Regulating bacterial biofilms in food and biomedicine: unraveling mechanisms and Innovating strategies

Abstract

Bacterial biofilm has brought a lot of intractable problems in food and biomedicine areas. Conventional biofilm control mainly focuses on inactivation and removal of biofilm. However, with robust construction and enhanced resistance, the established biofilm is extremely difficult to eradicate. According to the mechanism of biofilm development, biofilm formation can be modulated by intervening in the key factors and regulatory systems. Therefore, regulation of biofilm formation has been proposed as an alternative way for effective biofilm control. This review aims to provide insights into the regulation of biofilm formation in food and biomedicine. The underlying mechanisms for early-stage biofilm establishment are summarized based on the key factors and correlated regulatory networks. Recent developments and applications of novel regulatory strategies such as anti/pro-biofilm agents, nanomaterials, functionalized surface materials and physical strategies are also discussed. The current review indicates that these innovative methods have contributed to effective biofilm control in a smart, safe and eco-friendly way. However, standard methodology for regulating biofilm formation in practical use is still missing. As biofilm formation in real-world systems could be far more complicated, further studies and interdisciplinary collaboration are still needed for simulation and experiments in the industry and other open systems.

Keywords:

• Biofilm formation
• regulatory mechanisms
• biofilm control
• emerging technologies

1. Introduction

Biofilms are considered the predominant bacterial lifestyle in nature. Distinct from planktonic bacteria, these aggregates of surface-attached or non-surface-attached cells are embedded in self-produced extracellular polymeric substances (EPS) and live as heterogeneous communities with three-dimensional (3D) microbial structures (Karygianni et al. 2020). The development of bacterial biofilms is a cyclic and highly dynamic process. Based on the investigation of Pseudomonas aeruginosa (P. aeruginosa) biofilm, the classic five-step biofilm model defined biofilm development as a cyclic process consisting of five specific stages: reversible attachment, irreversible attachment, maturation-1, maturation-2 and dispersion (Figure 1A) (Sauer et al. 2002). As biofilm development in real-world systems can be far more complicated, the five-step model has been continuously challenged and expanded (Sauer et al. 2022), with variations of this model for different species (Otto 2013; Rendueles and Ghigo 2012). In the life cycle of biofilm development, biofilm formation refers to the transition from the planktonic to the biofilm mode of growth, which involves three major steps: attachment, EPS production and micro-colony formation. Recent studies revealed that the ability of bacteria to form biofilms depends on a number of factors such as surface appendages (e.g., flagella and pili), EPS components, external environments and expression of related genes (Persat et al. 2015; Yin et al. 2019). Meanwhile, the whole process of biofilm formation is controlled by sophisticated regulatory networks consisting of two-component systems (TCSs), second messengers, small non-coding RNAs (sRNAs) and quorum sensing (QS) systems (Condinho et al. 2023).

Figure 1. Schematic of the biofilm formation process and related factors. A five-step biofilm model. Stage 1: reversible attachment. Stage 2: irreversible attachment. Stage 3: maturation-1. Stage 4: maturation-2. Stage 5: dispersion. B roles of surface appendages during initial attachment. C composition of EPS components. D environmental factors during biofilm formation.

Biofilm formation has both negative and positive effects in the food industry and biomedicine area. On the one hand, bacteria can spontaneously get attached and establish biofilm on both biotic and abiotic surfaces, leading to a series of problems including biofouling, cross-contamination, chronic infection, antibiotic resistance, etc. (Patra et al. 2022; Stewart and Bjarnsholt 2020; Uruén et al. 2020; He and Sun 2015a, 2015b; Jayan, Pu, and Sun 2022). A public announcement from the National Institutes of Health indicates that biofilm could account for over 60% of microbial infections, and most of such infections are hard to treat or frequently recurring (Lewis 2001). Similarly, in the food industry, biofilm formation can happen easily. For example, from ready-to-eat food, which could be easily contaminated by microorganisms during transportation and storage, 33 of 54 Staphylococcus aureus (S. aureus) isolates have been reported to show biofilm formation capacity, including two strong biofilm producers with multi-drug resistance (Lin et al. 2019). On the other hand, with enhanced resistance and self-immobilization properties, the established biofilm can be widely applied in fermentation, biosynthesis, and biomaterial development to improve biochemical production productivities during continuous processing (Ghosh et al. 2022; Hayta et al. 2021; Wu et al. 2022). Moreover, mediated by biofilm formation, microbial fermentation can complete more complex tasks, such as the direct lignocellulose degradation through the microbial co-culture of aerobic fungi and anaerobic or facultative bacteria (Jiang et al. 2021).

Considering the significance of biofilms in food and biomedicine, effective strategies for biofilm control have been continuously proposed. In previous reviews, biofilm formation, inhibition and eradication have been discussed thoroughly (Sauer et al. 2022; Yin et al. 2021). For the removal of pathogenic biofilms, current strategies can be generally classified into physical methods (e.g., ultrasound treatment, photodynamic or photothermal therapy) and biochemical methods (e.g., phage lysins, degradative enzymes and metabolites). However, since biofilms show higher resistance than planktonic bacteria, it remains challenging to eradicate mature biofilms (Van Acker, Van Dijck, and Coenye 2014). A promising alternative is to prevent or delay the establishment of biofilms. Strategies such as surface modification and antimicrobial agents have been widely used for the prevention of biofilm formation, but most of the existing strategies only focus on minimizing the number of surface-attached cells (Desrousseaux et al. 2013; Paluch et al. 2020). From the view of biofilm development and related regulatory networks, a preferred approach for biofilm control, whether to prevent the establishment of unexpected biofilms or to promote the growth of commensal bacteria, is to regulate the biofilm formation process by intervening in the regulatory systems and the key process of biofilm development. Progress in novel strategies for the regulation of biofilm formation such as anti/pro-biofilm agents, nanomaterials, surface materials and physical methods has contributed to effective biofilm control in a smart, safe and eco-friendly way. However, few reviews focused on the updated mechanisms and strategies for the regulation of the biofilm formation process. In this review, current knowledge about mechanisms for the regulation of biofilm formation is summarized by illustrating major factors and regulatory networks participating in early-stage biofilm establishment. Some interesting findings from research in microbiology, molecular biology, micromechanics and materials science are reported to help better understand the roles of bacterial appendages, EPS components and environmental factors during biofilm formation, as well as the participation of common bacterial regulatory networks in the phenotype switch from planktonic cells to sessile biofilms. Based on the relationship between regulatory networks and biofilm formation, newly developed strategies for regulation of biofilm formation such as natural anti-biofilm agents, surface coating and modification, cold plasma technique, etc. are also discussed.

2. Mechanisms for regulating biofilm formation

2.1. Factors influencing biofilm formation

2.1.1. Surface appendages

Bacteria possess a wide variety of surface appendages such as flagellum and pili, which can mediate the interaction between bacteria cells and biotic or abiotic substrates. These appendages not only have motility properties but also enable bacteria to maintain their attachment to surfaces in a flow environment (Figure 1B).

Flagella are long helical filaments possessed by motile bacteria which allow them to swim in liquid or swarm over solid surfaces. Flagellum-mediated motility is critical for biofilm formation and development (Duan et al. 2013). In the process of initial attachment, alterations in flagellar load and function can initiate potential signals that are subsequently coupled to changes in cellular behaviors, resulting in switches between swimming and swarming, swimming and adhesion as well as biofilm initiation (Schniederberend et al. 2019). Moreover, increasing interest in flagellum-meditated attachment revealed that flagella and their properties could improve initial attachment and biofilm formation in many bacterial species. In the case of Escherichia coli (E. coli) biofilm formation on plastic surfaces, flagella, especially sticky flagella, have been demonstrated to help E. coli pierce the energy barrier, attach onto plastics and form a rigid attachment layer (Zhang et al. 2023). Similarly, for biotic substrates, it has been reported that purified flagella of E. coli performed the ability to bind mucins, contributing to colonization and biofilm formation on the mucosal surface (Erdem et al. 2007).

Pili (or fimbriae) are long filamentous structures found on cell surfaces both in Gram-negative and Gram-positive bacteria. In Gram-negative bacteria, pili are typically formed by non-covalent interactions between pilin subunit proteins (pilins), while in Gram-positive pathogens most discovered pili are formed by covalent polymerization of adhesive pilin subunits (Danne and Dramsi 2012; Telford et al. 2006). Pili perform motility and adherence functions during biofilm formation. Although pili can be divided into several groups based on their assembly pathway, type IV pili (T4P), which are mainly found in Gram-negative bacteria, represent the most widespread and well-characterized class. Biofilm formation is influenced by the T4P, which functions as motorized surface appendages and adhesive fibers. During early-stage biofilm formation, P. aeruginosa and other bacteria employ T4P-dependent twitching motility to crawl or walk on surfaces (Nieto et al. 2019). This motility mode contributes to cell aggregation and micro-colony formation. Additionally, in P. aeruginosa, T4P mechanics play a key role in controlling bacterial attachment to biotic and abiotic surfaces (Beaussart et al. 2014). Meanwhile, in Clostridioides difficile (a Gram-positive bacillus), T4P and related structures can also exhibit extracellular DNA (eDNA) recognition ability, providing further insight into the relationship between pili function and biofilm formation (Ronish et al. 2022).

2.1.2. Extracellular biomolecules

In the process of early-stage biofilm formation, extracellular biomolecules, whether surface-associated adhesive substances or extracellular components, can also make significant contributions. In many studies, due to the inextricable link between surface-associated and extracellular substances in adhesion, cohesion, and scaffolding, both surface-associated molecules (such as surface proteins in Gram-positive bacteria and outer membrane proteins in Gram-negative bacteria) and extracellular molecules are considered as EPS component (Karygianni et al. 2020). This self-produced sticky matrix has played multifaceted roles in biofilm development, mechanical stability and resistance (Flemming and Wingender 2010).

The composition and structure of the biofilm matrix vary with bacterial species and growth conditions. Generally, the major EPS components include polysaccharides, proteins, and eDNA (Figure 1C). These components are important biofilm-building materials forming the three-dimensional polymer network structure to interconnect and immobilize bacteria cells (Dragoš and Kovács 2017; Flemming and Wingender 2010). Polysaccharides are considered the predominant extracellular biopolymer in biofilms (Das 2022). In P. aeruginosa, for example, the key extracellular polysaccharides implicated in biofilm formation and biofilm structuring are alginate, Psl and Pel (Ryder, Byrd, and Wozniak 2007). Colvin et al. (2012) demonstrated the importance of Psl in surface attachment for clinical and environmental P. aeruginosa isolates, indicating the adhesion function of Psl. An interesting founding in P. aeruginosa biofilm was that the cationic exopolysaccharide Pel could cross-link eDNA via ionic interactions (Jennings et al. 2015). The ability of cationic polysaccharides to interact with anionic molecules was also found in S. aureus (Formosa-Dague et al. 2016).

Another indispensable composition of EPS is protein. Unlike the well-studied composition and function of exopolysaccharides, the protein content of EPS is more complicated. In the aspect of composition, the proteomic study revealed that the matrix proteome in P. aeruginosa biofilm contained secreted proteins (13.3%), cytoplasmic proteins (28.9%), periplasmic proteins (11.1%), cytoplasmic membrane proteins (2.2%), and most abundantly, outer membrane proteins (35.6%) (Toyofuku et al. 2012). In the aspect of function, surface proteins are emerging as important elements promoting primary adhesion and biofilm formation (Lasa and Penadés 2006; Schiffer et al. 2022). In S. aureus, polysaccharide intercellular adhesion (PIA), encoded by the ica operon, has been considered as the critical EPS component for bacteria accumulation, while cell wall-anchored (CWA) proteins are significant for biofilm formation in an ica-independent way (Foster et al. 2014; Schilcher and Horswill 2020). Meanwhile, S. aureus can also utilize biofilm-associated proteins (Baps), which self-assemble into functional amyloid aggregates, to build biofilm under acidic culture conditions (Taglialegna et al. 2016). In addition, there has been increasing attention on the roles of eDNA during biofilm formation. Produced by active secretion or controlled cell lysis, eDNA promotes bacterial adhesion and maintains the structural stability of biofilms (Okshevsky and Meyer 2015). Moreover, Gloag et al. (2013) demonstrated that eDNA could mediate the twitching motility of P. aeruginosa and facilitate the self-organization of biofilm.

2.1.3. Environmental factors

Biofilm formation is considered a strategy adopted by bacteria to allow their adaption to changing environments. In turn, the external environment can also affect or even regulate the biofilm formation process. The common environmental factors, basically, can be grouped into three major categories: physical factors, chemical factors and biological factors (Figure 1D) (Dwyer, Kohanski, and Collins 2009; Gu et al. 2021; Kim et al. 2020; Persat et al. 2015; Plate and Marletta 2013; Xu et al. 2019; Pu et al. 2021). Some of these factors have been identified, while many remain unstudied. Here, the influence of environmental stresses and physical factors on biofilm formation is discussed, as they are more common in the food and biomedicine industry.

Biofilm is considered a successful way for bacteria to survive various stresses (Yin et al. 2019). For instance, in P. aeruginosa, increased biofilm formation and altered biofilm structure at temperatures lower than 25 °C were reported, followed by temperature-controlled exopolysaccharide biosynthesis (Kim et al. 2020). It was also found that after cold stress, surface adhesion and biofilm formation enhanced in Listeria monocytogenes (L. monocytogenes) and S. aureus (Lee, Hébraud, and Bernardi 2017; Qiao et al. 2021). In S. aureus, the enhanced biofilm formation after cold stress was ascribed to the increased polysaccharide content in the matrix, as well as hydrophobicity and the adhesion proteins (Qiao et al. 2021). Another example of the effects of environmental stress on biofilm formation is oxidative stress. It has been identified that antibiotics-induced oxidative stress plays a significant role in bacterial cell death as well as resistance development (Dwyer, Kohanski, and Collins 2009). Biofilm formation can be considered a common strategy for bacteria to enhance their resistance toward oxidative stress. In Campylobacter jejuni, the stimulated biofilm formation by Ferrous and Ferric could be attributed to oxidative stress (Oh, Andrews, and Jeon 2018). In Azotobacter vinelandii, chronic sub-lethal oxidative stress induced the increased production of polysaccharide-rich EPS and lower susceptibility to hydrogen peroxide, promoting sessile behavior and biofilm formation (Villa et al. 2012).

Unlike the well-established understanding of molecular mechanisms under biofilm formation, the influence of the physical environment is just starting to emerge. Fluid flow and surface properties are two ubiquitous external effects during biofilm formation (Persat et al. 2015). The process of surface attachment, which is considered the first step for biofilm establishment, is highly dependent on the characteristics of abiotic or biotic surfaces, such as their smoothness and hydrophilicity (Yin et al. 2021). Meanwhile, surface topographies can also mediate microbial attachment (Lee et al. 2021). It has been reported that L. monocytogenes biofilm formation on stainless steel 304 could be significantly affected by surface topography and composition (Gu et al. 2021). For surface attachment, a challenging question is how biofilm cells adapt to different contact surfaces. By comparative proteomics, Guo et al. (2020) revealed the physiological responses of Vibrio parahaemolyticus (V. parahaemolyticus) biofilm to different abiotic surfaces (stainless steel, glass and polystyrene). It was suggested that the adaption behavior of biofilm cells on different surfaces was affected by metal ion stress, nutrition, osmotic stress, and sugar utilization.

As current research about the effects of surface properties mainly focuses on flat surface models, it is hard to describe the biofilm formation process on non-planar surfaces only based on common understanding. Recent findings revealed that the colonization of P. aeruginosa and E. coli on curve surfaces was largely controlled by flow, indicating the participation of fluid flow in early-stage biofilm establishment (Secchi et al. 2020). Due to the deflection of the trajectories of swimming bacteria by the flow, attachment location and rate mainly depended on the interplay between local flow and bacterial motility. Additionally, it was also reported that the balance between motility and flow promoted the emergence of morphological patterns in Caulobacter crescentus biofilms, suggesting the influence of hydrodynamics on species interaction and evolution within surface-associated communities (Rossy, Nadell, and Persat 2019).

2.2. Regulatory networks

2.2.1. TCSs

For bacteria, a common regulatory mode to sense and respond to environmental signals is TCS, which, in the simplest form, consists of a sensor histidine kinase (HK) and a response regulator (RR) (Figure 2A). The fluctuant environmental signal, once sensed by HK, is relayed by phosphoryl group transfer from histidine domain of HK to conserved aspartic residue in receiver domain of RR (Liu et al. 2018). However, in reality, the signal transduction system of bacteria can be more complicated, as most of them consist of multiple components and hybrid kinase-response regulators (Francis and Porter 2019).

Figure 2. Regulatory networks for biofilm formation. A signal transduction in the TCS system. B metabolic pathway of c-di-GMP. C cell-density-dependent regulatory mode of QS. D roles of sRNA in transcriptional regulation.

TCSs are widely involved in the regulation of biofilm formation in many species, as a bridge connecting extracellular stimuli and intracellular response (Aslan et al. 2021; Kong et al. 2015). For instance, YycFG (also designated as WalRK) TCS in S. aureus is well-known for its participation in biofilm formation. The membrane-anchored HK YycG/WalK could monitor and respond to extracellular changes by phosphoryl group transfer, while the activated RR YycF specifically bound to promoter regions including icaA, agr, sarA and sarX, modifying the expression of downstream target genes, which promoted biofilm formation in an ica-dependent manner (Wu et al. 2021).

2.2.2. Second messengers

Cyclic dinucleotides are recognized as second messengers involved in important biological processes and behaviors of bacteria (Jenal, Reinders, and Lori 2017). These signal molecules can interact with target molecules (e.g., proteins and RNA) to adjust bacterial behaviors in a certain environment (Stülke and Krüger 2020). The best-studied example is bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP), one of the most common bacterial second messengers, especially in Gram-negative bacteria. The biochemistry of c-di-GMP synthesis, degradation and binding has been elaborated on in a previous review (Römling, Galperin, and Gomelsky 2013). In simplest terms, enzymes involved in c-di-GMP metabolic can be identified by GGDEF, EAL, and GD-GYP domains (Figure 2B). GGDEF domain proteins possess diguanylate cyclases (DGCs) activity, synthesizing c-di-GMP from 2 molecules of GTP. By comparison, EAL domain proteins function in phosphodiesterases (PDEs), degrading c-di-GMP into 5′-phosphoguanylyl-(3′-5′)-guanosine (pGpG) dinucleotide, which is subsequently broken down into two molecules of GMP by PDE-Bs. Besides, c-di-GMP can also be directly hydrolyzed into GMPs by the HD-GYP domain-containing PDEs (Liu, Huang, et al. 2022).

C-di-GMP has been recognized as the key regulator of bacterial lifestyle. Modulation of the c-di-GMP metabolic process allows bacteria to switch between motile and sessile phenotypes, mediating the formation and dispersal of biofilms for adaptation to environmental stress and signals (Jenal, Reinders, and Lori 2017). A typical example is bacterial surface adaption regulated by c-di-GMP. It has been shown that when bacteria were exposed to surfaces, c-di-GMP orchestrated the process by regulating pili and flagella and promoting the production of adhesin and EPS (Laventie and Jenal 2020). In P. aeruginosa, the activity of the DGC SadC could be modulated by the T4P alignment complex protein (PilO). Webster et al. (2021) proposed that in the planktonic mode of life, PilO could interact with SadC and suppress its activity, resulting in decreased biofilm formation and increased motility. Once cells adhered to the surface, however, the PilO-SadC interaction was reduced, relieving repression of SadC activity. The interaction between SadC and flagellar motor (MotC) promoted biofilm formation and inhibited motility. This novel model suggested that DGC SadC could act as a bridge to integrate surface-derived input signals from both T4P and flagella and mediate c-di-GMP regulation on bacteria phenotypes.

2.2.3. QS

QS is a bacterial cell-cell communication process based on production, detection and response to extracellular signaling molecules named autoinducers (AIs), enabling bacteria to coordinate their activities according to changes in the cell density (Whiteley, Diggle, and Greenberg 2017). In the QS circuit, AIs are produced by members of the community and accumulate locally in the environment with an increase in population density. Once the concentration of AIs exceeds a certain threshold, these signal molecules will be detected by receptors and the community members will collectively coordinate their activities by regulating the expression of target genes (Figure 2C). The QS systems are different between Gram-positive and Gram-negative bacteria. Gram-positive bacteria use autoinducing peptides (AIPs) and transmembrane two-component HKs or cytoplasmic AIP-binding transcription factors (Rutherford and Bassler 2012). Gram-negative bacteria use acyl-homoserine lactones (AHLs) or other small molecules and LuxI/LuxR-type systems or TCSs (Papenfort and Bassler 2016). It was also reported that P. aeruginosa could package Pseudomonas quinolone signal (PQS) with outer membrane vesicles, which promoted the growth of biofilm through changes in cell morphology, structure and components of EPS in biofilm formed by P. aeruginosa, and enhanced the inhibitory effect of PQS to the growth of S. aureus, resulting in decreased EPS production in the dual-species biofilm formed by P. aeruginosa and S. aureus (Zhao et al. 2022).

Bacterial community behaviors, which are shown during the biofilm formation process, largely rely on the regulation of QS. Although QS systems vary among different species present in biofilms, the link between QS and biofilm formation mainly depends on environmental conditions, like fluid flow and surface topography (Mukherjee and Bassler 2019). In the case of the LasR system in P. aeruginosa, it was investigated that surface-associated bacteria were more sensitive to AIs than planktonic cells, due to surface-dependent lasR induction initiating a positive feedback loop through the sRNA Lrs1 as well as the involvement of T4P retraction motors and the minor pilins (Chuang et al. 2019).

2.2.4. sRNA

While proteins have been widely studied as common regulators, RNA-mediated regulation is an emerging field of bacterial regulatory networks. With higher sensitivity and reduced metabolic cost, sRNA was found to conduct some signal transduction and regulation processes and thus provided an additional regulatory mode in response to environmental signals (Beisel and Storz 2010). As an important posttranscriptional regulator, sRNA is integrated into the complicated regulatory networks to participate in the regulation of biofilm development (Nitzan, Rehani, and Margalit 2017). Typically 50 to 400 nucleotides in length, sRNAs can be divided into two groups according to their mode of function (Figure 2D): mRNA-binding sRNAs, such as trans-encoded sRNAs and antisense RNAs (asRNAs), and protein-binding sRNA (Storz, Vogel, and Wassarman 2011). Most sRNAs are trans-encoded sRNAs. These sRNAs function in the transcription process by short, imperfect base pairing with their target mRNAs, facilitated by RNA chaperone Hfq. Furthermore, asRNAs, which are encoded on the opposite strand of established coding sequences, can also impact translation and mRNA stability. Conversely, other sRNA regulators act by binding specific proteins and modifying protein activity.

It has been suggested that during biofilm formation, the sRNA regulator is essential for the transition from planktonic cells to sessile cells. Based on sRNA, the regulatory networks enable concentration-specific responses to environmental cues through regulatory proteins, or by directly affecting the synthesis of proteins promoting or disfavoring the formation of biofilms. During the stationary phase of E. coli, the 3′ UTR-derived sRNA FimR2 is processed from the fimAICDFGH mRNA transcript by RNase E, facilitating biofilm formation for survival under nutrient-depleted conditions (Raad et al. 2022). On the one hand, FimR2 interacts with the translational regulator CsrA and antagonizes its effects, downregulating type I pilus and flagellar biosynthesis. On the other hand, FimR2 fine-tunes target mRNA levels through direct base-pairing, inhibiting flagellar synthesis.

2.2.5. Connected regulatory networks during biofilm formation

Biofilm formation is a highly complicated and variable process controlled by a series of highly connected regulatory networks encoded within the bacterial genome. The close correlation between different networks can help to efficiently integrate information in multiple pathways. A typical example is the participation of TCS in regulating intracellular c-di-GMP levels. In the instance of biofilm formation regulation by carbon source, sodium lactate was reported to negatively regulate biofilm formation in Shewanella putrefaciens CN32 via a three-component regulatory system (LrbS-LrbA-LrbR) consisting of one KK (LrbS) and two cognate RRs (LrbA and LrbR) (Liu et al. 2017). LrbS could respond to the signal of sodium lactate and subsequently activate transcription factor LrbA, which then promoted the expression of lrbR. As PDE contains an EAL domain, LrbR decreased the intracellular c-di-GMP level, thereby negatively regulating biofilm formation. Besides, QS is also involved in c-di-GMP regulation. In V. parahaemolyticus, OpaR is the master QS regulator under high cell density. Zhang et al. (2021) demonstrated that OpaR directly regulated the transcription of scrA, scrG, VP0117, VPA0198, VPA1176, VP0699, and VP2979, encoding a group of GGDEF and/or EAL-type proteins, decreasing the intracellular concentration of c-di-GMP, and thus repressing biofilm formation. Similarly, in Acidithiobacillus thiooxidans, AHLs were demonstrated to positively regulate the c-di-GMP effector PelD encoding gene (Díaz et al. 2021).

Vibrio cholera (V. cholera) has been widely studied as a model organism for understanding biofilm formation and the regulatory networks underlying this process (Teschler et al. 2015). In many Vibrios, QS relies on post-transcriptional gene regulation by sRNAs called quorum regulatory RNA (Qrr) (Lenz et al. 2004). Huber et al. (2022) proposed a regulatory model for QS-mediated gene expression control in V. cholera, identifying the QrrX sponge RNA as a key component of V. cholera QS architecture. It was suggested that CqsS, LuxPQ, CqsR, and VpsS, as QS-specific receptors, could respond to external AIs and modulate the phosphorylation status of the LuxO transcription factor via the LuxU phosphorelay protein. At low cell densities, LuxO was phosphorylated, activating Qrr1-4 expression. Then Qrr1-4 reduced the translation of luxO and inhibited the hapR mRNA, which encoded the negative regulator of biofilm formation, HapR. While Qrr2-4 activated AphA expression, which could antagonize HapR activity and promote biofilm formation. At high cell density, however, QrrT induced the transcription of the QrrX sponge RNA, which, together with Hfq, could base-pair with and sequester Qrr1-4 sRNAs, and therefore facilitated RNase E-mediated decay.

3. Innovating regulatory strategies for biofilm formation

With robust construction and high tolerance toward harsh environments, eradication and inactivation of mature biofilms are getting increasingly challenging in food and biomedicine areas. Given the close relationship between bacterial regulatory networks and the biofilm formation process, regulation of early-stage biofilm establishment would be a highly possible alternative for biofilm control. Moreover, by precise regulation of the biofilm formation process, both prevention of biofilm and large-scale production of biofilm-related microbial products can be realized (Table 1). On the basis of the biofilm formation process and related regulatory networks, the application of anti/pro-biofilm agents, nanomaterials, surface coating and modification as well as physical methods have made regulatory strategies more efficient and effective (Figure 3).

Figure 3. Strategies for regulation of biofilm formation.

Table 1. Emerging strategies for regulating biofilm formation in food and biomedicine.

Technology field	Strategy	Target	Bacteria species	Effects	Application potential	References
Anti-biofilm agents	Natural compound ferulic acid	Cell membrane, regulatory networks	S. flexneri	Destructing cell membrane; inhibiting cell adhesion and biofilm formation, altering gene transcription	Food	Kang et al. (2020)
	Natural compound (+)-terpinen-4-ol	QS	B. cereus	Upregulating diffusing signal factors (DSFs) and diketopiperazines (DKPs); inhibiting swarming motility, protease activity and exopolysaccharide synthesis; downregulating critical genes for DSFs turnover	Food, pharmaceutics	Zhao, Duan, et al. (2021)
	Methyl anthranilate	QS	A. sobria	Reducing biofilm formation, swinging motility, swarming motility, protease activity and AHL production; interfering with the biosynthesis of AHL and competitively binding with the receptor protein	Food	Li et al. (2020)
	Citrus peel extract	c-di-GMP	P. aeruginosa	Increasing PDE activity; reducing biofilm formation and inducing biofilm dispersion	Medicine, engineering fields	Kim et al. (2023)
	Coumarin derivate 3,3′-(3,4-dichlorobenzylidene)-bis-(4-hydroxycoumarin).	Arginine catabolic pathway	MRSA	Inhibiting biofilm formation; suppressing the biosynthesis of intracellular ornithine and polyamines; competitively binding to arginine repressor ArgR; reducing the production of biofilm-associated virulence factors	Medicine	Qu et al. (2020)
Nanomaterials	Tannin-AgNPs	Biofilm life-cycle, QS	E. coli	Modulating biofilm formation process with shortened growth period and extended dispersion period; decreasing QS AIs production	Medicine	Liu et al. (2020)
	Enzyme/biopolymer/metal NPs	QS, cell membrane	P. aeruginosa	Enhancing anti-bacterial and anti-biofilm activities of AgNPs; degrading the QS signals in the extracellular environment for QS disruption	Medicine	Ivanova et al. (2020)
	CH/ZnO nanocomposite	QS	P. aeruginosa	Preventing AHL production; decreasing biofilm formation, swimming ability and swarming ability; downregulating the expression of LasI and RhlI	Anti-virulence therapy	Badawy et al. (2020)
Surface materials	AIP-I-coated surface and TrAIP-II-coated surface	QS	S. aureus	AIP-I-coated surface for agr-I QS system activiation; TrAIP-II-coated surface for agr-I repression	Medicine and industry	Kim et al. (2017)
	Electrospun cellulose acetate nanofibrous membranes	Adhesion process, expression of biofilm-related genes	L. paracasei	Enhancing biofilm formation and tolerance; regulating gene expression related to cell adhesion, biofilm formation and tolerance	Large-scale production of probiotics	Hu et al. (2022)
	Dual functional surface based on HEMA, APBA and quercetin	Adhesion process, QS	P. aeruginosa	HEMA layer for suppression of adhesion and aggregation; poly(APBA) component for pH-responsive release of natural anti-biofilm molecule quercetin	Biomedicine	Zou et al. (2021)
	Anti-biofilm surface coating AGXX®	QS, gene expression	MRSA	Interfering with agr QS system; reducing biofilm formation; downregulating expression of biofilm-associated genes and genes related to virulence	Medicine	Vaishampayan et al. (2018)
Cold plasma	PAW	QS, membrane potential	E. faecalis	Interfereing with QS system; decreasing the membrane potential.	Medicine	Li et al. (2019)
Electrostatic field	HVPEF	EPS production, expression of biofilm-related genes	S. aureus	Direct effect on planktonic S. aureus; reducing the release of EPS key components; regulating the expression of genes related to biofilm formation.	Food	Qi et al. (2022)

Note: QS = Quorum sensing.

3.1. Anti/pro-biofilm agents for regulation of biofilm formation

The development and spread of antibiotic resistance have become a global problem. As mature biofilms always possess higher resistance against conventional antibiotics, exploitation of novel anti-biofilm agents for biofilm inhibition has become an important future direction. On the other hand, some newly found pro-biofilm agents can be applied to activate the establishment of commensal or beneficial biofilm (Jiang et al. 2023). Targeting the biofilm formation process, regulatory networks and crucial metabolic pathways, a large number of candidates have been designed, synthesized, or found as natural compounds for the regulation of biofilm formation (Figure 3) (Kang et al. 2020; Pompilio et al. 2023; Qu et al. 2020).

A key regulatory hub for biofilm formation is QS. QS inhibitors, as novel agents for anti-biofilm and anti-virulence therapy, have been intensively studied for inhibition of AIs synthesis, inactivation or enzymatic degradation of AIs, AIs’ antagonists and blocking of signal transduction cascades (Paluch et al. 2020). Recently, natural compounds have received researchers’ attention as a safe and effective way for biofilm inhibition. Methyl anthranilate, a plant spice extract, was found to down-regulate the expression levels of QS-related genes and inhibit the QS-regulated phenotypes in Aeromonas sobria (A. sobria) by competitively binding to receptors and interfering with AHL biosynthesis (Li et al. 2020). Similarly, sodium butyrate, which is widely used as a flavoring agent and dietary supplement for health, has been reported to inhibit biofilm formation at sub-inhibitory concentrations by interfering with the AHL QS system in Chromobacterium violaceum and AI-2 QS system in Vibrio harveyi (Zhu et al. 2022). Furthermore, it was found that some QS inhibitors could also enhance the susceptibility of biofilm toward conventional antibiotics (Zhou et al. 2018). However, although QS inhibitors have displayed potent regulatory functions for biofilm formation, reliable identification of newly discovered inhibitors as well as deeper investigation of QS-inhibitor-resistant mutants are still lacking (Defoirdt 2018).

Given the well-studied participation of c-di-GMP in bacterial phenotype switching, interfering with c-di-GMP metabolism and signaling has become a potential approach for the development of regulatory agents for early-stage biofilm formation (Liu, Cao, et al. 2022). The majority of c-di-GMP targeted strategies focus on the development of inhibitors of DGCs rather than PDEs, owing to the old view that inhibiting any c-di-GMP PDE would lead to biofilm formation (Zheng et al. 2016). However, Kim et al. (2023) found that citrus peel extract from Jeju island concentration-dependently inhibited biofilm formation and induced biofilm dispersion by reducing intracellular concentration of c-di-GMP through increased PDE activity. With the addition of citrus peel extract in different concentrations (0%–0.05%), P. aeruginosa biofilm formation under static conditions was significantly reduced by 13%–71% in a concentration-dependent manner without inhibition of bacterial growth. Similarly, under continuous flow conditions, P. aeruginosa biofilm developed without and with 0.0002%–0.02% citrus peel extract treatments showed significant differences in volume and thickness. This naturally derived extract provided an alternative way for the regulation of biofilm formation targeting c-di-GMP metabolism.

Not all the agents have specific targets for biofilm regulation. In fact, many of the newly reported agents, especially natural compounds derived from plants or microbes, still lack in-depth study on their regulatory mechanisms. Meanwhile, it was also found that some regulatory agents showed their regulatory function targeting multiple pathways, such as ferulic acid. This ubiquitous natural phenolic constituent was demonstrated to inhibit cell adhesion and biofilm formation of Shigella flexneri (S. flexneri) targeting cell membrane, transcription of adhesion-related genes as well as EPS synthesis and transportation (Kang et al. 2020). Moreover, some regulatory agents can simultaneously realize inhibition of harmful biofilm and activation of beneficial biofilm in multi-species biofilm models. Utilizing the pH-sensitivity of histidine, Jiang et al. (2023) designed a pH-responsive and dual-functional antimicrobial peptide, LH12, for the regulation of microbial communities containing both cariogenic bacteria (Streptococcus mutans) and commensal bacteria (Streptococcus gordonii). Once activated by the acidic microenvironment, LH12 showed the ability to inhibit biofilm formation in Streptococcus mutans and improve the ecological competitiveness of Streptococcus gordonii by promoting the production of H₂O₂, providing a bio-responsive approach for caries management (Figure 4A).

Figure 4. Schematic illustration of strategies for regulating biofilm formation. A dual-functional LH12 for regulation of oral biofilm (Jiang et al. 2023). B inhibition of P. aeruginosa biofilm formation by LbL Ag@AM_AC NPs. (Ivanova et al. 2020). C dual-functional surface for prevention of biofilm formation (Zou et al. 2021).

3.2. Nanomaterials for regulation of biofilm formation

The rapid development of nanotechnology has broadened the application of nanomaterials in biosensing platform establishment, drug delivery, disease therapy, and other aspects of food and biomedicine areas (Hassan et al. 2017; He, Deng, and Hwang 2019; Liang et al. 2020; Pu et al. 2021; Zhang et al. 2021). As a promising type of antimicrobial, nanomaterials have been widely used for microbial inactivation and biofilm control (Ndayishimiye et al. 2022; Zhao, Chen, et al. 2021). In most nano-based methods for biofilm eradication, the EPS matrix, which reduces the permeability and enrichment of therapeutic agents inside mature biofilms, is the most discussed target (Lv et al. 2023). In contrast, for regulatory strategies, the key is to inhibit initial attachment and EPS production by disrupting the regulatory network and group behaviors. Recently, the combination of nanomaterials with anti-biofilm agents has been adopted as an effective strategy to enhance the effectiveness of anti-biofilm agents, control drug release, increase permeability and endow responsive properties toward certain microenvironments (Figure 3) (Badawy et al. 2020; Liu et al. 2020; Sarveswari et al. 2023; Huang, Pu, and Sun 2023).

The broad-spectrum anti-bacterial and anti-biofilm activities of metal-based nanomaterials, such as silver nanoparticles (AgNPs) and zinc oxide nanoparticles (ZnONPs), have been demonstrated (Dai et al. 2017; Lee et al. 2014). With synergic biofilm inhibitory effect, nanocomposites of anti-biofilm agents and metal-based nanoparticles have been proposed for augmenting anti-biofilm activity. Based on the QS inhibitory activity of tannic acid and the anti-bacterial activity of AgNPs, the nanocomposite of tannic acid and silver nanoparticles (Tannin-AgNPs) showed the ability to modulate the biofilm formation process and decrease the production of AIs (Liu et al. 2020). Similarly, it was also reported that chitosan-zinc oxide (CH/ZnO) nanocomposite enhanced the effect of chitosan on decreasing AHL production, biofilm formation and motility of P. aeruginosa (Badawy et al. 2020). Furthermore, taking advantage of quorum quenching enzyme acylase and membrane-disrupting biopolymer amino cellulose, Ivanova et al. (2020) decorated AgNPs templates in a layer-by-layer fashion to obtain hybrid enzyme/biopolymer/metal NPs (LbL Ag@AM_AC NPs) with enhanced anti-bacterial and anti-biofilm activities, as well as decreased toxicity to mammalian cells. By degrading the AHL signaling molecules, the hybrid nanoparticles were able to inhibit the QS-regulated biofilm formation by P. aeruginosa at lower concentrations than the AgNP templates (Figure 4B). However, for these proposed nanomaterials, assessment of the long-term effects on human health and environmental safety is still required, which is considered one of the major barriers to application in food and biomedical use.

3.3. Functionalized surface materials for regulation of biofilm formation

As surface properties can significantly impact initial attachment, surface coating and surface modification have been widely used to diminish the formation of early-stage biofilms (Yin et al. 2021). Anti-biofilm surfaces can be generally divided into two modes: the “attack” mode and the “defence” mode (Ma et al. 2022). In the “attack” mode, antimicrobial agents such as AgNPs, enzymes, and peptides are attached to the surface to efficiently kill the bacteria for prevention of cell adhesion and colonization. In the “defence” mode, strategies such as anti-adhesive coatings based on polymer brushes or highly hydrated hydrogel networks have been widely applied to resist the initial attachment or release the previously attached cells into aqueous media (Ahmadabadi, Yu, and Kizhakkedathu 2020). Based on the conventional anti-biofilm surfaces, a new class of functionalized surface materials are emerging for the regulation of biofilm formation. Similar to the already proposed approaches, most of these materials also adopt the “attack” or “defence” modes for biofilm inhibition (Figure 3). However, rather than directly inactivating the bacteria or resisting the attachment stage, the newly developed materials are designed to modulate bacterial behaviors related to biofilm establishment (Hu et al. 2022; Vaishampayan et al. 2018; Zou et al. 2021). For instance, Vaishampayan et al. (2018) developed a novel surface coating AGXX® consisting of micro-galvanic elements of silver and ruthenium. Compared with conventional antimicrobial silver coating, AGXX® was demonstrated to down-regulate many biofilm-associated genes and the two-component agr QS system in Methicillin-resistant S. aureus (MRSA), suggesting that it might diminish biofilm formation by interfering with QS system.

Besides metal-based coating materials, signaling molecules are also applied in surface coating for biofilm regulation. Using click chemistry, Kim et al. (2017) covalently attached AIP-I (pro-QS molecule) and TrAIP-II (anti-QS molecule) to surfaces, realizing control of the agr-I system and biofilm development in S. aureus. Nitric oxide (NO), a well-established signaling molecule, has been demonstrated to participate in the regulation of communal behaviors by increasing c-di-GMP hydrolysis and influencing the QS system (Plate and Marletta 2013). Based on the regulatory function of NO, Woehlk et al. (2019) introduced a NO-functionalized catecholamine as a polymerisable coating agent to spatially control biofilm formation on a variety of surfaces. The prepared NO-coated surfaces completely suppressed surface colonization of P. aeruginosa, providing a bioinspired and versatile coating approach to reduce biofilm contamination and biofilm-related infections in the hospital. Meanwhile, recent advances in nanomaterials also supplied an unconventional option for regulating biofilm formation. With fiber size and pore size close to Lactobacillus paracasei, electrospun cellulose acetate nanofibrous membranes were shown to be powerful biofilm-enriching scaffolds (Hu et al. 2022). Compared to planktonic cells, residents embedded in biofilms showed superior pH and antibiotic tolerance with genes related to cell adhesion, biofilm formation and tolerance expressed differently.

Dual-functional surfaces are hypothesized to largely prevent initial attachment and biofilm development by integrating the advantages of “attack” and “defence” modes. Focusing on the initial and middle stages of biofilm formation, Zou et al. (2021) developed a dual-functional surface based on surface-tethered copolymer brushes of 2-hydroxyethyl methacrylate (HEMA) and 3-(acrylamide)phenylboronic acid (APBA). With anti-adhesion properties, the poly(HEMA) component enabled the surface to inhibit cell adhesion during the initial stages of biofilm formation, while the poly(APBA) component provided binding sites for attachment and pH-responsive release of quercetin (a natural anti-biofilm molecule). Compared with anti-bacterial and anti-adhesive surfaces, this dual-functional surface has significantly improved anti-biofilm performance to prevent biofilm formation involving both Gram-negative P. aeruginosa and Gram-positive S. aureus for up to 3 days.

The development of functionalized anti-biofilm surfaces, especially the stimuli-responsive surfaces, has provided new ideas for the prevention of biofouling and contamination of implants. Unfortunately, the limitations for practical applications also exist. For medical implants, surface functionalization can be easily realized, as the surface area is relatively small. But for the surfaces of pipes and production equipment, cost and operability should be taken into account. Meanwhile, as biofilm formation is a long-term process, evaluation of anti-biofilm capacity and potential toxicity in real-world application scenarios rather than laboratory is requisite.

3.4. Physical strategies for regulation of biofilm formation

In comparison with anti-biofilm agents and anti-biofilm surfaces, physical strategies, such as ultrasound, electric field and cold plasma, are regarded as a more eco-friendly solution for biofilm control and removal (Liu et al. 2022; Pan, Sun, and Han 2017; Pan et al. 2020; Esua, Cheng, and Sun 2021; Esua et al. 2022). Current physical strategies mainly focus on the eradication of biofilms (Gilmore et al. 2018; Josic et al. 2022). Many in-depth studies also partially revealed the mechanisms of anti-biofilm activity of these strategies (Erriu et al. 2014; Mai-Prochnow et al. 2021). Alternatively, the idea of applying physical strategies in regulating biofilm formation is still at the starting stage. A prominent characteristic of these strategies is the multiple regulatory functions arising from the various modes of action toward planktonic cells and biofilm. Based on the generation of reactive oxygen species (ROS), reactive nitrogen species (RNS) and other active particles during plasma discharge, cold plasma has exhibited great potential in biofilm inhibition and prevention through surface treatment and direct interaction with planktonic bacteria (Hage et al. 2022; Li et al. 2019; Pan et al. 2019; Esua, Cheng, and Sun 2020; Zhao et al. 2020a, 2020b, 2021; Zhao et al. 2023). Among the multiple active species, the possible roles of ROS as biofilm inhibitors and signaling molecules during biofilm development have been suggested (Čáp, Váchová, and Palková 2012; Pan, Cheng, and Sun 2021; Zhao et al. 2020). Li et al. (2019) evaluated the feasibilities and mechanisms of ultra-low dose ROS produced by plasma-activated water (PAW) to inhibit the Enterococcus faecalis (E. faecalis) biofilm development. It was shown that the ultra-low dose ROS treatment within 30 s effectively inhibited biofilm formation without lethal effect. The possible mechanisms mainly involved interfering with the QS system by down-regulating the related genes and decreasing the membrane potential.

Similar to cold plasma, other physical strategies also showed the ability to regulate the biofilm formation process. Qi et al. (2022) proposed a high voltage prick electrostatic field (HVPEF) as a novel method to inhibit biofilm formation in S. aureus. The suppressed biofilm formation was realized by the direct effect of HVPEF on planktonic cells, the reduced release of EPS components and regulated expression of biofilm-related genes. Conversely, recent studies suggested that low-power ultrasound might stimulate biofilm formation and improve the stability of the established biofilm, indicating the application potential in the modulation of probiotics in food and other related products (Bevilacqua et al. 2019). Meanwhile, it also indicated that insufficiently high power of ultrasound treatment could lead to an unexpected increase in pathogenic biofilm formation (Yu et al. 2021).

4. Conclusions and future trends

Biofilm has posed challenges for food safety and medical services, as well as tools for the production of biomaterials and probiotic products. Biofilm formation is a dynamic process with a transition from planktonic to community lifestyle in both phenotype and genotype. Although the underlying mechanisms are still not completely known, the review of surface appendages and EPS components suggests the participation of cellular and extracellular factors in early-stage biofilm establishment. The effects of environmental factors such as cold stress, oxidative stress, surface topographies and flow on biofilm formation are also discussed. However, current knowledge of these factors is mainly based on studies about single-species biofilm models in the laboratory, which are not suitable to mimic the biofilm formation process in real-world systems. Therefore, simulation and experiments of the biofilm development process in the industry and other open systems still require further exploration.

Regulation of biofilm formation is an active event driven by a series of complicated regulatory networks, involving TCSs, c-di-GMP, sRNAs and QS. Various models have been established to illustrate the function of a single regulator during the biofilm formation process. Moreover, it has been shown that these regulators are highly correlated, enabling bacteria to sense and respond to the changeable external environment through different pathways, integrate multiple signals, and then regulate their behaviors associated with biofilm formation and development. In order to reveal the regulatory mechanism of biofilm establishment, further studies on the synergetic effects of different regulatory networks should be carried out.

The current understanding of biofilm formation and regulation facilitates the exploitation of regulatory strategies as an alternative approach for biofilm control in food and biomedicine. Different from the conventional views which prefer to eliminate mature biofilm or inactivate the bacteria to prevent biofilm formation, the novel concept of regulation aims to inhibit the ability of planktonic bacteria to establish biofilms through modulating the biofilm formation process. On the other hand, by stimulating biofilm establishment in commensal and engineering bacteria, regulatory strategies can also promote the fermentation process and production of probiotics. Based on the progress in anti/pro-biofilm agents, nanomaterials, surface materials and physical methods, novel strategies for the regulation of biofilm formation have contributed to effective biofilm control in an effective, smart, safe and eco-friendly way. The advantages, limitations and suggested application of the proposed strategies are summarized in Table 2. Despite the promising results obtained with these methods in laboratory studies, the standard methodology for regulating biofilm formation in practical use is still missing. For some of these regulatory strategies, the precise regulation mechanisms are still unclear. Meanwhile, most of the proposed strategies are designed for specific single-species models, while their effect on multi-species biofilm and other open systems remains to be verified. Furthermore, in real-world systems, cost, operability, potential toxicity and long-term functional durability should also be evaluated. Therefore, it would require interdisciplinary communication and collaboration among chemists, microbiologists, and engineers to generate a standard methodology for the regulation of biofilm formation and realize the practical application in food, biomedicine, and other related areas.

Table 2. Advantages, limitations and suggested application of innovating strategies for regulating biofilm formation.

Innovating strategy	Advantages	Limitations	Suggested fields of application
Anti/pro-biofilm agents	Natural compounds: low-toxicity, broad-spectrum and highly effective, with multiple functions and targets Synthesized agents: usually designed for specific targets and function	Difficult to identify the active compounds and specific targets of naturally derived extracts Cost and viability remain to be assessed	Food preservation, infection treatment, modulation of microbial communities
Nano­materials	High efficiency, designable for specific targets and function, ability to enhance the effect of common anti/pro-biofilm agents	Long-term effects on human and systematic safety Cost and viability remain to be assessed	Infection treatment, usually combined with other anti/pro-biofilm agents
Functionalized surface materials	Multiple modes of action, designable for a specific function	Cost and operability for large-size surfaces should be considered Long-term durability and potential toxicity	Implanted devices, surfaces of pipes and equipment (cost and operability permitting)
Physical strategies	Safe, low toxicity, eco-friendly, easy to operate in practical application	Potential risk under insufficient treatment	Food processing factory, food preservation, infection treatment, potential enhancement of probiotics production

Abbreviation
EPS	=	Extracellular polymeric substances
TCS	=	Two-component system
sRNA	=	Small non-coding RNA
QS	=	Quorum sensing
eDNA	=	Extracellular DNA
P. aeruginosa	=	Pseudomonas aeruginosa
S. aureus	=	Staphylococcus aureus
E. coli	=	Escherichia coli
PIA	=	Polysaccharide intercellular adhesion
CWA proteins	=	Cell wall-anchored proteins
Baps	=	Biofilm-associated proteins
L. monocytogenes	=	Listeria monocytogenes
V. parahaemolyticus	=	Vibrio parahaemolyticus
HK	=	Histidine kinase
RR	=	Response regulator
c-di-GMP	=	Bis-(3’-5’)-cyclic dimeric guanosine monophosphate
DGC	=	Diguanylate cyclase
PDE	=	Phosphodiesterase
pGpG	=	5’-phosphoguanylyl-(3’-5’)-guanosine
AI	=	Autoinducer
AIP	=	Autoinducing peptide
AHL	=	Acyl-homoserine lactone
V. cholera	=	Vibrio cholera
Qrr	=	Quorum regulatory RNA
A. sobria	=	Aeromonas sobria
S. flexneri	=	Shigella flexneri
AgNPs	=	Silver nanoparticles
ZnONPs	=	Zinc oxide nanoparticles
MRSA	=	Methicillin-resistant Staphylococcus aureus
ROS	=	Reactive oxygen species
RNS	=	Reactive nitrogen species
E. faecalis	=	Enterococcus faecalis
PAW	=	Plasma-activated water
HVPEF	=	High voltage prick electrostatic field

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The authors are grateful to the National Natural Science Foundation of China (32272466) for its support. This research was also supported by Guangdong Basic and Applied Basic Research Foundation (2021A1515010644, 2021A1515110396), Guangzhou Basic and Applied Basic Research Foundation (202201010008), the Contemporary International Collaborative Research Centre of Guangdong Province on Food Innovative Processing and Intelligent Control (2019A050519001), and the Common Technical Innovation Team of Guangdong Province on Preservation and Logistics of Agricultural Products (2023KJ145).