(M0930-04-20) An Innovative Dual Action Antibiotic Adjuvant Strategy Using Ionic Liquid toward Synergistic Restoration of Activity in MDRO and Enhancing Antimicrobial Activity Against MRSA

Purpose: Antimicrobial resistance has emerged as a critical issue that needs to be addressed, it develops when microorganisms develop resistance and tend to stop responding to treatments in humans. [1] Multi Drug Resistant Organisms (MDRO) such as Methicillin Resistant Staphylococcus Aureus (MRSA) and gram-negative bacteria are nonresponsive to various classes of antimicrobial agents thereby limiting the diverse treatment options for patients. These organisms are also responsible for around 23,000 deaths annually in US alone along with 70,837 inpatient deaths caused by MDRO sepsis. [2] Ionic liquids (IL) have recently surfaced as the salts possessing antimicrobial properties with varying degrees of solubility and they are used as adjuvants in this study to enhance the antibiotic activity. [3] Cetylpyridinium chloride is a quaternary ammonium compound possessing antimicrobial activity due to it’s hydrophobicity, [4] and the effective concentration against microbes is between 0.05% and 0.1%. [5] In this research Cetylpyridinium chloride is used as IL adjuvant.  This study elucidates how novel ionic liquids, either alone or in combination with current antibiotics restore their effect against MDRO, thereby enhancing antimicrobial activity.

Methods: To synthesize the Ionic Liquid- Antibiotic complex Cetylpyridinium chloride was initially dissolved in methanol and passed through an ion exchange column to form a Cetylpyridinium hydroxide solution which was then added to the Antibiotic- Colistin, similar process was performed for Amoxicillin. Bacterial strains such as S. aureus, MRSA, and both Colistin-resistant and -sensitive MCR-1 encoded E. coli were utilized to investigate bacterial growth. Colonies from the strains were collected and suspended using PBS as diluent to read the absorbance at 0.1@ 600nm. Various concentrations of IL, ranging from 1.5 µg/ml to 20 µg/ml, were tested to determine the minimum inhibitory concentration of the antimicrobial agent.  Serial dilutions were performed for each treatment group and the bacterial suspension is added to it. The treated bacteria are now incubated at 37°C, and the absorbance values are read at 600 nm at various time points. After incubating the cells for 4hours the samples were taken into a centrifuge tube and the suspension was centrifuged, and the pellet was collected and preserved in glutaraldehyde. Electron microscope was used to study the bacterial surface. To monitor bacterial growth curves against Colistin sensitive & resistant E.Coli, S.Aureus and MRSA the groups established include a control group, an antibiotic group, antibiotic-Ionic liquid group and an Ionic liquid-only group. All groups were monitored from 4 hours up to overnight for both strains of bacteria.

Results: The antibiotic adjuvant effects on E. coli membrane have shown physical changes in bacteria when treated with different antibiotics. While untreated cells showed normal shape of bacteria, E. coli treated with Colistin and IL adjuvant were clumped and showed damaged cell integrity under the electron microscope. There were minimal changes such as elongation when treated with colistin alone and IL adjuvant alone. Based on the growth curve results for both Colistin resistant and sensitive bacteria, it is observed that the IL when combined with antibiotic Colistin a strong synergistic effect was observed after 24hours indicating a notably lower bacterial growth i.e p < 0.0001. However, the results also show that the IL alone has moderate effect when compared with combination of IL and antibiotic in inhibiting the growth of both sensitive and resistant bacteria. Similar effects were observed when Amoxicillin was added to antibiotic adjuvant with peak effects at 4hours i.e  p < 0.0001. The data obtained suggests that the novel antibiotic adjuvant IL possesses the bactericidal activity and restores colistin action against MDRO.

Conclusion: Ionic liquids possessing antimicrobial properties are now emerging as effective adjuvants. The in-vitro assessment of the Novel antibiotic IL demonstrated its dual-purpose functionality as an adjuvant for possessing bactericidal activity and exerting synergistic activity against E. Coli strains when combined with Colistin. Quantitatively, the most significant effect was observed at 24hrs for IL and Colistin complex and at 4hrs for IL and Amoxicillin complex, mitigating the antibiotic resistance. These results successfully demonstrated that the IL and antibiotic complex combat the antimicrobial resistance effectively.

References: [1] Tang KWK, Millar BC, Moore JE. Antimicrobial Resistance (AMR). Br J Biomed Sci 2023;80:11387. https://doi.org/10.3389/bjbs.2023.11387.
[2] Burnham JP, Olsen MA, Kollef MH. Re-estimating annual deaths due to multidrug-resistant organism infections. Infect Control Hosp Epidemiol 2019;40:112–3. https://doi.org/10.1017/ice.2018.304.
[3] Guzmán L, Parra-Cid C, Guerrero-Muñoz E, Peña-Varas C, Polo-Cuadrado E, Duarte Y, et al. Antimicrobial properties of novel ionic liquids derived from imidazolium cation with phenolic functional groups. Bioorganic Chemistry 2021;115:105289. https://doi.org/10.1016/j.bioorg.2021.105289.
[4] Maris P. Modes of action of disinfectants. Rev Sci Tech 1995;14:47–55. https://doi.org/10.20506/rst.14.1.829.
[5] Pahwa N, Kumar A, Gupta S. Short term clinical effectiveness of a 0.07% cetylpyridinium chloride mouth rinse in patients undergoing fixed orthodontic appliance treatment. The Saudi Dental Journal 2011;23:135–41. https://doi.org/10.1016/j.sdentj.2011.03.001.

Acknowledgements: Acknowledging the Central Lab Unit (CLU) in QU for the electron microscopy imaging.


Figure 01- Represents the method involved in carrying out the Growth Curve for bacterial strains.


Figure 02- Absorbance is read at 600nm indicating the bacterial growth for various treatment groups over 24 hours. A.) Colistin sensitive E.coli **** p < 0.0001 peak effects noted at 24 hours for IL and Colistin complex. B.) Colistin Resistant E.coli- Enhanced effects for IL and Colistin complex at 24 hours with p < 0.0001 C & D.) S.aureus and MRSA- Notable reductions in AMOX Adjuvant complex with p < 0.0001.


Represents the Electron microscopic imaging of the bacterial cells with different treatment groups, D.) shows the synergistic effect of colistin and the antibiotic adjuvant on MCR-1 positive E.coli membrane.