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Bacterial biofilms significantly cause persistent exacerbation of infections in the clinical setting. These groups of microorganisms are highly resistant to host immune responses and conventional antibiotic therapies, whereas they are embedded in an extracellular matrix. This chapter provides more detailed information on the mechanism of biofilm formation involving key stages of adherence, maturation, and spread, including the composition and structure of a biofilm matrix. This chapter further explores how biofilms contribute to antibiotic resistance, including physical barriers to drug penetration, quorum sensing mechanisms, and adaptive resistance strategies such as genetic adaptation, stress responses, and the formation of persister cells. The role of horizontal gene transfer in the spread of antibiotic resistance within biofilm communities is also discussed. The chapter discusses the clinical challenges posed by biofilm-associated infections, focusing on the challenges of diagnosing and treating chronic and recurrent infections, the role of host factors in biofilm persistence, and the limitations of current therapeutic options. Finally, we address emerging countermeasures to counter resistance mediated by biofilms, such as enzymatic therapies, nanomedicine technologies, natural product-based inhibitors, quorum sensing inhibitory agents, photodynamic and sonodynamic antimicrobial therapy, and combinatorial therapies.
Department of Microbiology, Quaid-i-Azam University, Islamabad, Pakistan
Hafiza Sehrish Kiani
Department of Microbiology, Quaid-i-Azam University, Islamabad, Pakistan
Faryal Gohar
Department of Microbiology, Quaid-i-Azam University, Islamabad, Pakistan
Shama Zahra
Department of Microbiology, Quaid-i-Azam University, Islamabad, Pakistan
Alisha Javed
Department of Microbiology, Quaid-i-Azam University, Islamabad, Pakistan
Shahzar Khan
Department of Microbiology, Quaid-i-Azam University, Islamabad, Pakistan
Dilawaiz Khan
Department of Animal Sciences, Quaid-i-Azam University, Islamabad, Pakistan
*Address all correspondence to: habbas@bs.qau.edu.pk
1. Introduction
For over a century, various implications have been made by scientists and microbiologists regarding uniform bacterial cultures in portraying a faulty image of microbial life in terms of genetics and physiology. Initially, it was thought bacteria existed in the natural environment as free-floating (plankton) structures. Still, many studies later showed the presence of multicellular bacterial cells or communities that can live in biofilms that consist of extracellular substances and are fixed in extracellular polymers where one cell communicates with another and forms biofilm [1]. With the aid of a basic microscope, a Dutch researcher named Antoni van Leeuwenhoek made the basic observation of an “animalcule” on tooth surfaces in 1683–1708, which was called the discovery of the microbial biofilm for the first time. Over the past decade, the concept of biofilm has been very well-known and sufficiently studied. Further investigation proved that almost 99.8% of bacterial cells show the process of biofilm formation at least in some stage of their life. But the question arises: Why do bacterial cells need to accumulate and result in biofilm formation? This concept has many better reasons, such as to survive many harsh and unbearable environments and a variety of phenotypical variations to survive and resist the antibiotic’s wide range [2].
Several types of bacterial communities interact and communicate with each other to live in the form of colonies. However, multispecies biofilms, having various species of bacteria, are better adapted to the environment, such as nutritional deficiency and instability, than single-species biofilms. We can say that a monofilm is defined as a biofilm made by a single species of bacteria. On the other hand, multifilms are those constructed by more than one species of bacteria [3]. Many medical illnesses, such as native valve endocarditis, lung infections in cystic fibrosis patients, infections linked to medical devices, infections from ocular implants, middle ear infections, carditis, and osteomyelitis, have been difficult to deal with because of the development of biofilm in all such illnesses. It is still very difficult to remove from the human body, as it can easily tolerate the required concentrations of antimicrobial treatment (10–1000 times) compared to the actual strength of the simple bacterial cells [4]. Moreover, a mature biofilm is unaffected by phagocytosis (cell eating). That is why, even after treating certain diseases due to biofilm, the disease can return after weeks or months. For this, the painful process of surgically removing affected tissues is more emphasized [5].
Microorganisms coexist with humans and play a vital role in influencing the host’s overall health and physiology. Some bacteria, which live in harmony with the host, form biofilms that can be formed on various surfaces, such as the skin, vagina, intestines, and mucosa of the mouth [6]. Pathogenic bacteria that colonize hosts and form detrimental biofilms can lead to persistent infections and recurrence. Biofilms can evade the host’s immune system and demonstrate the remarkable capacity to endure antibiotic treatment attributed to tolerance and resistance mechanisms. Resistance often occurs due to acquired mutations, which involve mechanisms such as modifications to antibiotic targets, efflux pumps, or enzymes that degrade antibiotics [7]. These changes enable bacteria to resist antibiotics outside biofilms by eliminating their molecular targets. However, within biofilms, antibiotic-resistant cells can survive exposure to high concentrations of antibiotics. Since Fleming discovered penicillin in 1928, numerous antibiotics have been developed to treat bacterial infections, saving countless lives. Unfortunately, the misuse and overuse of antibiotics have led to bacterial resistance, resulting in multi-drug resistance, marking the onset of a “post-antibiotic era.” Biofilm infections are responsible for numerous diseases, posing a significant and persistent threat to public health and the economy due to their novel resistance mechanisms and high mortality rates. It has been observed that bacteria within biofilms often exhibit increased resistance due to the reduced effectiveness of antibiotics against biofilm-associated infections [8].
The biofilm usually includes grouping single bacterial cells together to make the community, which is further covered by the outer extracellular material that protects the biofilm and is attached to the solid surface for further development. The maximum dry mass, i.e., 90–91%, is constituted by the semi-liquid matrix; the remaining microorganisms occupy 9–10%. However, cell-cell contact, firm growth of microbial species on a surface, and intimate cell contact and communication are done by different physiological processes inside the cell [9]. Eventually, five basic stages of biofilm formation and development are highlighted. In the first step of biofilm formation, the adhesion mechanism helps the planktonic (single cell) attach to the surface material. In the second step of biofilm formation, the cell adjusts itself to adhere and multiply further. In the third step, the complexity starts as the biofilm formation is constructed by cell-to-cell communication with the help of certain molecules that play a major role in signaling. Later on, the architecture of a very mature biofilm is formed that also produces the outer extracellular polymeric substance [10]. Lastly, in the final step, the single small cells from the mature biofilm are released into their surrounding environment. It has been found that both environmental and genetic factors contribute their role in the formation and maturation of biofilm, as shown in Figure 1. Extracellular Polymeric substance, an important constituent of biofilm, comprises various components such as lipids, nucleic acids, and proteins. The biofilm’s attachment to the surface and its architectural stability are usually contributed to by certain molecules such as lipopolysaccharides, glycopeptides, and lipids [11].
The first step involves the attachment of the planktonic cell to its substrate by the adhesion process. The free-flowing microorganisms in the isolated form get assembled into the proper community structures [12]. During this stage, microorganisms can go back to their planktonic state as they are loosely connected to the surfaces and are reversible. The bacteria then change their orientation, and they begin synthesizing the adhesion molecules called extracellular polymeric substance (EPS), which enables the attachment of bacterial cells to their surface form, facilitating irreversible attachment. This irreversible attachment also makes them resistant to various environmental factors that hinder the formation of biofilms [13, 14]. The studies indicated that bis-(3′–5′)-cyclic dimeric guanosine monophosphate (c-di-GMP), which is an intracellular signaling molecule, plays a crucial role in the early stages of biofilm formation as it promotes the formation of a biofilm matrix and inhibits flagella-mediated swimming motility. Microorganisms have the Pil-Chp system, a surface-sensing mechanism; as bacteria attach or detach from surfaces, the concentration of c-di-GMP, a signaling molecule, increases. This increase in c-di-GMP levels triggers the transformation of planktonic bacteria into surface-sensing bacteria, leading to irreversible attachment and the initiation of biofilm formation [14].
2.1.2 Expansion or creation of microcolonies
Bacteria attach to the biological tissue or physical surface, and this binding eventually becomes stable, creating microcolonies. Chemical signals released by bacterial cells signaled the beginning of bacterial multiplication. These signals serve as a communication channel between the bacterial cells, creating the biofilm. When the signal strength crosses a particular level, the genetic mechanism for exopolysaccharide synthesis is started [15]. Therefore, the bacterial cell divisions occur within the implanted exopolysaccharide matrix in response to the chemical signal and in the presence of a high concentration of c-di-GMP, ultimately creating a microcolony. It is also believed that flagella and type IV pili-mediated motilities are essential for interacting microbes with surfaces and forming microcolonies by cell-cell aggregations [16].
2.1.3 Maturation
Some biofilm-related genes can be expressed after the microcolony stage of biofilm development. These genetic products are required for the EPS, the primary structural component of the biofilm. It has been studied that bacterial adhesion alone can cause the extracellular matrix to develop. Water-filled tubes are formed after matrix development to carry nutrients within the biofilm [17]. According to research, these water channels act as a kind of circulatory system, supplying various nutrients and eliminating waste by the groups in the microcolonies of biofilm. Extracellular polymeric substances (EPS) not only help the microorganisms to adhere to surfaces but also stabilize biofilm three-dimensional structure, cluster cells, protect the biofilm from a variety of stresses, including the host immune system response, antimicrobials, oxidative damage, and metallic cations, and encapsulate signaling molecules required for quorum sensing, metabolic products, and enzymes [18]. These factors make EPS crucial for biofilm maturation. Biofilm in a maturation phase consists of three layers: an inner layer that controls the biofilm, an outer layer that harbors microorganisms that are ready to be again in their planktonic/isolated condition, and a middle layer known as the microbial basement layer [19].
2.1.4 Spread
After maturation, bacterial cells regularly depart from biofilms either passively due to physical factors like liquid flow-dependent dispersion or actively (due to motility and EPS degradation-dependent dispersion). Bacteria can occasionally become isolated from the colony and enter the environment due to stress. In more instances, some bacteria stop the production of EPS and release it into the environment. Cells express genes for motility during dissociation of motile bacterial cells, including transcription of ribosomal protein and pilus, and are almost visible in planktonic cells [20]. Biofilms disperse because of various factors, such as lack of nutrients, intense competition, and outgrown population, which encourage the production of dispersion-related genes, for example, upregulation of genes involved in EPS degradation and cell motility and a downregulation of genes essential for polysaccharide and fimbriae synthesis.
Biofilm is a bacteria colony wrapped in a self-produced polymeric matrix. The protective layer includes polysaccharides, proteins, and DNA, collectively called extracellular polymeric substances (EPS). Biofilms are important in the sense that they cause chronic and persistent infections. This is achieved by their incredible ability to reduce the effectiveness of antibiotics. In the biofilm state, the efficiency of antibiotics is reduced by 1000 times more than in the planktonic state. Biofilm-mediated antibiotic resistance is a relatively new clinical issue. Studying biofilms and resulting chronic infections is important because biofilms are not easily treatable with antibiotics, immune systems, or surfactants.
3.1 Physical barrier: EPS - Reducing the penetration of antibiotics
External nucleic acids (eDNA, eRNA), proteins, and polysaccharides are known to form a matrix. This matrix is called an extracellular polymeric substance (EPS). It is common in biofilms because it offers mechanical stability to biofilms. EPS is the physical barrier that is believed to be the first control against the use of antibiotics. EPS is a diverse matrix in terms of its structure and its roles in the process of amplification of antibiotic resistance. This matrix prevents the diffusion of antibiotics within the desired community and can also prevent the penetration of wanted antibiotics by rendering them useless. For example, the EPS layer reduces the ability of β-lactams to penetrate, affecting cell wall synthesis. This makes it possible for the bacteria to survive in biofilm [21]. Likewise, aminoglycosides that pass through porin channels and diffuse across the cell membrane are substantially less effective because of low mobility within the matrix against bacteria that develop biofilm [22]. The EPS layer is not necessarily uniform in composition, charge, and size for different types of bacteria and for the same type of bacteria. This is explicable in light of recent developments in imaging and microscopic technology due to the heterogeneous nature of biofilms, where some parts are less, and others are more resistant to treatment [23]. However, some are connected to biofilms as factors such as β-lactamases that break down the β-lactam antibiotics, increasing antibiotic resistance [24].
3.2 Quorum sensing
Bacteria have a method to communicate within an environment. This method is known as quorum sensing (QS). Communication is possible through the release of tiny molecules. This communication mechanism depends on the expression, regulation, and increase of antibiotic resistance. Both Gram-negative and positive bacteria have specialized molecules that enable them to communicate. Gram-negative QS systems depend on AHLs, and Gram-positive have peptide-based signals. These enable bacteria to form biofilms for regulating genes involved in pathogenicity, EPS synthesis, stress response, and synchronization. QS alters the rigidity of bacterial structures and controls its behavior, enhancing antibiotic resistance. Antibiotic resistance of Pseudomonas aeruginosa is higher toward Ciprofloxacin because QS systems enable it to activate the virulence factors and biofilm formation by using Las and Rhl systems for signaling [25]. QS also regulates the formation of efflux pumps besides matrix formation. These pump the antibiotics out of the cell, therefore increasing antibiotic resistance. It is important to alter quorum sensing to make commercially available antibiotics effective against bacteria and to limit antibiotic resistance [26].
3.3 Strategies of adaptive resistance
Through adaptive mechanisms, bacteria can overcome antibiotic stress, especially in biofilm. The distinct environment of biofilm facilitates the adaptive mechanism. These strategies that enable bacteria to go in such harsh environments include changes in the physiological environment, genetic changes as well as phenotypic changes.
3.3.1 Genetic mechanisms
Some of the genetic processes by which biofilm bacteria acquire adaptive resistance are now known from experimental investigations. One such strategy is the Horizontal Gene Transfer (HGT), or genetic modifications of overexpression of resistance genes, to give microbes a new set of functions. We can also notice that bacteria in a biofilm can be genetically different. Genetic mutants within certain subpopulations can contain antibiotic-resistance genes. These mutations can occur in antibiotic-degrading enzyme genes or antibiotic target genes of DNA gyrase or penicillin-binding protein [27]. Furthermore, the biofilms favor the exchange of genetic material among bacteria and thus the rate of emergence of resistance. In this mechanism, horizontal gene transfer, particularly conjugation, plays a significant role. Biofilm matrices enhance the transfer of the resistance plasmid and other genes because cells are in close contact. For example, plasmids with ESBL genes in Escherichia coli can be dispersed using biofilms, causing multi-drug resistance [28].
3.3.2 Physiological modifications
Besides, bacteria residing in biofilms display certain metabolic changes that will help them become antibiotic-resistant. Downregulation of metabolic activity, slow division, and nutrient restriction in the biofilm mode of growth are often cited causes for reduced antibiotic susceptibility [29]. It turns out that fluoroquinolones and β-lactams primarily target only actively dividing bacteria. These are less effective in biofilms because many cells are in a slow-growing phase or quiescent, aiding the biofilm population. Susceptibility is frequently the result of low metabolic activity, decreased cell division, and nutrition limitations in the biofilm environment [29]. The fluoroquinolones and β-lactams target the actively growing bacteria. As many cells are slowly growing and dormant, these drugs are less effective in biofilms. This helps the biofilm population to thrive even in the presence of antibiotics.
3.3.3 Mechanisms of stress response
Biofilm bacteria that form in difficult operating environments, including an environment containing antibiotics, activate stress response signal transduction pathways. Such processes may include lowered growth, metabolic variations, or the switching on of protective enzymes. The SOS response is one of the pathways, a deeply conservative bacterial response to DNA damage that leads to a higher probability of obtaining antibiotic resistance genes and a higher mutation rate [30].
3.3.4 Persister cells: Leading figure in biofilm resistance
It is important to understand that bacterial cells entering a dormant stage, a metabolically inactive state, are called persister cells. They are one of the toughest to handle from the aspect of being treatable with drugs. Such cells can be found most often in biofilms; they can also be found in the planktonic cultures. Antibiotics that act on bacteria that require an active metabolic state or bacterial cell division present a problem because persisters are not replicating or metabolizing. Stress response pathways, which include toxin-antitoxin systems that control cell growth and survival in unfavorable conditions, are involved in controlling the synthesis of persister cells [31]. Accumulation of toxins produced by certain bacterial species may enhance the formation of persister cells in biofilms. For instance, Pseudomonas aeruginosa forms a subpopulation of persisters more resistant to antimicrobial agents, hence the persistent biofilm-associated infections. These cells are very stubborn; thus, biofilms can regrow once the antibiotics are stopped, hence recurrent infections. In conclusion, recent research has shown that if the process of persister cell formation is controlled, this could lead to a fresh combating strategy to avoid infections connected to biofilms [32].
3.4 Horizontal gene transfer (HGT)
Recent studies have shown a connection between horizontal gene transfer and biofilm formation. During biofilm, conjugation is enhanced. Through this, the bacteria in biofilm are more resistant to antibiotics, and horizontal gene transfer helps the bacteria gain antibiotic-resistant genes from the surroundings. Three main transfer mechanisms, namely transduction, transformation, and conjugation, are involved [33]. During biofilm, the main process of transfer is via conjugation. The features of overlapping and the compact arrangement of bacterial cells in biofilms foster genetic exchange efficiently [34]. The mobile genetic elements that facilitate the exchange of antibiotic-resistance genes in biofilms include plasmids, transposons, and integrons, which may harbor multiple resistance genes [35]. Likewise, bacteria residing in biofilms are likely to develop resistance against many classes of antibiotics by horizontal gene transfer between species or strains [36]. Biofilm environments also contribute to the selection of antibiotic-resistant strains because, in microbial communities containing both susceptible and resistant strains, resistance is rapidly selected by the powerful adherence forces exerted by biofilms.
Biofilms are also involved in acquiring chronic and recurrent infections [37]. These microbial communities are protected and maintained in a suitable environment and favorable circumstances, surrounded by an EPS matrix produced by the microbes. Pathogens often colonize host tissues or medical devices, leading to persistent infections [38]. This biofilm allows microorganisms to remain protected from immune responses and antimicrobial intervention. This persistence is due to the EPS matrix shielding immune recognition and antibiotic penetration [39]. Furthermore, when bacteria develop biofilms, they become dormant or slow-growing, and this also plays a role in their resistance to antimicrobial agents, which target actively proliferative cells. For instance, in pulmonary infections due to cystic fibrosis, biofilms allow a chronic infection with Pseudomonas aeruginosa to adhere to the surfactant layer of the lung. The situation worsens because these biofilms secrete toxins and inflammatory mediators, leading to lung disease [40].
4.1 Recurrent infections
Biofilms are difficult to eradicate since they contain persister cells, dormant forms of bacteria that exist after antibiotic use. They reside inside the biofilm structure, and on the withdrawal of antibiotics or any weakening of the immune system, they can wake up and grow back to give a relapse [41]. A good example of biofilm-mediated infection is recurrent urinary tract infections (UTIs). Uropathogenic Escherichia coli (UPEC) attaches to the bladder epithelium, forming biofilms that create reservoirs protected from antibiotics and host defenses [42]. However, these reservoirs can reinfect new cycles after removing stresses like antibiotics and host immune responses, particularly in patients with underlying diseases such as diabetes or anatomic deviations.
Recurrent infection with biofilms is also commonly related to chronic sinusitis. The bacteria Haemophilus influenzae and Staphylococcus aureus cause biofilm formation in the nasal mucosa, thus causing chronic inflammation and recurrent symptoms despite the treatment. The ability of biofilm to exist for long periods is attributed to its ability to withstand mucociliary clearance and antimicrobial agents [43]. CF is a disease resulting from a cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation, leading to thick mucus in the respiratory and digestive systems. Due to biofilm formation in the thick mucus in the lungs, chronic and relapsing infections occur. Biofilm-forming bacteria are highly susceptible to colonization of the respiratory tract in CF patients [44]. The thick mucus provides the microbes with nutrients they can attach to and multiply. Bacteria that adhere to the mucus grow in biofilms, a shield from the host’s immune system and other antimicrobial substances. Biofilms release extracellular polymeric substances (EPS) that create a physical barrier against the entry of antibiotics and shield bacteria from immune cells [45]. This invariably leads to worsening, poorer lung functioning, and enhanced mortality rates. P. aeruginosa biofilms are relatively hard to eliminate and are linked to the deterioration of lung function over time [46]. Moreover, CF patients also have biofilms of other pathogens, such as S. aureus and H. influenzae, which makes the problem worse and results in chronic infections.
Bacterial colonization is also a common characteristic of chronic wounds such as diabetic foot ulcers [47], venous leg ulcers [48], and pressure ulcers [49] due to the presence of biofilm. Biofilms are significant in the context of the inhibition of wound healing [48]. Biofilm formation especially prevails in chronic wounds since the microenvironment where biofilms are produced is highly suitable [50]. Moreover, bacterial colonization and formation of biofilms are further enhanced by factors such as moist conditions, necrotic tissue, and decreased blood circulation. As biofilms develop, they change the local inflammatory pattern, interfere with the healing of the tissues, and create conditions favorable for repeated infections. Bacteria can also form biofilm, and it can adhere to tissue base, dead tissue, and wound secretion to form a vast and long-lasting microbial conglomerate [50].
4.2 Role of host factors in chronicity and recurrence of biofilm infections
The duration and frequency of biofilm-associated infections have also been established to be host factors. Exopolysaccharide matrix hinders phagocytosis and cytokine signaling so microbial communities can survive in a secure niche; immune evasion is essential [51]. While inflammation is intended to eradicate infection, it can also destroy tissue and lead to biofilm persistence with external conditions. Host-related risks such as immune suppression, diabetes, or using indwelling medical devices also increase the risk of chronic and recurrent infections [52]. For instance, the biofilm on the catheter surface is the site of infection that leads to repeated bacterial colonization in the case of CAUTIs.
4.3 Challenges
Bacterial localization in a biofilm makes it hard to diagnose because of its structural and functional properties and the current diagnostic tools. Biofilm infections are challenging to diagnose because they cannot be detected by the immune system or standard microbiological assays [53]. The protective matrix produced by EPS also shields the bacteria and makes them less available for diagnostic procedures [54]. When in biofilm, bacteria have a different behavior; they are dormant, while the planktonic bacteria are free-floating and metabolically active. Traditional diagnostic methods like culture-based depend on the growth of microorganisms for identification [55] and cannot identify these metabolically dormant bacteria. Furthermore, biofilms are considered to have high-density structures. A bacterium in polymicrobial biofilms can be in various physiological statuses or in contact with other microorganisms, including fungi, in the same biofilm [58]. The diversity of the biofilm poses a challenge to the identification process since such diagnostic methods may not be able to identify the whole spectrum of microbial activity or even the microbial population density of the biofilm [56].
Biofilm-related infections are challenging to manage because biofilm bacteria are highly resistant to antibiotics than planktonic (free-living) bacteria. Due to biofilms’ structural, metabolic, and genetic characteristics, this resistance is a multifactorial phenomenon. The EPS matrix, which is the outer layer of the biofilm, acts as a physical barrier that does not allow the antibiotics to penetrate the deeper layers of the biofilm. The matrix is gel-like and dense, hindering the diffusion of antibiotics, particularly big molecules [57]. For instance, aminoglycosides and beta-lactams are often entrapped or delivered slowly into the matrix, reducing their access to bacteria in deeper layers [58]. Furthermore, the EPS matrix can also adsorb with antibiotics, making them ineffective before interacting with the target cells. For instance, the negatively charged parts of the EPS, eDNA, or specific polysaccharides bind positively charged antibiotics, such as aminoglycosides [59]. This binding effect leads to a concentration gradient in which sublethal concentrations in the deeper zones can help bacteria survive.
Another characteristic is the metabolic heterogeneity of bacterial cells within the biofilm. Cells at the surface of the biofilm are in contact with nutrients and oxygen and are metabolically active. In contrast, the deeper cells are starved of nutrients [60] and become nonmetabolically active or even enter a dormant state. The metabolic stratification has profound significance for antibiotic resistance. Most antibiotics, including beta-lactams and fluoroquinolones, work on some cellular functions such as cell wall synthesis, protein synthesis, or DNA replication. These antibiotics’ implementation methods are constrained because these cells are less metabolically active and thus less vulnerable to these antibiotics in biofilms where they are stationary or slow dividing within the matrix. Steady-state hypoxia is often observed in biofilms developed on mucosal tissues or the surface of medical implants [61]. When exposed to hypoxic conditions, the bacteria change their metabolic activity to glycolysis fermentation or nitrate reduction [62]. This metabolic switch changes bacterial phenotype and is a factor that contributes to resistance. The developing alterations under anaerobic growth led to the decreased absorption of some antibiotics requiring an electron transport chain, such as the aminoglycoside antibiotics.
4.4 Limited treatment options
Biofilm-related infections are challenging in clinical medicine because they are recurrent and resistant to traditional antimicrobial agents. The existing antibiotic therapies cannot remove biofilms completely, leading to chronic and recurring infections, which are dangerous to patients and a significant cost to the healthcare system. Due to the properties of biofilm bacteria, such as limited penetration and metabolic activity, antibiotics cannot reach bactericidal concentrations across the biofilm [63]. Not only can bacteria survive this sublethal exposure, but this exposure will also favor the emergence of antibiotic-resistant mutants. Antibiotics target fundamental bacterial functions, including synthesizing proteins and cell walls or replicating DNA [64]. Nonetheless, these antibiotics are ineffective in biofilms because bacteria within them are embedded in a layer of extracellular polymeric substances and metabolically distinct. In addition, quorum sensing systems control the formation and establishment of biofilms. However, current antibiotics cannot act on quorum-sensing pathways important for biofilm stability and synchronization, as shown in Figure 2 [65].
5. Defensive strategies against biofilm-associated resistance
5.1 Novel approaches to preventing and treating biofilm infections
Recently, many advanced and novel methods are being analyzed to fight and remove the biofilms affecting different natural vicinities. These methods that are being focused on and effective for biofilm studies include the disruption of quorum sensing, certain natural substances, antimicrobial sonodynamic or photodynamic therapies, nanotechnology-based techniques, and enzymatic degradation. These approaches have shown promising results by targeting the physiological structure and functioning of biofilm and disturbing the single-cell bacterial populations in biofilm, proving the older method of antibiotic treatment to be efficient and still effective.
5.2 Enzymatic breakdown of biofilms
In enzymatic treatments, the mature biofilms normally divide into smaller fragments by the action of specific enzymes, which help their easy degradation. This treatment is not only safe to handle. Still, it is also applicable to different conditions and is nontoxic in nature, so it can be effectively implemented for various medical illnesses at hospitals [1]. Enzymatic biodegradation is an effective tool for biofilm removal that cannot be easily removed by using conventional cleaning methods. The areas that are difficult to deal with in medical cases, such as biomedical devices and medical implants, are useful for breaking the biofilm in Table 1 [66]. The biofilm’s cellular components, such as proteins, lipids, and carbohydrates, which provide the potential to the biofilm structure, are acted upon by various enzymes, such as lipases, amylases, and proteases, during the enzyme degradation strategy. Using the standard cleaning technique by cells, the waste is usually removed from the cells after enzymatic action. For instance, the lysozyme enzyme is responsible for unwinding the components of bacterial cell walls, thus releasing the biofilm layer. By breaking the peptidoglycan layer of the bacterial cell wall, the bacterial cells become much more at risk of being targeted by the respective immune system and antibiotics [67]. Moreover, one of the enzymes, Dispersion B, known for its biodegradable nature, targets the cell by making the Extrapolysaccharides soluble and easier to eradicate. The various factors on which the action of enzymes depends are the biofilm’s composition and nature, the prevailing surrounding environment, and the biofilm and enzymatic types utilized [68]. Furthermore, one more important enzyme responsible for degrading is DNAse, which degrades the bacterial DNA in biofilm and helps efficiently remove DNA components [69].
Aspect
Description
Clinical implications
Biofilm structure
Biofilms consist of a bacterial community encased in a self-produced matrix of EPS containing polysaccharides, proteins, and eDNA.
Biofilms reduce the efficacy of antibiotics and immune responses, leading to persistent infections.
Resistance enhancement
Tight biofilm-dense structure limits antibiotic penetration and increases bacterial survival.
Increases resistance up to 1000 times compared to planktonic (free-floating) bacteria.
Biofilms are difficult to treat with conventional antibiotics.
Role of Quorum Sensing (QS)
Bacteria in biofilms communicate through QS, which regulates gene expression for biofilm formation, stress response, and resistance.
Increases biofilm stability, virulence factor production, and antibiotic resistance.
Targeting QS could enhance antibiotic effectiveness.
Adaptation mechanisms
Biofilms allow bacteria to adapt through genetic, physiological, and stress responses.
Genetic mutations, metabolic changes, and stress response pathways enable bacteria to survive harsh conditions like antibiotics.
Table 1.
Biofilm formation and its role in chronic infections [66].
5.3 Advanced strategies utilizing nanotechnology
Nanotechnology-based strategies offer substantially promising avenues in addressing biofilm-based infections. In this approach, biofilms are usually degraded using components such as specified nanoparticles that can have a powerful impact on biofilm eradication. This emerging strategy works by focusing on the nanoparticle’s actions to treat and stop the biofilm formation [70]. Nanomaterials have also been studied to detect biofilm formation at the initial stage and diagnose and identify various biofilm-related infections and illnesses. For example, a more rapid and easy diagnosis is made by binding bacterial cells with real-time signals with nanomaterial-based biosensors. Different factors such as infection dissemination, bacterial cell attachments with the surface, and biofilm formation and maturation can be stopped by coating nanomaterials with certain medical devices to make them functional properly against bacterial cells. A thin layer of nano-sized particles is being applied on various coatings to make it more antimicrobial and protective against the biofilm consisting of mature cells. These nanoparticles form a strong barrier that stops the bacterial cell from penetrating the medical devices by highlighting their power to remove and degrade the biofilm components [71]. They are also preferred to be used in the drug delivery process to treat and stop the infections related to biofilm to combat the other side effects of older methods of antibiotics, such as allergic reactions, organ toxicity, and gastrointestinal or stomach-related issues in the body [72]. Several metals are associated with their action in biofilm destabilization and degradation. Certain key mechanisms are related to these nanoparticle methods for their anti-biofilm effects, such as disturbance of outer polysaccharides having various components, attachment of biofilm, penetration of cell wall and membrane, and disruption of quorum sensing. By studying these aspects, the nanoparticles have potential anti-biofilm progress against a wide range of pathogenic and disease-causing bacterial species. For instance, the catheters coated with the Ag particles help stop the Gram-positive bacteria antibiotic-resistant settlement, such as Staphylococcus. aureus and S. epidermis. But, it is more effective against gram-negative bacteria such as E. coli and Pseudomonas aeruginosa. Similarly, Cu metal has also been found to effectively target the fungal cells while grouping by impacting and blocking the quorum sensing [73].
5.4 Natural compounds
Natural compounds, including plant extracts, essential oils, and substances derived from marine sources, have demonstrated considerable potential in preventing biofilm formation. Due to their lower likelihood of side effects and natural origin than synthetic drugs, these compounds are emerging as promising anti-biofilm agents [74]. Plant-based compounds such as eucalyptus (Eucalyptus globulus), garlic (Allium sativum), oregano (Oreganum vulgare), and grape (Vitis vinifera) possess antimicrobial activity against a variety of pathogens, including drug-resistant strains. Their antibacterial effects result from mechanisms such as interfering with metabolic processes, disrupting bacterial membranes and walls, and inhibiting protein synthesis. These natural compounds also present a more environmentally friendly, cost-effective, and sustainable alternative to synthetic antimicrobials. Additionally, their ability to selectively target specific bacteria enhances the precision of anti-biofilm treatments, making them effective against a broad spectrum of bacterial strains [75]. Thymol, a natural compound derived from Thymus vulgaris, has proven effective against antibiotic-resistant bacteria such as Escherichia coli and Staphylococcus aureus. It achieves this by inhibiting survival and growth by disrupting bacterial cell walls and preventing biofilm formation. Plant-derived terpenoids, known for their antibacterial properties, are potential sources for developing new antibiotics. These compounds exhibit strong antioxidant effects that damage bacterial cell walls, interfere with quorum sensing and biofilm formation, and inhibit bacterial replication, thereby curbing infection spread. Similarly, polyphenols from Vitis vinifera are effective against infections caused by antibiotic-resistant bacteria. They exhibit potential anti-biofilm activity and possess anti-inflammatory and antioxidant properties that may be beneficial for treating various health conditions [76]. Quercetin, a flavonoid with antibacterial activity, is effective against Gram-negative and Gram-positive bacteria, including resistant strains. It functions as an antioxidant, disrupts bacterial membranes, and inhibits bacterial growth and reproduction while preventing the formation of biofilms that protect bacteria under harsh conditions. Curcumin, a polyphenolic compound obtained from the rhizome of Curcuma longa, exhibits broad-spectrum antimicrobial activity. Its anti-inflammatory properties help reduce infection risks, especially in individuals with chronic inflammation. Additionally, curcumin exhibits biofilm formation, making it a promising candidate for combating infections caused by antibiotic-resistant bacteria [77].
5.5 Quorum-sensing inhibition
The inhibition of quorum sensing is a new strategy adopted to curtail the formation of biofilms. This method interferes with bacterial communication, which hinders the formation of biofilms. Quorum-sensing molecules are chemical signals released by the biofilm-forming organisms that are essential for coordinating behavior within bacterial communities. These chemicals signal the start of biofilm development when a specific bacterial threshold is achieved. Quorum-sensing inhibitors serve by averting the synthesis of auto-inducer molecules, which is vital for bacterial communication. Bacteria cannot form biofilms when the synthesis of these molecules is inhibited, hence stopping the spread of diseases. Additionally, by reducing bacterial contamination in the environment, these inhibitors can aid in preventing infections. Quorum-sensing inhibitors are, therefore, significant tools for controlling and treating bacterial diseases. Moreover, quorum-sensing inhibitors have demonstrated efficacy in reducing antibiotic resistance in bacterial strains, providing a viable strategy to fight infections caused by multidrug-resistant bacteria. Notably, quorum-sensing inhibition is nontoxic and environmentally safe, making it a desirable substitute for traditional therapeutic approaches. Various quorum-sensing inhibitors have been created, including phages and small compounds to fight bacterial infections. For example, the small chemical acyldepsipeptide (ADEP) has demonstrated potential as a strong inhibitor for bacterial infections by recently being found to be effective in blocking quorum-sensing in multidrug-resistant Acinetobacter baumannii as shown in Figure 3 [78]. ADEP inhibits the virulence factors that cause infection by interfering with bacterial communication. Incredibly, ADEP is harmless to mammalian cells, making it a promising option for new treatments for biofilm infections. Numerous other small peptide molecules, such as furanone, AHL, 2-heptyl-4-hydroxyquinoline sulfonamide (HQSA), and N-(3-(4-fluorophenyl)-2-propynyl)-4-quinolone, are being studied as possible anti-biofilm agents as they exhibit the ability to inhibit the production of quorum-sensing molecules in a variety of bacterial species [79]. A subclass of bacteriophages known as quorum-sensing inhibitor phages works by interfering with the quorum-sensing systems of bacteria. These phages impede the synthesis of quorum-sensing signals, which are essential for bacterial communication and the formation of biofilm. Notably, this approach is more persistent than previous anti-biofilm therapies since phages are less likely to acquire resistance.
Antimicrobial photodynamic therapy (aPDT) and antimicrobial sonodynamic therapy (aSDT) are innovative approaches for combating biofilms, utilizing sound waves and light to eliminate bacteria and inhibit biofilm formation. In aPDT, light-sensitive compounds known as photosensitizers are activated by specific wavelengths of light, generating reactive oxygen species (ROS) that destroy bacterial cells. Conversely, aSDT uses ultrasound waves to create vibrations in bacterial cell walls, ultimately causing cell death. These therapies are considered safe and have diverse applications in medical settings, particularly in managing biofilm-associated infections. aPDT is a non-invasive method well-suited for treating infections in hard-to-reach areas, while aSDT, being minimally invasive, is effective for addressing infections located in deeper tissues [80]. Both treatments demonstrate effectiveness against antibiotic-resistant bacteria due to their unique mechanism of action. The reactive oxygen species (ROS) generated by aPDT are toxic to biofilm-forming bacteria. However, prolonged exposure to light may lead to bacterial resistance, necessitating regular monitoring and periodic treatments every few months to maintain its effectiveness. Combating aPDT with lipid-coated nanoparticles containing peptide nucleic acid (PNA) has emerged as a promising approach for managing biofilm-associated infections. This method effectively combats biofilm-related infections by utilizing gene-specific PNAs to target pathogens. The purine and pyrimidine components in PNAs enable them to bind with DNA and RNA, expanding their applications in biological research. Antimicrobial sonodynamic therapy (aSDT) is a valuable complement to biofilm treatments. Ultrasound waves can disrupt the structural integrity of biofilms, enhancing the efficacy of antimicrobial photodynamic therapy (aPDT) by activating photosensitizers. Additionally, ultrasound can be paired with antibiotics to improve their penetration into biofilm matrices, increasing their effectiveness. The mechanism by which ultrasound disrupts biofilms involves breaking hydrogen bonds between biofilm cells, thereby boosting the efficiency of other anti-biofilm methods and promoting bacterial cell destruction [81]. Combating aPDT and aSDT leverages the strengths of both therapies to provide a more comprehensive treatment strategy. While aPDT is particularly effective at eradicating bacteria, aSDT excels at reducing the size and density of biofilms. The synergistic use of these therapies maximizes their potential, offering a more effective solution for managing biofilm-associated infections [82].
5.7 Combination anti-biofilm therapies
Combination therapies, an integration of multiple treatments against the diseases, help researchers to attack multiple pathways simultaneously, enabling a more thorough approach to treating different infectious diseases [83]. These treatments have more significance than individual treatments, as they are frequently less harmful and more cost-effective, making them a desirable choice for infection prevention. Combination therapies can be used to reduce the alarming issue of antibiotic resistance by customizing their levels. It has been observed that combining antibiotics with other agents, such as probiotics and surfactants, reduced biofilm production levels. Antibiotics work by limiting the growth of bacteria, while surfactants and probiotics aid in disrupting the biofilms. Due to their ability to target multiple aspects of biofilms, they are effective in treating different chronic infections. Some examples of effective combination therapies are demonstrated in Table 2 [84].
Sr. No.
Combination therapies
Effect
References
l
Combining ambroxol with vancomycin
Enhanced effectiveness of vancomycin against Staphylococcus epidermidis biofilms
Advanced nanomaterial-based solutions offer a promising and innovative approach to tackling biofilms and other persistent substances. The unique properties of nanomaterials have revolutionized numerous fields, including medicine. These materials can be engineered to mimic the size and structure of bacterial internal components and proteins. In chronic wound infections, effectively eliminating biofilms and managing infections are crucial steps for promoting improved wound healing. To achieve improved therapeutic outcomes, focusing on targeting specific stages of the biofilm life cycle is essential. For persistent wound infections associated with biofilms, one strategy involves disrupting the initial attachment of microorganisms to the wound site. This can be achieved by inhibiting their interaction with the wound surface, such as cell surface-associated adhesions and polymeric extracellular substances (EPS), including proteins and appendages. The early stages of biofilm production can be prevented by targeting cell and EPS production. Established biofilms can be disrupted through various strategies such as breaking down the EPS, interfering with microbial interactions, physically removing the biofilms, eradicating dominant cells, or altering the pathogenic environment (e.g., addressing hypoxia or reducing pH). Bacteriophages, viruses specifically engineered to target bacteria by recognizing bacterial cell surface receptors, are emerging as a promising alternative in the fight against antimicrobial resistance.
No conflicts of interest are associated with this publication.
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Written By
Syed Hamza Abbas, Hafiza Sehrish Kiani, Faryal Gohar, Shama Zahra, Alisha Javed, Shahzar Khan and Dilawaiz Khan
Submitted: 04 December 2024Reviewed: 03 February 2025Published: 13 March 2025
Open access peer-reviewed chapter
