Approaches to Enhance Therapeutic Activity of Drugs against Bacterial Biofilms

Abstract

Biofilm may be a consortium of microbial species where the cells of microbes attach to both life form and inanimate surfaces inside a self-made matrix of extracellular polymeric substance (EPS). Biofilm matrix surrounding the polymicrobial environment makes them highly resistant to harsh conditions and antibacterial treatments. The two significant factors that differentiate planktonic from biofilm resident microbes are EPS containing a variety of macromolecules and a diffusible molecule for transferring signals known as quorum sensing (QS). Against this backdrop of microbial resistance and cell signaling, different approaches have been developed to interfere with the specific mechanisms of intracellular and extracellular targets that include herbal active compounds and synthetic nanoparticles. This chapter outlines the features of biofilm development and the approaches with the evidence that can be incorporated into clinical usage.

1. Introduction

In seventeenth century, Antonie von Leeuwenhoek saw microbial aggregates on the scrapings of the plaque from his teeth that was termed as “biofilm” by Bill Costerton in 1978 [1]. The biofilms were not characterized for their physical and chemical properties until the end of 1960 [2]. The evolution of scanning electron microscopy and transmission electron microscopy allowed for identifying the biofilm from wastewater treatment plant [3] after when Heukelekian and Heller identified the “Bottle effect” on marine microbes where there is a significant difference in the microbial population between in situ and in vitro due to environmental or man-made changes. Biofilm is an aggregation of microbially derived sessile communities having various bacterial colonies or individual cells in the group, which adheres to the surface. This group of cells attaches on an extracellular polymeric substance (EPS), a matrix that is mostly comprised of environmental DNA (eDNA), proteins, and polysaccharides, which provides significantly excessive resistance to antibiotics [4]. Bacterial biofilm can be formed in response to various factors such as high salt concentration, restricted nutrients, high pH and pressure, and UV radiation. Biofilm life process is depicted in Figure 1.

Figure 1.

Biofilm life process. (1) Planktonic bacteria attaches to the exterior face. (2) Adhesion, irreversible attachment occurs at this phase. (3) EPS is secreted and results in a matrix that forms the basis for biofilm’s structure and initiates the onset of biofilm maturation. (4) The biofilm becomes completely matured, with the tower-like structures dispersed with water channels for the movement of oxygen, nutrients, and for discharging waste products.

The biofilm formation can be described in three steps:

1. Flexible attachment of bacteria to the surface subsequently irreversible attachment with the help of adhesive structures of bacteria.

2. Production of EPS and development of an organized structure entrapped inside an EPS matrix.

3. Finally, bacterial cell starts to break out from the biofilm and spread into the habitat through chemical signaling [5].

2. Biofilm: a threat to antibiotics and infections caused by biofilm

Around 80% of chronic and periodic microbial infections in the human bodies are caused by bacterial biofilm. Bacteria’s present inside the biofilm aids to the chronic phase of infection, when released from the biofilm can cause an acute phase of infection [6]. The infections caused by bacterial biofilm can be placed in two broad categories such as device and non-device-associated infections. They can develop on or inside medical devices that are built in body such as central venous catheters, mechanical heart valves, pacemakers, urinary catheters, which cover both Gram-positive and Gram-negative bacteria or yeasts. These organisms on the medical devices may cause blood stream and urinary tract infections in the patient [7]. Table 1 shows the microbial species that colonizes the devices based on the type of medical device and time taken for their action.

S. No	Medical device	Microbial organisms
1.	Contact lenses	Escherichia coli, Staphylococcus epidermidis, Pseudomonas. aeruginosa, species of Candida, Serratia and Proteus, Staphylococcus aureus. [8]
2.	Central venous catheters	Klebsiella species, P. aeruginosa, and Enterobacter species [9]
3.	Mechanical heart valve	Enterococcus and Candida spp, Streptococcus species, S. aureus, S. epidermidis, Bacillus [10]
4.	Urinary catheters	E. coli, Enterococcus faecalis, S. epidermidis, P. aeruginosa, Proteus mirabilis, Klebsiella pneumonia [11]

Table 1.

Medical devices and associated biofilm organisms.

Microbial biofilm show 10–1000 times more antibiotic resistance than the planktonic species [12]. Bacterial biofilm offers huge evolutionary advantage for the bacteria including changes in environmental pH, resistance to antimicrobial agents, and phagocytic attack [13].

3. Quorum sensing (QS) and interaction

The bacterial cells have intercellular communication that is delivered through the extracellular signaling molecules known as autoinducers. The collection of signaling molecules enables individual bacterial cells to analyze the total number of bacteria, that is, cell density known as quorum sensing. In low-density planktonic populations, bacteria releases low-molecular-weight, highly diffusible, signal molecules (autoinducers, such as oligopeptides in Gram-positive bacteria and N-acyl-L-homoserine lactones in Gram-negative bacteria) at very low levels to produce changes in gene expression. When critical mass of bacterial population becomes high, the concentration of autoinducer molecules increases in the EPS followed by allowing individual bacteria to sense the presence of other bacterial species [14].

4. Conventional treatments and antimicrobial resistance

Biofilms are considered to be important owing to their potency in showing resistance toward antibiotics and antifungals. Once routed within the wound infection, biofilm shows enhanced tolerance to conventional treatments. Antibiotics work by deranging the cell wall of bacteria and affecting the DNA replication, repair, and protein synthesis. Apparently biofilm has various mechanisms through which they resist the effectiveness of antibiotics [15]. The primary defense mechanism involves EPS, which is capable of restricting the permeability of antibiotics into the cell thereby trapping them in the pores, followed by acidic internal environment and lack of oxygen. Ultimately, the lysis of genetic component can be carried between the cells to extend antimicrobial resistance.

The persister cells within the biofilm have the potency to restrict the effects of antibiotics targeting the cell division [16]. Figure 2 illustrates the mechanism through which biofilm develops resistance to conventional antibiotics [17].

Figure 2.

Antibiotic resistance associated with biofilm.

The resistance can be developed through persistent cells, phenotype of the biofilm, inhibition in antibiotics penetration, production of enzymes that resist the action of antimicrobial agents.

5. Nanoparticles (NPs) as antibiofilm agents

Nanotechnology is fascinating, which likely benefited the field of biomedical and became widely conceded for the treatment of various diseases. Numerous resistance mechanisms set biofilm as one of the major disputes in infection treatment, which can be addressed by the strategy of using nanoparticles. NPs have two or three dimensions in the size range of 1 to 100 nm. They are of various types based on their size, shape, and composition [18]. Their higher surface area built them as suitable drug career, which has the capability to immobilize the compounds on their surface to increase their solubility and targeted delivery [19]. They can be of two types, polymer NPs and metallic NPs. Polymer NPs also possess the advantage of retaining the drug inside the cavity and delivers the drug at the target area in either entangled or immobilized form. Reports suggest that NPs disrupt the integrity of biofilm by interacting with EPS, eDNA, proteins, lipids, and biofilm release reactive oxygen species (ROS) on interaction with NPs that can damage the cell envelope, cell membranes, cell structures, and biomolecules of the microbes. Figure 3 represents the general mechanism involved in combating biofilm through NPs [20].

Figure 3.

Depiction of various mechanisms involved in combating biofilm through NPs.

The nanoparticles can restrict biofilm by disruption electron transport between cell membrane, damaging the peptidoglycan layer, breaking through the cell membrane, denaturation of proteins, and DNA damage.

6. Synthesis of nanoparticles

Nanoparticles can be synthesized in laboratory broadly using two different approaches, that is, bottom-up and top-down techniques. The top-down approach implies breaking the bulk material into nanosized structures, which is based on miniaturizing the bulk substance through fabrication process and produces the NPs of appropriate properties. Bottom-up technique is an alternative approach because it creates less waste and involves building up of a material from the bottom [21].

7. Types of nanoparticles

Polymeric NPs can be engineered to release antibiotics, antibacterial agents, and bacteriostatic peptides or by modifying their chemical surface. The antibacterial activity of these organic NPs is due to polycationic groups accountable for cell damage through ion exchange interaction between bacteria and polymer surface with charges [22]. Metals are used in the synthesis of nanoparticles because of their antibacterial property broadly used in managing infections. Metallic nanoparticles can exert physical disruption to bacterial biofilms. Table 2 enlists the types of metallic nanoparticles and their potential antimicrobial property [23].

S. No	Metallic nanoparticles	Properties
1.	Zinc oxide	These NPs gets accumulated inside the cell releasing H2O2 and zinc ions thereby causing cell wall disruption.
2.	Titanium dioxide	Generation of reactive oxygen species.
3.	Copper oxide	Lipid oxidation takes place through reactive oxygen species and hydroxyl free radicals.
4.	Carbon nanotube	Reactive oxygen species results in cell wall disruptionthereby oxidizing lipids and proteins.
5.	Gold	They produce strong electrostatic effects and reacts with cell membrane.
6.	Silver	Releases silver ions and causes electron impairment DNA damage.

Table 2.

Metallic nanoparticles and their antimicrobial property.

The pH of micro-environment, magnetic field, or light can be used to turn on the nanomaterials or transform it to more active species enhancing their antibiofilm activity. These are often metallic nanoparticles due to their broad-spectrum antimicrobial activity and rich surface chemistry [24]. For negatively charged bacteria, the adhesion property rises because of the positively charged surface of NPs and the binding takes place through electrostatic interactions and Van der Waals interaction especially to cell membrane proteins [25].

8. Metal NPs against biofilm

CuO NPs inhibit formation of biofilm that was studied by Agarwal et al. that concluded eradication of biofilm formed by MRSA and E. coli with the exposure period of 4 days to CuO NPs at the concentration of 50 μg/ml [26]. ZnO NPs can exhibit antibacterial action between the concentration of 20–500 μg/ml for E. coli and S. aureus that can be enhanced by additional physical exposure and amplified by ultrasound [27]. MgO NPs can act against Gram-positive and Gram-negative bacteria, bacterial spores, and viruses at higher concentration of 100–1200 μg/ml. TiO₂ NPs can destroy biofilms of both Gram-positive and Gram-negative bacteria but the latter being more sensitive due to the sturdy layer of peptidoglycan that increases the absorption of reactive radicals [28]. However, their toxicity to humans and environment outweighs their advantages. Gold and silver NPs offer huge advantages such as higher surface area to volume ratio, small size, amenability, cheaper method of synthesis. Extensive research studies have been accomplished over the recent areas involving AgNPs and AuNPs. Three important steps involved in their antimicrobial action are a) interaction with biofilm when it comes into contact with the surface, b) subsequent penetration of NPs into the cell based on this interaction, and c) NPs as a whole or ions (Au⁺ and Ag⁺) reacts with cellular and biofilm components. The factor that plays significant role in penetration includes particle size, surface chemistry, surface charge, and concentration.

8.1 Silver nanoparticles: a biofilm buster

Silver has been used since remote time because of their therapeutic properties and their antibacterial activity and also explored through extensive research in medical field. Topical ointments and creams contain silver for treating burn wound infection. Several approaches are involved in synthesis of AgNPs, which include the use of microorganisms and plants but one of the easiest and convenient methods is through chemical synthesis [29]. Table 3 enlists some of the sources that can be used for the synthesis of AgNP.

S.No	Approaches	Sources for synthesis
1.	Microbial approach	Cedecea sp., Pseudomonas sp., Lactobacillus plantarum, Aspergillus fumigatus, Aeromonas sp., Klebsiella pneumonia, Corynbacterium sp., Enterobacter cloacae, Verticillium sp., Fusarium semitectum, Fusarium oxysporium. [30, 31, 32]
2.	Plant synthesis	Aloe vera leaf extract, Azadirachta indica, Cinnamomum camphora, Emblica officinalis, Pinus eldarica, Cassia auriculata leaf extract, Geranium leaf extract, Ficus benghalensis leaf extract, Aqueous fruit extract of Syzygium alternifolium, fruit extract of Sambucus nigra [33,34]
3.	Chemical reduction	DMF, NaBH4, Trisodium citrate, ascorbic acid, dextrose,; ethylene glycol, glucose.

Table 3.

Sources used for synthesis of AgNP.

The synthesis of AgNPs can also be done by utilizing other physical methods such as evaporation-condensation and laser ablation, UV-initiated photo reduction, electrochemical synthetic method, irradiation methods [35]. Studies concluded that geometric mean diameter, shape, pH, and source for synthesis of AgNPs influence their efficiency. The synthesized AgNP can be characterized using UV-visible spectroscopy, X-ray diffraction, scanning electron microscopy, transmission electron microscopy for their structural properties. Although AgNPs were remarkably noted for their potential in pathogenic control, their effect on EPS has not been given sufficient attention [36].

8.2 Natural compounds as antibiofilm agents

Herbal compound aids the determination of novel constituents with interesting structures and biological activity. The antibiofilm properties of natural products rely on the inhibition of polymer matrix formation, resisting cell adhesion and attachment, breaking in ECM generation, and reducing virulence factors generation, thereby obstructing QS network and biofilm development [37]. The natural compounds that possess antibiofilm properties can be broadly classified into phenolics, essential oils, terpenoids, lectins, alkaloids, polypeptides, and polyacetylenes [38]. They either act merely or synergistically by different mechanisms. Various researches have been carried out with natural products that are discussed below:

8.2.1 Garlic

Allium sativum L has been extensively used in treating numerous diseases such as wound infection, malaria, common cold, sexually transmitted diseases [39]. Garlic possibly has a QS-interfering compound. DNA microarray analysis disclosed that Ajoene, a garlic-derived sulfur-containing compound, restricted QS-regulated gene expression in P. aeruginosa. Reasonable designing and biological screening of all compounds from garlic was carried out, resulting in the identification of a potent QS inhibitor N-(heptylsulfanylacetyl)-l-homoserine lactone. This element was found to disrupt the QS signaling by inhibiting transcriptional regulators LuxR and LasR. Recent studies have proved the antiswarming, anti-adherence, and antibiofilm activity of the aquatic extracts of garlic [40]. Ethanolic and methanolic extracts of garlic against six different bacterial species (Escherichia coli, Staphylococcus aureus, Bacillus cereus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Klebsiella pneumonia) show antibacterial activity at the concentration of 125–500 mg/mL through disc diffusion, and the A. sativum L extracts were potent enough restrict biofilm structures and the concentrations of each extract depend on the inhibitory effect [41].

8.2.2 Onion

Extracts of onion contains pharmaceutical properties that can be used as one of the promising therapies for the treatment of neoplastic, metabolic, and immunological diseases, which also involves bacterial, viral, and other fungal infections [42]. The anti-adherence, antibacterial, antibiofilm, and antimotility role of aqueous extracts of fresh or powdered onion and onion oil were studied from which the aqueous extracts of fresh and powdered onion showed more powerful inhibitory effects on biofilm than onion oil on the growth of both Gram-positive and Gram-negative bacteria [43]. Systematic assessment of quercetin, total phenolics, flavonoids, antioxidants, antibacterial, and antibiofilm or antibiofouling properties of methanolic extracts of fresh and aging onions of six varieties was studied by Kavitha et al., which concluded that the onions that had been stored for 3 months showed the best antibiofilm effects. The red variety of Allium cepa extract was found to have higher antimicrobial activity when compared with the white and yellow varieties. At the range of about 50 μg mL^–1, the extracts were observed to reduce the biofilm growth of P. aeruginosa and S. aureus [44].

8.2.3 Rhubarb

Rhubarb is one of the most traditionally available medicinal materials included in Pharmacpoeia due it its bacteriostatic and anti-inflammatory properties. Emodin is the bioactive compound that has the ability to reverse multi-drug resistance. Natural emodin is obtained from Rheum palmatum L., Rheum tanguticum Maxim ex Balf, and Rheum ocinale [44]. Yan et al. studied the activity of emodin against S. aureus biofilm and confirmed the molecular mechanism that they decrease the release of eDNA and represses the biofilm-forming genes such as cidA, icaA, dltB, agrA, sortaseA, and sarA [45].

8.2.4 Banana

Studies concluded the antibacterial properties of banana in traditional medicine across the world. Generally, stem juice, flowers, and fruits of the banana plant are utilized for treating diarrhea and dysentery [46]. Vijayakumar et al. studied the antibiofilm properties of Musa acuminata Colla. against P. aeruginosa and described the mechanism of inhibiting the secretion of biofilm proteins and cell surface hydrophobicity productions [47].

8.2.5 Ginger

Ginger had been used in food and medicine for thousand years with the evidence demonstrating that it has antibacterial activity against the commercially available antibiotics by inhibiting QS signaling pathway [48]. Kim et al. initially investigated the inhibition of biofilm with ginger extract in P. aeruginosa. The biofilm assay demonstrated that the ginger extract decreased the biofilm development by 39–56% by reducing the formation of extracellular polymeric substances (EPS), which was associated with the suppression in secondary messenger, bis-(3c-5c)-cyclic dimericguagranosine [49]. Studies have shown that ginger essential oil at the biofilm inhibitory concentration (BIC) of 1.56 μL mL⁻¹, S. aureus had 94% inhibition of biofilm, and at BIC 0.78 μL mL⁻¹Enterococcus faecalis, K. pneumonia, and E. coli showed 91, 89, and 83% inhibition of biofilm [50].

Table 4 enlists the natural compounds with antibiofilm activity.

S.No	Source	Active compound	Mechanism of action
1.	Origanum vulgare (oregano)	Carvacrol	Post-translational inhibition againstlasI, which effects N- acyl-homoserine lactone secretion. It mostly acts on QS machinery against P. aeruginosa [51].
2.	Apis mellifera (Honey)	Defensin-1	Manuka and Honey dew significantly reduce cell viability of S. aureus, P. aeruginosa, S. agalactia [52]
3.	Curcuma longa L (Turmeric)	Curcumin	Restricts pellicle formation, Pilli motility and ring biofilm formation by interaction with biofilm response regulator BfmR [53].
4.	Camellia sinesis (L)	Epigallocatechin-3-gallate	Reduce the curli production and expression of curli-related proteins csgA, csgB, and csgD increases the degradation of sigma factor (RpoS) by ClpXP protease [54].
5.	Capsicum annum (Bell pepper)	Capsicum storage peptide 37 (CSP37)	Inhibited the formation and development of biofilm in common pathogenic strains at the concentration of 5 and 10 mg/ml through CSP [55].

Table 4.

Natural compounds with antibiofilm activity.

9. Conclusion

In recent times, the concept of biofilm has influenced almost every treatment step of infection due to high level of protection against antibiotics and antimicrobial agents, being the thrust to human medical management. Hence, there is a crucial demand to develop novel strategies to surpass the antibiotic resistance after understanding the clear mechanisms behind it. The plant compounds, phytochemicals, and nanoparticles can be fused with antimicrobial agents, which have substantial research evidence for their antibiofilm effects through their synergism. In spite of the clinical trials being done on such compounds, further study is required to prove their safety and effectiveness to support the clinical systems.

Acknowledgments

We would like to thank our principal Dr.M. Ramanathan D.Sc., for providing us with sufficient facilities to complete this book chapter.

Conflict of interest

The authors declare no conflict of interest.