In vitro antibacterial and antibiofilm activity of phycocyanin against Klebsiella pneumoniae: effects on growth kinetics and antibiotic susceptibility

The increasing clinical burden of antimicrobial-resistant Klebsiella pneumoniae , together with its capacity for biofilm formation and persistence, has intensified the search for alternative and adjunctive antimicrobial strategies. Phycocyanin is a cyanobacteria-derived phycobiliprotein with emerging biological and antimicrobial properties; however, its activity against K. pneumoniae , particularly across planktonic, biofilm, and antibiotic-response phenotypes, remains insufficiently characterized. Klebsiella pneumoniae ATCC 700,603 was used as the experimental strain, and phycocyanin obtained from Sigma was evaluated for antibacterial and antibiofilm activity. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by broth microdilution and subsequent subculture, respectively. Bacterial growth kinetics were monitored for 72 h under sub-inhibitory phycocyanin exposure, while direct antibacterial activity was evaluated using an agar well diffusion assay. Biofilm formation was assessed using the crystal violet microtiter-plate method. Changes in antibiotic inhibition-zone diameters following sub-inhibitory phycocyanin exposure were evaluated using ampicillin, ceftriaxone, and imipenem. All experiments were performed in triplicate. Phycocyanin exhibited measurable antibacterial activity against K. pneumoniae , with MIC and MBC values of 10 and 20 mg/mL, respectively. Growth-kinetic analysis showed comparable bacterial growth during the initial hours, followed by sustained attenuation of growth in phycocyanin-exposed cultures from approximately 6 h onward. In the agar well diffusion assay, inhibition-zone diameters increased in a concentration-dependent manner, from approximately 7 mm at the lowest tested amount to 12 mm at the highest. Phycocyanin also inhibited biofilm formation in a concentration-dependent manner, with maximum inhibition reaching 41.3% at the highest tested concentration. Following sub-inhibitory phycocyanin exposure, increased inhibition-zone diameters were observed for ampicillin, ceftriaxone, and imipenem, with the greatest changes generally occurring after 24 h of exposure. Phycocyanin demonstrated antibacterial and concentration-dependent antibiofilm activity against K. pneumoniae ATCC 700,603 and altered the bacterial antibiotic-response phenotype following sub-inhibitory exposure. The observed effects on bacterial growth, biofilm formation, and antibiotic inhibition-zone diameters suggest that phycocyanin may have potential as an adjunctive antimicrobial or antibiofilm agent. However, these findings do not establish pharmacodynamic synergy or reversal of antimicrobial resistance. Further studies using quantitative combination assays, resistant clinical isolates, mechanistic approaches, and in vivo models are required to determine the translational relevance of these effects.