Loss of outer membrane protein A (OmpA) impairs the survival of Salmonella Typhimurium by inducing membrane damage in the presence of ceftazidime and meropenem.

Abstract

Salmonella enterica serovar Typhimurium is one of the significant non-typhoidal Salmonella serovars that causes gastroenteritis. The rapid development of antimicrobial resistance necessitates studying new antimicrobials and their therapeutic targets in this pathogen. Our study aimed to investigate the role of four prominent outer membrane porins of S. Typhimurium, namely OmpA, OmpC, OmpD and OmpF, in developing resistance against ceftazidime and meropenem. The antibiotic-mediated inhibition of bacterial growth was determined by measuring the absorbance and the resazurin assay. DiBAC4 (Bis-(1,3-Dibutylbarbituric Acid)Trimethine Oxonol), 2,7-dichlorodihydrofluoroscein diacetate (DCFDA) and propidium iodide were used to determine the outer membrane depolarization, reactive oxygen species (ROS) generation and subsequent killing of Salmonella. The expression of oxidative stress-response and efflux pump genes was quantified by quantitative RT-qPCR. HPLC was done to determine the amount of antibiotics that entered the bacteria. The damage to the bacterial outer membrane was studied by confocal and atomic force microscopy. The in vivo efficacy of ceftazidime and meropenem were tested in the C57BL/6 mouse model. Deleting ompA reduced the survival of Salmonella in the presence of ceftazidime and meropenem. Massive outer membrane depolarization and reduced expression of oxidative stress-response genes in S. Typhimurium ΔompA hampered its growth in the presence of antibiotics. The enhanced uptake of antibiotics and decreased expression of efflux pump genes in S. Typhimurium ΔompA resulted in damage to the bacterial outer membrane. The clearance of the S. Typhimurium ΔompA from C57BL/6 mice with ceftazidime treatment proved the role of OmpA in rendering protection against β-lactam antibiotics. OmpA protects S. Typhimurium from two broad-spectrum β-lactam antibiotics, ceftazidime and meropenem, by maintaining the stability of the outer membrane.