Background: As of 2022, 80% of all documented microbial infections are biofilm-associated: communities of microorganisms adhered to a surface and enclosed in a complex extracellular polymeric substance (EPS). The EPS acts as a physical barrier protecting the bacteria from antimicrobial agents and host immune responses. To combat this hurdle, the application of glycoside hydrolases (GH) has been investigated due to their ability to cleave particular structural polysaccharides within the EPS, thus breaking down the protective barrier and improving antibiotic clearance. While various studies demonstrate the capacity of GHs to improve antibiotic efficacy against biofilms in combination, there is clear differential success between these treatments depending on the GH and antibiotic chosen. Due to the overlap of GH targets and antibiotic structures, it is imperative to ensure that the antibiotics in combinatorial treatments are not degraded by the GH. Methods: This study aimed to screen the GH α-amylase produced from Aspergillus oryzae (AO) and Bacillus subtilis (BS), combined with various antibiotics from different classes, charges, and mode of actions by determining MICs. against the bacterium Pseudomonas aeruginosa (PA) of 6 antibiotics with or without α-amylase and treat 2-day PA biofilms with antibiotics with or without GHs. Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) stability assays and Differential Scanning Fluorimetry (DSF) were conducted to determine antibiotic and GH degradation as well as antibiotic sequestration. Results: Increased MICs in the presence of GHs as well as decreased antibiotic clearance against 2-day biofilms were suggestive of antibiotic degradation. LC-MS/MS stability assays of tetracycline and ciprofloxacin in the presence and absence of α-amylase further demonstrated the α-amylase-mediated antibiotic sequestration. Differential scanning fluorimetry (DSF) assays confirmed α-amylase-antibiotic interactions. Conclusions: This study suggests that α-amylase is capable of degrading and sequestering a variety of antibiotics, and the degree to which these phenomena occur varies depending upon the source of the GH. As a potential treatment for biofilm-associated infections, it is imperative that the GH + antibiotic combinations are determined compatible prior to clinical use.

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Pseudomonas aeruginosa is a prominent biofilm-forming pathogen associated with chronic infections that are difficult to eradicate due to the protective biofilm matrix. Inducing biofilm dispersion has emerged as a potential therapeutic strategy. Although researchers have compared selected biofilm dispersion mechanisms, the characteristics of cells generated by mechanistically distinct release strategies, particularly enzymatic matrix degradation, remain uncharacterized. In this study, we compared P. aeruginosa cells released from biofilms using four distinct dispersion mechanisms: An external signaling molecule (cis-2-decenoic acid; cis-DA), a nutrient cue (glutamate), an environmental toxin (sodium nitroprusside; SNP), and a matrix-degrading enzyme (amylase). Released cells exhibited distinct antibiotic susceptibility, attachment, and gene expression depending on the release mechanism. Glutamate-released cells exhibited greater tobramycin tolerance and attachment, whereas glutamate and amylase-released populations exhibited differences in the expression of selected genes. Additionally, the release mechanism influenced subsequent biofilm recovery and reformation, and cis-DA significantly enhanced tobramycin-mediated killing of mature biofilms. Collectively, these findings demonstrate that the mechanism used to induce release may influence subsequent bacterial behavior and antibiotic susceptibility. These findings highlight the importance of considering release mechanism when developing dispersion-based strategies for treating biofilm-associated infections.