Comparative evaluation of antimicrobial peptides: effect on formation, metabolic activity and viability of <i>Klebsiella pneumoniae</i> biofilms.

Abstract

<h4>Introduction</h4><i>Klebsiella pneumoniae (K. pneumoniae)</i> is a major human nosocomial infectious agent and an important veterinary pathogen, frequently resistant to various antibiotics. It causes diseases such as pneumonia, urinary tract infections, surgical wound infections and septicemia. Biofilm formation of <i>K. pneumoniae</i> promotes persistent infection and contributes to resistance against antimicrobial agents. The objective of this study was to comparatively evaluate the effect of selected AMPs on the formation, metabolic activity and viability of <i>Klebsiella pneumoniae</i> biofilms of veterinary and human origin.<h4>Methods</h4>Biofilm formation of three <i>K. pneumoniae</i> strains was quantified using the crystal violet assay and visualized by scanning electron microscopy (SEM). The inhibitory effects of eight different AMPs on the formation and metabolic activity of <i>K. pneumoniae</i> biofilms, as well as on planktonic growth, were examined using crystal violet, resazurin and broth microdilution assays, respectively. The effect on living and dead bacteria in mature biofilms was investigated using the fluorescent dyes SYTO™ 9 and propidium iodide. In addition, the distribution of rhodamine B-labeled peptide DJK-5 in mature biofilms of strain 17349 was visualized by confocal laser scanning microscopy (CLSM).<h4>Results</h4>Biofilm formation was confirmed for all three <i>K. pneumoniae</i> strains. Depending on the strain, we found that planktonic growth was affected by the AMPs DJK-5, DJK-6, Onc72, and Onc112. Biofilm formation of all three strains was inhibited by hbD3, LL-37, DJK-5, and DJK-6, with biofilm mass reduced to less than 40% of the untreated control. In addition to the inhibition of biofilm formation, a reduction in the metabolic activity of the biofilm-associated bacteria was also observed. These four AMPs also showed an effect on mature biofilms by reducing the number of both viable and dead bacteria in 22 h-old biofilms. Rhodamine B-labeled DJK-5 took 7 h to visibly accumulate in the planktonic bacteria. Multi-layered biofilm aggregations were mainly negative for rhodamine B-labeled DJK-5, even 44 h after AMP treatment, indicating that certain parts of mature <i>K. pneumoniae</i> biofilms are not accessible for this AMP.<h4>Conclusion</h4>In conclusion, we found differences in the effect of AMPs on biofilms including both increases and decreases in biofilm mass and viability.