Escherichia coli is a Gram-negative, facultatively anaerobic bacterium commonly found in the gut of humans and animals, where most strains are harmless and play a beneficial role in the gut microbiome. However, pathogenic strains of E. coli are an important cause of disease in humans (Braz et al., 2020). These pathogenic strains possess virulence factors that allow them to attach to host tissues, evade immune responses, and produce toxins, leading to various clinical consequences ranging from mild gastrointestinal discomfort to severe conditions such as hemolytic uremic syndrome (HUS), urinary tract infections (UTIs), and sepsis (Kaper et al., 2004).

E. coli is a well-known biofilm-forming bacterium, particularly in its pathogenic forms, and this characteristic contributes significantly to its role in persistent infections (Ballén et al., 2022). In healthcare settings, E. coli biofilms are a major cause of catheter-associated urinary tract infections (CAUTIs), ventilator-associated pneumonia, and other device-related infections, as biofilms can easily form on indwelling medical devices (Hamdy Mohamed et al., 2024). Catheter-associated urinary tract infections account for more than 40 % of hospital-acquired infections and more than 80 % of all urinary tract infections (Milo et al., 2019). Uropathogenic E. coli (UPEC), which are frequently responsible for UTIs, can also invade bladder epithelial cells and form intracellular biofilm-like communities, that lead to persistent infections and frequent relapses after antibiotic treatment (Goller and Seed, 2010).

The biofilm matrix of E. coli is a complex and dynamic structure composed of extracellular polymeric substances (EPS), which include polysaccharides, proteins, lipids, and extracellular DNA (eDNA). Major constituents include polysaccharides such as cellulose, poly-β-1,6-N-acetyl-D-glucosamine (PGA) and colanic acid, a capsular heteropolysaccharide with hexasaccharide repeating units, consisting of glucose, fucose, galactose and glucuronic acid (Sande and Whitfield, 2021). Polysaccharides, such as the capsular polysaccharides (CPS) and the lipopolysaccharide (LPS) layer found on the outer membrane of E. coli, serve as a protective barrier against the host’s immune defenses and protect the bacteria from phagocytosis and complement-mediated lysis (Arredondo-Alonso et al., 2023, Liu et al., 2019). Polysaccharides and oligosaccharides are thus key factors in the pathogenesis of E. coli, promoting bacterial survival, colonization and formation of biofilms, which are often resistant to conventional therapies. Targeting polysaccharides can disrupt the protective barriers, including the biofilm, that help the bacteria resist host defenses and make them more susceptible to clearance by the immune system and antibiotics (Lim et al., 2019, Park and Park, 2021, Wu et al., 2019).

An effective approach to combat E. coli biofilms is the use of enzymes that target the major biofilm components. Glycoside hydrolases (e.g., cellulase) degrade polysaccharides, proteases (e.g., proteinase K, subtilisin) degrade structural proteins, and DNases (e.g., DNase I) target extracellular DNA, weaken the biofilm structure and improve the efficacy of antibiotics (Lim et al., 2019, Van Nederveen and Melton-Celsa, 2024, Villa et al., 2015). The disintegration of the biofilm matrix supports antibiotic and immune responses and helps in the treatment of persistent E. coli infections.

Polygalacturonases belong to the group of pectinolytic enzymes that are widely used in various biotechnological industries (Jayani et al., 2005). The commercially available pectinase from Aspergillus niger, which is known for its polygalacturonase activity, has previously been successfully used to prevent biofilm formation in the laboratory strain E. coli K-12 (Villa et al., 2015). Since polygalacturonases can utilize glucuronic acid oligosaccharides as substrates (Tan et al., 2020), LPS, colanic acid and other CPS containing glucuronic acid are possible targets in the E. coli biofilm (Klein et al., 2013, Sande and Whitfield, 2021).

The role of CPS in biofilm formation is complex and not fully understood due to the variability of CPS production between different bacterial strains and stages of infection. It is generally believed that CPS inhibit bacterial adherence and biofilm formation (Beloin et al., 2008), but they play an important role also in biofilm maintenance (Limoli et al., 2015). In uropathogenic E. coli, CPS is involved in the formation of intracellular biofilm-like communities in bladder cells (Goller and Seed, 2010). In addition, LPS contributes to the adhesive properties of the biofilm by interacting directly with adhesion molecules on the cell surface, which helps the bacteria to adhere more effectively to surfaces and initiate the process of biofilm formation (Sharma et al., 2016). Colanic acid primarily contributes to biofilm maturation and matrix thickening (Prigent-Combaret et al., 2000). Recent studies have shown that inhibition of colanic acid biosynthesis effectively prevents biofilm formation, demonstrating its important role even in the early stages of biofilm formation (Zhang and Poh, 2018).

In this study, we describe a novel polygalacturonase named PG-BG31 from Pedobacter sp. BG31 that effectively inhibits E. coli biofilm formation. The recombinantly expressed enzyme was purified, characterized and its ability to inhibit biofilm formation in clinical E. coli strains was investigated.