Norfloxacin salts of carboxylic acids curtail planktonic and biofilm mode of growth in ESKAPE pathogens

• Published in: Journal of applied microbiology 2018-02, Vol.124 (2), p.408-422
• Main Authors: Lowrence, R.C., Ramakrishnan, A., Sundaramoorthy, N.S., Shyam, A., Mohan, V., Subbarao, H.M.V., Ulaganathan, V., Raman, T., Solomon, A., Nagarajan, S.
• Format: Article
• Language: English
• Subjects: Acids; Acinetobacter baumannii - drug effects; Acinetobacter baumannii - growth & development; Acinetobacter baumannii - physiology; Animals; Anti-Bacterial Agents - chemistry; Anti-Bacterial Agents - pharmacology; antibacterial; antibiofilm; Antiinfectives and antibacterials; Bacteria; Biofilms; Biofilms - drug effects; Carboxylic acids; Carboxylic Acids - chemistry; Carboxylic Acids - pharmacology; Colonization; Deoxyribonucleic acid; DNA; DNA topoisomerase; Drug resistance; Enterobacter - drug effects; Enterobacter - growth & development; Enterobacter - physiology; Enterococcus faecium - drug effects; Enterococcus faecium - growth & development; Enterococcus faecium - physiology; Escherichia coli - drug effects; Escherichia coli - growth & development; Escherichia coli - physiology; ESKAPE; Gram-Negative Aerobic Bacteria - drug effects; Gram-Negative Aerobic Bacteria - growth & development; Gram-Negative Aerobic Bacteria - physiology; Gram-Negative Bacterial Infections - microbiology; In vivo methods and tests; Infections; Integrity; Klebsiella pneumoniae - drug effects; Klebsiella pneumoniae - growth & development; Klebsiella pneumoniae - physiology; Membrane permeability; Microbial Sensitivity Tests; Minimum inhibitory concentration; Molecular docking; Muscles; Norfloxacin; Norfloxacin - chemistry; Norfloxacin - pharmacology; norfloxacin salts; Pathogens; Permeability; Pseudomonas aeruginosa; Pseudomonas aeruginosa - drug effects; Pseudomonas aeruginosa - growth & development; Pseudomonas aeruginosa - physiology; Salts; Staphylococcal Infections - microbiology; Staphylococcus aureus - drug effects; Staphylococcus aureus - growth & development; Staphylococcus aureus - physiology; Zebrafish
• ISSN: 1364-5072; 1365-2672
• DOI: 10.1111/jam.13651

Aims To enhance the antimicrobial and antibiofilm activity of norfloxacin against the planktonic and biofilm mode of growth in ESKAPE pathogens using chemically modified norfloxacin salts. Methods and Results Antimicrobial testing, synergy testing and time‐kill curve analysis were performed to evaluate antibacterial effect of norfloxacin carboxylic acid salts against ESKAPE pathogens. In vivo efficacy to reduce bacterial bioburden was evaluated in zebrafish infection model. Crystal violet assay and live–dead staining were performed to discern antibiofilm effect. Membrane permeability, integrity and molecular docking studies were carried out to ascertain the mechanism of action. The carboxylic acid salts, relative to parent molecule norfloxacin, displayed two‐ to fourfold reduction in minimum inhibitory concentration against Staphylococcus aureus and Pseudomonas aeruginosa, in addition to displaying potent bacteriostatic effect against certain members of ESKAPE pathogens. In vivo treatments revealed that norfloxacin tartrate (SRIN2) reduced MRSA bioburden by greater than 1 log fold relative to parent molecule in the muscle tissue. In silico docking with gyrA of S. aureus showed increased affinity of SRIN2 towards DNA gyrase. The enhanced antibacterial effect of norfloxacin salts could be partially accounted by altered membrane permeability in S. aureus and perturbed membrane integrity in P. aeruginosa. Antibiofilm studies revealed that SRIN2 (norfloxacin tartrate) and SRIN3 (norfloxacin benzoate) exerted potent antibiofilm effect particularly against Gram‐negative ESKAPE pathogens. The impaired colonization of both S. aureus and P. aeruginosa due to improved norfloxacin salts was further supported by live–dead imaging. Conclusion Norfloxacin carboxylic acid salts can act as potential alternatives in terms of drug resensitization and reuse. Significance and Impact of the Study Our study shows that carboxylic acid salts of norfloxacin could be effectively employed to treat both planktonic‐ and biofilm‐based infections caused by select members of ESKAPE pathogens.