Bacterial Biofilm Eradication in Human Infections

Abstract

Microbial biofilms are elaborate and highly resistant aggregates formed on surfaces or medical devices, causing two-thirds of infections and leading to a serious threat to public health. Their presence increases the rate of infections and mortality in the affected individuals. The strategies and eradication patterns are necessary to be established or implemented to eliminate them in human beings. This chapter highlights recent approaches for combating bacterial biofilms, including the methods used by promising antibiofilm compounds to enhance the total elimination of bacterial biofilms involved in some specific human infections. Biofilms must be eradicated to ensure efficient treatment of the infections.

1. Introduction

Bacterial drug resistance and biofilm infections can result in a wide range of diseases and associated complications, such as sepsis, endocarditis, pneumonia, and even death in the worst scenarios [1, 2]. Bacterial biofilms are complex and elaborate microbial communities that are very resistant and readily colonizing the surfaces of organs or medical implants to cause intractable and recurring infections [3]. They have a large spectrum of activities ranging from nosocomial setting, especially linked to lower respiratory, urinary tract, and surgical wound infections as well as the medical devices used during treatment resulting to a serious challenge to patients’ health [4]. Bacteria tend to work in synergy and create groups to achieve resistance about 10–1000 times on antibiotics and the human immune system [3, 4], while also secreting various virulence factors in certain cases [2]. The arrangement of bacteria in the biofilm in a micro-colony shape enclosed in an extracellular polymeric substance (EPS) of the matrix, is the highest surviving mechanisms of biofilms giving them more resilience and versatility [5]. Fleming’s discovery of penicillin in 1928 marked the advent of antibiotics and the subsequent production of several antibiotics has been a lifesaving against bacterial infections [6]. Many strategies are being used today to control and eradicate important biofilms that ranges from drugs and cell methods to non-biological modern technologies. These include novel antibiotics and their carriers, bacteriophage and its components, antiseptics and disinfectants, small molecule anti-biofilm agents, surface treatment strategies, ultrasound-induced microbubbles, nanomaterials and nanostructure functionalization, as well as multifunctional coating [4, 7, 8]. This chapter therefore, highlights recent approaches for combating bacterial biofilms, including methods used by promising antibiofilm compounds to enhance the total elimination of bacterial biofilms involved in some specific human infections.

1.1 Bacterial biofilms in human infections: Examples and consequences

Bacteria can grow to a biofilm during favorable condition. Notwithstanding certain species seem to have a preference to form biofilms and examples of these are given in Table 1 [9]. Centuries ago it was thought that bacteria only existed in free floating forms or as planktonic organisms until the 1970s when they were observed adhering and growing on surfaces [10].

Organism	Site of biofilm formation
Actinomyces spp.	Teeth
Escherichia coli and other enterobacteria	Urinary catheters
Escherichia coli	Intestinal tract
Lactobacillus spp.	Vagina, Teeth
Pseudomonas aeruginosa	Lungs of cystic fibrosis patients
Staphylococcus aureus	Implantable medical devices
Staphylococcus epidermidis and other coagulase-negative staphylococci	Implantable medical devices
Streptococcus spp.	Teeth

Table 1.

Examples of representative bacterial pathogens that frequently form biofilms.

Most of these species form biofilms at their natural sites and constitute the microflora in the human body (Figure 1) [9]. Biofilms are extremely resistant to orthodox antimicrobial treatment and to the host immune response. It is reported to play a key role in various chronic infections in human diseases, thereby representing a challenge in clinical settings [9, 11]. The control and treatment of infections caused by biofilms are challenging in medical settings and this has led to the development of novel technology and new strategies to combat microbial biofilms [9].

Figure 1.

Bacterial biofilm in human infections.

1.2 Bacterial biofilm eradication mechanisms

Biofilms are difficult to eradicate even with extended treatment and important attention is given to less conventional treatments, especially those that employ macromolecular species [12, 13]. Because of this failure of conventional therapeutics biofilms require other strategies and mechanism for their elimination [14]. Many strategies are presently under investigation, aiming to effectively eradicate biofilm-related infections and several agents have been known to have anti-biofilm activity, including some natural products, synthetic compounds, enzymes, peptides, chelating agents, polyphenols, as well as some antibiotics [13, 14]. These biofilms have different mechanisms of action.

2. N-acyl homoserine lactones mediated quorum sensing inhibition

Quorum sensing (QS) is a system where bacterial cells communicate through the activation of specific signals, with key objective of enabling the adaptation of bacteria hostile environmental conditions, including bacterial population densities. This process involves reacting to extracellular chemical signaling molecules called auto-inducers (AIs) through synthetization and sensing. Gram-negative bacteria communicate using AIs, most commonly acyl-homoserine lactones (AHLs) and other small molecules [14]. The mechanism is the disruption of AIs and then mitigate quorum sensing controlled responses for biofilm control. Most of the anti-biofilm chemical structures under studies are: N-acyl homoserine lactones (AHL) (Figure 2a), triazole dihydro furanone (Figure 2b), synthetic halogenated furanone (Figure 2c), EGCG (Figure 2d), and ellagic acid (Figure 2e) [14]. Numerous AHLs disrupt biofilm formation. Important biofilm inhibitory effect against P. aeruginosa and Serratia marcescens were observed when the lactone moiety of the native AHL molecules is replaced by cyclohexanone or cyclopentyl [15, 16].

Figure 2.

Chemical structures of some anti-biofilm compounds that inhibit AHL-mediated QS. (a) AHL. (b) Triazole dihydro furanone. (c) Synthetic halogenated furanone. (d) Epigallocatechin gallate (EGCG). (e) Ellagic acid.

3. Membrane permeabilization and potential alteration

Pore formation and destruction of the cytoplasmic membrane is as a result of bacterial membrane modification. There are three possible mechanisms of bacterial membrane disruption by antimicrobial peptides (AMPs): (a) pore-induced barrel-stave pathway, (b) toroidal pathway, and (c) carpet (non-pore) mode (Figure 3) [17]. Peptides that inhibit bacteria by disrupting their membranes and consequently inhibiting enzyme production are produced and post-translationally modified. These peptides are lantibiotics that are ring-structured peptide antibiotics containing thioether amino acids (methyllanthionine or lanthionine) or unsaturated amino acids (2-amino isobutyric acids or dehydro-alanine) [18]. A pore-forming lantibiotic called subtilin, produced from a Gram-positive bacteria B. subtilis strain ATCC6633, induces the dissipation of transmembrane electrostatic-potential releasing cytoplasmic solutes from B. subtilis and Staphylococcus simulans membrane vesicles [19]. In Figure 2, AMP outreaches the cytoplasmic membrane via permeabilizing the outer membrane in Gram-negative bacteria, while in Gram-positive bacteria, the AMP directly disperses through nano-ranged pores of the peptidoglycan layer. After binding to the inner membrane, AMPs can create three types of pores, that is, barrel-stave pore, toroidal pore, and carpet model [17].

Figure 3.

Mechanism of action of AMPs on the membrane system of Gram-negative and Gram-positive bacteria [17].

4. Peptidoglycan cleavage

Peptidoglycan, the cleavage of which is also known to inhibit biofilm formation, is a layer located in the cell walls of many bacteria and originates from amino acids and sugars [20]. Peptidoglycan cleavage causes a change in protein composition and amount of teichoic acid in the bacterial cell wall resulting to biofilm inhibition [20]. An example of peptidoglycan hydrolases is endolysin encoded by bacteriophages [21]. Endolysin can work on multidrug-resistant strains, by disrupting biofilms in vitro e.g., PlyC (specific Streptococcal bacteriophage) [22].

5. Inhibition of bacterial cell division

One mechanism used to stop biofilms from growing is to inhibit cell division. Peptides having antimicrobial activity inhibit cytoplasmic proteins that play a big role in cell division and also promote cell growth by penetrating the bacterial cytosol through formation of channels at outer membrane or via a flip-flop of phospholipids (when the cell is ready to divide, then the nuclear membrane melts) [14]. Drosocin, pyrrhocoricin, and apidaecin are proline-rich antimicrobial peptides (AMPs). They can impede the initiation of chromosomal DNA (cDNA) replication by binding with a shock protein of bacteria DnaK [23, 24] or bacterial death [25].

6. Biofilm’s inhibitors based on nucleotide second messenger molecules

Nucleotide second messenger molecule cyclic di-GMP (c-di-GMP) is involved in biofilm development and the growth of biofilm can be altered by modifying the c-di-GMP signaling pathway (Figure 4) [14, 26]. c-di-GMP is synthesized from two molecules of GTP by diguanylate cyclases (DGCs). Its mechanism of action is achieved by microbial cells reducing the level of c-di-GMP via phosphodiesterase activation due to nitrosative and starvation conditions [27] leading to biofilm dispersion. However, c-di-GMP has three main mechanisms of biofilm formation regulation:

1. Weakening bacterial movement to promote bacterial attachment onto a solid surface. Transition of bacteria from motility to attachment is an important stage in biofilm formation. In E. coli, the c-di-GMP-bound form of the flagellar brake protein YcgR interacts with the flagellar motor protein Mot A, thus regulatory motor output in a brake-like fashion [28]. Bacterial surface attachment is enhanced by, c-di-GMP with a role in the suppression of bacteria motility (Figure 4A).

2. Regulating pilus development. Mannose-sensitive haemagglutinin (MsHA) pilus found in Vibrio cholera promotes bacteria attachment on solid surfaces during the early stages of biofilm formation and controlled by MshE, an ATPase responsible for pilus polymerization [29, 30]. Once c-di-GMP binds to MshE, it enhances the assembly of MsHA pilus and thereby increases biofilm formation [29] (Figure 4B). The number of MsHA pili on the bacterial surface can also increase proportionally with increase in intracellular c-di-GMP concentration, leading to rapid biofilm formation.

3. Regulation of biofilm components production. Curli fibers is regulated by the diguanylate cyclases (DGC YdaM) and the phosphodiesterases (PDE YciR) through controlling the c-di-GMP concentrations in E. coli K-12 strain W3110, production of biofilm matrix components [31]. At very high concentrations of c-di-GMP, YciR begins to apply its PDE function to release the inhibition of YdaM and MlrA and concomitantly, YdaM can activate MlrA to enhance the central curli regulator CsgD, thus prompting the transcription of curli genes and enabling the curli formation [31] (Figure 4C). c-di-GMP also regulates another essential component of biofilm matrix called bacteria cellulose [32]. Bacterial cellulose synthase (BcsA), is attached in the inner membrane of the cell and contains a catalytic glycosyltransferase domain and a c-di-GMP-binding PilZ domain in its intracellular part [33] (Figure 4D). Glycosyltransferase domain is activated when c-di-GMP binds to the PilZ domain of BcsA which allows the bacterial cell to assemble the nascent polysaccharide with the help of the BcsB/BcsC/BcsZ complex to form extracellular cellulose [33] (Figure 4D).

Figure 4.

Regulation of cyclic di-GMP on biofilm formation by inhibiting bacterial motility and increasing EPS production [26].

7. Application of external pressures to eradicate biofilm

Several biochemical and physical methods can be used to eradicate formed biofilms as described in Figure 5.

1. Physical methods: This include treatment with ultrasound and magnetic fields as shown in Figure 5A [34]. The forces produced by cavitation bubbles and fluid movement are primarily what cause biofilm dispersion during ultrasonic biofilm removal.

2. Biochemical methods: Application of phage lysins, degradative enzymes, and microbial metabolites (Figure 5B). Humans’ enemies are pathogenic bacteria, and bacteria’s enemies are bacteriophages. Phage lysins, the weapons of bacteriophages, could be used to combat detrimental biofilms and multidrug-resistant microorganisms (Figure 5B). At a later stage of infection, bacteriophages express peptidoglycan hydrolases known as phage lysins. They have the ability to destroy bacteria by cleaving the peptidoglycan layer of the cell wall (Figure 5B) [35].

3. Degradative enzymes: Through the degradation of EPS and removing this protective clothing (Figure 5C) [26]. Exopolysaccharides, proteins, lipids, and nucleic acids make up the majority of the biofilm matrix in EPS. Degrading the EPS and eliminating the “protective clothing” that biofilm provides for microorganisms allows one to get rid of the hazardous biofilm. Emerging nosocomial pathogen Corynebacterium auris has a mannan-glucan-rich matrix in its biofilms. Infections brought on by this organism were successfully treated by hydrolyzing mannan-glucan in the biofilm matrix using mannosidase or glucanase. Additionally, cystic fibrosis patients’ sputum and human lung tissue have both been found to have Pseudomonas cells encased in alginate-rich biofilms. By eliminating exopolysaccharide from the P. aeruginosa cell surface and converting alginate into unsaturated uronic acid-containing oligosaccharides, alginate lyase has been used to aid in the removal of biofilms [26].

4. Microbial metabolites: Various microbial physiological processes, including the development of biofilms, have been revealed to be regulated by secondary metabolites acting as intercellular signals. As a result, metabolites can also be used to regulate the growth of biofilms (Figure 5D) [36]. By generating cell chain elongation, morphological alterations, and even cell death in S. mutans biofilms, carolacton, a secondary metabolite isolated from Sorangium cellulosum, shown excellent eradication activity against S. mutans biofilms. The biosurfactant made by Pseudomonas spp., rhamnolipid, demonstrated the capacity to dislodge and eliminate S. aureus biofilms.

5. Nitric oxide: At low and non-toxic concentrations, NO produced by the anaerobic respiration activities inside the P. aeruginosa biofilm can start the dispersal of the biofilm. Further investigation indicated that P. aeruginosa biofilm NO signaling can increase PDE activity, lowering intracellular c-di-GMP levels and promoting biofilm dispersion. NO-induced biofilm dispersal was also seen in several other bacteria, including E. coli and S. aureus, in addition to P. aeruginosa. Similarly, exogenous NO addition therapy can promote biofilm dispersion. For instance, NO-releasing polymers have the ability to dose-dependently lower the metabolic activity of different biofilms. Additionally, regardless of the matrix’s composition, NO-releasing cyclodextrins can destroy P. aeruginosa biofilm. A supramolecular nanocarrier was created by combining the NO prodrug with the glutathione-sensitive a-cyclodextrin and chlorin e6 prodrug demonstrate quick NO release when glutathione is overexpressed in the biofilm, effectively destroying the S. aureus biofilm [37].

Figure 5.

Application of external pressures to eradicate mature biofilm, which include (A) ultrasound, (B) phage lysins, (C) degradative enzymes, and (D) microbial metabolites [26].

7.1 Biofilm eradication novel perspectives

Bacteria resistance to antibiotics is fast expanding and orthodox treatment of biofilms with antibiotics is ineffective. Combating biofilms therefore requires the development of different approaches and biologists looking for alternative ways to eradicate biofilms. Some recent approaches for combating biofilms include electrochemical methods, promising antibiofilm compounds and drug delivery strategies to enhance the bioavailability and potency of antibiofilm agents [38].

7.1.1 Antimicrobial compounds to eradicate biofilms: Substrates with antibiofilm activity

In recent approaches many antimicrobial substances are screened with variable results on their ability to eradicate biofilms [38]. Before the discovery of antibiotics, silver (Ag) was used for decades as an anti-bacterial agent for food and water preservation [39]. After its ionization from Ag to Ag⁺, silver ions are capable of irreversibly denaturing key enzymes and in the process successfully killing mature biofilms after 24 hours of treatment [39, 40] as well as biofilms grown after 4–6 days though higher concentrations are needed. Biofilms that have been successfully eradicated by silver oxynitrate include E. coli, S. aureus, and P. aeruginosa biofilms [40].

7.1.2 Modulation of the biofilm architecture to eradicate biofilms

Biofilm extracellular polymeric substances (EPS) called the matrix is composed of proteins, polysaccharides, and eDNA and loosely links bacteria within the biofilm. They are responsible for irreversible cellular attachment, improve mechanical stability, and maintain secreted enzymes [41]. Theoretically, biofilm EPS matrix targeted agents have the possibility to interfere with the growth of biofilm, their dislocation, destabilization, detachment, sensitization, and increase access of antibiotics [38]. An example is the inhibition of biofilm formation by a variety of Gram-positive and Gram-negative organisms, e.g., S. epidermidis and P. aeruginosa. Specifically, deoxyribonuclease I (DNase I) cleaves single-stranded or double-stranded DNA at phosphodiester bonds that make up the phosphate backbone during the addition of DNase I [41, 42, 43].

7.1.3 Electrochemical biofilm eradication

One of the new perspectives in eradicating biofilms is through electrochemical techniques. The mechanism of action involved is the formation of H₂O₂, which is a result of the partial reduction of oxygen on metal surfaces [44]. This stimulates an electric current that affects the organization of biological membranes, cellular processes [45], cell behavior [46], bacterial respiratory rate, and oxidation of proteins, likewise cell electrophysiology [47]. The eradication of biofilm using electrochemically generated biocides varies depending on biocide concentration, exposure time, biofilm thickness and/or growth stage, and bacterial strain, as observed [44]. An example of experiment carried out in vivo on Acinetobacter baumannii grown as biofilms on porcine explants showed it could be overlaid with the same e-scaffold, and this significantly reduced viable bacteria by about 1000 folds [48].

7.2 Strategies for combating bacterial biofilm infections

Strategies to combat biofilm formation range from the control by surface adhesins to the control by cell-to-cell communication pathways [49]. Three strategies have been identified, which include (a) altering abiotic surface characteristics to prevent biofilm formation; (b) regulating the signaling pathways to inhibit biofilm formation and stimulate biofilm dispersal; and (c) applying external forces to eradicate the biofilm (Figure 6) [26].

7.2.1 Altering abiotic surface characteristics to prevent biofilm formation

Here two strategies are characterized, that is, treating abiotic surfaces and coating surfaces (Figure 6A–B) [26]. Treatment relies on changing the characteristics of surface material like smoothness and wettability through thermal cycling and UV irradiation or hydrophilicity [50] and coating surfaces with polymers, trimethyl-silane (TMS)/O₂, and antimicrobial peptides in order to prevent biofilm attachment [26].

7.2.2 Regulation of signaling pathways to inhibit biofilm

Examples of pathways inhibition are those based on quorum sensing that triggers a cascade of intracellular signaling events, eventually regulating different physiological phenotypes after binding to their matching receptors [51], inhibiting biofilm-related genes expression through the interfering with the QS signaling pathway [26], and inhibition based on nucleotides (Figure 6C) [27].

7.2.3 Application external forces to eradicate the biofilm

Both physical and biochemical methods are used to eradicate already formed biofilm. Physical methods entail use of UV radiation whereas biochemical methods use phage lysins, degradative enzymes, metabolites and nitric oxides (Figure 6D) [26].

Figure 6.

Strategies for controlling biofilm infections. (A) Surface treatment. (B) Surface coating. (C) Chemical agents that influence QS. (D) External force [26].

7.3 Treatment methods for biofilm infections

Biofilm infections can be reduced by using conventional antibiotics, either alone or in combination with additional medicines (Figure 7). For example, it was observed that sub-minimum inhibitory concentrations (MICs) of ceftazidime repressed the expression of genes involved in P. aeruginosa bacterial adherence and matrix synthesis, decrease biofilm volume, and impede twitching motility [7, 52]. Colistin also greatly decreased E. coli biofilms and planktonic cells in a concentration-dependent manner [53]. According to an in vitro study, gentamycin released by bone graft replacements can inhibit E. coli adhesion at 12 μg/mL and can remove biofilms that have been present for 24 hours at 23 μg/mL [54].

Figure 7.

Schematic illustration of antibiotics-based tactics for preventing the growth of clinically-significant bacterial biofilms [7].

However, because biofilm has emerged, the majority of antibiotics are now given in clinical settings in mixtures with other antibiotics. Despite the fact that vancomycin is still the antibiotic most frequently recommended for S. aureus biofilm-associated infections, the rise of vancomycin-resistant S. aureus has made it necessary to combine vancomycin with other antibiotics, such as rifampin. Additionally, colistin and other antibiotics, such tigecycline, have demonstrated synergistic effects in vitro, indicating the possibility of their use in clinical settings. Also, it was proven that various strains of E. coli linked to UTIs had their biofilm biomass drastically decreased by amikacin, ciprofloxacin, and third-generation cephalosporins. Additionally, it was shown that Staphylococcal biofilms developed on titanium devices may be removed within 72 hours with the help of a combined antibiotic therapy of clarithromycin and daptomycin [7].

8. Conclusion

Complex and dynamic interactions between the surface, microorganisms, and EPS are necessary for the development of a biofilm. In addition, biofilms contain a form of bacteria that is common in nature. Their resistance is a major barrier that traditional methods must overcome. However, antimicrobials have not received enough attention at current stage. The spatial heterogeneity in the chemical and microbial composition of biofilms has made it more challenging to execute eradication strategies. For the purpose of preventing infections, this paper highlighted a number of cutting-edge antimicrobials based on nanotechnology and delivery techniques, especially in the context of better penetration and targeted antimicrobial administration inside the biofilm for its eradication.