Antibiotics Resistance of Bacterial Biofilms





Amna Butt
Aisha Khan


Fatima Jinnah Women University
Rawalpindi
Pakistan


Corresponding author:
Aisha Khan
Fatima Jinnah Women University,
the mall,
Rawalpindi
Pakistan
Email: aishahayat85@yahoo.com

Abstract


Biofilms are microorganism communities that get attached to a surface. It is evident through various research that cells grown in a biofilm express distinct properties from that of planktonic cells. One such property which is of high significance is an increased resistance to antimicrobial/ antibiotic agents. Bacterial biofilms are a cause of chronic infections worldwide because of their increased antibiotic tolerance. Their resistance to the antibiotics is due to mutation, resistant phenotypes, adaptations to stress, quorum sensing, stratified activity, nutrient gradients, oxidative stress, failure of antibiotic penetration and heterogeneity. Thus in order to control the bacterial infections caused by these biofilms, there is a need of novel drug delivery approaches and enhanced therapeutic use of quorum sensing inhibitors.

Key words: Biofilms, antibiotic resistance, quorum sensing, quorum sensing inhibitors

1. Introduction
A biofilm is a complex structure that adheres to surfaces regularly in contact with water. Microorganisms secreting mucilaginous protective coating in which they are encased usually form biofilms. Generally colonies of bacteria and other microorganisms such as fungi, yeasts and protozoa form biofilms. Biofilms generally form on liquid or solid surfaces in addition to soft tissues in living organisms. Thus they show aspects of both liquids and solids (much similar to slug slime) and come under a category named "viscoelastic". Conversely, as biofilms become scaled with calcium deposits or rust or collect sediment they become more like a brittle solid and less fluid. Typically biofilms show considerable resistance to conventional disinfection methods. Examples of biofilms include dental plaque, algal mats on water bodies and the slimy coating that fouls pipes and tanks. Van Leeuwenhoek was the first to observe microorganisms on tooth surfaces by making use of his simple microscopes and thus was the one who made the discovery of microbial biofilms. Over the years several other scientists studied biofilms including H. Heukelekian and A. Heller in 1940 and C. E. Zobell in 1943 however detailed assessment of biofilms had to await the development of the electron microscope, which permitted high-resolution photo-microscopy at magnifications that were much higher than that of light microscope (Donlan, 2002).

Biofilms are extremely heterogeneous in nature. Several observations and measurements on various biofilms have been made; all of them point to the diversity found in individual biofilm colonies. Typically, naturally occurring biofilms almost always have a quantitatively large number of different kinds of organisms living in colonial form. Additionally, different biofilms seemingly show different electrical properties, chemical properties, internal structures, and, definitely, different properties of pretty nearly any other observation that can be made. These properties contribute to differential characteristics of the biofilm (e.g., hard to kill) compared to individual microorganisms in isolation (in a planktonic environment) (Cunningham, Lennox and Ross, 2008).

1.1. Biofilms Formation Process
Generally development of a biofilm occurs in different chronological phases, which are illustrated in Figure 1 below:

Figure 1. Major steps involved in biofilm formation (Source: Harrison, 2007)


1.1.1. Surface conditioning
Surfaces on which biofilms are attached are first conditioned by adsorption of inorganic and organic nutrients. This adsorption is required to influence the subsequent bacterial attachment. Surface conditioning can increase tolerance to antibiotics for example; Pseudomonas aeruginosa biofilms developed better tolerance to tobramycin (antibiotic agent) and large cellular aggregates when attached to surfaces conditioned with the glycoprotein mucin and allowed to grow there.

1.1.2. Reversible attachment
The preliminary transport of bacterial cells and their reversible attachment to a surface usually occurs by brownian motion of the cells, active movement of motile bacteria, physical and electrostatic interactions between the cell surface, convection currents within a bulk liquid responsible for transporting bacteria to the surface, substratum and sedimentation. This state of bacterial adhesion to surface may result in symmetrical distribution between suspended and adhering cells, and is considered as the weakest link in the series of events that connect the bacterial cells to the surface conditioned beforehand (Landry et al. 2006).

1.1.3. Irreversible attachment
Because of stimulation of membrane bound sensory proteins, the cells which were attached reversibly to surface now produce extracellular polymeric substances which allows the cell-to-cell bridges development that attach the cells to the surface irreversibly.

1.1.4. Colonization
Surface colonization is the final phase of biofilm formation. Attached bacteria form micro-colonies by growing and dividing. These micro-colonies are considered as the fundamental units of organization of a biofilm. Other planktonic cells also get entrapped in the extracellular polymeric substances, resulting in the establishment of a biofilm. Primary colonizers i.e. one bacterium that colonizes a surface, often influence the secondary colonizers i.e. attachment of others to the same surface.

1.1.5. Detachment
Initially the researchers suggested that the detachment of clumps of biofilm cells and subsequent transfer and attachment to other surfaces might be due to turbulent shear forces. Such a mechanism of detachment can only be accurate for biofilms which grow under laminar shear forces and are seemingly detached due to turbulent shear forces. Nevertheless, recent studies have suggested that detachment, frequently termed as 'dissolution' or 'dispersion', is an active and highly regulated process controlled by the attached cell populations (Lindsey and Holy, 2006). (Figure 1)

1.2. Properties of Biofilms
Some apparent characteristics common to almost all biofilms being observed are as follows:
• Biofilms are responsive to their environment and are dynamic in nature; that is, they are very adaptive to environmental changes.
• Detachment phenomenon is common among all biofilms. Through this phenomenon individual or clumps cells of bacteria become able to detach themselves from the biofilm colony.
• Detached individual microorganisms are comparatively easy to kill in isolated form with specifically designed chemicals for this purpose (antibiotics).
• The detached microorganisms of the biofilm are the cells in clump form that are just not attached to the biofilm at that time. However, these clumps are able to maintain the original properties of the parental biofilm and thus are a lot harder to kill.
• In favorable conditions, biofilms can migrate from surface to surface in a variety of ways over a period of time. This migration may be via streaming, rippling, detaching, seeding dispersal and rolling as illustrated below in Figure 2.

Figure 2. Biofilm migration (Source: Cunningham, Lennox, and Ross, 2008)

• Another main characteristic of the cells that are found in a biofilm is their ability to communicate with one another. Bacterial communities communicate with each other using different chemical signals. These chemicals are produced and passed by outer membranes of these cells and can be interpreted by members of the same cell species as well as other microbial species present in the same biofilm community.
• These chemical signals are sensed by adjacent cells in the biofilm and can induce different behavior of neighboring cells due to occurring of different genetic expression in those cells (Figure 3).

Figure 3: Communication mechanism within a Biofilm community. (Source: Cunningham, Lennox, and Ross, 2008)


• In Figure 3, different colors represent different bacterial species. Bacteria can "talk" to others and they "listen" or respond to the chemical signals produced by the other. This type of interaction produces behavioral changes because in biofilms the population is numerous enough to initiate genetic activity. Although the same signals are produced in planktonic populations they are not concentrated enough to cause genetic expression change while passing through water.
• There is a population recognition system present in biofilm which is termed as "quorum sensing". For example, bacterial pathogens only produce toxins when they sense that a sufficient population is present to survive host defenses (e.g., antibodies). Vibrio fischeri, a marine bacterium was the first one in which this phenomenon was observed which produced light after ensuring that an adequate population of this bacterium has been developed in biofilm.
• The coordinated behavior of biofilm is responsible for the survival strategies against host immune system and antimicrobial agents.

2. Bacterial Biofilms
Formation of bacterial biofilms occurs when unicellular organisms join each other to shape a community which attaches itself to a solid surface and sheathed in an exopolysaccharide matrix. Single or multiple bacterial species can make up biofilms. For instance, the estimations reveal that dental biofilms contain more than 500 different bacterial taxa; on the contrary, the primary bacterium found in latter stages of cystic fibrosis (CF) patients' lung is Pseudomonas aeruginosa. The bacteria of the same kind act or behave differently (or show different properties) when they are in a biofilm in comparison to their isolated or planktonic form (that is, freely floating bacteria as single cells in water). Some of the very important characteristics of bacteria growing in biofilm are dissimilar to the planktonic bacteria and this has noteworthy therapeutic and diagnostic consequences. This difference is due to the location of bacteria in biofilm infections in aggregates surrounded by the self-produced matrix. Biofilms can be recognized in clinical specimens (biopsies, septum, pus) by making use of light microscopy, though all the bacteria within a biofilm cannot be identified precisely. Their identification requires specialized staining techniques. Additionally the bacteria growing in a biofilm cannot be cultured by traditional sampling techniques unless they are released by ultrasonic pre-treatment (Moskowitz et al. 2004; Bjarnsholt et al. 2007; Hoiby et al. 2010).

It has been observed that the resistance of bacteria to an antibiotic is also amplified in comparison to what is seen usually with planktonic cells. In effect, when these cells exist in a biofilm, they utilize 10-1000 times more resistance to the effects of antibiotic agents (Evan and Holmes, 1987; Mah and O'Toole, 2001). These antimicrobial drugs are traditionally developed to kill planktonic bacteria (free bacteria) by assuming that they would destroy the same bacterial species irrespective of wherever or in what form they were found. However studies reveal that:

1. Planktonic bacteria are more prone to antibiotic chemicals intended to destroy them than are the bacteria present in biofilms, and
2. Numerous infections that effect humans are in fact caused by bacterial colonies present in the biofilm state, not the bacteria in planktonic state.
With this information along with the fact that traditional antibiotics have been tested and designed for bacterial population in their planktonic growth mode which is relatively unprotected, we can begin to understand the basic reason behind the antibiotics resistance of same bacteria when they exist in a biofilm (Cunningham, Lennox, and Ross, 2008).

3. Antibiotic Tolerance/Resistance Of Bacterial Biofilms
Since the discovery of penicillin in 1938, there has been a tremendous success in control of acute bacterial infections by the help of antibiotics. Microbiologists have predicted in vivo effects of antibiotic effects in vitro evaluation of the minimal bactericidal concentration (MBC) and minimal inhibitory concentration (MIC). MBC and MIC help in assessing the effect of antibiotics against planktonic bacteria in the exponential phase of growth and consequently predict effectiveness of antibiotic against rapidly dividing bacteria in acute infections quite correctly. However, biofilms show a resistance against these antibiotics and thus are of major importance from a clinical point of view as more than 60% of the infections due to bacteria, currently treated in the developed world by physicians involve the bacteria responsible for biofilm formation, some of which are given in Table 1. This increased tolerance of bacterial biofilms to disinfectant chemicals and antibiotics as well as resistance to phagocytosis and other mechanisms of the defensce system are a cause of many chronic infections. For instance, persistence of staphylococcal infections related to foreign bodies is due to formation of biofilm. Similarly, chronic lung infection due to Pseudomonas aeruginosa in CF patients is caused by biofilm-growing mucoid strains. (Table 1)

Table 1. Human infections involving biofilms

(Source: Fux et al. 2005)

Biofilms have a triple role to play in the spread of antibiotic resistance such as:
• The treatment of infections caused by biofilms requires long-term and recurrent therapy with antibiotics.
• Physiology of biofilm enables bacteria embedded in them to survive long term exposure of antibiotic to acquire specific resistance to the particular antibiotic agent.
• The accumulated mobile genetic elements within biofilms, high cell density and increased genetic competence provide an ideal condition for efficient horizontal gene transfer (Fux et al. 2005).

The major factors that are involved in rendering antibiotic resistance to bacterial biofilms are explained in Figure 4 given below. (Figure 4)

Figure 4. Antibiotic resistance of bacterial biofilms (Source: Stewart and Costerton, 2001)


3.1. Mutation
The frequency of mutations in bacteria growing in biofilms is significantly higher in comparison to bacteria growing in planktonic environment and additionally the horizontal gene transmission is also found to be higher in biofilms. These physiological conditions explain the reason behind multidrug resistance of bacterial biofilms. These bacteria easily become resistant to aminoglycosides, fluoroquinolones and lactam antibiotics. Bacterial biofilms may simultaneously produce antibiotic degradation enzymes. It has been shown experimentally that mutations in bacterial biofilms establish the surfacing of antibiotic resistance, especially due to expression of multidrug efflux pumps. The cause of enhanced mutability is considered to be a result of oxidative stress to which the bacterial biofilms are subjected. Researchers show that due to oxidative stress within the biofilms, there are specific sites within them for superior genetic adaptation and evolutionary change. Thus the hypermutability of bacterial biofilms promotes the antibiotic resistance to biofilms conferred by mutations (Driffield et al 2008; Conibear et al. 2009).

3.2. Presence of Resistant Phenotype
There is a biofilm-specific phenotype which is resistant to antibiotics. This phenotype is present within a subpopulation of the biofilm community which results in the expression of active mechanisms for making the biofilm antimicrobial resistant. Generally when the biofilm attaches to a surface their expression is a general phenotype representative of that biofilm and the subpopulation genes are repressed. However, it is a possibility that just a subset of these biofilm cells could express genes for augmented resistance to antibiotics (Mah and O'Toole, 2001; Fux et al. 2005).
3.2.1. Induction of a Biofilm Phenotype
The resistant phenotype is maybe induced by certain types of environmental stress, nutrient limitation, high cell density or an amalgamation of all of these happenings. Another mechanism for inducing antibiotic resistance in biofilm cells is the membrane-protein composition alteration in response to exposure of antibiotic agents. The resultant change could decrease the permeability of the cell to these substances (Mah and O'Toole, 2001; Fux et al. 2005).

3.3. Bacterial Adaptation to Stress and Damage
Another reason for antibiotic resistance of bacterial biofilms is the ability of bacteria to adapt itself to stress conditions. For instance, organisms present in a biofilm increase their capacity to neutralize and withstand monochloramine, induce the expression of chromosomal betalactamases or stimulate catalase production when subjected to prolonged and concurrent subjection to or treatment with the respective antibiotic compounds. Bacterial biofilms can switch themselves to more tolerant phenotypes upon facing prevalent environmental stresses such as temperature alterations, alterations in osmolarity, pH, cell density and nutritional quality by turning on stress-response genes. Stress-response genes produced by the bacterial biofilms can not only antagonize the deleterious effects of antibiotics but also that of the environmental toxins and immune system on bacteria. These genes are regulated by the interacting signals, i.e. quorum sensing (Boaretti 2003).

3.4. Quorum Sensing and High Cell Density
As mentioned earlier, bacteria in a biofilm communicate with others present in the same biofilm by synthesizing and reacting on some chemical signals. This phenomenon is referred to as Quorum Sensing (QS) and it allows bacteria to sense when a sufficient amount (or concentration) of bacterial cells are in attendance in a confined space of biofilm environment and thus act in response to it by certain gene activation that results in production of virulence factors such as toxins or enzymes. This relationship between biofilms and QS is termed as sociomicrobiology. The QS molecules (signals) are peptides of small size in many Gram-positive bacteria, while N-acyl-l-homoserine lactones present in Gram-negative bacteria are the most well described QS molecules (Jensen et al. 2007). An example of QS's role in designating antibiotic resistance to biofilms is that for Pseudomonas aeruginosa; QS is responsible for regulating the virulence factors production such as cellular lysins and extracellular enzyme, which are important for the pathogenesis of infections by functioning as a protective shield against phagocytes. In addition QS has also been shown to determine the resistance or tolerance of biofilms of Pseudomonas aeruginosa to antibiotic therapy and to the innate inflammatory response (Gennip et al. 2009; Alhede et al. 2009).

3.5. Stratified Activity and Low Oxygen Concentration
Examination of environmental and in-vitro biofilms exposed that the concentration of oxygen and other nutrients might be high at the surface but moving towards the centre of the biofilm anaerobic conditions might exist and nutrient concentration may decrease thus forming gradients on nutrients and oxygen. This gradient formation is related to the decreased metabolic activity of bacteria and thus increases doubling time. It is this somewhat dormant state that is responsible for some of antibiotic tolerance of biofilms. This could be explained by the fact that planktonic organisms, though they utilize nutrients, do not have adequate metabolic activity required for their depletion. Thus when they are present in biofilm, in form of a group, their collective metabolic activity reduces further and leads to formation of concentration gradients of substrates and localized chemical microenvironments leading to stratified protein synthesis, growth and metabolic activity in biofilms, i.e. a high level activity at the surface of biofilm while a low level activity along with slow or no-growth persistent in the center of biofilm. Thus stratified activity along with low concentration of oxygen and reduced growth rate may result in less liability of biofilms to antibiotics or antimicrobials (Werner et al. 2004; Hoiby et al. 2010).

3.6. Failure of Penetration of Antibiotic in the Biofilm
The glycocalyx or exopolysaccharide matrix production is one of the distinctive traits of biofilms. This matrix, among performing other functions, prevents antibiotics' access to the bacterial cells entrenched in the community. Usually, it is either compound's sorption to or its reaction with the components of the biofilm, which limits the transportation of antimicrobial agents into the cells embedded in biofilm. Although it is suggested by various mathematical models that there should be no barrier to their diffusion into a biofilm for the antibiotics, some researchers have revealed a noticeable failure of penetration of certain antibiotic agents to the biofilm. For instance, as measured by a chlorine-detecting microelectrode, Chlorine which is a common disinfectant, failed to reach 20% of the concentration of bulk media within a mixed Klebsiella pneumoniae and Pseudomonas aeruginosa biofilm. Indeed, the penetration profile suggested that the substrate got consumed within the matrix. Other researchers made use of infrared spectroscopy to show that the transportation rate of the antibiotic ciprofloxacin to the colonized surface was reduced in comparison to a sterile surface. It was suggested that the low penetration of ciprofloxacin was due to its binding to the biofilm components. Even the antibiotic agents that get adsorbed into the biofilm matrix have shown to have a retarded penetration. Slow penetration of aminoglycoside antibiotics is one such example. These antibiotics are positively charged agents that bind themselves to negatively charged polymers in the matrix of biofilm (Stewart and Costerton, 2001).

3.7. Heterogenity
Any bacterial cell present within the biofilm experiences a somewhat different environment as compared to the neighboring cells present in the same biofilm, and therefore has a different growth rate. The nutrients, signaling factor and waste products gradients that are formed within a biofilm allow for the heterogeneity to develop within the biofilm. This heterogeneity can also be one of the reasons for the antibiotic resistance of biofilms. For example in an experiment when biofilm cells were subjected to the antibiotic treatment with fleroxocin, cell elongation occurred which was at extreme level in exposed cells i.e. the ones located on the uncovered side of the biofilm. These studies disclose that the response to antibiotics can vary greatly, depending on the location of a particular cell within the community of biofilm. Thus the distribution of bacterial survival is irregular within seemingly identical microenvironments.

4. Anti-Biofilms Approaches
Anti-biofilm approaches have to put a stop to more than one antimicrobial mechanism concurrently to be effective clinically, as due to the heterogeneity of biofilms an antibiotic or antimicrobial agent may be able to destroy some of the bacterial cells present in a biofilm, but it is very improbable for it to efficiently kill them all. All the currently used antibiotics were developed on account of their activity against bacterial cultures growing in a planktonic environment. Thus, development of new screens of currently accessible and potential antibiotics might yield clinically effective antimicrobial agents against biofilm infections if they are selected for their activity against biofilm or non-growing cells. As genes and the products of genes responsible for antibiotic resistance of biofilm are identified and characterized, these will become chemotherapeutic targets to augment the effectiveness of currently in use antibiotics against infections caused by biofilms.

As the antibiotic resistance of a biofilm depends on multicellular communities of bacteria, one of the anti-biofilm strategies may be development of therapies that can disrupt this multicellularity of the biofilm. If that happens, defenses of the host will be able to resolve the infection, and the effectiveness of antibiotic agents may be enhanced (Stewart and Costerton, 2001).

Some of the other potential antibiofilm strategies include QSIs and improvement of drug delivery systems which are explained in the sections below.

4.1. Use of Quorum Sensing Inhibitors
There exists in nature several compounds that can act as Quorum Sensing Inhibitors (QSI). A case of such a naturally existing QSI is found to be present in garlic extract, which renders otherwise resistant biofilms liable to antibiotic therapy both in vivo and in vitro. The naturally occurring QSI compounds can also be synthesized and subjected to structural modification for inhibition of QS in experimental animal infections (in vivo). Since it is well known that the bacteria present in a biofilm lead to chronic infections due to their communication, QSIs can be used to cure these infections. Some antibiotics such as azithromycin, ceftazidime and ciprofloxacin inhibit QS in Pseudomonas aeruginosa leading to inhibition of the bacterial virulence. Thus QSIs have improved the synergistic weak effects of antibiotics on bacterial biofilms leading to elimination of biofilms. Current research states that resistance against QSI can only occur due to mutations (Hoiby et al. 2010). If this holds true, then conventional problems of antibiotic resistance that we face nowadays will not be a clinical problem.

4.2. Improvement of Drug Delivery System
Numbers of strategies have been proposed for prevention of biofilm formation and colonization, drug accumulation at the surface of biofilm and its delivery into the biofilm. An effective approach to counter antibiotic resistance of biofilms is the use of a better or more effective drug delivery system which could be done by the processes illustrated in Figure 5 (Smith, 2005).

Figure 5. Antibiofilm strategies (Source: Smith, 2005)


4.2.1. Liposomal Delivery to Biofilms
Liposomes are considered as attractive vehicles for drug targeting/delivery because of their compatibility with biological components. In the context of infection treatment via antibiotics liposomes have been studied for their ability to deliver these antibiotic agents into the biofilm. Research shows that if a drug is targeted to the biofilm by anionic liposomes, each of the bacteria in the biofilm adsorbs the drug separately to a smaller extent. Interestingly when it targets mixed biofilms then the drug is most effective against one but ineffective against the other, for example when liposomes laden with the bactericide triclosan targeted mixed biofilms of Streptococcus salivarius and Streptococcus sanguis, they were most useful against Streptococcus sanguis, but relatively useless against Streptococcus salivarius (Robinson et al. 2001).

5. Conclusion
Antibiotic resistance of biofilms depends not only upon the bacterial colony of the biofilm but also to the antibiotic agent used to destroy that biofilm along with inherent characteristics of biofilms including their mode of growth, environmental heterogeneity leading to the generation of heterogeneous population, signaling, mutations etc. Nonetheless, further studies are required for further elucidation of how the bacterial biofilms protect themselves against microbial agents. Additional cause of antibiotic resistance of biofilms is that almost all the currently available antibiotics have shown to be less effective for treating biofilms due mainly to the reason that they were designed on account of their activity against planktonic cells in conventional laboratory culture and have a small range of cellular targets. In the meanwhile eradication of bacterial biofilm depends on combined treatment strategies, quorum sensing inhibition and modern drug delivery approaches.

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