the mature biofilm may contain water-filled channels and thereby resemble primitive, multicellular organisms and the attachment is mediated by extracellular polymers that extend outward from the bacterial cell wall. This polymeric material, or glycocalyx, consists of charged and neutral polysaccharides groups that not only facilitate attachment, but also act as an ion-exchange system for trapping and concentrating trace nutrients from the overlying water. The glycocalyx also acts as a protective coating for the attached cells, thereby mitigating the effects of biocides and other toxic substances [18, 19]. As well as trapping nutrient molecules, the glycocalyx net also snares other types of microbial cells through physical restraint and electrostatic interaction. According to Borenstein (1994), other bacteria and fungi can become associated with the surface following colonization by the pioneering species over a matter of days [20]. In a mature biofilm, more volume is occupied by the loosely organized glycocalyx matrix (75-95%) than by bacterial cells (5-25%) [9-11]. In most cases, the base of the biofilm is a bed of dense, with thickness up 5 to 50 m, composed of a sticky mix of polysaccharides, other polymeric substances and water, all produced by the bacteria. Soaring 100 to 200 m upwards are colonies of bacteria, shaped like mushrooms or cones. According to Mittelman (1985), the development of a mature biofilm may take from several hours to several weeks, depending on the system [19]. Biofilms are permeated at all levels by a network of channels through which water, bacterial garbage, nutrients, enzymes, metabolites and oxygen move to and fro, with gradients of chemicals and ions between microzones providing the power to shunt the substances around the biofilms [15]. Oxygen may be depleted within only 30-40 m of the water/biofilm interface. Although the precise depth of the oxygen gradient in the biofilm varies depending on the oxygen content in the bulk water, water temperature, and water flow, this gives a rough idea of how far oxygen can diffuse [3].

3. Why is it important to study biofilms?

Biofilms in nature are generally beneficial and are frequently established on hydrous solid and semi-solid surfaces, such as soil, rock material, or the surfaces of animals and plants. Microbial communities natively populate human mucous membranes and epithelial surfaces, for example, the gastrointestinal tract, oral cavity, and skin. Despite our bodies being colonized with a mixed microbial community of characteristic compositions and they are important and beneficial to us as they can degrade nutrients and thereby make these accessible [21, 22]. In addition, they can synthesize some vitamins which we are unable to synthesize on our own. In particular, these communities play key roles in the development of our immune systems and in the anatomy of the mucosal surfaces, and also provide protective functions against exogenous pathogens [4, 5]. The relationship between the host and its microbial communities is delicately balanced, but under certain conditions it can break down and result in infectious diseases. According to a recent public announcement from the National Institutes of Health, more than 60% of all microbial infections are caused by biofilms. Although the planktonic form of

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the bacteria has been very useful in understanding acute infections, chronic ones are more related to the presence of biofilms, with current research indicating an important role for bacterial biofilms in recurrent or chronic infection, including those which are not responsive to a culture-appropriate antibiotic therapy [23]. Biofilm-growing bacteria cause chronic infections, including foreign-body infections, that are characterized by persistent inflammation and tissue damage despite antibiotic therapy and the innate and adaptive immune and inflammatory responses of the host and persisting pathology. Some general features of biofilm infections in humans compared with acute planktonic infections are shown below [24]:

1. Aggregates of bacteria embedded in a self-produced polymer matrix 2. Tolerant to both innate and adaptive immune responses 3. Tolerant to clinically dosing of antibiotics despite susceptibility of planktonic cells 4. Chronic infections

The surface-associated microorganisms are responsible for a several chronic infections as: periodontitis [25], heart valves (endocarditis) [26], in lung infection in patients with cystic fibrosis (CF) causing chronic bronchopneumonia by Pseudomonas aeruginosa [27], child middle-ear infections (caused by Haemophilus influenzae, for example) [28], in chronic rhinosinusitis [29], in chronic osteomyelitis and infections caused by a variety of surgical implants [30], wound infection in burn patients [6], urinary tract infections (caused by Escherichia coli and other pathogens) [31], in intravenous catheters and stents (caused by Staphylococcus aureus and other gram-positive pathogens) [32], among others [4, 9]. To date, the link between the concept of biofilms and chronic infectious disease is still the subject of a lot of many studies.

4. How do biofilms provide protection from antibiotics?

In the traditional antibiotic resistance of planktonic bacteria, usually involves inactivation of the antibiotic, modification of targets, and exclusion of the antibiotic [33]. These actions typically require the acquisition of specific genetic factors, such as genes for -lactamase or efflux pumps. One of the most important aspects of bacterial biofilm formation is the increased resistance of the constituent microbes to antibiotics and other stressors. The structural nature of the biofilms and the characteristics of the sessile cells, produce resistance towards the antimicrobial agents, leading to a protected environment against adverse conditions and the host´s defenses [23, 33-35]. Free living bacteria, on the other hand, are generally susceptible to antibiotic treatment and to host defense mechanisms. However, the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of antibiotics to biofilm-growing bacteria being up to 100–1000 fold higher than for planktonic bacteria, and possibly 150-3000 times more resistant to disinfectant [6, 33]. Despite decades of research, very little is known about the molecular mechanisms of antibiotic resistance in biofilms. Although several theories have been proposed, the precise mechanism of how this sensitivity is altered has still not been clarified. Nevertheless, it is possible to separate these mechanisms into intrinsic (or innate) and extrinsic (or induced) resistance factors to biofilms [36].

4.1 Intrinsic or innate resistance factors to biofilms

The intrinsic factors of resistance are activated as part of the biofilm developmental pathway, which are integral parts of the biofilm structure and physiology resulting from conversion to a biofilm lifestyle. The influences of several different intrinsic biofilm factors affecting antibiotic resistance have been identified. For example: the biofilm matrix may act as a diffusion barrier; microenvironments within biofilms can be established; small subpopulations of bacteria within the biofilms may differentiate into persisters; an increased production of oxidative stress causes might changes in the physiology of bacteria; and an antagonist of antibiotics and degradation mechanisms may be active in some parts of the biofilms [4, 6, 24, 33, 36-40].

4.1.1 The biofilm matrix may act as a diffusion barrier

Biofilms can act as physical diffusion barriers to prevent antibiotics from reaching their targets. Antibiotic was shown to be able to penetrate these structures through a thick mixture of exopolysaccharide, DNA, and protein in order to reach the targets [36, 37]. However, this mixture was not able to achieve an effective concentration in some all parts due to the physical and/or chemical properties of the matrix, which resulted in an apparent increase in resistance. Indeed, recent mathematical modeling has predicted that while limited antibiotic diffusion may lead to the death of the outer layer of bacteria, it also stimulates subpopulations of bacteria buried deeper within the biofilm to enact adaptive changes, thereby countering the insult [41]. However, limited antibiotic diffusion does not appear to be a universal trait shared by all biofilms, and, there is conflicting data about whether the biofilm matrix is a major contributing factor influencing biofilm resistance [33]. A decreased penetration and diffusion of antimicrobials through the biofilm matrix has been shown to influence biofilm survival in some cases. For example, at sub-MIC concentrations of -lactam

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antibiotics, an increased alginate synthesis in P. aeruginosa biofilms was induced and also an enhancement of the biofilm matrix of some slime-producing coagulase-negative staphylococci. Moreover, although it was originally it was thought that tolerance of biofilms to aminoglycoside was the result of transport limitation due to the binding of these positively charged antibiotics to the negatively charged exopolysaccharide matrix, repeated dosing of antibiotics during therapy probably leads to saturation of the binding sites [42-44].

4.1.2 Establishment of microenvironments within biofilms

The depletion of nutrients and oxygen inside biofilms might cause an altered metabolic activity and lead to slow growth of the bacteria. Several studies have revealed oxygen limitation and the presence of hypoxic zones deep within biofilms, with nutrient diffusion through biofilms being restricted [33, 38]. Inspection of environmental as well as in vitro biofilms has revealed that the oxygen concentration may be high at the surface, but low in the centre of the biofilm where anaerobic conditions may be present. Likewise, growth, protein synthesis and metabolic activity are stratified in biofilms, for example, there is a high level of activity at the surface but a low level in the centre, resulting in slow or no growth. This fact is one of the explanations put forward for the reduced susceptibility of biofilms to antibiotics [3, 27, 45]. In general, all antimicrobials are more effective in killing rapidly growing cells. For example, penicillin and ampicillin have an absolute requirement for cell growth to occur in order to kill, be able to, with the mortality rate being proportional to the rate of growth. Also, although other advanced -lactams such as cephalosporins, aminoglycosides, and fluoroquinolones can kill nongrowing cells, they are distinctly more effective in killing rapidly dividing cells. Therefore, slow growth undoubtedly contributes to biofilm resistance to the killing of bacteria [4].In addition, slow growth is a major factor in the increased resistance of stationary planktonic cells to being killed. Related to this, the existence of microenvironments that antagonize the action of antibiotics (pH variations) and the degradation mechanisms active in some parts of the biofilms could also play a role [24, 33].

4.1.3 Differentiation into persister cells

Persister cells are considered to be either nongrowing or slow-growing, and also have a greatly reduced susceptibility to antibiotics [46]. In the persister’s theory, these small subpopulations of bacteria within the biofilms differentiate into dormant cells, are able to survive extreme antibiotic treatment and have been hypothesized to be the result of phenotypic variations rather than due to stable genetic changes [45]. Indeed, the formation of persisters might represent a common mechanism used by a wide range of bacteria during biofilm formation. This persistent population within biofilms may drastically inhibit the complete eradication of the biofilms, even after prolonged, high-level antibiotic treatment. However, it is unclear what relationship exists between planktonic persisters and biofilm resistance, and the mechanisms(s) by which persisters form and/or confer increased antibiotic tolerance are still unknown.

4.1.4 Increased production of oxidative stress

Oxidative stress is caused by an imbalance between the production of oxidants, such as the free radicals, peroxide and nitric oxide, with the levels of antioxidant defenses. A disturbance in the prooxidant-antioxidant balance in favor of the overproduction of reactive oxygen species (ROS) can result in damage to the cellular components, including the matix, DNA, proteins and lipids [47-49]. Diverse stresses, including nutrient availability, low oxygen, high osmolarity, ethanol

and sub-inhibitory antibiotic concentrations, can alter the cellular functions associated with the oxidative metabolism [49], thereby stimulating the production of ROS and the highly reactive hydroxyl radicals (HO.), which are generated by the presence of hydrogen peroxide (H2O2) and iron (Fenton reaction) either by the superoxide anion (O2

-) or by the superoxide anion, hydrogen peroxide and a metal catalyst (Haber–Weiss reaction) [50, 51]. Another form of stress is termed nitrosative stress, where reactive nitrogen intermediates (RNI), such as nitrate (NO3-) and nitrite (NO2-) are used as terminal electron acceptors under anaerobic conditions. Nitric oxide (NO) is a short-lived free radical produced enzymatically by nitric oxide synthesis in various types of cells, and has a great diffusion across membranes. It plays a key role in a variety of biological processes; for example, as an important effector of host innate immunity, where it acts as an antimicrobial compound and signaling molecule. Moreover, when NO is produced by microorganisms as a physiological response to medium oxidants, the effect of NO is also beneficial, with NO having been also observed to be able to immediately protect bacteria from ROS, thus playing a critical role in the adaptation to oxidative stress associated with rapid metabolic changes [52, 53]. Nevertheless, ROS and RNI having been extensively studied in planktonic bacterial physiology, the precise role of cellular stress in biofilm development is still unclear. In the antioxidant defense system, the main enzymes involved in the detoxification of ROS are superoxide dismutase (SOD) and catalase (CAT), among others [47]. However, in oxidative imbalance with due to an overproduction of ROS, a reduction in the oxidative defenses is insufficient to remove the free radicals, and therefore, the antioxidant system plays a very important role in the control of this process [54] (Equation 1).

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The increased production of oxidative stress causes changes in the physiology of bacteria, with specific phenotypic alterations occurring , and we have observed that biofilm development is influenced by the balance between the production of oxidants (ROS and NO) and the levels of antioxidant defenses (SOD), which can be significantly affected by different environmental stresses. Our results suggest that when this balance is broken, under unfavorable conditions, an increase in the ROS production or a reduction in the antioxidant defenses (or both) produces an overproduction of cellular stress, resulting in a reduction of the extracellular matrix of the biofilms [49].

(1)

Oxidative stress is considered to cause enhanced mutability in biofilms. Recent data suggest that microcolonies structures, due to endogenous oxidative stress, are specific sites within biofilms where enhanced genetic adaptation and evolutionary change take place [55, 56]. In addition, Boles and Singh showed that endogenous oxidative stress in biofilms promotes antibiotic resistance and that the addition of antioxidants reduces the diversity in biofilms [57].

4.1.5 Antagonist action of antibiotics and degradation mechanisms active in some parts of biofilms

Microenvironments exits that can antagonize the action of antibiotics, and the degradation mechanisms active in some parts of biofilms may also be involved. Sociomicrobiology is defined as the relation existing between quorum sensing (QS) and biofilms. Bacteria communicate by means of synthesizing and reacting on signal molecules in order to sense when a concentration of bacteria is present in a limited space in the environment, and then respond by activating certain genes that produce, for example, virulence factors such as enzymes or toxins. The most well described QS molecules in Gram-negative bacteria are the N-acyl-l-homoserine lactones, and in many Gram-positive bacteria the QS molecules are small peptides. QS can regulate the production of virulence factors such as extracellular enzymes and cellular lysins, which are important for the pathogenesis of infections, where the bacteria functions as a protective shield against phagocytes [58-61]. QS may also influence the development of the biofilm and determine the tolerance of biofilms to antibiotic therapy and to the innate inflammatory response [62].

4.2 Extrinsic or induced resistance factors to biofilms

Other groups resulting from transcriptional induction by treatment with antibiotics agent itself, is extrinsic or induced resistance factors. The mutation frequency of biofilm-growing bacteria is significantly an increased compared with planktonically growing isogenic bacteria and there is an increased horizontal gene transmission in biofilms. These physiological conditions may explain why biofilm-growing bacteria easily become multidrug resistant by means of traditional resistance mechanisms against -lactam antibiotics, aminoglycosides and fluoroquinolones, which can be detected by routine susceptibility testing in the clinical microbiology laboratory when planktonic bacterial growth is investigated. Thus, bacterial cells in biofilms may simultaneously produce enzymes that degrade antibiotics, have antibiotic targets of low affinity and over express efflux pumps that have a broad range of substrates. The source of -lactamase in biofilms has been considered to come from the layer of lysed bacteria resulting from exposure to antibiotic, and is related to the release of defensive enzymes into the extracellular space. Imipenem and piperacillin were able to induce overproduction of -lactamase in P. aeruginosa biofilms, which hydrolysed the -lactam antibiotics before reaching the bacterial cells. Bacteria expressing a high level of chromosomal -lactamase in biofilms could be exposed to a reduced concentration of -lactam antibiotics, owing to the accumulation of the enzyme in the polysaccharide matrix. Also, the extracellular -lactamase may inactivate the antibiotic as it penetrates, thereby protecting the deeper-lying cells. In stratified biofilms, the surface sessile cells of biofilms can be killed by high antibiotic concentrations, and a have limited time to adapt. However, slowed antibiotic diffusion through the

Antioxidant defenses

RNI

ROS

CAT H2O + ½ O2

NO

O2

ONOO-

H2O2

SOD

O2-

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microcolonies, due to matrix inhibition to the other factors explained, might lead to the establishment of an antimicrobial gradient that may produce differential gene expression of antibiotic induced factors throughout the biofilm [4, 23, 22]. Antibiotic treatment can result in an increased production of oxidative stress within a biofilm. Consequently, one would expect that there must be some antibiotic-regulated genes that can influence antibiotic resistance or sensitivity of biofilm bacteria, and it is likely that numerous genetic loci are activated upon treatment with antibiotics. These induced factors may work synergistically with intrinsic factors to enhance survival in the face of strong antimicrobial stresses.

5. Which methods can be used to study biofilms?

A single standard method for the study of biofilm susceptibility is not available, several methods are available; each with its own advantages and disadvantages. Thus, it is very difficult, to compare the results obtained with biofilms of even the same species, when cultured and assayed under vastly different conditions. Determination of the MIC, based on antimicrobial activity against planktonic organisms, is the standard assay for susceptibility testing and serves as an important reference in the treatment of many acute infections. However, the application of MICs in the treatment of chronic or device-related infections involving bacterial biofilms is often ineffective [6, 23, 33] and several different techniques have been used to study biofilm populations [63, 64]. A popular method used to study biofilms being the Robbins device, which is based on passing a bacterial suspension through a flow cell that has 24 detachable coupons on which cells can adhere and grow into biofilms. A modified version of the Robbin’s device (MRD) has also provided important information regarding biofilm physiology and antibiotic susceptibility [65]. Morck et al. [66] demonstrated an important correlation between the antibiotic susceptibilities of biofilms in vitro using the MRD and the efficacy of antibiotic treatment in vivo. While the MRD has proven to be an effective model of biofilm formation, it is not suited for rapid antibiotic susceptibility testing in a clinical laboratory setting once the biofilm is formed the feeding liquid is switched to a culture medium that contains test compounds. Then, after a period of incubation, the device is taken apart and the cells are dislodged by sonication and plated. This method enables reproducible biofilm formation and the observation of biofilm dynamics, and the coupons can also be used for microscopic studies the biofilm structure. The strengths of this approach derive from the well-controlled conditions that emulate in vivo biofilm formation and from the possibility to be able formed biofilm by a variety of techniques. However, this method is ill suited for susceptibility studies, which require hundreds and often thousands of samples to be examined. A microtiter plate-based method has also been utilized for the study of biofilm. Wells of microtiter plates are inoculated with a bacterial suspension, and biofilms then form on the surface of these wells. After 24- to 48-h incubation, the planktonic cells are removed by rinsing the wells. A solution of crystal violet is then added to the wells to stain the cells. The wells are then rinsed, and the bound dye is extracted with acetone-ethanol and quantified spectrophotometrically. This provides a quantitative measure of the mass of biofilm cells, and it could be very useful to adapt this simple method to be able to perform antimicrobial susceptibility measurements [67]. A recently developed, high-throughput approach to the antibiotic and biocide susceptibility testing of microbial biofilms is the Calgary Biofilm Device (CBD) [68, 69]. This batch culture method of biofilm and planktonic cell susceptibility testing yields the following three internally consistent, comparative measurements from a single experiment: the planktonic MIC, the planktonic MBC, and the minimum biofilm eradication concentration (MBEC). The device looks like a 96- prong replicator with plastic pins, and is inserted into a grooved tray filled with growth medium inoculated with cells. The apparatus is then placed on a tilting shaker platform, and the growing cell suspension washes the pins on which the biofilms grow. Importantly, any cell or cell mass that is not clinging strongly to the pin is washed away. As a result a robust biofilm can be formed that is able to be rinsed without losing its integrity. Once the biofilm is formed, the lid with pins can be placed into a microtiter plate for susceptibility testing. Then, after a period of incubation with antibiotics, the cells can be dislodged from the pins by mild sonication and plated for determination of colony counts. Although the round pins do not make it easy to perform microscopic observations of the biofilms, one can envisage a simple modification in which the pins are made flat and thin with a perforated thin base for easy detachment. It is possible to use different mean numbers of bacterial cells and compare susceptibility data of different strains, alternate exposure times and diverse growth media formulations. Biofilms on the pegs of the MBEC device may be examined in situ using scanning electron microscopy, epifluorescent microscopy, and confocal laser-scanning microscopy [70]. The reproducibility of the results for biofilms formed on each of the pegs of the CBD demonstrates the equivalence of the biofilms formed at each of the sites for susceptibility testing. The CBD requires no pumps or tubing, making the process much simpler to set up than the MRD, and eliminates a major source of possible contamination. The availability of multiple testing sites greatly reduces the time required to determine the antibiotic susceptibilities of biofilms, from weeks with the MRD to 3 days with the CBD [68-70]. The antibiotic susceptibilities of planktonic populations, as determined by MIC methodologies, are not necessarily applicable to the effective treatment of the same organism once a biofilm has been established. One problem faced in selecting an alternative antibiotic treatment has been the lack of an easy, reproducible assay which could provide a measure of the antibiotic activity against a biofilm. In the future, the CBD or other a new technology that can be applied

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to recalcitrant, recurrent, or device-related infections caused by organisms for which MICs have not provided clinically relevant information [70].

6. Conclusions

During their evolution, bacteria have been able to develop successful strategies for survival, which include attachment to surfaces and the development of protective biofilms where bacteria behave very differently to the free-floating types. These successful strategies make it difficult to control biofilm growth, with a biofilm providing bacteria with a 10- to 1,000-fold increase in antibiotic resistance compared to free ones. Some mechanisms of resistance appear to be intrinsic to growth in a biofilm, with the inhibited diffusion through the matrix, reduced metabolism by nutrient limitation, formation of dormant persisters, and increased production of oxidative stress all appearing to impact on the development of a protective environment within the biofilm; in addition to extrinsic factors such as -induced gene expression by antibiotics in biofilm cells. Due to the heterogeneous nature of biofilms, it is likely that multiple mechanisms of antimicrobial resistance are useful in order to explain biofilm survival in a number of cases, with antibiotic resistance being the result of an intricate mixture of intrinsic and extrinsic factors. However, additional mechanisms must also exist to be able account for increased biofilm antibiotic resistance. Although methods to test biofilm-growing bacteria have already been developed, their clinical relevance with regard to the prediction of clinically successful therapies still awaits confirmation. It is clear that much more research is needed to reveal additional and/or novel antibiotic-induced factors in biofilms, as the multifactorial nature of biofilm antibiotic resistance has hindered identification of these pathways, with much still being unknown about the induced factors in biofilm resistance. Discovery of these factors should lead to new and better treatments for biofilm related infections. New strategies are required to overcome extreme biofilm antibiotic resistance by through the development of novel therapies aimed at disrupting biofilms and killing the constituent bacteria, with manipulation of intrinsic and extrinsic resistance pathways providing much promise for future treatment of biofilm infections.

Acknowledgments: The assistance of Inés Albesa, Julio Arce Miranda, Natalia Angel Villegas and Soledad Ravetti is gratefully acknowledged. The author thanks Dr. P. Hobson (native speaker) for revision of this manuscript. This work was supported by the following Grants: Secretaría de Ciencia y Tecnología, FONCyT, CONICET and Ministerio de Ciencia y Tecnología de la Pcia de Córdoba. Dr. María Gabriela Paraje is a member of the Research Career of CONICET.

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