Increasing antimicrobial susceptibility of MDR Salmonella with the efflux pump inhibitor 1-(1-Naphthylmethyl)-piperazine

Authors: PRICE, EUAN - OAK RIDGE INSTITUTE FOR SCIENCE AND EDUCATION (ORISE); Dassanayake, Rohana; Bearson, Shawn

Submitted to: Biochemical and Biophysical Research Communications
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/12/2023
Publication Date: 8/6/2023
Citation: Price, E.D., Dassanayake, R.P., Bearson, S.M. 2023. Increasing antimicrobial susceptibility of MDR Salmonella with the efflux pump inhibitor 1-(1-Naphthylmethyl)-piperazine. Biochemical and Biophysical Research Communications. 668:49-54. https://doi.org/10.1016/j.bbrc.2023.05.035.
DOI: https://doi.org/10.1016/j.bbrc.2023.05.035

Interpretive Summary: Salmonella is a leading cause of human foodborne illness, and multidrug-resistance (MDR; resistance to =3 antimicrobial classes) in Salmonella is a prevalent issue worldwide. In the United States, recent human outbreaks associated with consumption of food animal products have involved Salmonella isolates that are resistant to multiple antibiotics, including those that have been used in animal production such as tetracycline. Because few new antibiotics are being developed, it is becoming increasingly necessary to improve the effectiveness of existing antibiotics. One mechanism that bacteria use to develop resistance to an antibiotic (like tetracycline) is to rapidly pump the antibiotic out of the bacterial cell before it can exert its antimicrobial effect. We evaluated a pump inhibitor (efflux pump inhibitor, EPI) called 1-(1-Naphthylmethyl)-Piperazine (NMP) for its ability to recover tetracycline sensitivity in four MDR outbreak isolates of Salmonella. NMP reduced resistance in tetracycline-resistant Salmonella to levels that would no longer classify the isolates as ‘resistant’ to tetracycline, thereby restoring the effectiveness of the antibiotic against the MDR Salmonella isolates. Thus, efflux pump inhibitors, such as NMP, have the potential to contribute to novel treatment options that are effective against antibiotic resistance in highly relevant bacteria.

Technical Abstract: Salmonella is a common foodborne pathogen that can exhibit multidrug resistance (MDR; resistance to =3 antimicrobial classes). Therefore, the development of new preventative measures against MDR Salmonella is highly important. Bacterial antibiotic resistance is commonly mediated by efflux pumps. In this study, two compounds that block efflux pump activity, 1-(1-Naphthylmethyl)-Piperazine (NMP) and Phenylalanine-arginine ß-naphthylamide (PaßN), were tested with the antibiotic tetracycline to determine if a synergistic reduction in resistance could be achieved in tetracycline-resistant Salmonella. Both efflux pump inhibitors (EPIs) reduced Salmonella resistance to tetracycline by 16 to 32-fold in several tetracycline resistant isolates. For example, the tetracycline minimum inhibitory concentration (MIC) for MDR Salmonella enterica serovar I 4,[5],12:i:- USDA15WA-1 (SX 238) was 256 µg/mL. However, in the presence of NMP, the MIC dropped to 8 µg/mL which is below the Clinical Laboratory Standards Institute (CLSI) breakpoint for tetracycline resistance in Salmonella (=16 µg/mL). Confocal and transmission electron microscopy revealed NMP-mediated damage to Salmonella membranes, implying that the EPI disrupts membrane morphology which can lead to cell death; however, this effect was dependent on NMP concentration, as NMP blocked efflux activity without the membrane-disrupting effect at a lower concentration. These findings suggest that the use of EPIs can reduce the MIC of tetracycline and restore the effectiveness of the antibiotic against tetracycline-resistant Salmonella.