Antibiotic Resistant Biofilms and the Quest for Novel Therapeutic Strategies

Abstract

Antimicrobial resistance (AMR) is one of the major leading causes of death around the globe. Present treatment pipelines are insufficient to overcome the critical situation. Prominent biofilm forming human pathogens which can thrive in infection sites using adaptive features results in biofilm persistence. Considering the present scenario, prudential investigations into the mechanisms of resistance target them to improve antibiotic efficacy is required. Regarding this, developing newer and effective treatment options using edge cutting technologies in medical research is the need of time. The reasons underlying the adaptive features in biofilm persistence have been centred on different metabolic and physiological aspects. The high tolerance levels against antibiotics direct researchers to search for novel bioactive molecules that can help combat the problem. In view of this, the present review outlines the focuses on an opportunity of different strategies which are in testing pipeline can thus be developed into products ready to use.

Introduction

Despite the fact, that our bodies contain ten times more bacteria than human cells, many of these bacteria are innocuous or even beneficial [1]. However, the spread of hazardous illnesses, from minor inconveniences and extending to deadly epidemics due to bacteria is a matter of concern. The development of antibiotics over the course of the twentieth century has rendered many dangerous diseases easily treatable [2–4]. Bacterial infections kill millions of people worldwide instead of huge pharmaceutical research. Due to less effectiveness, medical practitioners worldwide are losing, one of the most useful weapons from their outreach posing a threat to the modern world. Additionally, evolutionary changes are making things related to resistance more complicated. Bacteria have adapted to environmental threats for over billions of years and have evolved many ways to protect themselves. They do this either by genetic mutations or by horizontal gene transfer [5, 6]. Another strategy that evolved with time for survival involves the existence of bacteria within a self-produced polymeric matrix, termed as biofilm. A biofilm constitutes a highly organized microsphere of bacteria that lives together in communes [7, 8]. Bacteria that live in biofilms are embedded in a slime-like matrix comprised of extracellular polymeric components such as polysaccharides, nucleic acids, and proteins. Most bacteria live in biofilms regardless of what kind of species or type they are. It is presently known that approximately eighty percent of bacterial species are found as biofilms, which is, in fact, their natural way of life [8]. Inside the biofilm, the bacteria can transfer themselves, biological material such as antibiotic resistance genes [9].

The fungal biofilm further burden the issue due to formation of polymicrobial ecosystems but imposes a significant effect on the host immune response [10]. While biofilm show increased tolerance to many antibiotics, it is becoming increasingly apparent that biofilms also impact the evolution and dissemination of antimicrobial resistance [11]. According to WHO, antibiotic resistance is one of the ten biggest global public health threats we are facing today [12]. Many research investigations on planktonic microorganisms are being carried out worldwide to overcome the problem of antibiotic resistance [13–15]. However, as biofilms are known to be hotspots for genetic evolution, which cause both the emergence and the spread of resistance genes, it should be given considerable importance to control the development and spreading of antimicrobial resistance. Many research groups have spent years trying to figure out what elements contribute to biofilm formation. As a result, scientists have discovered that unrelated bacteria produce: (i) the same exopolysaccharides (cellulose, poly-β-1,6-N-acetylglucosamine) to build the matrix, (ii) the same secondary messenger, c-di-GMP, to regulate the production of biofilm matrix exopolysaccharides, and (iii) a group of surface proteins that are homologous to Biofilm associated protein (Bap) of Staphylococcus aureus [16]. Many fungal biofilms have also been reported in clinical infections which protects fungal pathogens from the host immune system, but biofilm formation in Candida spp., Aspergillus spp., and Cryptococcus spp. have been investigated extensively [17].

In biofilms, poor antibiotic penetration, nutrient limitation and slow growth, adaptive stress responses, and formation of persister cells are hypothesized to constitute a multi-layered defence [15]. The genetic and biochemical details of these biofilm defences are only now beginning to emerge. Thus, each gene and gene product contributing to the present resistance could also be a target for the latest antimicrobial agents [18]. Understanding the microbial gene modulation at different stages of biofilm formation, and how these genes get altered during the normal course of evolution is very important to manage and eradicate problems related to biofilm. Many bioactive molecules can be developed as therapeutic agents for the treatment of numerous human diseases [19]. Antimicrobial peptides (AMPs) can be deployed to combat biofilms as they have potent broad-spectrum activity against different disease-causing bacterial pathogens. They act on by disorganizing the molecular pathways which are responsible for biofilm formation. Thus, active research to develop AMP-containing treatment strategies must be promoted as anti-biofilm agents for therapy and prophylaxis. The idea of proteins as a therapeutic strategy to prevent or control biofilm may enhance the potential of existing antibiotics to fight against infections caused by biofilm that are obstinate to current treatments (Fig. 1).

Fig. 1.

Various mechanisms of action of antibiotics on planktonic forms [1], biofilm matrix resisting the action of antibiotics against the residing bacterial communes [2], promiscuous peptides to combat biofilms [3]

This article reviews the development of biofilms and their response towards host immune system and in use antibiotics. Also, the article discussed the using of antimicrobial peptides and nanotechnology as a novel approach to bring out new molecules with enhanced effectiveness for the treatment of biofilms and associated complications.

Data Compilation and Exclusions

The review has been done following the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) rules and guidelines. The data was sourced by searching in the databases including PubMed, Google Scholar and Web of Science using keywords such as biofilm, antimicrobials, biofilm killing by antimicrobial agents, nanoparticles-biofilms, stringent response and promiscuity were gathered from publications from the above-mentioned databases [from the year 1993 to 2021]. All the published materials were extracted from May 20, 2021, with an update on November 5, 2021. The restricted inclusion was followed to a countable set of biofilms forming pathogens with respect to matrix related parameters like pH, nutrient availability, competition, and immune landscapes. The data extracted from the included articles contained the following: the author’s name and the year of publication; the type of disease; study design; random methods; treating method of antimicrobial involvement; treatment method and primary outcomes. The duplicate records and unrelated literature were excluded from the selection process. The PRISMA flowchart of the study plan is shown in Fig. 2.

Fig. 2.

PRISMA flowchart showing the study design process

Biofilm Microenvironment

The accumulation of antibiotic-degrading enzymes in the matrix of formed biofilms has been screened in Pseudomonas aeruginosa and Klebsiella pneumoniae biofilms. β-lactamases have been found in the biofilm matrix being utilized for this defence mechanism. The accumulation of β-lactamases increases hindrance in activity of antibiotic and also prevent antibiotics to reach the deeper layers of biofilms as well [20, 21]. Another significant component of the bacterial biofilm matrix is extracellular DNA (eDNA) formed from the outer membrane and components of degraded integrity of the microorganisms [22, 23]. Due to the presence of eDNA in the matrix, anionic environment formation takes place which in turn chelate cationic peptides and aminoglycosides. This chelation reaction offers less penetrance to the antimicrobial agents that are positively charged. Also, eDNA has provided resistance to antibiotics through horizontal gene transfer in biofilm forming bacteria, playing a key role as a physical attribute.

Physiological heterogeneity also contributes to biofilm stability with the gradients of oxygen and other nutrients in different biofilms depending upon the microenvironment. In context to microenvironment responses, competitive strains growing with each other either delete the least competitive species or stimulate potent strains for the gradual development of biofilm for protection purposes. During the competitive growth phenomenon, low-molecular-weight compounds from the micro-metabolism is implemented to establish exclusion of surrounding species. Some of the best described elimination methods are based on Hydrogen peroxide (H₂O₂), secretion of antimicrobial compounds and contact dependent growth inhibition [24].

Moreover, data also suggest that biofilm formation increases the resistance/tolerance by orders of magnitude in bacterial strains. Therefore, the ability to form biofilms has serious implications for antibiotic therapy.

Factors Responsible for Biofilm Formation

Variability in pH

The pH influences the species of bacteria and leads to biofilm development. It has been found that some advancement of biofilm is possible at very high acidic and alkaline pH among Acetobacter and Candida species respectively [25]. It has been studied that during biofilm development, the thickness of biofilm is directly proportional to the stimulation of pH in the cellular environment. Also, some recent investigations have shown that pH influence the formation and maintenance of biofilms in urinary tract infections (UTIs) and dental plaques [26–28]. Some studies have also been carried out to enquire the role of pH in infections associated with healthcare facilities bringing severe complications in hospitalized conditions. The infections caused by Vibrio cholera, Klebsiella spp. and Pseudomonas aeruginosa persist for a long period and result in the failure of conventional antibiotics for elimination [27]. In S. aureus and Staphylococcus epidermidis, it is seen that at very high acidic and high alkaline conditions (pH below 3 and above 12), the formation of biofilm is not possible. The variations describe the relation between pH conditions and biofilm formation within species of bacteria in different microenvironments. To date, no studies have been reported which describes the role of pH in stimulating the formation of biofilm and the growth of bacteria inside the host. This research requires greater attention to understand the exact knowhow of underlying roles of pH biofilm development and maintenance.

Mechanical Forces

Dissecting the mechanical factors that contribute to attachment and adhesion finally leading to biofilm formation follows a very complex procedure as the factors vary for different bacterial species. The involvement of electrostatic and surface energy plays a significant role in bacterial adhesion to the surfaces [29]. Also, interactions like steric forces, hydrophobic interactions, hydrations forces which are non-specific in nature have been studied for their involvement in the regulation of physiology of bacteria and their growth into biofilms [30]. Electrostatic interactions play the foremost role in the attachment of bacteria to surfaces. As most of the bacteria carry negative charges, they preferably attach faster to the surfaces with net positive charges making the attachment very swift and tighter as well. Due to the repulsions from the positive surfaces, it is then overcome by preference-based alignment of functional groups (hydrophobic and hydrophilic). It is seen that bacterial attachment on the surface energy basis is preferentially species-specific as described for bacterium Vibrio cholerae which prefer a collective cascade of cells reorientation [31]. However, the underlying mechanisms of cell/surface interactions is still a limitation and thus brings the difficulty of reaching the exact scenario of relative importance in cell attachment on basis of physical mechanics. For a better understanding, the design of materials can be very helpful to uncover the biofilm formation related mechanisms. The studies of these types will surely bring control over bacteria-surface attachment and biofilm formation [32].

Nutrient Availability

During the starvation period of bacteria, the reduction in nutrient availability slows its growth. In a condition like this log phase to the stationary phase transition in bacterial species results in reductional antibiotic susceptibility [33, 34]. It has been clear from investigations that effect of the antibiotics remains same for S. epidermidis, P. aeruginosa, Escherichia coli when growing either freely or residing in clusters due to biofilm response [35]. Another fact which adds to the ineffectiveness of antibiotics in the biofilm is the variation in presence of oxygen in biofilm layers. It is recorded that residing deep in the biofilm matrix give exposure to less or no oxygen for the bacterial communities. This availability of oxygen offers growth at slower levels or even no growth in some bacterial communities. The declining growth patterns accounts for less antibiotic susceptibility as many commercial antibiotic’s favours targeting the pathways in which synthesis of essential macromolecules occurs favouring the survival of species [36, 37].

Competitive Rule

The stimulation of resistance might exist in cells with competitive exclusion behaviour. According to Darwin, the most severe competition for survival is between the communities of the same species in regard to the fact that preferences for food, habits and habitats are the same for each of them. This invariable struggle leads to the formation of protective weaponry which is seen as ‘biofilm response’ instead of showing any type of cooperativity benefitting the presence of each other. During the competitive response, the less dominant strain may not be able to completely get involved in the whole scenario. Also, the knowledge related to the formation of biofilm formation in bacterial species is still lacking due to meagre exploration in the field. The amount of formed biofilms is based on the stimulation caused by the levels of damage done to cells caused between the competitive strains [38, 39]. During competitive growth, bacteria use a unique type of adaptive system which make them efficient for food and space availability. The type 6 secretion system (T6SS) is the macromolecular system that enables the bacteria for their survival. The biofilms for defense used by bacteria to protect themselves from the noxious, small macromolecules provides them more resistance against effective antimicrobials. Due to the toxic nature of T6SS and its role in the induction of biofilm formation is a keen platform of research which can bring better understanding of the mechanisms involved. Vibrio cholerae protected itself against the exogenous attack by the formation of biofilm but still mechanistic knowledge of how it occurred is still limited. The blockade against enzymatic activities of T6SS prevents permeabilization of the outer membrane of the formed biofilm [40].

Role of Antibiotics

The usage of antibiotics for eliminating bacterial colonization in patients has produced controversial benefits that needs to be precisely investigated. In clinical setting use of selective media for identifying the level of susceptibility or resistance in pathogens has its own limitations. The classical culturing method only evaluates the planktonic forms while the biofilm present high resistance to antibiotics. Determination of the minimum inhibitory concentration values for planktonic forms has led to successful evaluation of susceptibility breakpoint and important pharmacodynamics parameters [41]. On the other hand, biofilms can become fractious to activity of drugs due to the adaptive factors in physical, metabolic, and genetic mechanisms of tolerance [42, 43].

The conjoint works have emphasized the biofilm responses through different modes of action by clinical antibiotics, ciprofloxacin (inhibition of deoxyribonucleic acid replication), rifampicin (transcription), or tetracycline (translation and disruption of membrane potentials) [44–48]. During the antibiotic treatment, bacterial cells enter into a metabolically inactive state bringing prevention of the antibiotic action which cause infection persistence and recurrence [49–51]. Biofilm formation is the primary causative reason for life-threatening diseases infecting respiratory, urinary and other systems of the host. Also, interventions in medical device research have faced complications due to the involvement of biofilm-forming bacteria which causes contamination of surfaces used during implantations. Bacteria withstand any stress caused by the administered antibiotics with the help of biofilm and its complex integrations which allows secondary metabolite and genetic material exchange processes [52, 53]. Also, the biofilm matrix brings down the activity of antibiotics by inactivation or entrapment techniques but some antimicrobials can surpass the biofilm matrix as they have negligible interactions with the components present in extra polymeric substances (EPS), for example Dispersin B, LL-37, and indolicidin [54–56]. Another explanation for the permeability barrier against antibiotics through the EPS matrix can be the chelation of antibiotics. The presence of numerous enzymes in the biofilm matrix brings down the activity of antibiotics in the way through to the susceptible target by on the way degradation mechanism that prevents the effective concentration from reaching the desired target site [57].

Quorum Sensing (QS) Systems

During the phase of growth when the density if bacterial population fluctuates, the condition triggers the release of signalling molecules ‘autoinducers’ that affects the bacterial communication [58]. The recognition of autoinducers after secretion is regulated by the receptor proteins present on the membrane or intracellular matrix of the bacteria. The biofilm maturation occurs along with the accumulation of extracellular polymeric substances. The typical QS systems in bacteria which influence the formation of biofilms represent the lasI/lasR gene system where homoserine lactone molecule are formed in gram-negative species [59]. In gram-positive bacteria like Streptococcus spp., comE gene is responsible for the formation of competence stimulating peptide (CSP) as the primary autoinducer which is responsible for both the virulence and biofilm formation [60].

Involvement of Immune Response

Antibiotic resistance has been developed by pathogenic bacteria as a result of increased usage. The majority of antibiotics are therefore no longer effective against the established resistance mechanisms. Antimicrobial resistance currently poses a challenge to the successful treatment and prevention of an ever-widening spectrum of infections. As new resistance mechanisms appear and spread quickly over the world, it poses an ever-greater threat to public health worldwide and necessitates fast action.

It has become clear in recent years that biofilms—what we call community level resistance—can display stress tolerance to external stimuli that single cells cannot. A biofilm community is formed when cells are immersed in spatially complex matrices of extracellular DNA and polysaccharides, which are created as a result of cell–cell interactions. Therefore, fighting bacterial infections necessitates knowledge of both intracellular genetics and biochemistry as well as how antibiotic uptake and resistance are impacted by the biofilm mode of life.

The participation of the host immune system in biofilm associated infections is also thought to be involved through a synergistic pathway based on the involvement of innate and acquired immune responses as well. The establishment of biofilms and related infections includes immune cells playing a key role in epithelial/mucosal barrier disruption. The bacteria are protected from environmental adversities by the biological properties of the biofilm structure, which also provide the microbe with intrinsic resistance to the host complement system. The complement system is widely recognised for being one of the first lines of defence against invading pathogens by recognition and clearance mechanisms. Whilst evading complement immunity and phagocytosis is considered beneficial for bacterial dispersion, it is also possible that some infections use this immune evasion strategy to allow long-term colonisation and carriage. The deployment of active neutrophils presents the first line of defence against invading bacterial species. During this response, reactive oxygen species (ROS) or nitric oxide (NO) is generated. In an investigation, change in O₂ dynamics (limited supply) brings a significant stratification of P. aeruginosa biofilm. As a result, the quantity of surrounding neutrophils that deplete O₂ and indirect anaerobic respiration determines the growth rate of P. aeruginosa biofilms. The context for a relative result of immune response is yet unknown, particularly in light of the predicted production of biofilms and needs extensive research [61].

Biofilm Proteins

The link between biofilms and disease persistence was discovered considerably later after the Dutch scientist Antonie van Leeuwenhoek discovered biofilms in the form of dental plaque under the microscope in 1684 and named the clusters of cells ‘scurf’. The morphological properties of distinct bacterial species are defined by the conditioned stimuli that cause them to form biofilms. Antibiotics, environmental stress, and host immunity are all protected by extracellular matrix (ECM) proteins and other components, and the development of well-organized assemblies increases the chances of participating microbial populations surviving. Competition between microbial species occurs through chemical warfare, in which competing species release various complex compounds either to hinder the growth of other species or to kill them. Antibiofilm chemicals derived from bioactives are a promising approach that can be used to combat dangerous biofilm-forming bacterial species using modern medical technology [62, 63].

Proteins present in bacterial biofilms are responsible for nutrient adsorption, structural integrity and protection of extracellular matrix from the actions developed by immune antimicrobials [64]. Biofilm lattices' elasticity in the extracellular environment allows them to modify and fight protease action, resulting in denaturation locking. Bacterial escape and antibiotic resistance are both aided by the functional specialised mechanisms involved in the deployment and polymerization of protein subunits of biofilms. Protein aggregates have been found in a range of unrelated bacterial species, allowing them to adapt and respond to changes in their environment. The major protein group that makes up the extracellular matrix of numerous unrelated bacteria has recently been identified as amyloid fibres. The aggregation of biofilm-associated protein communicates mostly through amyloid-like characteristics that occur in particular circumstances during biofilm lattice creation. Microscopic methods, cell-based investigations, and in vitro experiments have all been used to explain these communication characteristics. So far, two unique forms of amyloids machinery linked to biofilms have been identified. The first group is intrinsic bacterial amyloids, with the amyloid state being the most common functional form of the proteins. The second kind are facultative bacterial amyloid-like proteins, which have a functional globular folded shape that can convert to an amyloid conformation in response to proteolytic processing and environmental signals [65]. For intrinsic type of aggregates, curli, Fap and chaplins have been studied in enterobacteria, Pseudomonas spp. and Streptomyces coelicolor respectively [66, 67]. Also, facultative bacterial amyloid-like proteins have been recorded as Bap in S. aureus to modulate the immunological response of the host (Fig. 3).

Fig. 3.

Biofilm proteins in bacteria that forms amyloid structures

Facultative amyloids appear to play a dual role in biofilm development, both as adhesins in their natural state and as matrix scaffolds when polymerized into fibrillar structures that look like amyloids. Bap from S. aureus is an example of this class of protein. After being secreted Bap gets anchored through the covalent bond formation to its own cell wall. Bap is engaged in initial adhesion to an abiotic surface and host cell ligand in its native conformation [68]. Then Bap is processed, with part of the C-terminal repeat region remaining tethered to the membrane and fragments comprising the N-terminal area released to the media. Acidic pH promotes the transition of Bap's N-terminal domain from its partially ordered native state to an aggregation-prone conformation that favours polymerization into amyloid-like fibrillar structures. The behaviour of the proteins speculates the role in complex bacterial infectious processes and capacitation of bacterial species in the formation of biofilm [69]. The trait is favoured because the matrix is continuously formed and remodelled throughout the biofilm's life cycle. The examination of dynamical changes in the microenvironment that occur during reorganisation has been limited to single time points or static settings. The existence of competitive bacterial species, which adds to the polymicrobial/promiscuous kind of matrix, complicates the situation. Despite the fact that we only have a rudimentary grasp of the molecular complexities of competitively induced matrices, research is still underway to gain a better understanding. As a result, real-time insights about microenvironment changes in biofilm architectural features are necessary [70].

Obstacles to Therapeutic Options

Because of their resistance, biofilm-forming pathogens are difficult to treat with coventional antibiotics. Biofilms differ from planktonic cells in that they have a specific development phase and reaction to their surroundings. Most microbial biofilms include and flourish not just as a single microbial species, but also as harmful and non-pathogenic microbial commensals. Biofilms can confront and endure environmental stress such as hunger and desiccation because of their elastic nature, which is a critical benefit for their existence [71]. Antibiotic permeability is reduced due to the accumulation of microorganisms inside the extracellular matrix. Any particular cell in the biofilm will have a somewhat different environment than other cells in the biofilm, resulting in a varied rate of growth. To allow for this variability, nutrients and signalling components establish gradients within the biofilm [72, 73]. Within biofilms, protein synthesis and respiratory activity have been found to be diverse. The reaction to antimicrobial drugs varies substantially depending on the position of a single cell within a biofilm community, according to studies [74].

Bacteria in biofilms reversibly convert into persistent or inactive cells, which proliferate slowly. These cells are extremely resistant to antibiotics and are formed stochastically or under endogenous stress (e.g., oxidative stress and antibiotic exposure) [75]. When compared to active and rapidly developing bacteria, these cells have a reduced metabolic rate, making them less sensitive to antibiotics. Toxin/antitoxin (TA) systems generated by environmental stimuli or DNA damage promote the production of large numbers of persistent cells. Bacteria have a variety of stress responses that allow them to improve their survival and antimicrobial resistance [76].

To alter the microenvironment and effect the covered cells, it is critical to prevent antibiotic molecules from blocking their way to the protected cells across the biofilm matrix [77]. The interaction of the antibiotic moieties with the extracellular polymeric matrix's inner layer components causes an anti-effectiveness reaction in the overall quantity of antibiotic transmitted. Despite several research, the act of reduced antibiotic penetration in biofilms remains unknown. It's also likely that biofilm susceptibility will be lowered as a result of the time delay for adaptive phenotypic response caused by reduced antibiotic penetration [78]. As a defensive strategy, the accumulation of antibiotic-degrading enzymes in the matrix, such as -lactamases in biofilm spheres, prevents the deeper layers from being exposed to antibiotics [79].

Because biofilms are a heterogeneous repository of microbial communities' protective spheres, an increase in horizontal gene transfer (HGT) is a major factor in biofilm resistance to commercial antibiotics [80]. In HGT, biofilms have a greater mutation frequency than planktonic cells, which contributes to enhanced antibiotic resistance [81]. The transfer of plasmids among microorganism cells in a biofilm has been established by research, which show that plasmid movement among bacterial cells in biofilms may be more successful than in planktonic cells [82].

Strategies to Combat Biofilm Mediated Infections

Antimicrobial Peptides

Cross-infection risks are reduced by early and vigorous antibiotic treatment to remove disease-causing pathogen colonisation in accordance to current therapeutic standards for preventing biofilm-mediated chronic infections. However, with ongoing treatment procedures, the effects of antibiotic excess become apparent, resulting in increases in populations of resistant/persistent bacteria and downstream events [83]. When the microorganism is devoid of matrix formation or related matrix contributing components, the bacterial species' lost resistance to antibiotics can be reclaimed. The need to understand how to assemble sensing inhibitors from natural sources with complex composition has generated interest in extending research efforts to study potential interference with biofilm growth and the generation of persisters [84, 85]. Other non-biological agents like an electrical current and antimicrobial coatings have been additionally added to the battle for elimination of biofilm resistance [86].

Mechanism of Action of AMPs

The AMPs act on biofilms through pore formation or little deformities that disperse the transmembrane potential, which brings about entry into the bacterial cell [19, 87–89]. The mixed cationic and hydrophobic makeup of AMPs make them capable of interacting with Teichoic acids and lipopolysaccharides that are found on the outer surfaces of Gram-positive and Gram-negative bacteria, and possessing negative charge which makes it possible for initial electrostatic interaction with cationic AMPs. The interactions that establish the foundational architecture of bacterial membranes and the host membrane complex are usually the focus of AMPs. The outer monolayer (leaflet) of bacterial membranes is primarily composed of lipids with negatively charged head groups, such as phosphatidylglycerol and cardiolipin, whereas the outer leaflet of animal membranes is primarily composed of zwitterionic phospholipids, such as phosphatidylcholine and sphingomyelin, as well as other neutral components, such as cholesterol [90]. In animal membranes, the majority of lipids with negatively charged head groups are found in the inner leaflet facing the cytoplasm. The negatively charged phospholipids on the outside leaflet of the bacterial membrane have strong electrostatic interactions with the positively charged AMPs (Fig. 3). Following the initial electrostatic and hydrophobic contacts, the AMPs accumulate at the surface and, once sufficiently concentrated, self-assemble on the bacterial membrane [91, 92].

At this point, a variety of models have been used to explain how AMPs work. The models can be categorised into two categories: pore and non-pore models (Fig. 4).

Fig. 4.

Selectivity based interaction of cationic antimicrobial peptides (AMPs) with bacterial membrane and animal membrane (host)

In pore models, film traversing pores and formation of hydrophilic channels have been reported which in response have been potentially influencing the curve of the biofilms. The barrel-stave and toroidal pore models are two types of transmembrane pore models. The AMPs in initial phases are orientated parallel to the membrane in the barrel stave model, but subsequently enter the lipid bilayer in perpendicular manner. This is analogous to membrane protein ion channels where similar peptide-peptide interactions occur. The peptides in the toroidal pore model likewise insert perpendicularly in the lipid bilayer, but there are no specific peptide-peptide interactions [93] (Fig. 5).

Fig. 5.

Mechanism of action explained by models for AMPs against bacterial pathogens

On the other hand, non-pore models involve destabilization effect, cataclysmic breakdown of the film by combining with matrix proteins, prompt lipid stage division, film preceding disturbance by AMPs through moving across the matrix by restricting and irritation and clarify the underlying action. The components and processes of dynamic peptide activity must be determined using biocomplex-based cooperative concentrates with biofilm matrix. The potentiation of these biomolecules for use in medical research domains is directed by the distinctiveness of their instrumentive activity. Antibiofilm activities of AMPs have been studied extensively in vitro and in vivo in a number of studies (Table 1).

Table 1.

Antibiofilm peptides and related microbes

S. No	Biofilm forming microbe	AMP used	Evaluation method used	Action on microbe	Reference
1	Pseudomonas aeruginosa	NovispirinG10, LL–37, 1037, IDR–1018, DJK-5, DJK-6, Indolocidin, [Lys]7-Pol-CPNH2, (P)PAP-A3, EcDBS1R5, PaDBS1R6F10, mastoparan-R1, mastoparan-R4, DRGN1, tachyplesin III, HPA3NT3-A2, EC1-17 kV, BMAP27, Melittin, P5, P6.2, ZY4, Esc (1 − 21), Esc (1 − 21)-1c, AS-10, Battacin, RN3, SPLUNC1	In vitro/In vivo/In silico	Inhibition/Eradication	[54–56, 94, 95, 97, 98, 107, 114–135]
2	Escherichia coli	Cecropin A, FLIP7, IDR-1018, AS-10, DJK-5, DJK-6,	In vitro/In vivo	Inhibition/Eradication	[98, 107, 121, 122, 124, 127, 133, 136–138]
3	Staphylococcus aureus	Citropin, BMAP27-melittin, Tet–20, CAMA, IDR–1018, Battacin, hBD–3, FLIP7, Kn2-7, RP557, RIP, DRGN1, Nisin Z, Nisin A, Nukasin ISK-1, Laticin Q, WRL3, peptide 73, peptide 73-C, BMAP-28, IB-367, WLBU2, 17tF-W, cys-melimine, DD13-RIP, Dispersin B	In vitro/In vivo	Inhibition/Eradication	[50, 54, 94, 96, 98, 114, 120, 122, 124, 127, 136, 139–150]
4	Staphylococcus epidermidis	BP2, hBD–3, hep–20, temporin B, RIP	In vitro/In vivo	Inhibition/Eradication	[150–155]
5	Streptococcus mutans	P1, LN-7, GH12	In vitro/In vivo	Inhibition/Eradication	[156–158]
6	Klebsiella pneumoniae	IDR–1018, IK8L, DJK-5, DJK-6, SPLUNC1	In vitro/In vivo	Inhibition/Eradication	[98, 124, 127, 159–161]
7	Listeria monocytogenes	P1	In vitro	Inhibition/Eradication	[156]
8	Acienetobacter baumannii	FLIP7, Cecropin A, HHC-10, DJK-5, DJK-6	In vitro/In vivo	Inhibition/Eradication	[123, 133, 136, 137, 162]
9	Porphyromonas gingivalis	BAR, Nal-P-113	In vivo	Inhibition/Eradication	[163–165]
10	Burkholderia cenocepacia	SPLUNC1, 1018	In vitro	Inhibition	[166, 167]

Biofilm Matrix Disruption

Studies have been conducted to better understand the disruption of generated biofilms by chemicals that have the potential to increase the efficacy of traditional antibiotics due to their adaptability in bridging biofilms. The chemicals work by disintegrating the biofilm matrix, allowing for greater flexibility in reducing microbe numbers in body fluids. Dispersin B, a substance that engages dispersion processes within the barrier of microorganism-induced illnesses, has also been linked to biofilm breakup [54, 94]. Cationic peptides like LL-37, a natural human ion amide, and indolicidin, a bovine white corpuscle amide, have been isolated, identified, and artificially synthesised for biofilm suppression in vitro have a considerably low dosage-based efficacy on pre-identified P. aeruginosa biofilms [55, 56]. Also, β-lactam, macrolides, fluoroquinolones, have been examined using transcriptomic and phenotyping approaches for their underlying biofilm assemblages (quorum sensing) related to inhibitory activity [55]. The use of metabolic inhibitors is being considered as a good potentiation alternative to help with the present antibiotic resistance challenge. Antibiotics used in conjunction with other antibiotics or naturally generated AMPs, on the other hand, are predicted to function in the same way when bacteria perform resistance activity.

Membrane Potential Disruption of Biofilm Participating Cells

The idea of membrane depolarization for the breakdown of cells in biofilms has been established in studies to have a fundamentally distinct method of action. AMPs damage the cell membrane, causing attachment glycolipids to delocalize and autolysins to be released. The potential of esculentin (Esc (1–21) generated from frog skin was documented in P. aeruginosa biofilms as the release of beta-galactosidase following rapid penetration into the cytoplasmic membrane [95]. In another research, the application of nisin A, nukasin ISK-1, and laticin Q in S. aureus resulted in the release of ATP from inside the bacterial cells that creates a bed inside the biofilm [96]. The bactericidal efficacy of the synthetic peptide RN3(5-17P22-36), an eosinophil granule based cationic peptide, has also been investigated in biofilm producing species [97].

Bacterial Signalling Interference

Biofilm development in bacterial cells has been targeted by cellular signalling that plays a role in cell mobility. Many AMPs have been demonstrated to increase the expression of genes necessary for the creation of type IV pilli, which are entirely responsible for the cells' locomotory activity. The increased mobility of cells caused by the accelerated production of genes keeps the cells moving continually [98]. The phenomena of quorum sensing have also piqued the curiosity of researchers. Rhl and Las systems regulated by the human cathelicidin LL-37 and indolicidins have been studied extensively in the case of P. aeruginosa [99].

Nanosytems Based Drug Delivery

The well documented AMPs with potent in vitro activity need to be sent to clinical use so that their impact can be potentiated. Although many AMPs have been described and investigated but not all have reached the clinical trials till date. The reason behind this shortcoming is the subjection of these peptides to system toxicity, proteolytic degradation by the gut enzymes [100]. During the systemic administration of the major challenge faced by the AMPs is their short half-life and protease degradation as well [101]. Many investigations have been done to stabilize these AMPs by chemical modulations during the synthesis and use of different delivery platforms for attacking the target site. The use of nanotechnology can be used to overcome the problem of system toxicity, degradation by proteolysis, short half-life and fast clearance of the AMPs in the renal system. The use of nano-based delivery systems to deliver drugs into biofilms is a promising method that needs to be further investigated. High permeability, inhibition of antibiotic-EPS binding, inhibiting enzymatic inactivation, and transporting medications to the infection site in an optimum dose are all benefits of nanoparticles for pharmaceutical administration [102, 103]. This method results in a large reduction in the dose of commercial antibiotics, which reduces medication-related toxicity and the development of drug resistance. With technological advancements, the combination of nanotechnology and sophisticated biology offers a new avenue in the field of effective therapies for biofilm-related illnesses and dangers. Extensive research and development for newer agents/compounds with comparable greater biocompatibility and biosafety, as well as new delivery systems that support various operating principles and routes, might be highly useful in the removal of difficult biofilm generating species. In a study, AuNPs–indolicidin complex increased the susceptibility of C. albicans by penetrating the biofilm matrix and the participating cells. Also, a significant down-regulation of the genes (ALS1, ALS3, EFG1, and HWP1) involved in biofilm formation, and drug transporter encoding genes (CDR1, and CDR2) as well, suggesting eradication potential of nano-AMP against the mature biofilms [104].

Inhibiting Bacterial Stringent Response

Genes are responsible for the production of biofilm as a stress response in both Gram-positive and Gram-negative bacteria. The creation of guanosine 5'-diphosphate 3'-diphosphate nucleotide, a key signalling factor produced by bacteria, influences gene expression regulation [105, 106]. The reduction of biofilm formation by AMPs like DJK-5, DJK-6 and 1018 either by blocking synthesis or inducing degradation has been investigated [107].

In staphylococcus epidermidis bacteria interference in biofilm formation has been studied by the action of Human β-defensin 3 (hBD-3) peptides. The peptide is responsible for reductional synthesis of polysaccharide intercellular adhesion (PIA) molecule which is controlled by the downstream regulation of icaA, icaD and icaR [108]. A superfamily of integral membrane proteins that are responsible for the ATP-powered translocation of numerous substrates across membranes facilitates cell-to-surface and cell-to-cell contacts in biofilm formation [108–110]. The activity of an AMP, Nal-P-113, modified by the substitution of histidine residues with bulky amino acid-naphthylalanine in P-113 structure, was shown to down-regulate genes such as PG0282 (ABC transporter) and PG1663 (ATP-binding protein) in a research [111].

Promiscuity Based Approach

The immune system is based on the process of "recognition and display," according to traditional biochemical ideas. Specificity or particular reactivity are terms used to describe such events. During the study of protein interactions, the concept of restricting combinations to specified sites has been intensively investigated. Proteins are thought to bind to their designated domains in a certain fashion, according to biochemical research. Promiscuous behaviour refers to a variety of departures from mainstream beliefs that have been discovered. Surprisingly, the capacity of multi-specific AMPs to target complex biofilm lattices suggests that they might be a viable alternative in the drug discovery pipeline. However, a considerable emphasis should be placed on determining the structural processes used by antimicrobial proteins/peptides to achieve promiscuity at the molecular level.

The great majority of antimicrobial items developed so far are drawn from known mixes and target the same component of resistance in the hopes of improving pathogen activity. Currently, research is focusing on AMPs, which stand out due to their much more complex compositions and higher hit rate, as studies have been conducted using a computational approach. However, traditional practises involving the use of AMPs in combination with conventional antibiotics are still lacking [112]. AMPs are found in a variety of complicated composites and come from a variety of sources. Because of their unknown routes of dispersion across biological systems, AMPs appear to play a critical role in pathogenic and immunological cascades in various living forms. AMPs have remained effective defences against a wide range of infections and their complications. Without a doubt, it has been suggested that AMPs and AMP-coordinated antibiotics have the ability to overcome commercial antimicrobial and antibiotic resistance, as well as leave inadequate microorganism balance for infection to grow successfully [113]. In contrast to traditional antimicrobials, which are primarily active against bacteria and their growth, AMPs are active against a broad spectrum of microorganisms and biofilms. The problem of drug resistance has gotten worse in the case of antibiotics/bacterial infections due to gene transfer, which is a typical occurrence, and the production of efflux pumps (proteins), which operate as a carrier medium for removing antibiotics from bacterial cells. In nosocomial situations, there is also a high level of malignancy and medication resistance. Promiscuity has been defined in numerous studies as the similarity of binding sites in diverse targets. For the promiscuous proteins that are now accessible, the currently available protein datasets have been rigorously examined. A report recorded that due to equal K_cat value of nucleotide triphosphate to a protein dethiobiotin synthetase, found in Mycobacterium tuberculosis bacteria. On a broader part many promiscuous drugs have been screened till date against harmful pathogens (Trypanosomia, Leishmania major) and conditions (cancer, diabetes and skin diseases) thus making the approach of promiscuity for better drug treatment therapies [114].

Conclusions

Antibiotic resistance is emerging as one of the most significant dangers to global health, despite the fast-paced research conducted by health specialists all over the world. Antibiotic resistance owing to biofilms and associated recalcitrance in homo and heterospecies microbial microenvironments is a subject of medical inquiry due to the complexity of composition. This complicated growth stage aids bacteria's survival amid extracellular stress caused by antibiotic usage and abuse. Biofilm-based antibiotic resistance plays a critical role in departures from the antibiotic specificity rule, giving a fresh platform for learning about the underlying concerns. AMPs have wide antibacterial action and can be used in conjunction with first-line antibiotics. AMPs have a variety of fascinating qualities that can be used to inhibit the production of biofilms. According to the study, AMPs contain a number of active anti-biofilm mechanisms that might be exploited to eradicate biofilms in nosocomial situations. AMPs might be used as a stand-alone treatment or in combination with other antimicrobials to remove biofilms. Furthermore, AMPs' diversion from one target one choice, both directly on biofilms and for potentiation of existing antibiotics, opens up a new avenue for drug research progress. Nonetheless, additional research into the anti-biofilm efficacy of AMPs on growing biofilms is needed. More study is needed to better understand the microenvironments in which reduced toxicity, high stability, and improved effectiveness are important therapeutic components. As a result, research into alternatives to standard antibiotics that directly inhibit and/or remove biofilms is urgently needed.

Author Contributions

SS: Conceptualization, investigation, visualization, writing-original draft, review and editing. AKL and VG: Investigation, visualization, writing, review and editing.

Declarations

Competing interests

The author declared that there is no conflict of interest.