Multidrug Efflux Pump Research Articles

Protistan predation selects for antibiotic resistance in soil bacterial communities

Understanding how antibiotic resistance emerges and evolves in natural habitats is critical for predicting and mitigating antibiotic resistance in the context of global change. Bacteria have evolved antibiotic production as a strategy to fight competitors, predators and other stressors, but how predation pressure of their most important consumers (i.e., protists) affects soil antibiotic resistance genes (ARGs) profiles is still poorly understood. To address this gap, we investigated responses of soil resistome to varying levels of protistan predation by inoculating low, medium and high concentrations of indigenous soil protist suspensions in soil microcosms. We found that an increase in protistan predation pressure was strongly associated with higher abundance and diversity of soil ARGs. High protist concentrations significantly enhanced the abundances of ARGs encoding multidrug (oprJ and ttgB genes) and tetracycline (tetV) efflux pump by 608%, 724% and 3052%, respectively. Additionally, we observed an increase in the abundance of numerous bacterial genera under high protistan pressure. Our findings provide empirical evidence that protistan predation significantly promotes antibiotic resistance in soil bacterial communities and advances our understanding of the biological driving forces behind the evolution and development of environmental antibiotic resistance.

Microbial Primer: Multidrug efflux pumps.

Multidrug efflux pumps are molecular machines that sit in the bacterial cell membrane and pump molecules out from either the periplasm or cytoplasm to outside the cell. While involved in a variety of biological roles, they are primarily known for their contribution to antibiotic resistance by limiting the intracellular accumulation of antimicrobial compounds within bacteria. These transporters are often overexpressed in clinical isolates, leading to multidrug-resistant phenotypes. Efflux pumps are classified into several families based on their structure and understanding the characteristics of each family is important for the development of novel therapies to restore antibiotic potency.

The multidrug efflux pump regulator AcrR directly represses motility in Escherichia coli.

Efflux and motility are two key biological functions in bacteria. Recent findings have shown that efflux impacts flagellum biosynthesis and motility in Escherichia coli and other bacteria. AcrR is known to be the major transcriptional repressor of AcrAB-TolC, the main multidrug efflux pump in E. coli and other Enterobacteriaceae. However, the underlying molecular mechanisms of how efflux and motility are co-regulated remain poorly understood. Here, we have studied the role of AcrR in direct regulation of motility in E. coli. By combining bioinformatics, electrophoretic mobility shift assays (EMSAs), gene expression, and motility experiments, we have found that AcrR represses motility in E. coli by directly repressing transcription of the flhDC operon, but not the other flagellum genes/operons tested. flhDC encodes the master regulator of flagellum biosynthesis and motility genes. We found that such regulation primarily occurs by direct binding of AcrR to the flhDC promoter region containing the first of the two predicted AcrR-binding sites identified in this promoter. This is the first report of direct regulation by AcrR of genes unrelated to efflux or detoxification. Moreover, we report that overexpression of AcrR restores to parental levels the increased swimming motility previously observed in E. coli strains without a functional AcrAB-TolC pump, and that such effect by AcrR is prevented by the AcrR ligand and AcrAB-TolC substrate ethidium bromide. Based on these and prior findings, we provide a novel model in which AcrR senses efflux and then co-regulates efflux and motility in E. coli to maintain homeostasis and escape hazards. IMPORTANCE Efflux and motility play a major role in bacterial growth, colonization, and survival. In Escherichia coli, the transcriptional repressor AcrR is known to directly repress efflux and was later found to also repress flagellum biosynthesis and motility by Kim et al. (J Microbiol Biotechnol 26:1824-1828, 2016, doi: 10.4014/jmb.1607.07058). However, it remained unknown whether AcrR represses flagellum biosynthesis and motility directly and through which target genes, or indirectly because of altering the amount of efflux. This study reveals that AcrR represses flagellum biosynthesis and motility by directly repressing the expression of the flhDC master regulator of flagellum biosynthesis and motility genes, but not the other flagellum genes tested. We also show that the antimicrobial, efflux pump substrate, and AcrR ligand ethidium bromide regulates motility via AcrR. Overall, these findings support a novel model of direct co-regulation of efflux and motility mediated by AcrR in response to stress in E. coli.

EFFLUX PUMPS AS A PHARMACOLOGICAL TARGET TO COMBAT ANTIBIOTIC RESISTANCE

Backround. Antibiotic resistance is the resistance of bacteria to antimicrobial drugs to which they were previously sensitive. A particular danger is the spread of multidrug-resistant healthcare-associated infections of "ESKAPE" group. Among the mechanisms of antibiotic resistance of these bacteria, the activation of efflux pumps attracts special attention.
 Aim: to review the current data on the possibility of using efflux pumps as targets for pharmacological action to overcome antibiotic resistance.
 Materials and methods. Analysis of the data presented in PubMed by keywords "antimicrobial resistance", "efflux pumps", "efflux pump inhibitors".
 Results. Multidrug efflux pumps are transmembrane transporter proteins that are located in the bacterial cell membrane and periplasm, they are naturally expressed, removing most of the clinically relevant antibiotic from the internal environment of the cell to the external environment, reducing its intracellular concentration. It is promising to combine certain antibiotics with efflux pump inhibitors, this allows to reduce the therapeutic dose of antibiotics and prevent the formation of bacterial biofilms. Data on the study of the effectiveness of well-known drugs sertraline, trimethoprim, thioridazine, diclofenac sodium, ibuprofen, nitroglycerin, metformin, and verapamil as efflux pump inhibitors are presented. The use of compounds obtained from plants (for example, berberine, artesunate and curcumin inhibit the activity of the efflux pump of gram-negative bacilli E. coli and P. aeruginosa), vegetables (in particular, Momordica balsamina), seeds of thistle, pepper, cumin, essential oils, etc. is promising.
 Conclusion. Thus, inhibition of efflux pumps may be a potential way to improve antibiotic activity. The advantage of efflux pumps inhibitors is the difficulty of developing resistance against them in bacteria. It is advisable to conduct in vivo studies to confirm their activity and clinical effectiveness.

Transcriptome Analysis of Streptococcus mutans Quorum Sensing-Mediated Persisters Reveals an Enrichment in Genes Related to Stress Defense Mechanisms.

Persisters are a small fraction of growth-arrested phenotypic variants that can survive lethal concentrations of antibiotics but are able to resume growth once antibiotics are stopped. Their formation can be a stochastic process or one triggered by environmental cues. In the human pathogen Streptococcus mutans, the canonical peptide-based quorum-sensing system is an inducible DNA repair system that is pivotal for bacterial survival. Previous work has shown that the CSP-signaling peptide is a stress-signaling alarmone that promotes the formation of stress-induced persisters. In this study, we exposed S. mutans to the CSP pheromone to mimic DNA damage conditions and isolated the antibiotic persisters by treating the cultures with ofloxacin. A transcriptome analysis was then performed to evaluate the differential gene expression between the normal stationary-phase cells and the persisters. RNA sequencing revealed that triggered persistence was associated with the upregulation of genes related to several stress defense mechanisms, notably, multidrug efflux pumps, the arginine deaminase pathway, and the Opu/Opc system. In addition, we showed that inactivation of the VicK kinase of the YycFG essential two-component regulatory system abolished the formation of triggered persisters via the CSP pheromone. These data contribute to the understanding of the triggered persistence phenotype and may suggest new therapeutic strategies for treating persistent streptococcal infections.

Genomic Profiling of Multidrug Efflux Pumps and Heavy Metal Proteins in Multidrug-resistant Campylobacter fetus Isolated from Sheath Wash Samples of Bulls in South Africa

A substantial evolution of resistance mechanisms among zoonotic bacteria has resulted from anthropogenic factors related to the application of antibiotics in human and veterinary medicine, particularly in contemporary agriculture. This issue associated with the presence of heavy metal-laced protein in zoonotic bacteria should be taken seriously with regard to the health of animals and the general people. To address this issue, the present study employed whole genome sequencing to identify the antimicrobial resistance patterns of Campylobacter fetus subsp. fetus (Cff) and Campylobacter fetus subsp. venerealis (Cfv), resistance and virulence genes, as well as heavy metal protein. Based on culture method biochemical testing and PCR amplification using particular primer pairs (MG3F-MG4R and VenSF-VenSR), bacteria were isolated and identified as C. fetus subsp. fetus and C.fetus subsp. venerealis. Subsequently, antimicrobial disc diffusion tests and whole genome sequencing were performed. Isolated bacteria were resistant to tetracycline at 65%, amoxicillin, and doxycycline at 60%. The resistance was also observed against neomycin at (55%), streptomycin (60%), and gentamycin (55%). Through comprehensive genome sequencing analysis and PCR, multiple efflux pumps linked to multidrug resistance were identified, including the broad-specificity multidrug efflux pump (YkkD), along with CmeA, CmeB, CmeC, and gryA.The genome sequence also revealed genes associated with the production of Cytotoxin (Cdt A, B, and C), adhesion and colonization (VirB10 and VirB9), and invasion (CiaB). In addition, different genomic features in heavy metal resistance included Cobalt-zinc-cadmium resistance protein (CzcD), Tellurite resistance protein (TehA), and arsenic efflux pump protein. The findings of the current study revealed that the emergence of bacterial multidrug resistance is increasingly associated with the substantial and growing contribution of Multidrug resistance efflux pumps, as evident in Cff and Cfv. Therefore, it is crucial to tighten the control of Cff and Cfv in livestock production to prevent the transfer of genes resistant to humans through the food chain.

Metagenome meta-analysis reveals an increase in the abundance of some multidrug efflux pumps and mobile genetic elements in chemically polluted environments.

Many human activities contaminate terrestrial and aquatic environments with numerous chemical pollutants that not only directly alter the environment but also affect microbial communities in ways that are potentially concerning to human health, such as selecting for the spread of antibiotic-resistance genes (ARGs) through horizontal gene transfer. In the present study, metagenomes available in the public domain from polluted (with antibiotics, with petroleum, with metal mining, or with coal-mining effluents) and unpolluted terrestrial and aquatic environments were compared to examine whether pollution has influenced the abundance and composition of ARGs and mobile elements, with specific focus on IS26 and class 1 integrons (intI1). When aggregated together, polluted environments had a greater relative abundance of ARGs than unpolluted environments and a greater relative abundance of IS26 and intI1. In general, chemical pollution, notably with petroleum, was associated with an increase in the prevalence of ARGs linked to multidrug efflux pumps. Included in the suite of efflux pumps were mexK, mexB, mexF, and mexW that are polyspecific and whose substrate ranges include multiple classes of critically important antibiotics. Also, in some instances, β-lactam resistance (TEM181 and OXA-541) genes increased, and genes associated with rifampicin resistance (RNA polymerases subunits rpoB and rpoB2) decreased in relative abundance. This meta-analysis suggests that different types of chemical pollution can enrich populations that carry efflux pump systems associated with resistance to multiple classes of medically critical antibiotics. IMPORTANCE The United Nations has identified chemical pollution as being one of the three greatest threats to environmental health, through which the evolution of antimicrobial resistance, a seminally important public health challenge, may be favored. While this is a very plausible outcome of continued chemical pollution, there is little evidence or research evaluating this risk. The objective of the present study was to examine existing metagenomes from chemically polluted environments and evaluate whether there is evidence that pollution increases the relative abundance of genes and mobile genetic elements that are associated with antibiotic resistance. The key finding is that for some types of pollution, particularly in environments exposed to petroleum, efflux pumps are enriched, and these efflux pumps can confer resistance to multiple classes of medically important antibiotics that are typically associated with Pseudomonas spp. or other Gram-negative bacteria. This finding makes clear the need for more investigation on the impact of chemical pollution on the environmental reservoir of ARGs and their association with mobile genetic elements that can contribute to horizontal gene transfer events.

Evolution of antimicrobial resistance in E. coli biofilm treated with high doses of ciprofloxacin.

The evolution of antimicrobial resistance (AMR) has mainly been studied in planktonic bacteria exposed to sub-inhibitory antimicrobial (AM) concentrations. However, in a number of infections that are treated with AMs the bacteria are located in biofilms where they tolerate high doses of AM. In the present study, we continuously exposed biofilm residing E. coli at body temperature to high ciprofloxacin (CIP) concentrations increasing from 4 to 130 times the minimal inhibitory concentration (MIC), i.e., from 0.06 to 2.0 mg/L. After 1 week, the biofilms were full of CIP resistant bacteria. The evolutionary trajectory observed was the same as described in the literature for planktonic bacteria, i.e., starting with a single mutation in the target gene gyrA followed by mutations in parC, gyrB, and parE, as well as in genes for regulation of multidrug efflux pump systems and outer membrane porins. Strains with higher numbers of these mutations also displayed higher MIC values. Furthermore, the evolution of CIP resistance was more rapid, and resulted in strains with higher MIC values, when the bacteria were biofilm residing than when they were in a planktonic suspension. These results may indicate that extensive clinical AM treatment of biofilm-residing bacteria may not only fail to eradicate the infection but also pose an increased risk of AMR development.

Characterization of pyridylpiperazine-based efflux pump inhibitors for Acinetobacter baumannii.

In Acinetobacter baumannii, multidrug efflux pumps belonging to the resistance-nodulation-division (RND) superfamily result in decreased antibiotic susceptibility. Improving the activity of current antibiotics via efflux pump inhibitors (EPIs) represents an attractive alternative approach to control this bacterium. Pyridylpiperazines (PyrPips) are a new class of EPIs that can effectively inhibit the Escherichia coli RND efflux pump AcrAB-TolC and boost the activity of several antibiotics. Here we have evaluated and characterized whether the PyrPip chemical family is also able to boost antibiotic activity through inhibition of the RND efflux pumps in A. baumannii. Comparative structural modelling and docking, structure-activity relationship studies alongside molecular genetic approaches were deployed to improve, characterize and validate PyrPips' target. We showed that two enhanced PyrPip EPIs are capable of rescuing the activity of different classes of antibiotics in A. baumannii. By expressing A. baumannii main efflux pumps (AdeB, AdeG and AdeJ) individually in E. coli recombinant strains, we could gain further insights about the EPIs' capacity to act upon each pump. Finally, we showed that PyrPip EPIs are mostly acting through AdeJ inhibition via interactions with two key charged residues, namely E959 and E963. Our work demonstrates that PyrPip EPIs are capable of inhibiting RND efflux pumps of A. baumannii, and thus may present a promising chemical scaffold for further development.

Nutrient condition modulates the antibiotic tolerance of Pseudomonas aeruginosa

The variation in nutrient content across diverse environments has a significant impact on the survival and metabolism of microorganisms. In this study, we examined the influence of nutrients on the antibiotic tolerance of the PAO1 strain of Pseudomonas aeruginosa. Our findings indicate that under nutrient-rich conditions, this strain exhibited relatively high tolerance to ceftazidime, chloramphenicol, and tetracycline, but not aminoglycosides and fluoroquinolones. Transcriptome analysis revealed that genes associated with antibiotic tolerance were expressed more efficiently in nutrient-rich media, including ribosomal protein genes and multidrug efflux pump genes, which conferred higher tetracycline tolerance to the strain. Furthermore, the genes responsible for translation, biosynthesis, and oxidative phosphorylation were suppressed when nutrients were limited, resulting in decreased metabolic activity and lower sensitivity to ciprofloxacin. Artificial interference with ATP synthesis utilizing arsenate confirmed that the curtailment of energy provision bolstered the observed tolerance to ciprofloxacin. In general, our results indicate that this strain of P. aeruginosa tends to activate its intrinsic resistance mechanisms in nutrient-rich environments, thereby enhancing resistance to certain antibiotics. Conversely, in nutrient-limited environments, the strain is more likely to enter a dormant state, which enables it to tolerate antibiotics to which it would otherwise be sensitive. These findings further suggest that antibiotics released in environments with varying eutrophication levels may have divergent effects on the development of bacterial antibiotic resistance.

Unveiling the microbial diversity and functional dynamics of Shiv Kund, Sohna hot spring, India through a shotgun metagenomics approach.

In this research, we examined the microbial diversity in Sohna hot spring, Haryana, India using shotgun metagenome sequencing based on the Illumina Hiseq 4000 sequencing technology. The raw sequence data from metagenomic paired-end libraries were analysed for taxonomic classification, diversity, and functional annotation using MG-RAST online server. The results showed the presence of total of 57 phyla, 931 genera, and 2068 species, predominantly occupied by Moraxellaceae (Gammaproteobacteria). However, at the species level, we reported the presence of some representative pathogenic taxa, such as Acinetobacter baumannii and Moraxella osloensis. The functional annotation predicted at various levels based on SEED-based subsystem, KEGG ortholog identity (KO), Cluster of Orthologous Groups (COGs) database identified the predominance of genes associated with primary and secondary metabolism along with a crucial role in environmental and genetic signals, cellular communication, and cell signalling. Comparative Genome Analysis (CGA) using The Pathosystem Resource Integration Centre (PATRIC) tool based on genome annotation and assembly of the metagenomic libraries for representative taxon Acinetobacter baumannii (NCBI tax id:470) characterized the reads with a unique genome identifier of 470.20380 (A. baumannii DDLJ4) which is evolutionary closer to A. baumannii ATCC 470.17978 400667.7. In addition, the CARD database results about the presence of potential AMR pathotypes and the prevalence of adeABC, adeIJK, abeM gene-specific clusters that function as multidrug efflux pumps. Overall, the results provided a comprehensive insight into virulence and anti-microbial resistance mechanism and could be useful for developing potential drug targets against the possible AMR pathotypes.

Identification of antibiotic resistant bacteria communities and a GeoChip based study of resistome in Fusarium wilt diseased and healthy soil

Soil plays a crucial role as a significant reservoir for antibiotic resistance genes (ARGs). Despite the extensive research conducted in this area, there remains a need for further investigation, particularly concerning diverse types of agricultural soil. This comprehensive study examined Fusarium wilt diseased and healthy soil samples to investigate the spectrum of ARGs and identify bacteria potentially harboring these genes. The level of antibiotic resistance (AR) in the two soil types was detected by using culture plate counts (PC). We quantified and identified antibiotic resistant bacteria (ARB) against nine antibiotics. From the PC results, the relative number of ARB (number of ARB/total number culturable bacteria) resistant to erythromycin, lincomycin, sulfonamides, streptomycin sulfate, kanamycin, and enrofloxacin in the diseased soil was significantly higher than that in the healthy soil (One-way ANOVA, p < 0.01). The GeoChip (version 5.0) was employed to identify ARGs and antibiotic biosynthetic genes (ABGs). Interestingly, despite the presence of similar ARGs in both soil types, the Fusarium wilt diseased soil demonstrated a significantly elevated abundance of the tetracycline (tetX) resistance gene compared to the healthy soil. A robust correlation (r ≥ 0.8) between multidrug efflux pumps and ARGs indicates that natural antibiotics as selective pressures for transportation mechanisms. Our analysis identified diverse ARB phylotypes and multiple ARGs in both soil types. Incorporating these soil types into ARGs risk assessments and relevant monitoring is essential for a comprehensive understanding of AR dynamics in various environments.

Structural insights into the transcriptional regulator NalC, a key component of the MexAB-OprM efflux pump system, from Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen with significant public health implications due to its multi-drug resistance (MDR). Among the mechanisms that mediate MDR, the NalC protein, a member of the TetR family of transcriptional regulators, modulates the mexAB-oprM operon, thus facilitating the efflux pump system. The resistance-nodulation-division (RND) family of multidrug efflux pumps plays a crucial role in expelling a broad spectrum of antimicrobial compounds, serving as a key adaptive mechanism. Structural analyses revealed that NalC adopts a modular architecture consisting of distinct domains involved in ligand recognition and transcriptional regulation. The N-terminal domain of NalC contains a DNA-binding helix-turn-helix motif, which interacts with specific DNA sequences in the PA3720-armR operon region. This interaction initiates the transcriptional activation of the efflux pump system. On the other hand, the C-terminal domain of NalC exhibits a highly dynamic structure and is implicated in ligand sensing and signal transduction. Our findings suggest potential binding sites for small molecules that could act as allosteric modulators, thereby providing new avenues for the development of therapeutic strategies targeting MDR Pseudomonas aeruginosa.

PTCH1 and CTNNB1 emerge as pivotal predictors of resistance to neoadjuvant chemotherapy in ER+/HER2- breast cancer.

Endeavors in the molecular characterization of breast cancer opened the doors to endocrine therapies in ER+/HER2- breast cancer, increasing response rates substantially. Despite that, taxane-based neoadjuvant chemotherapy is still a cornerstone for achieving breast-conserving surgery and complete tumor resection in locally advanced cancers with high recurrence risk. Nonetheless, the rate of chemoresistance is high, and deselecting patients who will not benefit from chemotherapy is a significant task to prevent futile toxicities. Several multigene assays are being used to guide decisions on chemotherapy. However, their development as prognostic assays but not predictive assays limits predictive strength, leading to discordant results. Moreover, high costs impediment their use in developing countries. For global health equity, robust predictors that can be cost-effectively incorporated into routine clinical management are essential. In this study, we comprehensively analyzed 5 GEO datasets, 2 validation sets, and The Cancer Genome Atlas breast cancer data to identify predictors of resistance to taxane-based neoadjuvant therapy in ER+/HER2- breast cancer using efficient bioinformatics algorithms. Gene expression and gene set enrichment analysis of 5 GEO datasets revealed the upregulation of 63 genes and the enrichment of CTNNB1-related oncogenic signatures in non-responsive patients. We validated the upregulation and predictive strength of 18 genes associated with resistance in the validation cohort, all exhibiting higher predictive powers for residual disease and higher specificities for ER+/HER2- breast cancers compared to one of the benchmark multi-gene assays. Cox Proportional Hazards Regression in three different treatment arms (neoadjuvant chemotherapy, endocrine therapy, and no systemic treatment) in a second comprehensive validation cohort strengthened the significance of PTCH1 and CTNNB1 as key predictors, with hazard ratios over 1.5, and 1.6 respectively in the univariate and multivariate models. Our results strongly suggest that PTCH1 and CTNNB1 can be used as robust and cost-effective predictors in developing countries to guide decisions on chemotherapy in ER +/HER2- breast cancer patients with a high risk of recurrence. The dual function of PTCH1 as a multidrug efflux pump and a hedgehog receptor, and the active involvement of CTNNB1 in breast cancer strongly indicate that PTCH1 and CTNNB1 can be potential drug targets to overcome chemoresistance in ER +/HER2- breast cancer patients.

Cryo-Electron Microscopy Structures of a Campylobacter Multidrug Efflux Pump Reveal a Novel Mechanism of Drug Recognition and Resistance.

Campylobacter jejuni is a bacterium that is commonly present in the intestinal tracts of animals. It is also a major foodborne pathogen that causes gastroenteritis in humans. The most predominant and clinically important multidrug efflux system in C. jejuni is the CmeABC (Campylobacter multidrug efflux) pump, a tripartite system that includes an inner membrane transporter (CmeB), a periplasmic fusion protein (CmeA), and an outer membrane channel protein (CmeC). This efflux protein machinery mediates resistance to a number of structurally diverse antimicrobial agents. A recently identified CmeB variant, termed resistance enhancing CmeB (RE-CmeB), can increase its multidrug efflux pump activity, likely by influencing antimicrobial recognition and extrusion. Here, we report structures of RE-CmeB in its apo form as well as in the presence of four different drugs by using single-particle cryo-electron microscopy (cryo-EM). Coupled with mutagenesis and functional studies, this structural information allows us to identify critical amino acids that are important for drug resistance. We also report that RE-CmeB utilizes a somewhat unique subset of residues to bind different drugs, thereby optimizing its ability to accommodate different compounds with distinct scaffolds. These findings provide insights into the structure-function relationship of this newly emerged antibiotic efflux transporter variant in Campylobacter. IMPORTANCE Campylobacter jejuni has emerged as one of the most problematic and highly antibiotic-resistant pathogens, worldwide. The Centers for Disease Control and Prevention have designated antibiotic-resistant C. jejuni as a serious antibiotic resistance threat in the United States. We recently identified a C. jejuni resistance enhancing CmeB (RE-CmeB) variant that can increase its multidrug efflux pump activity and confers an exceedingly high-level of resistance to fluoroquinolones. Here, we report the cryo-EM structures of this prevalent and clinically important C. jejuni RE-CmeB multidrug efflux pump in both the absence and presence of four antibiotics. These structures allow us to understand the action mechanism for multidrug recognition in this pump. Our studies will ultimately inform an era in structure-guided drug design to combat multidrug resistance in these Gram-negative pathogens.

A brief update on the controversial and opposing roles of Pseudomonas aeruginosa efflux pumps in virulence regulation

Pseudomonas aeruginosa is highly adaptable and constantly mutates to resist natural and synthetic antibiotic stresses. Listed as a serious threat by the Centers for Disease Control, novel antimicrobials are urgently needed for drug resistant P. aeruginosa infections. Multidrug efflux pumps which contribute to antibiotic resistance are genetically encoded, highly conserved, and have evolved long before the rampant clinical use of antibiotics. Hence, efflux pumps may have been selected for functions beyond the mere exclusion of antibiotics. In this review, we discuss recent updates and controversies surrounding how alternative functions of multidrug efflux pumps can influence the virulence of P. aeruginosa. We conclude by highlighting unexpected consequences of targeting efflux pumps with therapies, including potential risks and benefits. Understanding these consequences will be critical to the development of successful therapeutic strategies that consider aspects of both antimicrobial resistance and bacterial pathogenesis.

Bacteria exposed to antiviral drugs develop antibiotic cross-resistance and unique resistance profiles

Antiviral drugs are used globally as treatment and prophylaxis for long-term and acute viral infections. Even though antivirals also have been shown to have off-target effects on bacterial growth, the potential contributions of antivirals to antimicrobial resistance remains unknown. Herein we explored the ability of different classes of antiviral drugs to induce antimicrobial resistance. Our results establish the previously unrecognized capacity of antivirals to broadly alter the phenotypic antimicrobial resistance profiles of both gram-negative and gram-positive bacteria Escherichia coli and Bacillus cereus. Bacteria exposed to antivirals including zidovudine, dolutegravir and raltegravir developed cross-resistance to commonly used antibiotics including trimethoprim, tetracycline, clarithromycin, erythromycin, and amoxicillin. Whole genome sequencing of antiviral-resistant E. coli isolates revealed numerous unique single base pair mutations, as well as multi-base pair insertions and deletions, in genes with known and suspected roles in antimicrobial resistance including those coding for multidrug efflux pumps, carbohydrate transport, and cellular metabolism. The observed phenotypic changes coupled with genotypic results indicate that bacteria exposed to antiviral drugs with antibacterial properties in vitro can develop multiple resistance mutations that confer cross-resistance to antibiotics. Our findings underscore the potential contribution of wide scale usage of antiviral drugs to the development and spread of antimicrobial resistance in humans and the environment.

Exploring α, β-unsaturated carbonyl compounds against bacterial efflux pumps via computational approach

Antibiotic resistance has become a pressing global health crisis, with bacterial infections increasingly difficult to treat due to the emergence of multidrug resistance. This study aims to identify potential chalcone molecules that interact with two key multidrug efflux pumps, AcrB and EmrD, of Escherichia coli, using advanced computational tools. In silico ADMET (absorption, distribution, metabolism, excretion, and toxicity), drug-likeness prediction, molecular docking, and molecular dynamics simulation analyses were conducted on a ligand library comprising 100 chalcone compounds against AcrB (PDB: 4DX5) and EmrD (PDB: 2GFP). The results demonstrated that Elastichalcone A (PubChem CID 102103730) exhibited a remarkable binding affinity of −9.9 kcal/mol against AcrB, while 4'-methoxy-4-hydroxychalcone (PubChem CID 5927890) displayed a binding affinity of −9.8 kcal/mol against EmrD. Both ligands satisfied drug-likeness rules and possessed favorable pharmacokinetic profiles. Molecular dynamics simulation of the AcrB-Elastichalcone A complex remained stable over 100 ns, with minimal fluctuations in root-mean-square deviation and root-mean-square fluctuation. The screened ligand library demonstrated good drug-likeness and pharmacokinetic properties. Moreover, the MM/PB(GB)SA calculation indicated the tight binding and thermodynamic stability of the simulated protein-ligand complexes. Overall, this study highlights the potential of chalcones as promising candidates for targeting multidrug efflux pumps, offering a potential strategy to overcome antibiotic resistance. Further exploration and optimization of these compounds may lead to the development of effective therapeutics against multidrug-resistant bacterial infections. Communicated by Ramaswamy H. Sarma

Conformational restriction shapes the inhibition of a multidrug efflux adaptor protein

Membrane efflux pumps play a major role in bacterial multidrug resistance. The tripartite multidrug efflux pump system from Escherichia coli, AcrAB-TolC, is a target for inhibition to lessen resistance development and restore antibiotic efficacy, with homologs in other ESKAPE pathogens. Here, we rationalize a mechanism of inhibition against the periplasmic adaptor protein, AcrA, using a combination of hydrogen/deuterium exchange mass spectrometry, cellular efflux assays, and molecular dynamics simulations. We define the structural dynamics of AcrA and find that an inhibitor can inflict long-range stabilisation across all four of its domains, whereas an interacting efflux substrate has minimal effect. Our results support a model where an inhibitor forms a molecular wedge within a cleft between the lipoyl and αβ barrel domains of AcrA, diminishing its conformational transmission of drug-evoked signals from AcrB to TolC. This work provides molecular insights into multidrug adaptor protein function which could be valuable for developing antimicrobial therapeutics.

Rhizosphere bacteria show a stronger response to antibiotic-based biopesticide than to conventional pesticides

The plant microbiota can substantially contribute to various functions related to host health, fitness, and productivity. Therefore, maintaining the integrity of the microbiota is beginning to be seen as a crucial factor in modern agriculture. Here, we evaluated the effects of two chemical pesticides (azoxystrobin and carbendazim) and an antibiotic-based biopesticide (wuyiencin) on the rhizosphere microbiome of tomato plants. It was found that all treatments resulted in changes in the bacterial community structure to varying degrees. The most pronounced changes were observed with the biopesticide, which resulted in an enrichment of Streptomyces in the microbiome. In contrast, the relative abundance of Actinobacteria decreased in samples that were treated with low and high dosages of carbendazim. Clostridia were enriched after the applications of azoxystrobin and wuyiencin. When functioning of the microbiome was assessed, it was shown that genes encoding multidrug efflux pumps and ABC transporters related to nutrient uptake were enriched. This enrichment is likely to overcome potentially negative effects linked to the exposure to the employed substances. The study provides new insights into the potential of different pesticides to modulate native plant microbiomes, and thus highlights the importance to include such evaluations when new active agents are developed.