Novel Approaches to Combat Medical Device-Associated BioFilms

Biofilms are aggregates formed as a protective survival state by microorganisms to adapt to the environment and can be resistant to antimicrobial agents and host immune responses due to chemical or physical diffusion barriers, modified nutrient environments, suppression of the growth rate within biofilms, and the genetic adaptation of cells within biofilms. With the widespread use of medical devices, medical device-associated biofilms continue to pose a serious threat to human health, and these biofilms have become the most important source of nosocomial infections. However, traditional antimicrobial agents cannot completely eliminate medical device-associated biofilms. New strategies for the treatment of these biofilms and targeting biofilm infections are urgently required. Several novel approaches have been developed and identified as effective and promising treatments. In this review, we briefly summarize the challenges associated with the treatment of medical device-associated biofilm infections and highlight the latest promising approaches aimed at preventing or eradicating these biofilms.


Introduction
A biofilm is a special existence state with a three-dimensional tower structure formed by a microbial community attached to a biological or nonbiological surface and embedded in the matrix of an extracellular polymeric substance (EPS). An EPS is secreted by attached cells and is mainly composed of polysaccharides, extracellular DNA (eDNA), proteins, and other secreted substances ( Figure 1) [1][2][3]. These substances act as a shield that often makes microorganisms resistant to physical removal, host immune responses, and antimicrobial treatments [1][2][3]. The development of biofilms is linked to quorum sensing (QS), which is a cell-to-cell communication mechanism that regulates a variety of physiological and biochemical functions [4,5]. It can influence the biological cycle of biofilms by targeting matrix compounds and regulate the production of multiple virulence factors to resist phagocytosis [4,6,7]. Biofilms have their own properties that change dynamically, and their biological cycle involves four stages, namely, adhesion, sessile growth, maturation, and dispersal [1,8]. The adhesion and sessile growth stages are reversible, wherein the cells aggregate loosely, but can also become detached and return to a free-floating state [3,8,9]. The maturation of biofilms is related to the production of EPS, which encapsulates cells in a thick and stable layer of complex biomolecules. This stage is irreversible [3,8,10,11]. Cells in the dispersion stage can secrete destruction factors to promote detachment of the biofilm and the colonization of surrounding sites [3,8,10,12]. Biofilms exhibit spatial heterogeneity [10,13]. The concentrations of various chemical substances in the biofilm, such as metabolic products, signal molecules, and nutrients, change stepwise from the surface to the inside, which makes the physiological activities of bacteria different in different parts of the biofilm [10,13]. The outer-layer cells remain metabolically active, while the inner-layer cells are persister bacteria, which are usually dormant [14]. Because of the uneven metabolism of bacteria in biofilms, regardless of  Up to 80% of microbial infections in the human body involve biofilm formation, especially in hospital settings, which greatly promotes the incidence rate and mortality [15,16]. With the high standards for the quality of life of patients, the improvement of medical standards, and the widespread application of medical devices, medical device-associated biofilms pose a serious threat to human health, and these biofilms are the most important source of nosocomial infections, despite the comprehensive prevention and control measures taken, such as disinfection and hygiene monitoring. According to statistics, about 2 million nosocomial infection cases occurred in the United States each year at the beginning of the 21st century, of which 50%-70% of nosocomial infections were related to indwelling medical devices [17][18][19]. Given the challenges associated with medical deviceassociated biofilm control, new strategies are urgently needed for the treatment of these biofilm-specific infections. Several novel approaches have been developed and identified as effective and promising treatment strategies. Although previous reviews have already summarized novel approaches to combat medical device-associated biofilms [20][21][22][23], we briefly summarize the challenges associated with the treatment of medical device-associated biofilm infections and provide a more comprehensive summary of strategies that can be employed to combat medical device-associated biofilms. In particular, we discuss the progress and limitations of the latest studies on antimicrobial peptides (AMPs) and nanotechnology in detail. Moreover, bacteriophage therapy is included in our review, though it is often ignored by most other review articles. Table 1. The main microorganisms involved in the formation of biofilms and infection rates for different medical devices.
Each of these mechanisms can only partially explain the increase in antimicrobial resistance observed in biofilms. However, in the face of the synergistic effects of these mechanisms, even the most aggressive antibiotic treatment options or combination schemes involving different kinds of antibiotics cannot completely eliminate biofilm infections because of the limited dose of antimicrobial agents and their side effects [105]. In addition, the removal of medical devices can effectively prevent the recurrence of biofilm infections. Although many studies have emphasized the importance of implant removal to avoid the recurrence of infection at the surgical site, not all patients need or can undergo surgical revision surgery for local infections [32]. Implant exchange surgery, that is, the replacement of infected implants with new implants, may not be the best choice for the treatment of biofilm infection due to the high degree of trauma and high infection rate [106], which not only cause great physical and mental damage to patients, but also increase the economic burden on patients and the medical and healthcare system [32,106]. A study on the treatment of infection with surgical site implants revealed that the average number of surgeries required for infection treatment was 4 (range 1-16), resulting in an average cost of treatment ranging from two hundred and fifty thousand to one million dollars per patient [107]. Approximately 2 million healthcare-related infections occur in the United States each year, resulting in more than $5 billion in additional medical expenses each year. Among them, 50%-70% of nosocomial infections are related to indwelling medical devices [17][18][19]. Additionally, the mortality from nosocomial infections associated with medical devices is largely dependent on the type of devices, ranging from <5% for dental implants and foley catheters to >25% for mechanical heart valves [108]. In the intensive care unit, the incidence of catheter-related bloodstream infections is as high as 2.5~6.9/1000 catheter days, depending on the type of catheter and the site of catheter insertion [17,109]. The additional economic cost of treatment for each catheter-related infection may be as high as $30,000, with the potential for increased mechanical ventilation, an additional 1-week stay in the ICU, and an additional 2-3-week hospital stay [110,111]. Therefore, the constant improvement of prevention strategies and the development of novel therapeutic approaches are needed to overcome these weaknesses. Currently, we still need to emphasize the basic role of antimicrobial therapy in combatting medical device-associated biofilms, while the combination of novel promising approaches and antimicrobial therapy may help us eradicate existing biofilms.

Physical Therapy
The removal of medical devices from the body and continuous irrigation and debridement are the most successful treatment options for eradicating biofilms when standardized antibiotic treatment fails to effectively eliminate medical device-related biofilm infections [32]. Because of technical difficulties and a high morbidity, the removal of implants infected with biofilms, such as prosthetic joints and pacemakers, may not be the best option.
Photodynamic therapy (PDT) is an effective physical method for removing biofilms. It is composed of three main components: Light; a chemical molecule called a photosensitizer; and oxygen [112]. Light at a specific wavelength has an antibacterial effect. Antibacterial PDT generates highly cytotoxic reactive oxygen species (ROS) or excitable singlet oxygen to oxidize biological molecules on or within the cell membrane, such as proteins, nucleic acids, and lipids, causing cell DNA damage, cell membrane and organelle destruction, and even cell death [113] (Figure 1). A recent study has shown that PDT can not only inhibit the formation of E. faecalis biofilms formed on root canals by producing ROS, but also fight against biofilm infections by inhibiting the expression of toxic factors that affect early bacterial adhesion and promote the formation of the biofilm matrix structure [114] ( Figure 1). Additionally, nanomaterials (NMs) can be used in PDT to enhance the effect of PDT by generating heat or transforming NMs into biofilm-destroying species [115,116]. PDT technology is mostly used in research on the treatment of dental plaque-related diseases [117]. PDT technology can be used to combat other implant-related biofilm infections, such as prosthetic joint infections and infections caused by ventilator-associated pneumonia biofilms [118,119]. Although preclinical studies have suggested that PDT is a promising technique for the treatment of biofilm infections, there remain many measures that need to be taken before it can be safely applied in clinical practice. The short-term side effects of PDT are usually mild and self-limiting, such as erythema, edema, and pain in the surrounding skin, while long-term exposure to light may cause cataracts, skin cancer, and other side effects [120]. During PDT, protection measures should be taken to protect the patient's eyes from laser exposure and to ensure that the photosensitizer and photochemical reactions do not affect the patient's surrounding tissues [11].
Studies have shown that low-intensity ultrasound at the physiotherapy level can enhance the activity of antibacterial agents, resulting in a powerful biofilm removal effect [121]. Moreover, ultrasound or ultrasound-targeted microbubble destruction (UTMD) can destroy the matrix structure of bacterial biofilms by using cavitation to promote drug entry into the biofilm and can also directly destroy cells and increase the metabolic activity of cells to enhance the bactericidal ability of antibiotics [122][123][124] (Figure 1). These findings provide new insights for the use of ultrasound or UTMD to improve the effectiveness of antibiotic therapy for prosthesis-or catheter-related biofilms, and these approaches have shown promising effects in vivo [124,125]. However, ultrasound-mediated antibiotic activity on bacteria in biofilms is affected by many factors, including the acoustic intensity, duty cycle, frequency, and duration [126]. For example, a greater ultrasound intensity and longer duration may cause skin or tissue damage or cause bacteria to spread to surrounding tissues and affect blood flow [127,128]. Cavitation can also potentially damage the implant [129]. Considering that this technique may be applied to clinical practice in the future, further research is needed to select the optimal ultrasound parameters.
The use of water jets is another effective physical method for removing biofilms on the surface of implants through the mechanical action of pulse and pressure [130]. Water jets can wash away loosely attached plaques, eliminate bacterial cells, and interfere with biofilm maturation [131] (Figure 1). A previous study has shown that under different pressure conditions, the ability of dental water jets to remove biofilms differs. The research results showed that, under pressures of 707, 350, and 102 kPa, the rate of reduction in biofilm thickness was 85.5%, 85.1%, and 63.4%, respectively [132]. A recent study has shown that the combination of PDT and water jets has good application prospects for removing scale from the surface of traditional implants. Compared with an untreated biofilm, the biofilms of Streptococcus mutans grown on a titanium surface treated with PDT were more easily removed by the impact force of a water jet. This showed that PDT can weaken biofilms in different stages, making them easier to separate with a simple washing force [133]. Another study demonstrated that the cavitating jet can also effectively clean biofilms formed on the rough surface of implant screws, especially on the root sector [134].
In addition, mechanical methods, such as the use of a plastic curette, an air-abrasive device (Perio-Flow), a titanium brush (TiBrush), and implantoplasty, and electrochemical treatment can effectively clean dental biofilms and improve hygiene around the implant [135,136]. However, it has been found that the mechanical treatment strategies used to remove implant biofilms may cause titanium dissolution and increase the cytotoxicity. Therefore, the dissolution of implant material should be an important consideration in the clinical choice of treatment of peri-implant inflammation [137].

Surface Modification
Applying antibacterial agents or antiadhesion agents to the surface of medical devices through surface engineering can effectively inhibit the adhesion and growth of microorganisms and prevent the formation of biofilms, which has become an important strategy for combatting biofilms related to medical devices. Antibacterial coating approaches prevent the formation and development of biofilms on medical devices through bacteriostatic or bactericidal effects. Traditional antibacterial coatings, such as chlorhexidine, gentamicin, minocycline, rifampicin, silver sulfadiazine, amikacin, and vancomycin, have been widely used in clinical practice, showing the efficacy of preventing catheter-related and other implant-related infections [138][139][140]. The combination of multiple antimicrobials, such as the combination of the antibiotics minocycline and rifampicin, has also shown good results in preventing single-drug resistance [138]. On the other hand, the extensive use of antimicrobial coatings may lead to bacterial resistance to antibiotics. To date, studies of antibacterial coatings have focused on the development of novel compound coatings with antibacterial effects or antibiotic delivery functions to prevent the formation of biofilms. We have summarized the novel surface-modifying compounds used against biofilms and mechanisms of action in Table 2.
Antimicrobial compound coatings or antiadhesion coatings are used to modify the surfaces of medical device materials to inhibit biofilm formation by killing bacteria or reducing bacterial adhesion ( Figure 1). An isoeugenol coating has been shown to prevent the formation of biofilms on stainless steel and polyethylene surfaces due to its good antibacterial activity [141]. N-acetyl cysteine is an Food and Drug Administration (FDA) -approved drug that is widely used to treat many different clinical diseases, such as chronic bronchitis, cardiac ischemia-reperfusion injury, and paracetamol poisoning [142]. The drug has been suggested to not only have antibacterial activity [143], but also reduce bacterial adhesion and prevent biofilm formation. Because of its excellent safety performance, it has been widely studied as a coating for medical implants, such as bone-related implants [144]. Existing methods of coating biomaterials with antibiotics and other reagents produce surfaces that are prone to degradation over time. Metal-based materials have a long history as antibacterial agents [145], such as the silver coatings used on a variety of medical devices. NMs exhibit enhanced antibacterial activity and a reduced cytotoxicity [146]. Silver nanoparticle (AgNP) coatings are also used to prevent bacteria from becoming attached to medical devices [147]. A recent study has shown that the topographical effects of nanostructure surfaces may have significant impacts on the adherence pattern and stretching degree of the bacterial cell membrane [148]. New alloys and metal-ceramic compounds are also used in anti-medical device biofilms. For example, a copper-bearing titanium alloy has been shown to efficiently kill the attached bacteria by damaging cell membranes and cell walls and strongly inhibit the biofilm formation and prevent bacterial infection associated with dental implants, meaning that it has great potential for application in dental implants with an excellent antibacterial viability and positive effect against bone resorption induced by peri-implantitis [149]. The tantalum processed by micro-Arc oxidation increased its surface roughness, hydrophilicity, and corrosion resistance, and improved its antibacterial activity against S. aureus, meaning that it exhibits great potential for application as a material for hard tissue implants [150]. Polycrystalline Zirconium Dioxide Ceramic presents the same characteristics for S. mutans biofilm formation as polymer-infiltrated ceramic, which can inhibit biofilm formation, and they both have a good biocompatibility with human fibroblasts. Therefore, they have great potential in the application of indirect restoration and implant abutment [151]. Antiadhesion coatings have also been realized by changing the physical and chemical properties of surfaces so that the bacteria no longer adhere easily. Hydrophobin has been used to coat different medically relevant materials and has been shown to be effective in preventing biofilm formation by S. epidermidis on polystyrene surfaces through the formation of a self-assembled amphiphilic layer [152]. The most common denture material, heat-polymerized polymethyl methacrylate, exhibits a high surface roughness, hydrophobicity, and surface free energy, which contribute to the initial adhesion of microorganisms. Cyanoacrylate adhesives can change the characteristics of the resin surface, reduce the surface roughness, increase the hydrophilicity, and change the surface free energy, thus inhibiting the formation of biofilms. Adhesives also have a good biocompatibility, so they are used to coat medical devices [153]. Ultrahigh-molecularweight polyethylene (UHMWPE) is a kind of solid, inert, and stable polymer and is the most commonly used polymer in total hip and knee arthroplasty. However, its strong hydrophobic surface makes it vulnerable to bacterial adhesion and biofilm formation. Polymer hydrophilic coatings are grafted to the surface of UHMWPE by polymer coating technology, such as covalent chemical bonds, which change the surface chemistry from hydrophobic to hydrophilic and prevent bacterial adhesion and biofilm formation [154]. Many studies on the prevention of biofilm formation have been carried out by changing the surface properties of materials, such as the roughness, hydrophobicity/hydrophilicity, surface free energy, and electric charge, to find more suitable materials that can be implanted into the human body and function favorably. For example, modification of the surface of orthopedic implants, in addition to effective resistance to bacterial adhesion, the functional retention of raw material implants, biocompatibility, the ability to resist tribological corrosion of coatings, and nontoxicity to host cells, should also be considered and evaluated [155]. S. aureus is one of the most important pathogens associated with medical device biofilms and poses a major medical challenge due to its high incidence and drug resistance. The virulence factor surface proteins of S. aureus can specifically bind to host extracellular matrix proteins, such as fibrinogen, fibronectin, and collagen. These proteins have been shown to have a high affinity for implants and become easily attached to the implant surface. Therefore, these proteins mediate the attachment of S. aureus to the surface of the implant [156]. Targeting these mediator proteins to degrade or inhibit their production or reduce their adsorption capacity may provide a viable option for preventing S. aureus biofilm formation on implant surfaces. Fibrinolytic coatings (e.g., with tissue plasminogen activator (tPA)) reduce S. aureus biofilm formation on indwelling medical devices both in vitro and in vivo. The tPA coating prevents the early attachment of bacteria and the subsequent accumulation of biomass by splitting local fibrin. In addition, the tPA coating increases the sensitivity of biofilm infections to antibiotics [157]. Monomeric trimethylsilane and oxygen plasma coatings cause chemical changes on the surface of the material, resulting in changes in the adsorption of intermediate proteins on the coating surface, which can reduce the adhesion of bacteria to the surface, thereby inhibiting the formation of and infection caused by biofilms [158]. It has recently been demonstrated that device-related infections caused by S. aureus biofilms rely on the coagulation pathway to activate prothrombin and fibrin production. Direct thrombin inhibitors can be used to coat biomaterial surfaces to prevent staphylococcal binding and subsequent device-related infections [159].
Another approach is to inhibit the colonization of microorganisms and the formation of biofilms by affecting the structural integrity of the biofilm. The immobilization of biofilm-dispersing enzymes on material surfaces is regarded as one of the most promising strategies for preventing biofilm formation ( Figure 1). For example, a deoxyribonuclease I (DNase I) coating has a good anti-infection ability and biocompatibility, which is beneficial for improving the application success rate of implants [160]. Polypyrrole is an organic conductive polymer formed from a pyrrole ring structure that affects the integrity of biofilms by promoting a structure with repeated and positive charge-regulated ionic cross-bridging and subsequent aggregation so that biofilms can be easily washed away by water [161]. The combined application of the above measures may lead to stronger antibiofilm activity. Regardless of the measures taken, the safety of the host must be considered, and the functionality of the raw materials must be ensured.
As mentioned above, in order to better protect implanted medical devices from bacterial invasion, preclinical studies on the surface modification of medical devices have made very successful progress, but there is still a significant discrepancy between the proposed strategies and their clinical application. Most of the coatings being studied may not be suitable for medical implants due to cytotoxicity, immunoreactivity, or genotoxicity problems. However, some technologies have shown sufficient evidence to prove the effectiveness, safety, and durability of antibacterial drug delivery coatings [146,162], such as AgNP coatings, which could be proposed in clinical practice. Some have been applied in clinical practice. For example, the antimicrobial hydrogel-coated prosthesis can safely protect the prosthesis and reduced early surgical site infections without adverse effects at a mean follow-up of 2 years [163]. Another study showed that silver-coated megaprosthesis is effective in terms of a reduction in prosthetic joint infections, with 2.2% local argyria and without systemic side effects [164]. In order to be better applied in clinical practice, the development of device surfaces with excellent antibacterial properties, high biocompatibility, and a long-lasting antibacterial efficacy still needs a breakthrough.

Antimicrobial Peptides
Antimicrobial peptides (AMPs), which are a type of small-molecular peptide and are also known as host defense peptides (HDPs) [10], widely exist in nature and are a part of the innate immunity of almost all living organisms. AMPs can be extracted from bacteria, fungi, plants, insects, amphibians, fish, birds, mammals, and even the human body and can also be produced by chemical synthesis. AMPs have broad-spectrum and rapid antibacterial activity and do not easily result in drug resistance. In particular, their killing effect on certain drug-resistant pathogens has attracted attention. They can be used as single antibacterial agents or combined with traditional antibiotics.
To date, a variety of AMPs have been used in in vivo and in vitro studies, and some have been used in clinical practice, such as colicins and daptomycin. AMPs can not only exhibit powerful bactericidal activity, inhibit the formation of biofilms, and reduce or eliminate mature biofilms when the concentration is equal to or higher than the minimum inhibitory concentration (MIC) of the corresponding planktonic cells, but also display antibiofilm activity when the concentration is lower than the MIC of the corresponding planktonic cells [10]. This may be related to the mechanism underlying the multimode antibiofilm activity of AMPs. AMPs act on the bacterial cell membrane, forming different complex structures on the membrane through different mechanisms, such as the formation of barrel-state, toroidal pore, carpet, and aggregate models, which destroy the integrity and stability of the membrane, cause leakage of the cellular contents, and lead to the death of bacterial cells [165][166][167][168][169][170][171]. In addition, there are other potential antibiofilm mechanisms (Figure 1). Some AMPs can interfere with cellular metabolic processes and inhibit cell wall, nucleic acid, or protein biosynthesis by interacting with intracellular targets [172]. For example, B22, which is a derivative of BMAP-27B, can penetrate and kill established Vibrio cholerae and P. aeruginosa biofilms. After treatment with nanomolar concentrations of B22, P. aeruginosa and V. cholerae cells display expression changes in a variety of physiological metabolic processes, including protein synthesis, peptide glycan biosynthesis, respiration, and the detoxification of ROS. The main role of B22 has been shown to be the induction of ROS production, which destroys bacterial molecules, resulting in slowed cell growth or death [172]. LL-37 can downregulate the expression of the biofilm-related genes rhlA and rhlB, inhibit the synthesis of rhamnosyltransferases, reduce biofilm formation, and degrade existing biofilms formed by P. aeruginosa [173]. In addition, LL-37 also has antibacterial effects on biofilms formed by other pathogens. The anti-biofilm effects and mechanisms of LL-37 and LL-37 derivatives against biofilms formed by different pathogens (such as S. epidermidis, E. coli, S. aureus, C. albicans, and A. baumannii) have been well-summarized by Zhang Kaiyu and other colleagues [1]. HBD3 and HBD3-derived peptides interfere with the expression of the adhesin gene and reduce the expression of adhesion proteins, which leads to a decrease in biofilm formation [174,175]. In vivo, HBD3 has also been demonstrated to inhibit Staphylococcus biofilm formation [176]. HBD3 and its derivatives are most commonly used to study anti-biofilm activity, and other defensins have also shown anti-biofilm activity in some studies. For example, HBD2 can inhibit P. aeruginosa biofilm formation independent of biofilm regulatory pathways [177]. HNP-1 derivatives can also inhibit and eradicate the biofilm formation of multidrug-resistant uropathogenic E. coli, which may be used as a potential source for the treatment of biofilm-associated bacterial diseases [178]. The peptide Nal-P-113 inhibits Porphyromonas gingivalis biofilm formation by inhibiting the synthesis of ATP-binding cassette (ABC) transporters and ATP-binding proteins [24,179]. ABC transporters play a role in the cell adhesion phase of biofilm formation [180]. Temporin L (TL), derived from frog skin, has been confirmed to have antibiofilm activity [181]. It has been shown to act on the bacterial FtsZ protein, which is a protein involved in Z-ring formation, to inhibit cell division, thereby slowing bacterial growth [182]. Some AMPs can inhibit microbial cell adhesion or disperse cellular aggregates in the early stages of biofilm formation. LL-37 can also promote twitching in P. aeruginosa and cause the bacteria to move instead of forming biofilms [173,183]. Twitching is a form of surface motility that is regulated by type IV pili [184]. In S. aureus, lL-37-and LL-37-derived peptides have also been proven to inhibit the formation and degradation of existing biofilms [185]. In P. aeruginosa, the antibiofilm peptide 1037 can also directly inhibit biofilms by reducing swimming and swarming motilities, stimulating twitching motility, and suppressing the expression of a variety of genes involved in biofilm formation [186]. Polymyxin B (PmB), which is a cationic bacterial AMP produced by Bacillus polymyxa, can affect V. cholerae flagella, resulting in a decrease in motility and a compromised ability to form biofilms [187]. The formation of biofilms can also be inhibited by interruption of the signaling system of bacterial cells. QS is a cell-cell communication process that regulates multiple physiological and biochemical functions [1]. The human cathelicidin AMP LL-37 can prevent biofilm formation by inhibiting QS in microorganisms [183]. The immunomodulatory peptide innate defense regulator (IDR)-1018 was able to potently prevent biofilm formation and eradicate preformed biofilms formed by diverse species of Gram-negative bacteria and the Gram-positive bacterium S. aureus by blocking (p)ppGpp and signaling nucleotides [188]. Persister cells are a small subgroup of bacterial cells that are in a dormant state and are highly resistant to antibiotics. Some AMPs can prevent the formation of biofilms and promote the degradation of biofilms by interfering with the formation of persister cells [189]. The representative AMP is SAAP-148. S. aureus, which survives under the action of high-dose rifampicin, can be completely cleared within 2 h by low concentrations of SAAP-1448 [190]. Temporin 1Tb, derived from frog skin, can also kill both developing and mature S. epidermidis biofilms [191]. Some AMPs can impair EPS production to cause structural changes and degradation of the biofilm matrix. The synthetic peptide P1, derived from a tick antifreeze protein, significantly reduced S. mutans biofilm formation in an in vitro microplate assay and in a saliva-coated hydroxyapatite disc tooth model through the degradation of EPS and destruction of the biofilm structure [192]. TL can also affect mature biofilms in Pseudomonas fluorescens, and a large portion of the preformed biofilm architecture was clearly perturbed by addition of the peptide, with a significant decrease in all biofilm surface parameters and the overall biomass [181]. Hepcidin 20, which is an antimicrobial peptide derived from the human liver, can reduce the mass of the extracellular matrix and change the structure of biofilms of S. epidermidis by targeting the intracellular polysaccharide adhesion [193]. The fish-derived AMP piscidin-3 has nucleosidase activity and can destroy the extracellular DNA of P. aeruginosa through its Nterminal coordination with Cu 2+ [194]. (LIN-SB056-1) 2 -K was also proven to disrupt the P. aeruginosa biofilm structure in an ex vivo model [195]. Some AMPs, as immune modulators, recruit immune cells at the site of infection, enhance the activity of host immune cells, and regulate the inflammatory response [196]. Some AMPs can resist endotoxin activity and neutralize proinflammatory factors, which may cause serious complications and damage to the body [197]. Some AMPs also display a synergistic effect when combined with traditional antibiotics via the promotion of antibiotic uptake [198]. We have listed the representative AMPs and their antibiofilm effects and mechanisms of action in Table 3.
Some AMPs have been applied to multi-drug resistance in clinical practice, such as colistin and polymyxin B. The modes of their application include intravenous or topical administration (such as intrathecal injection and atomization inhalation) [199]. International consensus guidelines have recommended intravenous doses of colistin and polymyxin B based on the kidney function of patients [200]. Some AMPs are prepared as ointments for infections of superficial tissues or medical device surfaces [190]. Most of the new AMPs have not been applied to humans and the experiments have been carried out in vitro or in animal experiments. However, many studies have provided inspiring results and the in vivo data have provided valuable references for the therapeutic dose, safety, and stability of AMPs for further clinical practice in humans. For example, in an animal experiment, 50 mg/kg administration of the four cathelicidins (Cathelicidin-BF, Pc-CATH1, Cc-CATH2, Cc-CATH3) could significantly reduce the fungal counts in a murine oral candidiasis model induced by clinically isolated C. albicans with slight hemolytic activity. Cathelicidin-BF strongly inhibited C. albicans biofilm formation at 20 µg/mL [171]. In an in vitro experiment simulating real flow conditions in the human body, Temporin-L(TL) had an anti-biofilm effect without increasing cell damage, demonstrating that it has great potential for clinical application [181]. The anti-biofilm doses of AMPs in the above experiments are shown in Table 3. Prevent the formation of the extracellular matrix and promote its breakdown and eradication, eradicate persister cells [190]  AMPs represent promising compounds for antibiofilm strategies. However, the activity of AMPs is related to many factors, including the size, sequence, charge, structure, conformation, hydrophobicity, and amphiphilicity, which may lead to toxicity and side effects on host cells under some conditions [11]. Although these AMPs have good antibiofilm activity in vitro, their potential toxicity and poor stability under physiological conditions limit their application in the clinical treatment of biofilm infections [201]. To date, many studies have examined the relationship between the structure of AMPs and their antibiofilm activity and safety. The structure with the best antibacterial activity is determined through the artificial modification and synthesis of AMPs, with the aim of seeking suitable AMPs for clinical application. Many methods have been adopted, for example, substitution, cyclization, hybridization techniques, or modification of the C-and N-termini via acetylation and myristoylation [173,[202][203][204][205][206]. The bactericidal activity of some AMPs can be increased by truncating or increasing the overall positive charge [172,207]. In addition, the application of some drug delivery systems can not only help AMPs increase the antibacterial activity, but also improve their stability under physiological conditions and reduce their cytotoxicity [168]. For example, the conjugation of AMPs with nanoparticles (NPs) leads to the higher penetration of biofilms and improves the antibacterial activity. At the same time, NPs provide protection to AMPs, preventing their degradation in different environments, such as enzymatic degradation [208].
In conclusion, AMPs have a variety of active antibiofilm mechanisms and have potential applications in clinical treatment to remove biofilms. Although a large number of preclinical and clinical studies are underway, focusing on improving the activity, reducing the host cell toxicity, and improving the stability under physiological conditions of AMPs, further in vivo studies are needed due to the complexity of the host environment.

Nanotechnology
Nanotechnology is used to study the properties and applications of NMs. Nanometer is a unit of length that is equal to one billionth of a meter or 10 −9 m. NMs are materials with at least one dimension at the nanometer scale (1-100 nm) in three-dimensional space, and these materials exhibit some special physical and chemical properties that are different from those of macrolevel materials. NMs have a wide range of applications in the fields of electronics, optics, manufacturing, and biomedicine, and they have also been developed for use in a variety of medical diagnosis and treatment applications [209]. NMs, especially NPs, have an increased specific surface area and surface functionalization that enable them to interact closely with microbial membranes and exhibit antimicrobial activity, even at very small doses [210]. These properties contribute to the development of antibacterial drugs [210]. The antibacterial and antibiofilm activities of NPs have been widely demonstrated in different pathogens, and their biological activities depend on different characteristics, including the size, shape, surface charge, structure, material type, and concentration [210,211]. The antibiofilm mechanisms of NPs are as follows. NPs can come into contact with bacterial cell walls and damage them through different interactions, induce changes in the membrane shape and function, induce oxidative stress and act as catalysts to interact with microbial cells, disturb the respiratory chain function, cross microbe membranes, interfere with metabolic pathways, interact with the microbial cellular machinery to inhibit enzymes, inactivate proteins, or modify gene expression levels [212,213]. NPs can also be used as drug delivery carriers or as catalysts to promote the penetration of drugs into biofilms; improve the solubility, stability, and biocompatibility of drugs; or enhance the antibiofilm activity by physically destroying the structure of the biofilm [22]. The antibiofilm mechanism of NPs is well-described in the study by Zhang Kaiyu and others [1] (Figure 1). Nanotechnology has developed rapidly. As mentioned above, this technology has been applied for the surface modification and improvement of AMPs. In this section, we will focus on the research progress in NMs that have exhibited promising results against biofilms.
Antibiofilm NMs include inorganic materials (such as gold and silver) [214][215][216][217][218][219], polymers (such as the natural antibacterial substance chitosan, biodegradable polymer polylactic acid, and carbon-based polymer fullerene) [220][221][222][223], lipids (such as liposomes) [224,225], and molecular complexes (such as protein nanocomplexes and cyclodextrin nanocomplexes) [226]. The following studies are examples of different NMs that are used against biofilms. Metal NPs exhibit bactericidal activity against a variety of microorganisms by releasing metal ions and are effective bactericides. At the same time, they have also been proven to have an impact on biofilms. Silver is currently one of the most widely studied metals in nm research. Biosynthetic AgNPs from Phanerochaete chrysosporium with a diameter of 45 nm exhibited a high negative surface charge close to 30 mV and showed colloidal stability from pH 3 to 9 under conditions of a high ionic strength ([NaCl] = 10-500 mM). It was shown that the AgNPs could kill bacteria by acting on fatty acids of E. coli cells to destroy the integrity of cell membranes. Moreover, the results further demonstrated that these biosynthetic AgNPs displayed biofilm disruption activity. These biogenic AgNPs were added to preformed biofilms and incubated for 48 hours, leading to a 29% reduction in the biomass of E. coli biofilms and an 80% reduction in the biomass of C. albicans biofilms [212]. Another study showed that P. aeruginosa biofilms treated with green synthesized seabuckthorn AgNPs (SBT@AgNps) can significantly reduce the bacterial load in biofilms, disrupt the matrix structure and cell wall, and interact with the cell membrane. The results showed a significant reduction in the bacterial load in each biofilm stage. On the peak day, i.e., the 4th day of biofilm formation, a significant reduction of approximately 33% (MIC) and 54% (MBC) in the bacterial load of the SBT@AgNps-treated biofilms was observed compared to that of the control biofilms. A biofilm of P. aeruginosa was established on a coverslip and analyzed by field emission scanning electron microscopy (FESEM) on the peak day. The results showed that the intact three-dimensional structure of the biofilm treated at the MIC and MBC was disrupted to form plaques embedded with biofilm cells. A concentration-dependent biofilm matrix structure was observed upon treatment with SBT-AgNps. In addition, the interaction of SBT@AgNPs with the bacterial cell membrane and disruption of the bacterial wall were also observed. The biofilm disruption resulted from SBT@AgNPs-mediated bacterial killing, and these NPs interacted with bacterial cells. Moreover, SBT@AgNPs showed an excellent anti-QS ability, which may be helpful in controlling bacterial infection at initial stages by blocking bacterial communication [227]. AuNPs are also commonly used nanoparticles, and biosynthesized AuNPs from brown seaweed at sub-MICs could inhibit P. aeruginosa biofilm formation by more than 80% and eradicate mature biofilms. Furthermore, sub-MICs of biosynthesized AuNPs also attenuated the production of several important virulence factors, such as pyoverdine, pyocyanin, and rhamnolipids, and impaired bacterial swarming, swimming, and twitching motilities [228]. The antibacterial activity and antibiofilm potential of chitosan and its nanometer-scale derivatives have been demonstrated in a variety of microorganisms [229]. A recent study proved that chitosan NPs (CNPs) can inhibit S. aureus biofilm formation, damage mature biofilms, reduce EPS production, and lower the cell surface hydrophobicity. The results showed that the biofilms were inhibited by approximately 50% upon treatment with CNPs compared to the control group. The treatment of a preformed biofilm with CNPs also resulted in a reduction of approximately 15% in the biofilm mass. Chrysin, which is a flavone constituent of the Orocylumineicum vent, has already been demonstrated to have antibacterial properties. However, its low water solubility, low bioavailability, and rapid metabolism in the body limit its therapeutic applications. Chitosan was used as a nanocarrier loaded with chrysin to enhance the antibiofilm activity through a synergistic effect [220]. In addition to their inherent antibiofilm activity, NMs can also be used as carriers for antibiofilm agents or other biofilm scavengers. Rifampicin-conjugated silver (Rif-Ag) NPs enhance the biofilm penetrating power compared to rifampicin alone. The synthesized NPs inhibit >90% of biofilms formed by K. pneumoniae and methicillin-resistant Staphylococcus aureus (MRSA) at low doses and show 1.5-2 times more penetrating potential, as observed by biofilm eradication and percentage viability reduction, compared to rifampicin alone [230]. Some NMs, as response carriers, exert antibiofilm effects. For example, mesoporous polydopamine NPs can cooperate with PDT to combat biofilms. These NPs, when integrated on titanium surfaces with an attached indocyanine green photosensitizer, can reduce S. aureus biofilm formation by 95% in murine specimens exposed to light with a wavelength of 808 nm [115]. Another study showed, that under laser irradiation, AuNPs produce vapor nanobubbles (VNB) to interfere with the structure of biofilms, promote the penetration of tobramycin, and improve the bactericidal effect [214]. Some NMs have inherent antibiofilm activity, which can prevent microorganisms from adhering to devices and the formation of biofilms, destroy EPS, and kill biofilm microorganisms through different mechanisms. Some NMs, as carriers of antibiofilm agents or other biofilm scavengers, can enhance the solubility and stability of the drug, overcome the drug resistance mechanism, and increase the biocompatibility of the drug at the target site. However, some NMs act as responsive carriers of external forces such as light to exert antibiofilm effects [22]. nm composites can simultaneously exert antibiofilm effects through all of the above methods. We have summarized the methods and effects of different nanomaterials against biofilms in Table 4.
Nanomaterials have a broad prospect in the application of anti-biofilms for medical devices. Although a large number of studies have been devoted to the antimicrobial activity of nanomaterials, the heterogeneity of these studies (e.g., the study design, synthesis methods, study methods of antimicrobial properties, and bacterial species selection) makes it impossible to cross-interpret between them, which limits nanomaterial application prospects in antibacterial aspects. Therefore, studies on nanomaterials need to be homogenized to obtain more reliable data to provide a solid theoretical basis for clinical treatment [231]. According to the above studies, the application methods can be summarized as device coatings [147], topical applications [223], intraperitoneal injections [221], etc., all of which are in vitro and animal experiments.

Agents for Degradation of the Extracellular Matrix of Biofilms
The extracellular matrix plays an important role in the formation, development, and survival of biofilms. It is not only a protective barrier against external factors, but also a source of nutrients and enzymes, as well as connectors between cells [232]. Degradation of the matrix is a way to weaken biofilms, and several strategies have been proposed ( Figure 1).
First, enzymes can degrade various EPS components. The bacterial extracellular polysaccharide polyβ-1,6-N-acetylglucosamine (PNAG) is the main extracellular matrix component of the biofilms of Gram-positive bacteria and Gram-negative bacteria [233]. Dispersin B, which is a β-N-acetylglucosaminosidase produced by the Gram-negative species Actinobacillus actinomycetemcomitans, shows great potential for hydrolyzing PNAG to disperse biofilms formed by various bacteria, but does not kill the bacteria [234,235]. Due to the biofilm dispersal activity of dispersin B, it is used to generate specific probes for monitoring the production and localization of PNAG in the process of biofilm formation and elucidating the early stages of PNAG-dependent biofilm formation [233]. eDNA contributes to the structural integrity of the matrix [236]. Deoxyribonuclease (DNase) can degrade the eDNA present in the matrix, thus disrupting the stability of the biofilm. The antibiofilm effect of DNase has been studied for several kinds of microorganisms, such as S. aureus, P. aeruginosa, E. coli, A. baumannii, Haemophilus influenzae, and K. pneumonia [237,238]. DNase I immobilized on a titanium surface can prevent the adhesion and biofilm formation of S. mutans and S. aureus and exhibits good biocompatibility [160]. DNase I added to mature biofilms can also degrade biofilms. DNase I was further found to be less effective against mixed-species biofilms than individual biofilms [238]. Many other enzymes, such as proteinase K, lysozyme [239], α amylase [240], and the glycoside hydrolases PelAh and PslGh [241], have been proven to have promising antibiofilm activity.
Second, cation chelators can not only breakdown the extracellular matrix and eradicate biofilms, but also act as enhancers for antimicrobial agents, thereby disabling resistance mechanisms. For example, branched polyethylenimine (BPEI) is a cationic polymer with a low molecular weight of 600 Da. A recent study on treatment with BPEI combined with oxacillin demonstrated that this combination therapy could eradicate methicillinresistant Staphylococcus epidermidis (MRSE) biofilms and reduce the cell density of the biofilms by 100,000-fold. The strong synergistic effect between BPEI and lactam antibiotics is because BPEI can neutralize the anionic character of the biofilm, resulting in rupture of the EPS matrix, as well as destruction of the hydrophobic barrier, which prevents the permeation of hydrophilic antibiotics, allowing the antibiotics to enter and kill the bacteria [14]. Ethylene diamine tetraacetic acid (EDTA), which is a cationic chelating agent, has been shown to affect the structural integrity of biofilms through the chelation of calcium, iron, and magnesium in staphylococci and Candida spp. [242]. A study revealed that EDTA alone significantly decreased fungal metabolic activity in preformed C. albicans biofilms. Additionally, EDTA combined with fluconazole significantly reduced the growth of biofilms compared with fluconazole alone. In addition, the study also showed that the cell density decreased when biofilm cells were treated with EDTA alone or in combination with fluconazole [243].
In addition, a few compounds, such as ambroxol and N-acetylcysteine, have been reported to have active matrix destruction properties and can be used for antibiofilm purposes [232]. Therefore, matrix disruptive agents such as enzymes, cationic chelators, and compounds provide us with another effective treatment strategy against biofilm-associated infections. These biofilm-dispersing agents can be used to functionalize the surface of medical devices to inhibit the formation of biofilms and can also be combined with other antibiofilm agents or technologies to improve antibiofilm activity [234,235,244,245].
Moreover, several new compounds have also been shown to have antibiofilm activity (Figure 1). These compounds can be extracted from organisms or produced by chemical synthesis. Farnesol, which is a QS molecule, can be secreted by a variety of organisms, mainly Candida, and can also be produced by chemical synthesis [246]. A recent study demonstrated that farnesol has significant antibiofilm activity, disrupting the mycelia of Fusarium keratoplasticum and the biofilm extracellular matrix and preventing conidial adhesion, filamentation, and biofilm formation [247]. In addition, a study on the effects of farnesol on Candida dubliniensis biofilms indicated synergy between farnesol and fluconazole in resistant strains. This led to a reversal of fluconazole resistance, which is a crucial result that suggests a possible application of farnesol as an adjuvant therapeutic agent [248]. Another study showed that caffeine, which is a xanthine alkaloid compound, significantly inhibited the formation of P. aeruginosa biofilms at sub-MIC concentrations by interfering with QS [249]. β-Lapachone and lapachol oxime are semisynthetic derivatives of lapachol that were originally isolated from species of the Bignoniaceae family and have also been proven to be effective in inhibiting biofilm formation [250]. In addition, Warburgia ugandensis Sprague subspecies ugandensis is a plant that is widely distributed in Eastern, Central, and Southern Africa. Warburganal, polygodial, and alpha-linolenic acid (ALA) were the major bioactive compounds isolated from the acetone extract of W. ugandensis. These compounds have been shown to inhibit the formation of C. albicans and S. aureus biofilms and exhibit synergistic effects with azole compounds [251]. However, some compounds have also been shown to enhance biofilm formation. Nicotine, for example, promotes S. aureus biofilm formation by promoting initial bacterial attachment and extracellular DNA release [252]. These new compounds provide new perspectives and possibilities for antibiofilm agents, requiring further verification of the efficacy and safety in clinical applications.

Bacteriophage Therapy
Bacteriophages (or phages) are viruses that can infect and replicate in bacterial cells. They are widely found in soil and water bodies and play an important role in microbial physiology. Bacteriophages have strict host specificity, reside in susceptible host bacteria, and can lyse bacteria, so they are sometimes used as adjuvant therapy for local infections. However, because of the emergence of bacterial infections for which antibiotics and surgery are not sufficient for eradication, bacteriophage therapy has attracted attention, and it has become a possible choice for the treatment of refractory bacterial infections (Figure 1).
A recent study conducted by Di Luca's group showed that phages can lyse bacterial cells in vitro, even inside biofilms. In particular, the antibiofilm properties of some phages have been observed, such as the ability to kill persistent cells and degrade the extracellular matrix. These properties may be due to the depolymerase activity of the virus-encoded enzyme, which degrades the extracellular polysaccharide of the matrix, allowing phages or antibiotics to reach the bacteria and kill them [253]. Phage lysins have also been effectively used to remove bacterial biofilms. For example, in vitro, the phage lysin LysGH15 can efficiently prevent the biofilm formation of staphylococcal species. LysGH15 also showed notable disruption of 24-h and 72-h biofilms formed by S. aureus and coagulase-negative species [254]. The ability of LysGH15 to inhibit biofilm formation is most likely attributable to its rapid bactericidal activity prior to biofilm formation. At higher doses, LysGH15 can also destroy biofilms because it can dissolve the bacteria in the biofilm, thereby destroying the integrity of the biofilm instead of destroying the biofilm matrix [254]. Similarly, the effectiveness of phages against biofilms was demonstrated in vivo. In the established murine model of prosthetic joint infection, the combination of PlySs2 (a bacteriophage-derived lysin) (2.5 mg/kg, Intraperitoneal, q24hr) with vancomycin (110 mg/kg, Subcutaneous, q12hr) can significantly reduce the bacterial load in periprosthetic tissues and on implant surfaces [255]. In a recent case reported by Mario Corbellino et al., bacteriophages cured urinary tract infections that were recurrent despite previous treatment with effective antibiotics and the replacement of ureteral stents. After two weeks of lytic phage therapy, the presence of the KPC producer K. pneumoniae was no longer found in the urine and ureteral stent of the patient, and no microbial colonization was observed during the following two consecutive years [256]. However, due to host specificity, phage tolerance, and the complexity of the mature biofilm structure, phages have difficulty completely removing biofilms. Moreover, personalized phage cocktail (mixed phages) therapy has been developed. These phages work together to expand the scope of action to increase the potential of phage therapy. A study of phage cocktails composed of five phages against orthopedic implant biofilm infections caused by S. aureus showed that viable bacterial numbers within biofilms on titanium surfaces were significantly reduced in vitro [257]. In another case reported by Qin Jinhong et al., a cocktail of bacteriophages (5 × 10 8 pfu/mL, 50 mL bladder irrigation and kidney irrigation, q48hr) was selected for activity against all 21 heterogeneous K. pneumoniae strains to eradicate multifocal colonization, combined with piperacillin/tazobactam treatment. Finally, the patient recovered, with an obviously improved bladder [258].
Bacteriophages are species specific against bacterial infections. Antibiotics can easily cause dysregulation or resistance in the bacterial population. Bacteria are less likely to develop tolerance to bacteriophages. Therefore, bacteriophage therapy has good prospects alone or in combination with antibiotics in the treatment of biofilm infections. However, more scientific and clinical studies are needed to understand its true efficacy, pharmacokinetics, and mode of administration to establish appropriate clinical treatment regimens, and it is also necessary to determine which pathogen infections can be treated with bacteriophage therapy [259].

Conclusions
Medical device-related biofilm infections remain a serious problem in clinical services. The high resistance of biofilms to current antibiotic treatments appears to be a major challenge in this field. Antibiotic treatment alone often fails to eradicate microbial biofilms. The surgical removal of implants is effective in preventing the recurrence of biofilm infections; however, not all patients need or can undergo surgical revision for the treatment of local infections. In recent years, with an in-depth understanding of the mechanism of biofilm resistance, many novel approaches have been developed to combat device-related biofilm infections, such as coatings, AMPs, and nanotechnology. Some of these strategies exhibit antibiofilm activities against multiple targets and have been applied to the clinical treatment of medical device-associated biofilms. By combining these promising agents with antibiotics, it may be possible to eliminate device-related biofilms in the future. However, to develop safe, effective, practical, and economically viable strategies to combat medical device-related biofilm infections, further careful study is needed in this area to establish appropriate clinical treatment protocols.