Current Strategies for Combating Biofilm-Forming Pathogens in Clinical Healthcare-Associated Infections

Abstract

The biofilm formation by various pathogens causes chronic infections and poses severe threats to industry, healthcare, and society. They can form biofilm on surfaces of medical implants, heart valves, pacemakers, contact lenses, vascular grafts, urinary catheters, dialysis catheters, etc. These biofilms play a central role in bacterial persistence and antibiotic tolerance. Biofilm formation occurs in a series of steps, and any interference in these steps can prevent its formation. Therefore, the hunt to explore and develop effective anti-biofilm strategies became necessary to decrease the rate of biofilm-related infections. In this review, we highlighted and discussed the current therapeutic approaches to eradicate biofilm formation and combat drug resistance by anti-biofilm drugs, phytocompounds, antimicrobial peptides (AMPs), antimicrobial lipids (AMLs), matrix-degrading enzymes, nanoparticles, phagebiotics, surface coatings, photodynamic therapy (PDT), riboswitches, vaccines, and antibodies. The clinical validation of these findings will provide novel preventive and therapeutic strategies for biofilm-associated infections to the medical world.

Introduction

Before the discovery of antibiotics, microbial infections posed a top global threat to public health and were responsible for causing numerous diseases and even mortality. Following Alexander Fleming's discovery of penicillin, multiple antibiotics were developed to treat infectious diseases, eventually saving countless lives [1]. On the other hand, due to the misuse and overuse of antibacterial agents, multi-drug resistance (MDR) and bacterial resistance development has led to the post-antibiotic era [2].

Biofilm formation is a clinically major threat to human health because of host defense mechanisms, antimicrobial resistance, and other stresses. As a result, it causes persistent bacterial infections worldwide [3, 4]. In 1971, Marshall first introduced the concept of biofilm, further described by Fletcher, Characklis, and Costerton [5]. Biofilms are complex and sessile communities of microorganisms, primarily bacteria but fungi, viruses, protozoans, and other microbes can also form biofilm. It produces extra polymeric substances (EPS) such as exopolysaccharides (1–2%), extracellular DNA (< 1%), RNA (< 1%) and proteins (< 1–2%). In addition to these, water (> 97%) is the primary element of biofilm. It is distributed non-homogenously and is primarily responsible for the movement of nutrients within the biofilm matrix [6]. The EPS provides mechanical strength, tolerance to dehydration, protection against antimicrobial agents and host immune cells, clumping and adhesion of biofilm cells, assimilation of various substances, and acts as a source of carbon at nutrient-deficient conditions [7]. The extracellular DNA (e-DNA) enhances drug resistance, nutritional provision, biofilm stability, genetic information exchange, and structural integrity of biofilms [8].

Biofilm-mediated diseases are challenging to treat with antimicrobial agents and contribute to the spread of the infection. These diseases comprise both surface-located biofilms and tissue or secretion-located biofilms that can affect millions of individuals worldwide each year, resulting in numerous deaths [9]. Surface-located biofilms can be formed on biotic (gingiva, tooth) or abiotic (catheters, contact lenses, sutures, prosthetic joints, prosthetic heart valves, vascular grafts, pacemakers, voice prostheses, and intrauterine devices) surfaces. Tissue or secretion-located biofilms can be formed when bacteria accumulate within infected tissues or secretion (mucus) [10, 11]. Both Gram-negative (Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae) and Gram-positive (Listeria monocytogenes, Enterococcus faecalis, Staphylococcus aureus, Clostridium difficile, Streptococcus viridans, Staphylococcus epidermidis, Streptococcus mutans) bacteria can develop biofilms [12]. Infections related to indwelling medical devices are mostly caused by P. aeruginosa and staphylococci spp. (particularly S. epidermidis and S. aureus) [13]. It was anticipated that P. aeruginosa caused cystic fibrosis, S. aureus and S. epidermis caused infective endocarditis, prosthetic heart valve infections (40%-50%), and catheter biofilm infections (40%-50%) and Helicobacter pylori caused gastric ulcer [14]. It was estimated that staphylococci were responsible for 87% of bloodstream infections [15]. This review provides contemporary knowledge about effective strategies designed to combat bacterial biofilms-associated infections.

Process of Biofilm Formation

The process of biofilm development is complex and dynamic. Generally, the formation of biofilm by any microbial pathogens on any surface or layer includes four distinct phases: (1) attachment, (2) colonization, (3) proliferation and maturation, and (4) dispersion [16, 17]. During the early stage, free-swimming planktonic cells reversibly bind and adhere to the surfaces of host tissues or implants to form biofilms. In this phase, several surface-associated proteins are involved such as SasG, fibronectin-binding protein (FnBP), protein A, OmpA, and biofilm-associated protein (BAP) [18–21]. Many factors govern bacterial deposition, including hydrodynamic forces, Van der Waals forces, sedimentation, Brownian motions, and hydrophobic or electrostatic interactions [22]. Adhesion is a process by which microbes adhere to a surface or the host via adhesin, lipopolysaccharide, flagella, and pili. During the colonization phase, bacterial cells begin to divide and produce an exopolymeric substance (EPS), which increases adherence and colonization of microbes. During the third phase, 3D biofilm assemblies are formed in which the EPS provides multifunctional and concealing micro-environments in which multiple microbes may survive and communicate via a quorum sensing (QS) system [23]. Dispersion of mature biofilm is the last phase, during which the microbial cells are released from the biofilm and again go back into the planktonic phase [24]. The different phases of biofilm development are illustrated in Fig. 1.

Fig. 1.

Process of biofilm formation

Different Therapeutic Strategies for the Prevention of Biofilms

In healthcare-associated infections, biofilms play a crucial role in tooth surfaces, living tissues, medically relevant devices, and implants (heart valves, catheters, sutures, prosthetic joints, contact lenses, and pacemakers). Biofilms are complex, so more than a single approach is required. To combat the different phases of biofilm formation, it is necessary to use a combination of various strategies. Recently, many novel approaches have been proposed as antibiofilm, such as phytochemicals, drug repurposing, antibiotics, nanoparticles, antimicrobial peptides and antimicrobial lipids, matrix-degrading enzymes, riboswitches, and surface modifications [25]. The various novel approaches to combat biofilm formation are highlighted in Fig. 2.

Fig. 2.

Novel approaches to combat biofilm formation in various pathogens

Anti-Biofilm Drugs to Combat Biofilm Formation

The development of anti-biofilm drugs is essential for the prevention and treatment of infectious diseases since pathogenic biofilm formation is a significant clinical issue in terms of morbidity, mortality, and economic losses.

Novel antibiotic or antibacterial drug development is a time and cost-consuming process. An alternative would be to repurpose the medications approved by the Food and Drug Administration (FDA) for multiple diseases based on preclinical and clinical trials data and their toxicology and pharmacokinetic and pharmacodynamics data [26]. Auranofin an anti-rheumatic agent, terfenadine an antihistaminic drug, and some NSAIDs (Non-Steroidal Anti-Inflammatory Drugs) like naproxen sodium, acetylsalicylic acid, piroxicam, and diclofenac sodium have been repurposed to treat S. aureus biofilm-mediated infections [27–29]. Recently, it has been reported that penfluridol an antipsychotic drug repurposed to inhibit the biofilm formation of E. faecalis [30]. Several publications have provided information about antibiotics with antibiofilm activities. It has been found that the antibiotic rifampin, azithromycin, clindamycin in S. aureus, and vancomycin in methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) inhibit and disrupt biofilm formation [31, 32]. Mitomycin C and cisplatin are FDA-approved antineoplastic drugs commonly used to treat numerous types of cancer and can eradicate S. aureus and P. aeruginosa biofilm [33, 34].

Metal cations (Mg²⁺, Fe²⁺, Ca²⁺, Zn²⁺) are pivotal in maintaining the matrix integrity. These divalent cations may promote intracellular adhesion and aggregation via their interactions with cell-wall teichoic acid [35]. For example, disodium-EDTA in combination with sodium citrate and gentamicin or tigecycline efficiently eradicates biofilm formation in staphylococcus species. In contrast, tetrasodium-EDTA prevents biofilm formation in in vitro, and calcium-EGTA (ethylene glycol tetraacetic acid) inhibits biofilm formation and inter-bacterial interaction [36].

Sulfhydryl compounds such as β-mercaptoethanol, dithiothreitol, and cysteine significantly reduce biofilm formation in S. aureus by inhibiting polysaccharide intercellular adhesin (PIA) biosynthesis [37]. UDP-GlcNAc (Uridine diphosphate N-acetyl glucosamine) is required for the development of S. aureus biofilm during PIA or poly-N-acetylglucosamine (PNAG) biosynthesis [38]. These sulfhydryl compounds promote glucose metabolism and induce several protein expressions in the Embden Meyerhof and Pentose Phosphate pathways. This decreases the amount of UDP-GlcNAc that is available for PIA/PNAG synthesis, eventually reducing the biofilm formation [37]. A few antibiofilm agents and their sources are summarized in Table 1.

Table 1.

Some antibiofilm agents with their sources

Type	Antibiofilm Molecule	Source	Susceptible Microorganism	References
Antibiotics and other drugs	Penfluridol	–	E. faecalis	[30]
	Subtilin	Bacillus subtilis ATCC6633	Lactococcus lactis	[39, 40]
	Polymyxin B	Paenibacillus polymyxa	P. aeruginosa, S. aureus, E. coli
	Polymyxin E (Colistin)	Paenibacillus polymyxa	Stenotrophomonas maltophilia
	Mitomycin C	–	S. aureus, P. aeruginosa	[33, 34]
	Cisplatin	–
	Colistin	–	K. pneumoniae	[41]
Chelating agents	Sodium citrate, Disodium-EDTA, Tetrasodium EDTA	–	Staphylococcus species, P. aeruginosa,	[35, 42]
Surfactants	Triton X-100	–	MRSA	[43, 44]
	Tween 80	–	P. aeruginosa, S. aureus	[45]
Sulfhydryl Compounds	Dithiothreitol	–	S. aureus	[37]
Biosurfactants	Rhamnolipids	Burkholderia thailandensis E264 P. aeruginosa MN1	S. aureus, Salmonella enteritidis, L. monocytogenes	[46]
	Coryxin	Corynebacterium xerosis	S. aureus, P. aeruginosa, S. mutans, E. coli,	[47]
	Pontifactin	Pontibacter korlensis	Salmonella typhi, S. aureus, Vibrio cholera, B. subtilis	[48]

Phytochemicals Mediated Biofilm Inhibition

Plant extracts and plant-derived compounds (polyphenolic compounds, flavonoids, alkaloids, and terpenoids) have been used to counter infectious diseases. Some phytochemicals such as ferulic acid, gallic acid, caffeic acid, chlorogenic acid [49], tannic acid [50], xanthohumol, carvacrol, eugenol, resveratrol, berberine, genistein, catechin, hydroquinone, p-hydroxybenzoic acid, protocatechuic acid have promising antimicrobial and antibiofilm activity against various pathogens [51]. These substances combat biofilm through six primary processes which include (1) substrate depletion, (2) cell membrane disruption, (3) adhesin complex binding, (4) protein and cell wall binding, (5) interaction with eukaryotic DNA, and (6) blocking viral fusion [52]. For biofilm-mediated infections, phytocompounds having fewer side effects may be a more effective therapeutic agent; however, current studies demonstrate that a combination strategy is always preferable to an individualistic one. Examples of some plant-based antibiofilm molecules along with their mechanism of action are depicted in Table 2.

Table 2.

Anti-biofilm activity of phytochemicals with their mode of action

Plant Extracts	Source	Structure	Target Strains	Anti-Biofilm Effects	References
Carvacrol	Origanum vulgare		P. aeruginosa S. aureus	Reduces the cviI gene expression, violacein production, and chitinase activity	[53]
Quercetin	Usnea longissima		S. pneumoniae	Inhibits Sortase A functioning and sialic acid synthesis	[54]
Reserpine	Rauwolfia vomitoria, Rauwolfia serpentine		K. pneumoniae	Inhibits EPS production and virulence factor production and reduces cell viability	[55]
Curcumin	Curcuma longa		K. pneumoniae S. mutans A. baumannii C. albicans	Inhibits pellicle formation, pili motility	[56]
Berberine	Berberis aquifolium, Berberis aristate, Berberis vulgaris		K. pneumoniae	Reduce EPS formation and downregulate the biofilm-related genes	[50]
Eugenol	Ocimum plants, Syzigium aromaticum		S. mutans K. pneumoniae P. aeruginosa PAO1	Inhibits swimming and swarming motility and EPS production	[50]
Epigallocatechin- 3-gallate	Camellia sinesis		E. coli	Suppresses curli production and gene (csgD, csgB and csgA) expression	[57]
Ajoene	Allium sativum		P. aeruginosa, S. aureus	Downregulates rhamnolipid production	[51]
7- Epiclusianone	Rheedia brasiliensis		S. mutans	Disrupts insoluble EPS and intracellular polysaccharides	[58, 59]

Nanoparticles Mediated Biofilm Control

In recent days, the use of nanotechnology has acquired immense significance in preventing bacterial adhesion. It is used as a therapeutic agent because they are smaller in size (less than 1 μm) and have a high surface-to-mass ratio which increases the ability to interact with different biological systems [60]. To tackle biofilm-associated infections nanotechnology provides novel approaches such as nanoparticles, nanomaterials, and drug-encapsulated nanoparticles that possess good antibacterial and antibiofilm activities [61].

It was discovered that the adhesion and biofilm development of S. aureus were disrupted by silver (Ag-NPs), zinc oxide (ZnO-NPs), stannic oxide (SnO₂-NPs), cerium oxide (CeO₂-NPs), and sustained nitric oxide-releasing nanoparticles (NO-NPs) [62]. Ramasamy et al. reported that cinnamaldehyde-immobilized gold nanoparticles (Au-NPs) have superior biofilm reduction against MRSA and MSSA [63]. Biologically synthesized Ag-NPs using β-1,3 glucan binding protein have shown immature biofilm reduction against P. aeruginosa and E. faecalis (80 and 85% respectively) [64].

Nanoparticles of non-toxic polymer chitosan effectively prevent MRSA and MSSA biofilm development. It was noted that nanoparticles of chitosan-coated with iron oxide also inhibit biofilm formation in S. aureus [65]. The crystal violet-based assay demonstrated that the Ag-NPs at a very low concentration (0.125 μg/ml) can decrease the biofilm formation of Salmonella enterica and L. monocytogenes [66]. When nanoparticles were used in combination with antibiotics, synergistic antibiofilm activity was observed. Chaudhari et al. demonstrated that the synergistic effect of Ag-NPs with gentamicin and chloramphenicol resulted in enhanced dispersion of S. aureus biofilms [67]. It was reported that Ag-NPs mainly decrease the expression levels of virulence and biofilm-related genes and it has a significant antibacterial effect on MDR K. pneumoniae [68]. ZnO-NPs were reported to efficiently reduce biofilm development and the synthesis of pyochelin, pyocyanin, Pseudomonas quinolone signal (PQS), and hemolytic activity of P. aeruginosa without interfering with the planktonic cell’s proliferation [69].

Furthermore, it was reported that the antimicrobial activity of fluoride nanomaterials reduced S. aureus colonization on catheter surfaces coated by magnesium fluoride (MgF₂) nanoparticles and yttrium fluoride (YF₃) nanoparticles [70, 71]. Kulshrestha et al. reported that calcium fluoride (CaF₂) nanoparticles have an inhibitory effect on S. mutans virulence-related genes (ftf, gtfC, comDE, vicR, and spaP). Additionally, these nanoparticles also inhibit biofilms by suppressing the enzymatic activities associated with quorum sensing, glucan synthesis, bacterial cell adherence, acid production, and tolerance [72].

Antimicrobial Peptides and Antimicrobial Lipids as Biofilm Inhibitors

Antimicrobial peptides (AMPs) are a class of short and cationic peptides produced by the innate immune system that have been considered as a novel and promising approach to prevent biofilm formation and disrupt mature biofilms [73]. These peptides reduce biofilm formation on various medical devices including stents, catheters, heart valves, pacemakers, dentures, etc. involved in hospital-acquired diseases caused by both ESKAPE pathogens (E. faecium, S. aureus, K. pneumoniae, A. baumanii, P. aeruginosa, and Enterobacter spp.), and non-ESKAPE pathogens [74]. A broad range of antibiofilm effects are exhibited by AMPs by (1) peptidoglycan cleavage, (2) altering bacterial membrane potential, (3) degradation of lipopolysaccharides, (4) prevention of bacterial cell survival and division, (5) modulating the production and function of adhesion molecules, and (6) suppression of the intense response of the bacteria [75]. AMPs provide as an alternative to conventional antibiotics that are less susceptible to microbial resistance by targeting the bacterial cell membrane [76].

Yang et al. synthesized 9-fungal defensin-like peptides that bind to bacterial DNA and are permeable to the cytoplasmic membrane, disturbed the outer membrane, and showed biofilm inhibition in S. aureus (99%) [77]. Recently, it has been reported that Staphylococcus lugdunensis produces a non-ribosomal cyclic peptide lugdunin with excellent antimicrobial activity against MRSA (21.2–84.8 μM) [78]. AMP such as hepcidin 20 can lower the EPS concentration and affect the S. epidermidis biofilm formation via suppressing the PIA synthesis [79]. The EPS produced by S. mutans can be disrupted by another AMP called P1, which would lessen the production of biofilms on polystyrene and saliva-coated hydroxyapatite [80]. Lytic peptides (PPT-7) are another family of AMPs that have been extensively studied for their ability to inhibit S. aureus biofilm formation. These peptides bind to bacterial cell membrane’s lipopolysaccharides and destroy the membrane stability [81].

Another peptide, S4 (1–16), a derivative of dermaseptin S4 caused P. aeruginosa biofilm reduction by inducing bacterial dispersal and movement of membrane lipids [82]. Insect-derived AMPs apidaecin, drosocin, and pyrrhocoricin mainly target bacterial heat-shock protein (DnaK) by hindering chaperone-assisted folded proteins and decreasing the activity of DnaK ATPase [83–85]. Similarly, an AMP LL-37 effectively disrupts the biofilm production of P. aeruginosa by increasing the expression of genes required for type IV pili synthesis and function [86, 87]. An AMP 1037 reduces the biofilm formation of L. monocytogenes, Burkholderia cenocepacia, and P. aeruginosa by inhibiting the swimming and swarming motilities, downregulating biofilm formation-related genes and promoting twitching motility [87]. Some antibiofilm peptides with their anti-biofilm activities are summarized in Table 3.

Table 3.

Antimicrobial peptides with their modes of actions

Susceptible Microbes	AMPs	Amino Acid Sequence	Mode of Action	References
S. aureus	HC5	VGXRYASXPGXSWKYVXF	Alters surface hydrophobicity	[88]
	Nisin A	MSTKDFNLDLVSVSKKDSGASPR	Depolarizes cell membrane
	Lacticin	MAGFLKVVQLLAKYGSKAVQMAWANKGKILDWLNAGQAIDKVVSKIKQILGIK
	Nukacin ISK-1	KKKSGVIPTVSHGCHMNSFQFVFTCC
S. epidermidis	Human β defensin 3 (HBD-3)	GIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK	Target icaA, icaD, and icaR genes	[79, 89]
	Hepcidin 20	ICIFCCGCCHRSHCGMCCKT	Acts on PIA
E. coli	Pyrrhocoricin	VDKGSYLPRPTPPRPIYNRN	Binds with DNaK	[83–85]
	Drosocin	GKPRPYSPRPTSHPRPIRV
	Apidaecin	GNNRPVYIPQPRPPHPRI
	Microcin B17	VGIGGGGGGGGGGSCGGQGGGCGGCSNGCSGGNGGSGGSGSH	Inhibits DNA replication by hindering type II DNA topoisomerase	[90]
	PR-39	RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRF PPRFP	Stops synthesis of DNA and protein	[91]
S. mutans	P1	PARKARAATAATAATAATAATAAT	Interferes and degrades EPS	[80]
P. aeruginosa	1018	VRLIVAVRIWRR	Binds and degrades (p)ppGpp	[92, 93]
	DJK-5	VQWRAIRVRVIR
	1037	KRFRIRVRV	Decreases swarming and swimming motilities, downregulates biofilm forming related genes, promotes twitching motility, and enhances QS system	[86, 87]
	LL-37	LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES
	S4 (1–16)	ALWKTLLKVLKAAAK	Disintegrates and releases membrane lipids	[82]

AMLs are single-chain lipid amphiphiles composed of monoglycerides and fatty acids. AML functions through various mechanisms, including increased membrane permeability, membrane pore formation, disruption of the electron transport chain, targeting the microbial surface signal transduction system, cell lysis, and microbial enzyme inhibition [94].

It has been investigated that glycerol monolaurate (GML), a fatty acid composed of lauric acid and glycerol has antibiofilm and antibacterial activity against L. monocytogenes, Haemophilus influenzae and S. aureus [95, 96]. Lopes et al. used Atomic Force Microscopy to demonstrate the biofilm inhibitory capability of GML nano-capsules against P. aeruginosa biofilm [97]. Furthermore, a number of unsaturated fatty acids, including linoleic acid, petroselinic acid, oleic acid, vaccenic acid, and palmitoleic acid have been demonstrated to successfully prevent the biofilm formation of S. epidermidis, S. aureus, and S. mutans [98, 99]. According to the research carried out by Sun et al. two fatty acids eicosapentaenoic acid and docosahexaenoic acid, diminish the outermost layer of resident biofilm cells (58.8% and 62.5%, respectively) and exhibit anti-biofilm activity against S. mutans. Therefore, the thickness of the biofilm is reduced (19% and 42%, respectively) [100]. Additionally, these two fatty acids are also effective against Porphyromonas gingivalis (61% and 47%, respectively) biofilm formation by the same research group [101]. The chemical structure of some antimicrobial lipids (AMLs) that can inhibit biofilm formation is illustrated in Fig. 3.

Fig. 3.

Chemical structures of various antimicrobial lipids that disrupt biofilm formation (a) Glycerol Monolaurate (b) Linoleic acid (c) Petroselinic acid (d) Oleic acid (e) Vaccenic acid (f) Palmitoleic acid

Biofilm Disruption by Enzymes

Enzymes are used to remove biofilms with minimal environmental impacts and are biodegradable. Enzymes generally target the EPS of the biofilm and interfere in their formation [102]. Enzymes act by damaging the biofilm's architecture by reducing its physical stability [103]. Enzymes can also be used to decrease the biofilm formation and the attachment of the cells to various biotic and abiotic surfaces [104].

For biofilm removal mainly four types of enzymes are used: oxidative enzymes, anti-QS (quorum sensing) enzymes, polysaccharide-degrading enzymes, and proteolytic enzymes. Oxidative enzymes produce reactive oxygen species to destroy pathogens. Some oxidative enzymes are glucose oxidase and peroxidase used in biofilm removal [105]. Lactonases are the anti-QS enzymes that hydrolyze the bond of the homoserine ring and prevent acyl homoserine lactones (AHLs) from binding to transcriptional activators and regulators. Proteases are proteolytic enzymes that hydrolyze proteins and polysaccharide-degrading enzymes such as amylases, lysozymes, hydrolases, and lyases are responsible for disrupting or degrading biofilm formation [106]. Furthermore, enzymes like lysostaphin, nucleases, hyaluronate lyase, DNase I (Deoxyribonuclease I), and dispersin B can also disrupt and hinder the biofilm production of various pathogens such as S. aureus, E. coli, K. pneumoniae, P. aeruginosa, etc. [107]. Lysostaphin, an endopeptidase breaks the pentaglycine (PG) bridges the cell wall of the staphylococcal spp., and disrupts biofilm formation [108, 109]. Nucleases and DNase I mainly degrades the eDNA, and dispersin B and α-amylase (glycoside hydrolase) degrades the polysaccharide matrix component PNAG [110–113]. Researchers in Japan have demonstrated that a serine protease (Esp) derived from S. epidermidis can disperse and inhibit S. aureus biofilm formation in an in vitro biofilm model. Moreover, in in vivo, it minimizes nasal infection caused by S. aureus [114, 115]. Some of the biofilm-disrupting enzymes along with their mechanism of biofilm reduction are depicted in Table 4.

Table 4.

Biofilm-disrupting enzymes against numerous pathogenic strain

Name of the enzyme	Source of the enzyme	Pathogen Strains	Mechanism of biofilm reduction	References
DNase I	Human stratum corneum	S. aureus, E. coli, V. cholerae, Streptococcus pyogenes, P. aeruginosa, A. baumannii, Shewanella oneidensis, E. faecalis, K. pneumoniae, L. monocytogenes, Campylobacter jejuni	Degrades the preformed biofilm by disrupting eDNA	[113]
Dispersin B	Aggregatibacter actinomycet-emcomitans	S. aureus, S. epidermidis, K. pneumoniae, E. coli	Disrupts preformed biofilm by degrading β 1–6 N-acetylglucosamine polymers	[107]
Lysostaphin	Staphylococcus simulans	MRSA	Disrupts the EPS matrix and breaks the peptidoglycan pentaglycine cross-bridges	[108, 109]
Trypsin	Pancreatic serine end protease	P. aeruginosa, Streptococcus mitis, S. epidermidis	Destroys the protein content of the biofilm matrix	[116]
Alginate lyase	Bacillus circulans ATCC 15518	P. aeruginosa	Destroys the EPS	[117]
α-Amylase	B. subtilis S8-18	MRSA	Disrupts the pre-formed mature biofilm through degrading the EPS	[112]
Serine protease (Esp)	S. epidermidis	S. aureus	Rupture surface-linked proteins and host-specific receptors	[114, 115]
Cellulase	Penicillium funiculosum, Trichoderma reesei	S. aureus, P. aeruginosa	Hydrolyzes the β (1,4) glycosidic linkage and induces the dispersal of biofilm	[118]

Surface Modifications of Surgical Implants and Catheters

In the development of biomaterials, bacterial biofilms are a major concern. Hydrophobicity plays an essential role in bacterial adhesion, mainly on plastic and metal surfaces. Coating with some materials on the surface of medical implants reduced the adhesion of bacteria. Surface modifications with polyethylene glycol, polyethene oxide (PEO), polyvinyl pyrrolidone, methylcellulose, and polypropylene oxide (PPO) were shown to have reduced bacterial adherence [119, 120].

Cobalt-chromium-molybdenum alloy, ultra-high-molecular-weight polyethylene, and titanium alloy stainless steel are used as biomaterials for implants. Titanium oxide nanopatterning rough implant surfaces significantly reduced the binding of S. aureus with the implant [121]. Physical topographic surface modification of nitric oxide (NO) hinders and controls bacterial adhesion and biofilm formation on polymeric surfaces [122]. AGXX® is a new antibacterial surface coating that is composed of ruthenium and silver and is interface-conditioned with ascorbic acid to prevent MRSA biofilm growth and formation (40%) [123]. Hydrophobin is used in the coating of several medical devices. It effectively reduces the biofilm formation of S. epidermidis on polystyrene surfaces by forming a self-assembled amphiphilic layer [124]. The chemical alterations caused by monomeric trimethylsilane and oxygen plasma coatings on the material's surface result in changes in the adhesion of intermediary proteins on the coating surface. This may diminish the bacterial adhesion to the coating surface, preventing the biofilm production and reduce biofilm-related infections [125]. Polycrystalline Zirconium Dioxide Ceramic and polymer-infiltrated Ceramic both are able to suppress S. mutans biofilm formation. They are biocompatible with human fibroblasts, and have a high potential for use in implant abutment and indirect restoration [126].

Antibiotics conjugated with biomaterial surfaces are also under investigation such as vancomycin and titanium on the surface of biomaterials are covalently attached which resists the S. aureus surface colonization [127].

Phagebiotics Mediated Biofilm Control

Phage therapy is one of the novel therapeutic strategies that describe the utility of lytic phages to treat bacterial diseases and biofilm inhibition. Phage therapy offers a number of noteworthy benefits, including the ability to kill the target bacterium with minimal impact on the surrounding microbiota due to its high selectivity and narrow inhibition spectrum [128].

Myoviridae phages are remarkably effective on MRSA cells. It has been found that lytic phages belonging to the Myoviriade family can infect different staphylococcal species and coagulase-negative species (CoNS) [129, 130]. Schuch et al. demonstrated that bacteriophage lysine CF-301 is a potent anti-staphylococcal biofilm agent that primarily targets the biofilms that grow on catheters, glass, polystyrene, and surgical mesh. CF-301, when combined with enzymes such as hydrolase and lysostaphin enhanced antibiofilm activity against S. aureus [131]. In catheter models, the phages (PSTCR4 and PSTCR6) effectively reduced the amount of Providencia stuartii biofilms [132]. The human saliva bacteriophage (SMHBZ8) displayed antibiofilm activity against S. mutans biofilm in a cariogenic dentin model [133].

Phage cocktails are produced by mixing two or more lytic phages to target one or more pathogens. These are the new strategy to target biofilms. Phage cocktails mainly target the bacterial receptors involved in several antibacterial pathways. According to numerous study models, phage cocktails are more effective in eliminating biofilms than mono phage therapy [134]. Alves et al. examined that combining a broad host range phage (DRA88) and Bacteriophage K can effectively reduce S. aureus biofilm formation [135]. A cocktail of two pages, AB-SA01 and AB-PA01 target against S. aureus and P. aeruginosa biofilm and efficiently decreased mixed-species biofilm accumulation as compared to their respective individual treatments [136]. Combining the three phages (FKpnM-vB1, FKpnM-vB2, and FKpnP-vB3) cocktails showed significant biofilm inhibition against K. pneumoniae biofilm [137].

According to recent reports, using phages in combination with antibiotics may be one therapeutic strategy to enhance treatment efficacy and increase bacterial death. For instance, co-administration of ciprofloxacin with phages demonstrates a remarkable synergistic effect, eradicating almost 6 log CFUs/g of fibrin clumps within six hours and successfully treat 64% (n = 7/11) rats suffering from acute endocarditis caused by P. aeruginosa [138].

Certain bacteriophage-encode enzymes in their genome that exhibit excellent activity against bacterial biofilms and pathogens. Depolymerases and lysins are the two primary phage degradation enzymes that are used to target bacterial biofilms [139]. For instance, a study found that a novel depolymerase (Dpo10) demonstrates an outstanding ability to inhibit biofilm development on a variety of abiotic surfaces and the capacity to bind and degrade the lipopolysaccharide of E. Coli O157 [140]. Additionally, Wu and his colleagues demonstrated a novel phage-derived depolymerase (Dep42) encoded by the phage SH-KP152226 in controlling infections caused by the K47 capsule of K. pneumoniae. They also suggested that using Dep42 and polymyxin together had a synergistic effect on MDR K. pneumoniae biofilm inhibition [141]. Further research found that LysAB2 effectively against MDR A. baumannii. Additionally, the engineered LysAB2 demonstrated outstanding activity against A. baumannii and a remarkable ability to disrupt the formation of biofilms. [142]. A few phage endolysins and antibiofilm activities are summarized in Table 5.

Table 5.

Biofilm inhibitory activity of some phages (endolysins) against their target pathogens

Name of the phage (endolysin)	Target Pathogen	Results	References
Phi84 (Lys84)	S. aureus	90% of the biofilm was destroyed	[143]
ECD7 (LysECD7)	E. coli, K. pneumoniae	Demonstrated biofilm inhibition against a broad spectrum of bacterial biofilms notably K. pneumoniae biofilm	[144]
PA26 (LysPA26)	P. aeruginosa	Biofilm reduction of P. aeruginosa and biofilm matrix disruption	[145]
phi68 (Lys68)	Salmonella spp.	Biofilm reduction when combined with citric or malic acid	[146]
ClyR (LysClyR)	S. mutans, S. sobrinus	Reduced the viable cell counts in S. mutans and S. sobrinus mature biofilms	[147]
C1(PlyC)	Streptococcus pyogenes	Degradation of biofilm matrix	[148]
PlyF307	A. baumannii	Significant biofilm reduction	[149]

Riboswitches to Disrupt Biofilm Formation

Riboswitches are gene control units located in the 5′ untranslated regions (UTRs) of messenger RNAs (mRNAs), where they bind to ion ligands or small molecules [150, 151]. Since they are lacking in humans but present in the genomes of several bacteria, makes them suitable candidates for antibacterial drug development [152].

The important and common nucleotide second messenger bis- (3'-5)-cyclic dimeric guanosine monophosphate (c-di-GMP) was discovered 25 years ago and found in many bacteria. It plays a pivotal role in the formation of biofilm, colonization, virulence, and EPS synthesis [153]. It can bind with different proteins as well as riboswitches in some mRNA. C-di-GMP riboswitches are involved in regulating genes related with motility, virulence. Additionally, alteration of the intracellular concentration of c-di-GMP governs the production of biofilm [154].

Photodynamic Approach to Eradicate Biofilm

Photodynamic therapy (PDT) is one of the most innovative strategies that use non-toxic photosensitizers or photosensitizing (PS) agents to treat various bacterial, viral, fungal, and parasitic infections. It is known that PDT has significantly reduced the biofilm formation of various clinically significant drug-resistant Gram-positive and Gram-negative bacteria [155]. These compounds got photoactivated by absorbing energy from a certain visible light wavelength. Then it generates reactive oxygen species (ROS), which include singlet oxygen (¹O₂), superoxide, and free radicals which cause rapid lipid oxidation of the bacteria [156].

A number of photosensitizers such as malachite green, methylene blue (MB), toluidine blue ortho (TBO) [157, 158], sinoporphyrin sodium, chlorin e6 [159], and 5-aminolevulinic acid [160] are capable in disrupting staphylococcal biofilm. The biofilm inhibitory effect of 5-aminolevulinic acid was observed when used in combination with netilmicin, vancomycin, and cefaclor antibiotics [161]. In recent years, it has been shown that PDT can diminish the S. mutans biofilms-mediated infections [8].

Novel Therapeutic Vaccines and Antibodies to Control Biofilms

A lot of research has been put into developing antibodies and vaccinations that can be used to treat and prevent diseases associated with biofilms. The novel approach of using vaccines offers several challenges in targeting microbial biofilms because they are microorganism-specific and show significant variations in the expression of vaccine-targeted epitopes [162].

Various conjugative vaccines are developed and they mainly target the EPS components of the biofilm matrix. Staphvax, a conjugative vaccine made of proteins and polysaccharides targets S. aureus virulence factor capsular polysaccharides (CP5 and CP8) [163]. Alternatively, using the Virus-like Particles (VLPs) is increasing to recognise immunised mimics of a QS peptide. According to some findings, it was concluded that diagnosis with VLP-based epitope as a vaccine mainly disrupts agr signal and effective against combatting S. aureus skin and soft tissue infections (SSTI) [164, 165].

Previously, it was reported that affinity-purified polyclonal antibodies have antibiofilm activities against the PhnD antigen, and these PhnD-specific antibodies prevent the development of S. aureus biofilms at the initial attachment and aggregation stages [166]. In other studies, it has been demonstrated that TRL1068, a human monoclonal antibody could combat S. aureus biofilm formation. TRL1068 has a strong specificity towards DNABII proteins, which assist to retain the eDNA within the extracellular matrix (ECM) of the mature biofilm [167, 168]. It has been discovered that monoclonal antibodies (mAbs) bind to Psl, an EPS of P. aeruginosa, inhibiting the production of biofilms [169]. It also reduces the binding of P. aeruginosa to host cells and offers adequate defense in various P. aeruginosa-infected animal models, including a mouse model with acute pneumoniae and heat injury [170].

Conclusion and Future Prospects

To date, bacterial biofilm formation has been extensively studied and understood. Most microorganisms can form multicellular biofilm colonies. Treating biofilm-related infections is one of the significant concerns in biomedical research as they have posed a great challenge to tackle because of their complexity and resistance to antibiotic treatment. Hence, hindering their colonization on various biotic and abiotic surfaces to restrict biofilm development. In this review, we have discussed several innovative therapeutic strategies that could be adopted to prevent biofilm-mediated bacterial infections caused by the notorious pathogenic bacterium. However, extensive efforts are still needed to create safe, effective, and economically feasible methods against biofilm infections. Future studies could focus on removing whole biofilms to improve therapeutic potential and reduce toxicity and the emergence of resistance.

Declarations

Conflict of interest

The authors declare no conflict of interest.