Efficacy of outer membrane permeabilization in promoting aromatic isothiocyanates-mediated eradication of multidrug resistant Gram-negative bacteria and bacterial persisters.

Multidrug resistant (MDR) bacteria are recognized to be one of the most important problems in public health. The outer membrane permeability is a critical intrinsic mechanism of bacterial resistance. In addition, bacteria produce a small number of dormant persister cells causing multidrug tolerance that reduces antimicrobial efficacy. This study aimed to evaluate the inhibitory effects of the combination of aromatic isothiocyanates (ITCs) with membrane-active agents on bacterial persisters and MDR Gram-negative bacteria. Our study demonstrated that membrane-active agents, particularly ethylenediaminetetraacetic acid (EDTA) synergistically enhanced the inhibitory activity of aromatic benzyl ITC and phenethyl ITC against most Gram-negative bacteria strains with fractional inhibitory concentration index values ranging from 0.18 to 0.5 and 0.16 to 0.5, respectively, and contributed to an 8- to 64-fold minimal inhibitory concentration reduction compared with those of aromatic ITCs alone. The EDTA-aromatic ITCs combination effectively reduced the survival rates of tested bacteria and significantly eradicated bacterial persisters (p = 0.033 and 0.037, respectively). The growth kinetics analysis also supported the enhanced inhibitory effect of EDTA-aromatic ITCs combination against tested bacteria. Our results suggested an alternate treatment strategy against Gram-negative bacteria, promoting the entry of aromatic ITCs into bacterial cytoplasm to facilitate bacterial clearance and thus preventing the development of bacterial resistance.

Synergistic action of indole-3-carbinol with membrane-active agents against multidrug-resistant Gram-negative bacteria.

The purpose of this study was to evaluate the antimicrobial activity of indole-3-carbinol (I3C) with membrane-active agents, namely carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and ethylenediaminetetraacetic acid (EDTA) against multidrug-resistant (MDR) Gram-negative bacteria and bacterial persisters. The determination of minimal inhibitory concentration (MIC) showed that I3C was effective against Acinetobacter baumannii (3.13‒6.25 × 10-3 mol l-1), Klebsiella pneumoniae (8 × 10-3 mol l-1), Pseudomonas aeruginosa (6.25‒12.5 × 10-3 mol l-1), and Escherichia coli (6.25‒12.5 × 10-3 mol l-1). Our study demonstrated that EDTA synergistically enhanced the bactericidal activity of I3C against most MDR Gram-negative bacteria isolates and contributed to an 8- to 64-fold MIC reduction compared with that of I3C alone, yet CCCP only displayed synergy with I3C against P. aeruginosa and A. baumannii. The EDTA-I3C combination also significantly reduced the viable number of testing bacteria (P = 7.2E-05), effectively reduced bacterial persisters, and repressed bacterial growth compared with that the use of I3C alone. Our data demonstrate that use of EDTA as adjuvant molecules can effectively improve the antibacterial activity of I3C and may help to reduce the development of antimicrobial resistance.

Overexpression of Resistance-Nodulation-Division Efflux Pump Genes Contributes to Multidrug Resistance in Aeromonas hydrophila Clinical Isolates.

Aeromonas hydrophila is a Gram-negative bacterium that is a critical causative agent of infections in fish and is occasionally responsible for human infections following contact with contaminated water or food. Currently, the extensive use of antibiotics in clinical practice has led to increased number of isolates of multidrug-resistant (MDR) Aeromonas and has posed a serious public health challenge. The efflux pump system is a critical mechanism of antibiotic resistance in most Gram-negative bacteria. However, the role of resistance-nodulation-division (RND)-type efflux pumps in MDR A. hydrophila is not fully understood. We aimed to evaluate the contribution of the RND efflux pump system to MDR A. hydrophila clinical isolates. PCR results indicated a considerable variation in the presence of RND efflux pump genes in clinical isolates compared to that of the environmental reference strain ATCC7966T. Compared to non-MDR clinical isolates, the expression levels of three putative RND efflux pump genes, AHA0021, AHA1320, and AheB, were significantly elevated in MDR strains. The minimal inhibitory concentrations of piperacillin/tazobactam, imipenem, erythromycin, and polymyxin B were significantly reduced by phenylalanine-arginine ß-naphthylamide (PAßN), further supporting the contribution of the RND efflux system in MDR A. hydrophila. We provided evidence supporting the contribution of the RND efflux system to multidrug resistance in A. hydrophila clinical isolates. Further studies are warranted to elucidate the detailed mechanisms that confer intrinsic resistance to antimicrobials in A. hydrophila.

Synergistic Actions of Benzyl Isothiocyanate with Ethylenediaminetetraacetic Acid and Efflux Pump Inhibitor Phenylalanine-Arginine ß-Naphthylamide Against Multidrug-Resistant Escherichia coli.

The aim of this study was to assess the efficacy of benzyl isothiocyanate (BITC) in combination with efflux inhibitors and metal chelators against multidrug-resistant Escherichia coli. In vitro synergism between testing molecules was observed based on the minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), fractional inhibitory concentration index (FICI), bactericidal kinetics, and growth inhibition assay. BITC alone exhibited moderate antibacterial activity against E. coli strains with MIC and MBC values of 0.625-1.25 µM and 1.25-2.5 µM, respectively. In contrast, double and triple combinations of BITC, ethylenediaminetetraacetic acid (EDTA), and phenylalanine-arginine ß-naphthylamide (PAßN) resulted in synergistic activities with FICI values between 0.18 and 0.5, whereas combination of BITC with carbonyl cyanide m-chlorophenyl hydrazone or 2, 2'-dipyridyl revealed additive or indifference effect with FICI values of 0.75-1.5 and 1-1.5, respectively. Results of bactericidal kinetics and growth inhibition assays also supported the synergistic effects of EDTA and PAßN with BITC against E. coli strains. Our data demonstrate the possible use of adjuvant agents, such as the chelating agent EDTA and the efflux inhibitor PAßN to improve the antibacterial potential of isothiocyanate and may help to develop an alternative strategy for reducing the occurrence of multidrug resistance.