Keywords

9.1 Introduction

Biofilms are universal and found in a wide variety of environments, both natural, such as in rivers, streams, and alimentary canal or living tissues of mammals, and man-made like in wastewater treatment plant, tickling beds, indwelling medical devices (IMDs), and industrial or potable water system piping (Donlan 2002). Biofilms can evade host defense mechanisms that include both innate and adaptive immunity (Dunne Jr. 2002). It is the reason why biofilm formation is an increasing cause of concern throughout the world.

Bacterial biofilms not only contribute to hospital-acquired infections, but also are a leading cause of corrosion, fouling of water pipes, and food and pharmaceutical spoilage (Henderson 2010; Kumar and Anand 1998). Some of the health issues associated with biofilms are indirect such as in drinking water distribution system where biofilms corrode water pipes and weaken them and this loss of integrity weakens pipes aside from causing esthetic problems which may lead to a health concern. Microorganisms forming biofilms can cause infection in humans and animals and may be transmitted to each through cross-contamination. Biofilm-associated infection in animals can cause massive economic loss such as in livestock/poultry industry and others in terms of production (Chakraborty et al. 2018). Also, biofilms producing microorganisms contaminate foods and generate damage to the product, equipment, and consumers leading to economic loses.

In the food product manufacturing facilities, biofilm formation leads to deleterious hygiene issues due to adherence of a variety of microbes on food and degradation of equipment (Kabwanga et al. 2018). In the pharmaceutical industry, the development of biofilms and adherence of it into the production equipment and facilities are critical issues that need to be addressed (Kabwanga et al. 2018; Stewart 2015). Although most of the biofilm-forming microbes are harmful in many ways, some of them exhibit beneficial properties which have been put to use in several industrial processes (Morikawa 2006). The infections caused by biofilm-forming microbes are chronic, and for the treatment, antimicrobial agents need to be administered, but biofilms make the microbe resistant to antimicrobial agents compared to their planktonic counterparts (Costerton et al. 1999; Mah and Toole 2001; Stewart and Costerton 2001; Donlan and Costerton 2002).

Therefore, treatment of infections caused by biofilm-forming microbes is not resolved with the sole administration of antibiotics due to the problem of the development of resistance against them. Although highly sterile conditions and practices are fundamental to maintain a strategic distance from biofilm development, for proper resolution, some of the novel antibiofilm compounds should be explored as a potential antibiofilm agent in the near future. Some of them, which are already discovered or tested till date, are active herbal compounds such as essential oils, quorum-sensing inhibitors, antimicrobial peptide alone or in combination with antibiotics, and synthetic or genetically engineered compounds.

Out of these new control strategies which are continually emerging, most of the focus is on antibiofilm agents of biological origin such as enzymes, phages, AMPs, and QSIs. The present review will focus on describing in detail the various biocontrol agents explored till date for the eradication of biofilms from the site of its formation.

9.1.1 Biofilms

Biofilms are defined as the structural community of bacterial cells which are formed by a self-produced polymeric matrix known as exopolysaccharide (EPS), which takes around 85% of the volume of a biofilm. This community of cells adheres either to living or nonliving surfaces (Costerton et al. 1999) (Fig. 9.1).

Fig. 9.1
figure 1

Scanning electron microscopy photomicrograph of a 6 days old B. cereus biofilm formed on a stainless steel surface. 6330 magnification; bar ¼ 5 mm (Simões et al. 2010)

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The distinct levels in the process of biofilm formation can be divided into various steps (Crouzet et al. 2014). The following are the general stages for biofilm formation, though the precise details of the regulation of biofilm formation vary significantly from species to species.

  1. 1.

    Macromolecules in the liquid where biofilms are forming precondition the surface (living or nonliving) for adhesion.

  2. 2.

    Transportation of bacterial cells to the surface also occurs.

  3. 3.

    The cells transported are adsorbed to the surface.

  4. 4.

    Desorption of reversibly adsorbed cells and retention of irreversibly adsorbed cells occur.

  5. 5.

    Metabolism of the substrate by the biofilm-bound cells and then transportation of the by-products out of the biofilm.

  6. 6.

    The adsorbed cells produce cell-to-cell signaling molecules for monolayer/microcolony formation.

  7. 7.

    Maturation of biofilms occurs through the formation of extracellular matrix (EPS) and other cell materials. It forms a three-dimensional structure of cells known as a microcolony (O’ Toole et al. 2000).

  8. 8.

    Detachment or dispersal of bacteria to migrate and then colonize in new areas (Landini et al. 2010).

The main composition of biofilms is the EPS matrix which is formed by retaining water and other bacterially originated substances released by bacterial cells which get embedded in this EPS matrix, and it provides the following advantages to the cells (Crouzet et al. 2014; Donlan and Costerton 2002; Jamal et al. 2018):

  1. (a)

    Structural stability to the microbe due to aggregation and adhesion of cells to one another.

  2. (b)

    Transportation of the necessary nutrients becomes easy in closely associated cells.

  3. (c)

    Acts as an electron donor or receptor.

  4. (d)

    Storage of most of the energy.

  5. (e)

    Provides the binding or receptor site to enzymes.

  6. (f)

    Protects from external factors such as antimicrobials and other environmental changes.

  7. (g)

    Provides adaptation.

During biofilm formation, several species of bacteria communicate with one another through quorum sensing (Davies et al. 1998; Shirtliff et al. 2002). During biofilm formation, genetic information can be modified by horizontal gene transfer (HGT) within and between bacterial species and increase the adaptation in bacteria for changing environments. Moreover, this kind of higher gene transfer rates was observed more in biofilms than their counterparts. It confers protection and survival in adverse environmental conditions such as antibiotics (Costerton et al. 1999; Mah and Toole 2001), predators (Kadouri et al. 2007), and human immune system (Anderson and O’Toole 2008). This way biofilms enhance the virulence of microbes (Brooks et al. 2005).

HGT in biofilms is beneficial to microbes but are harmful to us because antimicrobial resistance and virulence genes get disseminated or new ones get emarginated, making multiple drug-resistant (MDR) strains which are known as multiresistant “superbugs.” Moreover, biofilms’ architecture is tuned under a specific environment with the help of different enzymes secreted by bacteria that modify its EPS composition when a change in nutrient availability occurs (Sauer et al. 2004; Ma et al. 2009).

In the natural environment, 99% of bacteria exist in biofilms. As per reports from the National Institutes of Health (NIH), up to 65% and 80% of all microbial and chronic infections, respectively, are related to biofilms which feature their immense clinical impact (Jamal et al. 2018). Biofilms are responsible for more than 65% of nosocomial infections (Böhme et al. 2009) and approximately 61% zoonotic human infections (García and Percival 2011). Not only human infections but most of the infections caused in animals like pneumonia, liver abscesses, enteritis, wound infections, and mastitis are caused by biofilm-forming microbes (Olson et al. 2002; Clutterbuck et al. 2007).

9.2 Biocontrol Agents Against Biofilms

9.2.1 Plant-Derived Essential Oils (EOs)

Essential oils (EOs) are derivatives of the various parts of the plants such as flowers, roots, leaves, seeds, fruits, bark, herbs, twigs, and seeds. They are hydrophobic and aromatic liquids. From ancient times, herbs and spices are commonly used in our homes, as flavoring agents of food, as a food preservative for its long-time storage, or as a medicinal plant product. EOs perform a significant function in the defense of crops from various microorganisms, insects, and animals (Kerekes et al. 2015). They are obtained either traditionally by methods like extraction, steam distillation, cold press/expressing, and enfleurage or by modern techniques employing microwave or ultrasound waves for extraction or pressurized extractions. Among the 3000 EOs known, 300 EOs are commercially explored which comprise more than 60 individual compounds (Van de Braak and Leijten 1999; De Martino et al. 2009; Cowan 1999). The amount of extracted EOs from plants depends upon factors like the part of the plant used for the purpose, its age, and the extraction method used (Lemberkovics et al. 2004; Reyes-Jurado et al. 2015). Essential oils are classified as depicted below (Kerekes et al. 2015).

Their mechanism of action involves the following: they are lipophilic in nature and therefore are permeable in the cell membrane, and they may inhibit ATP production and ATPase activity and bring about the outward flow of ions or other cellular content (Bakkali et al. 2008), disrupting the genetic (de Oliveira et al. 2010, 2012) as well as cellular material of the microorganisms (Perricone et al. 2015). It was found that aldehyde and phenolic EOs are the most effective in fighting against microbes such as cinnamaldehyde, carvacrol, eugenol, or thymol (Bakkali et al. 2008; Perricone et al. 2015). Gram-positive microorganisms showed more sensitivity to EOs when compared to their Gram-negative counterparts (Burt 2004; Lambert et al. 2001).

figure a

Some EOs can also act as quorum-sensing inhibitors (interfering with the communication and regulation of quorum-sensing genes) which leads to the reduced activity of biofilm formation and other virulence-related factors (Nazzaro et al. 2013). Some of the features which make EOs as future therapeutic agents are: they are easily extracted, are nontoxic to the tissue culture cell lines, are rapidly degraded when mixed in water, and have no side effects to health (Fabian et al. 2006; Warnke et al. 2006; Isman 2000). It has been observed that the presence of EOs modifies the antibiotic tolerance ability of the bacterial cell (Yap et al. 2014), and when the two antimicrobials, which target two different components of the bacterial cell, are combined, it changes the tolerance of the microorganism (Rosato et al. 2007; Cox et al. 1998; Langeveld et al. 2014; Longbottom et al. 2004; Cirino et al. 2014) (Table 9.1).

Table 9.1 Essential oil (EO) associated studies effective against biofilms
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9.2.2 Quorum-Sensing Inhibitors (QSIs)

Quorum sensing (QS) is an interaction strategy in the microbial community that is chemical in nature and is used to regulate various behaviors such as virulence and biofilm formation (Uroz et al. 2009). As soon as the population of bacteria becomes dense, QS compounds start accumulating for the recognition of the population density to activate a corresponding response. Quorum-sensing inhibitors target the QS molecules to reduce the formation of biofilms, and this disruption reduces the growth, virulence, and dispersion of microorganisms (Papenfort and Bassler 2016).

It was proposed that quorum-sensing inhibitors mainly target the following:

  1. 1.

    The signal generator

  2. 2.

    The quorum-sensing molecule

  3. 3.

    The signal receptor

The QS signal receptor mediates the pharmacological action. One of the modes of action that often facilitates the transformation of biofilm pathogenicity is reducing the biofilm’s resistance to conventional antimicrobial treatment. Rasamiravaka et al. (2015) reported several QS-inhibiting compounds, including penicillic acid, solenopsin A, catechin, ellagic acid derivatives, and curcumin. QSIs can be obtained from various sources, but their antibiofilm activity should be explored in future studies.

Most of the plant-derived QSIs have shown to exhibit remarkable antibiofilm activity. Several studies were performed related to QC-mediated inhibition of biofilm formation as shown in Table 9.2. These studies showed that the QSI when used alone or in synergism with various other antimicrobial agents can be used to control biofilms. Christensen et al. (2012) showed that antibiotic tobramycin, when combined with QS compounds including furanone and horseradish juice extract, disrupted the biofilms of Pseudomonas aeruginosa in mouse as experimental organism. The synergic effect of QS molecules and availability of QS inhibitors increased the susceptibility of the P. aeruginosa biofilm to tobramycin. Such methods create a less favorable surface for biofilms to reside on, and they reduce biofilm pathogenicity using QS inhibitors, demonstrating a promising and exciting potential avenue for further exploration. However, more work needs to be done to incorporate these ideas into an in vivo environment, particularly in the case of biofilm formation, as in vitro biofilm models may not mimic complex in vivo conditions.

Table 9.2 QSI associated with biofilm control
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9.2.3 Antimicrobial Peptide (AMP)

Antimicrobial peptides (AMPs) are also known as “host defense peptides.” In higher eukaryotic organisms, AMPs are “L”-shaped cationic molecules containing 15–50 amino acids having molecular weights between 1 and 5 KDa and are produced as part of an innate immune defense mechanism by eukaryotes and prokaryotes. They usually contain arginine and lysine residues in excess (Izadpanah and Gallo 2005; Rossi et al. 2008; de la Fuente-Núñz et al. 2012). They act on a wide variety of organisms like bacteria, yeasts, fungi, viruses, and even cancer cells to directly kill them. They show specific and diverse activities related to normal immune homeostasis, which includes a variety of cytokine and growth factor-like effects. They mainly target cell membranes because the peptides with a positive charge and cell membranes/biofilm surfaces of microbes with a negative charge attract each other, killing the active and slow-growing bacteria in biofilms (Melo et al. 2009; Jorge et al. 2012). However, AMPs at deficient concentrations change their activity from bactericidal to bacteriostatic (Beloin et al. 2014). Cationic peptides induce gene expression in microorganisms by binding to their DNA because they can pass through the cell membrane.

As per the literature review done by Yasir et al. (2018), the following mode of actions of antimicrobial peptides worked for biofilm removal (Table 9.3):

Table 9.3 Mode of action of AMP (Yasir et al. 2018)
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Various studies (Table 9.4) reported that AMPs are more effective when combined with various conventionally used antibiotics. Also, it was found that by changing the amino acid composition of AMPs, antimicrobial activity can be increased (Ma et al. 2012; Xu et al. 2014; Tiwari et al. 2015). One such example of genetic manipulation is the replacement of functional “defective” sequence RR7 in one of the AMP R-FV-I16 by inserting the antibiofilm sequence FV7 (Xu et al. 2014). Another way in which the manipulation of AMPs can be done is by designing STAMPs (specifically targeted AMPs). The benefit of these STAMPs is that they harm pathogenic bacteria but not nonpathogenic ones (Li et al. 2010; He et al. 2009). These AMPs rupture the cell membrane or act as membrane perturbers (Wimley and Hristova 2011). Genetically engineered peptide such as peptide RN3 (5-17P22-36) of eosinophil granules can also be explored as a potential antibiofilm agent (Venge 1999; Acharya and Ackerman 2014).

Table 9.4 Antimicrobial peptides associated with biofilm control
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9.2.4 Biofilm-Degrading Enzymes

Primarily, enzymes whose composition are proteins or RNAs are natural catalysts that either accelerate chemical reactions without being consumed or altered or increase reaction rates without changing the chemical equilibrium between the reactants and products. Based on their functional characteristics on the ENZYME database (https://www.expasy.org/enzyme/), there are mainly six classes of enzymes (Shen and Chou 2007) (Table 9.5):

Table 9.5 Different classes of enzymes with their mode of action (Shen and Chou 2007)
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The biofilms produce an extracellular polysaccharide substance (EPS). The main composition of EPS are proteins, polysaccharides, and nucleic acids (Low and White 1989, Bayles 2007). EPS adheres to surfaces and protects the associated microorganisms from various antimicrobials and other shearing stress due to its structural stability factors (Cooksey and Wigglesworth-Cooksey 1995; Ramasamy and Zhang 2005). Therefore, disorganization of EPS with certain classes of enzymes will lead to detachment of biofilm (Stewart 2015) and would expose the bacteria to these agents.

Various actions of enzymes involve biochemical breakdown of EPS, inhibition of QS signaling, degradation of the adhesive bonds between cells, and the toxic substance accumulation, the cumulative effect of which leads to lysis of affected cell and deactivation of necessary enzymes needed for cell development (Thallinger et al. 2013). Enzymes such as DNase I-amylase and dispersin B (DspB) minimize the exopolysaccharide layer of the microbe; thus, the number of biofilm cells is reduced (Eckhart et al. 2007; Whitchurch et al. 2002; Kalpana et al. 2012). Moreover, the specificity of enzymes and their activities are interfered or influenced by many environmental factors like availability or nonavailability of activators, cofactors, or inhibitors, temperature, substrate, and pH (Baidamshina et al. 2017). One of the critical characteristics of enzymes is that they are substrate-specific, i.e., they cleave the EPS at a specific site (Bridier et al. 2015).

EPS composition governs a significant role in deciding whether enzymes alone or a blend of enzymes in synergy with other treatment methods, physical (ultrasound, stress) or chemical (chelating agents, buffers, surfactants, and detergents), is required to remove the EPS altogether (Thallinger et al. 2013; Darouiche et al. 2009; Izano et al. 2007). Additionally, significant reduction of biofilm mass is obtained, when the active enzyme is immobilized by entrapping in substances like poly(ethylene-alt-maleic anhydride), ceramics, polycaprolactam, etc. (Regina et al. 2012). The resistance of biofilm-forming pathogens to enzymes is quite uncommon; however, there are few exceptions, like L. monocytogenes resistant to lysozyme (Nguyen and Burrows 2014), S. aureus mutant to lysostaphin (Gründling et al. 2006), and P. aeruginosa to peroxidase (Lewis 2001).

Though enzymatic therapies also have some limitations, the first one is that they are costly compared to several other antimicrobials. In the natural environment, biofilms are a composition of variably diversified microbial species; therefore, the EPS is also diverse (Jahid and Ha 2014). This diverse biofilm matrix is difficult to treat with substrate-specific enzymes. It is known that wrong selection of enzymes and their combinations sometimes leads to attenuation instead of killing (Baidamshina et al. 2017), or sometimes it does the reverse of increasing virulence factors and biofilm formation, i.e., induction of biofilm formation occurred in Pseudomonas aeruginosa and Enterococcus faecalis that is generated by a protease enzyme (Ołdak and Trafny 2005; Xu et al. 2014) (Table 9.6).

Table 9.6 Enzymes associated with biofilm control
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9.2.5 Bacteriophage

Bacteriophages were discovered by Frederick Twort in 1915 and Félix Bd’Hérelle in 1917 independently. These are viruses, shorter in size, and survive on host prokaryotes (d’Herelle 1917, 1918). Taxonomically, they are divided into Myoviridae, Siphoviridae, and Podoviridae (Ackermann 2009). Bacteriophages are bacterial viruses that exhibit two kinds of life cycles: the first one is lytic and the other is lysogenic. They have the ability to lyse the host bacterial cell or grow generation by generation with bacterial cell (Twort 1936). Bacteriophages have been applied medically to take care of human microbial diseases from the last 80 years in former Soviet Union and European countries (Clark and March 2006).

Bacteriophages penetrate biofilms (Pires et al. 2011; Vilas Boas et al. 2016); therefore, phages are active against both planktonic and biofilm form of bacteria (Kim et al. 2011; Gutiérrez et al. 2016). Antiphage refuges are formed in bacteria in biofilms, which establishes bacteria phage coexistence (Heilmann et al. 2012). The phage takes advantage of high cell density in biofilm and spreads rapidly; this weakens the biofilm structural integrity of bacterial cells and causes its lysis. Phages and antibiofilm substances can be applied together to target host bacteria for complete removal of biofilms (Uppuluri and Lopez-Ribot 2016). Alternatively, another method to enhance the broad host range of bacteriophages is that they can be genetically engineered. Dispersin B from Aggregatibacter actinomycetemcomitans is a biofilm-degrading enzyme expressed from engineered phages (Lu and Collins 2007).

The phage therapy has many advantages over conventional antibiotic therapy (Matsuzaki et al. 2005): it attacks the targeted microbe and does not affect the healthy microbial flora, is effective against MDR and phage-resistant bacterial mutants, is cheaper compared to antibiotics, and has minimum/rare side effects (Matsuzaki et al. 2003). One of the critical factors determining the efficacy of phage therapy is attaining high phage “killing titers” (Abedon and Thomas-Abedon 2010). However, Defence mechanisms and other host-mediated responses should be considered before adapting any conventional therapeutics methods in mammals. The phage therapeutics should be developed to have active, harmless, safe, and long-term treatment options (Szczaurska-Nowak et al. 2009). Phages also modulate the immune system. One of the primary example is respiratory burst induced by bacterial cell wall that is inhibited by phagocytes in human blood (Levin and Bull 2004). Another essential feature observed about phages is the normalization of cytokine production by blood cells isolated from patients (Weber-Dabrowska et al. 2000). All these studies showed that mammal–phage interactions should be explored in detail for their further use as a treatment option either alone or in synergism with antibiotics.

Many phage combinations can be applied to obtain broader activity, i.e., cocktails of phages (Chan et al. 2013). Alternatively, an excellent strategy to fight against older biofilms is the use of combinations of both bacteriophages and antibiotics. The combination of a bacteriophage with amoxicillin was much more effective in reducing a mature biofilm of Klebsiella pneumoniae B5055 than each of the agents alone. The advantage of using phage–antibiotic combinations are decreased with the emergence of resistant cells that would appear upon using phages or antibiotica lone (Chhibber et al. 2009a, b). The recent multidrug-resistant (MDR) strains found in clinical isolates of bacteria are emerging day by day, and it has become difficult to treat these infections causing endemics (Alisky et al. 1998; Carlton 1999).

The principal downside of the use of therapeutic phages in medical treatment is the introduction of resistance against phages by pathogenic bacteria. The resistance of bacteria to phage may be developed due to inactivation of phage by the immune system of the host, and it may occur when virulence genes get incorporated into the host bacterial genome (Dolan 2009). The bacteriophage therapy has limitations of specificity towards the host which limits the phage to have a narrow range of host bacteria except for some exceptions, e.g., Staphylococcal phage K, Sb-1, and Stau2 (Curtin and Donlan 2006; Sharma et al. 2005). Cross-infections in closely related species, for example, of Staphylococcus by polyvalent phage K, SK311, U16, ɸ131, and ɸ812 are also one of the problems while using phage therapy (Pantůček et al. 1998). However, if a phage uses a bacterial virulence factor as a receptor, it should target the “virulent” subpopulation only (Bedi et al. 2009). Some of the obstacles which come across in the commercial production of phage as therapeutic agents are their complex manufacturing and testing methodology, current regulations, patenting and efficacy problems, and costly clinical trials (Debarbieux et al. 2016; Vandenheuvel et al. 2015). Despite these limitations, it can be summarized that phages are quite safe and effective as a future antibiofilm agent. Table 9.7 summarizes some of the phage-associated biofilm control studies done in the past years.

Table 9.7 Bacteriophage associated with biofilm control
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9.3 Conclusion

The infections caused by biofilms are chronic, recurrent, and resistant to antibiotics. Also, the contamination caused by them in industrial systems is challenging to eradicate. As a result of strengthening antimicrobial drug resistance, conventionally used antibiotic therapy alone is not sufficient to control biofilm-related infections. Hence, another category of molecules/remedies to treat biofilm-associated threats is an appealing area and still has to be explored by researchers. Each new novel molecule has some advantages and limitations. Although in AMPs, enzymes, and bacteriophages, QSIs have broad-spectrum antibacterial function and tend to be protected from the occurrence of microbial resistance and could work synergistically with antibiotics, extensive research is needed such as chemical studies of the EPS matrix of various microbes and complex immunomodulatory activities inside the host cells which can reduce/enhance their efficacy. However, the effectiveness of biological control strategies might be affected through a range of physical and chemical factors. These factors include temperature or time applied in the biocontrol method, treatment of single species or multiple species biofilm, development strategy used by an organism to develop a biofilm, and composition of the surface matrix. Therefore, strategically defined control methods or validation studies of new emerging biocontrol assays against microbial biofilms need to be done before the commercialization of these products.