Bacterial attachment to surfaces favors in biofilm formation in initial step. Initially, the bacterium gets close enough to allow initial attachment, and the forces involved in this initial attachment are van der Waals forces, electrostatic and hydrophobic interactions. During this initial contact, the bacteria still exhibit Brownian motion and can be easily removed by the flow rate of the liquid. Subsequently, an irreversible step occurs, as the bacteria attach to the surface by producing EPS and or filamentous appendages, such as pili or flagella and non-fimbrial adhesions. At the end of this phase, much stronger physical or chemical forces are needed to remove the bacteria attached to the surface (Palmer et al., 2007). The size of the contact area between bacterial cell and material surface, the total of the repulsive or attractive forces between two surfaces, the hydrophobicity/hydrophilicity, roughness and surface charge, the physicochemical and topographic properties of a substrate surface can all influence the attachment of bacteria (Renner and Weibel, 2011). In S. aureus, atlE gene expression mediates bacterial adhesion and secretes autolysin. Strains lacking the atlE gene exhibit a marked reduction in adhesion capacity. The expression of fbe and sap genes leads to synthesis of fiber protein binding protein, which mediates the attachment of S. aureus and fibrin. During the accumulation phase, iapABD gene is expressed to synthesize the ECM and eventually form pileus-like multilayer structure in mature biofilms (Zhao and Ashraf, 2015).

Irreversible adhesion is promoted by the synthesis of EPS, which is controlled by the QS of the bacterial cells. Bacteria produce EPS, which is an important element of biofilm ECM. EPS can mediate bacterial adhesion–cohesion via hydrophobic and ion-bridging forces. Overall, EPS directly mediates adherence to microbial cell surfaces, cell-to-cell adhesion, biofilm structure, biofilm formation, water retention, cell–cell signaling, cell protection, nutrient trapping capability, and genetic adaptation. In addition, the cyclic messenger di-GMP (c-di-GMP) is considered to be one of the triggers for the switching of reversible to irreversible attachment via EPS and cell surface structures. EPS are mainly enzymes and structural proteins, DNAs, polysaccharides, phospholipids, and glycoproteins (Czaczyk and Myszka, 2007). Polysaccharides are a key fraction of the EPS matrix and are necessary during the biofilm development process and growth of the bacteria (Flemming, 2016).

Microbial cells begin to multiply and divide after they connect to a biotic or abiotic surface and this attachment becomes stable. This process is started by specific chemical signaling within the EPS. The next step in this process is the formation of microcolonies. The different micro-communities that make up the bacterial colonies in a biofilm usually coordinate with each other in different ways. The exchange of substrate, the distribution of important metabolic products, and the excretion of metabolic end products all depend on this coordination (Gestel et al., 2015).

The EPS-embedded bacteria conduct to microcolonies formation and biofilms maturation. The EPS then acts as a biological ‘glue’ between the embedded bacterial cells through changes in gene expression. Through matrix formation, nutrients are transported to the cell communities and unwanted products are removed through the formation of water-filled channels. Often a mushroom-shaped multicellular structure of microcolonies is displayed. During the maturation process, inhibition of the production of surface structures restricts motility in microcolonies and the pattern of gene expression differs significantly from that of planktonic cells (Muhammad et al., 2020).

The dispersal process is a tactic used by bacterial cells to breakdown biofilms and begin a new biofilm life-cycle. The complicated process of dispersal is controlled by effectors, signal transduction pathways, and environmental signals. Genes responsible for the EPS generation, initial attachment, and fimbriae synthesis are often reduced during dispersal, but genes related to EPS degradation and flagella synthesis are typically enhanced. Inhibition of c-di-GMP signaling pathways is another effective method of biofilm dispersion. Furthermore, environmental factors (i.e., temperature, nutrients, and pH) increase in glucose supply, and lack of oxygen can contribute to biofilm dispersal (Toyofuku et al., 2016).

Ag NPs are widely used in healthcare and biomedicine, food storage, and environmental applications. Ag NPs are remarkable for their unique physicochemical features such as excellent catalytic activity and stability, high conductivity, and significant anti-inflammatory and antibacterial activities (Lee and Jun, 2019). Interference with cell-membrane function and intracellular ROS generation have been the key pathways for antibacterial activity (Liao et al., 2019).

Ag NPs probably destabilize the cell membrane and produce Ag ions that react with membrane proteins. These NPs have the ability to pass through bacterial cell walls and/or membranes, attach to bacterial DNA, and obstruct DNA replication. Furthermore, they have the ability to disrupt ribosomal function to translate mRNA into protein forms, thereby activating cytochrome B proteins (Yan et al., 2018). The antibacterial mechanism of Ag NPs works by inhibiting O₂ metabolism, eventually killing microorganisms (Shahverdi et al., 2007). Furthermore, Ag NPs can efficiently diminish bacterial biofilm biomass (Montazeri et al., 2020).

The anti-biofilm activity and downregulation of bacterial biofilm-related genes of Ag NPs against a variety of bacteria has been reported in several research papers (Table 1). Ag NPs have the ability to block the synthesis of bacterial EPS and then biofilm. The ability of biofilm inhibition by Ag NPs may be due to the existence of water channels throughout the biofilm (Kalishwaralal et al., 2010; Montazeri et al., 2020; Pourmbarak Mahnaie and Mahmoudi, 2020). They can enter biofilms and inhibit biofilm development by suppressing gene expression. These ions are internalized and prevent the penetration of amines, thiols, or carboxylates. Ag NPs tend to agglomerate, which negatively affects their antimicrobial efficacy. Therefore, their surface must be functionalized before application (Le Ouay and Stellacci, 2015).

Ag has a strong inhibitory effect on the strong biofilms produced by Acinetobacter baumannii. In presence of Ag NPs, the expression of major adhesion virulence factors of A. baumannii such as group 2 capsule synthesis (kpsMII) and afa/draBC adhesin genes decreased by 4.6-folds and 3.4-folds, respectively. Furthermore, in comparison to the control, the transcription of biofilm-related genes (bap, OmpA, and csuA/B) was drastically reduced by 4.5-, 3.1-, and 3.1-folds, respectively (Hetta et al., 2021).

Miola et al. (2015) assert that Ag NPs mixed with poly methyl methacrylate (PMMA)-based bone cement significantly reduced biofilm formation. The main mechanism of this Ag NP–PMMA is inhibition of bacterial colonization. Ag hydroxyapatite nanocomposite (Ag/HA-NC) can obviously inhibit S. aureus biofilm formation. Bacterial adhesion and biofilm formation on the Ag/HA-NC coating were significantly lower than on the HA coating. In addition, the expression of atlE, fbe, sap, and iapB genes, all involved in initial attachment of S. aureus was inhibited with Ag/HA-NC. Therefore, it can be concluded that the antibacterial effect of Ag/HA-NC is achieved due to the release of Ag NPs, which supports the clinical use of Ag/HA-NC (Zhao and Ashraf, 2015).

Silencing of QS is a novel approach to combat the pathogenicity of P. aeruginosa. Mycofabricated Ag NPs with metabolites of the soil fungus Rhizopus arrhizus BRS-07 were able to decrease LasIR-RhlIR levels, inhibit biofilm formation, and significantly reduce the expression of QS-regulated genes (lasI, lasR, rhlI, rhlR; Singh et al., 2015). In addition, Ag NPs stabilized with glutathione (GSH-Ag NPs) at a concentration of ½ MIC was found to have anti-biofilm activity in P. aeruginosa by reducing the expression of lasR and lasI genes (Pourmbarak Mahnaie and Mahmoudi, 2020). One study found that in resistant Klebsiella pneumoniae strains, the expression of the efflux pump gene OxqAB was decreased by both commercial and biosynthesized Ag NPs (Dolatabadi et al., 2021). What is more, Ag NPs were potential NPs that showed excellent reduction of biofilm-related genes (fimH, rmpA, and mrkA) in K. pneumoniae (Mousavi et al., 2023).

Wang et al., developed the Ag NP-functionalized titanium implant surface. The Ag NP-doped surface reduced the expression of icaA gene for Staphylococcus epidermidis and fnbA and fnbB genes for methicillin-resistant S. aureus to decrease the production of intercellular polysaccharide adhesin and fibronectin-binding proteins, thereby reducing bacterial adhesion and biofilm formation (Wang et al., 2016). Although Ag NPs have been extensively studied for antibacterial applications, their production and use are subject to many limitations, including the inability to control size and shape, aggregation, colloidal stability, and potential toxicity at high doses (Jena et al., 2012; Zhang et al., 2019).

ZnO NPs are widely known for their antibacterial and anti-biofilm activity against a wide range of bacteria such as P. aeruginosa, Streptococcus pneumoniae, Listeria monocytogens, Salmonella enteritidis, and E. coli with low toxicity to human cells (Mahamuni-Badiger et al., 2020). Thanks to their potent antibacterial action, these NPs can reduce microbial adhesion, proliferation, and biofilm growth (Campoccia et al., 2013). However, their antibacterial activity is influenced by various factors such as UV illumination, size, shape, concentration, surface modifications, and surface defects (Mahamuni-Badiger et al., 2020).

Recently, ZnO NPs have gained even more attention as they possess the highest toxicity against drug resistant microorganisms. ZnO NPs exhibit a different type of antimicrobial action compared to other NPs. They first destroy the bacterial cell wall, then enter the cell, and finally accumulate in the cell membrane, leading to death (Li et al., 2012). These NPs can alter the microenvironment near the bacteria and liberate ROS species and Zn²⁺ ions cause damaging lipids, proteins, carbohydrates, and DNA by oxidative stress, lipid peroxidation, and disrupting vital cellular functions (Sondi and Salopek-Sondi, 2004). Among NPs, they are strongly preferred because ZnO is recognized as a safe material by the US Food and Drug Administration (De Romana et al., 2002). ZnO meets the requirements for an ideal anti-biofilm agent (Hou et al., 2018).

QS inhibitory effect of ZnO NPs has been demonstrated by reducing the production of P. aeruginosa virulence factors such as rhamnolipids, pyocyanin, pyoverdin, hemolysins, elastase, and proteases. In addition, the inhibitory effect of ZnO NPs on the QS regulatory genes lasI, lasR, rhlI, rhlR, pqsA, and pqsR, which control the secretion of virulence factors, was confirmed (Saleh et al., 2019).

Further, norA, norB, norC, and tet38 are important efflux pump genes that contribute to the MDR phenotype in S. aureus. The simultaneous use of ZnO@glutamic acid–thiosemicarbazide NP at sub-MIC concentrations in combination with ciprofloxacin decreased the expression of norA, norB, norC, and tet38 by 5.4-, 3.8-, 2.1-, and 3.4-fold, respectively, compared to ciprofloxacin alone (Nejabatdoust et al., 2019).

A previous study showed that ZnO/zeolite NC at concentrations below the MIC in combination with chitosan reduced the expression of gtfB, gtfC, and ftf genes in S. mutans by 4.16-, 4.40-, and 2.88-fold, respectively (Afrasiabi et al., 2021). Along these lines, inhibition of the esp gene five-fold in Enterococcus faecalis decreased the ability to form biofilm (Partoazar et al., 2019). According to Khan et al. (2016), the expression level of the gtfB gene decreased 0.93-fold when Streptococcus mitis was grown with 30 μg/mL ZnO NP. Abdelghafar et al. also found a significant decrease in icaA and sarA genes after treatment of S. aureus with ZnO NPs (Abdelghafar et al., 2022). Similarly, sub-MIC of ZnO NPs had a significant effect on the biofilm formation rate of S. aureus and the gene expression of ica A, ica D, and fnb A (Abdelraheem et al., 2021).

TiO₂ NPs have been found as antimicrobial agents against various microorganisms that exert anti-QS activity. TiO₂ NPs exhibited improved antibacterial and anti-biofilm activity in comparison to bulk form. In addition, it reduces expression of the efflux pump genes (MexY, MexB, MexA) and QS-regulated genes (lasR, lasI, rhll, rhlR, pqsA, pqsR) of P. aeruginosa. TiO₂ NPs can increase the effectiveness of conventional antibiotics (Ahmed et al., 2021). The diminutive size, large surface area, and their ability to penetrate the cell wall are the factors determining the antimicrobial activities (Saranya et al., 2018). Moreover, exposure to UV light leads to the generation of ROS, which may enhance the antibacterial activity of TiO₂ NPs (de Dicastillo et al., 2019). Several studies reported the antibacterial and antifungal activities of TiO₂ NPs (Anupong et al., 2023; Younis et al., 2023). Abdulazeem et al. found that biofilm growth is completely reduced in A. baumannii, P. aeruginosa, Proteus vulgaris, and Serratia marcescens as TiO₂ NPs target sulfhydryl groups in the cell membrane to form S–TiO₂ bond. This reaction inhibits the electron transport chain and enzymes needed for biofilm formation (Abdulazeem et al., 2019). However, Abdel-Fatah et al. stated that TiO₂ NPs had no bactericidal effect against S. aureus, E. coli, and Bacillus subtilis (Abdel-Fatah et al., 2016). These variations among different results may reflect differences in concentration, zeta potential, particle shape, size, and the examined pathogen (Skandalis et al., 2017).

The overexpression of efflux pump genes was dramatic in non-treated P. aeruginosa, especially the MexY gene. TiO₂ NPs significantly reduce the expression of efflux pump genes with its efflux pump inhibitor activity (Ahmed et al., 2021). The mxdABCD complex is a new set of genes that are important essential for biofilm formation and growth and can be influenced by TiO₂ NPs. Of these, mxdA gene encoding diguanylate cyclase and mxdB gene encoding glycosyl transferase, both of which confer to cell attachment and EPS synthesis. These genes are required for the three-dimensional of biofilm architecture (Maurer-Jones et al., 2013). In Liu et al. (2015) reported that TiO₂ NPs on the surface and inside titania nanotubes reduced gtfB, gtfC, and gtfD genes of S. mutans, which improves the efficacy of orthopedic and dental implants. In a recent research, the antibacterial activity of TiO₂ NPs and Ganoderma extract against P. aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) was investigated. As a result, the algD gene was reduced when TiO₂ NPs were used alone or in combination with Ganoderma extract. None of them affected the iacA gene (Marzhoseyni et al., 2023). However, DNA damage induced by TiO₂ NPs limits the efficacy of them (Chen et al., 2014).

Cu NPs have proven to be efficient antibacterial agents due to their low cost, ease of mixing with polarized liquids such as water, and their relatively stable physical and chemical properties (LewisOscar et al., 2015). Graphene oxide-Cu NC has been shown to alter biofilm architecture, inhibit EPS formation and distribution, and dysregulate the expression of EPS-related genes (Mao et al., 2021). The gtf gene family, a glucosyltransferase (Gtf) encoding gene in S. mutans, can synthesize EPSs directly from sucrose. GTF-produced glucan maintains the integrity of the matrix structure. In exposure to graphene oxide-Cu NCs, the expression of gtfB, gtfC, and gbpB decreased, while the expression of the rnc gene increased. In addition, the expression of vicRKX was suppressed by rnc genes. Cell wall homeostasis and expression of gtfB/C genes can be enhanced by the VicRK two-component system. The group treated with graphene oxide and Cu NCs showed a significant increase in rnc expression, which is consistent with the negative effect of the rnc gene (Mao et al., 2021). Further, it was also reported that Cu/graphitic carbon nitride NCs downregulated icaA gene of S. aureus to reduce biofilm formation (Rashki et al., 2023b).

Cu NPs lowered the biofilm of P. aeruginosa by reducing the ppyR gene. In addition to this, the presence of Cu NPs led to an increase in the expression of the biofilm dispersion locus gene (bdlA). Also, Cu NPs caused a decrease in the rsaL gene as well as the mexA and mex B efflux genes (Singh et al., 2019). Aziz et al. found that the virulence gene-related to adhesion (fimH, papC) of Klebsiella oxytoca was reduced 8.5- and 9.0-fold by using Cu cobalt oxide NPs (Aziz et al., 2023).

The high toxicity of Cu NPs causes oxidative lesions, which limits the effectiveness of these NPs (Naz et al., 2020). Quercetin is a natural substance with anti-QS potential that has anti-biofilm properties. However, it has some disadvantages, including low water solubility and bioavailability issues. Quercetin/Cu NPs have been found to prevent biofilm formation by disrupting the integrity of the cell membrane and processes involved in QS, and by reducing the expression of lasI, lasR, rhlI, and rhIR genes in P. aeruginosa and agrA and icaA in S. aureus, and are non-toxic to mouse fibroblast cells (Cheng et al., 2024).

Fe₃O₄ NPs have attracted attention due to their unique properties, which include larger surface area, superparamagnetic nature, surface-to-volume ratio, and easy separation process. These NPs have antibacterial and anti-biofilm properties via various mechanisms such as intracellular ROS generation, electrostatic attraction with the cell membrane and its proteins, leading to physical destruction and eventual death of the microbes. In addition, there are a number of advantages, including low toxicity, biocompatibility for medical applications and environmental friendliness (Das et al., 2021).

The clinical management of antibiotic-resistant S. aureus and E. coli raises a number of public health issues. Furthermore, increasingly serious antimicrobial resistance and biofilm formation pose a barrier to conventional therapy. Rhamnolipid (RHL)-coated Fe₃O₄ NPs- p-coumaric acid (p-CoA) and gallic acid (GA)-polymer common coatings (PVA; RHL-Fe₃O₄@PVA@p-CoA/GA) can strangely inhibit bacterial growth and biofilm formation via downregulating icaABCD and csgBAC operons, which encode the production of slime layer (icaA and icaD) and the production of curli fimbriae (csgA, csgD, and crl) in S. aureus and E. coli, respectively (Sharaf et al., 2022). Analysis of fimA and csgA genes for biofilm formation of E. coli confirmed the ability of clay halloysite nanotubules with tannic acid and Fe₃O₄ NPs to prevent bacterial adhesion and biofilm formation (Bu et al., 2024). A summary of the effect of the other NPs on the downregulation of bacterial biofilm-related genes can be found in Table 2.

Although metal NPs have been shown to be toxic in human cells, efforts are being focused on reducing their toxicity through various strategies, e.g., by producing composites with clays, doping with polymers and proteins, etc. (Odatsu et al., 2020). The limited use of NPs in clinical applications is due to the incomplete understanding of their side effects, and further studies are needed to utilize them more effectively. The routine use of NPs in the fight against bacterial infections will be possible once the toxicity of NPs is clarified by detailed in vivo and clinical studies. The correct determination of the threshold dose and exposure time is essential for the safe administration of NPs (Ozdal and Gurkok, 2022).