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76000.9813The underling mechanism of bacterial TetR/AcrR family transcriptional repressors. Bacteria transcriptional regulators are classified by their functional and sequence similarities. Member of the TetR/AcrR family is two-domain proteins including an N-terminal HTH DNA-binding motif and a C-terminal ligand recognition domain. The C-terminal ligand recognition domain can recognize the very same compounds as their target transporters transferred. TetRs act as chemical sensors to monitor both the cellular environmental dynamics and their regulated genes underlying many events, such as antibiotics production, osmotic stress, efflux pumps, multidrug resistance, metabolic modulation, and pathogenesis. Compounds targeting Mycobacterium tuberculosis ethR represent promising novel antibiotic potentiater. TetR-mediated multidrug efflux pumps regulation might be good target candidate for the discovery of better new antibiotics against drug resistance.201323602932
903110.9812EmrR-Dependent Upregulation of the Efflux Pump EmrCAB Contributes to Antibiotic Resistance in Chromobacterium violaceum. Chromobacterium violaceum is an environmental Gram-negative bacterium that causes infections in humans. Treatment of C. violaceum infections is difficult and little is known about the mechanisms of antibiotic resistance in this bacterium. In this work, we identified mutations in the MarR family transcription factor EmrR and in the protein GyrA as key determinants of quinolone resistance in C. violaceum, and we defined EmrR as a repressor of the MFS-type efflux pump EmrCAB. Null deletion of emrR caused increased resistance to nalidixic acid, but not to other quinolones or antibiotics of different classes. Moreover, the ΔemrR mutant showed decreased production of the purple pigment violacein. Importantly, we isolated C. violaceum spontaneous nalidixic acid-resistant mutants with a point mutation in the DNA-binding domain of EmrR (R92H), with antibiotic resistance profile similar to that of the ΔemrR mutant. Other spontaneous mutants with high MIC values for nalidixic acid and increased resistance to fluoroquinolones presented point mutations in the gene gyrA. Using DNA microarray, Northern blot and EMSA assays, we demonstrated that EmrR represses directly a few dozen genes, including the emrCAB operon and other genes related to transport, oxidative stress and virulence. This EmrR repression on emrCAB was relieved by salicylate. Although mutation of the C. violaceum emrCAB operon had no effect in antibiotic susceptibility or violacein production, deletion of emrCAB in an emrR mutant background restored antibiotic susceptibility and violacein production in the ΔemrR mutant. Using a biosensor reporter strain, we demonstrated that the lack of pigment production in ΔemrR correlates with the accumulation of quorum-sensing molecules in the cell supernatant of this mutant strain. Therefore, our data revealed that overexpression of the efflux pump EmrCAB via mutation and/or derepression of EmrR confers quinolone resistance and alters quorum-sensing signaling in C. violaceum, and that point mutation in emrR can contribute to emergence of antibiotic resistance in bacteria.201830498484
61920.9811Inactivation of farR Causes High Rhodomyrtone Resistance and Increased Pathogenicity in Staphylococcus aureus. Rhodomyrtone (Rom) is an acylphloroglucinol antibiotic originally isolated from leaves of Rhodomyrtus tomentosa. Rom targets the bacterial membrane and is active against a wide range of Gram-positive bacteria but the exact mode of action remains obscure. Here we isolated and characterized a spontaneous Rom-resistant mutant from the model strain Staphylococcus aureus HG001 (Rom(R)) to learn more about the resistance mechanism. We showed that Rom-resistance is based on a single point mutation in the coding region of farR [regulator of fatty acid (FA) resistance] that causes an amino acid change from Cys to Arg at position 116 in FarR, that affects FarR activity. Comparative transcriptome analysis revealed that mutated farR affects transcription of many genes in distinct pathways. FarR represses for example the expression of its own gene (farR), its flanking gene farE (effector of FA resistance), and other global regulators such as agr and sarA. All these genes were consequently upregulated in the Rom(R) clone. Particularly the upregulation of agr and sarA leads to increased expression of virulence genes rendering the Rom(R) clone more cytotoxic and more pathogenic in a mouse infection model. The Rom-resistance is largely due to the de-repression of farE. FarE is described as an efflux pump for linoleic and arachidonic acids. We observed an increased release of lipids in the Rom(R) clone compared to its parental strain HG001. If farE is deleted in the Rom(R) clone, or, if native farR is expressed in the Rom(R) strain, the corresponding strains become hypersensitive to Rom. Overall, we show here that the high Rom-resistance is mediated by overexpression of farE in the Rom(R) clone, that FarR is an important regulator, and that the point mutation in farR (Rom(R) clone) makes the clone hyper-virulent.201931191485
75130.9809Global transcriptomics and targeted metabolite analysis reveal the involvement of the AcrAB efflux pump in physiological functions by exporting signaling molecules in Photorhabdus laumondii. In Gram-negative bacteria, resistance-nodulation-division (RND)-type efflux pumps, particularly AcrAB-TolC, play a critical role in mediating resistance to antimicrobial agents and toxic metabolites, contributing to multidrug resistance. Photorhabdus laumondii is an entomopathogenic bacterium that has garnered significant interest due to its production of bioactive specialized metabolites with anti-inflammatory, antimicrobial, and scavenger deterrent properties. In previous work, we demonstrated that AcrAB confers self-resistance to stilbenes in P. laumondii TT01. Here, we explore the pleiotropic effects of AcrAB in this bacterium. RNA sequencing of ∆acrA compared to wild type revealed growth-phase-specific gene regulation, with stationary-phase cultures showing significant downregulation of genes involved in stilbene, fatty acid, and anthraquinone pigment biosynthesis, as well as genes related to cellular clumping and fimbrial pilin formation. Genes encoding putative LuxR regulators, type VI secretion systems, two-partner secretion systems, and contact-dependent growth inhibition systems were upregulated in ∆acrA. Additionally, exponential-phase cultures revealed reduced expression of genes related to motility in ∆acrA. The observed transcriptional changes were consistent with phenotypic assays, demonstrating that the ∆acrA mutant had altered bioluminescence and defective orange pigmentation due to disrupted anthraquinone production. These findings confirm the role of stilbenes as signaling molecules involved in gene expression, thereby shaping these phenotypes. Furthermore, we showed that AcrAB contributes to swarming and swimming motilities independently of stilbenes. Collectively, these results highlight that disrupting acrAB causes transcriptional and metabolic dysregulation in P. laumondii, likely by impeding the export of key signaling molecules such as stilbenes, which may serve as a ligand for global transcriptional regulators.IMPORTANCERecent discoveries have highlighted Photorhabdus laumondii as a promising source of novel anti-infective compounds, including non-ribosomal peptides and polyketides. One key player in the self-resistance of this bacterium to stilbene derivatives is the AcrAB-TolC complex, which is also a well-known contributor to multidrug resistance. Here, we demonstrate the pleiotropic effects of the AcrAB efflux pump in P. laumondii TT01, impacting secondary metabolite biosynthesis, motility, and bioluminescence. These effects are evident at transcriptional, metabolic, and phenotypic levels and are likely mediated by the efflux of signaling molecules such as stilbenes. These findings shed light on the multifaceted roles of efflux pumps and open avenues to better explore the complexity of resistance-nodulation-division (RND) pump-mediated signaling pathways in bacteria, thereby aiding in combating multidrug-resistant infections.202540920493
65740.9807Mycobacterial HflX is a ribosome splitting factor that mediates antibiotic resistance. Antibiotic resistance in bacteria is typically conferred by proteins that function as efflux pumps or enzymes that modify either the drug or the antibiotic target. Here we report an unusual mechanism of resistance to macrolide-lincosamide antibiotics mediated by mycobacterial HflX, a conserved ribosome-associated GTPase. We show that deletion of the hflX gene in the pathogenic Mycobacterium abscessus, as well as the nonpathogenic Mycobacterium smegmatis, results in hypersensitivity to the macrolide-lincosamide class of antibiotics. Importantly, the level of resistance provided by Mab_hflX is equivalent to that conferred by erm41, implying that hflX constitutes a significant resistance determinant in M. abscessus We demonstrate that mycobacterial HflX associates with the 50S ribosomal subunits in vivo and can dissociate purified 70S ribosomes in vitro, independent of GTP hydrolysis. The absence of HflX in a ΔMs_hflX strain also results in a significant accumulation of 70S ribosomes upon erythromycin exposure. Finally, a deletion of either the N-terminal or the C-terminal domain of HflX abrogates ribosome splitting and concomitantly abolishes the ability of mutant proteins to mediate antibiotic tolerance. Together, our results suggest a mechanism of macrolide-lincosamide resistance in which the mycobacterial HflX dissociates antibiotic-stalled ribosomes and rescues the bound mRNA. Given the widespread presence of hflX genes, we anticipate this as a generalized mechanism of macrolide resistance used by several bacteria.202031871194
62050.9803Transcriptomic Responses and Survival Mechanisms of Staphylococci to the Antimicrobial Skin Lipid Sphingosine. Sphingosines are antimicrobial lipids that form part of the innate barrier to skin colonization by microbes. Sphingosine deficiencies can result in increased epithelial infections by bacteria including Staphylococcus aureus. Recent studies have focused on the potential use of sphingosine resistance or its potential mechanisms. We used RNA-Seq to identify the common d-sphingosine transcriptomic response of the transient skin colonizer S. aureus and the dominant skin coloniser S. epidermidis. A common d-sphingosine stimulon was identified that included downregulation of the SaeSR two-component system (TCS) regulon and upregulation of both the VraSR TCS and CtsR stress regulons. We show that the PstSCAB phosphate transporter, and VraSR offer intrinsic resistance to d-sphingosine. Further, we demonstrate increased sphingosine resistance in these staphylococci evolves readily through mutations in genes encoding the FarE-FarR efflux/regulator proteins. The ease of selecting mutants with resistance to sphingosine may impact upon staphylococcal colonization of skin where the lipid is present and have implications with topical therapeutic applications.202234902269
65660.9803HflXr, a homolog of a ribosome-splitting factor, mediates antibiotic resistance. To overcome the action of antibiotics, bacteria have evolved a variety of different strategies, such as drug modification, target mutation, and efflux pumps. Recently, we performed a genome-wide analysis of Listeria monocytogenes gene expression after growth in the presence of antibiotics, identifying genes that are up-regulated upon antibiotic treatment. One of them, lmo0762, is a homolog of hflX, which encodes a heat shock protein that rescues stalled ribosomes by separating their two subunits. To our knowledge, ribosome splitting has never been described as an antibiotic resistance mechanism. We thus investigated the role of lmo0762 in antibiotic resistance. First, we demonstrated that lmo0762 is an antibiotic resistance gene that confers protection against lincomycin and erythromycin, and that we renamed hflXr (hflX resistance). We show that hflXr expression is regulated by a transcription attenuation mechanism relying on the presence of alternative RNA structures and a small ORF encoding a 14 amino acid peptide containing the RLR motif, characteristic of macrolide resistance genes. We also provide evidence that HflXr is involved in ribosome recycling in presence of antibiotics. Interestingly, L. monocytogenes possesses another copy of hflX, lmo1296, that is not involved in antibiotic resistance. Phylogenetic analysis shows several events of hflXr duplication in prokaryotes and widespread presence of hflXr in Firmicutes. Overall, this study reveals the Listeria hflXr as the founding member of a family of antibiotic resistance genes. The resistance conferred by this gene is probably of importance in the environment and within microbial communities.201830545912
77970.9802The menaquinone pathway is important for susceptibility of Staphylococcus aureus to the antibiotic adjuvant, cannabidiol. Emergence of antibiotic resistant bacteria is evolving at an alarming pace; therefore, we must start turning to alternative approaches. One of these, could be the use of antibiotic adjuvants that enhances the effect of antibiotics towards resistant bacteria. A novel antibiotic adjuvant is cannabidiol (CBD), which we have previously shown can enhance the effect of bacitracin (BAC). BAC targets cell wall synthesis by inhibiting dephosphorylation of the lipid carrier undecaprenyl pyrophosphate prior to recycling across the membrane. However, the mechanism underlying this CBD mediated potentiation of BAC has remained unknown. To explore this, we examined resistance to CBD in Staphylococcus aureus through daily exposures to CBD. By subsequent whole genome sequencing, we observed multiple genes to be mutated, including the farE/farR system encoding a fatty acid efflux pump (FarE) and its regulator (FarR). Importantly, recreation of mutations in these genes showed decreased susceptibility towards the combination of CBD and BAC. Furthermore, we searched the Nebraska Transposon Mutant Library for CBD susceptible strains and identified menH encoding a protein participating in menaquinone biosynthesis. Strains containing deletions in this and other menaquinone related genes showed increased susceptibility towards CBD, while addition of exogenous menaquinone reversed the effect and reduced susceptible towards CBD. These results suggest that CBD potentiates BAC by redirecting the isoprenoid precursor isopentenyl pyrophosphate towards production of menaquinone rather than the lipid carrier undecaprenyl pyrophosphate, which dephosphorylation is inhibited by BAC. This in turn might decrease the level of undecaprenyl pyrophosphate thus enhancing the effect of BAC. Our study illustrates how antibiotic adjuvants may apply to enhance efficacy of antimicrobial compounds.202235091344
79880.9801Involvement of the SCO3366 efflux pump from S. coelicolor in rifampicin resistance and its regulation by a TetR regulator. Overexpression of efflux pumps represents a key mechanism of resistance in bacteria. Soil bacteria such as Streptomyces harbour a vast array of efflux genes that are transcriptionally silent under laboratory conditions. However, dissemination of many of these genes into clinical pathogens via horizontal gene transfer results in conferring resistance to multiple drugs. In this study, we have identified the role of a MFS transporter, SCO3366 from Streptomyces coelicolor, in governing multidrug resistance. Overexpression and knockout studies revealed that SCO3366 provides resistance to several structurally unrelated drugs including ciprofloxacin, chloramphenicol, rifampicin and EtBr, with rifampicin being the major substrate. Beyond multidrug resistance, SCO3366 was efficient in providing tolerance towards oxidative stress. A combinatorial mechanism of increased oxidative stress tolerance decreased intracellular drug levels and decreased permeability act synergistically to provide resistance towards rifampicin. Shedding light on the regulation of SCO3366, we find the pump to be directly regulated by the TetR regulator SCO3367 in a negative manner and the repression was found to be relieved in presence of different compounds recognized as substrates of SCO3366. KEY POINTS: • First reported rifampicin efflux pump in Streptomyces coelicolor • Resistance to rifampicin is the result of a synergistic action of increased efflux with increased oxidative stress tolerance and decreased permeability, which can potentially arise in clinically relevant bacteria • SCO3366-SCO3367 to be a novel system that operates to protect the bacteria under varied environmental stress conditions.202235194656
469690.9800High-throughput transcriptomics of 409 bacteria-drug pairs reveals drivers of gut microbiota perturbation. Many drugs can perturb the gut microbiome, potentially leading to negative health consequences. However, mechanisms of most microorganism-drug responses have not been elucidated at the genetic level. Using high-throughput bacterial transcriptomics, we systematically characterized the gene expression profiles of prevalent human gut bacteria exposed to the most frequently prescribed orally administered pharmaceuticals. Across >400 drug-microorganism pairs, significant and reproducible transcriptional responses were observed, including pathways involved in multidrug resistance, metabolite transport, tartrate metabolism and riboflavin biosynthesis. Importantly, we discovered that statin-mediated upregulation of the AcrAB-TolC efflux pump in Bacteroidales species enhances microbial sensitivity to vitamin A and secondary bile acids. Moreover, gut bacteria carrying acrAB-tolC genes are depleted in patients taking simvastatin, suggesting that drug-efflux interactions generate collateral toxicity that depletes pump-containing microorganisms from patient microbiomes. This study provides a resource to further understand the drivers of drug-mediated microbiota shifts for better informed clinical interventions.202438233648
786100.9798The Varied Role of Efflux Pumps of the MFS Family in the Interplay of Bacteria with Animal and Plant Cells. Efflux pumps represent an important and large group of transporter proteins found in all organisms. The importance of efflux pumps resides in their ability to extrude a wide range of antibiotics, resulting in the emergence of multidrug resistance in many bacteria. Besides antibiotics, multidrug efflux pumps can also extrude a large variety of compounds: Bacterial metabolites, plant-produced compounds, quorum-sensing molecules, and virulence factors. This versatility makes efflux pumps relevant players in interactions not only with other bacteria, but also with plant or animal cells. The multidrug efflux pumps belonging to the major facilitator superfamily (MFS) are widely distributed in microbial genomes and exhibit a large spectrum of substrate specificities. Multidrug MFS efflux pumps are present either as single-component transporters or as tripartite complexes. In this review, we will summarize how the multidrug MFS efflux pumps contribute to the interplay between bacteria and targeted host cells, with emphasis on their role in bacterial virulence, in the colonization of plant and animal host cells and in biofilm formation. We will also address the complexity of these interactions in the light of the underlying regulatory networks required for the effective activation of efflux pump genes.201931443538
759110.9798The WblC/WhiB7 Transcription Factor Controls Intrinsic Resistance to Translation-Targeting Antibiotics by Altering Ribosome Composition. Bacteria that encounter antibiotics can efficiently change their physiology to develop resistance. This intrinsic antibiotic resistance is mediated by multiple pathways, including a regulatory system(s) that activates specific genes. In some Streptomyces and Mycobacterium spp., the WblC/WhiB7 transcription factor is required for intrinsic resistance to translation-targeting antibiotics. Wide conservation of WblC/WhiB7 within Actinobacteria indicates a critical role of WblC/WhiB7 in developing resistance to such antibiotics. Here, we identified 312 WblC target genes in Streptomyces coelicolor, a model antibiotic-producing bacterium, using a combined analysis of RNA sequencing and chromatin immunoprecipitation sequencing. Interestingly, WblC controls many genes involved in translation, in addition to previously identified antibiotic resistance genes. Moreover, WblC promotes translation rate during antibiotic stress by altering the ribosome-associated protein composition. Our genome-wide analyses highlight a previously unappreciated antibiotic resistance mechanism that modifies ribosome composition and maintains the translation rate in the presence of sub-MIC levels of antibiotics.IMPORTANCE The emergence of antibiotic-resistant bacteria is one of the top threats in human health. Therefore, we need to understand how bacteria acquire resistance to antibiotics and continue growth even in the presence of antibiotics. Streptomyces coelicolor, an antibiotic-producing soil bacterium, intrinsically develops resistance to translation-targeting antibiotics. Intrinsic resistance is controlled by the WblC/WhiB7 transcription factor that is highly conserved within Actinobacteria, including Mycobacterium tuberculosis Here, identification of the WblC/WhiB7 regulon revealed that WblC/WhiB7 controls ribosome maintenance genes and promotes translation in the presence of antibiotics by altering the composition of ribosome-associated proteins. Also, the WblC-mediated ribosomal alteration is indeed required for resistance to translation-targeting antibiotics. This suggests that inactivation of the WblC/WhiB7 regulon could be a potential target to treat antibiotic-resistant mycobacteria.202032291305
6181120.9798Two distinct major facilitator superfamily drug efflux pumps mediate chloramphenicol resistance in Streptomyces coelicolor. Chloramphenicol, florfenicol, and thiamphenicol are used as antibacterial drugs in clinical and veterinary medicine. Two efflux pumps of the major facilitator superfamily encoded by the cmlR1 and cmlR2 genes mediate resistance to these antibiotics in Streptomyces coelicolor, a close relative of Mycobacterium tuberculosis. The transcription of both genes was observed by reverse transcription-PCR. Disruption of cmlR1 decreased the chloramphenicol MIC 1.6-fold, while disruption of cmlR2 lowered the MIC 16-fold. The chloramphenicol MIC of wild-type S. coelicolor decreased fourfold and eightfold in the presence of reserpine and Phe-Arg-beta-naphthylamide, respectively. These compounds are known to potentiate the activity of some antibacterial drugs via efflux pump inhibition. While reserpine is known to potentiate drug activity against gram-positive bacteria, this is the first time that Phe-Arg-beta-naphthylamide has been shown to potentiate drug activity against a gram-positive bacterium.200919687245
6179130.9796Structures of Class I and Class II Transcription Complexes Reveal the Molecular Basis of RamA-Dependent Transcription Activation. Transcription activator RamA is linked to multidrug resistance of Klebsiella pneumoniae through controlling genes that encode efflux pumps (acrA) and porin-regulating antisense RNA (micF). In bacteria, σ(70) , together with activators, controls the majority of genes by recruiting RNA polymerase (RNAP) to the promoter regions. RNAP and σ(70) form a holoenzyme that recognizes -35 and -10 promoter DNA consensus sites. Many activators bind upstream from the holoenzyme and can be broadly divided into two classes. RamA acts as a class I activator on acrA and class II activator on micF, respectively. The authors present biochemical and structural data on RamA in complex with RNAP-σ(70) at the two promoters and the data reveal the molecular basis for how RamA assembles and interacts with core RNAP and activates transcription that contributes to antibiotic resistance. Further, comparing with CAP/TAP complexes reveals common and activator-specific features in activator binding and uncovers distinct roles of the two C-terminal domains of RNAP α subunit.202234761556
665140.9795Functional versatility of Zur in metal homeostasis, motility, biofilm formation, and stress resistance in Yersinia pseudotuberculosis. Zur (zinc uptake regulator) is a significant member of the Fur (ferric uptake regulator) superfamily, which is widely distributed in bacteria. Zur plays crucial roles in zinc homeostasis and influences cell development and environmental adaptation in various species. Yersinia pseudotuberculosis is a Gram-negative enteric that pathogen usually serves as a model organism in pathogenicity studies. The regulatory effects of Zur on the zinc transporter ZnuABC and the protein secretion system T6SS have been documented in Y. pseudotuberculosis. In this study, a comparative transcriptomics analysis between a ∆zur mutant and the wild-type (WT) strain of Y. pseudotuberculosis was conducted using RNA-seq. This analysis revealed global regulation by Zur across multiple functional categories, including membrane transport, cell motility, and molecular and energy metabolism. Additionally, Zur mediates the homeostasis not only of zinc but also ferric and magnesium in vivo. There was a notable decrease in 35 flagellar biosynthesis and assembly-related genes, leading to reduced swimming motility in the ∆zur mutant strain. Furthermore, Zur upregulated multiple simple sugar and oligopeptide transport system genes by directly binding to their promoters. The absence of Zur inhibited biofilm formation as well as reduced resistance to chloramphenicol and acidic stress. This study illustrates the comprehensive regulatory functions of Zur, emphasizing its importance in stress resistance and pathogenicity in Y. pseudotuberculosis. IMPORTANCE: Bacteria encounter diverse stresses in the environment and possess essential regulators to modulate the expression of genes in responding to the stresses for better fitness and survival. Zur (zinc uptake regulator) plays a vital role in zinc homeostasis. Studies of Zur from multiple species reviewed that it influences cell development, stress resistance, and virulence of bacteria. Y. pseudotuberculosis is an enteric pathogen that serves a model organism in the study of pathogenicity, virulence factors, and mechanism of environmental adaptation. In this study, transcriptomics analysis of Zur's regulons was conducted in Y. pseudotuberculosis. The functions of Zur as a global regulator in metal homeostasis, motility, nutrient acquisition, glycan metabolism, and nucleotide metabolism, in turn, increasing the biofilm formation, stress resistance, and virulence were reviewed. The importance of Zur in environmental adaptation and pathogenicity of Y. pseudotuberculosis was emphasized.202438534119
9028150.9795Efflux Pumps in Chromobacterium Species Increase Antibiotic Resistance and Promote Survival in a Coculture Competition Model. Members of the Chromobacterium genus include opportunistic but often-fatal pathogens and soil saprophytes with highly versatile metabolic capabilities. In previous studies of Chromobacterium subtsugae (formerly C. violaceum) strain CV017, we identified a resistance nodulation division (RND)-family efflux pump (CdeAB-OprM) that confers resistance to several antibiotics, including the bactobolin antibiotic produced by the soil saprophyte Burkholderia thailandensis Here, we show the cdeAB-oprM genes increase C. subtsugae survival in a laboratory competition model with B. thailandensis We also demonstrate that adding sublethal bactobolin concentrations to the coculture increases C. subtsugae survival, but this effect is not through CdeAB-OprM. Instead, the increased survival requires a second, previously unreported pump we call CseAB-OprN. We show that in cells exposed to sublethal bactobolin concentrations, the cseAB-oprN genes are transcriptionally induced, and this corresponds to an increase in bactobolin resistance. Induction of this pump is highly specific and sensitive to bactobolin, while CdeAB-OprM appears to have a broader range of antibiotic recognition. We examine the distribution of cseAB-oprN and cdeAB-oprM gene clusters in members of the Chromobacterium genus and find the cseAB-oprN genes are limited to the nonpathogenic C. subtsugae strains, whereas the cdeAB-oprM genes are more widely distributed among members of the Chromobacterium genus. Our results provide new information on the antibiotic resistance mechanisms of Chromobacterium species and highlight the importance of efflux pumps for saprophytic bacteria existing in multispecies communities.IMPORTANCE Antibiotic efflux pumps are best known for increasing antibiotic resistance of pathogens; however, the role of these pumps in saprophytes is much less well defined. This study describes two predicted efflux pump gene clusters in the Chromobacterium genus, which is comprised of both nonpathogenic saprophytes and species that cause highly fatal human infections. One of the predicted efflux pump clusters is present in every member of the Chromobacterium genus and increases resistance to a broad range of antibiotics. The other gene cluster has more narrow antibiotic specificity and is found only in Chromobacterium subtsugae, a subset of entirely nonpathogenic species. We demonstrate the role of both pumps in increasing antibiotic resistance and demonstrate the importance of efflux-dependent resistance induction for C. subtsugae survival in a dual-species competition model. These results have implications for managing antibiotic-resistant Chromobacterium infections and for understanding the evolution of efflux pumps outside the host.201931324628
781160.9795Efflux as a mechanism of resistance to antimicrobials in Pseudomonas aeruginosa and related bacteria: unanswered questions. Pseudomonas aeruginosa is an opportunistic human pathogen exhibiting innate resistance to multiple antimicrobial agents. This intrinsic multidrug resistance is caused by synergy between a low-permeability outer membrane and expression of a number of broadly-specific multidrug efflux (Mex) systems, including MexAB-OprM and MexXY-OprM. In addition to this intrinsic resistance, these and three additional systems, MexCD-OprJ, MexEF-OprN and MexJK-OprM promote acquired multidrug resistance as a consequence of hyper-expression of the efflux genes by mutational events. In addition to antibiotics, these pumps export biocides, dyes, detergents, metabolic inhibitors, organic solvents and molecules involved in bacterial cell-cell communication. Homologues of the resistance-nodulation-division systems of P. aeruginosa have been found in Burkholderia cepacia, B. pseudomallei, Stenotrophomonas maltophilia, and the nonpathogen P. putida, where they play roles in resistance to antimicrobials and/or organic solvents. Despite intensive studies of these multidrug efflux systems over the past several years, their precise molecular architectures, their modes of regulation of expression and their natural functions remain largely unknown.200312917802
8799170.9795The membrane-active polyaminoisoprenyl compound NV716 re-sensitizes Pseudomonas aeruginosa to antibiotics and reduces bacterial virulence. Pseudomonas aeruginosa is intrinsically resistant to many antibiotics due to the impermeability of its outer membrane and to the constitutive expression of efflux pumps. Here, we show that the polyaminoisoprenyl compound NV716 at sub-MIC concentrations re-sensitizes P. aeruginosa to abandoned antibiotics by binding to the lipopolysaccharides (LPS) of the outer membrane, permeabilizing this membrane and increasing antibiotic accumulation inside the bacteria. It also prevents selection of resistance to antibiotics and increases their activity against biofilms. No stable resistance could be selected to NV716-itself after serial passages with subinhibitory concentrations, but the transcriptome of the resulting daughter cells shows an upregulation of genes involved in the synthesis of lipid A and LPS, and a downregulation of quorum sensing-related genes. Accordingly, NV716 also reduces motility, virulence factors production, and biofilm formation. NV716 shows a unique and highly promising profile of activity when used alone or in combination with antibiotics against P. aeruginosa, combining in a single molecule anti-virulence and potentiator effects. Additional work is required to more thoroughly understand the various functions of NV716.202236008485
774180.9795The 2019 Garrod Lecture: MDR efflux in Gram-negative bacteria-how understanding resistance led to a new tool for drug discovery. The AcrAB-TolC MDR efflux system confers intrinsic MDR and overproduction confers clinically relevant resistance to some antibiotics active against Gram-negative bacteria. The system is made up of three components, namely AcrA, AcrB and TolC, otherwise known as the AcrAB-TolC tripartite system. Inactivation or deletion of a gene encoding one of the constituent proteins, or substitution of a single amino acid in the efflux pump component AcrB that results in loss of efflux function, confers increased antibiotic susceptibility. Clinically relevant resistance can be mediated by a mutation in acrB that changes the way AcrB substrates are transported. However, it is more common that resistant clinical and veterinary isolates overproduce the AcrAB-TolC MDR efflux system. This is due to mutations in genes such as marR and ramR that encode repressors of transcription factors (MarA and RamA, respectively) that when produced activate expression of the acrAB and tolC genes thereby increasing efflux. The Lon protease degrades MarA and RamA to return the level of efflux to that of the WT. Furthermore, the levels of AcrAB-TolC are regulated by CsrA. Studies with fluorescent reporters that report levels of acrAB and regulatory factors allowed the development of a new tool for discovering efflux inhibitors. Screens of the Prestwick Chemical Library and a large library from a collaborating pharmaceutical company have generated a number of candidate compounds for further research.201931626705
735190.9794The Pseudomonas aeruginosa flagellum confers resistance to pulmonary surfactant protein-A by impacting the production of exoproteases through quorum-sensing. Surfactant protein-A (SP-A) is an important antimicrobial protein that opsonizes and permeabilizes membranes of microbial pathogens in mammalian lungs. Previously, we have shown that Pseudomonas aeruginosa flagellum-deficient mutants are preferentially cleared in the lungs of wild-type mice by SP-A-mediated membrane permeabilization, and not by opsonization. In this study, we report a flagellum-mediated mechanism of P. aeruginosa resistance to SP-A. We discovered that flagellum-deficient (ΔfliC) bacteria are unable to produce adequate amounts of exoproteases to degrade SP-A in vitro and in vivo, leading to its preferential clearance in the lungs of SP-A(+/+) mice. In addition, ΔfliC bacteria failed to degrade another important lung antimicrobial protein lysozyme. Detailed analyses showed that ΔfliC bacteria are unable to upregulate the transcription of lasI and rhlI genes, impairing the production of homoserine lactones necessary for quorum-sensing, an important virulence process that regulates the production of multiple exoproteases. Thus, reduced ability of ΔfliC bacteria to quorum-sense attenuates production of exoproteases and limits degradation of SP-A, thereby conferring susceptibility to this major pulmonary host defence protein.201121205009