It has long been known that microbiological infections exist, and since then, scientists have worked tirelessly to eradicate both established and new infections that cause infectious diseases and create antimicrobial drugs to cure and get rid of the contagious illness. Antimicrobials are a diverse group of substances that can fight a broad range of pathogenic microbes, including bacteria, protozoa, fungi, viruses and parasites (Dutt et al., 2022; Ahmad et al., 2023). From the early 20^th century, these substances have been employed to treat infected individuals and they have greatly assisted in reducing the most infectious rates of morbidity and mortality (Dutt et al., 2022). In 1928, penicillin was discovered by Alexander Fleming, and it entered clinical usage in the 1940s at the perfect moment for the second world war (Gaynes, 2017). In just four years of usage, the first strains of penicillin-resistant bacteria, a new species of bacteria appeared, which led to the development of antibiotic resistance. As a result, AMR has quickened and expanded to include more harmful species because of the continuous exposure and indiscriminate use of antibiotics in clinical and farming environments. Numerous modern antibiotics have lost their efficacy because of the development of AMR and scientists are working continuously to comprehend how AMR works to create new antimicrobials (Dutt et al., 2022).

Looking towards bacteria, it feels that the capacity to form biofilm is shared by mostly all the bacteria and it is considered a universal attribute. In biofilms, the extracellular matrix formed by the cells themselves holds the groups of bacteria or multicellular communities together. Different bacteria use different ways to create biofilms, these mechanisms soften depending on the environment in which they are found as well as strain-specific characteristics (Percival et al., 2011). He was Antonie van Leeuwenhoek who first saw “animalcules “on his teeth in the 17^th century. The bottle effect was first noted in marine microorganisms in 1940. This demonstrates that germs proliferate more frequently on surfaces (Percival et al., 2011; Dutt et al., 2022).

Then, in 1943, Zobell created biofilms and discovered that the number of bacteria on surfaces was higher than that of the surrounding seawater (Zobell, 1943). Today, in scientific language, we define biofilms as the microbial communities that are attached to a substrate and covered in extracellular polymeric substance (EPS), which is secreted by these bacteria (Costerton et al., 1987; Donlan, 2001; Zuberi et al., 2017b). Microbial biofilms can be found on many surfaces in aquatic environments, damp structures, plant roots, human tooth or dental implants, catheters, medical equipment, sutures, etc. They can even be found in human and animal tissues in pathogenic forms that can release toxins into the surrounding extracellular matrix (Donlan, 2001; Percival et al., 2011). They can evade the human response (Cangui-Panchi et al., 2022, Cangui-Panchi et al., 2023).

In addition, biofilms can be found in symbiotic form in aquatic bodies, wastewater filters and the alimentary canals of humans and animals (Costerton et al., 1981; Dutt et al., 2022). Because of their resistance to antibiotics, biofilms, which are the most common in natural settings, can infect both humans and animals (Mah, 2012; Sinclair, 2019). Thus, it is crucial to comprehend the mechanism of biofilm-led resistance to antibiotics. This review will cover antimicrobial resistance (AMR), specifically the mechanism underlying biofilm-led AMR, possible pharmacological or drug candidates and present modalities that are used to target bacteria within the biofilm. We will also focus on current ongoing research like the CRISPR/Cas9 gene editing system to fight bacterial biofilm infections.

Bacterial biofilms are groups of sessile microbes that are entrenched and connected to substratum, within the self-generated non-crystalline pool of the extracellular matrix (Zuberi et al., 2017a, Zuberi et al., 2017b). These bacterial communities are distinct from planktonic ones concerning several methods like transcription, gene expression and growth rate since they live in various stressed environments with increased cell density, osmolarity, nutrient shortage, etc (Sharma et al., 2019). Bacterial biofilm is a dynamic three-dimensional structure that is processed by a heterogenous group of bacterial communities and the bacteria living in these dynamic strictures are shielded from many environmental stressors, including desiccation, immune attack, protozoan ingestion, antimicrobial target, etc., hence making them more resistant and superior over the planktonic form of bacteria (Wilkins et al., 2014; Sharma et al., 2019). The process of developing this three-dimensional structure is a multi-step procedure. It begins when bacteria first attach themselves to the surface and create an unbreakable bond that is followed by other bacterial colonization (Sharma et al., 2019). The bacteria that colonize first are called primary colonizers in biofilm and the secondary colonizers are the ones that attach to the primary colonizers (MaChado and Cerca, 2015). This stage led to changes in the expression of genes or proteins by bacteria. The second stage of the exponential growth phase is characterized by the secretion of exopolysaccharides and the creation of water channels by these bacteria, enabling the nutrient supply to mature biofilms. Finally, the cell surface detachment begins the relaunched or recycled biofilm development on the fresh surfaces (Sharma et al., 2019). This detachment step is usually triggered by stress factors such as a limited nutritional environment and antibiotics. Usually, the cells in the inner or deep layers of biofilm become latent or dormant cells, while the ones on the top layers are metabolically active.

Biofilms can host multiple bacterial species and can lead to a complex system that can accommodate bacterial cell densities between 10⁸–10¹¹ cells g^-1 wet weight (Morgan-Sagastume et al., 2008). Water makes up to 97% of its matrix major constituents and the other contents include proteins, soluble or gel-forming polysaccharides, extra-cellar DNA (eDNA) and non-soluble elements like cellulose, amyloids, pili, fimbria and flagella that deliver structural and functional properties to these biofilms (Flemming and Wingender, 2010; Flemming et al., 2016).

Because of their increased resistance to antibiotics and disinfectants, bacterial biofilms are a major contributing factor to chronic infections. They can also interfere with phagocytosis and other immune system functions. As a result, microorganisms within biofilms become less vulnerable to various antibiotic medicines, hence posing an imminent challenge in the field of therapeutics (Høiby et al., 2010; Amato et al., 2014; Flemming et al., 2016; Dutt et al., 2022).

The term antimicrobials is used for substances that kill microorganisms, inhibit their growth, and prevent or treat diseases or infections in animals, humans, and plants. They included a wide variety of antivirals, antifungals, and antiparasitics. The capacity of microorganisms to withstand an antimicrobial at a higher dose for an extended duration is known as antimicrobial resistance and is usually measured in terms of its minimum inhibitory concentration (Brauner et al., 2016; Ahmad et al., 2017, Ahmad et al., 2018). In biofilms, antibiotic tolerance or resistance may occur simultaneously. Through the introduction of foreign genetic material that codes for resistance genes by horizontal gene transfer (HGT) between the bacterial cells of the biofilm or through genetic mutation, microorganisms within the biofilm develop antibiotic resistance. As a subset of antimicrobial resistance (AMR), antibiotic resistance (ABR) occurs when bacteria develop resistance to antibiotics despite the drug’s effectiveness against them (Dutt et al., 2022). Usually, antibiotic resistance can be acquired extrinsically, adaptively or intrinsically. The classic example of intrinsically acquired resistance is the susceptibility of gram-positive bacteria against antibiotics like daptomycin or vancomycin over gram-negative bacteria due to the difference in their cell wall composition. Conversely, acquired resistance results from either mutation or horizontal gene transfer (HGT). In addition, during adaptation, the bacteria may quickly modify their pattern of gene expression and translation in response to other environmental conditions or stimuli including stress (Blair et al., 2015; Dutt et al., 2022).

On the other hand, tolerance refers to a microorganism’s capability to endure antibiotics at concentrations greater than their inhibitory effect for a specific period (Arzanlou et al., 2017; Hall and Mah, 2017). For a brief duration, tolerance is a form of adaptation that represents shifts in cellular activity from active to latent state. Like antibiotic entrapment to the extracellular polymeric substance (EPS) in the absence of target attachment induces tolerance and causes bacteria cell dormancy. Persistence is an exceptional form of tolerance, where persisters refer to the tolerant form of cells within that bacterial population that can survive the antibiotics but can be killed at long exposure (Wilmaerts et al., 2019). Biofilm-mediated resistance is a multifaceted type of resistance that necessitates tolerance in addition to the antibiotic resistance mechanism. Furthermore, the state and developmental stage of the biofilm, its growth conditions, and the microbial species present within it also contribute to this process (Hall and Mah, 2017; Dutt et al., 2022). Some of the mechanisms of antimicrobial resistance (AMR) that are related to biofilm-mediated resistance are limiting the permeability or blocking access to antimicrobials; altering the targets of antibiotics through mutations; and breaking down the antimicrobials through enzymatic hydrolysis or chemical alteration. In this review, we tried to discuss each of them under the following headings and they have been vividly illustrated in Figure 1 .

Roughly 80% of recurring and chronic microbial illnesses are caused by biofilms of bacteria. Biofilm-containing microbial cells have demonstrated 10–1000 times greater tolerance to drugs than planktonic cells. Infections linked to biofilms can be widely separated into two categories (Mah, 2012). Either biofilm might grow on the abiotic surfaces that include knee replacements, implants, dental units, catheters, contact lenses, screws, pins or prosthetic valves and joints or they are host tissue related that leads to chronic wounds, endocarditis, cystic fibrosis lings or chronic otitis media (Donlan, 2001; Burmølle et al., 2010). The infections related to the urinary tract and bloodstream are usually caused via a biofilm that initially developed on the medical implants associated with them and the only way to treat such infection is to get rid of those implants that not only raise the price of the therapy, but it also causes other health-related issues to the patients (Costerton et al., 2005; Sharma et al., 2019). Several of the primary infections associated with bacterial biofilms that are responsible for human illness are mentioned in Table 1 .

S. aureus, S. pneumoniae, K. pneumoniae, P. aeruginosa, E. coli and E. faecium are the most persistent and common multidrug-resistant bacteria that are linked to significantly high rates of death and morbidity worldwide (Dutt et al., 2022). Additionally, according to some reports, high levels of antibiotic resistance have also been linked to cancer-related neutropenia. Furthermore, because of biofilm biofilm-forming tendency of antibiotic-resistant bacteria, managing and treating newborn sepsis becomes a challenge in many clinical settings. In healthcare and hospital settings almost all procedures, surgeries, transplantation and intensive care are not typically carried out without antibiotics. However, with the increasing failure of first and second-generation antibiotics, the demands of expensive and time-consuming research for the next generation of antibiotics are increasing. Treating such resistant bacteria-mediated infections is complicated and requires the use of more costly and hazardous alternative treatments or higher doses (Magiorakos et al., 2012; Nesher and Rolston, 2014). Below we discuss some mechanisms that bacteria employ to confer antibiotic resistance:

The fact that biofilm functions as a self-motivation mechanism during the pathogenic process helps to explain it. There have been several approaches and methodologies that have been employed to understand its antibiotic resistance nature and to target its residing bacteria. Some of its common approaches are the use of natural products, plant extracts, surface coatings, antibiotics, hydrogels, peptides, lasers during photodynamic therapy (PDT) and nanomedicines or nanoparticles (Hernández-Sierra et al., 2008; Lonn-Stensrud et al., 2008; Kolodkin-Gal et al., 2010; Hochbaum et al., 2011; Iannitelli et al., 2011; Kulshrestha et al., 2014; Muñoz-Egea et al., 2016; Misba and Khan, 2018; Misba et al., 2019). The dramatic portrayal of these treatment strategies i.e. Photodynamic therapy (PDT), small molecules as polypeptides or quorum sensing inhibitors, antibiotics, hydrogels and nanoparticles are shown in Figure 2 .

The present treatment modalities utilize mainly the traditional methods to combat these biofilms that mainly focus on the dispersal of biofilm or its eradication or inhibition through antibiotics, small molecules inhibitors, enzymes, quorum sensing inhibitors, hydrogels etc (Dutt et al., 2022). Table 2 shows the list of potential drug candidates and small molecules that are found effective in the mitigation of biofilm-related infections.

Biofilm growth on implanted medical devices, prosthetic surfaces or biomaterials may be controlled by modifying the attachment surface, like coating the external surfaces. Many coating materials and biomaterials have been established, that make the target surface unfavourable for bacterial attachment. Moreover, the use of therapeutic agents and inhibitors against biofilm formation on dental implants or dental filling material has also demonstrated positive effects in combating biofilm-associated infections (Sharma et al., 2019).

As mentioned earlier too, the bacteria in biofilm form are more resistant and tolerant to antibiotics as compared to their planktonic form. Quorum sensing is a principal pathway that leads to biofilm-mediated bacterial maintenance and survival. Hence certain strategies that lead to the dispersal of biofilm or targeting quorum sensing mechanism have exhibited promising results in addressing biofilm-mediated infections (Lonn-Stensrud et al., 2008; McDougald et al., 2012; Guilhen et al., 2017; Roy et al., 2018; Sharma et al., 2019). Moreover, co-treatment or combination therapy that consists of antibiotics or drugs along with a biofilm dispersal agent has also demonstrated efficacy in these cases but has some limitations due to inappropriate concentration of both components and still research is going on (Barraud et al., 2006; Marvasi et al., 2014; Reffuveille et al., 2015; Roizman et al., 2017).

With the new research development and advancements in technologies, the future proposed methods to combat biofilm-mediated antibiotic infections include the use of nanoparticles, antimicrobial peptides, photodynamic therapy and implementation of gene editing technologies like CRISPR/Cas9 ( Figure 2 ). Antimicrobial peptides are also considered as an alternative to antibiotics in eliminating biofilm-mediated infections, they are well-studied biofilm-eradicating agents (Flemming et al., 2008; Baltzer and Brown, 2011). They are ubiquitous in nature and cationic in nature. They consist of 5–90 amino acids (van Boxtel et al., 2017). Despite their exceptional potential to disrupt cell membranes, their mechanism of action is yet to be studied in the case of biofilms.

As reported, nanoparticles are also promising drug delivery systems that tend to penetrate deep due to their small size. Among all drug carriers, they are one of the most effective and explored drug carriers. In the case of biofilm-mediated infections, these particles enter the cells break the biofilm barrier and increase the availability of drugs or antibiotics to the bacterial cells. They have high efficacy, low toxicity, efficient penetration power and high site-specificity for drug release when they are given along with the drug (Hernández-Sierra et al., 2008; Harris et al., 2009; Kulshrestha et al., 2014).

In studies involving photodynamic therapy, some researchers have demonstrated its notable efficiency in fighting against biofilm-mediated antibiotic resistance, attributed to their tendency to generate reactive oxygen species (ROS) (Misba and Khan, 2018; Misba et al., 2019). On the other hand, considering gene editing technologies, numerous reports highlight the promising data regarding CRISPR/Cas9 and its derivatives like CRISPRi, in thwarting biofilms and their related infections (Zuberi et al., 2017a, Zuberi et al., 2017b; Azam et al., 2020; Zuberi et al., 2022). In the subsequent section, we will aim to delineate the potential avenues for the advancement of CRISPR/Cas9 and its barriers to overcome.

According to reports, CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeat) exists approximately in 50% of bacterial genomes and 87% of archaeal genomes and has been acknowledged as an adaptive immune system in bacteria (Ran et al., 2015; Hille et al., 2018; Watson et al., 2021; Mayorga-Ramos et al., 2023). The CRISPR/Cas9 system has exhibited promising potential in recent years in the advancement and development of next-generation antimicrobial medicines or drugs to fight infections that are bought out by antibiotic resistance bacteria (Getahun et al., 2022; Mayorga-Ramos et al., 2023).

Targeting the genes that confer virulence and antibiotic resistance in bacteria has been a common application of this mechanism. CRISPR/Cas9 can be employed in two different ways: a pathogen-focused strategy and a gene-focused approach, depending on where the target gene is located (Li et al., 2016; Shabbir et al., 2018; Tang et al., 2019). Targeting chromosome regions to cause bacterial cell death is one pathogen-focused method. On the other hand, the gene-focused strategy includes focusing on the plasmids that may carry antibiotic-resistance genes (Palacios Araya et al., 2021; Nie et al., 2022). In such cases, the plasmid is eliminated, and the bacteria becomes antibiotic susceptible. The role of CRISPR/Cas9 has come across many times to target the genes that are implicated in antibiotic resistance (Nie et al., 2022; Tao et al., 2022). A study published by Bikard et al, used CRISPR/Cas9 to target the mecA gene (responsible for methicillin resistance) in USA300, the clinical isolates of S. aureus. His results revealed a marked decrease in the pollution of S. aureus in the mixed bacterial population as compared to the control group (Bikard et al., 2014).

In another research, a mouse skin colonization model was used to demonstrate that CRISPR/Cas9 was successful in specifically decreasing the colonization of Staphylococcus bacteria, in contrast to alternative treatment scenarios (Bikard et al., 2014; Wang et al., 2019). Moreover, in a separate study published by Ates et al., it was illustrated that resistance genes (aacA, grlA, grlB and mecA) in MRSA strains when targeted through CRISPR/Cas9 by designed CRISPR plasmids harbouring specific sgRNA, increase their susceptibility against antibiotics, hence changes their resistance profile (Juszczuk-Kubiak, 2024). Moreover, the use of pCasCure plasmids also came across in reversing the susceptibility of Enterobacteriaceae against carbapenems. pCasCure was reported successful in especially cleaving genes like bal _KPC, bla _OXA-48 and bla _NDM and targeting their corresponding plasmids (Hao et al., 2020).

The other team led by Yosef used the CRISPR/Cas9 system to eliminate plasmids containing bla _CTXM-15 and bla _NDM-1 (beta-lactamase genes) to eradicate E. coli that produce extended-spectrum beta-lactamases (ESBLs) (Yosef et al., 2015). The CRISPR/Cas system that targets are, the virulence factor in E. coli O157:H7 (EHEC), subsequently resulted in a 20-fold drop in viable cell counts, as shown by Citorik et al (Citorik et al., 2014). Rodrigues et al. tried to specifically eliminate the tetracycline (tetM)and erythromycin (ermB) in E. faecalis in both vitro and vivo conditions. His in vivo data demonstrated a considerable reduction in the percentage of antibiotic-resistant E. faecalis within the gut of mice (Rodrigues et al., 2019).

Askoura et al, revealed that S. enterica biofilm development, cell adhesion and cell invasion were impacted by CRISPR/Cas9 system that targeted sdiA (Askoura et al., 2021). Additionally, it’s noteworthy to mention the two of our studies that were published in 2017 manifesting the role of CRISPRi (CRISPR interference: derivative of CRISPR/Cas9) in restraining biofilm-mediated infections that are caused by clinical strains of E. coli. In one study we targeted luxS, the main quorum sensing gene in E. coli and in another article we tried to knockdown fimH gene and targeted bacterial adherence property through CRISPRi (Zuberi et al., 2017a, Zuberi et al., 2017b). Quorum sensing is one of the most important mechanisms that govern bacterial biofilm resistance against antibiotics, while the fimH gene plays a crucial role in bacterial virulence by contributing the fimbriae production. In both of our studies, CRISPRi demonstrated its highest level of effectiveness and exhibited optimal performance in targeting its specific genes and its related mechanisms, hence manifesting its lead role in biofilm-induced infections like urinary tract infections (UTI).

Additionally, in another study published by our group, we illustrated the role of CRISPRi in targeting the bolA gene (Azam et al., 2020). It has been discovered already that curli and fimbria formation have a role in the production of biofilms and are directly related to bacterial pathogenicity. BolA is a conserved protein and a transcriptional factor that is involved in bacterial motility and biofilm formation so by targeting this gene through CRISPR gene silencing we tried to combat these biofilm-mediated infections. We also targeted the OmpR/EnvZ, a two-component regulatory mechanism that is involved in transcriptional regulation when osmolarity changes. The main aim of that study was to elucidate the function of OmpR/EnvZ in controlling biofilm through curli and fimbriae production (Zuberi et al., 2022).

All our studies demonstrated exceptional outcomes and obtained outstanding success regarding the use of CRISPRi against biofilm-mediated infections. Not only this but these findings generated fresh insights and sparked innovative scientific discussion platforms regarding the use of this gene editing technology in combating biofilm-mediated infections, which represents a significant barrier within the realm of antibiotic resistance.

Analogous to the protective mechanism of thorns on a rose this gene editing technology also has certain intrinsic limitations that necessitate scientific scrutiny and consideration like the delivery challenge, which is the primary concern (Mayorga-Ramos et al., 2023). However, recent scientific researchers have suggested and hypothesized several different potential solutions to use this technology, such as employing the use of nanoparticles in conjugation with the CRISPR system (Li et al., 2018; Duan et al., 2021; Wan et al., 2021; Yan et al., 2021). Not only this but some studies have also proposed the concept of direct delivery of CRISPRi-edited bacterial cells to the infection site. This approach may facilitate the transfer of this gene editing mechanism to other virulent or antibiotic-resistant bacterial cells at the site of infection via natural conjugation of horizontal gene transfer mechanism (Ji et al., 2014; Watson et al., 2018; Barrangou et al., 2022).

It is a known fact that Biofilm complicates the infections that are associated with communicable as well as non-communicable diseases. Other than that, the role of biofilm in post-operative infections, digestive disorders, cystic fibrosis, atherosclerotic arteries and infective endocarditis also has been documented in various studies and reports. Surface adherence is considered the prevailing mode of bacterial growth culminating in biofilm formation. Consequently, the biofilm environment fosters the emergence of antibiotic resistance or antimicrobial resistance. The understanding of how this lifestyle or environment influences the antimicrobial resistance evolution is still limited. Different hypotheses and explanations have emerged in this context like the proximity of bacterial cells in biofilms may facilitate the horizontal transfer and persistence of resistance genes within these bacterial populations.

The role of Biofilms in antibiotic resistance opens avenues and new vistas for repurposing existing drugs targeting biofilm. Forecasting antibiotic resistance by analysing the population of biofilm formers would be helpful in this scenario. Moreover, the use of nanoparticles in biomaterials and different drug delivery modes could also be considered as alternative options. Furthermore, to develop new and effective anti-biofilm agents computational and new sequencing technologies involving bioinformatic tools are also needed.

Currently, most of the studies focus on in silico screening, omics studies, and machine learning to identify the specific targets for anti-biofilm agents. To predicate antibiotic resistance and formulate therapeutic strategies it is essential to address potential protein targets, biofilm formation mechanisms and pathways. Integrated labs and computational science can lead to the development of successful anti-biofilm agents. RNA-Seq, a high-throughput technology, can assay gene regulation and expression, identifying transcriptomic signatures distinct to biofilms and bacterial dispersion.

As mentioned earlier, in addition to the advancements further research in the field of gene editing technology like CRISPR, is essential. It is imperative to address barriers in a CRISPR-like delivery system, as the advantages of utilizing this gene editing technology far outweigh its limitations, provided the delivery issue can be effectively resolved (Mayorga-Ramos et al., 2023). Moreover, through this mechanism, it could also become possible to target various genes associated with other pathways leading to bacterial biofilm formation and antibiotic resistance. For instance, genes responsible for efflux pumps could be the ones that can be specifically targeted, thereby enhancing our ability to combat antibiotic resistance.

AZ: Software, Supervision, Writing – original draft, Writing – review & editing. NA: Conceptualization, Formal analysis, Investigation, Methodology, Supervision, Visualization, Writing – original draft, Writing – review & editing. HA: Validation, Writing – review & editing. MS: Methodology, Writing – review & editing. IA: Validation, Writing – review & editing.