The activity of hydrolytic enzymes and antibiotics against biofilms of bacteria isolated from industrial-scale cooling towers.

Background: Cooling towers (CTs) are crucial to myriad industrial processes, supporting thermal exchange between fluids in heat exchangers using water from lakes and rivers as coolant. However, CT water can sometimes introduce microbial contaminants that adhere to and colonize various surfaces within the CT system. These microorganisms can form biofilms, significantly hindering the system's thermal exchange efficiency. Current treatment strategies employ oxidizing biocides to prevent microbial growth. However, despite their affordability, they do not eliminate biofilms effectively and can lead to corrosive damage within the system. Herein, we aim to devise an anti-biofilm strategy utilizing hydrolytic enzymes (such as α-amylase, glucoamylase, pectin-lyase, cellulase, protease, and DNase) alongside antibiotics (including meropenem, ciprofloxacin, gentamicin, erythromycin, chloramphenicol, and ceftriaxone) to combat microbial growth and biofilm formation in cooling systems.

Results: All enzymes reduced the development of the biofilms significantly compared to controls (p < 0.05). The polysaccharidases exhibited biomass reduction of 90%, except for pectin-lyase (80%), followed by DNAse and protease at 43% and 49%, respectively. The antibiotics reduced the biofilms of 70% of isolates in concentration of > 2 mg/mL. The minimal biofilm eradication concentration (MBEC) lower than 1 mg/mL was detected for some 7-day-old sessile isolates. The enzymes and antibiotics were also used in combination against biofilms using the modified Chequerboard method. We found six synergistic combinations, with Fractional inhibitory concentrations (FIC) < 0.5, out of the ten tested. In the presence of the enzymatic mixture, MBECs presented a significant decrease (p < 0.05), at least 4-fold for antibiotics and 32-fold for enzymes. Moreover, we characterized high molecular weight (> 12 kDa) exopolysaccharides (EPS) from biofilms of ten isolates, and glycosyl composition analysis indicated a high frequency of glucose, mannose, erythrose, arabinose, and idose across isolates EPS contrasting with rhamnose, allose, and those carbohydrates, which were detected in only one isolate.

Conclusion: The synergistic approach of combining enzymes with antibiotics emerges as a highly effective and innovative strategy for anti-biofilm intervention, highlighting its potential to enhance biofilm management practices.