AcrAB-TolC is the paradigm resistance-nodulation-division (RND) multidrug resistance efflux system in Gram-negative bacteria, with AcrB being the pump protein in this complex. We constructed a nonfunctional AcrB mutant by replacing D408, a highly conserved residue essential for proton translocation. Western blotting confirmed that the AcrB D408A mutant had the same native level of expression of AcrB as the parental strain. The mutant had no growth deficiencies in rich or minimal medium. However, compared with wild-type SL1344, the mutant had increased accumulation of Hoechst 33342 dye and decreased efflux of ethidium bromide and was multidrug hypersusceptible. The D408A mutant was attenuated in vivo in mouse and Galleria mellonella models and showed significantly reduced invasion into intestinal epithelial cells and macrophages in vitro. A dose-dependent inhibition of invasion was also observed when two different efflux pump inhibitors were added to the wild-type strain during infection of epithelial cells. RNA sequencing (RNA-seq) revealed downregulation of bacterial factors necessary for infection, including those in the Salmonella pathogenicity islands 1, 2, and 4; quorum sensing genes; and phoPQ. Several general stress response genes were upregulated, probably due to retention of noxious molecules inside the bacterium. Unlike loss of AcrB protein, loss of efflux function did not induce overexpression of other RND efflux pumps. Our data suggest that gene deletion mutants are unsuitable for studying membrane transporters and, importantly, that inhibitors of AcrB efflux function will not induce expression of other RND pumps.

A simple, environmentally friendly and cost-effective synthetic method has been developed to prepare highly stable aqueous colloidal solutions of small (2–14 nm) silver nanoparticles using aminocellulose (AMC) as a combined reducing and capping reagent. The effects of temperature, reaction time, the concentration of silver nitrate and AMC are systematically investigated and the reaction conditions optimised. The AMC-stabilized silver nanoparticles can be deposited on the surfaces of cotton fibres and microporous cellulose acetate (CA) filters, without affecting the permeability of the filters. The AMC-stabilized aqueous silver colloidal solutions and silver nanoparticle coated CA filters and cotton fibres all show significant antibacterial action against all the bacterial isolates tested, with the antibacterial levels between “Sufficient” and “Good”, although some of solutions have been stored at room temperature for 18 months

Colistin resistance in Acinetobacter baumannii is of great concern and is a threat to human health. In this study, we investigate the mechanisms of colistin resistance in four isogenic pairs of A. baumannii isolates displaying an increase in colistin MICs. A mutation in pmrB was detected in each colistin-resistant isolate, three of which were novel (A28V, I232T, and ΔL9-G12). Increased expression of pmrC was shown by semi-quantitative reverse transcription-PCR (qRT-PCR) for three colistin-resistant isolates, and the addition of phosphoethanolamine (PEtN) to lipid A by PmrC was revealed by mass spectrometry. Interestingly, PEtN addition was also observed in some colistin-susceptible isolates, indicating that this resistance mechanism might be strain specific and that other factors could contribute to colistin resistance. Furthermore, the introduction of pmrAB carrying the short amino acid deletion ΔL9-G12 into a pmrAB knockout strain resulted in increased pmrC expression and lipid A modification, but colistin MICs remained unchanged, further supporting the strain specificity of this colistin resistance mechanism. Of note, a mutation in the pmrC homologue eptA and a point mutation in ISAba1 upstream of eptA were associated with colistin resistance and increased eptA expression, which is a hitherto undescribed resistance mechanism. Moreover, no cost of fitness was observed for colistin-resistant isolates, while the virulence of these isolates was increased in a Galleria mellonella infection model. Although the mutations in pmrB were associated with colistin resistance, PEtN addition appears not to be the sole factor leading to colistin resistance, indicating that the mechanism of colistin resistance is far more complex than previously suspected and is potentially strain specific.

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BackgroundAcinetobacter baumannii is an opportunistic human pathogen often associated with life-threatening infections in the immunocompromised and the critically ill. Strains are often multidrug-resistant (MDR) and due to the lack of new synthetic antimicrobials in development for treatment, attention is increasingly focused on natural compounds either as stand-alone or adjunctive agents. Curcumin (CCM) is a natural polyphenol found in turmeric and isolated from the plant, Curcuma longa. Curcumin has been found to possess many biological properties, including antibacterial activity. In this study the antimicrobial activity of CCM and synergistic effects with epigallocatechin gallate (EGCG) against multidrug-resistant strains of A. baumannii were investigated and assessed via checkerboard and time-kill assays.ResultsThe MIC of CCM was >256 μg/mL against all strains of A. baumannii whilst those for EGCG ranged from 128-1024 μg/mL. In checkerboard studies synergy was observed against 5/9 isolates, with an additive effect noted in the remaining 4. The addition of EGCG reduced the MIC of CCM by 3- to 7-fold, with the greatest interaction resulting in a CCM MIC of 4 μg/mL. Time-kill curves indicated that a CCM-EGCG (1:8 and 1:4) combination was bactericidal with a 4 to 5-log reduction in viable counts after 24 h compared to the most effective polyphenol alone.ConclusionsThis study demonstrates that despite little antibacterial activity alone, CCM activity is greatly enhanced in the presence of EGCG resulting in antibacterial activity against MDR A. baumannii. The combination may have a potential use in medicine as a topical agent to prevent or treat A. baumannii infections.