Resistance-nodulation-cell Division Research Articles

Deciphering the impact of Acinetobacter baumannii on human health, and exploration of natural compounds as efflux pump inhibitors to treat multidrug resistance.

Acinetobacter baumannii is an ESKAPE pathogen and threatens human health by generating infections with high fatality rates. A. baumannii leads to a spectrum of infections such as skin and wound infections, endocarditis, meningitis pneumonia, septicaemia and urinary tract infections. Recently, strains of A. baumannii have emerged as multidrug-resistant (MDR), meaning they are resistant to at least three different classes of antibiotics. MDR development is primarily intensified by widespread antibiotic misuse and inadequate stewardship. The World Health Organization (WHO) declared A. baumannii a precarious MDR species. A. baumannii maintains the MDR phenotype via a diverse array of antimicrobial metabolite-hydrolysing enzymes, efflux of antibiotics, impermeability and antibiotic target modification, thereby complicating treatment. Hence, a deeper understanding of the resistance mechanisms employed by MDR A. baumannii can give possible approaches to treat antimicrobial resistance. Resistance-nodulation-cell division (RND) efflux pumps have been identified as the key contributors to MDR determinants, owing to their capacity to force a broad spectrum of chemical substances out of the bacterial cell. Though synthetic inhibitors have been reported previously, their efficacy and safety are of debate. As resistance-modifying agents, phytochemicals are ideal choices. These natural compounds could eliminate the bacteria or interact with pathogenicity events and reduce the bacteria's ability to evolve resistance. This review aims to highlight the mechanism behind the multidrug resistance in A. baumannii and elucidate the utility of natural compounds as efflux pump inhibitors to deal with the infections caused by A. baumannii.

RND Efflux Pump Induction: A Crucial Network Unveiling Adaptive Antibiotic Resistance Mechanisms of Gram-Negative Bacteria.

The rise of multi-drug-resistant (MDR) pathogenic bacteria presents a grave challenge to global public health, with antimicrobial resistance ranking as the third leading cause of mortality worldwide. Understanding the mechanisms underlying antibiotic resistance is crucial for developing effective treatments. Efflux pumps, particularly those of the resistance-nodulation-cell division (RND) superfamily, play a significant role in expelling molecules from bacterial cells, contributing to the emergence of multi-drug resistance. These are transmembrane transporters naturally produced by Gram-negative bacteria. This review provides comprehensive insights into the modulation of RND efflux pump expression in bacterial pathogens by numerous and common molecules (bile, biocides, pharmaceuticals, additives, plant extracts, etc.). The interplay between these molecules and efflux pump regulators underscores the complexity of antibiotic resistance mechanisms. The clinical implications of efflux pump induction by non-antibiotic compounds highlight the challenges posed to public health and the urgent need for further investigation. By addressing antibiotic resistance from multiple angles, we can mitigate its impact and preserve the efficacy of antimicrobial therapies.

Microbiological and molecular studies on a multidrug-resistant Pseudomonas aeruginosa from a liver transplant patient with urinary tract infection in Egypt.

Pseudomonas aeruginosa is an opportunistic pathogen responsible for complicated UTIs and exhibits high antibiotic resistance, leading to increased mortality rates, especially in cases of multidrug-resistant strains. This study aimed to investigate the antibiotic susceptibility patterns and genomic characterization of XDR strains identified in end-stage liver disease patients who underwent liver transplants. In this study, a number of 30 individuals who underwent liver transplants were registered. Ninety urine and 60 wound site swab samples were collected and processed for culturing, identification, and antimicrobial sensitivity. Extensively drug-resistant strain EMARA01 was confirmed through Sanger sequencing and was then processed for whole genome sequencing to characterize the genomic pattern. Sequencing data were processed for de novo assembly using various tools and databases, including genome annotation, serotype identification, virulence factor genes, and antimicrobial resistance gene. Pangenome analysis of randomly selected 147 reference strains and EMAR01 sequenced strain was performed using the Bacterial Pan Genome Analysis (BPGA) software. Of these total examined samples, nosocomial infection due to P. aeruginosa was detected in twelve patients' samples. AST analysis showed that P. aeruginosa strains exhibit resistance to tobramycin, erythromycin, and gentamicin, followed by piperacillin and ofloxacin, and no strains exhibit resistance to meropenem and imipenem. The CARD database identified 59 AMR genes similar to the EMAR01 strain genome and mostly belong to the family involved in the resistance-nodulation-cell division (RND) antibiotic efflux pump. Five genes; nalC, nalD, MexR, MexA, and MexB, exhibit resistance to 14 classes of antibiotics, while two AMR; CpxR, and OprM, exhibit resistance to 15 classes of drugs. Pangenome analysis revealed that the pan-genome remained open, suggesting the potential for acquiring accessory and unique genes. Notably, the genes predominantly involved in amino acid transport metabolism were identified using the KEGG database. This study provides valuable insights into the antimicrobial resistance profile, genetic features, and genomic evolution of P. aeruginosa strains causing UTIs in liver transplant patients. The findings emphasize the significance of comprehending AMR mechanisms and genetic diversity in P. aeruginosa for developing effective treatment strategies and infection control measures.

Molecular epidemiology and resistance mechanisms of tigecycline-non-susceptible Acinetobacter baumannii isolated from a tertiary care hospital in Chongqing, China.

We studied 34 isolates of Tigecycline-Non-Susceptible A. baumannii (TNAB) obtained from clinical specimens at a large tertiary care hospital in Chongqing, China. These 34 strains belonged to 8 different clones including ST195 (35.3%) and ST208 (17.7%). EBURST analysis found that these 8 ST types belonged to the Clonal Complex 92. Tigecycline resistance-associated genes adeR, adeS, adeL, adeN, rrf, rpsJ, and trm were detected in most strains. The expression level of the resistance-nodulation-cell division (RND) efflux pumps in TNAB strains was higher than the reference strain ATCC19606. 58.8% of strains had a decrease in the tigecycline minimum inhibitory concentration (MIC) after the addition of carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The TNAB strains in our hospital have a high degree of affinity and antibiotic resistance. Regular surveillance should be conducted to prevent outbreaks of TNAB epidemics.

Pyrrole-based inhibitors of RND-type efflux pumps reverse antibiotic resistance and display anti-virulence potential.

Efflux pumps of the resistance-nodulation-cell division (RND) superfamily, particularly the AcrAB-TolC, and MexAB-OprM, besides mediating intrinsic and acquired resistance, also intervene in bacterial pathogenicity. Inhibitors of such pumps could restore the activities of antibiotics and curb bacterial virulence. Here, we identify pyrrole-based compounds that boost antibiotic activity in Escherichia coli and Pseudomonas aeruginosa by inhibiting their archetype RND transporters. Molecular docking and biophysical studies revealed that the EPIs bind to AcrB. The identified efflux pump inhibitors (EPIs) inhibit the efflux of fluorescent probes, attenuate persister formation, extend post-antibiotic effect, and diminish resistant mutant development. The bacterial membranes remained intact upon exposure to the EPIs. EPIs also possess an anti-pathogenic potential and attenuate P. aeruginosa virulence in vivo. The intracellular invasion of E. coli and P. aeruginosa inside the macrophages was hampered upon treatment with the lead EPI. The excellent efficacy of the EPI-antibiotic combination was evidenced in animal lung infection and sepsis protection models. These findings indicate that EPIs discovered herein with negligible toxicity are potential antibiotic adjuvants to address life-threatening Gram-negative bacterial infections.

Structural analysis of resistance-nodulation cell division transporters.

SUMMARYInfectious bacteria have both intrinsic and acquired mechanisms to combat harmful biocides that enter the cell. Through adaptive pressures, many of these pathogens have become resistant to many, if not all, of the current antibiotics used today to treat these often deadly infections. One prominent mechanism is the upregulation of efflux systems, especially the resistance-nodulation-cell division class of exporters. These tripartite systems consist of an inner membrane transporter coupled with a periplasmic adaptor protein and an outer membrane channel to efficiently transport a diverse array of substrates from inside the cell to the extracellular space. Detailed mechanistic insight into how these inner membrane transporters recognize and shuttle their substrates can ultimately inform both new antibiotic and efflux pump inhibitor design. This review examines the structural basis of substrate recognition of these pumps and the molecular mechanisms underlying multidrug extrusion, which in turn mediate antimicrobial resistance in bacterial pathogens.

Regulation of overexpressed efflux pump encoding genes by cinnamon oil and trimethoprim to abolish carbapenem-resistant Acinetobacter baumannii clinical strains

Resistance mechanisms are a shelter for Acinetobacter baumannii to adapt to our environment which causes difficulty for the infections to be treated and WHO declares this organism on the top of pathogens priority for new drug development. The most common mechanism that develops drug resistance is the overexpression of the efflux pump, especially Resistance-nodulation-cell division (RND) family, to almost most antibiotics. The study is designed to detect RND efflux pump genes in A. baumannii, and its correlation to multidrug resistance, in particular, the carbapenems resistance Acinetobacterbaumannii (CRAB), and using different inhibitors that restore the antibiotic susceptibility of imipenem. Clinical A. baumannii isolates were recovered from different Egyptian hospitals in Intensive care unit (ICU). The expression of genes in two strains was analyzed using RT-PCR before and after inhibitor treatment. About 100 clinical A. baumannii isolates were recovered and identified and recorded as MDR strains with 75% strains resistant to imipenem. adeB, adeC, adeK, and adeJ were detected in thirty- seven the carbapenems resistance Acinetobacterbaumannii (CRAB) strains. Cinnamomum verum oil, Trimethoprim, and Omeprazole was promising inhibitor against 90% of the carbapenems resistance Acinetobacterbaumannii (CRAB) strains with a 2-6-fold decrease in imipenem MIC. Downregulation of four genes was associated with the addition of those inhibitors to imipenem for two the carbapenems resistance Acinetobacterbaumannii (CRAB) (ACN15 and ACN99) strains, and the effect was confirmed in 24 h killing kinetics. Our investigation points to the carbapenems resistance Acinetobacterbaumannii (CRAB) strain’s prevalence in Egyptian hospitals with the idea to revive the imipenem activity using natural and chemical drugs as inhibitors that possessed high synergistic activity.

Impact of low-dose ozone nanobubble treatments on antimicrobial resistance genes in pond water.

Antimicrobial resistance (AMR) poses a significant global health threat as the silent pandemic. Because of the use of antimicrobials in aquaculture systems, fish farms may be potential reservoirs for the dissemination of antimicrobial resistance genes (ARGs). Treatments with disinfectants have been promoted to reduce the use of antibiotics; however, the effect of these types of treatments on AMR or ARGs is not well known. This study aimed to evaluate the effects of low dose ozone treatments (0.15 mg/L) on ARG dynamics in pond water using metagenomic shotgun sequencing analysis. The results suggested that ozone disinfection can increase the relative abundance of acquired ARGs and intrinsic efflux mediated ARGs found in the resistance nodulation cell division (RND) family. Notably, a co-occurrence of efflux and non-efflux ARGs within the same bacterial genera was also observed, with most of these genera dominating the bacterial population following ozone treatments. These findings suggest that ozone treatments may selectively favor the survival of bacterial genera harboring efflux ARGs, which may also have non-efflux ARGs. This study underscores the importance of considering the potential impacts of disinfection practices on AMR gene dissemination particularly in aquaculture settings where disinfectants are frequently used at low levels. Future endeavors should prioritize the evaluation of these strategies, as they may be associated with an increased risk of AMR in aquatic environments.

Structures and Efflux Mechanisms of the AcrAB-TolC Pump.

The global emergence of multidrug resistance (MDR) in gram-negative bacteria has become a matter of worldwide concern. MDR in these pathogens is closely linked to the overexpression of certain efflux pumps, particularly the resistance-nodulation-cell division (RND) efflux pumps. Inhibition of these pumps presents an attractive and promising strategy to combat antibiotic resistance, as the efflux pump inhibitors can effectively restore the potency of existing antibiotics. AcrAB-TolC is one well-studied RND efflux pump, which transports a variety of substrates, therefore providing resistance to a broad spectrum of antibiotics. To develop effective pump inhibitors, a comprehensive understanding of the structural aspect of the AcrAB-TolC efflux pump is imperative. Previous studies on this pump's structure have been limited to individual components or in vitro determination of fully assembled pumps. Recent advancements in cellular cryo-electron tomography (cryo-ET) have provided novel insights into this pump's assembly and functional mechanism within its native cell membrane environment. Here, we present a summary of the structural data regarding the AcrAB-TolC efflux pump, shedding light on its assembly pathway and operational mechanism.

A TetR family regulator of an RND efflux system that directs artemisinin resistance in Vibrio cholerae.

Artemisinin (ARS) displayed bactericidal activity against Vibrio cholerae. To assess the mechanistic details of its antibacterial action, we have isolated V. cholerae mutants with enhanced ARS resistance and identified a gene (VCA0767) whose loss-of-function resulted in the ARS resistance phenotypes. This gene (atrR) encodes a TetR family transcriptional regulator, and its deletion mutant displayed the reduction in ARS-induced ROS formation and DNA damage. Transcriptomic analysis revealed that the genes encoding a resistance-nodulation-cell division (RND) efflux pump operon (vexRAB) and the outer membrane component (tolC) were highly upregulated in the artR mutant, suggesting that AtrR might act as a negative regulator of this operon and tolC. Gene deletion of vexR, vexB, or tolC abrogated the ARS resistance of the atrR mutant, and more importantly, the ectopic expression of VexAB-TolC was sufficient for the ARS resistance, indicating that the increased expression of the VexAB-TolC efflux system is necessary and sufficient for the ARS resistance of the atrR mutant. The cytoplasmic accumulation of ARS was compromised in the vexBtolC mutant, suggesting that the VexAB-TolC might be the primary efflux system exporting ARS to reduce its toxicity inside of the bacterial cells. The atrR mutant displayed resistance to erythromycin as well in a VexR-dependent manner. This result suggests that AtrR may act as a global regulator responsible for preventing intracellular accumulation of toxic chemicals by enhancing the RND efflux system.IMPORTANCEDrug efflux protein complexes or efflux pumps are considered as the major determinants of multiple antimicrobial resistance by exporting a wide range of structurally diverse antibiotics in bacterial pathogens. Despite the clinical significance of the increased expression of the efflux pumps, their substrate specificity and regulation mechanisms are poorly understood. Here, we demonstrated that VexAB-TolC, a resistance-nodulation-cell division (RND) efflux pump of V. cholerae, is responsible for the resistance to artemisinin (ARS), an antimalarial drug with bactericidal activity. Furthermore, we newly identified AtrR, a TetR family repressor, as a global regulator for VexRAB and the common outer membrane channel, TolC, where VexR functions as the pathway-specific regulator of the vexAB operon. Our findings will help improve our insight into a broad range of substrate specificity of the VexAB-TolC system and highlight the complex regulatory networks of the multiple RND efflux systems during V. cholerae pathogenesis.

Antibiotic resistance in Campylobacter jejuni and Campylobacter coli: significant contribution of an RND type efflux pump in erythromycin resistance

In this study, we aimed to determine the antibiotic resistance status of Campylobacter spp. isolated from human infections in our region, including the role of mechanisms involved in erythromycin resistance. Standard methods were used for the isolation, identification and antibiotic susceptibility testing of Campylobacter spp. isolates. Erythromycin-resistant mutants were selected from erythromycin-susceptible clinical isolates, and the erythromycin resistance mechanisms were investigated phenotypically by determining the erythromycin MICs of isolates in the presence and absence of the resistance nodulation cell division (RND) type efflux pump inhibitor, phenylalanine-arginine β-naphthylamide dihydrochloride (PAβN), and genotypically by determining ribosomal and cmeABC alterations using PCR and DNA sequence analysis. Campylobacter spp., including 184 C. jejuni and 20 C. coli in a two-year period, were the most frequently isolated gastrointestinal bacterial pathogens in our region. However, in both C. jejuni and C. coli, resistance to tetracycline and ciprofloxacin were found to be high, erythromycin resistance was especially high (20%) in C. coli. With a ribosomal alteration, A2075G, which was found to be associated with high-level erythromycin resistance in clinical isolates, PAβN significantly reduced the erythromycin MICs in both clinical isolates and mutants. An important finding of this study, while considering cmeABC operon, is the explanation of why erythromycin resistance is more common among C. coli than C. jejuni, bearing in mind the specific deletions and alterations in the intergenic region of the operon in all erythromycin-resistant C. coli isolates. Ultimately, these findings revealed the significant role of RND-type efflux activity in increased erythromycin MICs of the isolates.

Abundant resistome determinants in rhizosphere soil of the wild plant Abutilon fruticosum

A metagenomic whole genome shotgun sequencing approach was used for rhizospheric soil micribiome of the wild plant Abutilon fruticosum in order to detect antibiotic resistance genes (ARGs) along with their antibiotic resistance mechanisms and to detect potential risk of these ARGs to human health upon transfer to clinical isolates. The study emphasized the potential risk to human health of such human pathogenic or commensal bacteria, being transferred via food chain or horizontally transferred to human clinical isolates. The top highly abundant rhizospheric soil non-redundant ARGs that are prevalent in bacterial human pathogens or colonizers (commensal) included mtrA, soxR, vanRO, golS, rbpA, kdpE, rpoB2, arr-1, efrA and ileS genes. Human pathogenic/colonizer bacteria existing in this soil rhizosphere included members of genera Mycobacterium, Vibrio, Klebsiella, Stenotrophomonas, Pseudomonas, Nocardia, Salmonella, Escherichia, Citrobacter, Serratia, Shigella, Cronobacter and Bifidobacterium. These bacteria belong to phyla Actinobacteria and Proteobacteria. The most highly abundant resistance mechanisms included antibiotic efflux pump, antibiotic target alteration, antibiotic target protection and antibiotic inactivation. antimicrobial resistance (AMR) families of the resistance mechanism of antibiotic efflux pump included resistance-nodulation-cell division (RND) antibiotic efflux pump (for mtrA, soxR and golS genes), major facilitator superfamily (MFS) antibiotic efflux pump (for soxR gene), the two-component regulatory kdpDE system (for kdpE gene) and ATP-binding cassette (ABC) antibiotic efflux pump (for efrA gene). AMR families of the resistance mechanism of antibiotic target alteration included glycopeptide resistance gene cluster (for vanRO gene), rifamycin-resistant beta-subunit of RNA polymerase (for rpoB2 gene) and antibiotic-resistant isoleucyl-tRNA synthetase (for ileS gene). AMR families of the resistance mechanism of antibiotic target protection included bacterial RNA polymerase-binding protein (for RbpA gene), while those of the resistance mechanism of antibiotic inactivation included rifampin ADP-ribosyltransferase (for arr-1 gene). Better agricultural and food transport practices are required especially for edible plant parts or those used in folkloric medicine.

The genome of Symbiodiniaceae-associated Stutzerimonas frequens CAM01 reveals a broad spectrum of antibiotic resistance genes indicating anthropogenic drift in the Palk Bay coral reef of south-eastern India.

An increase in antibiotic pollution in reef areas will lead to the emergence of antibiotic-resistant bacteria, leading to ecological disturbances in the sensitive coral holobiont. This study provides insights into the genome of antibiotics-resistant Stutzerimonas frequens CAM01, isolated from Favites-associated Symbiodiniaceae of a near-shore polluted reef of Palk Bay, India. The draft genome contains 4.67 Mbp in size with 52 contigs. Further genome analysis revealed the presence of four antibiotic-resistant genes, namely, adeF, rsmA, APH (3")-Ib, and APH (6)-Id that provide resistance by encoding resistance-nodulation-cell division (RND) antibiotic efflux pump and aminoglycoside phosphotransferase. The isolate showed resistance against 73% of the antibiotics tested, concurrent with the predicted AMR genes. Four secondary metabolites, namely Aryl polyene, NRPS-independent-siderophore, terpenes, and ectoine were detected in the isolate, which may play a role in virulence and pathogenicity adaptation in microbes. This study provides key insights into the genome of Stutzerimonas frequens CAM01 and highlights the emergence of antibiotic-resistant bacteria in coral reef ecosystems.

Molecular determinants of avoidance and inhibition of Pseudomonas aeruginosa MexB efflux pump.

Transporters of the resistance-nodulation-cell division (RND) superfamily of proteins are the dominant multidrug efflux power of Gram-negative bacteria. The major RND efflux pump of Pseudomonas aeruginosa is MexAB-OprM, in which the inner membrane transporter MexB is responsible for the recognition and binding of compounds. The high importance of this pump in clinical antibiotic resistance made it a subject of intense investigations and a promising target for the discovery of efflux pump inhibitors. This study is focused on a series of peptidomimetic compounds developed as effective inhibitors of MexAB-OprM. We performed multi-copy molecular dynamics simulations, machine-learning (ML) analyses, and site-directed mutagenesis of MexB to investigate interactions of MexB with representatives of efflux avoiders, substrates, and inhibitors. The analysis of both direct and water-mediated protein-ligand interactions revealed characteristic patterns for each class, highlighting significant differences between them. We found that efflux avoiders poorly interact with the access binding site of MexB, and inhibition engages amino acid residues that are not directly involved in binding and transport of substrates. In agreement, machine-learning models selected different residues predictive of MexB substrates and inhibitors. The differences in interactions were further validated by site-directed mutagenesis. We conclude that the substrate translocation and inhibition pathways of MexB split at the interface (between the main putative binding sites) and at the deep binding pocket and that interactions outside of the hydrophobic patch contribute to the inhibition of MexB. This molecular-level information could help in the rational design of new inhibitors and antibiotics less susceptible to the efflux mechanism. IMPORTANCE Multidrug transporters recognize and expel from cells a broad range of ligands including their own inhibitors. The difference between the substrate translocation and inhibition routes remains unclear. In this study, machine learning and computational and experimental approaches were used to understand dynamics of MexB interactions with its ligands. Our results show that some ligands engage a certain combination of polar and charged residues in MexB binding sites to be effectively expelled into the exit funnel, whereas others engage aromatic and hydrophobic residues that slow down or hinder the next step in the transporter cycle. These findings suggest that all MexB ligands fit into this substrate-inhibitor spectrum depending on their physico-chemical structures and properties.

Amphiphilic peptide Mastoparan-B induces conformational changes within the AdeB efflux pump, down-regulates adeB gene expression, and restores antibiotic susceptibility in an MDR strain of Acinetobacter baumannii.

Mastoparan B (MP-B) is an amphiphilic peptide with a potent antimicrobial activity against most Gram-negative bacteria. However, there is little information available on the inhibition of the Acinetobacter baumannii resistance-nodulation-cell-division (RND) efflux pump using this antimicrobial peptide. Here, we carried out a series of in-silico experiments to find the mechanisms underlying the anti-efflux activity of MP-B using a multi-drug resistant (MDR) strain of A. baumannii (AB). According to our findings, MP-B demonstrated a potent antibacterial activity against an MDR-AB (minimum inhibitory concentration [MIC] = 1 μg/mL) followed by a 20-fold reduction in the adeB gene expression in the presence of sub-MIC of this peptide. Using Groningen Machine for Chemicals Simulation (GROMACS)via PyMOL Graphical User Interface (GUI), (we observed that, the AdeB transporter had conserved helix-turn-helix regions and a tight pore rich in Phe and Ala residues. To understand how inhibition of the AdeB is achieved, we generated 20 apo-MP-B poses using the InterPep and SiteMap tools. The high-quality model was created by homology modeling and used for docking via AutoDock/Vina to identify the MP-B binding sites. We established that the most apo-MP-B formed H-bonds to the backbone of five amino acids in the Helix-5. As a result, the dihedral angles of the involved amino acids shift by 9.0-9.6 Ǻ, causing a change in the conformation of the AdeB protein. This led to helix conformation stereoisomerization and block the AdeB activity. MP-B presumably has dual mechanisms. (1) It blocks the AdeB transporter by changing its conformation. (2) MP-B influences the adeB gene expression by binding to G-protein which laterally controls efflux regulators like MarA, RamA, SoxS, and Rob proteins.

Temocillin Resistance in the Enterobacter cloacae Complex Is Conferred by a Single Point Mutation in BaeS, Leading to Overexpression of the AcrD Efflux Pump.

The Enterobacter cloacae complex (ECC) has become a major opportunistic pathogen with antimicrobial resistance issues. Temocillin, an "old" carboxypenicillin that is remarkably stable toward β-lactamases, has been used as an alternative for the treatment of multidrug-resistant ECC infections. Here, we aimed at deciphering the never-investigated mechanisms of temocillin resistance acquisition in Enterobacterales. By comparative genomic analysis of two clonally related ECC clinical isolates, one susceptible (Temo_S [MIC of 4 mg/L]) and the other resistant (Temo_R [MIC of 32 mg/L]), we found that they differed by only 14 single-nucleotide polymorphisms, including one nonsynonymous mutation (Thr175Pro) in the two-component system (TCS) sensor histidine kinase BaeS. By site-directed mutagenesis in Escherichia coli CFT073, we demonstrated that this unique change in BaeS was responsible for a significant (16-fold) increase in temocillin MIC. Since the BaeSR TCS regulates the expression of two resistance-nodulation-cell division (RND)-type efflux pumps (namely, AcrD and MdtABCD) in E. coli and Salmonella, we demonstrated by quantitative reverse transcription-PCR that mdtB, baeS, and acrD genes were significantly overexpressed (15-, 11-, and 3-fold, respectively) in Temo_R. To confirm the role of each efflux pump in this mechanism, multicopy plasmids harboring mdtABCD or acrD were introduced into either Temo_S or the reference strain E. cloacae subsp. cloacae ATCC 13047. Interestingly, only the overexpression of acrD conferred a significant increase (from 8- to 16-fold) of the temocillin MIC. Altogether, we have shown that temocillin resistance in the ECC can result from a single BaeS alteration, likely resulting in the permanent phosphorylation of BaeR and leading to AcrD overexpression and temocillin resistance through enhanced active efflux.

Cryo-EM Structures of AcrD Illuminate a Mechanism for Capturing Aminoglycosides from Its Central Cavity.

The Escherichia coli acriflavine resistance protein D (AcrD) is an efflux pump that belongs to the resistance-nodulation-cell division (RND) superfamily. Its primary function is to provide resistance to aminoglycoside-based drugs by actively extruding these noxious compounds out of E. coli cells. AcrD can also mediate resistance to a limited range of other amphiphilic agents, including bile acids, novobiocin, and fusidic acids. As there is no structural information available for any aminoglycoside-specific RND pump, here we describe cryo-electron microscopy (cryo-EM) structures of AcrD in the absence and presence of bound gentamicin. These structures provide new information about the RND superfamily of efflux pumps, specifically, that three negatively charged residues central to the aminoglycoside-binding site are located within the ceiling of the central cavity of the AcrD trimer. Thus, it is likely that AcrD is capable of picking up aminoglycosides via this central cavity. Through the combination of cryo-EM structural determination, mutagenesis analysis, and molecular simulation, we show that charged residues are critically important for this pump to shuttle drugs directly from the central cavity to the funnel of the AcrD trimer for extrusion. IMPORTANCE Here, we report cryo-EM structures of the AcrD aminoglycoside efflux pump in the absence and presence of bound gentamicin, posing the possibility that this pump is capable of capturing aminoglycosides from the central cavity of the AcrD trimer. The results indicate that AcrD utilizes charged residues to bind and export drugs, mediating resistance to these antibiotics.

دور مضخة الدفق في مقاومة البكتيريا للمضادات الحيوية

The multi-drug resistant efflux pump is a glycoprotein pump whose function is to push foreign substances. The efflux pump is found in humans, animals. It also has wide-ranging properties in bacteria and fungi. They are found in all species of bacteria, and efflux pump genes can be found in bacterial chromosomes or mobile genetic elements, such as plasmids. The most sensitive function that leads to a global problem is its resistance to antibiotics in bacterial cells, which increases the ability to bacteria from becoming strong virulence factors that most or all antibiotics cannot kill. It also has other important functions. It is related to the defense mechanism of the horse by pushing all harmful substances. This pump is divided into five families. The division depends on the source of energy, symmetry, and the general structure. The major facilitator (MFS) superfamily; the multidrug and toxic compound extrusion (MATE) family and the adenosine-triphosphate (ATP)-binding cassette (ABC); superfamily the resistance-nodulation-cell division (RND) superfamily and the small multidrug resistance (SMR). The ABC family uses ATP as a source of energy for export, while other flow pumps use the driving force of the proton. These hosts participate in many cellular processes such as intercellular communication, formation of cell membranes, virulence factors of bacterial cell, extrusion of toxic substances, disinfectants, toxic metabolic by-products, and antibiotics. It is also one of the important formations in bacteria that give bacteria the ability to expel antibiotics through various and diverse genes that are located either on the chromosome or plasmid of different pump families. Evidence indicates that the efflux pump has physiological functions in bacteria, and its overexpression has a role in inducing multi-drug resistance and biofilm formation. Hence, the regulation of efflux pump action is an orderly, strict action in response to physiological and environmental signals.

TCA and SSRI Antidepressants Exert Selection Pressure for Efflux-Dependent Antibiotic Resistance Mechanisms in Escherichia coli.

Microbial diversity is reduced in the gut microbiota of animals and humans treated with selective serotonin reuptake inhibitors (SSRIs) and tricyclic antidepressants (TCAs). The mechanisms driving the changes in microbial composition, while largely unknown, is critical to understand considering that the gut microbiota plays important roles in drug metabolism and brain function. Using Escherichia coli, we show that the SSRI fluoxetine and the TCA amitriptyline exert strong selection pressure for enhanced efflux activity of the AcrAB-TolC pump, a member of the resistance-nodulation-cell division (RND) superfamily of transporters. Sequencing spontaneous fluoxetine- and amitriptyline-resistant mutants revealed mutations in marR and lon, negative regulators of AcrAB-TolC expression. In line with the broad specificity of AcrAB-TolC pumps these mutants conferred resistance to several classes of antibiotics. We show that the converse also occurs, as spontaneous chloramphenicol-resistant mutants displayed cross-resistance to SSRIs and TCAs. Chemical-genomic screens identified deletions in marR and lon, confirming the results observed for the spontaneous resistant mutants. In addition, deletions in 35 genes with no known role in drug resistance were identified that conferred cross-resistance to antibiotics and several displayed enhanced efflux activities. These results indicate that combinations of specific antidepressants and antibiotics may have important effects when both are used simultaneously or successively as they can impose selection for common mechanisms of resistance. Our work suggests that selection for enhanced efflux activities is an important factor to consider in understanding the microbial diversity changes associated with antidepressant treatments. IMPORTANCE Antidepressants are prescribed broadly for psychiatric conditions to alter neuronal levels of synaptic neurotransmitters such as serotonin and norepinephrine. Two categories of antidepressants are selective serotonin reuptake inhibitors (SSRIs) and tricyclic antidepressants (TCAs); both are among the most prescribed drugs in the United States. While it is well-established that antidepressants inhibit reuptake of neurotransmitters there is evidence that they also impact microbial diversity in the gastrointestinal tract. However, the mechanisms and therefore biological and clinical effects remain obscure. We demonstrate antidepressants may influence microbial diversity through strong selection for mutant bacteria with increased AcrAB-TolC activity, an efflux pump that removes antibiotics from cells. Furthermore, we identify a new group of genes that contribute to cross-resistance between antidepressants and antibiotics, several act by regulating efflux activity, underscoring overlapping mechanisms. Overall, this work provides new insights into bacterial responses to antidepressants important for understanding antidepressant treatment effects.

Common recognition topology of mex transporters of Pseudomonas aeruginosa revealed by molecular modelling.

The secondary transporters of the resistance-nodulation-cell division (RND) superfamily mediate multidrug resistance in Gram-negative bacteria like Pseudomonas aeruginosa. Among these RND transporters, MexB, MexF, and MexY, with partly overlapping specificities, have been implicated in pathogenicity. Only the structure of the former has been resolved experimentally, which together with the lack of data about the functional dynamics of the full set of transporters, limited a systematic investigation of the molecular determinants defining their peculiar and shared features. In a previous work (Ramaswamy et al., Front. Microbiol., 2018, 9, 1144), we compared at an atomistic level the two main putative recognition sites (named access and deep binding pockets) of MexB and MexY. In this work, we expand the comparison by performing extended molecular dynamics (MD) simulations of these transporters and the pathologically relevant transporter MexF. We employed a more realistic model of the inner phospholipid membrane of P. aeruginosa and more accurate force-fields. To elucidate structure/dynamics-activity relationships we performed physico-chemical analyses and mapped the binding propensities of several organic probes on all transporters. Our data revealed the presence, also in MexF, of a few multifunctional sites at locations equivalent to the access and deep binding pockets detected in MexB. Furthermore, we report for the first time about the multidrug binding abilities of two out of five gates of the channels deputed to peripheral (early) recognition of substrates. Overall, our findings help to define a common "recognition topology" characterizing Mex transporters, which can be exploited to optimize transport and inhibition propensities of antimicrobial compounds.