Species-Specific Optimisation and Environmental Regulation of Biofilm Formation in Enterobacter cloacae: Inhibitory Role of Glucose in Biofilm Development

Bacterial biofilms are prevalent in clinical environments, contributing to persistent infections associated with medical devices. Enterobacter cloacae forms biofilms on nonliving surfaces, leading to drug-resistant, recurrent infections that are difficult to treat. Biofilm development in Enterobacter species, including E. cloacae, occurs through five stages: reversible attachment, irreversible attachment, microcolony formation, maturation and dispersal. Initial attachment is mediated by adhesins, including fimbriae and lipopolysaccharides, which interact with surfaces. This is followed by secretion of an extracellular polymeric substance matrix composed of polysaccharides, extracellular DNA and proteins, providing stability and protection. This study aimed to establish a standardised in vitro 0.5% crystal violet staining method to quantify biofilm production in E. cloacae isolates and classify isolates by biofilm-forming capacity. Biofilm was quantified by optical density at 570–600 nm. A 96-well microtiter plate assay quantified biofilm formation in 40 E. cloacae strains collected between July 2021 and April 2023. Growth conditions were optimised, including culture media, fixation techniques and additive concentrations of glucose and sodium chloride. Brain heart infusion broth was optimal, and heat fixation was superior; glucose had no effect, whereas 1%–2% sodium chloride enhanced biofilm production. These findings improve understanding of environmental regulation of biofilm formation and microbial persistence across habitats.