Synergism of cationic antimicrobial peptide WLBU2 with antibacterial agents against biofilms of multi-drug resistant Acinetobacter baumannii and Klebsiella pneumoniae

Infection and Drug Resistance Dovepress open access to scientific and medical research Infection and Drug Resistance downloaded from https://www.dovepress.com/ by 88.198.20.149 on 25-Sep-2019 For personal use only. Open Access Full Text Article ORIGINAL RESEARCH Synergism of cationic antimicrobial peptide WLBU2 with antibacterial agents against biofilms of multi-drug resistant Acinetobacter baumannii and Klebsiella pneumoniae This article was published in the following Dove Press journal: Infection and Drug Resistance Samer Swedan 1 Zaina Shubair 1 Ammar Almaaytah 2 1 Jordan University of Science and Technology, Department of Medical Laboratory Sciences, Irbid, Jordan; 2 Jordan University of Science and Technology, Department of Pharmaceutical Technology, Irbid, Jordan Correspondence: Samer Swedan Jordan University of Science and Technology, Department of Medical Laboratory Sciences, PO Box 3030, Irbid 22110, Jordan Tel +962 2 720 1000 ext. 26886 Fax +962 2 720 1087 Email Introduction Bacterial resistance to conventional antimicrobials is increasing year after year and is a global health emergency.1 Biofilm formation is a common mechanism to overcome the activity of antimicrobials and the host immune response. Due to treatment difficulties, biofilm-associated infections have led to significant rates of morbidity and mortality among the community and the health-care settings.2,3 Hence, 2019 submit your manuscript | www.dovepress.com Infection and Drug Resistance 2019:12 2019–2030 DovePress © 2019 Swedan et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms. php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). http://doi.org/10.2147/IDR.S215084 Powered by TCPDF (www.tcpdf.org) Purpose: The activity of the cationic antimicrobial peptide WLBU2 was evaluated against planktonic cells and biofilms of multi-drug resistant (MDR) Acinetobacter baumannii and Klebsiella pneumoniae, alone and in combination with classical antimicrobial agents. Methods: Control American Type Culture Collection (ATCC) strains and MDR clinical isolates of A. baumannii and K. pneumoniae were utilized. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of WLBU2 alone and in combination with antimicrobials were determined by classical methods. The Calgary biofilm device was used to determine the minimum biofilm eradication concentration (MBEC). The MTT assay was used to determine the cytotoxicity of agents on eukaryotic cells. The electrophoretic mobility shift assay was used to evaluate the ability of WLBU2 to bind bacterial DNA. Results: The WLBU2 MIC and MBC values were identical indicating bactericidal activity. The MIC/MBC values ranged from 1.5625 to 12.5 µM. At these concentrations, Vero cells and human skin fibroblasts were viable. The MBEC of WLBU2 ranged from 25 to 200 µM. A significant loss of eukaryotic cell viability was observed at the MBEC range. The combination of sub-inhibitory concentrations of WLBU2 with amoxicillin-clavulanate or ciprofloxacin for K. pneumoniae, and with tobramycin or imipenem for A. baumannii, demonstrated synergism, leading to a significant decrease in MIC and MBEC values for some isolates and ATCC strains. However, all combinations were associated with considerable loss in eukaryotic cells’ viability. WLBU2 did not demonstrate the ability to bind bacterial plasmid DNA. Conclusion: WLBU2 in combination with antimicrobials holds promise in eradication of MDR pathogens. Keywords: antimicrobial peptide, synergy, combination therapy, biofilm, multi-drug resistance, bacteria Dovepress Infection and Drug Resistance downloaded from https://www.dovepress.com/ by 88.198.20.149 on 25-Sep-2019 For personal use only. Swedan et al developing safe alternate therapies with diverse mechanisms of action research is urgently needed.4–6 Antimicrobial peptides (AMPs) are immune effector molecules that are being considered as potential alternatives for conventional antimicrobial agents. AMPs demonstrate multiple mechanisms of action, including formation of transmembrane pores, which lead to lysis of microorganisms, the deterioration of bacterial viability by interfering with cell wall biosynthesis,7,8 the disruption of biochemical processes, and enhancement and activation of the immune response. Some AMPs are effective against biofilm and multi-drug resistant (MDR) bacteria, with rapid killing kinetics.9 Hence, AMPs have advantages over antimicrobial agents, as the bacteria are less likely to produce and transfer resistance genes against the peptides.7 Most AMPs are cationic peptides with an amphipathic structure that selectively targets bacterial membranes via electrostatic forces.10 WLBU2 is a 24-residue engineered cationic amphipathic peptide (eCAP) that consists of only three types of amino acids: tryptophan, valine, and arginine. The WLBU2 sequence was rationally designed by amino acid substitutions from precursor peptides to have an ideal amphipathic helix conformation to maximize antimicrobial properties, while minimizing epithelial cell cytotoxicity.11–13 Acinetobacter baumannii is a significant Gramnegative MDR pathogen associated with urinary tract infections, pulmonary infections, wound infection, and infections of other tissues and organs. A. baumannii has the ability to form biofilms, leading to increased virulence.14,15 Klebsiella pneumoniae is a Gram-negative, encapsulated, opportunistic pathogen associated with pneumonia, meningitis, urinary tract infections, sepsis, and surgical wound site infections. K. pneumoniae is the main cause of health care-associated Klebsiella infections, mostly involving the respiratory and urinary tracts.16 This study provides insights on the activity of WLBU2 against planktonic and biofilm-producing MDR A. baumannii and K. pneumoniae, and in combination with classical antimicrobial agents. This study also examined the potential toxicity of WLBU2antimicrobials combinations on human skin fibroblast cells and Vero cells, and the ability of WLBU2 to bind bacterial DNA. The findings pave the way for future investigations that can potentially culminate in the development of treatments for difficult to treat and biofilm-associated infections. Materials and methods The study was approved by Jordan University of Science and Technology (JUST) research committee. Requirement for approval by the institutional review board of JUST was waived as the study did not involve the study of human subjects (...truncated)