Study On The Antibacterial Activity And Mechanism Of Squaramides

Objective:In this study,the antibacterial activity of novel squaramides was screened to solve the infection caused by methicillin-resistant Staphylococcus aureus(MRSA).The antibacterial activity and spectrum of the screened squaramide SA2 were systematically evaluated in vitro,and the in vivo effect of SA2 on infectious animal models induced by drug-resistant bacteria was verified.Furthermore,the specific antibacterial mechanism was explored,aiming to provide new therapeutic strategies and agents against infection caused by resistant bacteria.Methods:In order to develop novel antibacterial agents against MRSA-induced infection,we synthesized several new squaramides and evaluated the antibacterial activity by the minimum inhibitory concentration(MIC)assay.In addition,the antibacterial profiles of SA2 were further determined by growth curves,bactericidal curves and an established mouse model of MRSA-induced skin infection.Scanning electron microscopy and transmission electron microscopy were used to observe the effect SA2 on MRSA morphology.The effects of SA2 on gene expression in MRSA were detected and determined by transcriptome sequencing and fluorescence quantitative PCR,and the potential antibacterial mechanism was analyzed accordingly.Results:1)The investigated squaramide SA2 showed promising antibacterial activity in vitro against standard sensitive and clinically isolated resistant strains of Staphylococcus aureus(S.aureus),Staphylococcus epidermidis(S.epidermidis),and Enterococcus faecalis(E.faecalis),all of which were Gram-positive bacteria.The MICs of SA2 against the standard sensitive strain of S.aureus(ATCC 29213)and the resistant strains MRSA(ATCC 43300),MRSA(USA 300),MRSA(Mu 50)and a methicillin-resistant S.epidermidis MRSE(XJ1537)were 2-4μg/m L,4μg/m L,4μg/m L,4μg/m L,and 4μg/m L,respectively.2)The compound SA2 completely inhibited the survival of the standard sensitive strain of S.aureus(ATCC 29213)and the resistant strains MRSA(ATCC 43300),MRSA(Mu 50)and a clinical MRSE(XJ 1537)under the concentrations of 2-fold and 4-fold MIC within 4 to 8 h.3)The compound SA2 showed significant growth inhibition in a concentration-dependent manner against the standard sensitive strain of S.aureus(ATCC29213)and the resistant strains MRSA(ATCC 43300),MRSA(Mu 50)and a clinical MRSE(XJ 1537),ranging from 1-to 4-fold MIC.4)The compound SA2 led to morphological changes in MRSA such as rough surface,wrinkled cell membrane,bacterial fragmentation and significant reduction of bacterial population.5)The compound SA2 effectively reduced the area of skin ulceration in MRSA-induced skin infection models,decreased the bacterial load at the infection site,attenuated the symptom,and shorten the course of disease.6)The compound SA2 altered the gene expression of alanine dehydrogenase(ald),and SA2 could further disrupt the NAD~+/NADH balance,leading to the generation of oxidative damage induced by the increased NAD~+/NADH ratio.Conclusion:1)The compound SA2 has promising antibacterial activity against Gram-positive bacteria as well as resistant strains,among which the standard sensitive strain of S.aureus(ATCC 29213)displays the highest susceptibility with MIC of 2-4μg/m L.However,SA2has little effect on Gram-negative bacteria.2)The disruption of bacterial membranes is the potential mechanism that the compound SA2 exerts antibacterial activity against S.aureus.3)In the treatment of MRSA-induced infection,the compound SA2 shows promising in vivo antimicrobial activity and therapeutic effect.Compared to model controls,the compound SA2 effectively attenuates the symptom,decreases the area of skin ulceration,and shortens the course of disease.4)The compound SA2 altered the gene expression of alanine dehydrogenase ald,disturbed the NAD~+/NADH balance,and led to the generation of oxidative damage caused by the increased NAD~+/NADH ratio.