is a Gram-negative nosocomial pathogen that causes soft tissue infections in patients who spend a long time in intensive care units. This recalcitrant bacterium is very well known for developing rapid drug resistance, which is a combined outcome of its natural competence and mobile genetic elements. Successful efforts to treat these infections would be aided by additional information on the physiology of Toward that end, we recently reported on a small RNA (sRNA), AbsR25, in this bacterium that regulates the genes of several efflux pumps. Because sRNAs often require the RNA chaperone Hfq for assistance in binding to their cognate mRNA targets, we identified and characterized this protein in The homolog in is a large protein with an extended C terminus unlike Hfqs in other Gram-negative pathogens. The extension has a compositional bias toward glycine and, to a lower extent, phenylalanine and glutamine, suggestive of an intrinsically disordered region. We studied the importance of this glycine-rich tail using truncated versions of Hfq in biophysical assays and complementation of an deletion mutant, finding that the tail was necessary for high-affinity RNA binding. Further tests implicate Hfq in important cellular processes of like metabolism, drug resistance, stress tolerance, and virulence. Our findings underline the importance of the glycine-rich C terminus in RNA binding, ribo-regulation, and auto-regulation of Hfq, demonstrating this hitherto overlooked protein motif to be an indispensable part of the Hfq.

Bacterial persisters represent non-inheritable drug tolerant population that are linked to recalcitrance of infections in healthcare settings. The rise of antibiotic resistance and depletion of new antibiotics in drug discovery pipeline has made the task of persister eradication more daunting. In the present study, we report that treatment of Acinetobacter baumannii with the last resort antibiotic polymyxin B displays continuous variation in tolerance among different clinical isolates. Mechanistically, Polymyxin B persisters exhibit disruption of proton motive force led delocalisation of cell division protein to attain a growth arrested phenotype. Tolerance studies on mutant strains revealed that superoxide dismutase (sodB) activity is a major contributor in tolerance of A. baumannii to polymyxin B. Using a dual fluorescence based persister detection system, screening of various antibiotics was performed to eradicate polymyxin B induced persisters of A. baumannii. Rifampicin exhibited eradication of polymyxin B tolerant population by log reduction of 6 in magnitude against different clinical isolates of A. baumannii. We establish that enhanced generation of ROS by rifampicin leads to clearance of these polymyxin B persisters. It was further demonstrated, as a proof of concept, that rifampicin potentiates the killing of polymyxin B persisters in murine wound infection model. We found that the effects were linked to significant down regulation of sodB by rifampicin, which contributes to higher generation of ROS in polymyxin B tolerant cells. In view of these results, we propose that the combination of polymyxin B and rifampicin is an effective antipersister strategy in clearing polymyxin B induced A. baumannii persisters.

Objectives To evaluate the in vitro and in vivo efficacy of the FDA-approved drug disulfiram in combination with meropenem against MBL-expressing carbapenem-resistant Acinetobacter baumannii. Methods Chequerboard and antibiotic resistance reversal analysis were performed using 25 clinical isolates producing different MBLs. Three representative strains harbouring NDM, IMP or non-MBL genes were subjected to a time–kill assay to further evaluate this synergistic interaction. Dose-dependent inhibition by disulfiram was assessed to determine IC50 for NDM-1, IMP-7, VIM-2 and KPC-2. Further, to test the efficacy of meropenem monotherapy and meropenem in combination with disulfiram against NDM- and IMP-harbouring A. baumannii, an experimental model of systemic infection and pneumonia was developed using BALB/c female mice. Results Chequerboard and antibiotic reversal assay displayed a synergistic interaction against MBL-expressing A. baumannii strains with 4- to 32-fold reduction in MICs of meropenem. In time–kill analysis, meropenem and disulfiram exhibited synergy against NDM- and IMP-producing carbapenem-resistant A. baumannii (CRAb) isolates. In vitro dose-dependent inhibition analysis showed that disulfiram inhibits NDM-1 and IMP-7 with IC50 values of 1.5 ± 0.6 and 16.25 ± 1.6 μM, respectively, with slight or no inhibition of VIM-2 (<20%) and KPC-2. The combination performed better in the clearance of bacterial load from the liver and spleen of mice infected with IMP-expressing CRAb. In the pneumonia model, the combination significantly decreased the bacterial burden of NDM producers compared with monotherapy. Conclusions These results strongly suggest that the combination of disulfiram and meropenem represents an effective treatment option for NDM- and IMP-associated CRAb infections.

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