Biofilm-associated healthcare-associated infections: mechanisms, clinical burden, and emerging therapeutic frontiers

dgkh000675 10.3205/dgkh000675 urn:nbn:de:0183-dgkh0006751 Review Article Biofilm-associated healthcare-associated infections: mechanisms, clinical burden, and emerging therapeutic frontiers – a narrative review Biofilm-assoziierte nosokomiale Infektionen: Mechanismen, klinische Belastung und therapeutische Herausforderungen – ein narratives Review Anand Anand Gargee G MD

Senior Resident, All India Institute of Medical Sciences, Phulwari Sharif, Patna, Bihar, India, 801507; Phone: +91 969118052All India Institute of Medical Sciences, Department of Microbiology, Patna, Bihar, India

gargeea3@gmail.com author Lahariya Lahariya Rijhul R

All India Institute of Medical Sciences, Patna, Bihar, India

author German Medical Science GMS Publishing House

Düsseldorf

610 antimicrobial stewardship central venous catheter urinary catheters endotracheal tubes prosthetic implants mortality Antibiotic Stewardship zentraler Venenkatheter Harnwegkatheter Endotrachealtuben Gelenkimplantationen Mortalität 20260910 engl This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 License. Dieser Artikel ist ein Open-Access-Artikel und steht unter den Lizenzbedingungen der Creative Commons Attribution 4.0 License (Namensnennung). 2196-5226 21 GMS Hygiene and Infection Control GMS Hyg Infect Control 66 Biofilme sind hochorganisierte mikrobielle Gemeinschaften, die in eine extrazelluläre Polymermatrix eingebettet sind und einen kritischen, aber unterschätzten Auslöser nosokomialer Infektionen (NI) darstellen. Ihre Fähigkeit, Devices und Wirtsgewebe zu besiedeln, ist Ursache für schwere Infektionen, darunter ZVK-assoziierte Blutstrominfektionen, Katheter-assoziierte Harnwegsinfektionen, Beatmungs-assoziierte P</PlainText></TextGroup>neumonien und postoperative Wundinfektionen. </Pgraph><Pgraph>Die Bildung von Biofilmen ist ein dynamischer, mehrstufiger Prozess, der Anlagerung, Reifung und Ausbreitung von Mikroorganismen umfasst, wobei jede Phase die Persistenz und das Therapieversagen verstärkt. Die Matrix behindert die Immunabwehr und die antimikrobielle Penetration, während Quorum Sensing, metabolische Heterogenität und Persistenzzellbildung die Toleranz erhöhen. Klinisch wichtige bakterielle und pilzliche Biofilme, insbesondere solche, die von <Mark2>Staphylococcus aureus</Mark2>, <Mark2>Pseudomonas aeruginosa</Mark2> und Candida-Arten gebildet werden, weisen eine Resistenz auf, die weit über die von Planktonzellen hinausgeht. Diagnostische Einschränkungen verschleiern die tatsächliche Krankheitslast, und aktuelle Strategien, die auf antimikrobiellen Mitteln und der Entfernung von Devices basieren, bleiben angesichts zunehmender Resistenzen unzureichend. </Pgraph><Pgraph>Das Manuskript bietet eine Zusammenführung bestehender und neuer Strategien zur Reduzierung von NI mit besonderem Schwerpunkt auf der translationalen Relevanz.</Pgraph></Abstract> <Abstract language="en" linked="yes"><Pgraph>Biofilms are highly organized microbial communities embedded within an extracellular polymeric matrix and represent a critical yet underappreciated driver of healthcare-associated infections (HAIs). Their ability to colonize medical devices and host tissues underlies major infections, including central line-associated bloodstream infections, catheter-associated urinary tract infections, ventilator-associated pneumonia, and surgical site infections. </Pgraph><Pgraph>Biofilm formation is dynamic, multistage process involving attachment, maturation, and dispersal of microorganisms, each phase reinforcing persistence and therapeutic failure. The matrix impedes immune clearance and antimicrobial penetration, while quorum sensing, metabolic heterogeneity, and persister cell formation further enhance tolerance. Clinically important bacterial and fungal biofilms, particularly those formed by <Mark2>Staphylococcus aureus</Mark2>, <Mark2>Pseudomonas aeruginosa</Mark2>, and Candida species, exhibit resistance far exceeding planktonic cells. Diagnostic limitations obscure true disease burden, and curren<TextGroup><PlainText>t s</PlainText></TextGroup>trategies relying on antimicrobials and device removal remain inadequate amid rising resistance. </Pgraph><Pgraph>This manuscript provides comprehensive synthesis of existing and novel strategies to mitigate HAIs, with particular emphasis on translational relevance.</Pgraph></Abstract> <TextBlock name="Introduction" linked="yes"> <MainHeadline>Introduction</MainHeadline><Pgraph>Healthcare-associated infections (HAIs) remain among the most frequent and devastating complications in modern medicine, representing a major global public health challenge <TextLink reference="1"></TextLink>. Microbial biofilms underlie the majority of device-associated infections, are implicated in up to 70% of all microbial diseases and represent a major driver of HAIs <TextLink reference="2"></TextLink>. Once established on indwelling medical devices such as central venous catheters (CVCs), urinary catheters, endotracheal tubes (ET), and prosthetic implants, biofilms form a highly resistant microbial reservoir that is nearly impossible to eradicate with standard antibiotic regimens <TextLink reference="3"></TextLink>, <TextLink reference="4"></TextLink>, <TextLink reference="5"></TextLink>, <TextLink reference="6"></TextLink>, <TextLink reference="7"></TextLink>. This persistent mode of infection not only complicates clinical management but also accelerates the development and dissemination of antimicrobial resistance (AMR), with an estimated annual economic burden exceeding USD 5 trillion <TextLink reference="8"></TextLink>, <TextLink reference="9"></TextLink>. </Pgraph><Pgraph>With the increasing use of invasive medical devices in contemporary healthcare, the incidence of biofilm-associated infections has escalated globally <TextLink reference="10"></TextLink>. Virtually all implanted or indwelling devices are susceptible to microbial colonization, turning life-saving interventions into potential sources of chronic infection <TextLink reference="4"></TextLink>. Biofilm-associated infections now account for the majority of persistent bacterial infections in humans and are implicated in nearly half of all HAIs linked to medical devices <TextLink reference="11"></TextLink>.</Pgraph><Pgraph>Understanding biofilms is essential to tackling HAIs, as nearly 80% of chronic infections are biofilm-associated <TextLink reference="12"></TextLink>. Their inherent resistance to antibiotics fuels persistent infections and the rise of multidrug-resistant pathogens, projected to cause 10 million deaths annually by 2050 <TextLink reference="13"></TextLink>. However, current infection control and treatment strategies, largely designed for planktonic bacteria, fail to adequately address biofilm-associated infections, particularly on medical devices and sterile sites <TextLink reference="14"></TextLink>, <TextLink reference="15"></TextLink>, <TextLink reference="16"></TextLink>. The interplay between HAIs and biofilms represents a silent yet escalating threat to patient safety and healthcare systems worldwide. This review seeks to close the critical gap between microbiological insight and clinical practice by translating the complex biology of biofilms into actionable strategies for the prevention, diagnosis, and treatment of HAIs. Addressing this challenge will require not only improved infection prevention protocols and device stewardship but also urgent innovation in anti-biofilm strategies and antimicrobial development.</Pgraph></TextBlock> <TextBlock name="Method" linked="yes"> <MainHeadline>Method</MainHeadline><Pgraph>A narrative review was conducted to evaluate the role of biofilms in HAIs, AMR, and emerging anti-biofilm strategies. Relevant articles were identified through search of PubMed using keywords such as “biofilm”, “healthcare-associated infections”, “device-associated infections”, “antimicrobial resistance”, “quorum sensing”, and “anti-biofilm therapy”. Original research articles, reviews, clinical studies, and guideline documents published in English were included. The retrieved literature was screened for relevance, and eligible studies were critically reviewed and synthesized to provide an overview of biofilm biology, clinical significance, diagnostic challenges, and current and emerging management approaches.</Pgraph></TextBlock> <TextBlock name="Results" linked="yes"> <MainHeadline>Results</MainHeadline><SubHeadline>The biofilm life cycle: a dynamic survival strategy of pathogens</SubHeadline><Pgraph>Biofilms represent a unifying pathogenic strategy across bacterial and fungal pathogens implicated in HAIs <TextLink reference="10"></TextLink>. They enable microbial persistence on indwelling medical devices, hospital surfaces, and host tissues, thereby driving chronicity, therapeutic failure, and recurrence <TextLink reference="17"></TextLink>. Unlike planktonic organisms, biofilm-embedded pathogens display recalcitrance, a coordinated, multicellular phenotype of tolerance to antibiotics even at high concentrations and immune evasion that is central to the epidemiology of ventilator-associated pneumonia (VAP), catheter-associated urinary tract infection (CAUTI), central line-associated bloodstream infection (CLABSI) and periprosthetic joint infections (PJI) <TextLink reference="18"></TextLink>.</Pgraph><SubHeadline2>Formation and maturation of bacterial biofilm</SubHeadline2><Pgraph>Bacterial biofilm development (Figure 1 <ImgLink imgNo="1" imgType="figure" />) follows a stepwise progression:</Pgraph><Pgraph><UnorderedList><ListItem level="1">Initial attachment (reversible docking): Microbes transiently adhere to host or device surfaces through weak physicochemical interactions – hydrophobic forces, van der Waals attractions, and electrostatic charges, guided by environmental cues and bacterial motility <TextLink reference="19"></TextLink>.</ListItem><ListItem level="1">Stable adhesion (irreversible locking): Bacterial surface structures such as pili, fimbriae, and adhesins anchor the cells firmly. This molecular “handshake” triggers the secretion of extracellular polymeric substances (EPS), marking the transition from a free-living to a sessile lifestyle <TextLink reference="19"></TextLink>.</ListItem><ListItem level="1">Biofilm maturation (structural and functional expansion): Rapid bacterial replication, EPS accumulation, and spatial organization form complex three-dimensional communities with nutrient channels and protective architecture. These mature biofilms resist immune clearance and antimicrobial penetration <TextLink reference="19"></TextLink>.</ListItem><ListItem level="1">Dispersal (seeding of new niches): Environmental and quorum-sensing (QS) signals activate biofilm dispersal, releasing planktonic cells to colonize new surfaces. Dispersal may occur via erosion (gradual shedding), sloughing (bulk release), or active seeding (from within) <TextLink reference="19"></TextLink>.</ListItem></UnorderedList></Pgraph><SubHeadline2>Quorum sensing – the molecular orchestration of bacterial biofilms </SubHeadline2><Pgraph>Biofilm formation is not a random bacterial aggregation but a highly regulated, multicellular behaviour, coordinated through QS, a chemical communication system that enables bacteria to sense and respond to population density <TextLink reference="20"></TextLink>. As bacterial numbers rise, diffusible signaling molecules, such as N-acyl homoserine lactones in Gram-negative species, autoinducing peptides in Gram-positives, and the interspecies autoinducer-2, accumulate in the extracellular environment <TextLink reference="20"></TextLink>. Once a critica<TextGroup><PlainText>l t</PlainText></TextGroup>hreshold is reached, these molecules trigger synchronized gene expression across the community. QS regulates both biofilm maturation and dispersal, facilitating colonization of new niches <TextLink reference="20"></TextLink>. In clinical settings, this network contributes to persistent device-associated infections (DAI) and therapeutic failure. However, most mechanistic insights into QS are derived from controlled in vitro systems, and the complexity of polymicrobial interactions along with host immune factors in vivo may significantly alter QS dynamics, thereby limiting the translational applicability of these findings to real-world infection settings <TextLink reference="21"></TextLink>.</Pgraph><SubHeadline2>The extracellular matrix (ECM) – architect and shield of the bacterial biofilm</SubHeadline2><Pgraph>EPS is the biochemical framework that transforms bacterial aggregates into resilient biofilms. Composed of polysaccharides, proteins, lipids, and extracellular DNA, it anchors cells, maintains structural integrity, and protects against environmental stress <TextLink reference="22"></TextLink>. EPS facilitates nutrient retention, immune evasion, and antibiotic tolerance by limiting drug penetration and host defenses <TextLink reference="22"></TextLink>. It also promotes QS and horizontal gene transfer, enhancing antimicrobial resistance. Thus, EPS functions as both the structural scaffold and protective barrier underlying biofilm persistence <TextLink reference="22"></TextLink>. Nevertheless, the relative contribution of EPS-mediated resistance versus metabolic dormancy in clinical infections remains debated, as antibiotics can penetrate biofilms yet fail due to physiological heterogeneity <TextLink reference="23"></TextLink>, <TextLink reference="24"></TextLink>.</Pgraph><SubHeadline2>Candida biofilms: a parallel fungal survival strategy</SubHeadline2><Pgraph>Fungal pathogens, particularly Candida species, mirror bacterial biofilm strategies but with following unique features (Figure 2 <ImgLink imgNo="2" imgType="figure" />) relevant to HAIs:</Pgraph><Pgraph><UnorderedList><ListItem level="1">Initial adhesion: Yeast cells rapidly adhere to biotic or abiotic surfaces, including mucosal epithelia and medical implants. This initial contact is mediated by adhesins, cell wall mannoproteins, and electrostatic interactions <TextLink reference="25"></TextLink>.</ListItem><ListItem level="1">Early biofilm development: Adherent yeast cells proliferate to form a basal monolayer. Hyphal transformation begins, a critical virulence trait, accompanied by the secretion of an ECM, rich in β-glucans, proteins, and extracellular DNA <TextLink reference="25"></TextLink>.</ListItem><ListItem level="1">Maturation: The biofilm architecture becomes increasingly complex, with dense networks of yeast, hyphae, and pseudo hyphae encased in ECM. Water channels form within the matrix, facilitating nutrient flow and metabolic cooperation. Antifungal resistance escalates sharply at this stage <TextLink reference="25"></TextLink>.</ListItem><ListItem level="1">Dispersal: Mature biofilms release yeast cells or hyphal fragments into the bloodstream or surrounding tissues. These disseminated cells exhibit an enhanced capacity to colonize new niches, contributing to recurrent and invasive candidiasis <TextLink reference="25"></TextLink>.</ListItem></UnorderedList></Pgraph><SubHeadline2>Quorum sensing in Candida – a driver of biofilm resilience</SubHeadline2><Pgraph>In <Mark2>Candida (C.) albicans</Mark2>, QS orchestrates biofilm development and drug resistance through population-dependent signaling <TextLink reference="26"></TextLink>. The QS molecule farnesol inhibits hyphal formation by repressing hypha-associated genes (TUP1, CRK1) and upregulating efflux transporters (CDR1, PDR16), tipping the balance toward a biofilm-embedded phenotype <TextLink reference="26"></TextLink>. Histidine kinase CHK1 mediates farnesol responsiveness, with CHK1 mutants forming biofilms despite QS inhibition <TextLink reference="26"></TextLink>. Farnesol also contributes to biofilm dispersal under nutrient stress, enabling colonization of new sites. QS may regulate surface adhesion via anti-adhesins like YWP1, dampening biofilm adherence in response to population cues <TextLink reference="26"></TextLink>.</Pgraph><SubHeadline2>Extracellular matrix – the structural and protective backbone of Candida biofilms</SubHeadline2><Pgraph>In <Mark2>C. albicans</Mark2>, the ECM forms a dense, polymeric shield critical to biofilm architecture and defense. Composed of proteins, chitin, extracellular DNA, and β-1,3-glucans, this matrix stabilizes the biofilm’s complex 3D structure, spanning 50 to 350 µm, and anchors cells against shear forces and antifungal insults <TextLink reference="26"></TextLink>. Beyond physical integrity, the ECM serves as a chemical fortress, impeding immune cell access and sequestering antifungal drugs, thus enabling persistent infection <TextLink reference="26"></TextLink>.</Pgraph><SubHeadline>Molecular basis of biofilm-mediated resistance</SubHeadline><Pgraph>Three interdependent mechanisms form the backbone of biofilm resilience <TextLink reference="27"></TextLink>: </Pgraph><Pgraph><UnorderedList><ListItem level="1">Physical barrier – the biofilm matrix impairs antibiotic penetration.</ListItem><ListItem level="1">Microenvironmental protection – nutrient gradients, acidic pH, hypoxia, and waste accumulation suppress antibiotic efficacy.</ListItem><ListItem level="1">Persistence – dormant subpopulations (“persister cells”) evade killing due to metabolic inactivity.</ListItem></UnorderedList></Pgraph><Pgraph>This resistance is further amplified by biochemical defences (extracellular polysaccharides, eDNA, antibiotic-degrading enzymes, efflux pumps), molecular adaptations (horizontal gene transfer, mutational plasticity), and host–pathogen interactions (sub-inhibitory drug exposure, oxidative stress, QS) <TextLink reference="27"></TextLink>. Collectively, these layered defences drive chronic infection and therapeutic failure in HAI.</Pgraph><SubHeadline2>Why conventional antibiotics fail against biofilms</SubHeadline2><Pgraph>Despite being genetically identical, bacteria within biofilms exhibit markedly reduced susceptibility to antibiotics compared to planktonic cells. This is not primarily due to classical resistance mechanisms but results from the structural, physiological, and metabolic heterogeneity of the biofilm state <TextLink reference="19"></TextLink>.</Pgraph><Pgraph>The EPS matrix acts as a diffusion barrier, limiting antibiotic penetration, particularly for aminoglycosides. However, incomplete efficacy cannot be explained by penetration alone <TextLink reference="19"></TextLink>. Microenvironmental gradients of oxygen, nutrients, and pH create metabolically inactive zones, where bacteria become intrinsically tolerant to antibiotics targeting active cellular processes. In addition, biofilms contain persister cells – dormant, non-replicative variants that survive antibiotic exposure without genetic resistance and regain susceptibility upon regrowth <TextLink reference="19"></TextLink>.</Pgraph><Pgraph>However, most of these mechanisms have been predominantly demonstrated in <Mark2>in vitro</Mark2> models, and their relative contribution in complex <Mark2>in vivo </Mark2>biofilm-associated infections remains incompletely understood <TextLink reference="28"></TextLink>, <TextLink reference="29"></TextLink>. In clinical settings, this multifactorial tolerance contributes to persistent DAI, often necessitating device removal and prolonged combination antimicrobial therapy, thereby increasing healthcare costs and patient morbidity <TextLink reference="30"></TextLink>, <TextLink reference="31"></TextLink>.</Pgraph><SubHeadline2>Why conventional antifungals fail against biofilms</SubHeadline2><Pgraph>Fungal biofilms, particularly those of Candida species, show markedly increased resistance to antifungals compared to planktonic cells <TextLink reference="26"></TextLink>. This is due to a protective extracellular matrix, metabolic dormancy, and the presence of persister cells. QS and high cell density further alter antifungal susceptibility <TextLink reference="26"></TextLink>. These adaptive, non-genetic mechanisms render conventional therapies largely ineffective. In clinical settings, Candida biofilms frequently exist as part of polymicrobial communities, particularly with <Mark2>Staphylococcus (S.) aureus</Mark2>, where cross-kingdom interactions enhance biofilm robustness, antifungal tolerance, and virulence <TextLink reference="32"></TextLink>, <TextLink reference="33"></TextLink>.</Pgraph><SubHeadline2>Mechanisms of tolerance and “persister” cells</SubHeadline2><Pgraph>Biofilm-associated antibiotic tolerance arises predominantly from non-genetic, physiological adaptations rather than conventional resistance mechanisms. The biofilm’s ECM limits antimicrobial penetration, while nutrient gradients and microenvironmental stressors create zones of low metabolic activity that reduce antibiotic efficacy <TextLink reference="22"></TextLink>, <TextLink reference="34"></TextLink>, <TextLink reference="35"></TextLink>. Additionally, changes in cellular physiology, such as slowed growth, altered redox states, and stress response activation, further impair antibiotic action. These features collectively confer a reversible, phenotypic tolerance that resolves upon biofilm dispersion. In addition to these innate defenses, a critical subpopulation of cells, termed persisters, enter a dormant, non-replicative state that renders them transiently tolerant to antibiotics <TextLink reference="22"></TextLink>, <TextLink reference="34"></TextLink>, <TextLink reference="35"></TextLink>. Unlike genetically resistant strains, persister cells do not harbor resistance mutations and regain susceptibility upon regrowth. However, their survival under antimicrobial pressure enables recurrence and may facilitate the eventual emergence of resistant mutants <TextLink reference="22"></TextLink>, <TextLink reference="34"></TextLink>, <TextLink reference="35"></TextLink>. Together, these mechanisms make biofilm infections uniquely refractory to treatment.</Pgraph><SubHeadline>Clinically relevant biofilm-forming pathogens</SubHeadline><Pgraph>Most commonly associated biofilm-forming microorganisms linked with device-associated infections are <Mark2>S. aureus, S. epidermidis, S. capitis, S. lugdunensis, Enterococcus (E.) faecalis, E. coli, Klebsiella (K.) pneumoniae, Enterobacter</Mark2> spp., <Mark2>Proteus (P.) mirabilis, Pseudomonas (P.) aeruginosa, Candida (C.) auris, C. albicans </Mark2>and <Mark2>C. rugosa</Mark2> <TextLink reference="19"></TextLink>, <TextLink reference="36"></TextLink>, <TextLink reference="37"></TextLink>, <TextLink reference="38"></TextLink>, <TextLink reference="39"></TextLink>, <TextLink reference="40"></TextLink>.</Pgraph><SubHeadline>Where biofilms thrive – clinical hotspots and devices</SubHeadline><Pgraph>An estimated 65% of microbial infections and up to 80% of chronic infections are linked to biofilm formation. These highly structured microbial communities colonize both host tissues and indwelling medical devices, including prosthetic implants, CVCs, urinary catheters, and ET – as well as hospital environmental reservoirs such as sinks, tubing, and surfaces <TextLink reference="41"></TextLink>. In clinical environments, biofilms act as persistent reservoirs for nosocomial pathogens, driving AMR and fuelling recurrent infections despite targeted therapy <TextLink reference="42"></TextLink>.</Pgraph><SubHeadline2>Common device-associated niches</SubHeadline2><Pgraph>Medical devices serve as prime substrates for biofilm development, transforming life-saving interventions into persistent infection reservoirs. Biofilm-associated infections are central to the burden of CLABSI, CAUTI, VAP and SSI, particularly those linked to prosthetic implants.</Pgraph><Pgraph>CVCs are major sites of biofilm formation and account for a significant proportion of CLABSI <TextLink reference="43"></TextLink>. Biofilms form rapidly on catheter surfaces, with short-term use (<10 days) associated with extraluminal colonization and long-term use (>30 days) favouring intraluminal biofilm development <TextLink reference="44"></TextLink>. Common pathogens include <Mark2>S. aureus, S. epidermidis, E. faecalis, K. pneumoniae, P. aeruginosa</Mark2>, and <Mark2>C. albicans</Mark2> <TextLink reference="38"></TextLink>. These biofilms evade host immunity and antimicrobial therapy, often leading to septicemia and complications such as endocarditis, making prevention through aseptic techniques and antimicrobial-impregnated catheters essential <TextLink reference="45"></TextLink>.</Pgraph><Pgraph>The ET play a central role in VAP by impairing mucociliary clearance, suppressing cough reflexes, and facilitating entry of oropharyngeal flora into the lower airways <TextLink reference="46"></TextLink>, <TextLink reference="47"></TextLink>. Biofilms forming on the internal lumen act as persistent reservoirs that protect pathogens from antibiotics and host defenses, contributing to relapse and prolonged infection <TextLink reference="48"></TextLink>, <TextLink reference="49"></TextLink>. VAP is predominantly caused by highly virulent and multidrug-resistant ESKAPE pathogens: <Mark2>E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa</Mark2>, and <Mark2>Enterobacter</Mark2> spp. – with <Mark2>E. coli</Mark2> increasingly implicated <TextLink reference="50"></TextLink>. Despite advances in infection control practices, complete eradication of device-associated biofilms remains challenging, and clinical management often relies on device removal rather than pharmacological eradication alone <TextLink reference="51"></TextLink>.</Pgraph><Pgraph>Urinary catheters are a key risk factor for CAUTI, with most long-term catheterized patients developing bacteriuria <TextLink reference="52"></TextLink>. Biofilms rapidly form on catheter surfaces, facilitating ascending infection by pathogens such as <Mark2>E. coli, P.s mirabilis, P. aeruginosa, E. faecalis</Mark2>, and <Mark2>S. epidermidis</Mark2>, often in polymicrobial communities <TextLink reference="53"></TextLink>. These biofilms can obstruct urinary flow, induce inflammation, and predispose to urosepsis, highlighting the importance of minimizing catheter duration, maintaining closed systems, and using antimicrobial-coated devices <TextLink reference="52"></TextLink>.</Pgraph><Pgraph>Orthopaedic implants are highly prone to biofilm-associated SSIs, which may present as acute, chronic, or hematogenous infections <TextLink reference="54"></TextLink>. Biofilm-embedded pathogens, including<Mark2> S. aureus</Mark2> (including MRSA), <Mark2>S. epidermidis, E. faecalis, P. aeruginosa</Mark2>, and <Mark2>K. pneumoniae</Mark2>, form polymicrobial consortia that resist host defences and antibiotic therapy <TextLink reference="54"></TextLink>, <TextLink reference="55"></TextLink>. Persistent infection leads to chronic inflammation, osteolysis, implant loosening, and prosthetic failure, necessitating preventive strategies including perioperative prophylaxis, strict asepsis, and development of anti-biofilm implant surfaces <TextLink reference="41"></TextLink>, <TextLink reference="56"></TextLink>, <TextLink reference="57"></TextLink>.</Pgraph><SubHeadline>Device surface determinants of biofilm formation</SubHeadline><Pgraph>Surface roughness, material composition, and interfacial physicochemical properties are not passive features but key drivers of microbial adhesion and biofilm maturation. Increased surface roughness promotes microbial retention by expanding surface area and creating protective niches that reduce shear-mediated detachment, leading to greater early biofilm accumulation on rougher implant surfaces <TextLink reference="58"></TextLink>. Beyond topography, material composition and surface chemistry, such as surface free energy, hydrophobicity, and charge, govern the strength and specificity of bacterial attachment, with hydrophobic interactions often favoring pathogen adherence <TextLink reference="59"></TextLink>. Additionally, differences in biomaterials (e.g., titanium, zirconia, polymers) and their surface modifications significantly influence biofilm burden, highlighting the absence of a universally biofilm-resistant material <TextLink reference="60"></TextLink>. Following implantation, rapid conditioning by host proteins like fibrinogen and fibronectin further enhances microbial colonization <TextLink reference="59"></TextLink>. Collectively, these factors highlight how device-related properties shape microbial colonization, directly defining where biofilms thrive.</Pgraph><SubHeadline>Biofilms as hidden drivers of healthcare-associated infections</SubHeadline><SubHeadline2>Diagnostic blind spots – why biofilms escape detection</SubHeadline2><Pgraph>Despite their critical role in DAI, biofilms remain diagnostically elusive due to fundamental limitations in current detection methods as shown in Table 1 <ImgLink imgNo="1" imgType="table" /> <TextLink reference="61"></TextLink>. Standard cultures may yield false negatives due to low metabolic activity and poor recovery of biofilm-embedded organisms; moreover, even when positive, they detect only planktonic cells and fail to identify the sessile biofilm phenotype, including EPS matrix and altered physiology <TextLink reference="61"></TextLink>, <TextLink reference="62"></TextLink>, <TextLink reference="63"></TextLink>. Routine imaging similarly lacks sensitivity for detecting biofilms on indwelling devices such as catheters, prostheses, and ETs. These limitations contribute to underrecognition of biofilm-mediated infections and may explain the discordance between in vitro susceptibility results and clinical outcomes <TextLink reference="61"></TextLink>, <TextLink reference="62"></TextLink>, <TextLink reference="63"></TextLink>. Although standardized in vitro models (e.g., American Society for Testing and Materials) exist, they fail to replicate the dynamic and heterogeneous nature of in vivo biofilms, and laboratory strains may lose biofilm-forming capacity over time <TextLink reference="64"></TextLink>. Emerging approaches, including experimental biomarkers and artificial intelligence, show promise for early non-invasive detection; however, the lack of validated biofilm-specific biomarkers remains a major limitation <TextLink reference="65"></TextLink>, <TextLink reference="66"></TextLink>, <TextLink reference="67"></TextLink>, <TextLink reference="68"></TextLink>, <TextLink reference="69"></TextLink>, <TextLink reference="70"></TextLink>, <TextLink reference="71"></TextLink>, <TextLink reference="72"></TextLink>. While advanced molecular and imaging techniques – such as 16S rRNA sequencing, next-generation sequencing, sonication, Confocal Microscopy, cryo-SEM, and atomic force microscopy – provide improved insights, their limited scalability restricts routine clinical use, leaving a persistent gap in the early and accurate diagnosis of biofilm-associated infections <TextLink reference="73"></TextLink>, <TextLink reference="74"></TextLink>, <TextLink reference="75"></TextLink>, <TextLink reference="76"></TextLink>, <TextLink reference="77"></TextLink>, <TextLink reference="78"></TextLink>, <TextLink reference="79"></TextLink>.</Pgraph><SubHeadline2>Epidemiology and clinical burden of biofilm-associated HAIs</SubHeadline2><Pgraph>Biofilm-associated infections account for up to 80% of all bacterial infections globally, with 60–70% of nosocomial infections directly linked to biofilm formation <TextLink reference="80"></TextLink>, <TextLink reference="81"></TextLink>, <TextLink reference="82"></TextLink>. WHO and Centers for Disease Control and Prevention surveillance highlight increasing prevalence in ICU settings, where biofilm-forming pathogens like MRSA and <Mark2>E. coli</Mark2> exhibit MDR rates ranging from 17.9% to 100% <TextLink reference="83"></TextLink>. The burden is amplified in patients with diabetes, chronic kidney disease, and multiple comorbidities, where biofilm-driven infections prolong ICU stay, escalate treatment costs, and increase mortality <TextLink reference="2"></TextLink>, <TextLink reference="83"></TextLink>.</Pgraph><SubHeadline2>Therapeutic roadblocks: why conventional therapy fails</SubHeadline2><Pgraph>Empirical monotherapy, a mainstay of early HAI management, fails against biofilm-embedded pathogens due to their fortified multicellular architecture <TextLink reference="84"></TextLink>. Encased in a dense extracellular matrix, these bacteria exhibit up to 1,000-fold increased antibiotic tolerance via impaired drug penetration, metabolic dormancy, and phenotypic persistence <TextLink reference="85"></TextLink>. Furthermore, biofilms act as genetic reservoirs for horizontal gene transfer, accelerating the spread of antimicrobial resistance across hospital ecosystems <TextLink reference="86"></TextLink>. These adaptive mechanisms collectively render monotherapy ineffective – driving chronicity, relapse, and therapeutic failure, thus mandating a shift toward precision diagnostics and multi-targeted interventions <TextLink reference="87"></TextLink>.</Pgraph><SubHeadline2>Sub-inhibitory antibiotic exposure – a hidden catalyst of biofilm growth</SubHeadline2><Pgraph>Paradoxically, antibiotics may aggravate biofilm-associated infections <TextLink reference="88"></TextLink>. Sub-inhibitory concentrations (sub-MICs), commonly encountered in poorly perfused tissues or between dosing intervals, not only facilitates biofilm maturation but can also modulate virulence, surface properties, and gene expression <TextLink reference="89"></TextLink>. Notably, cell wall-active agents such as ampicillin and vancomycin induce envelope stress and enhance extracellular DNA (eDNA) release, reinforcing biofilm architecture <TextLink reference="90"></TextLink>. Moreover, sub-MIC exposure can drive genetic adaptation through increased mutation, recombination, and horizontal gene transfer, thereby facilitating persistence and antimicrobial resistance <TextLink reference="91"></TextLink>.</Pgraph></TextBlock> <TextBlock name="Discussion" linked="yes"> <MainHeadline>Discussion</MainHeadline><SubHeadline>Anti-biofilm preventive strategies in clinical use</SubHeadline><SubHeadline2>Antibacterial and antifouling coatings</SubHeadline2><Pgraph>Surface coatings incorporating antibiotics, antiseptics, hydrophilic polymers, silver nanoparticles, and antimicrobial peptides inhibit initial microbial adhesion and early biofilm formation <TextLink reference="92"></TextLink>. Polihexanide covalently bound to a titanium alloy is bactericidal against both Gram-positive and Gram-negative bacteria. Modelling suggests that concentrations effective for antiseptic purposes are maintained near the surface for periods exceeding 6 months. Crucially, the polyhexanide film has no adverse effects on MG63 cells within a 48-hour cell culture. Across all measurement time points, viability and proliferation in the uncoated control group typically ranged from ≥90–95% with unchanged collagen synthesis. Within 15 minutes, the initial adhesion and spreading of osteoblasts on the test specimens were promoted <TextLink reference="93"></TextLink>, <TextLink reference="94"></TextLink>, <TextLink reference="95"></TextLink>, <TextLink reference="96"></TextLink>, <TextLink reference="97"></TextLink>. Nanomaterials, with high surface-to-volume ratio and tunable physicochemical properties, disrupt biofilm structure, enhance antibiotic penetration, and reduce resistance development <TextLink reference="98"></TextLink>, <TextLink reference="99"></TextLink>. These coatings act via release-based or contact-killing mechanisms while also preventing protein and cellular deposition critical for biofilm initiation <TextLink reference="100"></TextLink>, <TextLink reference="101"></TextLink>, <TextLink reference="102"></TextLink>. Nanocoatings (e.g., nanosilver, titanium, copper, zinc oxide) show promise but face challenges in biocompatibility and long-term safety <TextLink reference="103"></TextLink>, <TextLink reference="104"></TextLink>. Advances in lipid-coated nanoparticles, polymer-functionalized surfaces, and hydrogel or biodegradable alloy coatings have improved drug delivery and surface compatibility <TextLink reference="38"></TextLink>, <TextLink reference="105"></TextLink>, <TextLink reference="106"></TextLink>, <TextLink reference="107"></TextLink>. Recent advances in cell membrane-coated nanocarriers illustrate the versatility of lipid nanotechnology in enhancing implant safety and performance <TextLink reference="108"></TextLink>. Emerging systems including cell membrane-coated nanocarriers, enzyme-functionalized nanoparticles, and biofilm-responsive platforms, enable targeted EPS disruption and improved efficacy against polymicrobial biofilms, although approaches like nanoscale bacterial debridement remain experimental <TextLink reference="109"></TextLink>, <TextLink reference="110"></TextLink>, <TextLink reference="111"></TextLink>. Emerging approaches like “nanoscale bacterial debridement”, selectively detaching bacteria from biofilms, hold future potential but require further investigation <TextLink reference="112"></TextLink>. </Pgraph><SubHeadline2>Surface engineering to resist adhesion</SubHeadline2><Pgraph>Antiadhesive surfaces reduce bacterial-surface interactions, allowing microorganisms to be easily removed before biofilm maturation <TextLink reference="113"></TextLink>. Techniques include anchoring polymer brushes to device surfaces using barnacle cement or polydopamine <TextLink reference="114"></TextLink>. Such modifications on stainless steel have shown reduced protein adsorption and robust stability <TextLink reference="115"></TextLink>. Tannic acid–based bifunctional coatings further enhance biofouling resistance <TextLink reference="116"></TextLink>.</Pgraph><SubHeadline2>Antimicrobial hydrogel and alloy coatings</SubHeadline2><Pgraph>Hydrogel coatings and antibacterial surface modifications have demonstrated efficacy in preventing orthopedic implant-associated infections <TextLink reference="117"></TextLink>, <TextLink reference="118"></TextLink>. In parallel, biodegradable metal alloys with intrinsic antimicrobial properties are emerging as viable alternatives to conventional implants <TextLink reference="119"></TextLink>. These innovations reduce microbial colonization and offer promise for clinical translation pending further validation.</Pgraph><SubHeadline2>Surface modification of biomaterials</SubHeadline2><Pgraph>Modifying implant surface properties (e.g., energy, roughness, hydrophilicity) without coatings has shown to reduce bacterial adhesion <TextLink reference="120"></TextLink>. By altering surface characteristics, these strategies prevent bacterial settlement and biofilm initiation <TextLink reference="121"></TextLink>. Techniques like pulsed laser evaporation help engineer inherently anti-adhesive surfaces.</Pgraph><SubHeadline2>Natural products in biofilm control: emerging strategies beyond antibiotics</SubHeadline2><Pgraph>Natural products inhibit biofilms by targeting adhesion, QS, and maturation <TextLink reference="7"></TextLink>. Plant-derived compound<TextGroup><PlainText>s (f</PlainText></TextGroup>lavonoids, terpenoids, and phenolics) disrupt biofilms by inhibiting surface attachment and QS without direc<TextGroup><PlainText>t b</PlainText></TextGroup>actericidal action <TextLink reference="122"></TextLink>.</Pgraph><Pgraph>Phytochemicals like emodin, curcumin, and citrus flavonoids reduce virulence and biofilm integrity <TextLink reference="123"></TextLink>. Natural agents like honey and cranberry proanthocyanidins further reduce mature biofilm biomass <TextLink reference="124"></TextLink>, <TextLink reference="125"></TextLink>. Probiotic strains, especially <Mark2>Lactobacillus</Mark2> spp., further inhibit adhesion and destabilize biofilms via bacteriocins and biosurfactants <TextLink reference="19"></TextLink>.</Pgraph><SubHeadline2>Next-generation antimicrobials (NGAs) – disrupting medical device biofilms</SubHeadline2><Pgraph>NGAs combat medical device biofilms through multi-targeted mechanisms that overcome AMR <TextLink reference="126"></TextLink>. Enzymatic agents such as DNase I and proteases (e.g., Proteinase K, dispersin B) degrade EPS components, promoting biofilm dispersal and enhancing antibiotic penetration <TextLink reference="127"></TextLink>, <TextLink reference="128"></TextLink>, <TextLink reference="129"></TextLink>, <TextLink reference="130"></TextLink>. Anti-adhesion strategies (pilicides, glycomimetic) prevent initial bacterial attachment while while QS inhibitors disrupt biofilm maturation. Additionally, cyclic di-GMP modulators and nitric oxide donors induce biofilm dispersal via intracellular signaling pathways <TextLink reference="126"></TextLink>, <TextLink reference="131"></TextLink>, <TextLink reference="132"></TextLink>, <TextLink reference="133"></TextLink>.</Pgraph><SubHeadline2>Quorum sensing inhibition and biofilm disruption</SubHeadline2><Pgraph>Targeting QS pathways with small molecules, enzymes (e.g., DNase I), or natural extracts (e.g., rosmarinic acid, ginger) can prevent biofilm formation <TextLink reference="134"></TextLink>, <TextLink reference="135"></TextLink>. These methods interfere with bacterial communication and matrix integrity, thereby impairing biofilm stability and maturation <TextLink reference="121"></TextLink>. NSAIDs (e.g., meloxicam, aspirin) and antibiotics (e.g., azithromycin, ciprofloxacin) exhibit QS inhibitors activity <TextLink reference="136"></TextLink>. Synergistic combinations, such as resveratrol with aminoglycosides, enhance biofilm disruption and prevent QS inhibitors resistance <TextLink reference="137"></TextLink>. Adjuncts like D-amino acids and proteases enhance antibiotic efficacy against resistant biofilms <TextLink reference="138"></TextLink>, <TextLink reference="139"></TextLink>. </Pgraph><SubHeadline2>Plant-based strategies for control of biofilm associated infections</SubHeadline2><Pgraph>Phytochemicals (phenolics, flavonoids, terpenoids, alkaloids, essential oils) act via QS inhibition, membrane disruption, EPS degradation, and nutrient sequestration. They are eco-friendly, often synergistic, and less pron<TextGroup><PlainText>e t</PlainText></TextGroup>o resistance development <TextLink reference="140"></TextLink>.</Pgraph><Pgraph>Examples include <Mark2>Hypericum lydium</Mark2> extracts (anti-MRSA activity), <Mark2>Cochlospermum regium</Mark2> (phenol-rich) against MRSA biofilms, <Mark2>Persea americana</Mark2> seed extracts (enhanced wound healing and antibiofilm effect), isoflavonoid- and xanthone-rich <Mark2>Iris pseudacorus, Curcuma aromatica</Mark2> flavonoids/alkaloids for Gram-positive biofilms and <Mark2>Frangula angus</Mark2> and <Mark2>Hymenocallis littoralis</Mark2> with broad-spectrum antibiofilm activity.</Pgraph><SubHeadline2>Nanotechnology-based strategies for control of biofilm associated infections</SubHeadline2><Pgraph>Nanoparticles (NPs) offer high surface reactivity, biofilm penetration, and multi-mechanistic antimicrobial action (membrane disruption, ROS generation, DNA damage) <TextLink reference="140"></TextLink>. Examples are:</Pgraph><Pgraph><UnorderedList><ListItem level="1">Silver NPs (AgNPs) are effective suture/device coatings with broad-spectrum action; TiO<Subscript>2</Subscript>–Ag hybrids zwitterionic AgNP dressings works by ROS generation (TiO<Subscript>2</Subscript>) with AgNP bactericidal action <TextLink reference="140"></TextLink>.</ListItem><ListItem level="1">Composite and functionalized NPs such as amphora-shaped porous Ti implants deters bacterial attachment, CaP–Ag coatings prevent adhesion on bone implants and release bactericidal Ag<Superscript>+</Superscript> for orthopedic devices <TextLink reference="140"></TextLink>.</ListItem><ListItem level="1">Other nanomaterials are polymeric NPs, dendrimers, and liposomes for targeted delivery and improved biofilm penetration <TextLink reference="140"></TextLink></ListItem></UnorderedList></Pgraph><Pgraph>However, translation into clinical practice remains limited due to concerns regarding long-term toxicity, scalability, regulatory approval, and cost-effectiveness, particularly in low-resource healthcare settings <TextLink reference="141"></TextLink>.</Pgraph><SubHeadline>Established and clinically applicable therapeutic anti-biofilm approaches</SubHeadline><SubHeadline2>Physical chemical and biological approaches</SubHeadline2><Pgraph>Physical methods such as ionizing/UV radiation and ultrasonic cavitation disrupt biofilms through mechanical and oxidative stress <TextLink reference="142"></TextLink>. Cold atmospheric plasma enhances eradication through reactive oxygen species (ROS)/reactive nitrogen species, mediated damage, with synergistic effects when combined with ultrasound <TextLink reference="143"></TextLink>. Microneedles further improve antimicrobial penetration by breaching the EPS matrix <TextLink reference="144"></TextLink>. In contrast, chemical agents are limited by toxicity, instability, and resistance, whereas biological strategies, such as enzymes, and bacteriophages, offer greater specificity and safety <TextLink reference="145"></TextLink>.</Pgraph><SubHeadline2>Bacteriocins</SubHeadline2><Pgraph>These are ribosomally synthesized antimicrobial peptides (AMPs), have emerged as promising antibiofilm agents with multifaceted mechanisms <TextLink reference="146"></TextLink>, <TextLink reference="147"></TextLink>. They inhibit bacterial adhesion and biofilm formation, reduce EPS production, and disrupt mature biofilms <TextLink reference="146"></TextLink>, <TextLink reference="148"></TextLink>. Studies demonstrate significant reductions in biofilm biomass and matrix integrity across key pathogens, including <Mark2>S. aureus, P. aeruginosa</Mark2>, and <Mark2>E. faecalis</Mark2> <TextLink reference="149"></TextLink>. Additionally, bacteriocins can eradicate biofilm-associated cells, highlighting their potential in managing biofilm-mediated healthcare-associated infections <TextLink reference="150"></TextLink>.</Pgraph><SubHeadline2>Conventional management of biofilm-associated infections</SubHeadline2><Pgraph>Biofilm-associated infections require combined surgical and antimicrobial management. Superficial infections may respond to drainage, whereas deep or device-related infections often require debridement, prosthesis removal, and targeted therapy <TextLink reference="151"></TextLink>. Negative pressure wound therapy augments debridement by promoting granulation and reducing bioburden <TextLink reference="152"></TextLink>. Antibiotic-loaded spacers (e.g., vancomycin or gentamicin) with rifampin-fluoroquinolone regimens improve eradication of staphylococcal biofilms, whereas resistant infections may require agents such as linezolid, daptomycin, tigecycline, or carbapenems <TextLink reference="140"></TextLink>. Polymicrobial biofilms further complicate treatment by accelerating tissue damage and reducing antibiotic efficacy <TextLink reference="153"></TextLink>.</Pgraph><SubHeadline2>Antimicrobial peptides (AMPs)</SubHeadline2><Pgraph>AMPs disrupt bacterial membranes, suppress biofilm-related gene expression, and modulate QS, offering potent activity against both planktonic and biofilm-embedded pathogens <TextLink reference="154"></TextLink>. Peptides like LL-37 and lactoferrin derivatives exhibit broad-spectrum antibiofilm effects, particularly in respiratory infections, and synergize with antibiotics to enhance efficacy <TextLink reference="155"></TextLink>, <TextLink reference="156"></TextLink>. Lactoferrin, an innate immune component, prevents <Mark2>P. aeruginosa</Mark2> biofilm formation by stimulating bacterial twitching motility <TextLink reference="10"></TextLink>. Metal-binding AMPs, such as Gaduscidin-1, are effective in hostile microenvironments, including those seen in <Mark2>P. aeruginosa</Mark2> biofilms <TextLink reference="157"></TextLink>, <TextLink reference="158"></TextLink>. </Pgraph><SubHeadline>Established biological therapies</SubHeadline><SubHeadline2>Bacteriophage therapy</SubHeadline2><Pgraph> Bacteriophages disrupt biofilms through enzymatic degradation (e.g., haemolysinase, depolymerases) and lytic replication, effectively targeting both antibiotic-sensitive and resistant bacteria <TextLink reference="10"></TextLink>, <TextLink reference="159"></TextLink>, <TextLink reference="160"></TextLink>. Specific phages (e.g., Φ15, Φ29, PD1, PE2, T4) have demonstrated efficacy against Pseudomonas, Staphylococcus, Salmonella, and Klebsiella biofilms <TextLink reference="161"></TextLink>, <TextLink reference="162"></TextLink>. Phage-antibioti<TextGroup><PlainText>c c</PlainText></TextGroup>ombinations (e.g., T4 with tobramycin, or phage with amoxicillin) enhance biofilm eradication and limit resistance emergence <TextLink reference="163"></TextLink>. Phage cocktails further expand host range and reduce resistance development <TextLink reference="164"></TextLink>.</Pgraph><SubHeadline2>Enzyme-based disruption of bacterial biofilms</SubHeadline2><Pgraph>Enzymes (oxidases, proteases, and polysaccharide-degrading hydrolases) disrupt biofilms by degrading EPS, interfering with QS, and inhibiting maturation <TextLink reference="164"></TextLink>, <TextLink reference="165"></TextLink>. Their high specificity makes them ideal biological anti-biofilm agents. However, their stability is limited; immobilization strategies, such as crosslinked enzyme aggregates (CLEA), enhance stability and reusability <TextLink reference="166"></TextLink>. Magnetic CLEA formulation (m-combi-CLEA) have shown >75% inhibition of <Mark2>E. coli and</Mark2> <Mark2>S. aureus</Mark2> biofilms, highlighting their therapeutic potential <TextLink reference="167"></TextLink>. </Pgraph><SubHeadline2>Vaccines</SubHeadline2><Pgraph>Vaccines incorporating biofilm-derived antigens represent a promising strategy to enhance protection against persistent infections <TextLink reference="168"></TextLink>. In <Mark2>Bordetella pertussis</Mark2>, biofilm-derived outer membrane vesicles induce stronger immunogenicity and protection than planktonic counterparts, including against pertactin-deficient strains, and generate durable mucosal CD4<Superscript>+</Superscript> memory responses <TextLink reference="169"></TextLink>, <TextLink reference="170"></TextLink>. While still under investigation, biofilm-based vaccines offer a compelling direction for future anti-biofilm immunotherapies <TextLink reference="14"></TextLink>.</Pgraph><SubHeadline2>Monoclonal antibodies as therapeutics for fungal biofilm infections</SubHeadline2><Pgraph>Monoclonal antibodies (MAb) targeting biofilm-specific antigens enable radioimmunotherapy through MAb-guided alpha radiation, allowing in situ treatment when device removal is contraindicated <TextLink reference="171"></TextLink>. Prophylactic MAb administration prevents Cryptococcal biofilm establishment post-implantation <TextLink reference="172"></TextLink>. Chitosan device coatings provide biocompatible protection by disrupting microbial membrane integrity and preventing surface colonization <TextLink reference="173"></TextLink>. <Mark2>In vivo</Mark2> studies demonstrate efficacy against <Mark2>Candida</Mark2> biofilms on CVCs without host cell toxicity <TextLink reference="174"></TextLink>. Optimized antifungal approaches – targeting early biofilm stages, novel formulations (amphotericin B lipid complex), and combination therapies – significantly enhanc<TextGroup><PlainText>e t</PlainText></TextGroup>reatment efficacy compared to conventional methods <TextLink reference="175"></TextLink>.</Pgraph><SubHeadline>Emerging and advanced therapeutic strategies</SubHeadline><Pgraph>The growing threat of antimicrobial resistance underscores the need for novel approaches. </Pgraph><SubHeadline2>Energy-based methods</SubHeadline2><Pgraph>Electric fields (e.g., DC, AC, pulsed electric fields) induce membrane disruption and enhance antibiotic uptake through a “bioelectric effect” <TextLink reference="176"></TextLink>, <TextLink reference="177"></TextLink>. These approaches help destabilize the biofilm matrix and enhance drug penetration into biofilm-embedded bacteria <TextLink reference="121"></TextLink>. In vitro and animal studies show synergy with antibiotics, reducing biofilm burden, but further clinical validation is needed <TextLink reference="178"></TextLink>.</Pgraph><Pgraph>Low-frequency ultrasound enhances antibiotic penetration and disrupts EPS matrices. Devices delivering surface acoustic waves weakens biofilm cohesion and promotes antimicrobial access and have demonstrated >85% reduction in common pathogens’ biofilms when combined with antimicrobials <TextLink reference="179"></TextLink>. Although ultrasound enhances drug penetration, its clinical applicability is limited by optimization challenges, device standardization, and gaps in clinical translation <TextLink reference="180"></TextLink>.</Pgraph><Pgraph>Antimicrobial photodynamic therapy (aPDT) leverages light-activated photosensitizers to generate ROS and directly damages biofilm components and microbial cells, leading to effective biofilm disruption <TextLink reference="121"></TextLink>. Laser and LED-based systems (405–940 nm) have successfully eradicated biofilms on various medical substrates. Clinical use is constrained by limited light penetration, challenges in effective photosensitizer delivery, and difficulty in treating deep-seated infections, with further safety data required for clinical translation <TextLink reference="181"></TextLink>, <TextLink reference="182"></TextLink>, <TextLink reference="183"></TextLink>.</Pgraph><SubHeadline2>Molecular and genetic tools</SubHeadline2><Pgraph>CRISPR/Cas systems enable precise disruption of biofilm formation by targeting key regulatory genes, such as <Mark2>icaA</Mark2> in <Mark2>S. aureus</Mark2>, <Mark2>lasR/rhlR</Mark2> in <Mark2>P. aeruginosa</Mark2>, and <Mark2>pelA</Mark2> in <Mark2>P. aeruginosa</Mark2> and <Mark2>E. coli</Mark2>, thereby reducing biofilm biomass and adhesion <TextLink reference="184"></TextLink>, <TextLink reference="185"></TextLink>, <TextLink reference="186"></TextLink>, <TextLink reference="187"></TextLink>. Additionally, CRISPR-associated nucleases can selectively degrade AMR determinants, re-sensitizing biofilm-embedded pathogens to conventional antibiotics <TextLink reference="188"></TextLink>. To overcome delivery barriers, engineered bacteriophages carrying CRISPR constructs have shown efficacy against<Mark2> K. pneumoniae</Mark2> biofilms, while nanocarrier and liposomal systems further enhance penetration and therapeutic efficiency within the biofilm matrix <TextLink reference="189"></TextLink>. Clinical translation is hindered by delivery barriers, off-target effects, biosafety concerns, and complex regulatory approval pathways <TextLink reference="190"></TextLink>.</Pgraph><Pgraph>Aptamers, synthetic single-stranded oligonucleotides or peptides, exhibit high-affinity, target-specific binding via defined 3D structures <TextLink reference="191"></TextLink>. They disrupt biofilms by depolarizing bacterial membranes and enhancing antibiotic delivery. Aptamer-functionalized nanomaterials, such as aptamer-graphene oxide complexes, have shown >90% inhibition of <Mark2>Salmonella typhimurium</Mark2> biofilms <TextLink reference="192"></TextLink>. Their therapeutic application is limited by rapid degradation <Mark2>in vivo</Mark2>, poor stability, and challenges in targeted delivery and large-scale production <TextLink reference="193"></TextLink>.</Pgraph><Pgraph>Peptide nucleic acids (PNAs) bind bacterial DNA with high specificity and affinity, showing promise against MDR pathogens and biofilms <TextLink reference="194"></TextLink>. Although limited by poor penetration, delivery strategies such as conjugation with cell-penetrating peptides enhance efficacy. PNAs targeting <Mark2>ftsZ, efaA</Mark2>, or <Mark2>acpP</Mark2> genes inhibit bacterial division and biofilm formation in <Mark2>Escherichia coli, Enterococcus</Mark2> spp., and <Mark2>Hemophilus influenzae</Mark2> <TextLink reference="195"></TextLink>, <TextLink reference="196"></TextLink>, <TextLink reference="197"></TextLink>. Synergistic combinations with antibiotics (e.g., polymyxin B) potentiate anti-biofilm effects, making PNAs a compelling NGA strategy <TextLink reference="198"></TextLink>. PNAs face significant barriers including poor cellular uptake, delivery challenges, and potential toxicity at higher concentrations <TextLink reference="199"></TextLink>.</Pgraph><Pgraph>Real-time biofilm detection via biosensors (e.g., impedance-based systems, Raman spectroscopy, qPCR) allows for early identification enabling timely therapeutic intervention before biofilm maturation, improving treatment outcomes <TextLink reference="121"></TextLink>, <TextLink reference="200"></TextLink>, <TextLink reference="201"></TextLink>. Techniques like SERS and interdigitated microelectrodes offer high sensitivity for detecting pathogens on medical surfaces <TextLink reference="121"></TextLink>. Despite high sensitivity, widespread clinical use is limited by high cost, need for specialized infrastructure, and challenges in real-time clinical integration <TextLink reference="180"></TextLink>.</Pgraph><SubHeadline2>Targeted molecular anti-biofilm strategies</SubHeadline2><Pgraph>Catabolite control protein A (CcpA) is a key regulator of biofilm formation in <Mark2>S. aureus</Mark2>, promoting adhesin and eDNA production (cidA/icaA) while repressing the sak gene, which encodes staphylokinase and enhancing virulence via a-hemolysin <TextLink reference="202"></TextLink>. Inhibiting CcpA–DNA binding reduces toxin expression against <Mark2>S. aureus</Mark2> biofilm-related infections, highlighting the CcpA–Sak axis as a promising low-toxicity therapeutic potential <TextLink reference="203"></TextLink>, <TextLink reference="204"></TextLink>.</Pgraph><Pgraph>Functional amyloids play a central role in biofilm development across several bacterial species <TextLink reference="10"></TextLink>. Small molecules such as FN075 and BibC6 in <Mark2>E. coli</Mark2>, and AA-861 or parthenolide in <Mark2>Bacillus subtilis</Mark2>, inhibit amyloid fiber formation or disrupt established biofilms, thereby reducing virulence <TextLink reference="205"></TextLink>, <TextLink reference="206"></TextLink>. These findings highlight that targeting amyloid assembly can effectively weaken biofilm structure and persistence. Table 2 <ImgLink imgNo="2" imgType="table" /> summarizes all biofilm-eradicating agents and their action.</Pgraph><SubHeadline>Clinical translation and real-world challenges</SubHeadline><Pgraph>Although all these strategies demonstrate significant antibiofilm activity in vitro and in preclinical models, robust clinical evidence supporting their routine use remains limited, highlighting a critical gap between experimental innovation and bedside application <TextLink reference="207"></TextLink>, <TextLink reference="208"></TextLink>. Most approaches, including NPs, AMP, QS inhibitors, and enzymatic therapies, are supported primarily by in vitro or animal model data, with a paucity of large-scale randomized clinical trials <TextLink reference="209"></TextLink>, <TextLink reference="210"></TextLink>. Current management of biofilm-associated HAIs continues to rely heavily on device removal, prolonged antimicrobial therapy, and infection control measures <TextLink reference="51"></TextLink>.</Pgraph><Pgraph>Cost, scalability, toxicity, and regulatory barriers further hinder clinical adoption, particularly in low- and middle-income countries where the burden of HAIs is highest <TextLink reference="141"></TextLink>, <TextLink reference="211"></TextLink>. Even promising interventions such as antimicrobial coatings and lock therapies have demonstrated variable efficacy in clinical settings. Bridging this gap requires well-designed clinical trials, standardized evaluation models, and integration of biofilm-specific strategies into existing infection control frameworks <TextLink reference="212"></TextLink>.</Pgraph></TextBlock> <TextBlock name="Conclusion" linked="yes"> <MainHeadline>Conclusion</MainHeadline><Pgraph>Biofilms are a critical yet often underrecognized driver of HAIs, contributing to persistence, recurrence, and antimicrobial resistance. Through coordinated processes such as adhesion, extracellular matrix formation, and quorum sensing, biofilm-embedded pathogens evade host defenses and exhibit marked tolerance to conventional antimicrobial therapies. As a result, standard treatment strategies designed for planktonic organisms are frequently ineffective, particularly in device-associated infections such as CLABSI, CAUTI, VAP, and SSI.</Pgraph><Pgraph>The intrinsic tolerance conferred by the biofilm matrix, metabolic heterogeneity, and persister cell populations necessitates a paradigm shift toward biofilm-targeted prevention, early detection, and multi-modal treatment approaches. Preventive strategies particularly surface engineering, antimicrobial coatings, and quorum sensing inhibition, offer the most effective opportunity to reduce biofilm establishment, especially in device-associated settings.</Pgraph><Pgraph>Therapeutically, while established approaches such as surgical debridement, device removal, and combination antimicrobial therapy remain the clinical cornerstone, a broad spectrum of emerging interventions, including nanotechnology-based systems, antimicrobial peptides, bacteriophages, enzyme-based matrix disruption, and molecular tools such as CRISPR – demonstrate promising anti-biofilm activity. However, a critical translational gap persists, as most of these strategies remain confined to in vitro and preclinical models, with limited validation in large-scale clinical trials.</Pgraph><Pgraph>Importantly, this review underscores the need for integration of biofilm-specific diagnostics, preventive strategies, and targeted therapeutics into existing infection control frameworks. Advancing the field will require standardized models for biofilm evaluation, robust clinical studies, and interdisciplinary collaboration bridging microbiology, material science, and clinical medicine.</Pgraph><Pgraph>Addressing biofilm-associated HAIs demands a shift from reactive treatment to proactive prevention and precision-targeted therapy. Bridging the gap between mechanistic understanding and clinical application is essential to reduce the burden of HAIs, combat AMR, and improve patient outcomes in modern healthcare systems.</Pgraph></TextBlock> <TextBlock name="Notes" linked="yes"> <MainHeadline>Notes</MainHeadline><SubHeadline>Authors’ ORCIDs </SubHeadline><Pgraph><UnorderedList><ListItem level="1">Anand G: <Hyperlink href="https://orcid.org/0009-0008-0473-389X">https://orcid.org/0009-0008-0473-389X</Hyperlink></ListItem><ListItem level="1">Lahariya R: <Hyperlink href="https://orcid.org/0009-0003-5769-4509">https://orcid.org/0009-0003-5769-4509</Hyperlink></ListItem></UnorderedList></Pgraph><SubHeadline>Funding</SubHeadline><Pgraph>None. </Pgraph><SubHeadline>Competing interests</SubHeadline><Pgraph>The authors declare that they have no competing interests.</Pgraph><SubHeadline>Generative AI statement</SubHeadline><Pgraph>The author(s) declare that generative AI (ChatGPT) was used exclusively to assist with language editing, grammatical refinement, and improvement of clarity of expression. The author(s) reviewed and edited the manuscript and take full responsibility for its content. The figures were generated using Google’s generative AI tools based on original text developed by the authors. The generated images were created specifically for this manuscript and are not reproduced from any previously published source.</Pgraph></TextBlock> <References linked="yes"> <Reference refNo="1"> <RefAuthor>Lowe H</RefAuthor> <RefAuthor>Woodd S</RefAuthor> <RefAuthor>Lange IL</RefAuthor> <RefAuthor>Janjanin S</RefAuthor> <RefAuthor>Barnet J</RefAuthor> <RefAuthor>Graham W</RefAuthor> <RefTitle>Challenges and opportunities for infection prevention and control in hospitals in conflict-affected settings: a qualitative study</RefTitle> <RefYear>2021</RefYear> <RefJournal>Confl Health</RefJournal> <RefPage>10</RefPage> <RefTotal>Lowe H, Woodd S, Lange IL, Janjanin S, Barnet J, Graham W. 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