Decoding Bacterial Persistence: Mechanisms and Strategies for Effective Eradication.

The ability of pathogenic bacteria to evade antibiotic treatment is an intricate and multifaceted phenomenon. Over the years, treatment failure among patients due to determinants of antimicrobial resistance (AMR) has been the focal point for the research and development of new therapeutic agents. However, the survival of bacteria by persisting under antibiotic stress has largely been overlooked. Bacterial persisters are a subpopulation of sensitive bacterial cells exhibiting a noninheritable drug-tolerant phenotype. They are linked to the recalcitrance of infections in healthcare settings, in turn giving rise to AMR variants. The importance of bacterial persistence in recurring infections has been firmly recognized. Fundamental work over the past decade has highlighted numerous unique tolerance factors contributing to the persister phenotype in many clinically relevant pathogens. This review summarizes contributing factors that could aid in developing new strategies against bacterial antibiotic persisters.

Small Molecule IITR08367 Potentiates Antibacterial Efficacy of Fosfomycin against Acinetobacter baumannii by Efflux Pump Inhibition.

Fosfomycin is a broad-spectrum single-dose therapy approved for treating lower urinary tract infections. Acinetobacter baumannii, one of the five major UTI-causing pathogens, is intrinsically resistant to fosfomycin. Reduced uptake and active efflux are major reasons for this intrinsic resistance. AbaF, a major facilitator superfamily class of transporter in A. baumannii, is responsible for fosfomycin efflux and biofilm formation. This study describes the identification and validation of a novel small-molecule efflux pump inhibitor that potentiates fosfomycin efficacy against A. baumannii. An AbaF inhibitor screening was performed against Escherichia coli KAM32/pUC18_abaF, using the noninhibitory concentration of 24 putative efflux pump inhibitors. The inhibitory activity of IITR08367 [bis(4-methylbenzyl) disufide] against fosfomycin/H+ antiport was validated using ethidium bromide efflux, quinacrine-based proton-sensitive fluorescence, and membrane depolarization assays. IITR08367 inhibits fosfomycin/H+ antiport activity by perturbing the transmembrane proton gradient. IITR08367 is a nontoxic molecule that potentiates fosfomycin activity against clinical strains of A. baumannii and prevents biofilm formation by inhibiting efflux pump (AbaF). The IITR08367-fosfomycin combination reduced bacterial burden by > 3 log10 in kidney and bladder tissue in the murine UTI model. Overall, fosfomycin, in combination with IITR08367, holds the potential to treat urinary tract infections caused by A. baumannii.

Chemical genetic approaches for the discovery of bacterial cell wall inhibitors.

Antimicrobial resistance (AMR) in bacterial pathogens is a worldwide health issue. The innovation gap in discovering new antibiotics has remained a significant hurdle in combating the AMR problem. Currently, antibiotics target various vital components of the bacterial cell envelope, nucleic acid and protein biosynthesis machinery and metabolic pathways essential for bacterial survival. The critical role of the bacterial cell envelope in cell morphogenesis and integrity makes it an attractive drug target. While a significant number of in-clinic antibiotics target peptidoglycan biosynthesis, several components of the bacterial cell envelope have been overlooked. This review focuses on various antibacterial targets in the bacterial cell wall and the strategies employed to find their novel inhibitors. This review will further elaborate on combining forward and reverse chemical genetic approaches to discover antibacterials that target the bacterial cell envelope.

Role of bacterial efflux pumps in antibiotic resistance, virulence, and strategies to discover novel efflux pump inhibitors.

The problem of antibiotic resistance among pathogenic bacteria has reached a crisis level. The treatment options against infections caused by multiple drug-resistant bacteria are shrinking gradually. The current pace of the discovery of new antibacterial entities is lagging behind the rate of development of new resistance. Efflux pumps play a central role in making a bacterium resistant to multiple antibiotics due to their ability to expel a wide range of structurally diverse compounds. Besides providing an escape from antibacterial compounds, efflux pumps are also involved in bacterial stress response, virulence, biofilm formation, and altering host physiology. Efflux pumps are unique yet challenging targets for the discovery of novel efflux pump inhibitors (EPIs). EPIs could help rejuvenate our currently dried pipeline of antibacterial drug discovery. The current article highlights the recent developments in the field of efflux pumps, challenges faced during the development of EPIs and potential approaches for their development. Additionally, this review highlights the utility of resources such as natural products and machine learning to expand our EPIs arsenal using these latest technologies.

Deciphering the virulence factors, regulation, and immune response to Acinetobacter baumannii infection.

Deciphering the virulence factors, regulation, and immune response to Acinetobacter baumannii infectionAcinetobacter baumannii is a gram-negative multidrug-resistant nosocomial pathogen and a major cause of hospital acquired infetions. Carbapenem resistant A. baumannii has been categorised as a Priority1 critial pathogen by the World Health Organisation. A. baumannii is responsible for infections in hospital settings, clinical sectors, ventilator-associated pneumonia, and bloodstream infections with a mortality rates up to 35%. With the development of advanced genome sequencing, molecular mechanisms of manipulating bacterial genomes, and animal infection studies, it has become more convenient to identify the factors that play a major role in A. baumannii infection and its persistence. In the present review, we have explored the mechanism of infection, virulence factors, and various other factors associated with the pathogenesis of this organism. Additionally, the role of the innate and adaptive immune response, and the current progress in the development of innovative strategies to combat this multidrug-resistant pathogen is also discussed.

Small Molecule IITR00693 (2-Aminoperimidine) Synergizes Polymyxin B Activity against Staphylococcus aureus and Pseudomonas aeruginosa.

The rise of antibiotic resistance among skin-infecting pathogens poses an urgent threat to public health and has fueled the search for new therapies. Enhancing the potency of currently used antibiotics is an alternative for the treatment of infections caused by drug-resistant pathogens. In this study, we aimed to identify a small molecule that can potentiate currently used antibiotics. IITR00693 (2-aminoperimidine), a novel antibacterial small molecule, potentiates the antibacterial activity of polymyxin B against Staphylococcus aureus and Pseudomonas aeruginosa. Herein, we investigated in detail the mode of action of this interaction and the molecule's capability to combat soft-tissue infections caused by S. aureus and P. aeruginosa. A microdilution checkerboard assay was performed to determine the synergistic interaction between polymyxin B and IITR00693 in clinical isolates of S. aureus and P. aeruginosa. Time-kill kinetics, post-antibiotic effect, and resistance generation studies were performed to assess the pharmacodynamics of the combination. Assays based on different fluorescent probes were performed to decipher the mechanism of action of this combination. The in vivo efficacy of the IITR00693-polymyxin B combination was determined in a murine acute wound infection model. IITR00693 exhibited broad-spectrum antibacterial activity. IITR00693 potentiated polymyxin B and colistin against polymyxin-resistant S. aureus. IITR00693 prevented the generation of resistant mutants against multiple antibiotics. The IITR00693-polymyxin B combination decreased the S. aureus count by >3 log10 CFU in a murine acute wound infection model. IITR00693 is a potential and promising candidate for the treatment of soft-tissue infections along with polymyxins.

Targeting Superoxide dismutase confers enhanced Reactive Oxygen Species mediated eradication of Polymyxin B induced Acinetobacter baumannii persisters.

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.

Double-Edged Nanobiotic Platform with Protean Functionality: Leveraging the Synergistic Antibacterial Activity of a Food-Grade Peptide to Mitigate Multidrug-Resistant Bacterial Pathogens.

While persistent efforts are being made to develop a novel arsenal against bacterial pathogens, the development of such materials remains a formidable challenge. One such strategy is to develop a multimodel antibacterial agent which will synergistically combat bacterial pathogens, including multidrug-resistant bacteria. Herein, we used pediocin, a class IIa bacteriocin, to decorate Ag° and developed a double-edged nanoplatform (Pd-SNPs) that inherits intrinsic properties of both antibacterial moieties, which engenders strikingly high antibacterial potency against a broad spectrum of bacterial pathogens including the ESKAPE category without displaying adverse cytotoxicity. The enhanced antimicrobial activity of Pd-SNPs is due to their higher affinity with the bacterial cell wall, which allows Pd-SNPs to penetrate the outer membrane, inducing membrane depolarization and the disruption of membrane integrity. Bioreporter assays revealed the upregulation of cpxP, degP, and sosX genes, triggering the burst of reactive oxygen species which eventually cause bacterial cell death. Pd-SNPs prevented biofilm formation, eradicated established biofilms, and inhibited persister cells. Pd-SNPs display unprecedented advantages because they are heat-resistant, retain antibacterial activity in human serum, and alleviate vancomycin intermediate Staphylococcus aureus (VISA) infection in the mouse model. In addition, Pd-SNPs wrapped in biodegradable nanofibers mitigated Listeria monocytogenes in cheese samples. Collectively, Pd-SNPs exhibited excellent biocompatibility and in vivo therapeutic potency without allowing foreseeable resistance acquisition by pathogens. These findings underscore new avenues for using a potent biocompatible nanobiotic platform to combat a wide range of bacterial pathogens.

Disulfiram enhances meropenem activity against NDM- and IMP-producing carbapenem-resistant Acinetobacter baumannii infections.

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.

Mechanistic insights into synergy between nalidixic acid and tetracycline against clinical isolates of Acinetobacter baumannii and Escherichia coli.

The increasing prevalence of antimicrobial resistance has become a global health problem. Acinetobacter baumannii is an important nosocomial pathogen due to its capacity to persist in the hospital environment. It has a high mortality rate and few treatment options. Antibiotic combinations can help to fight multi-drug resistant (MDR) bacterial infections, but they are rarely used in the clinics and mostly unexplored. The interaction between bacteriostatic and bactericidal antibiotics are mostly reported as antagonism based on the results obtained in the susceptible model laboratory strain Escherichia coli. However, in the present study, we report a synergistic interaction between nalidixic acid and tetracycline against clinical multi-drug resistant A. baumannii and E. coli. Here we provide mechanistic insight into this dichotomy. The synergistic combination was studied by checkerboard assay and time-kill curve analysis. We also elucidate the mechanism behind this synergy using several techniques such as fluorescence spectroscopy, flow cytometry, fluorescence microscopy, morphometric analysis, and real-time polymerase chain reaction. Nalidixic acid and tetracycline combination displayed synergy against most of the MDR clinical isolates of A. baumannii and E. coli but not against susceptible isolates. Finally, we demonstrate that this combination is also effective in vivo in an A. baumannii/Caenorhabditis elegans infection model (p < 0.001).

Draft Genome Sequence of Limosilactobacillus fermentum Strain NKN-51, Isolated from Fermented Yak Milk in the Western Himalayas of India.

Here, we report the draft genome sequence of Limosilactobacillus fermentum strain NKN-51, which was isolated from naturally processed yak cheese from the western Himalayas of India. The genome was assembled in 101 contigs with a total length of 1,879,705 bp and a GC content of 53.5%. Genome annotation predicted 1,730 protein-coding genes and 50 tRNA genes.

The Outer Membrane Proteins OmpA, CarO, and OprD of Acinetobacter baumannii Confer a Two-Pronged Defense in Facilitating Its Success as a Potent Human Pathogen.

Of all the ESKAPE pathogens, carbapenem-resistant and multidrug-resistant Acinetobacter baumannii is the leading cause of hospital-acquired and ventilator-associated pneumonia. A. baumannii infections are notoriously hard to eradicate due to its propensity to rapidly acquire multitude of resistance determinants and the virulence factor cornucopia elucidated by the bacterium that help it fend off a wide range of adverse conditions imposed upon by host and environment. One such weapon in the arsenal of A. baumannii is the outer membrane protein (OMP) compendium. OMPs in A. baumannii play distinctive roles in facilitating the bacterial acclimatization to antibiotic- and host-induced stresses, albeit following entirely different mechanisms. OMPs are major immunogenic proteins in bacteria conferring bacteria host-fitness advantages including immune evasion, stress tolerance, and resistance to antibiotics and antibacterials. In this review, we summarize the current knowledge of major A. baumannii OMPs and discuss their versatile role in antibiotic resistance and virulence. Specifically, we explore how OmpA, CarO, and OprD-like porins mediate antibiotic and amino acid shuttle and host virulence.

Assessment of polymyxin B-doxycycline in combination against Pseudomonas aeruginosa in vitro and in a mouse model of acute pneumonia.

The increasing prevalence of antibiotic resistance in Pseudomonas aeruginosa has created an urgent need for suitable therapy. This study explored the pairing of doxycycline with other antipseudomonal antibiotics, and found that polymyxin B in combination with doxycycline had a synergistic effect against clinical strains of P. aeruginosa. This synergistic combination was studied by checkerboard assays and time-kill curve analysis. Further, in-vitro biofilm disruption, pyoverdine inhibition assays were performed. The efficacy of polymyxin B-doxycycline in combination, administered by inhalation, was evaluated using a mouse model of acute pneumonia. The combination was found to have a synergistic effect in both in-vitro and in-vivo studies. The combination decreased biofilms of P. aeruginosa and reduced the level of pyoverdine, an important siderophore of P. aeruginosa. In addition, the combination decreased the P. aeruginosa population by 3 log10 (P<0.01) in the mouse model of acute pneumonia, and showed an improvement in lung function by inhalation. To the best of the authors' knowledge, this is the first in-vivo study to evaluate the efficacy of polymyxin B in combination with doxycycline against P. aeruginosa, showing a possible promising option for acute pneumonia due to multi-drug-resistant P. aeruginosa.

Antibacterial properties and in vivo efficacy of a novel nitrofuran, IITR06144, against MDR pathogens.

OBJECTIVES: The emergence of MDR Gram-negative pathogens and increasing prevalence of chronic infections presents an unmet need for the discovery of novel antibacterial agents. The aim of this study was to evaluate the biological properties of a small molecule, IITR06144, identified in a phenotypic screen against the Gram-negative model organism Escherichia coli. METHODS: A small-molecule library of 10956 compounds was screened for growth inhibition against E. coli ATCC 25922 at concentration 50 µM. MICs of lead compounds were determined by the broth microdilution method. Time-kill kinetics, anti-persister activity, spontaneous frequency of resistance, biofilm inhibition and disruption were assessed by standard protocols. Resistant mutants were generated by serial passaging followed by WGS. In vitro toxicity studies were carried out via the MTT assay. In vivo toxicity and efficacy in a mouse model were also evaluated. RESULTS: IITR06144 was identified as the most promising candidate amongst 29 other potential antibacterial leads, exhibiting the lowest MIC, 0.5 mg/L. IITR06144 belongs to the nitrofuran class and exhibited broad-spectrum bactericidal activity against most MDR bacteria, including the 'priority pathogen', carbapenem-resistant Acinetobacter baumannii. IITR06144 retained its potency against nitrofurantoin-resistant clinical isolates. It displayed anti-persister, anti-biofilm activity and lack of spontaneous resistance development. IITR06144 demonstrated a large therapeutic index with no associated in vitro and in vivo toxicity. CONCLUSIONS: In the light of excellent in vitro properties displayed by IITR06144 coupled with its considerable in vivo efficacy, further evaluation of IITR06144 as a therapeutic lead against antibiotic-resistant infections is warranted.

Design, synthesis and biological profile of heterocyclic benzimidazole analogues as prospective antimicrobial and antiproliferative agents.

BACKGROUND: Nitrogen containing heterocycles are widely used and investigated by pharmaceutical industry, as they are important in discovery and designing of new drug molecules. Drugs with a benzimidazole nucleus possess exclusive structural features and electron-rich atmosphere, which enable them to bind to a number of biologically important targets and result in a wide range of activities. This has served as the basis of the present study whereby new scaffolds with benzimidazole moiety were designed and synthesized. METHODS: The structures of synthesized compounds were confirmed by physicochemical and spectral means. The synthesized compounds were screened for their antimicrobial and antiproliferative activities by tube dilution and Sulforhodamine B (SRB) assays, respectively. RESULTS AND CONCLUSION: The in vitro biological screening results revealed that compound Z24 exhibited promising antimicrobial and anticancer activities which are comparable to standards.

Efflux pump inhibitors for bacterial pathogens: From bench to bedside.

With the advent of antibiotics, bacterial infections were supposed to be a thing of past. However, this instead led to the selection and evolution of bacteria with mechanisms to counter the action of antibiotics. Antibiotic efflux is one of the major mechanisms, whereby bacteria pump out the antibiotics from their cellular interior to the external environment using special transporter proteins called efflux pumps. Inhibiting these pumps seems to be an attractive strategy at a time when novel antibiotic supplies are dwindling. Molecules capable of inhibiting these pumps, known as efflux pump inhibitors (EPIs), have been viewed as potential therapeutic agents that can rejuvenate the activity of antibiotics that are no longer effective against bacterial pathogens. EPIs follow some general mechanisms of efflux inhibition and are derived from various natural as well as synthetic sources. This review focuses on EPIs and identifies the challenges that have kept these futuristic therapeutics away from the commercial realm so far.

Computational Prediction of sRNA in Acinetobacter baumannii.

Small RNAs in bacteria are noncoding RNAs that act as posttranscriptional regulators of gene expression. Over time, they have gained importance as fine-tuners of expression of genes involved in critical biological processes like metabolism, fitness, virulence, and antibiotic resistance. The availability of various high-throughput strategies enable the detection of these molecules but are technically challenging and time-intensive. Thus, to fulfil the need of a simple computational algorithm pipeline to predict these sRNAs in bacterial species, we detail a user-friendly ensemble method with specific application in Acinetobacter spp. The developed algorithms primarily look for intergenic regions in the genome of related Acinetobacter spp., thermodynamic stability, and conservation of RNA secondary structures to generate a model input for the sRNAPredict3 tool which utilizes all this information to generate a list of putative sRNA. We confirmed the accuracy of the method by comparing its output with the RNA-seq data and found the method to be faster and more accurate for Acinetobacter baumannii ATCC 17978. Thus, this method improves the identification of sRNA in Acinetobacter and other bacterial species.

A novel bi-functional chalcone inhibits multi-drug resistant Staphylococcus aureus and potentiates the activity of fluoroquinolones.

Staphylococcus aureus is the leading cause of bacteraemia and the dwindling supply of effective antibacterials has exacerbated the problem of managing infections caused by this bacterium. Isoliquiritigenin (ISL) is a plant flavonoid that displays therapeutic potential against S. aureus. The present study identified a novel mannich base derivatives of ISL, IMRG4, active against Vancomycin intermediate S. aureus (VISA). IMRG4 damages the bacterial membranes causing membrane depolarization and permeabilization, as determined by loss of salt tolerance, flow cytometric analysis, propidium idodie and fluorescent microscopy. It reduces the intracellular invasion of HEK-293 cells by S. aureus and decreases the staphylococcal load in different organs of infected mice models. In addition to anti-staphylococcal activity, IMRG4 inhibits the multidrug efflux pump, NorA, which was determined by molecular docking and EtBr efflux assays. In combination, IMRG4 significantly reduces the MIC of norfloxacin for clinical strains of S. aureus including VISA. Development of resistance against IMRG4 alone and in combination with norfloxacin was low and IMRG4 prolongs the post-antibiotic effect of norfloxacin. These virtues combined with the low toxicity of IMRG4, assessed by MTT assay and haemolysis, makes it an ideal candidate to enter drug development pipeline against S. aureus.

A Combination of Linalool, Vitamin C, and Copper Synergistically Triggers Reactive Oxygen Species and DNA Damage and Inhibits Salmonella enterica subsp. enterica Serovar Typhi and Vibrio fluvialis.

Inappropriate and disproportionate use of antibiotics is contributing immensely to the development of antibiotic resistance in bacterial species associated with food contamination. The use of natural products in combination can be a potent alternative hurdle strategy to inactivate foodborne pathogens. Here, we explored the pro-oxidant properties of essential oil linalool and vitamin C in combination with copper (LVC) in combating the foodborne pathogens Vibrio fluvialis and Salmonella enterica subsp. enterica serovar Typhi using a three-dimensional (3D) checkerboard microdilution assay. Antibacterial activity in terms of the MIC revealed that the triple combination exerted a synergistic effect compared to the effects of the individual constituents. The bactericidal effect of the triple combination was confirmed by a live/dead staining assay. Reactive oxygen species (ROS) measurements with the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay and scanning electron microscopy imaging strongly suggested that the increase in ROS production is the underlying mechanism of the enhanced antibacterial potency of the LVC combination (linalool [1.298 mM], vitamin C [8 mM], copper [16.3 µM]). In addition, the hypersensitivity of oxidative stress regulator mutants (oxyR, katG, ahpC, and sodA mutants) toward LVC corroborated the involvement of ROS in cell death. Live/dead staining and changes in cellular morphology revealed that oxidative stress did not transform the cells into the viable but nonculturable (VBNC) state; rather, killing was associated with intracellular and extracellular oxidative burst. Furthermore, the LVC combination did not display toxicity to human cells, while it effectively reduced the pathogen levels in acidic fruit juices by 3 to 4 log CFU/ml without adversely altering the organoleptic properties. This study opens a new outlook for combinatorial antimicrobial therapy.IMPORTANCE There is a need to develop effective antibacterial therapies for mitigating bacterial pathogens in food systems. We used a 3D checkerboard assay to ascertain a safe synergistic combination of food-grade components: vitamin C, copper, and the essential oil linalool. Individually, these constituents have to be added in large amounts to exert their antibacterial effect, which leads to unwanted organoleptic properties. The triple combination could exceptionally inhibit foodborne Gram-negative pathogens like Vibrio fluvialis and Salmonella enterica subsp. enterica serovar Typhi at low concentrations (linalool, 1.298 mM; vitamin C, 8 mM; copper, 16.3 µM) and displayed potent microbial inhibition in acidic beverages. We found increased susceptibility in deletion mutants of oxidative stress regulators (oxyR, katG, ahpC, and sodA mutants) due to ROS generation by Fenton's chemistry. The results of this study show that it may be possible to use plant-based antimicrobials in synergistic combinations to control microbial contaminants.

The unusual glycine-rich C terminus of the Acinetobacter baumannii RNA chaperone Hfq plays an important role in bacterial physiology.

Acinetobacter baumannii 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 A. baumannii 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 A. baumannii The homolog in A. baumannii 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 hfq deletion mutant, finding that the tail was necessary for high-affinity RNA binding. Further tests implicate Hfq in important cellular processes of A. baumannii 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 A. baumannii Hfq.