## 8 Challenges in Biofilm-Associated HAIs  
### Difficulty in Diagnosis and Detection  
Diagnosing HAIs associated with biofilms poses considerable difficulties. Biofilms provide a protective matrix that conceals bacteria from standard detection techniques used for planktonic bacteria, often leading to false-negative outcomes [356]. Acquiring samples from medical devices or tissues containing biofilms complicates the process, as the complicated composition and integration of bacteria within the EPS matrix obstruct the identification of individual pathogens by standard culture methods [357]. Another significant diagnostic challenge is the existence of bacteria in a viable but non-culturable (VBNC) form inside biofilms. Bacteria persist in this form without proliferating on regular culture medium, complicating detection efforts [358]. This presents a considerable challenge to correctly identifying and treating biofilm-associated illnesses since conventional microbiological techniques may not detect bacteria in VBNC forms [359]. To address these diagnostic issues, several innovative methodologies have been investigated. Molecular approaches have shown efficacy in identifying biofilm-associated genes and virulence factors, including DNA sequencing, metagenomics, multiplex PCR, and metatranscriptomics [297]. Recent improvements in fiber-tip ball resonator optical fiber sensors and speckle analysis provide accurate capabilities for biofilm identification [360,361]. Nonetheless, despite their promise, the restricted accessibility of these technologies in healthcare environments continues to hinder wider implementation. Overcoming the challenges of diagnosing biofilm-associated HAIs necessitates developing and adopting diagnostic tools that are more accessible, sensitive, and specialized. These innovations are essential for promptly and effectively managing biofilm-related infections, thereby alleviating their impact on healthcare systems. A key consideration in this process is implementing such advanced diagnostics' economic and operational feasibility. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Approach Category** & **Description** & **Examples** & **Citation** \\ \hline \multirow{4}{*}{Therapeutic Strategies} & Entails the utilization of precise & Combination antibiotic therapy, QS & \\  & pharmaceuticals and treatment & inhibitors, biofilm-dispersing & \\  & procedures, frequently customized to & medicines such as DNase, and & \\  & counteract the tenacious & RNAIII-inhibiting peptide (RIP) are & \\  & characteristics of biofilms. & also potential treatments and & \\  & & phage therapy. & \\ \hline \multirow{4}{*}{Antimicrobial Peptides} & Utilizes brief peptides with & Peptides such as nisin, colistin, & \\  & antibacterial characteristics. & cathelicidins, and defensins & \\  & & specifically target bacteria that & \\  & & produce biofilms. & \\ \hline \multirow{4}{*}{Nanoparticle Strategies} & The process entails utilizing & Gold, silver and liposomal & \\  & nanoparticles to infiltrate biofilms & nanoparticles containing antibiotics, & \\  & and administer & iron oxide nanoparticles, and & \\  & antibacterial substances. & quantum dots for precise & \\  & & medication delivery. & \\ \hline \multirow{4}{*}{Antimicrobial Coatings} & Surface antimicrobial application & Coatings of copper or silver, coatings & \\  & refers to the use of chemicals that & of quaternary ammonium polymers, & \\  & inhibit the growth of biofilms. & and coatings of titanium dioxide are & \\  & & applied to medical devices. & \\ \hline \multirow{4}{*}{Antimicrobial Lock Solutions} & Employed in medical devices to & & \\  & deliver a concentrated amount of & Available lock solutions include & \\  & antibacterial substances at the & ethanol, antibiotic, and & \\  & location susceptible to & taurolidine solutions. & \\  & biofilm development. & & \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Overview of strategies to prevent and manage biofilm-related infections.