Biofilm Biology Research Articles

Contemporary comprehension of root canal biofilms and future perspectives: Part 2

ABSTRACT In Part 1 of this commissioned review, we discussed the basic biology of biofilms, its assembly, biogeography, and physicomechanical properties. In the second part of this review, we critically discuss the literature pertaining to microbiology of endodontic diseases, types of root canal infections, microbial dynamics, and host evasion strategies and highlight the concept of functional redundancy. Then, we review the role of fungi in endodontic diseases and provide an important narrative on the role of biofilms in weakening dentin. Finally, we briefly review the goals of prevention and treatment of apical periodontitis and provide futuristic perspectives to reinforce critical points that will aid future research and discovery.

Role of biofilms in antimicrobial resistance of the bacterial bovine respiratory disease complex.

An increase in chronic, non-responsive bovine respiratory disease (BRD) infections in North American feedlot cattle is observed each fall, a time when cattle are administered multiple antimicrobial treatments for BRD. A number of factors are responsible for BRD antimicrobial treatment failure, with formation of biofilms possibly being one. It is widely accepted that biofilms play a role in chronic infections in humans and it has been hypothesized that they are the default lifestyle of most bacteria. However, research on bacterial biofilms associated with livestock is scarce and significant knowledge gaps exist in our understanding of their role in AMR of the bacterial BRD complex. The four main bacterial species of the BRD complex, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis are able to form biofilms in vitro and there is evidence that at least H. somni retains this ability in vivo. However, there is a need to elucidate whether their biofilm-forming ability contributes to pathogenicity and antimicrobial treatment failure of BRD. Overall, a better understanding of the possible role of BRD bacterial biofilms in clinical disease and AMR could assist in the prevention and management of respiratory infections in feedlot cattle. We review and discuss the current knowledge of BRD bacteria biofilm biology, study methodologies, and their possible relationship to AMR.

The Hidden World Within: Microbial Dynamics in Root Canal Systems.

Several hundred different microbial taxa have made the oral cavity their home because of their evolution in multiple species communities within the special ecosystem. On the other hand, the dental pulpor internal tissue of the tooth is a connective tissue that is physiologically sterile and where any microbial infiltration is a harmfulindication. It causes the pulp tissue to become inflamed, which leads to the death of the pulp and diffuses infection with inflammation to the peri-radicular tissues. Comprehending the biology of biofilms, the microbial makeup, and the host's reaction to infections in the pathobiology of root canal infectionshas received a lot of attention throughout the last few decades. Such comprehensive knowledge is required to design preventive medicines as well as clinically effective treatment regimens. Surprisingly, clinical approaches have concentrated more on radiographically perfecting channel preparation than on debridement of these intricate root canal systems, despite the clear realization that root canal infections are biofilm mediated. Since the present comprehension of the microbial etiopathogenesis of apical periodontitis highlights the significance of focusing on procedures such as "canal cleaning" and chemo-mechanical disinfection, the exclusive purpose of endodontic therapy is mainly missed while discussing "canal shaping."We thoroughly examine the state of our knowledge of the composition and functional traits of the root canal microbiome in this review. We also go into the difficulties with root canal disinfection and the cutting-edge approaches that try to solve these difficulties. In conclusion, we present essential guidance for prospective research areas, underscoring their significance as crucial considerations in the field of frontiers in oral health.

Antibacterial Effect of Silver Nanoparticles on Polymicrobial Biofilms in Endodontics: Systematic Review and Meta Analysis

This systematic review investigates the antibacterial effects of silver nanoparticles (AgNPs) on polymicrobial biofilms in endodontics, emphasizing their potential enhancement of traditional treatment methods. Recognizing the limitations of current endodontic disinfectants in fully eradicating complex biofilms, this study focuses on the synergistic potential of AgNPs to improve antimicrobial efficacy. Utilizing a systematic approach based on PRISMA guidelines, the review draws on studies from databases like PubMed, Web of Science, and SCOPUS up to 2024. The included research specifically examines the role of AgNPs in various endodontic applications against resilient biofilms, adhering to strict inclusion criteria that demand clear methodology and measurable outcomes. The results suggest that AgNPs can significantly augment the biofilm disruption capabilities of conventional antimicrobials, potentially due to their ability to modify the biofilm matrix and enhance agent penetration. However, the effectiveness varies depending on the nanoparticle concentration and application technique, indicating a need for further optimized studies. In conclusion, while AgNPs show promise in enhancing endodontic antimicrobial protocols, additional in vivo research is necessary to establish standardized, effective, and safe clinical applications. The review highlights the complex interactions between nanoparticle properties and biofilm biology, underscoring the importance of further investigation in this area.

Biofilms in mycobacterial infection

The ability to form biofilms in lung tissue destruction cavities has been described for both the causative agent of tuberculosis (Mycobacterium tuberculosis) and for non-tuberculous mycobacteria. This process is associated with the chronic infection. It is assumed that a change in the biology of the pathogen inside the biofilm (formation of an antibiotic-resistant phenotype) makes ineffective standard chemotherapy regimens, which based on the susceptibility test data of mycobacteria in the “free state”. This review describes the biology of mycobacterial biofilms and the main approaches to combat them. Available information suggests that control of mycobacterial biofilm formation is one potential strategy to improve the effectiveness of treatment for tuberculosis and mycobacteriosis.

Biofilm antimicrobial susceptibility testing: where are we and where could we be going?

Our knowledge about the fundamental aspects of biofilm biology, including the mechanisms behind the reduced antimicrobial susceptibility of biofilms, has increased drastically over the last decades. However, this knowledge has so far not been translated into major changes in clinical practice. While the biofilm concept is increasingly on the radar of clinical microbiologists, physicians, and healthcare professionals in general, the standardized tools to study biofilms in the clinical microbiology laboratory are still lacking; one area in which this is particularly obvious is that of antimicrobial susceptibility testing (AST). It is generally accepted that the biofilm lifestyle has a tremendous impact on antibiotic susceptibility, yet AST is typically still carried out with planktonic cells. On top of that, the microenvironment at the site of infection is an important driver for microbial physiology and hence susceptibility; but this is poorly reflected in current AST methods. The goal of this review is to provide an overview of the state of the art concerning biofilm AST and highlight the knowledge gaps in this area. Subsequently, potential ways to improve biofilm-based AST will be discussed. Finally, bottlenecks currently preventing the use of biofilm AST in clinical practice, as well as the steps needed to get past these bottlenecks, will be discussed.

Biofilm Models: Different Ways of Biofilm Characterization and Drug Discovery.

The ability of bacteria to develop biofilms and its added effect on antimicrobial resistance have been a concern for both animal and human medicine. The need to understand biofilm biology has been addressed with the help of three biofilm models, i.e., in vitro, ex vivo, and in vivo. Due to the implications of animal welfare involved in in vivo models, this article is mainly focused on in vitro and ex vivo study models to analyze biofilm biology. In in vitro biofilm models, the microtiter plate and Calgary biofilm device are the most commonly used techniques for biofilm analysis. Quantification of the biofilm biomass generated by these two techniques can be assessed with the help of a crystal violet assay. Although in vitro biofilm models help advance understanding of the biology of biofilm and are easy to perform, they fail to address certain important questions, such as the importance of the substrate on which biofilm grows and the interaction between the organisms and the substrate. To address this concern, an ex vivo model can be utilized to characterize the behavior and characteristics of biofilms on different substrates. Ex vivo biofilm models are considered a bridge between the in vitro and in vivo biofilm models. Although neither of the currently available biofilm assessment models is considered the gold standard, they have significantly increased understanding of biofilm behavior. Further studies are warranted to develop more refined biofilm models. © 2023 Wiley Periodicals LLC. Basic Protocol 1: In vitro biofilm models for microtiter plate/crystal violet assay for biofilm growth assessment Basic Protocol 2: Crystal violet assay/tissue culture plate method for testing of antibiofilm agents Alternate Protocol: Calgary biofilm device to determine biofilm susceptibility to antimicrobial agents Basic Protocol 3: Ex vivo biofilm skin models: canine/porcine skin explants.

Effect of various types of extracellular DNA on V. hyugaensis biofilm formation.

Marine bacteria face a constant influx of new extracellular DNA (exDNA) due to the massive viral lysis that occurs in the ocean on a daily basis. Generally, biofilms have shown to be induced by self-secreted exDNA. However, the effect of various types of exDNA with varying lengths, self vs non-self, as well as guanine-cytosine content (GC) content on biofilm formation has not been explored, despite being a critical component of the extracellular polymeric substance. To test the effect of such exDNA on biofilms, a marine bioluminescent bacterium (Vibrio hyugaensis) was isolated from the Sippewissett Salt Marsh, USA, and treated with various types of exDNA. We observed rapid pellicle formation with distinct morphologies only in cultures treated with herring sperm gDNA, another Vibrio spp. gDNA, and an oligomer of 61-80% GC content. With pH measurements before and after the treatment, we observed a positive correlation between biofilm formation and the change to a more neutral pH. Our study highlights the importance of studying DNA-biofilm interaction by carefully examining the physical properties of the DNA and by varying its content, length, and source. Our observation may serve as the basis for future studies that seek to interrogate the molecular explanation for the various types of exDNA and their effects on biofilm formation. IMPORTANCE Bacteria mostly exist as biofilm, a protective niche that promotes protection from the environment and nutrient uptake. By forming these structures, bacteria have caused recalcitrant antibiotic-resistant infections, contamination of dairy and seafood, and fouling equipment in the industry. A critical component that makes up the extracellular polymeric substances, the structural component of a biofilm, is the extracellular DNA secreted by the bacteria found in the biofilm. However, previous studies on DNA and biofilm formation have neglected the unique properties of nucleic acid and its high diversity. Our study aims at disentangling these DNA properties by monitoring their effect at inducing biofilm formation. By varying length, self vs non-self, and GC percentage, we used various microscopy techniques to visualize the structural composition of a Vibrio hyugaensis biofilm. We observed DNA-dependent biofilm stimulation in this organism, a novel function of DNA in biofilm biology.

Biology and Regulation of Staphylococcal Biofilm

Despite continuing progress in medical and surgical procedures, staphylococci remain the major Gram-positive bacterial pathogens that cause a wide spectrum of diseases, especially in patients requiring the utilization of indwelling catheters and prosthetic devices implanted temporarily or for prolonged periods of time. Within the genus, if Staphylococcus aureus and S. epidermidis are prevalent species responsible for infections, several coagulase-negative species which are normal components of our microflora also constitute opportunistic pathogens that are able to infect patients. In such a clinical context, staphylococci producing biofilms show an increased resistance to antimicrobials and host immune defenses. Although the biochemical composition of the biofilm matrix has been extensively studied, the regulation of biofilm formation and the factors contributing to its stability and release are currently still being discovered. This review presents and discusses the composition and some regulation elements of biofilm development and describes its clinical importance. Finally, we summarize the numerous and various recent studies that address attempts to destroy an already-formed biofilm within the clinical context as a potential therapeutic strategy to avoid the removal of infected implant material, a critical event for patient convenience and health care costs.

One Step Forward with Dry Surface Biofilm (DSB) of Staphylococcus aureus: TMT-Based Quantitative Proteomic Analysis Reveals Proteomic Shifts between DSB and Hydrated Biofilm.

The Gram-positive bacterium Staphylococcus aureus is responsible for serious acute and chronic infections worldwide and is well-known for its biofilm formation ability. Recent findings of biofilms on dry hospital surfaces emphasise the failures in current cleaning practices and disinfection and the difficulty in removing these dry surface biofilms (DSBs). Many aspects of the formation of complex DSB biology on environmental surfaces in healthcare settings remains limited. In the present study, we aimed to determine how the protein component varied between DSBs and traditional hydrated biofilm. To do this, biofilms were grown in tryptic soy broth (TSB) on removable polycarbonate coupons in the CDC biofilm reactor over 12 days. Hydrated biofilm (50% TSB for 48 h, the media was then changed every 48 h with 20% TSB, at 37 °C with 130 rpm). DSB biofilm was produced in 5% TSB for 48 h at 35 °C followed by extended periods of dehydration (48, 66, 42 and 66 h at room temperature) interspersed with 6 h of 5% TSB at 35 °C. Then, we constructed a comprehensive reference map of 12-day DSB and 12-day hydrated biofilm associated proteins of S. aureus using a high-throughput tandem mass tag (TMT)-based mass spectrometry. Further pathway analysis of significantly differentially expressed identified proteins revealed that proteins significantly upregulated in 12-day DSB include PTS glucose transporter subunit IIBC (PtaA), UDP-N-acetylmuramate-L-alanine ligase (MurC) and UDP-N-acetylenolpyruvoylglucosamine (MurB) compared to 12-day hydrated biofilm. These three proteins are all linked with peptidoglycan biosynthesis pathway and are responsible for cell-wall formation and thicker EPS matrix deposition. Increased cell-wall formation may contribute to the persistence of DSB on dry surfaces. In contrast, proteins associated with energy metabolisms such as phosphoribosyl transferase (PyrR), glucosamine--fructose-6-phosphate aminotransferase (GlmS), galactose-6-phosphate isomerase (LacA), and argininosuccinate synthase (ArgG) were significantly upregulated whereas ribosomal and ABC transporters were significantly downregulated in the 12-day hydrated biofilm compared to DSB. However, validation by qPCR analysis showed that the levels of gene expression identified were only partially in line with our TMT-MS quantitation analysis. For the first time, a TMT-based proteomics study with DSB has shed novel insights and provided a basis for the identification and study of significant pathways vital for biofilm biology in this reference microorganism.

Correlation Analysis between Chronic Osteomyelitis and Bacterial Biofilm.

Objective To study the role of bacterial biofilm (BBF) in the formation of chronic osteomyelitis and its prevention and treatment. Methods In this paper, a large amount of relevant literature was searched for analysis and summary, and the key words “chronic osteomyelitis,” “bacterial biofilm,” “infection,” and “debridement” were searched in databases, mainly CNKI, Wanfang, and Wipu. The search was conducted until December 2020. The role of bacterial biofilm formation in chronic osteomyelitis and its prevention were analyzed. Results Chronic osteomyelitis is formed mainly due to poor blood supply and drug-resistant bacteria, of which cellular biofilm is the most important cause. BBF forms on the surface of necrotic soft tissue and bone tissue, which has a protective effect on bacteria and greatly enhances their resistance to antibiotics, leading to difficulties in complete bacterial clearance and recurrent infections in osteomyelitis. Conclusion Through an in-depth study of the molecular biology and signal transduction of osteomyelitis biofilm, antibiotic biofilm treatment strategies and surgical debridement remain the focus of clinical translation of chronic osteomyelitis.

A Selective Review on the Novel Approaches and Potential Control Agents of Anti-biofouling and Anti-biofilming.

Specific aggregates of bacterial colonies, which, when embedded in a self-produced matrix, are known to cause biofouling. These complex structures are highly resistant to extreme stress conditions like antibiotics, complex cleaning strategies, or even the human immune system. The formation of biofilm matrices and biofouling is chosen as the preferred microbial environment owing to the advantages offered for the embedded cells for their long-term survival. This increased resistance and virulence emphasizes the urgency of finding newer alternatives for its complete eradication. Combined approaches using enzymes, biomimetic surface modification, and a fusion of physical and chemical methods are gaining more prominence. In brief, this review discusses the structure and biology of biofilms, their mechanism of action, and the new-age anti-biofilm and biofouling agents in food industries, bioelectric, and medical devices. Current anti-biofouling methods, including engineered polymers, surface coatings to antimicrobials, and antibiotics, have also been described.

Biofilm matrix proteome of clinical strain of P. aeruginosa isolated from bronchoalveolar lavage of patient in intensive care unit.

Extracellular matrix plays a pivotal role in biofilm biology and proposed as a potential target for therapeutics development. As matrix is responsible for some extracellular functions and influence bacterial cytotoxicity against eukaryotic cells, it must have unique protein composition. P. aeruginosa is one of the most important pathogens with emerging antibiotic resistance, but only a few studies were devoted to matrix proteomes and there are no studies describing matrix proteome for any clinical isolates except reference strains PAO1 and ATCC27853. Here we report the first biofilm matrix proteome of P. aeruginosa isolated from bronchoalveolar lavage of patient in intensive care unit. We have identified the largest number of proteins in the matrix among all published studies devoted to P. aeruginosa biofilms. Comparison of matrix proteome with proteome from embedded cells let us to identify several enriched bioprocess groups. Bioprocess groups with the largest number of overrepresented in matrix proteins were oxidation-reduction processes, proteolysis, and transmembrane transport. The top three represented in matrix bioprocesses concerning the size of the GO annotated database were cell redox homeostasis, nucleoside metabolism, and fatty acid synthesis. Finally, we discuss the obtained data in a prism of antibiofilm therapeutics development.

Insight to Pseudomonas aeruginosa biofilms

Pseudomonas aeruginosa is well known nosocomial pathogen. It is recognized for hospital-related infections in immuno-compromised patients. P. aeruginosa has a plastic genome known for developing faster multidrug-resistant strains. P. aeruginosa is identified for transiting lifestyle from free-swimming to encapsulated sessile biofilms. This bacterium is notorious to cause acute infection during free swimming lifestyle (planktonic). The infections may transit to chronic form which is difficult to treat. Chronic infections are generally represented by biofilm formation. P. aeruginosa causes device and non-device related biofilm-based infections. The problem arises when biofilm infection becomes antibiotic tolerant and resistant to immune response due to restricted penetration of antimicrobials. Thus, biofilm formation creates more problem during infection as it can become untreatable and more persistent. Therefore, a detailed study of biofilm biology is a necessity. In this review, we have described infections caused by P. aeruginosa biofilms, immune response against biofilms and antibiotic tolerance mechanisms. We have also discussed how in vitro biofilms are studied in lab. Further we have described composition of biofilm matrix and their sequential formation. In addition, P. aeruginosa biofilm formation is tightly regulated mainly by three ways- changing c-di-GMP level, two components signaling (GAC/RET/LAD) and quorum sensing (QS). This review gives insight to different aspect of P. aeruginosa biofilms, one of the topmost model organism studied in infection biology.

The Association between Biofilm Formation and Antimicrobial Resistance with Possible Ingenious Bio-Remedial Approaches.

Biofilm has garnered a lot of interest due to concerns in various sectors such as public health, medicine, and the pharmaceutical industry. Biofilm-producing bacteria show a remarkable drug resistance capability, leading to an increase in morbidity and mortality. This results in enormous economic pressure on the healthcare sector. The development of biofilms is a complex phenomenon governed by multiple factors. Several attempts have been made to unravel the events of biofilm formation; and, such efforts have provided insights into the mechanisms to target for the therapy. Owing to the fact that the biofilm-state makes the bacterial pathogens significantly resistant to antibiotics, targeting pathogens within biofilm is indeed a lucrative prospect. The available drugs can be repurposed to eradicate the pathogen, and as a result, ease the antimicrobial treatment burden. Biofilm formers and their infections have also been found in plants, livestock, and humans. The advent of novel strategies such as bioinformatics tools in treating, as well as preventing, biofilm formation has gained a great deal of attention. Development of newfangled anti-biofilm agents, such as silver nanoparticles, may be accomplished through omics approaches such as transcriptomics, metabolomics, and proteomics. Nanoparticles’ anti-biofilm properties could help to reduce antimicrobial resistance (AMR). This approach may also be integrated for a better understanding of biofilm biology, guided by mechanistic understanding, virtual screening, and machine learning in silico techniques for discovering small molecules in order to inhibit key biofilm regulators. This stimulated research is a rapidly growing field for applicable control measures to prevent biofilm formation. Therefore, the current article discusses the current understanding of biofilm formation, antibiotic resistance mechanisms in bacterial biofilm, and the novel therapeutic strategies to combat biofilm-mediated infections.

Recent Advances and Opportunities in the Study of Candida albicans Polymicrobial Biofilms.

It is well known that the opportunistic pathogenic yeast, Candida albicans, can form polymicrobial biofilms with a variety of bacteria, both in vitro and in vivo, and that these polymicrobial biofilms can impact the course and management of disease. Although specific interactions are often described as either synergistic or antagonistic, this may be an oversimplification. Polymicrobial biofilms are complex two-way interacting communities, regulated by inter-domain (inter-kingdom) signaling and various molecular mechanisms. This review article will highlight advances over the last six years (2016-2021) regarding the unique biology of polymicrobial biofilms formed by C. albicans and bacteria, including regulation of their formation. In addition, some of the consequences of these interactions, such as the influence of co-existence on antimicrobial susceptibility and virulence, will be discussed. Since the aim of this knowledge is to inform possible alternative treatment options, recent studies on the discovery of novel anti-biofilm compounds will also be included. Throughout, an attempt will be made to identify ongoing challenges in this area.

Biofilm Biology and Engineering of Geobacter and Shewanella spp. for Energy Applications.

Geobacter and Shewanella spp. were discovered in late 1980s as dissimilatory metal-reducing microorganisms that can transfer electrons from cytoplasmic respiratory oxidation reactions to external metal-containing minerals. In addition to mineral-based electron acceptors, Geobacter and Shewanella spp. also can transfer electrons to electrodes. The microorganisms that have abilities to transfer electrons to electrodes are known as exoelectrogens. Because of their remarkable abilities of electron transfer, Geobacter and Shewanella spp. have been the two most well studied groups of exoelectrogens. They are widely used in bioelectrochemical systems (BESs) for various biotechnological applications, such as bioelectricity generation via microbial fuel cells. These applications mostly associate with Geobacter and Shewanella biofilms grown on the surfaces of electrodes. Geobacter and Shewanella biofilms are electrically conductive, which is conferred by matrix-associated electroactive components such as c-type cytochromes and electrically conductive nanowires. The thickness and electroactivity of Geobacter and Shewanella biofilms have a significant impact on electron transfer efficiency in BESs. In this review, we first briefly discuss the roles of planktonic and biofilm-forming Geobacter and Shewanella cells in BESs, and then review biofilm biology with the focus on biofilm development, biofilm matrix, heterogeneity in biofilm and signaling regulatory systems mediating formation of Geobacter and Shewanella biofilms. Finally, we discuss strategies of Geobacter and Shewanella biofilm engineering for improving electron transfer efficiency to obtain enhanced BES performance.

Non-Antimicrobial Adjuvant Strategies to Tackle Biofilm-Related Staphylococcus aureus Prosthetic Joint Infections.

Staphylococcus aureus frequently causes community- and hospital-acquired infections. S. aureus attachment followed by biofilm formation on tissues and medical devices plays a significant role in the establishment of chronic infections. Staphylococcal biofilms encase bacteria in a matrix and protect the cells from antimicrobials and the immune system, resulting in infections that are highly resistant to treatment. The biology of biofilms is complex and varies between organisms. In this review, we focus our discussion on S. aureus biofilms and describe the stages of their formation. We particularly emphasize genetic and biochemical processes that may be vulnerable to novel treatment approaches. Against this background, we discuss treatment strategies that have been successful in animal models of S. aureus biofilm-related infection and consider their possible use for the prevention and eradication of biofilm-related S. aureus prosthetic joint infection.

Microbiological Aspects of Root Canal Infections and Disinfection Strategies: An Update Review on the Current Knowledge and Challenges.

The oral cavity is the habitat of several hundreds of microbial taxa that have evolved to coexist in multispecies communities in this unique ecosystem. By contrast, the internal tissue of the tooth, i.e., the dental pulp, is a physiologically sterile connective tissue in which any microbial invasion is a pathological sign. It results in inflammation of the pulp tissue and eventually to pulp death and spread of inflammation/infection to the periradicular tissues. Over the past few decades, substantial emphasis has been placed on understanding the pathobiology of root canal infections, including the microbial composition, biofilm biology and host responses to infections. To develop clinically effective treatment regimens as well as preventive therapies, such extensive understanding is necessary. Rather surprisingly, despite the definitive realization that root canal infections are biofilm mediated, clinical strategies have been focused more on preparing canals to radiographically impeccable levels, while much is left desired on the debridement of these complex root canal systems. Hence, solely focusing on “canal shaping” largely misses the point of endodontic treatment as the current understanding of the microbial aetiopathogenesis of apical periodontitis calls for the emphasis to be placed on “canal cleaning” and chemo-mechanical disinfection. In this review, we dissect in great detail, the current knowledge on the root canal microbiome, both in terms of its composition and functional characteristics. We also describe the challenges in root canal disinfection and the novel strategies that attempt to address this challenge. Finally, we provide some critical pointers for areas of future research, which will serve as an important area for consideration in Frontiers in Oral Health.

An Overview of Biological and Computational Methods for Designing Mechanism-Informed Anti-biofilm Agents.

Bacterial biofilms are complex and highly antibiotic-resistant aggregates of microbes that form on surfaces in the environment and body including medical devices. They are key contributors to the growing antibiotic resistance crisis and account for two-thirds of all infections. Thus, there is a critical need to develop anti-biofilm specific therapeutics. Here we discuss mechanisms of biofilm formation, current anti-biofilm agents, and strategies for developing, discovering, and testing new anti-biofilm agents. Biofilm formation involves many factors and is broadly regulated by the stringent response, quorum sensing, and c-di-GMP signaling, processes that have been targeted by anti-biofilm agents. Developing new anti-biofilm agents requires a comprehensive systems-level understanding of these mechanisms, as well as the discovery of new mechanisms. This can be accomplished through omics approaches such as transcriptomics, metabolomics, and proteomics, which can also be integrated to better understand biofilm biology. Guided by mechanistic understanding, in silico techniques such as virtual screening and machine learning can discover small molecules that can inhibit key biofilm regulators. To increase the likelihood that these candidate agents selected from in silico approaches are efficacious in humans, they must be tested in biologically relevant biofilm models. We discuss the benefits and drawbacks of in vitro and in vivo biofilm models and highlight organoids as a new biofilm model. This review offers a comprehensive guide of current and future biological and computational approaches of anti-biofilm therapeutic discovery for investigators to utilize to combat the antibiotic resistance crisis.