Natural Strategies as Potential Weapons against Bacterial Biofilms

Microbial biofilm is an aggregation of microbial species that are either attached to surfaces or organized into an extracellular matrix. Microbes in the form of biofilms are highly resistant to several antimicrobials compared to planktonic microbial cells. Their resistance developing ability is one of the major root causes of antibiotic resistance in health sectors. Therefore, effective antibiofilm compounds are required to treat biofilm-associated health issues. The awareness of biofilm properties, formation, and resistance mechanisms facilitate researchers to design and develop combating strategies. This review highlights biofilm formation, composition, major stability parameters, resistance mechanisms, pathogenicity, combating strategies, and effective biofilm-controlling compounds. The naturally derived products, particularly plants, have demonstrated significant medicinal properties, producing them a practical approach for controlling biofilm-producing microbes. Despite providing effective antibiofilm activities, the plant-derived antimicrobial compounds may face the limitations of less bioavailability and low concentration of bioactive molecules. The microbes-derived and the phytonanotechnology-based antibiofilm compounds are emerging as an effective approach to inhibit and eliminate the biofilm-producing microbes.


Introduction
A biofilm is a complex of micro-organisms that sustains a structured and organized pathway for their growth, proliferation, and survival on any surface [1,2]. A single bacterial species may develop the biofilm organizations, or the biofilm can also be of mixed microbial species adhered to a surface [3]. The survival of biofilm-forming bacterial cells depends upon the alignment of extracellular polymeric substance-encapsulated micro-colonies in the matrix [4]. The biofilm-forming microbial cells maintain their micro-environment by controlling temperature, nutrients, and pH, which can affect biofilm formation. Biofilms are known to cause several infections or diseases in humans [5,6]. Several antibiotics have been used against biofilm-associated infections, but increased antimicrobial resistance (AMR) has directly been linked to biofilm microbes [7]. This inefficacy of numerous antibiotics against several biofilm-linked infections has gradually enhanced the emergence of AMR.
The development of microbial resistance to antibiotics reduces or inhibits the efficacy of antibiotics. Consequently, it has been demonstrated that the improper use of antibiotics leads to the emergence of antibiotic resistance [8]. Many antibiotics are losing efficacy due to several micro-organisms' expeditious developments in multidrug resistance. Several factors responsible for causing AMR and intrinsic biofilm formation have been recognized as major critical factors [9]. Moreover, AMR caused by biofilm development may cause harmful recurrent chronic microbial infections. Therefore, the discovery of a significant treatment approach is much needed to combat AMR. Several researchers are designing and evaluating new combat strategies based on one health concept [10].
This study explores the new combat approaches that avoid existing resistance mechanisms. Using different natural products, such as plant-derived components, bee products, marine-derived components, and plant-based nanomaterials, is gaining great attention from researchers to design novel antibiofilm compounds to avoid AMR. In the current review, the emerging global concerns regarding the development of biofilm resistance and their control by describing some potential therapeutic compounds have been recapitulated.

Biofilm Formation
Biofilm can be defined as the complex aggregation of micro-organisms, firmly adhered to a surface and micro-colonized into extracellular polymeric substances (EPS) matrix. EPS is comprised of exopolysaccharides, nucleic acids, lipids, and proteins [11]. The emergence of biofilm formation is a multistep process in which EPS performs particularly required functional and structural roles. Microbial communities may attach to both abiotic and biotic surfaces facilitated by EPS. In order to maintain a biofilm lifestyle, the EPS matrix provides essentially required chemical microenvironments and mechanical stability [12].
In addition, the EPS also improves biofilm tolerance toward different antimicrobial agents and immune cells. The biofilm maturation needs several developmental phases with particular features, resulting in biofilm development on the surfaces. Additionally, understanding every developmental phase is essential for designing and applying proper antimicrobial agents against microbial biofilms. Each developmental phase is briefly elaborated on in Figure 1.

Surface Adhesion
Biofilm development is initiated with the attachment of microbial cells to any surface. Several sensing pathways (e.g., BasSR, BaeSR, and CpxAR) facilitate the microbes' identification of the surface [13]. After initial adhesion, the microbial cells start to multiply and divide expeditiously under favorable conditions [14]. The microbial cells produce adhesins (enzymes) that facilitate their attachment to the host surfaces. This phase explicitly targets the adherence mechanism between the microbes and the surfaces. Thus, in this phase, significant combating intervention can be achieved by entirely disrupting the attachment mechanisms of micro-organisms with the surfaces via mainly targeting cell surface-linked adhesins. Consequently, the initial biofilm development can be inhibited by disrupting the initial adhesion process [15].
Surface adhesion is essentially the required parameter for biofilm development. The adhesion initiation and the biofilm dispersal start with the adherence capacity of a specific bacterial species concerning the host surface. The surface attachment, along with biofilm development, is the survival strategy of the microbes that typically adhere to themselves in a particular way to meet their environmental and nutritional requirements. The surface adhesion consists of two phases: the primary/reversible phase and the secondary/irreversible attachment phase [16]. Both of these phases are entirely controlled by the gene's expression. The attachment processes require several points to be considered, including microbial species, environmental conditions, gene products, and surface composition [17]. In the reversible phase, microbes hydrophobically interact with the abiotic surfaces for adhesion, whereas adherence with biotic surfaces takes place by developing molecular interactions [18].

Biofilm Maturation
The formation of mature biofilm is followed by early biofilm development when the microbes begin to multiply and divide, creating micro-colonies incorporated with the EPS matrix [19]. The EPS matrix plays a multifunctional role, allowing the establishment of several physical and chemical microhabitats facilitating the microbes to build social and

Surface Adhesion
Biofilm development is initiated with the attachment of microbial cells to any surface. Several sensing pathways (e.g., BasSR, BaeSR, and CpxAR) facilitate the microbes' identification of the surface [13]. After initial adhesion, the microbial cells start to multiply and divide expeditiously under favorable conditions [14]. The microbial cells produce adhesins (enzymes) that facilitate their attachment to the host surfaces. This phase explicitly targets the adherence mechanism between the microbes and the surfaces. Thus, in this phase, significant combating intervention can be achieved by entirely disrupting the attachment mechanisms of micro-organisms with the surfaces via mainly targeting cell surface-linked adhesins. Consequently, the initial biofilm development can be inhibited by disrupting the initial adhesion process [15].
Surface adhesion is essentially the required parameter for biofilm development. The adhesion initiation and the biofilm dispersal start with the adherence capacity of a specific bacterial species concerning the host surface. The surface attachment, along with biofilm development, is the survival strategy of the microbes that typically adhere to themselves in a particular way to meet their environmental and nutritional requirements. The surface adhesion consists of two phases: the primary/reversible phase and the secondary/irreversible attachment phase [16]. Both of these phases are entirely controlled by the gene's expression. The attachment processes require several points to be considered, including microbial species, environmental conditions, gene products, and surface composition [17]. In the reversible phase, microbes hydrophobically interact with the abiotic surfaces for adhesion, whereas adherence with biotic surfaces takes place by developing molecular interactions [18].

Extracellular Proteins
The biofilm complex can have a substantial number of extracellular proteins [34]. Their interaction with exopolysaccharides and nucleic acids facilitates surface colonization and stabilization in the biofilm matrix [35]. Some proteins mediate the degradation and dispersion of biofilm matrix, such as glycosyl hydrolase, dispersin B induces polysaccharide degradation [36], proteases dissolve the proteins of matrix [37], and some DNases cause extracellular nucleic acid breakage [38]. Toyofuku et al. described that 30% of EPS-matrix proteins in P. aeruginosa were observed in outer-membrane vesicles as membrane proteins. Some of them were secreted and lysed by cell-derived proteins [39].
Several extracellular enzymes have also been found in microbial biofilms; some of them are involved in biopolymer degradation. The extracellular enzyme substrates contain waterinsoluble components (such as lipids, cellulose, and chitin), water-soluble compounds (such as proteins, polysaccharides, and nucleic acids), and the biofilm entraps organic particles [40]. Additionally, some extracellular enzymes can be used for EPS structural degradation to mediate the microbial detachment in biofilms.

Extracellular DNA (eDNA)
eDNA is one of the prime components of the EPS matrix, which is essential for the accumulation of microbes within the biofilm. The amount of eDNA production may vary even among closely linked microbial species. eDNA is the primary structural constituent in the matrix of a Staphylococcus aureus biofilm, while in S. epidermidis biofilms, it is produced as a minor constituent [41]. It has been revealed that eDNA is a vital component of the biofilm matrix and its mode of life [42,43]. eDNA has also been found as a major component in the biofilm matrix of P. aeruginosa and facilitates intercellular interactions [44].
Moreover, the eDNA was observed to inhibit the P. aeruginosa biofilm formation, while in Bacillus cereus the eDNA acts similar to adhesins [45,46]. Okshevsky and Meyer reported that eDNA is involved in cell adhesion, structural stability maintenance, and horizontal gene transfer and also protects the immune system and antimicrobials [47]. eDNA has been observed to facilitate cell adhesion and biofilm development in Listeria monocytogenes [48]. Wilton et al. found that eDNA causes acidification of the biofilm matrix and consequently enhances resistance of P. aeruginosa biofilms against different antibiotics [49].

Surfactants and Lipids
The extracellular polysaccharides, proteins, and eDNA are quite hydrated (hydrophilic) molecules, while the other components of EPS exhibit hydrophobic properties. Some bacterial species, such as Rhodococcus spp. generate hydrophobic EPS, and can attach to Teflon and may colonize the waxen surfaces with the help of hydrophobic EPS [50]. In the EPS matrix, a few lipids exhibiting surface active properties, such as surfactin, viscosin, and emulsan, increase the availability of hydrophobic molecules by causing their dispersion [24]. An important surfactant class, biosurfactants, begins the micro-colony formation, aids in biofilm structural integrity, and mediates biofilm dispersal [51].

Water
Water is recognized as the largest constituent (accounts for up to 97%) of the EPS matrix of most of the biofilms, and it retains the biofilm hydrated and protects against desiccation [24]. The water in the biofilm matrix may exist in the form of solvents or can also be bound inside the bacterial cells' capsules [52]. The binding and movement of water inside the biofilm matrix are essential to diffusion mechanisms that occur inside the biofilm and result in fine biofilm structure development [53]. The amount of available water is responsible for nutrient flow and availability within the microbial biofilms [52].

Biofilm Structural Stability Parameters
The antimicrobial resistance and the other functional characteristics of microbial biofilms are linked with the structure of the biofilm, matrix shape, and the 3D organization of microbes [54]. The local environmental heterogeneous conditions inside the biofilm matrix impact the microbial gene expression and the metabolic actions of biofilm-developing microbial cells [55,56]. The closely packed microbial cells and the water channels are the two major constituents of biofilm formation [57]. The structural familiarity of microbial biofilms is of greater concern in identifying their behavioral and survival strategies. The biofilm architecture and formation variability have been analyzed by applying specific parameters, such as substratum exposure, bio-volume, thickness, and roughness, and observing significant inter-and intra-species variability [56].
The cell-cell communication, environmental influences, and the secondary messengers, such as c-di-GMP and cAMP, structure the biofilms by providing microbes with better environmental adaptability [58]. Several other factors influence the biofilm architecture, such as nutrient availability, microbial motility, hydrodynamic conditions, exopolysaccharides and protein abundance, and anionic and cationic concentrations within the biofilm. In P. aeruginosa, an EPS known as alginate facilitates biofilm formation and its architectural stability [59].
The EPS in Vibrio cholera and E. coli facilitates biofilm development in a three-dimensional configuration [60,61]. An EPS and a secreted protein called TasA are vital for developing a fruiting body, such as a Bacillus subtilis biofilm, and maintaining the biofilm matrix's integrity [62]. The architecture of biofilm can be altered by the exopolysaccharides' substituents, such as acetyl groups, known to be responsible for enhanced cohesive and adhesive biofilm properties [24].

Proteins
Manifoili et al. determined the impact of three different mitogen-activated protein kinases (MAPKs) (such as SakA, MpkA, and MpkC) and protein phosphatases (PhpA) on Aspergillus fumigatus biofilm formation. MAPKs reduce the A. fumigatus adhesion in biofilm formation. The ∆pphA strain was found to be more susceptible to cell wall destroying antimicrobials, had less chitin, and enhanced β-(1,3)-glucan, resulting in reduced adherence and biofilm formation [63].
The bacterial cell wall-linked fibronectin-binding proteins (FnBPs) (FnBPB and FnBPA) mediate the biofilm development of methicillin-resistant S. aureus (strain LAC) by promoting bacterial adhesion and accumulation [64]. The outer membrane protein W (OmpW) contributes to Cronobacter sakazakii survival and biofilm formation under NaCl-stressed conditions [65]. A surface protein, BapA1, plays a significant role in bacterial adhesin and biofilm formation. BapA1 carries the nine putative pilin iso-peptide linker domains, which are significant for bacterial accumulation of pilus in several Gram-positive bacteria, such as Streptococcus parasanguinis [66].

Genes and Signaling Cascades
Intracellular cyclic dinucleotide and extracellular quorum sensing (QS) signaling cascades are crucial in biofilm development. It has been reported that these two signaling pathways may coincide or link up and synergistically mediate biofilm formation [67]. QS is the process of intercellular communication that enables the bacteria to adapt to harsh environmental conditions. They mediate biofilm formation by activating small signaling molecules, such as autoinducer-2 (AI-2), auto-inducing peptide (AIP), and Nacyl-homoserine lactones (AHL), in Gram-positive and -negative bacteria, respectively [68]. AI-2 mediates the QS and biofilm development with bhpand ica-dependent modes. AI-2 regulates the QS in S. epidermidis through increased transcription levels of bhp (biofilmlinked protein containing icaR) and ica operon [69].
QS signaling cascades observed in P. aeruginosa include integrated QS (IQS), PQS, rhl, and las. These QS systems activate each other by regulating QS-associated genes [70]. The small non-coding RNAs (sRNAs) regulate the bacterial transition from planktonic-sessile bacterial biofilms. The two-component regulatory systems (TCSs), such as RsmZ and RsmY targeting RsmA, are involved in P. aeruginosa biofilm formation [71]. Several sRNAs have been studied that regulate the activity or expression of different transcriptional regulators to mediate bacterial adhesion and increase biofilm formation ( Table 2).

Resistance Mechanism in Biofilms
Several antimicrobial resistance mechanisms have been identified, and biofilm development is one of the major factors of resistance emergence. Mechanisms allowing microbial biofilms to resist or tolerate the antimicrobials' actions are discussed here.

The Structural Complexity of Biofilms
As EPS is crucial for a biofilm's architectural stability, it also acts as a physical barrier, protecting or shielding the embedded microbes against antimicrobials, ultraviolet light, etc. [85,86]. The polysaccharides (negatively charged) can significantly bind to the aminoglycoside antibiotics (positively charged) and block their penetration [87]. The EPS barrier can reduce the diffusion of small compounds, such as H 2 O 2 (hydrogen peroxide), to microbial cells inside the biofilm. It has been observed that P. aeruginosa in the planktonic  [88].

The Heterogeneity of Biofilms
The heterogeneity inside the developed biofilms averts the entire eradication of all involved microbial cells by antimicrobials. There are oxygen and nutrient gradients from the top-bottom of microbial biofilms. From the top-bottom of the biofilm matrix, the oxygen and nutrient reduction leads to a reduced growth rate and metabolic activity [89]. The protein expression of bacterial cells in biofilm is diverse and quite different from planktonic cells, which may also contribute to microbial resistance development. For example, in its biofilm form, a rice endophytic bacterium, Pantoea agglomerans YS19, expresses SPM43.1 protein (acid-resistant) at high levels to resist harsh environmental conditions [90,91].

The Heterogeneity of Biofilms
The heterogeneity inside the developed biofilms averts the entire eradication of a involved microbial cells by antimicrobials. There are oxygen and nutrient gradients from the top-bottom of microbial biofilms. From the top-bottom of the biofilm matrix, the oxy gen and nutrient reduction leads to a reduced growth rate and metabolic activity [89]. Th protein expression of bacterial cells in biofilm is diverse and quite different from plank tonic cells, which may also contribute to microbial resistance development. For example in its biofilm form, a rice endophytic bacterium, Pantoea agglomerans YS19, expresse SPM43.1 protein (acid-resistant) at high levels to resist harsh environmental condition [90,91].

Quorum Sensing
Quorum sensing (QS) in microbes is a cell-cell communication mediated by activa ing specific signaling molecules, facilitating environmental adaptation to microbes [2]. Q is a crucial mechanism for regulating and developing biofilms by reducing or inhibitin the effectiveness of antimicrobials against biofilm bacteria [92]. Gram-negative and pos tive bacterial species communicate using these signaling molecules, also known as auto inducers (AIs) [2]. Some QS signaling molecules used by Gram-positive and negative bac teria include (a) N-acyl homoserine lactone (AHL), (b) autoinducer-2 (AI-2) by V. harvey (c) autoinducing peptide 1 (AIP-1) by S. aureus, (d) N-(3-oxoacyl)-l-homoserine lactone (3 oxo-AHL), (e) diffusible signaling factor (DSF), (f) N-(3-hydroxyacyl) homoserine lacton (3-hydroxy-AHL), (g) 2-heptyl-3-hydroxy-4(1H)-quinolone by P. aeruginosa, and (h) hy droxy-palmitic acid methyl ester (PAME). They are presented in Figure 2. Environmental factors, such as nutrient deficiency, pH, antimicrobials, and salt con centrations, regulate QS-mediated activity in bacterial biofilms [93]. The feed-forwar mechanism enhances the QS communication in the biofilm matrix [94]. The QS signal facilitate biofilm formation when their concentration reaches the threshold [95]. Subse quently, the QS signaling molecules are translated into cells for gene expression modula tion. These genes are crucial for environmental adaptation, leading to biofilm formatio Environmental factors, such as nutrient deficiency, pH, antimicrobials, and salt concentrations, regulate QS-mediated activity in bacterial biofilms [93]. The feed-forward mechanism enhances the QS communication in the biofilm matrix [94]. The QS signals facilitate biofilm formation when their concentration reaches the threshold [95]. Subsequently, the QS signaling molecules are translated into cells for gene expression modulation. These genes are crucial for environmental adaptation, leading to biofilm formation [96]. The QS signaling system regulates bacterial and EPS secretion systems [97] and multidrug efflux pumps [98]. The complete information about QS signaling molecules may offer significant information for developing novel methods or chemicals for combating microbial biofilms. This research era has grabbed the great attention of researchers for designing and developing significant molecules with the ability to neutralize or compete with the QS signaling molecules or their receptors [99].

Enhanced Efflux Pumps
The efflux pumps (proteinaceous) entrenched in the cytoplasmic membranes act as active transporters. The multidrug and toxic compound extrusion family (MATE), the ATP-binding cassette family (ABC), the small multidrug resistance family (SMR), the resistance-nodulation-division family (RND), and the major facilitator superfamily (MF) are commonly reported efflux pump classes in different bacteria [100,101].
Several molecular studies have revealed that increased efflux pumps are a popular and criticizing resistance mechanism in microbial biofilms [102]. This mechanism has been extensively studied in a commonly found biofilm-producing P. aeruginosa pathogen [103]. The PA1874-1877 (cluster of genes) involved in developing resistance in biofilms was discovered by Zhang and Mah. Overexpression of PA1874-1877 in biofilm cells facilitates resistance in a biofilm-specific manner [104]. Numerous efflux pump genes inducing biofilm-specific resistance through their overexpression have been identified. For example, in RND-3 efflux pumps, the overexpression of BCAL1672-1676 induces biofilm resistance against ciprofloxacin and tobramycin, while in RND-8 and RND-9, the overexpression of BCAM0925-0927 and BCAM1945-1947 provides resistance to Burkholderia cepacia against tobramycin [105].

Pathogenicity of Biofilm Microbes
Numerous factors are known to contribute to pathogenicity in biofilm-producing microbes. Microbial biofilms release different extracellular substances, altering the gene regulation of several microbial virulence factors. Moreover, the biofilm-producing microbes strengthen the maturation rate of biofilms to escape from host defenses, enhance the activity of β-lactamase, and for plasmid-mediated gene transfer resulting in intense virulence and antimicrobial resistance with enhanced mutation rate and efflux pump. The properties of the extracellular matrix contribute to the biofilm's pathogenicity, offering a defensive barrier with less antimicrobial and immune cell penetration [106].
The MIC of antibiotics is significant against planktonic microbes but not effective against biofilm microbes [107]. Microbial biofilms can induce several persistent biofilmassociated infections, such as urinary tract infections, middle-ear infections, dental caries, endocarditis, cystic fibrosis, osteomyelitis, and implant-induced infections. Numerous pathogenic microbes are involved in causing persistent biofilm infections; some of them are listed in Table 3. [111] Haemophilus influenza, Streptococcus pneumoniae, Moraxella catarrhalis.
Lungs (in patients with cystic fibrosis) Mucoviscidosis, lung infections [113] The pathogenic activity of microbes in the form of biofilms is significantly higher, and they can escape from host defense cells and antibiotics. In numerous infections, biofilm bacteria are concerned with the pathogenesis and clinical symptoms [114]. Opportunistic pathogenic bacteria, such as P. aeruginosa and S. aureus, can cause chronic biofilm infections, and hospitalized individuals (approximately 8-10%) are more vulnerable to carrying infections.

Health Problems and Infections Caused by Biofilm Bacteria
Biofilm-associated infections pose a threat to human health. Over the last few decades, innovative methods have been discovered to control microbial infections. Biofilm formation in the era of the food industry poses a serious threat to human health. Biofilms may contain only one type of bacteria, different bacterial species, or fungal species that may be pathogenic and may only target immunocompromised patients (cancer patients, organ recipients, HIV patients, etc.). Systematic diseases (E. coli, L. monocytogenes), food intoxication (P. aeruginosa, S. aureus, B. cereus), and gastroenteritis (Salmonella enterica, E. coli) can be caused by biofilm-producing pathogens [114].

Biofilms in the Food Industry
Foodborne infections may arise from microbial biofilm development on food processing equipment or food matrices. Biofilms formed on food processing equipment can secrete toxins and may result in food poisoning. Biofilm formation in any food industry may put human health at potential risk. The severity of the risk is directly dependent on the microbial species of the biofilm matrix.
Food processing plants provide suitable conditions for biofilm development on food surfaces due to the complexity of manufacturing or processing plants, mass product yield, long manufacturing durations, and large biofilm formation areas [115]. These biofilm formations may contribute to the emergence of biofilm-associated foodborne infections. Approximately 80% of microbial infections in the USA are considered to be specifically associated with biofilm-producing foodborne pathogens [116]. Mixed-species or polymicrobial biofilm formation is a highly diverse phenomenon and depends upon environmental conditions [117], adherence characteristics of surfaces [118], involved microbial cells [119], and components of the food matrix [120]. Adherence surface characteristics, such as electrostatic charge, topography, interface roughness, and hydrophobicity, impact biofilm development and consequently affect the surface's hygienic status [118,121].
Properties of microbial cells, such as components of cell membranes (e.g., lipopolysaccharides and proteins), hydrophobicity, exopolysaccharides (EPS) production by microbes, and the bacterial appendages (e.g., fimbriae, pili, and flagella), contribute to a crucial role in biofilm formation [118]. Some studies have reported that microbial adherence is more likely to develop on rough surfaces [122], and some experiments indicated no association between microbial adherence and surface roughness [123]. The components of the food matrix in food processing plants may influence microbial adhesion, such as food waste, e.g., carbohydrates, proteins, and fat-enriched meat and milk exudates, mediate microbial growth and proliferation, and facilitate the dual-species biofilm development by S. aureus and E. coli [124,125]. Biofilm-producing foodborne pathogens have emerged as a serious threat to human health. Some foodborne pathogens with biofilm-forming ability and their harmful effects are listed in Table 4. Biofilms have been found to be associated with different outbreaks or epidemics (Table 5). Table 4. Biofilm-associated foodborne pathogens along with their consequences.

Organism
Contaminated Food Items Consequences Refs.

Biofilm Control
The global rise in antibiotic resistance has led to the failure of antibiotics. The ABR has become a major threat to human health. Therefore, alternative therapies have been reported to eliminate or inhibit biofilm formation and their associated infections. Different points can be targeted for biofilm inhibition and eradication at different stages of biofilm formation (Figure 3). These combating strategies include inhibition of planktonic cells, inhibition of bacterial adhesion, surface alteration, biofilm removal, degradation of EPS, QS inhibition, dispersion of biofilms, and matrix degradation.

Biofilm Controlling Compounds
Many natural compounds can act as biofilm-controlling compounds by interfering with QS, possessing antiadhesive properties, and inhibiting biofilm formation (Figure 4).

Biofilm Controlling Compounds
Many natural compounds can act as biofilm-controlling compounds by interfering with QS, possessing antiadhesive properties, and inhibiting biofilm formation (Figure 4).

Biofilm Controlling Compounds
Many natural compounds can act as biofilm-controlling compounds by interfering with QS, possessing antiadhesive properties, and inhibiting biofilm formation (Figure 4).

Natural Plants and Bee Products
Several naturally occurring compounds can be used as antibiofilm molecules to eradicate or inhibit biofilm development. The garlic extract can significantly block QS and may promote rapid virulence attenuation (e.g., elastase, protease A, exo-and cytotoxin production or motility, and adhesion capacity reduction) of P. aeruginosa by polymorphonuclear leukocytes (PMNs) within the immune response of a mouse infection model [146]. Persson et al. synthesized some QSIs derived from garlic extracts and AHLs [147]. Chamaemelum nobile is a naturally occurring, well-known plant for its antimicrobial, anti-inflammatory, antiseptic, spasmolytic, anticatarrhal, sedative, and carminative properties. It can inhibit P. aeruginosa biofilm by disrupting QS [148]. Proanthocyanidins extracted from cranberries have significantly inhibited the adhesion of E. coli to uroepithelial cells [149]. Cranberry juice also significantly inhibited the site-specific adherence of Helicobacter pylori to the gastric mucous of humans [150], and it also prevented Streptococci spp. biofilm formation [151,152]. Eighty medicinal plants, more prominently Fritillaria verticillata, Rhus verniciflua, Cocculus trilobus, and Liriope platyphylla, have been analyzed for their antibiofilm activity. Comparatively, Cocculus trilobus (ethyl acetate fraction) has shown the highest antibiofilm activity against Gram-positive bacteria by providing effective antiadhesive activity [153]. A Chinese herb, Herba patriniae, with medicinal properties, has averted the gene expression of six genes linked with biofilm development and EPS production in P. aeruginosa [154]. The Ginkgollic acid isolated from a plant, Ginkgo biloba, exhibited antitumor, antimicrobial, and neuroprotective and is active against S. aureus strains and E. coli biofilm formation [155,156].
Elekhnawy et al. determined the antiquorum sensing and biofilm inhibitory potentials of Dioon spinulosum plant extract against the clinical isolates of P. aeruginosa. The in vitro analysis of antibiofilm activity showed a 77.1-34.3% reduction in biofilm formation at 250-500 µg/mL concentrations. Both in vitro and in vivo investigations revealed a significant reduction in P. aeruginosa biofilm formation. However, preclinical studies leading to clinical studies are recommended to allow its practical application in treating P. aeruginosa infections [157]. Obaid et al. studied the antibiofilm activity of six plant extracts, such as Apium graveolens, Plantago ovata, Vitis vinifera, Viscus album, Senna acutifolia, and Melissa officinalis, against Aggregatibacter actinomycetemcomitans. The A. actinomycetemcomitans was collected from patients with dental caries. The obtained results indicated that the A. actinomycetemcomitans was more sensitive to S. acutifolia and M. officinalis, with zone inhibitions of 33 and 35 mm, respectively [158]. Negam et al. evaluated the antifungal and antibiofilm potential of Encephalartos laurentianus (methanol extract) against C. albicans. The in vitro antibiofilm analysis revealed a 62.5-25% reduction in C. albicans cell percentage. The in vivo evaluations of E. laurentianus performed on C. albicans infected rats resulted in an increased survival rate with a protective effect against renal damage caused by C. albicans [159].
Olawuwo et al. investigated the in vitro antibiofilm activity of Acalypha wilkesiana, Alchornea laxiflora, Ficus exasperata, Jatropha gossypiifolia, Morinda lucida, and Ocimum gratissimum plant extracts against poultry pathogens (Aspergillus flavus, A. fumigatus, C. albicans, Campylobacter spp., Salmonella spp., E. coli, S. aureus, and Enterococcus faecalis). All plant extracts showed effective biofilm inhibition of approximately >50% against the tested micro-organisms [160]. Fathi et al. determined the antibiofilm potential of Malva sylvestris methanolic extract against some human pathogens, such as E. coli, S. aureus, P. aeruginosa, E. faecalis, and K. pneumoniae. The highest biofilm inhibition was examined against S. aureus Nazzaro et al. determined the antibiofilm activity of EOs of aerial parts and bulbs of two different cultivars of Allium sativum (Bianco del Veneto, Staravec) against nosocomial and food pathogens S. aureus, E. coli, L. monocytogenes, and Acinetobacter baumannii. The EOs from the bulbs and aerial parts of Bianco del Veneto showed significant inhibitory activity against all tested bacteria, more prominently against L. monocytogenes 64.29-60.55%, respectively. The EOs from the aerial parts of Staravec exhibited effective inhibition more effectively against Acinetobacter baumannii (45.61%), while EOs from the bulbs of Staravec showed no inhibition. The outcomes revealed their potential application as potential antibiofilm agents in the food industry and health sector as well [166].
Gamal El-Din et al. investigated the in vitro antibiofilm potential of EOs of three species of Jatropha flowering plant. The EOs were obtained from J. intigrimma, J. gossypiifolia, and J. roseae. The 7. 81, 15.63, 31.25, 62.5, 125, 250, 500, and 1000 µg/mL concentrations were used to evaluate the antibiofilm activity. J. intigrimma EO exhibited 100% biofilm inhibitory activity at 31.25 µg/mL. J. roseae EO showed 100% inhibition at 250 µg/mL, while J. gossypiifolia EO revealed less effective biofilm inhibition even at 1000 µg/mL. However, it can be suggested that J. intigrimma and J. roseae EOs can be used as promising antibiofilms and furthering in vivo investigations is also highly recommended [167]. Djebilli et al. determined the composition profile, antioxidant, and antibiofilm efficacy of EOs from Algerian aromatic plants, including Thymus algeriensis, Eucalyptus globulus, and Origanum glandulosum. The EOs from all three plants showed significant antibiofilm activity with low minimum inhibitory concentrations (MICs) ranging between 0.078-1.25 µg/mL against C. albicans, E. coli, E. faecalis, L. monocytogenes, S. aureus, and S. Typhimurium. The order of biofilm inhibition against the tested bacteria was revealed as T. algeriensis > O. glandulosum > E. globulus EOs. The T. algeriensis EO showed the highest inhibition against L. monocytogenes (80.95%), and E. coli (77.83%) at MICs [168]. Some other biofilm inhibiting plant extracts and essential oils are listed in Table 6. Bee products are effectively being studied for their wide range of antibacterial, antioxidant, antiviral, antifungal, and anticancerous activities. Honey and its bioactive components are well recognized for their potential antibacterial effects against a wide range of bacteria and even against several antibiotic-resistant bacteria [185][186][187].
Fadl et al. reported the antibiofilm potential of bee venom against biofilm-forming MDR bacteria, such as S. aureus, vancomycin-resistant S. aureus (VRSA), P. aeruginosa, Enterobacter cloacae, and S. haemolyticus. Bee venom showed a considerable reduction in biofilm formation, ranging between 63.8-92% [195]. Bouchelaghem et al. determined the anti-biofilm impact of Hungarian propolis. The ethanolic extract of propolis (EEP) was used to evaluate the antibiofilm effect against MSSA and MRSA by applying a crystal violet assay. The EEP alone and in combination with vancomycin were tested against MSSA and MRSA. The EEP significantly inhibited biofilm development and degraded MSSA and MRSA mature biofilms. The EEP, combined with vancomycin, synergistically enhanced the antibiofilm activity against MRSA [188].

Nanotechnology and Phyto-Nanotechnology
Nanotechnology-based nanomaterials (NMs) are very small in size (<100 nm) with a large surface area and may provide several biological, chemical, and biomedical applications [196]. NMs can easily enter into the outer membrane (EPS) to release the antimicrobials to targeted sites without damage. Several NM types have been designed and evaluated to inhibit or eradicate microbial biofilms. Nanoparticles are mainly categorized into two categories: organic nanoparticles (NPs) (including polymers, cyclodextrins (CDs), liposomes, dendrimers, and solid lipid NPs) and inorganic NPs (including metal oxides, quantum dots, metallic NPs, and fullerene) [197]. NMs provide potential microbicidal activity alone or in combination with encapsulated drugs [198]. The NMs are reported to provide a promising therapeutic potential for developing significant antibiofilm action [199]. The development of plant-derived nanoparticles (NPs) has emerged as an innovative approach in the era of nanotechnology by synthesizing environmentally friendly substances with little to no toxicity [200]. Several researchers have significantly reported the green synthesis of NPs and their antimicrobial potential against different bacterial biofilms.
Swidan et al. investigated the biofilm inhibition of Ag NPs against enterococcal clinical isolates of the urinary tract (biofilm producing). Three types of Ag NPs were prepared to investigate the antibiofilm activity, including cinnamon Ag NPs synthesized by Cinnamon cassia, ginger Ag NPs synthesized by Zingiber officinale, and the chemically synthesized Ag NPs. The outcomes demonstrated that the chemical and ginger Ag NPs decreased the biofilm formation to 65.32% and 39.14%, and the adhesion to the catheter surface to 69.84% and 42.73%, respectively, and the cinnamon Ag NPs were not as significant. The ginger Ag NPs showed the most effective antibacterial and antiadhesion effects against the enterococcal clinical isolates that can also produce biofilms [201]. Muthulakshmi et al. synthesized the Ag NPs using Terminalia catappa plant leaves and evaluated the in vitro and in vivo antibiofilm potential against the foodborne pathogen L. monocytogenes. The in vitro analysis showed 33-45.5% biofilm inhibition at 50-100 µg/mL, respectively. The in vivo evaluation of Ag NPs using Caenorhabditis Elegans revealed 90% antiadherent activity against L. monocytogenes [202]. Salem et al. synthesized and characterized selenium NPs with orange peel. The biosynthesized Se NPs were used to evaluate the antibiofilm potential against S. aureus, K. pneumonia, and P. aeruginosa. The Se NPs at 0.5 µg/mL concentration showed 95, 88, and 75.5% biofilm inhibition against K. pneumonia, S. aureus, and P. aeruginosa, respectively [203]. Some plant-based NPs, along with their biofilm inhibitory actions, are summarized in Table 7. Liposomes can significantly enhance the interaction with bacterial membranes and mediate penetration into mature biofilms due to their high biocompatibility. The fusogenic liposome can be significantly employed to encapsulate drugs or antimicrobial agents with optimized release to increase the antibiofilm efficacy ( Figure 5). Vancomycin encapsulated with fusogenic liposomes (able to fuse with bacterial membranes) enhanced the bactericidal efficacy against S. aureus biofilms [213]. Meropenem-loaded nanoliposome with a dosage of ≥1.5 µg/mL entirely eradicated the P. aeruginosa biofilm [214]. The drug encapsulated in liposomes showed more effective results than the pristine drug. Liposomes as nanocarriers allow optimized drug release and inhibit biofilm growth.
Life 2022, 12, 1618 18 of 33 drug encapsulated in liposomes showed more effective results than the pristine drug. Liposomes as nanocarriers allow optimized drug release and inhibit biofilm growth.

Solid Lipid NPs
Solid lipid NPs (SLNs) containing surfactants stabilized lipid cores are spherical colloidal NMs (with 10-1000 nm diameters) [215]. Cefuroxime axetil-containing SLNs (CA-SLNs) were designed and analyzed against S. aureus biofilms. CA-SLNs showed a twofold higher anti-biofilm activity against S. aureus biofilm. Ninety-seven percent of the free CA was released in 2 h, while it takes 12 h to reach 96% release when it is in the encapsulated form [216].

QS Inhibiting NMs
Numerous QS inhibitor (QSI) NMs have been developed to inhibit or eradicate biofilm formation [217]. These QSI-NMs have numerous benefits over conventional QSIs. NMs can penetrate the biofilm matrix due to their smaller size and high solubility, providing optimized and targeted drug release. A QSI entrapped inside solid-lipid NPs with an ultra-small size (˂100 nm diameter) exhibits a 7-fold higher anti-biofilm activity against P. aeruginosa than a free QSI [217]. Tellurium (Te) and selenium (Se) NPs have been designed and tested against P. aeruginosa biofilms. Se-NPs and Te-NPs disrupted QS signaling and significantly reduced the bacterial biovolume, resulting in biofilm formation inhibition of 70-80% [218]. However, they were observed to be less effective in removing mature biofilms.

Cyclodextrins
Nguyen et al. investigated the anti-biofilm activity of a ubiquitous flavanone, naringenin (NAR), encapsulated β-CD and chitosan against the biofilm of E. coli [219]. NAR nanoencapsulation showed an effective antibiofilm potential against E. coli. Miconazole encapsulated CDs attached to polypropylene and polyethylene showed 87-96% inhibition against the biofilm formation of C. albicans [220]. CDs coated with drugs covalently bind to the surfaces and effectively inhibit C. albicans biofilm. Thymol and anidulafungin encapsulated cyclodextrins showed significant antibiofilm activity against the biofilms of C. albicans with 75-64% inhibition, respectively [221].

Solid Lipid NPs
Solid lipid NPs (SLNs) containing surfactants stabilized lipid cores are spherical colloidal NMs (with 10-1000 nm diameters) [215]. Cefuroxime axetil-containing SLNs (CA-SLNs) were designed and analyzed against S. aureus biofilms. CA-SLNs showed a two-fold higher anti-biofilm activity against S. aureus biofilm. Ninety-seven percent of the free CA was released in 2 h, while it takes 12 h to reach 96% release when it is in the encapsulated form [216].

QS Inhibiting NMs
Numerous QS inhibitor (QSI) NMs have been developed to inhibit or eradicate biofilm formation [217]. These QSI-NMs have numerous benefits over conventional QSIs. NMs can penetrate the biofilm matrix due to their smaller size and high solubility, providing optimized and targeted drug release. A QSI entrapped inside solid-lipid NPs with an ultra-small size (<100 nm diameter) exhibits a 7-fold higher anti-biofilm activity against P. aeruginosa than a free QSI [217]. Tellurium (Te) and selenium (Se) NPs have been designed and tested against P. aeruginosa biofilms. Se-NPs and Te-NPs disrupted QS signaling and significantly reduced the bacterial biovolume, resulting in biofilm formation inhibition of 70-80% [218]. However, they were observed to be less effective in removing mature biofilms.

Cyclodextrins
Nguyen et al. investigated the anti-biofilm activity of a ubiquitous flavanone, naringenin (NAR), encapsulated β-CD and chitosan against the biofilm of E. coli [219]. NAR nanoencapsulation showed an effective antibiofilm potential against E. coli. Miconazole encapsulated CDs attached to polypropylene and polyethylene showed 87-96% inhibition against the biofilm formation of C. albicans [220]. CDs coated with drugs covalently bind to the surfaces and effectively inhibit C. albicans biofilm. Thymol and anidulafungin encapsulated cyclodextrins showed significant antibiofilm activity against the biofilms of C. albicans with 75-64% inhibition, respectively [221].

Hydrogels
Many hydrogels have been developed to inhibit or eradicate several microbial biofilms. Some of these, such as metal nanoparticle-based hydrogels [222,223], chitosan-based hydrogels [224,225], bovine serum albumin (BSA) protein-based hydrogels [226], and pentapeptide-based supramolecular hydrogels [227], have exhibited significant biofilm inhibitory and eradication activities against several bacterial biofilms [228]. Some bacterio- phages [229,230], drugs [231], and different antimicrobials or active compounds [232] have also been encapsulated into hydrogels, providing efficient anti-biofilm activities with the optimized release of active compounds against different multidrug-resistant bacteria, such as P. aeruginosa, S. aureus, Acinetobacter baumannii, etc. Hydrogels can be used as promising biomaterials to treat and control multidrug-resistant bacterial infections. Some other NMs have also been reported for combating bacterial biofilms (Table 8).

Microbes and Marine-Derived Anti-Biofilm Compounds
Many micro-organisms produce several types of bioactive molecules with anti-microbial properties to benefit from other micro-organisms. Several investigations have determined the various secondary metabolites with potential anti-biofilm properties extracted from different bacterial and fungal species. Streptomyces species are known as the most promising sources of biofilm-controlling compounds. Methanolic compounds extracted from Streptomyces sp. (strain MUC 125) showed potential anti-biofilm activity against MRSA due to its 2,3-dihydroxybenzoic acid-mediated iron chelating ability [260]. Ethyl-acetate secondary metabolites extracted from Streptomyces sp. (isolated from Iraqi marine sediment) exhibited significant potential for designing and developing new anti-biotics for treating urinary tract infections. The extract showed biofilm inhibition against P. mirabilis (uropathogenic bacteria) even at sub-MIC by interrupting the QS signals [261].
Marine-derived bacteria are well categorized as a prime microbial group found in the marine environment. Peach et al. discovered and characterized the auromomycin chromophore as a potential inhibitor against V. cholera biofilms at 60.1 µM (IC 50 ). Additionally, the inhibitory effect of auromomycin was significantly enhanced by adding some antibiotics (such as tetracycline, ciprofloxacin, and ceftazidime) at their sub-inhibitory concentrations [262]. A red algae-halogenated furanone, Dilsea pulchra, has been reported to have effective anti-biofilm properties [263]. Some synthetic and natural brominated furanones have been reported as significant QS inhibitors against gram-negative and positive bacterial species [264][265][266][267].
tam were observed to be most significant against P. aeruginosa with IC50 values o 0.6 µ g/mL, respectively. The compound-5 was most effective against S. aureus wi biofilm inhibition at 44 µ g/mL. The compound-2, 4, and 6 inhibited the biofilm fo of S. mutans [268]. Antibiofilm activity of natural and synthetic cembranoid com has been analyzed against P. aeruginosa, V. harveyi, S. aureus, and Chromobacte laceum [269].
Actinobacteria are gram-positive and the most versatile bacteria in nature, from aerobic_anaerobic, motile_nonmotile, and sporing_nonsporing bacteria [2 tinobacterial spp. produces several secondary metabolites that can act as antibact tiviral, antifungal, and anticancer agents. Some of the recently reported antibiofilm bacteria are listed in Table 9. Song et al. revealed that some bacterial strains deriv coral Pocillopora damicornis showed anti-biofilm activity by QS inhibition. Predom H12-Vibrio alginolyticus (a coral symbiotic bacteria) inhibited the biofilm format aeruginosa PAO1 by interfering with rhl and the las system [271]. Actinobacteria are gram-positive and the most versatile bacteria in nature, ranging from aerobic_anaerobic, motile_nonmotile, and sporing_nonsporing bacteria [270]. Actinobacterial spp. produces several secondary metabolites that can act as antibacterial, antiviral, antifungal, and anticancer agents. Some of the recently reported antibiofilm actinobacteria are listed in Table 9. Song et al. revealed that some bacterial strains derived from coral Pocillopora damicornis showed anti-biofilm activity by QS inhibition. Predominantly, H12-Vibrio alginolyticus (a coral symbiotic bacteria) inhibited the biofilm formation of P. aeruginosa PAO1 by interfering with rhl and the las system [271].

Methodology Data Collection Criteria
A bibliographic search was conducted to screen relevant scientific publications before August 2022, on bacterial biofilm formation, resistance development mechanisms, and biofilm control strategies. The online search engines of Google scholar, PubMed, ScienceDirect, and Web of Science Core Collection databases were used. The search strategy consisted of separate or simultaneous use, under different combination forms, of several particular keywords including "biofilm formation", "microbial biofilm composition", antimicrobial resistance", "plant-derived anti-biofilm compounds", "bee products", "marine derived compounds", and "phyto-nanotechnology". Firstly, all titles and abstracts of the search results were individually examined to evaluate whether the articles met inclusion criteria, meaning reporting results in evaluating the antibiofilm activity of plant products, bee products, and nanomaterials against biofilm-forming bacteria with significant outcomes. All the duplicated papers and those published in a language other than English or irrelevant publications that did not contribute to retrieving meaningful results in the goal of assessing natural strategies as potential weapons against bacterial biofilms were excluded. Furthermore, the selected articles were entirely read to obtain significant material based on their experimental outcomes.

Conclusions
Over the last three decades, biofilm formation has become a potential threat in the food and health sector. Many biofilm-forming microbial species can develop resistance to harsh environmental conditions. Several antibiotics and different disinfectants are used in hospitals and the food industry. Several biofilm-forming foodborne pathogens have been found to cause outbreaks. Several chronic infections are associated with biofilmforming microbes. Therefore, several strategies have been designed and analyzed to inhibit microbial growth. However, the emergence of antimicrobial and multidrug resistance forced researchers to study all growth features of microbes and their resistance mechanisms for developing effective combating strategies and significant biofilm-controlling compounds. Many researchers are discovering and developing effective anti-biofilm compounds using natural products. In this review, recent studies were reviewed, focusing on biofilm-controlling compounds, such as natural plants, bee products, nanomaterials, microbes, and marine-derived anti-biofilm compounds to reduce or eradicate microbial biofilms and their associated infections as well. These antibiofilm compounds possess a significant potential to overcome the antimicrobial resistance linked to biofilm formation. On the basis of their potential antibiofilm properties with less to no toxicity and increased bioavailability, they can be suggested to treat biofilm-associated infections. Funding: This review is supported by the project "Increasing the impact of excellence research on the capacity for innovation and technology transfer within USAMVB Timis , oara" code 6PFE, submitted in the competition Program 1-development of the national system of research-development, Subprogram 1.2-institutional performance, institutional development projects-development projects of excellence in RDI.
Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.