Intestinal biofilms: pathophysiological relevance, host defense, and therapeutic opportunities

SUMMARY

The human intestinal tract harbors a profound variety of microorganisms that live in symbiosis with the host and each other. It is a complex and highly dynamic environment whose homeostasis directly relates to human health. Dysbiosis of the gut microbiota and polymicrobial biofilms have been associated with gastrointestinal diseases, including irritable bowel syndrome, inflammatory bowel diseases, and colorectal cancers. This review covers the molecular composition and organization of intestinal biofilms, mechanistic aspects of biofilm signaling networks for bacterial communication and behavior, and synergistic effects in polymicrobial biofilms. It further describes the clinical relevance and diseases associated with gut biofilms, the role of biofilms in antimicrobial resistance, and the intestinal host defense system and therapeutic strategies counteracting biofilms. Taken together, this review summarizes the latest knowledge and research on intestinal biofilms and their role in gut disorders and provides directions toward the development of biofilm-specific treatments.

THE GASTROINTESTINAL ENVIRONMENT AND BIOFILMS

The human gastrointestinal tract and occurrence of biofilms

Within the first 24 hours of birth, infants establish a unique gut microbiota shaped by genetic predispositions, maternal flora, and birth environment (1, 2). As adults, the gastrointestinal (GI) tract hosts ~10¹⁴ microbes, a number comparable to the total human cell count, making it the most densely populated microbial environment in the body (3, 4). This diverse microbial community profoundly influences the host’s defense mechanisms against pathogens and contributes to the host’s well-being. Bacteria residing in the mucosa continuously engage with their surroundings, impacting host physiology and gut homeostasis. Additionally, external factors including diet, food quality, metabolism, stress, pathogens, and medications (i.e., antibiotics) significantly influence microbiota composition and phenotypic expression (Fig. 1) (2, 5–15). Disruptions leading to an altered and dysbiotic microbiota are often associated with disease onset (16–18).

Fig 1.

Gut microbiota influencing factors and the biofilm life cycle. (A) Different factors that can influence the gut microbiota. Changes in the gut microbiota can lead to dysbiosis and biofilm formation. (B) The biofilm life cycle. The four key stages of the biofilm life cycle include (1) Attachment: bacteria attach to the mucosal surface using surface-expressed adhesion proteins, flagella, and pili; (2) Microcolony: mucus-attached bacteria form microcolonies and start biofilm matrix production; (3) Maturation: biofilm maturation leads to a complex extracellular polymeric substance (EPS) matrix architecture with a heterogeneous chemical and physical microenvironment supporting multi-species co-existence and efficient communication; in a mature biofilm, persister cells are present at the bottom of the biofilm, characterized by a strong tolerance against environmental stress and antimicrobial exposure; (4) Dispersal: biofilm dispersal starts with EPS matrix remodeling, resulting in the release of biofilm aggregates that subsequently can colonize new surfaces. Dispersed biofilm is a distinct phenotype that can cause the spread of biofilm-associated infections.

Bacteria colonize various regions of the body, including the mouth, airways, and skin, but the highest abundance (~30% of the human microbiome) is found in the large intestine (19, 20). Meta-omics analysis, including 16S ribosomal RNA sequencing, whole-genome-shotgun metagenomic sequencing, reference genome mapping, metagenomic assembly, gene cloning, and metabolic reconstructions, has provided valuable insights into the composition and functionality of the gut microbiota (21, 22). In healthy individuals, the dominant microbial phyla include Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, and Verrucomicrobia, which collectively constitute over 90% of the human gut microbiota (20, 23, 24). These commensal microbes have an increased host immune tolerance and are highly relevant for the host’s metabolism due to their involvement in secondary bile acid synthesis and short-chain fatty acid metabolism (21, 25). For example, Bacteroidetes are enriched in genes related to carbohydrate metabolism, facilitating the breakdown of dietary fiber, while Firmicutes are enriched in genes facilitating bile acid metabolism (26).

Certain gut-residing bacteria form higher-ordered sessile structures termed biofilms (27–30). A biofilm is a distinct phenotype defined as a surface-attached microbial population that is enclosed in a protective matrix of extracellular polymeric substances (EPS). Bacterial biofilm communities differ from free-floating planktonic bacteria in growth rate, gene expression, transcription, and translation (31–33). Disruption of the gut microbiota’s homeostasis often involves an overgrowth of specific microorganisms, leading to reduced microbial diversity and contributing to GI disorders as well as diseases beyond the gut. Understanding the gut microbiota’s complexity, function, and therapeutic relevance is a prolific area of research, driven by technological advances such as next-generation sequencing, multi-omics, big data analysis, bioinformatics, and artificial intelligence that aid in deciphering the microbiota’s (patho)physiological relevance.

Biofilm formation is a survival strategy for microbial communities, driven by close interactions among embedded microorganisms or environmental threats (28, 34, 35). The unique composition and organization of biofilms offer competitive advantages, allowing microbes to evade host defense mechanisms and resist stressors such as mechanical shear forces or antibiotics (34, 36).

Mucosal biofilms are often considered early warning signals for transitioning into a disease state (37–40), and they have been linked to irritable bowel syndrome (IBS), inflammatory bowel diseases (IBD), and gastric and colorectal cancers (27, 30, 41–43). In IBD, dysbiosis of the gut microbiome and reduced microbial diversity are strongly correlated, with a compromised mucous layer facilitating bacterial colonization and biofilm formation (44, 45). Moreover, biofilm composition can influence the course of IBD (46, 47). However, despite significant research efforts, substantial knowledge gaps remain regarding the interactions between the gut microorganisms and their host and environment, as well as whether dysbiosis of the gut microbiota is a cause or a consequence of GI diseases.

Gut biofilm dynamics, architecture, and signaling networks

Gut biofilm life cycle

Biofilm adhesion, maturation, and dispersal are cornerstones of the biofilm life cycle (Fig. 1). The life cycle starts with the initial adhesion of free-floating bacterial cells to the mucosal layer, facilitated by bacterial cell-surface appendages such as flagella, fimbriae, and pili (48). Once attached, microbes form an early communitive network and produce EPS that envelop the bacteria (30, 49). This mucus-attached bacterial population progresses into a denser, better-functioning microcolony with a communication system called quorum sensing (QS) (50, 51). QS relies on the expression and detection of autoinducers, enabling bacterial cells to monitor cell density and regulate gene expression (50–53). As biofilms mature, metabolically less active persister/dormant cells form, and horizontal gene transfer occurs within the biofilm community (54, 55). In later stages, changes in the transcription machinery result in biofilm remodeling, allowing the biofilm to disperse, expand, and colonize other surface areas (56, 57). Due to continuous peristalsis in the small intestine, biofilm dispersion is directed from proximal to distal regions, with the cecum acting like a dead end resulting in high bacterial density, possibly explaining the high biofilm abundance in this area (39). While factors like nutrient availability, nitric oxide, oxygen, iron, and proteases are known to influence biofilm dispersion, the underlying mechanisms remain poorly understood (57).

Gut biofilm appearance, composition, and organization

Biofilms dominate free-floating planktonic bacteria in most habitats, including in humans (27, 58). The human GI tract is a nutrition-rich ecosystem with a large surface area ideal for bacterial colonization and biofilms. GI biofilms are highly heterogeneous, even in GI regions with a generally poor taxonomic diversity, such as the stomach (30, 58–60). These multi-species biofilms have a high cellular density, facilitating efficient communication and interactions between species. This microbial assembly plays a crucial role in their survival and proliferation strategy, with some microbes depending on the metabolic activity of others. Gut biofilms occur as (i) mucosal biofilms with a mucin-rich matrix, (ii) biofilm clusters around mucin aggregates inside the lumen, (iii) biofilms adherent to food particles, or (iv) intermingled versions of these forms (Fig. 2) (27, 30, 61–63).

Fig 2.

Microbiota distribution, biofilm prevalence in gastrointestinal disorders, key biofilm-forming pathogens, and the appearance of colonic biofilms. (A) Biofilm prevalence, key pathogenic biofilm bacteria, microbiota distribution, and the human digestive system. The human digestive system comprises the oral cavity, pharynx, esophagus, stomach, liver, gallbladder, small and large intestines, appendix, rectum, and anus. Key biofilm-forming and disease-associated pathogens are Clostridioides difficile, enterotoxigenic Bacteroides fragilis (64), adherent-invasive Escherichia coli, and Salmonella (65). (B) Biofilm appearance in a colon cross-section. Higher-ordered bacterial assemblies in the human gut are mucosal biofilms, luminal biofilms, and luminal floating biofilm clusters aggregated to food particles and mucins (27, 30, 61–63). CRC, colorectal cancer; UC, ulcerative colitis.

Biofilm production is a dynamic and energy-intensive process, resulting in intricate micrometer-scale spatial organization (66–69). A key energy-demanding step is the production and secretion of EPS, comprising polysaccharides, proteins, and extracellular DNA as well as water. The EPS matrix constitutes the bulk of the biofilm’s biomass (up to 90%) and self-organizes through various inter- and intramolecular interactions (49). The EPS matrix can vary significantly among different biofilm-producing species (49) and is influenced by the nutritional environment and stress exposure (70). It forms a three-dimensional structure that is in contact with the mucus, providing mechanical and chemical stability and acting as a protective layer against environmental stresses, host defense mechanisms, antibiotics, and other drugs (71, 72). Filling the space between the individual biofilm cells, the EPS matrix defines the cellular surrounding (73) and facilitates surface adhesion, interconnection, and immobilization of the biofilm cells to form biofilm clusters in microcolonies (74, 75). Polysaccharides play a central role in water retention, creating a hydrated and non-rigid environment conducive to bacterial movement within the matrix (29, 49, 76). Structural proteins such as amyloids and lectins stabilize the biofilm architecture by connecting bacterial cells to the surrounding matrix. Extracellular DNA promotes structural integrity and facilitates the exchange of genetic information among biofilm-residing bacterial cells (49, 76, 77). Matrix enzymes contribute to nutrient supply by breaking down EPS, such as polysaccharides. Regulatory mechanisms such as QS, stringent response [(p)ppGpp], or second messengers (e.g., cyclic-di-GMP) influence biofilm organization and microbial survival within biofilms (50–53, 78–80).

Biofilm architecture has been visualized by various imaging techniques, such as confocal laser scanning microscopy or scanning electron microscopy (81–84). Confocal laser scanning microscopy, in particular, has been useful for reconstructing three-dimensional images of biofilms and visualizing the cellular assembly and viability through live/dead staining using SYTO9 and propidium iodide, respectively (81, 83). These studies have provided crucial insights into the biofilm formation process, elucidating both biological factors (e.g., flagella, cell-surface hydrophobicity, and QS) and environmental factors (e.g., substrate properties, hydrodynamic conditions, and nutrients) (81, 85–88). Super-resolution imaging techniques such as super-resolution light microscopy and expansion microscopy revealed further insights into the molecular architecture and assembly principles of biofilms, and inter alia revealed three distinct levels of spatial organization: individual cells, cell clusters, and assembly of cell clusters (89, 90).

Regulatory networks

QS stands as arguably the most important regulatory network governing cell-to-cell communication and orchestrating cooperative shifts in bacterial gene expression to trigger and regulate physiological processes and bacterial behavior. This density-dependent mechanism regulates various crucial aspects, including spore formation, virulence factor production, bacteriocin synthesis, programmed cell death, and biofilm formation (51, 91, 92). Central to QS are extracellular secreted signaling molecules, termed autoinducers, along with a suite of sensing molecules and downstream regulatory proteins that together facilitate signal transmission through a dynamic process of release and monitoring. The biological compartments and signaling molecules vary between different bacterial species and environmental conditions. QS also regulates interactions within bacterial communities, where group-specific benefits are obtained at the expense of cooperating individuals, ultimately enhancing fitness or providing communal goods as an ecology-driven survival strategy (32, 93). In mature biofilms, these secreted autoinducers accumulate in the surrounding environment, diffusing through the biofilm matrix to effectively influence bacterial growth mode and biofilm formation (52, 94, 95). Notably, QS not only governs biofilm development but also plays a key role in biofilm dispersal (96, 97). Various autoinducer types exist, including autoinducing peptides, acylated homoserine lactone (AHL), Pseudomonas quinolone signal, autoinducer-2 (AI-2), and autoinducer-3 (AI-3) (50, 91, 98). AHL communication predominates in Gram-negative bacteria, exemplified by Escherichia coli utilization of AHL, AI-2, and AI-3 despite lacking AHL synthase. E. coli can respond to AHL via the AHL-responsive transcriptional regulator SdiA, which can activate crucial cell division clusters (99, 100). AI-2, on the other hand, facilitates interspecies communication and promotes active cellular movement and microbial autoaggregation within E. coli populations (101, 102).

Another critical regulatory network within biofilms is known as the stringent response. This stress signaling pathway enables bacteria to operate ubiquitously to swiftly reprogram transcription upon encountering environmental stresses, such as starvation. Regulated by the alarmones guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate, collectively described as (p)ppGpp (103), the stringent response governs various aspects of bacterial metabolism and physiology and, notably, biofilm formation (104, 105). Moreover, the (p)ppGpp pathway controls cellular activities such as DNA replication, transcription, nucleotide synthesis, and lipid metabolism (106). Hydrolysis of the alarmone ppGpp is facilitated by the RelA/SpoT homolog, a protein superfamily comprising several enzymes with such activity (107). Consequently, the RelA/SpoT system is an attractive therapeutic target for modulating the stringent response to hinder biofilm formation (108).

Synergistic effects

Strong interspecies interactions within multi-species biofilms can lead to coordinated microbial activities and unique synergistic effects that increase biofilm production and fitness (109, 110). For instance, 58% and 43% of transcriptional changes were observed in the genome of Bifidobacterium bifidum when the bacteria were subjected to co-cultures with Bifidobacterium longum subsp. infantis and Parabacteroides distasonis as a three-species biofilm consortium, and with B. longum subsp. infantis, P. distasonis, and Bacteroides ovatus as a four-species biofilm consortium, respectively (111). The co-culture of all four species led to a ~fivefold increase in biofilm mass compared to the monocultivation (112). Upregulated genes were involved in carbohydrate and amino acid metabolism as well as QS. Metatranscriptomic analysis of changes in the genetic expression of multi-species biofilms revealed enhanced survivability under nutrient limitation and disinfectant exposure (113). Candida albicans produces β-1,3-glucan that can bind to the antibiotic ofloxacin. E. coli co-embedded in C. albicans biofilm displays increased resistance to ofloxacin compared to monomicrobial E. coli biofilm (114). Similar synergy-related resistance-enhancing effects were observed for Staphylococcus aureus against vancomycin when co-cultured with C. albicans (115).

In summary, GI biofilms create a highly dynamic microbial organization endowed with numerous distinct properties compared to their planktonic counterparts. This microbial organization and biofilm architecture facilitate efficient communication and enable rapid responses to environmental stressors, including exposure to antimicrobials.

Biofilm prevalence in gastrointestinal disorders, key pathogens, and clinical relevance

GI disorders are a main health concern in modern society, with biofilms playing an essential role in their pathogenesis. Biofilms contribute to around 60% of chronic and recurrent microbial infections, presenting a substantial challenge in clinical management (116–124). Among the most prevalent GI disorders globally are IBS and IBD, affecting ~10% of the population (121–123, 125). IBS manifests with debilitating symptoms, such as post-prandial abdominal pain, bloating, and irregular bowel habits, without signs of chronic inflammation or neoplasia. IBD encompass conditions like ulcerative colitis (UC) and Crohn’s disease (CD), characterized by inflammation within the colon or throughout the GI tract, respectively, resulting in abdominal pain, cramping, diarrhea, rectal bleeding, reduced appetite, weight loss, and fatigue.

Significant variations in the gut microbiota pose challenges in defining gut homeostasis or dysbiosis solely based on the presence or absence of specific microbes (126, 127). However, imbalances between the gut microbes and the mucous layer heighten susceptibility to inflammation (128, 129). Serving as a critical defense system in the human gut, the intestinal mucus layer represents the primary barrier separating microbes from the epithelial cells of the host (40, 45). When the resident flora colonizes a compromised mucus layer, it can initiate biofilm formation, potentially leading to alterations in epithelial cell biology and triggering inflammatory responses (30, 130–132). This dysregulated state can transform into a chronic inflammatory state, as evidenced in IBD (40, 43). Experimental findings suggest that IBD-specific mucosal biofilms can induce pro-inflammatory responses through various pathways (133–136). Macroscopically visible mucosal biofilms have been observed as an endoscopic feature in 34% of UC patients, compared to only 6% in healthy individuals, and dysbiotic gut microbiomes, characterized by overgrowth of E. coli and Ruminococcus gnavus, have been associated with these disease states (39). While E. coli and R. gnavus demonstrate biofilm-forming capabilities in vitro, further research is needed to ascertain their potential roles as key strains underpinning biofilm formation (39). Moreover, IBD patients exhibit a gut flora with reduced diversity and a distinct taxonomy (137–139). Macroscopically visible mucosal biofilms have also been identified in 57% of IBS patients of a recent clinical cohort study (39). Despite mounting evidence of increased gut biofilm occurrence in individuals with GI disorders, the underpinning disease relevance of these biofilms remains to be fully elucidated (39, 43). Gut biofilms are also implicated in other GI diseases, including colorectal cancers (41, 64, 140–143). Given that many, if not most, persistent human infections are associated with such biofilms, they are of significant clinical relevance (42, 43, 142, 144).

Efforts to identify unique microbial profiles associated with IBD have been extensive (145–148), yet a distinct pathogenic fingerprint directly linked to IBD remains elusive. Nonetheless, several trends have emerged from these investigations. Notably, a decreased abundance of Faecalibacterium prausnitzii and an increased abundance of virulent E. coli are observed in CD (149, 150). Enterotoxigenic Bacteroides fragilis and Pseudomonas aeruginosa occur in greater quantities in both UC and CD patients (151–153). Samples from multiple GI sites of new-onset pediatric CD displayed an overgrowth of bacteria, including Enterobacteriaceae, Pasteurellacaea, Veillonellaceae, and Fusobacteriaceae, and decreased abundance of Erysipelotrichales, Bacteroidales, and Clostridiales, strongly correlating with disease progression (154). Moreover, epithelia-adherent bacteria increasingly occur in IBD patients compared to healthy controls (39, 46, 151). In addition, ex vivo-generated biofilms from the gut microbiota of IBD patients exhibit greater size and cell numbers than those from healthy individuals (133). Enterococcus spp. and adherent-invasive E. coli isolated from CD patients have an enhanced ability to form biofilms on both plastic surfaces and intestinal epithelial cell line cultures (155, 156). Such findings underscore the importance of alterations in the distribution and biogeographic positioning of mucolytic commensal microorganisms, potentially leading to a compromised mucous layer that facilitates access to the intestinal epithelium by neighboring commensal microbes, thereby exerting pathogenic effects (157–159). These insights have also fueled the exploration of microbiome- or biofilm-targeted therapies, which are gaining traction within the biotech industry (160–162).

Patients with chronic inflammation, such as UC and CD, face an increased risk of developing colitis-associated colorectal cancers (163, 164). Pronounced polymicrobial biofilms have a high prevalence in patients with colorectal cancer (64, 130, 165), and mucus-invasive bacterial biofilms on the colon mucosa are observed in ~50% of cases compared to ~13% of healthy controls (143, 166). Among the tumorigenic strains isolated from the mucosa-associated microbiota of colorectal cancer patients are Fusobacterium nucleatum (167, 168), enterotoxigenic B. fragilis, genotoxin-producing E. coli (genotoxins are DNA damaging or mutating compounds) (64, 130, 169), and Streptococcus gallolyticus (170). Intriguingly, homogenates of human biofilm-positive biopsies obtained from both healthy and colorectal cancer patients demonstrated cancerogenic properties in a colorectal cancer mouse model study, whereas biopsy homogenates from biofilm-negative patients did not exert such effects (143). However, it remains unclear whether the biofilms observed in approximately one-third of IBD patients pose an additional carcinogenic risk beyond the mutations induced by inflammation (171).

Adherent-invasive E. coli is a pathogen known for its ability to colonize epithelial cells and invade macrophages, triggering inflammatory responses and contributing to the onset of CD (172). Within the GI environment, adherent-invasive E. coli has strong proficiency in forming biofilms, with its EPS matrix enhancing bacterial survival for several days post-infection (173).

B. fragilis, a highly prevalent bacterial species in the human gut, can form dense biofilms on the mucosal membrane of the colon (174–176). Within the B. fragilis group, enterotoxigenic B. fragilis is of particular clinical relevance, representing the most common carcinogenic bacteria and a pivotal pathogen implicated in the initiation and progression of colorectal cancers (177–179).

Clostridioides difficile is an anaerobic spore-forming Gram-positive bacterium that can colonize the human intestine asymptomatically in healthy individuals. It is an opportunistic enteropathogen known to form biofilms in the intestine and cause post-antibiotic and nosocomial diarrhea. Alongside its capacity for spore and persister cell formation, biofilm formation contributes to recurrent C. difficile infection (CDI) by serving as a protective reservoir for the pathogen against antibiotics (180). Post-antibiotic overgrowth of C. difficile frequently leads to CDI symptoms in initially asymptomatic individuals. C. difficile overgrowth correlates with elevated levels of TcdA and TcdB, two toxins produced by the pathogen that drive the clinical CDI manifestations (181–183). CDIs pose a substantial burden on society, with the highest prevalence among healthcare-associated infections in the USA, averaging a case management cost of ~$42,000 per case (184) and representing the leading cause of antibiotic-based diarrhea (185, 186). The ability of C. difficile to form biofilms has been confirmed in a study where over 50% of clinical isolates from stool samples of CDI patients produced biofilms in vitro (187). These biofilms have been implicated in the pathogenesis and high reoccurrence rate of CDIs (188,187). In C. difficile, the biofilm phenotype is also associated with metabolic changes, and adhesins such as the surface adhesin CD2831 as well as type IV pili subunits including PilA1 are upregulated, likely contributing to biofilm formation (189). These biofilm-producing capabilities and the clinical relevance of C. difficile make it a good model strain to study biofilm-specific characteristics, including mucosal interactions (85). Additionally, a subset of CDI patients exhibit increased levels of Fusobacterium sp. (190). When cocultured, F. nucleatum and C. difficile aggregate, enhancing extracellular polysaccharide production and biofilm formation (190). Although F. nucleatum is associated with tumorigenesis and colorectal cancers, it does not form mucosal biofilms in mice (191, 192).

Salmonella is another significant pathogen capable of forming biofilms within the human intestine, thereby facilitating chronic infections (65). The bacteria can colonize the intestines of various animals, where it may cause diarrhea or persist asymptomatically, as observed in chickens (65). Contaminated meat and its consumption by humans is a common route for pathogenic transmission. In 2017 alone, there were an estimated 535,000 global cases of Salmonella infections, posing a serious public health threat (193). The formation of biofilms by Salmonella plays a significant role in its robust antimicrobial resistance, complicating treatment strategies (194). The ability of distinct Salmonella spp. to form biofilms was also demonstrated in vitro (195), and the expressed EPS matrix contributes to the long-term survival outside the host (65).

Campylobacter is another formidable foodborne pathogen associated with gastroenteritis and post-infectious IBS. Biofilm formation is believed to play a significant role in its persistence and survival mechanisms, particularly in animals and during transmission to humans (81, 196, 197). Similar to C. difficile, Campylobacter biofilms have been extensively studied in vitro, providing valuable insights into both internal (e.g., metabolism, enzymatic processes, and QS) and external factors (e.g., nutrients, co-cultures, atmospheric conditions, and surface materials) that influence biofilm growth and establishment (81, 85–87).

Overall, the ability of intestinal pathogens to form biofilms poses significant clinical challenges as they complicate eradication efforts, render antibacterial therapies ineffective, and increase the risk of disease recurrence and transmission through the food chain. Given the mounting evidence of disease relevance of gut biofilms in IBD, IBS, and colorectal cancers, along with their socioeconomic burden, it is critical to better understand and define the role of biofilms in disease development and progression. This includes investigating how biofilms evade and resist our innate immune response and exploring novel therapeutic interventions. The following sections will delve into these aspects.

THE INTESTINAL HOST DEFENSE SYSTEM

The average adult human gut has an impressive surface area of 200–300 m² (compared to ~2 m² of skin), vital for nutrient absorption, water uptake, and waste elimination. The gut also is an integral part of the immune system (Fig. 3). Constant exposure to a diverse array of microorganisms and foreign substances helps to train and refine the immune system, enhancing it to recognize and respond to potential threats more effectively. Central to this process is the gut’s ability to discriminate between beneficial entities, such as food ingredients and commensal bacteria, and harmful antigens, such as pathogenic bacteria, viruses, and toxins. This discrimination predominantly occurs at the mucosal surface, where an extensive network of cells orchestrates the activities of the mucosal immune system, ensuring the maintenance of homeostasis and defense against invading pathogens.

Fig 3.

The intestinal host defense system. The GI mucosa consists of the mucus layer, the epithelium, the lamina propria, and the muscularis mucosa. The epithelium comprises inter alia Paneth cells (predominantly in the crypts) and goblet cells. Paneth cells secrete antimicrobial peptides into the lumen, which play a crucial role in the host’s defense against bacterial invasion. Goblet cells produce a thick layer of protective mucus. Multi-potent stem cells are located at the base of the GI crypts. The lamina propria comprises several immune cells, including macrophages, T cells, and dendritic cells.

The intestinal barrier

The human intestine comprises four main layers (listed with increasing distance from the gut lumen), each with distinct functions contributing to the digestive process and overall gut health: (i) the mucosa, (ii) the submucosa, (iii) the muscularis propria, and (iv) the serosa.

1. The intestinal mucosa is the innermost layer, lining the lumen of the GI tract. It is in constant contact with the gut microbiota, providing a key barrier against the invasion of harmful microorganisms. This layer is essential for digestion, absorption, protection against pathogens, immune defense, and sensory functions and comprises the mucus layer, epithelium, lamina propria, and muscularis mucosa (Fig. 2 and 3). Among these, the mucus layer is the first layer of defense against pathogens as it represents the first contact point during biofilm formation. The epithelium includes specialized cells such as enterocytes for absorption, Paneth cells for host defense, goblet cells for mucus production, and enteroendocrine cells for hormone production. These cells are tightly interconnected via desmosomes, tight junctions, and adherens junctions, forming a robust protective layer. The lamina propria comprises connective tissue, supportive vessels, and immune cells such as lymphocytes, macrophages, and dendritic cells. The muscularis mucosa consists of a thin muscular layer of smooth muscle cells, which, when contracting, leads to compression at the bottom of the crypt and expulsion of the crypt content.

2. The submucosa is a layer of connective tissue that contains blood vessels, lymphatics, and nerves. It provides structural support, nourishment, transportation of absorbed substances, innervation, and protection, contributing to the proper functioning of the digestive system.

3. The muscularis propria is a layer of smooth muscle, responsible for peristaltic contractions that move luminal content (food items) through the intestine.

4. The serosa comprises connective tissue forming the outermost layer. It secretes a lubricating fluid called serous fluid, which facilitates the movement of the digestive organs during digestion and peristalsis (198).

The intestinal mucosa undergoes constant renewal, driven by rapidly proliferating cells and stem cells within the intestinal crypts within the small intestine and colon mucosa. This renewal process is remarkably fast, with mucus being replaced within hours and epithelial cells migrating from the crypt to the villous tip in less than five days. Apoptosis at the villous tip culminates in this swift turnover, releasing apoptotic material into the mucus layer. Disruptions in the intestinal mucosal barrier, such as inflammation or epithelial damage, can lead to increased permeability, allowing pathogens to enter the bloodstream. Such a breach triggers immune responses and contributes to the development of various diseases and conditions (199). The intestinal mucosa also hosts various types of rapidly proliferating cells, each with unique functions contributing to gut health and immune defense:

1. Enterocytes are the most abundant cells in the intestine, crucial for maintaining the gut barrier function. They secrete antimicrobial peptides (AMPs) and proteins to prevent bacterial invasion and kill pathogens. They work in conjunction with specialized immune cells such as dendritic cells, macrophages, and lymphocytes present in the gut-associated lymphoid tissue (200, 201). Additionally, enterocytes transport immunoglobulin A, the most important antibody in the gut, which neutralizes pathogens and allergens, modulates the microbiota, and promotes immune tolerance (202).

2. Globet cells are secretory cells that produce mucin proteins to form the protective layer over the intestinal epithelium. This mucous layer is a physical barrier protecting the intestinal cells from damage and invasion by harmful bacteria and other pathogens. Goblet cells are particularly abundant in the colon, where the mucous layer is especially thick. Goblet cells store mucins in granules within their cytoplasm and release them in response to various stimuli, including bacterial products. Goblet cells also secrete other factors, including the trefoil factor family (203, 204) and AMPs, contributing to gut protection and microbial balance.

3. Paneth cells are specialized cells in the small intestinal crypts of Lieberkühn that can secrete AMPs such as defensins to prevent overgrowth of harmful bacteria and other pathogens. Dysfunction or loss of Paneth cells is associated with IBD and small bowel bacterial overgrowth.

4. Enteroendocrine cells are specialized cells scattered throughout the lining of the small intestine. These cells indirectly contribute to gut defense against pathogens by secreting substances such as somatostatin, which modulates immune cell function and balances pro- and anti-inflammatory responses.

5. M cells are specialized epithelial cells located in the lymphoid tissues of the small intestine, such as Peyer’s patches. M cells possess a unique structure, enabling them to take up antigens and particles from the gut lumen and transport them to the underlying lymphoid cells to initiate immune responses (205). However, some pathogens evolved to exploit the M cells’ antigen uptake mechanism to gain access to the underlying lymphoid tissue and spread to other parts of the body (206).

6. Intestinal stem cells reside at the base of the crypts and have the remarkable ability to self-renew and differentiate into various cell types. Continuously dividing, intestinal stem cells give rise to progenitor cells, contributing to the constant renewal and maintenance of the intestinal epithelium.

Immunological responses against pathogenic bacteria

The gut mucosal immune system begins its development during fetal growth, marked by the emergence of vital structures like Peyer’s patches, along with the production of functional B and T cells, goblet cells, and Paneth cells in the colon and small intestine, starting as early as 10–16 weeks after gestation. During birth, the newborn encounters maternal microbes from the vaginal canal, skin, and feces, profoundly shaping the composition of the child’s microbiota composition and influencing their immune response. Throughout life, the immune system undergoes diverse changes related to aging and external factors, including dietary, stress, and bacterial-derived factors, all of which impact immune function (207). Age-related changes in the immune system result in reduced protection against infections and cancer, as well as prolonged and heightened inflammation.

The immune system is an important component of the host defense mechanism, including both the adaptive and innate immune systems. The innate immune system does not generate immunological memory or provide long-term protection against subsequent infections by the same pathogen. Instead, it provides immediate protection through various mechanisms, such as physical barriers, AMPs, and phagocytic cells. Neutrophils play an essential role in controlling bacterial infection in the intestine. They are the first line of defense against bacterial invasion and are recruited to the site of infection shortly after the bacterial invasion. In the intestine, neutrophils can kill luminal microorganisms that translocate across the intestinal epithelium and invade the mucosa through various mechanisms, including the production of reactive oxygen species and the formation of neutrophil extracellular traps (208, 209). Macrophages and dendritic cells are other innate immune cells in the subepithelial intestinal mucosa. Macrophages are phagocytic cells that engulf and digest microorganisms, foreign substances, and cellular debris. They also produce pro-inflammatory cytokines that contribute to the activation of the adaptive immune response. Dendritic cells are specialized antigen-presenting cells that capture antigens and present them to T cells to initiate an adaptive immune response. In the intestinal mucosa, dendritic cells are crucial for maintaining immune tolerance to commensal bacteria while still being able to mount an immune response against pathogens (210, 211). The innate immunity system uses toll-like receptors to recognize conserved pathogen-associated molecular patterns on the surface of bacteria and other microorganisms, triggering an inflammatory response and the production of cytokines and chemokines that attract immune cells to the site of infection.

The adaptive immune system provides a more specific response to pathogenic bacteria by producing antibodies and activating T and B cells. This system responds slower but generates long-lasting immunity to specific pathogens through memory cells. B lymphocytes are primarily known for their ability to produce and secrete antibodies, which can neutralize bacterial toxins and prevent colonization by pathogenic bacteria. B lymphocytes can also secrete cytokines such as interleukin (IL)-4, IL-10, and IL-21 that help regulate immune responses, whereas T lymphocytes are the primary source of pro-inflammatory cytokines such as interferon gamma, tumor necrosis factor alpha, and IL-17. These cytokines help to activate macrophages, promote the recruitment of other immune cells, and enhance the bactericidal activity of the immune system (208, 209). Secretory immunoglobulin A antibodies (sIgAs) are another component of adaptive immunity and play a crucial role in the mucosal immune system by preventing the attachment and colonization of pathogenic bacteria on the epithelial surface, thereby reducing the chances of infection. sIgAs achieve this by coating the surface of the bacteria, preventing them from adhering to the host epithelium, and neutralizing their virulence factors (212, 213). Additionally, sIgAs can agglutinate bacteria, preventing their translocation across the intestinal epithelial barrier. sIgA coating is also essential for controlling commensal gut bacteria to prevent overgrowth and maintain a balanced microbial community (214).

The role of antimicrobial gut peptides

Various AMPs play a crucial role in the human gut as an immediate defense against bacterial invasion. Expression of AMPs is tightly regulated in response to microbial presence, mediated by pattern recognition receptors that detect specific microbial molecules, such as lipopolysaccharides, flagellin, and peptidoglycans. Activation of these pattern recognition receptors triggers downstream signaling pathways, culminating in the production and release of AMPs to combat microbial threats. These small cationic peptides can directly kill or inhibit the growth of bacteria and trigger a defensive immune response. Their effectiveness against bacteria stems from being positively charged supporting interactions with negatively charged components on bacterial cell membranes. Through adopting an amphipathic conformation, AMPs can integrate into the lipid bilayer of bacterial membranes, causing structural disruption and permeabilization, leading to leakage of essential cell components and eventual cell death (215, 216). Commensal bacteria also play a role in bolstering host defenses through the production of AMPs called bacteriocins. Bacteriocins inhibit the growth and virulence of pathogenic bacteria, further fortifying the mucosal barrier. Additionally, certain commensal bacteria can stimulate the host immune system to ramp up AMP production, providing an additional layer of protection against pathogens (217). Impairment of AMP expression or function can lead to abnormal host responses to infection and contribute to the pathogenesis of IBD (218). For example, dysregulation of the host immune response to the gut can decrease the expression of AMPs, resulting in a compromised protective barrier and a heightened susceptibility to bacterial invasion and subsequent inflammation (Table 1) (217, 218).

TABLE 1.

Antimicrobial peptides and proteins produced by intestinal barrier cells

Antimicrobial peptides and proteins	Type of cells producing them
Lysozyme, Secretory phospholipase A2, C-type lectins, Human α-defensin 5 (HAD5) and α-defensin 6 (HAD6)	Paneth cells
Human α-defensins 1–4 (HAD1-4), Lipocalin 2	Neutrophils
Cathelicidin LL-37	Enterocytes
Human α-defensin 5 (HAD5)	Intermediate cells
Human β-defensins 1–4 (HBD1-4)	Effector B cells

Although AMPs are structurally diverse, one of the common structural features is their cationic and hydrophobic composition. Basic amino acids, lysine, and arginine are more abundant in AMPs, whereas acidic amino acids, aspartic acid, and glutamic acid are less abundant (219). Cationic residues support electrostatic interactions with lipopolysaccharides, while hydrophobic residues support interactions with the fatty acid chains of the membrane of Gram-negative bacteria (220). This enables them to efficiently penetrate and disassemble the bacterial cell membrane’s lipid bilayer, leading to leakage-induced cell lysis (210, 211). Several models, such as the barrel-stave (221), toroidal pore (222), or carpet (223) model, describe these membrane interactions (224).

Defensins

Defensins are cysteine (Cys)-rich AMPs (3.5–4.5 kDa) with three highly conserved intramolecular disulfide bonds. These disulfide bonds stabilize a tertiary conformation predominantly of β-sheet that confers antimicrobial activity and protease resistance. Defensins are subdivided into α- and β-defensins based on their disulfide bond pattern (225–228). The disulfide bond pattern is conserved in α-defensins and follows a Cys^I−Cys^VI, Cys^II−Cys^IV, and Cys^III−Cys^V connectivity, whereas that in β-defensins follows a Cys^I–Cys^V, Cys^II–Cys^IV, and Cys^III–Cys^VI connectivity.

Human α-defensins (HADs) are ~4-kDa cationic and amphiphilic peptides with broad-spectrum antimicrobial activity against Gram-negative to Gram-positive bacteria, mycobacteria, and fungi (229, 230). HAD1–4 are secreted by neutrophils, human monocytes, and natural killer cells and modulate innate immunity against infections. HAD5 and HAD6 are expressed in Paneth cells of the human duodenum, jejunum, and ileum (231). Among them, HAD5 is not expressed in the normal adult colon due to the lack of Paneth cells but is observed in metaplastic Paneth cells in the colon of IBD patients (231, 232). Structurally, HAD consists of a triple-stranded β-sheet structure arranged into antiparallel β-sheet, seven- to eight-amino acid residue long loop connecting β1 and β2, the β-hairpin formed by β2 and β3 sheets, and two termini with one to four amino acids located close to each other. These structural elements are confined to their relative orientations by three disulfide bridges and a salt bridge formed by the side chains of arginine and glutamic acid. The last two cysteine residues are adjacent, whereas the first two are separated by one amino acid. HAD1–3 have identical sequences except for the N-termini (Fig. 4). Despite having similarities in tertiary structure and overall charge, α-defensins exhibit different host defense functions. For example, HAD5 exhibits broad-spectrum microbicidal activity, whereas HAD6 self-assembles into a web-like structure that captures bacteria in the intestinal lumen, thereby preventing bacterial invasion of the host epithelium and subsequent dissemination to other organs (233–235).

Fig 4.

Human α- and β-defensin sequences, their disulfide pattern, and representative structures. (A) Six human α-defensin amino acid sequences. (B) Four human β-defensin amino acid sequences. (C) Structure of human α-defensin 4 (HAD4). (D) Structure of human β-defensin 2 (HBD2). Cysteine residues are highlighted in red; red lines represent conserved disulfide linkages; disulfide bonds are presented in yellow.

Human β-defensins (HBDs) 1–4 comprise 36–47 amino acids (236–238). HBD1 is present in high levels in the colon and ileum and low levels in the duodenum and jejunum (239–241). By contrast, HBD2–4 are only marginally expressed in the small and large intestines (239, 242–244). The expression of HBD1 in the ileal and colonic epithelium is not altered in UC (245), but the expression of HBD2 is significantly increased in the inflamed colonic epithelium of IBD patients (246). The expression of HBD3 and HBD4 is increased in the colonic crypts of UC but not CD patients (242). HBD2 has potent antimicrobial activity against enteric Gram-negative bacteria P. aeruginosa and E. coli but is only bacteriostatic against Gram-positive S. aureus at low ionic strength (247, 248). HBD3 has efficacy against enteric Gram-negative bacteria such as P. aeruginosa and E. coli, as well as Gram-positive bacteria like S. aureus and Streptococcus pyogenes (249). On the other hand, HBD4 exhibits a broad-spectrum antimicrobial activity (250). HBD1 displays poor antimicrobial activity, but once the disulfide bonds get reduced via thioredoxin, it displays potent activity against Gram-positive commensal bacteria Lactobacillus and Bifidobacterium spp. (251, 252).

Electrostatic interactions between the electronegative microbial membranes and cationic β-defensin peptides mainly initiate the antimicrobial mechanisms of β-defensins. The human intestinal membranes are rich in sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine and generally neutral in net charge. Therefore, β-defensins interact less with the intestinal membrane (253). Due to their electrostatic affinity, β-defensins accumulate and target microorganism surfaces at the site of infection rather than the host membrane. After the interaction of β-defensins, the microbial surface causes leakage of ions and metabolites, ensuing depolarization, loss of membrane-coupled respiration, impaired peptidoglycan synthesis integrity, and ultimately, cell death (254).

Cathelicidin LL-37

Cathelicidins are essential in innate and adaptive immunity and considered natural antibiotics in mammals (255, 256). LL-37 is the only cathelicidin peptide expressed in humans (257, 258) and derives from the C-terminal part of the 18-kDa precursor protein hCAP18 through proteolytic cleavage (258). LL-37 was discovered in human neutrophil-specific granules (259) but also expressed in macrophages, monocytes, mast cells, and epithelial cells prone to microbial infection (260–262). LL-37 is positively charged, rich in hydrophobic amino acids, and displays a random coiled conformation in water that becomes α-helical at high ion concentration and pH (263). LL-37 does not have cysteine residues nor does it form β-sheets (the classical feature of the defensin family). LL-37 is potent against both Gram-positive and Gram-negative bacteria (264, 265), and the antibacterial activity correlates with α-helical content and net-positive charge (264, 266). LL-37 can also inhibit the toxicity of lipopolysaccharides (267, 268), a constituent of the cell wall of Gram-negative bacteria accountable for causing sepsis (269, 270). In addition to its antimicrobial activity, LL-37 displays chemotactic activity for mast cells, neutrophils, and monocytes and can alter the expression of chemokines and chemokine receptors (271, 272).

Bacteriocins

Bacteriocins are AMPs produced by gut bacteria (e.g., Ligilactobacillus salivarius) contributing to the defense against pathogens. Bacteriocins are typically 30–60 amino acids long, ribosomally synthesized, and highly heterogeneous in their size, structure, and mechanism of action (273). They are present throughout the human GI tract, with 317 microbial genomes encoding bacteriocins of classes I (44%), II (38.6%), and III (17.3%) (Table 2).

TABLE 2.

Bacteriocin classes, their examples and antibacterial spectrum (274–279)

Class	Subclass	Bacteriocin	Producer strain	Antibacterial spectrum
I	Ia	Nisin A	Lactococcus lactis	Gram-positive
		Epidermin	Staphylococcus epidermidis
		Gallidermin	Staphylococcus gallinarum
	Ib	Lacticin 481	Lactococcus lactis CNRZ481	Gram-positive
		Lactosin S	Latilactobacillus sakei L45
		Cytolysin	Enterococcus faecalis
		Salivaricin A	Streptococcus salivarius 20P3
		Carnocin U149	Carnobacterium piscicola U149
II	IIa	Pediocin PA-I	Pediococcus acidilactici	Gram-positive Gram-negative
		Acidocin A	Lactobacillus acidophilus TK9201
		Leucocin A	Leuconostoc gelidum
	IIb	Lactococcin G	Lactococcus lactis	Gram-positive
		Lactococcin Q	Lactococcus lactis
		Plantaricin NC8	Lactiplantibacillus plantarum
	IIc	Lactococcin A	Lactococcus cremoris 9B4 Lactococcus cremoris LMG2130 Lactococcus lactis WM4	Gram-positive
		Divergicin A	Carnobacterium divergens LV13
		Acidocin B	Lactobacillus acidophilus M46
		Circularin A	Clostridium beijerinckii
III	Produced by Gram-positive	Lysostaphin	Staphylococcus simulans	Gram-positive Gram-negative
		Lactacin A and B	Lactobacillus acidophilus
		Helveticin J	Lactobacillus helveticus 481
		Helveticin M	Lactobacillus crispatus
	Produced by Gram-negative	Pyocins	Escherichia coli	Gram-positive Gram-negative
		Salmocins	Salmonella sp.

Class I bacteriocins, also known as lantibiotics (<5 kDa), are small, heat-resistant peptides with unusual amino acids, such as lanthionine and 3-methyllanthionine, and unsaturated amino acids, such as dehydroalanine and 2-amino isobutyric acid (280). This class is divided into two subclasses, Ia and Ib, depending on the charge. Subclass Ia, such as nisin, are positively charged flexible screw-shaped molecules that act via pore formation within the bacterial membrane, whereas subclass Ib includes negatively charged globular and structurally rigid peptides that interfere with cellular enzymatic reactions such as those involved in cell wall synthesis (281). Nisin is the only bacteriocin approved for food applications by the World Health Organization since 1969. Further, it is also accepted as a biopreservative ingredient by the European Union (282).

Class II contains heat-stable non-lantibiotic peptides without unusual amino acids. This peptide class is <10 kDa and is divided into four subclasses (283). They have an amphiphilic helical structure, allowing them to insert into the bacterial membrane, leading to depolarization and death. Subclass IIa is characterized by high antimicrobial activity against the Listeria sp.. The C-terminal of these peptides contains one or two α-helices, and the N-terminal has a β-sheet configuration. The C-terminal interacts with the bacterial cell membrane, promoting pore formation and death. Subclass IIb includes heterodimeric bacteriocins, which consist of two peptides. Members of this subclass need both peptides for full antibacterial activity, and individual peptides are devoid of or have little antibacterial potential. Lactococcin G is the first discovered bacteriocin of this group, and its antimicrobial activity depends on both α- and β-peptides. These subclasses show their antibacterial action via a decrease in the intracellular ATP concentration, ultimately leading to bacterial cell death (284, 285). Subclass IIc are N- to-C-terminal cyclic peptides that carry two transmembrane segments that facilitate the formation of pores in the target cells (286). Subclass IIc bacteriocins are produced in the human GI tract by Gram-positive bacteria of the phylum Firmicutes. Lactobacillus and Lactococcus are well-known bacterial genera that produce subclass Ilc bacteriocins (287).

Class III bacteriocins are larger (>30 kDa) and include heat-labile peptides. These bacteriocins can be categorized as either lytic or non-lytic and are further divided into two classes based on their origin, arising from either Gram-positive or Gram-negative bacteria. Regarding their mode of action, the peptide’s C-terminal region recognizes the bacterial cell, while the N-terminal portion of the molecule acts as an endopeptidase (282). Their antimicrobial activity is attributed to their ability to disrupt the bacterial cell wall (288, 289).

THERAPEUTIC OPPORTUNITIES AND STRATEGIES

The challenge of antibiotic resistance and current biofilm treatment

Antimicrobial resistance of biofilms

The discovery of penicillin by Fleming in 1928 marked a breakthrough in treating bacterial infections, ultimately saving an incredible number of human lives (290). However, improper applications and overuse of antibiotics have led to the emergence of antibiotic resistance (291–294). The post-antibiotic era is rapidly approaching and stands as one of the most significant health threats to modern society (294–297). Biofilms play a crucial role in this development, with microbial cells within biofilms being up to 1,000-fold more protected against the effects of antibiotics compared to their planktonic counterparts (71, 298). Biofilm’s recalcitrance to antimicrobial treatment and the host immune system contribute to the persistence of infections (144, 299, 300). Development of antibiotic resistance occurs faster in biofilms than in planktonic cells (301), and resistance mechanisms of biofilms are different and more effective from those of planktonic bacteria (71, 302–304). Resistance mechanisms include slow and incomplete drug penetration, altered chemical environments, and greater efficiency for signaling pathways prone to increased protection, drug target site mutations, lower cell permeability, efflux pumps, drug modifying enzymes, and drug-neutralizing proteins (305–310).

The EPS matrix is the most visible distinction between biofilms and planktonic bacteria and represents a considerable hurdle for antimicrobials (49, 310, 311). A mechanistic explanation for the limited penetration is the electrostatic interactions between cationic antibiotics (e.g., tobramycin or polymyxin B) with the anionic polysaccharides and extracellular DNA in the matrix (304, 312). Moreover, antibiotic-modifying enzymes enriched in the biofilm’s outer layers can inactivate antibiotics before reaching the bacteria (311).

Slow- or non-dividing persister cells further contribute to the antibiotic resistance of biofilms. The low metabolic activity of such persister cells results in fewer antibiotic targets and higher survival (55, 313–315), and persister cells can resume growth after antibiotic treatment, causing relapses.

Effective multi-drug efflux pumps can transport antibiotics out of the bacterial cell, thereby preventing intracellular drug accumulation and cell death (316). In P. aeruginosa, more PA1874–1877 efflux pumps were expressed in biofilms compared to the planktonic state of the same bacterial species (317, 318), a finding that was further observed in E. coli biofilms with several genes encoding putative efflux and transport proteins being overexpressed compared to planktonic cells (319, 320). Experimental evolution studies on mutations upon antibiotic exposure revealed efficient efflux pump upregulation and higher fitness of biofilm bacteria compared to planktonic bacteria (310). Efflux pumps might furthermore play a role in biofilm formation by affecting the secretion of intercellularly produced EPS components and signaling molecules for QS (319). This hypothesis is supported by efflux pump inhibitors being potent biofilm inhibitors (319, 321, 322). Recent cryo-electron microscopy investigations contributed to characterizing multi-drug efflux pumps in the foodborne gut pathogen Campylobacter (323), enhancing our understanding of antibiotic resistance mechanisms and aiding drug development (324, 325).

Another pronounced biofilm antibiotic-resistance mechanism is the distribution and uptake of resistance genes by horizontal gene transfer, which can defend the global biofilm population (307, 326). Polymicrobial biofilms present an ideal environment for horizontal gene transfer, considering the sessile state, the high cellular density, and the increased genetic competence and accumulation of genetic material (309). A key mechanism of horizontal gene transfer is plasmid-mediated bacterial conjugation (326, 327). Plasmids carrying multiple drug-resistance genes can conjugatively transfer genes between plasmid-bearing cells (donors) and plasmid-free cells (recipients) (328–330). Conjugative gene transfer requires close contact with the bacterial cells and minimal shear forces; since both requirements are facilitated with the biofilm matrix, horizontal gene transfer occurs with higher frequency and efficiency in biofilms than in planktonic cells (307, 326, 330). Moreover, when antibiotics were tested at sublethal concentrations, the mutation rate in biofilm bacterial cells was higher compared to their planktonic counterparts, providing further evidence that antibiotic resistance is achieved faster in biofilms than in planktonic cells (301, 331). Taken together, the biofilm architecture strongly promotes antibiotic tolerance, enhancing overall survival.

Bacterial biofilms are highly prevalent in IBD and IBS patients (57% in IBS patients, 34% in UC patients, 22% in CD patients, compared to 6% in healthy patients) (39), and mucus-invasive colonic biofilms have an increased appearance in colorectal cancer patients (50% of colorectal cancer patients compared to 13% of healthy patients) (143, 166). Current treatment strategies still rely on conventional antibiotics despite the complexity of biofilm-associated infections and their increased antibiotic resistance. Biofilms require higher dose treatments over an extended period, often without satisfactory results, leading to an elevated risk of side effects. Other treatment adjustments include using a combination of antibiotics or the co-administration of adjuvants to increase the potency of antibiofilm effects (332–334). However, antibiotic overuse, especially if not effective, further drives the development of antibiotic resistance (291–294). The presence of persister cells is another factor contributing to treatment failures and severe relapses due to resistance-associated mutations (335–337). Drugs to selectively clear biofilms is a relatively new strategy that has yet to gain clinical and pharmaceutical traction, with no drug on the market or in clinical development that can achieve this. In addition to biofilm-targeted treatment approaches, improving our understanding of biofilm architectures and compositions and their role in disease development is crucial as this knowledge will aid the development of new therapeutic strategies.

Biofilm targets and antibiofilm strategies

Improving antimicrobial potency against biofilms often involves increasing biofilm drug penetration and matrix disruption capabilities. Targeting biofilm cells remains challenging since these matrix-embedded cells display unique resistance against conventional antibiotics (310, 338). Some combinatorial and multi-targeting strategies show promising effects against biofilms (339, 340). A gut-specific antibiofilm treatment should ideally be biofilm-specific, not affect commensal bacteria, be stable in the gut, and be gut-restricted to reduce off-target effects. The following sections discuss promising biofilm targets and therapeutic approaches (Fig. 5). These include (i) suppression of bacterial adhesion, (ii) inhibition of biofilm formation, (iii) disruption of mature biofilms, and (iv) initiation of biofilm dispersal (341–344). Finally, host immunization and microbiome modulation strategies are discussed, and several compound classes with promising antibiofilm activities are assessed, including small molecules, natural products, AMPs, nanoparticles (NPs), and vaccines.

Fig 5.

Molecular targets and therapeutic strategies to combat gastrointestinal biofilms. Several targets for therapeutic interventions are outlined, including the biofilm matrix, regulatory networks, and molecular targets and mechanisms of biofilm cells.

Biofilm targeting strategies

Preventing bacterial adhesion and biofilm formation

Early-stage biofilm intervention and relapse prevention strategies concentrate on disrupting the interactions of microorganisms with the surface to hinder bacterial adhesion and surface colonization. Inhibiting the expression of bacterial adhesins, such as curli or pili, emerges as a potent approach to prevent bacterial surface attachment (345–347). The chaperone-usher pathway, for instance, is a conserved secretion system that regulates pilus biogenesis. Several small-molecule pili biogenesis inhibitors have been developed to interfere with this pathway to prevent surface adhesion (348). Other strategies use antiadhesive agents such as mannosides to target the production of adhesins to reduce intestinal colonization (349). After surface colonization, the microcolonies produce the EPS matrix, which can be inhibited by natural products, small molecules, AMPs, or NPs (350–354). This interference not only inhibits bacterial cell-cell adhesion and biofilm formation but also destabilizes the biofilm architecture.

Eradication or dispersal of mature biofilms

Mature biofilms have a complex, multi-layered EPS matrix that increases cellular adhesion and overall protection. Adding d-amino acids, such as d-leucine, d-methionine, d-tryptophan, and d-tyrosine, to mature biofilms displays promising antibiofilm activity by suppressing biofilm formation and triggering biofilm disassembly (355, 356). Acidic d-amino acids, such as d-aspartic acid and d-glutamic acid, promote biofilm dispersal and have synergistic effects with ciprofloxacin (357). Various surfactants such as viscosin (358), surfactin (359), and putisolvin (360) weaken the cell-surface, cell-matrix, and cell-cell interactions and initiate biofilm detachment and disassembly. Another promising compound class includes quaternary ammonium amphiphiles, characterized by a lipophilic alkyl chain and hydrophilic quaternary ammonium groups. Extensive exploration of diverse structures with varying alkyl chain lengths and numbers of positive charges has demonstrated strong biofilm eradication effects (361). Optimization of nitroxide functionalized antibiotics resulted in a ciprofloxacin-dinitroxide conjugate with good E. coli biofilm eradication at 12.5 µM (362). The redox-active phenazines represent another biofilm eradication class. Halogenated phenazide analogs are non-toxic to mammalian cells and can eradicate Gram-positive bacterial biofilms (363). Colicin antibiotics, another antibiofilm compound class, display effects through species-specific targeting of adherent-invasive E. coli (364), which is strongly associated with bacterial dysbiosis in CD.

Biofilm matrix and drug penetration

Biofilm penetration of antimicrobials is often minimal and depends on the thickness of the biofilm matrix and the agent’s concentration (365). Enzymes within the matrix can inactivate antimicrobials and increase biofilm tolerance (366), while electrostatic interactions with the negatively charged extracellular DNA in the EPS limit drug diffusion (367, 368). Therapeutic agents that induce EPS matrix remodeling and activate biofilm dispersal mechanisms can transition biofilms into a motile lifestyle and increase antimicrobial susceptibility (341, 369). For example, the fatty acid messenger cis-2-decenoic acid and its synthetic analogs induce biofilm dispersal in various microbial species (370, 371). Such universal dispersing activity is very interesting for therapeutic applications. In a clinical setting, matrix-dispersive agents should be co-administered with antimicrobials to enhance biofilm removal and avoid biofilm recolonization.

Biofilm targets and therapeutic interventions

Regulatory networks: quorum sensing, cyclic-di-GMP, and (p)ppGpp

Regulatory networks such as QS, stringent response, and secondary messengers that control the behavior, function, structure, and architecture of biofilms represent other attractive drug targets. QS cell-to-cell communication assists the bacterial colony in managing their population density, swarming motility, and biofilm formation, and interfering with QS can trigger changes in bacterial behavior, including biofilm dispersal (50, 53, 372). The chemical nature of QS systems, however, varies substantially between different bacterial species, rendering it challenging to generate broadly acting QS drugs. On the other hand, species-selective modulators might benefit from reducing off-target effects in the GI environment and might be suitable for specific indications associated with a specific pathogenic biofilm-forming species (52, 94, 95). Such pathogen-directed antibiofilm therapies, without broadly impacting the healthy gut microbiota, are highly desirable and could revolutionize GI disorder treatment. The QS system of E. coli is also involved in expressing multi-drug-resistant efflux pumps. For instance, the overproduction of SdiA, a protein that regulates cell division via QS in E. coli, is linked to increased expression levels of multi-drug resistance AcrAB pump (373). Therefore, targeting SdiA allows for influencing efflux pump expression through QS manipulation. Agents that inhibit the QS microbial communication system are also called quorum quenchers (372, 374, 375). Quorum quenching not only can impact the biofilm formation process but also can manipulate the biofilm architecture to loosen the biofilm structure (372, 376, 377). Modulation of AHL-mediated QS can be achieved by interfering at three stages of the QS process: (i) signal production, (ii) signal transduction, and (iii) signal detection (354, 378). For instance, S-adenosylmethionine forms the homoserine lactone ring moiety of AHL and is a substrate of the signaling molecule producing enzyme RhII synthase. Three S-adenosylmethionine analogs (S-adenosyl-homocysteine, sinefungin, and butyryl S-adenosylmethionine) that inhibit RhII synthase and block AHL production in vitro were identified (379). Direct enzymatic degradation is another strategy to target QS signaling. Enzymes that decompose AHL signaling molecules, such as AHL-lactonase, AHL-acylase, and paraoxonase, have been investigated (380). The enzyme MomL can degrade different AHLs, leading to reduced biofilm formation and increased antibiotic susceptibility (381). LuxR-type receptors are responsible for signal detection in many Gram-negative bacteria. Currently, a strong focus lies also on developing small molecules that act competitively with the native AHL ligands of the LuxR receptor. Several AHL analogs have been developed by modifying the acyl chain or the lactone moiety to activate or inhibit the receptor (382, 383). Receptor inhibition consequently leads to a disruption of the cell-to-cell signaling cascade and transcription repression of AHL-upregulated genes.

Biofilm dispersal is a regulated process that can be triggered via different signaling pathways, leading to the disassembly of the biofilm structure. One endogenous and highly conserved dispersal signaling molecule that regulates such disassembly pathways is the nucleotide second messenger, bis-(3′−5′)-cyclic dimeric guanosine monophosphate (cyclic-di-GMP). Cyclic-di-GMP binds to diverse cellular effector molecules, including transcription factors, riboswitches, and enzymes (80). The cyclic-di-GMP has a regulatory function that promotes either biofilm formation or a planktonic lifestyle in a concentration-dependent manner (79, 384). Increased levels of cyclic-di-GMP trigger biofilm formation, and lower levels promote the planktonic phenotype and biofilm disassembly. Besides that, cyclic-di-GMP influences other cellular processes, such as flagella motility and virulence, which are involved in mammalian cell and tissue infections (80, 385). For instance, when cyclic-di-GMP binds to the Ycgr protein of E. coli, the formed complex interacts with the flagellar motor compartment, impeding its function and leading to the transition into a sessile/biofilm state (386, 387). This close relationship between cyclic-di-GMP signaling and biofilm formation and dispersal renders inhibition of cyclic-di-GMP, signaling a promising approach for therapeutic intervention (80, 388–390). Diguanylate cyclase and phosphodiesterase are two enzymes that directly impact the cellular cyclic-di-GMP concentration and are responsible for the synthesis and hydrolysis of the signaling molecule, respectively. With that in mind, there are three perspectives for manipulating cyclic-di-GMP levels: (i) inhibition of diguanylate cyclases to lower cyclic-di-GMP formation, or (ii) activation of phosphodiesterases to increase cyclic-di-GMP degradation, or (iii) effectors that conduct the signaling cascade, a challenge also tackled via high-throughput screening approaches (354, 388, 391–394). For example, two small molecules, H6-355 and H6-355-P1, were identified via high-throughput screening of a library of 50,000 synthetic compounds as potent biofilm dispersal agents that act by stimulating phosphodiesterase activity (390, 395). Endogenously synthesized nitric oxide also stimulates phosphodiesterase activity and induces biofilm dispersal, making nitric oxide-donor molecules an attractive therapeutic approach (396, 397). Nitroxides are sterically hindered nitric oxide analogs that trigger biofilm dispersal in E. coli and P. aeruginosa. The combination treatment of nitroxides with ciprofloxacin potentiates the effect (384, 398, 399), and also, the covalent linkage of the nitroxide to ciprofloxacin reduced the minimal biofilm eradication concentration compared to ciprofloxacin as a control (400). Sulfathiazole is a prominent example of a diguanylate cyclase inhibitor that prevents biofilm formation, but the acute cytotoxic effects of this compound must be considered (401).

Stringent response is another essential bacterial regulatory signaling cascade predominantly responding to environmental stress such as nutrient deprivation. The accumulation of (p)ppGpp by activating the stringent response affects biofilm formation, maintenance, and dispersal by regulating transcriptional activation on a genetic level (78, 402, 403). Modulating this signaling system inhibits biofilm formation or can trigger dispersal (108, 404, 405). Rel enzymes, for instance, are ubiquitous bacterial proteins that play a vital role in many key cellular processes, including DNA replication, transcription, and translation, and trigger the stringent response by catalyzing the synthesis of (p)ppGpp. Hence, ppGpp analogs that inhibit Rel enzymes (e.g., relacin and analogs) have been developed as stringent response modulators to inhibit biofilm formation (108, 404, 406). Peptides directly targeting ppGpp also efficiently inhibited biofilm formation (407, 408).

Efflux pumps

Bacterial cells use efflux pumps to eliminate antimicrobials, and multi-drug-resistant strains often boost drug-specific and multi-drug efflux mechanisms through efflux pump overexpression (316, 409, 410). In a biofilm context, efflux pumps are important in secreting QS molecules and EPS and are promising antibiofilm drug targets. For instance, evaluating the three efflux pump inhibitors phenylalanine-arginine β-naphthylamide, thioridazine, and 1-(1-naphtylmethyl)-piperazine against E. coli revealed that all compounds reduced biofilm formation (321). The well-characterized AcrAB-TolC efflux pump system in E. coli is known for its clearance and extrusion of aminoglycosides, fluoroquinolones, tetracycline rifampicin, and carbapenem, and this efflux pump is overexpressed in several clinical biofilm isolates (411–414). The combination of efflux pump inhibitors and antibiotics displays enhanced antibiotic activity through reducing/inhibiting drug extrusion (321). The use of pyridylpiperazine-based compounds, for instance, can potentiate antibiotic activity via inhibition of the AcrAB-TolC efflux pump (415). Artesunate, an antimalarial drug, also enhances the antimicrobial effects of β-lactam antimicrobials against E. coli by inhibiting the AcrAB-TolC efflux system (416). The antirheumatic drug auranofin affects the biofilm formation of B. fragilis by reducing the expression of the bmB3 gene, which is responsible for resistance nodulation division-type efflux pump expression (417). The interference with efflux pump expression levels also renders the opportunity to influence bacterial communication as (resistance-nodulation-division type) efflux pumps in B. fragilis control the intracellular AHL concentration and AHL extrusion (418).

Persister cells

Persister cells are slow- or non-growing (dormant) subpopulations within a biofilm. This dormant state refers to a metabolically inactive state that results in an increased tolerance against antibiotics. These cells distinctly tolerate antibiotics to well-described antibiotic resistance mechanisms, frequently because the antibiotic targets within the cell are diminished or inhibited (313, 315). Persister cells are of clinical importance not only due to their elevated antimicrobial tolerance but also for the recalcitrance of chronic infections, as these cells can drive biofilm repopulation by reverting their metabolic activity after the termination of antibiotic treatment (419). Several compounds have been identified from natural sources that can kill such persister cells (420–422). Promising candidates interfere with metabolically independent targets, such as the bacterial cellular bilayer (423–425). Interfering with mechanisms that support persister cell formation is another approach (426), as well as waking persister cells through sugar treatment (427) to reconstruct antibiotic targets followed by antibiotic treatment (426). Moreover, cis-2-decenoic acid can also revert the metabolic activity of persister cells, making them more susceptible to antibiotics (370). Another effective strategy against persister cells uses engineered bacteriophages in combination with antibiotics to enhance antibiotic susceptibility in E. coli (428). These bacteriophages are engineered to overexpress proteins LexA3 and SoxR, which suppress the SOS regulatory network (429) and oxidative stress responses in E. coli, respectively. This combined approach significantly enhances the eradication of antibiotic-resistant bacteria, persister cells, and biofilm cells by quinolones in vitro and improves the survival rates of bacteria-infected mice in vivo (428).

Natural products

The vast chemical and structural diversity in nature remains a key resource for drug discovery, including for antibiofilm leads (430, 431). Extracts from, for example, garlic (432), cinnamon (433), ginger (434), ginkgo (435), cranberry (436), and citrus fruit (437, 438) have antibiofilm properties (431, 439). The structural diversity of natural products delivers many molecules engaging with distinct biofilm targets, including microbial adhesion, bacterial aggregation, regulatory networks, and EPS production (431). Various pilicides and curlicides have been identified from natural sources, efficiently inhibiting pili- and curli-dependent biofilm attachment and formation. The flavanol myricetin, for instance, inhibits the curli-driven biofilm formation (440). The investigation of other myricetin derivatives revealed that the major catechin in green tea, epigallocatechin gallate, inhibits E. coli biofilm in the lower micromolar range. Epigallocatechin gallate suppresses curli production and expression of the curli-related proteins csgA, csgB, and csgD (441). Moreover, tannic acid kills bacteria in E. coli biofilms and inhibits the production of matrix-associated polysaccharides (442, 443). Naturally isolated biosurfactants also display antibiofilm properties. Rhamnolipids, for example, comprise a rhamnose sugar moiety linked to an alkanoic fatty acid chain, such as myrmicacin, and can inhibit the biofilm formation of several pathogens, including Streptococcus mutans and Streptococcus sanguinis (444). Zerumbone and α-humulene are other natural products that modulate efflux pumps in B. fragilis by downregulating different efflux pump-related bmeB genes, leading to suppressed biofilm formation (175).

Antimicrobial peptides

AMPs mostly derive from organisms to boost their survival (445–447). Due to their vast structural and mechanistic diversity, they form a valuable compound class to combat biofilms, particularly considering that bacteria seem less prone to developing resistance against AMPs than against conventional antibiotics (448–450). The ease of peptide synthesis supports rapid hit-to-lead optimizations compared to more challenging to synthesize small-molecule natural products (451). AMP drug lead optimization approaches include peptide cyclization/stapling, lipidation, terminus modification, and incorporating unnatural amino acids to improve drug properties and therapeutic scope (452–455). Innovative medicinal chemistry approaches are critical for AMP-based drug development, particularly considering peptides’ poor proteolytic stability (456, 457). Besides the membrane-associated activity, some AMPs also target protein and nucleic acid synthesis, efflux pump regulation, surface attachment, and the EPS matrix (224, 450). Indolicidin, for example, a 13-residue AMP from the cytoplasmic granules of bovine neutrophils, can permeate the cell wall without disrupting its integrity to inhibit bacterial DNA synthesis (458) and inhibit biofilm formation (459). Another example is the cathelicidin-derived human host defense peptide LL-37, which prevents bacterial adhesion and downregulates genes responsible for QS (460). Overall, mammalian cathelicidins potently act against biofilms by modulating host immune responses (461). Optimization approaches via defensin fragment modifications on HBD1 revealed the AMP Pam-3, which is effective against GI pathogenic biofilms without harming the commensal microbiota (462). Various efforts in establishing multi-functional AMP-antibiotic conjugates have also led to promising antibiofilm activities (453, 463).

Nanoparticles

The field of nanomedicine is rapidly evolving, bringing forth more and more promising tools to overcome hurdles in drug development and delivery, including treating bacterial biofilms (464–466). NPs range from 1 to 100 nm in size and display an exciting compound class for therapeutic applications due to their highly reactive nature and large surface area, making them suitable as drug carriers (465, 467). Metal-based NPs comprising silver, iron, copper, zinc, magnesium, and rare earth metals display antimicrobial antibiofilm properties (466, 468, 469). The generation of reactive oxygen species through NP-microbe interaction can damage the cell membranes and cellular structures (470, 471). Silver NPs, for example, exhibit antimicrobial activity by releasing silver ions (472–474). NPs can be engineered to enhance biofilm penetration based on electrostatic interactions between the electrically charged surface of the carrier system and the negatively charged matrix components (76, 341, 475, 476). Polymer NPs have been designed to have additional features over metal NPs and can be decorated with or encapsulate a drug to improve antibiofilm activity (76, 466). However, the large surface area and the high reactivity of NPs can have considerable toxic effects (477, 478).

Vaccines

Recent advancements in active host immunization and antibiofilm vaccine research show promise as a potential future treatment approach. Many of these vaccination strategies focus on antigen development derived from biofilm cells and components of the biofilm matrix, yielding encouraging outcomes (479). A pioneering study in this domain effectively targeted F. nucleatum’s outer membrane protein FomA, essential in bacterial co-aggregation and biofilm formation (480). Notably, antibodies have been engineered to target members of the DNABII family of bacterial DNA-binding proteins, abundant within the biofilm matrix, and crucial for its structural integrity. This targeted approach demonstrates the capacity to disrupt biofilms, enabling subsequent host immune-mediated or antibiotic clearance (481–484). Expanding in this area, efforts have been made to develop a chimeric peptide immunogen and humanized monoclonal antibodies to prevent and treat biofilm infections (483). Another strategy redirected the natural adaptive immune response toward immunoprotective domains, which disrupted Haemophilus influenzae biofilms in vitro and an experimental model of otitis media (482). Antibodies targeting the type IV pili of H. influenzae PilA inhibit biofilm formation and trigger biofilm dispersal, as well as make biofilm cells more susceptible to antibiotics (485). Planktonic and biofilm antigens might be required to fully clear bacterial pathogens through immunization (470). Other potential immunization strategies target cyclic-di-GMP synthesis (486). A biofilm-specific quadrivalent vaccine against S. aureus has shown efficacy in eradicating biofilms without eliminating planktonic cells (487). These outcomes present an attractive prospect for translation into GI applications to facilitate selective biofilm removal. However, careful consideration must be given to antigen selection, as the use of antigens not exclusively produced by the target pathogen may lead to unwanted side effects, potentially challenging the beneficial influence of the host’s commensal microbiota. While the economic and social benefits of vaccines and immunization programs are widely recognized, their effectiveness in treating GI disorders remains largely unexplored and necessitates future research efforts to provide further insights into their applicability.

Gut microbiota: probiotics/paraprobiotics, prebiotics, and fecal microbiome transplants

Therapeutic use of specific probiotics can prevent pathogenic colonization and recolonization, biofilm formation, and mucosal barrier degradation (50, 488–492). Probiotics are live microorganisms that can provide health benefits to the recipient (493). Intensively studied probiotics display beneficial effects in various GI disorders and associated diseases, including infectious diarrhea, antibiotic-associated diarrhea, hepatic encephalopathy, UC, and IBS (488, 492). Desirable characteristics of a probiotic strain include good GI tract adhesion and prolonged residence time, required to maximize therapeutic effects by stabilizing or regenerating healthy gut microbiota and intestinal epithelial homeostasis. For instance, non-pathogenic E. coli is a biofilm former that outcompetes enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) (490), providing a probiotic treatment approach for GI disorders driven by EPEC and ETEC infections. In addition, probiotic Lactobacillus spp. that secrete molecules with antibacterial, antiadhesion, and antibiofilm properties have been identified (494–496). Potential side effects of probiotic use include overstimulation of the immune system, systemic infections, harmful metabolic activities, and the promotion of gene transfer. To overcome these health challenges, paraprobiotics (non-viable probiotics) are used. Other microbiota treatment approaches include prebiotics. Prebiotics are food ingredients that support the growth of healthy gut bacteria. Inulin, for example, is a widely studied prebiotic that positively impacts various medical conditions such as infectious colitis, diarrhea, and IBD (497).

Imbalances in the gut microbiome and pathogenic overpopulation have also been resolved by fecal microbiota transplants from healthy donors into the gut of diseased recipients to enable the regeneration of a healthy gut microbiome. Currently, fecal microbiota transplantation is only indicated for C. difficile treatment, but clinical trials investigate its therapeutic use for other GI disorders, including IBD, which resulted in cure rates of ~85% (498). This renders fecal microbiota transplantation promising, with broad application potential for various GI disorders. Despite these results, little is known about long-term effects, and the U.S. Food and Drug Administration has issued several warnings related to follow-up infections (499–501). Quality control measurements, such as a detailed questionary screening process and clinical evaluation, before becoming a qualified fecal microbiota transplant donor, are now implemented.

While so far no biofilm-specific drug has been introduced to the market, several therapeutic agents across various compound classes demonstrate promising effects in inhibiting or eradicating biofilms by targeting a diverse range of biofilm targets and signaling pathways, supporting a growing preclinical drug pipeline (Table 3).

TABLE 3.

Summary of potential therapeutic antibiofilm strategies

Compound	Compound class	Effects	References
Small molecules
Mannosides	Small molecule with sugar moiety	Adhesion inhibition	(349)
H6-335 and H6-335-P1	Small molecule	Biofilm dispersal	(390, 395)
Nitric oxide	Diatomic gas	Biofilm dispersal	(396, 397)
Sulfathiazole	Antimetabolite drug	Biofilm inhibition	(401)
Relacing and analogs	ppGpp analog	Biofilm inhibition	(108, 404, 406)
PAβN,a thioridazine, and NMPa	Efflux pump inhibitor	Biofilm inhibition	(319, 321, 322)
Auranofin	Antirheumatic drug	Biofilm inhibition	(417)
d-Leucine, d-methionine, d-tryptophan, and d-tyrosine	d-Amino acid	Biofilm inhibition and dispersal	(355, 356)
Viscosin, surfactin, and putisolvin	Surfactant	Biofilm detachment	(358–360)
Quaternary ammonium amphiphiles	Quaternary ammonium compound	Biofilm eradication	(361)
Ciprofloxacin-dinitroxide conjugate	Nitroxide functionalized antibiotic	Biofilm eradication	(362)
Phenazines	Halogenated phenazide analog	Biofilm eradication	(363)
Colicin	Antibiotic	Biofilm inhibition and eradication	(364)
Natural products
Garlic, cinnamon, ginger, ginko, and citrus fruit extracts	Natural product extract	Biofilm inhibition and eradication	(431–439)
cis-2-Decenoic acid	Fatty acid	Biofilm dispersal	(370, 371)
Zerumbone and α-humulene	Natural product	Biofilm formation inhibition	(175)
Myricetin	Flavanol	Curli-driven biofilm formation inhibition	(441)
Rhamnolipids	Glycolipid biosurfactant	Biofilm formation inhibition	(444)
Antimicrobial peptides
Indolicidin	AMP	Biofilm inhibition	(458, 459)
LL-37	AMP (cathelicidin)	Biofilm inhibition and immune modulation	(460, 461)
Pam-3	AMP (HBD1 analog)	Biofilm eradication	(462)
Nanoparticles
Metal-based nanoparticles (e.g., silver, iron, copper, zinc, magnesium, and rare earth metal)	Metal-based nanoparticle	Biofilm inhibition and eradication	(466, 468, 469)
Polymer nanoparticles with decorated or encapsulated antibiofilm drug	Polymer-based nanopaticle	Biofilm inhibition and eradication	(76, 466)
Vaccines
DNABII targeting antibodies	Antibody	Biofilm dispersion	(484)
Chimeric peptide immunogen and humanized monoclonal antibody	Peptide and antibody	Biofilm inhibition	(483)
Antibodies against type IV pilus	Antibody	Biofilm dispersion	(485)
Quadrivalent vaccine	Four different antigens	Biofilm eradication	(487)
Gut microbiota modulation
E. coli (Nissle 1917)	Probiotic	Biofilm inhibition	(490)
Lactobacillus sp.	Probiotic	Antiadhesive	(494–496)
Inulin	Prebiotic	Improves GI disorders conditions	(497)
Fecal microbiota transplantation	Fecal microbiota	Regeneration of a healthy gut microbiome	(498)

^a NMP, 1-(1-naphtylmethyl)-piperazine; PAβN, phenylalanine-arginine β-naphthylamide.

CONCLUSIONS

GI biofilms are a diverse and spatially organized polymicrobial community optimized for survival in the GI tract. They can be visible during GI endoscopy; drive GI diseases, including IBD, IBS, and gastric and colorectal cancers; and expose a potential mechanism of disease. They protect the embedded microorganisms from host defense and antibiotic strategies and display better drug resistance than planktonic bacteria. This renders biofilm-related infections hard to treat, with no biofilm-specific therapeutics on the market or in clinical development. Different therapeutic strategies that focus on critical stages or molecular targets of the biofilm life cycle are being pursued. Targeting the biofilm matrix or regulatory networks are two promising approaches. Significant knowledge gaps remain regarding the characterization of patient-derived biofilms, structural makeup, host interactions, relevance in disease development, how they can evade the host defense system, and what causes GI biofilms. Environmental factors and the excessive and inappropriate use of antibiotics are associated with dysbiosis of the intestinal microbiome, which seems to favor biofilm formation. Further research is necessary to elucidate the tipping points delineating a healthy from a diseased gut microbiome. The identification of biomarkers indicative of these tipping points holds significant value. Collecting and analyzing patient-derived biofilm samples will be essential in bridging current knowledge gaps. Together, these efforts are anticipated to significantly enhance our comprehension of gut biofilms and unveil novel opportunities for their control.

Biographies

Bernhard Jandl (University of Vienna, Austria) is a bioorganic and medicinal chemist pursuing a PhD in applied biofilm research at the University of Vienna. He obtained his Master of Science degree from the Vienna University of Technology and carried out his Master’s thesis project at the Technical University of Denmark, where he worked on novel nucleosides for the diagnosis and treatment of infectious diseases. He is interested in investigating and understanding biologically active molecules and optimizing them toward therapeutic applications. He has a strong passion for translational research covering the development of molecular diagnostic tools and future medicines and his PhD research focuses on chemical strategies for gut-specific biofilm targeting.

Satish Dighe (The University of Queensland, Australia) is an early career medicinal chemist with a strong passion for multidisciplinary and translational research. His research interests include molecular tool development for the fundamental study and biologically active molecules for infectious diseases. His research has contributed to discovering novel biologically active molecules with activity against important drug targets such as caseinolytic protease and DNA gyrase in infectious diseases.

Christoph Gasche (Medical University of Vienna, Austria) is a gastroenterologist and a Professor of Medicine at the Medical University of Vienna. He was trained in internal medicine and gastroenterology at the General Hospital Vienna and did an in-depth research training as a Visiting Professor at the University of California at San Diego (UCSD). He has over 30 years of clinical experience in gastroenterology and leads a research lab focusing on the pathogenesis of IBD, prevention of colorectal cancer, and treatment of iron deficiency. His lab was pioneering the first approach to visualize intestinal biofilms by endoscopy and to link this intestinal biofilm phenotype to IBS and IBD.

Athanasios Makristathis (Medical University of Vienna, Austria) is the Vice Operational Director of the Microbiome Facility at the Medical University of Vienna and Deputy Head of the Division of Clinical Microbiology. He has more than 30 years of experience in culture-based and molecular diagnostics in medical microbiology and in-depth analysis of patient-derived microorganisms and has published over 100 peer-reviewed publications (80% in the top 20% of journal categories).

Markus Muttenthaler (The University of Queensland, Australia, and University of Vienna, Austria) is a medicinal chemist working at the interface of chemistry and biology with a strong focus on developing new therapeutic strategies to address unmet medical needs. He obtained his PhD at the University of Queensland (Brisbane, Australia) followed by postdoctoral stays at the Scripps Research Institute (La Jolla, California, USA) and the Institute for Research in Biomedicine Barcelona (Spain). He leads two research labs, one at the University of Queensland and one at the University of Vienna. He is an international leader in peptide drug discovery and development for therapeutic applications in gastrointestinal disorders, autism, pain, and cancer. Gastrointestinal biofilms have captured his interests due to their clinical relevance, underexplored link to gastrointestinal pathologies, and therapeutic opportunities.