Elucidation of the Mode of Action of a New Antibacterial Compound Active against Staphylococcus aureus and Pseudomonas aeruginosa

Abstract

Nosocomial and community-acquired infections caused by multidrug resistant bacteria represent a major human health problem. Thus, there is an urgent need for the development of antibiotics with new modes of action. In this study, we investigated the antibacterial characteristics and mode of action of a new antimicrobial compound, SPI031 (N-alkylated 3, 6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol), which was previously identified in our group. This compound exhibits broad-spectrum antibacterial activity, including activity against the human pathogens Staphylococcus aureus and Pseudomonas aeruginosa. We found that SPI031 has rapid bactericidal activity (7-log reduction within 30 min at 4x MIC) and that the frequency of resistance development against SPI031 is low. To elucidate the mode of action of SPI031, we performed a macromolecular synthesis assay, which showed that SPI031 causes non-specific inhibition of macromolecular biosynthesis pathways. Liposome leakage and membrane permeability studies revealed that SPI031 rapidly exerts membrane damage, which is likely the primary cause of its antibacterial activity. These findings were supported by a mutational analysis of SPI031-resistant mutants, a transcriptome analysis and the identification of transposon mutants with altered sensitivity to the compound. In conclusion, our results show that SPI031 exerts its antimicrobial activity by causing membrane damage, making it an interesting starting point for the development of new antibacterial therapies.

Repurposing AM404 for the treatment of oral infections by Porphyromonas gingivalis

“…Effect of AM404 on (a) outer (OM) and (b) inner membrane (IM) permeabilization of Porphyromonas gingivalis , as assessed by N‐phenyl‐1‐napthylamine (NPN) and SYTOX green uptake, respectively; 1× the minimum inhibitory concentration (MIC) of SPI031 was used as a positive control for outer membrane permeabilization (Gerits et al, ). Ten micrograms per milliliter melittin (MEL) was used as a positive control for inner membrane permeabilization (Park et al, ).…”

The Antistaphylococcal Lysin, CF-301, Activates Key Host Factors in Human Blood To Potentiate Methicillin-ResistantStaphylococcus aureusBacteriolysis

“…Bactericidal activity was defined as a decrease of Ն3 log 10 CFU/ml relative to the initial inoculum. The time-kill curve data were, in every case, provided as arithmetic means and standard deviations from the results of at least 3 independent experiments, exactly according to standard time-kill methodology (17,(62)(63)(64)(65)(66)(67). For analyses in serum, blood, and plasma, all replicates were performed using the indicated number (n Ͼ 3) of biologically independent samples representing different lot numbers (Tables 1 and 2); thus, if 5 biologically independent replicates are indicated, the analysis was performed in the blood, sera, or plasma of 5 different individuals and/or pooled samples.…”

Repurposing Toremifene for Treatment of Oral Bacterial Infections

“…The frequency at which mutants of P. gingivalis and S. mutans that are resistant to antibacterial agents emerge was determined as described previously (34). Briefly, 500 l of an overnight culture of P. gingivalis or S. mutans was plated on agar plates containing antibacterial agents at 5ϫ the MIC (see Table S1 in the supplemental material).…”

“…The ability of toremifene to permeabilize the inner membrane of P. gingivalis and the membrane of S. mutans was determined using the fluorescent dye Sytox green (Invitrogen, USA), as previously described (34). Briefly, exponential-phase cells were washed and resuspended to an OD 595 of 0.5 in PBS.…”