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S-amlodipine (S-AM) is a commonly prescribed antihypertensive drug for patients with hypertension, and it is used for an extended duration in most hypertensive patients. However, it remains unclear whether long-term exposure to non-antibiotic drug S-amlodipine induces bacterial antibiotic resistance. To investigate the potential impact of S-AM on bacterial antibiotic resistance, we conducted a 60-day in vitro evolution experiment in which intestinal pathogen K. pneumoniae was exposed to 2 mg⋅L-1 S-AM. We determined the resistance of the evolved strains to six antibiotics through antibiotic susceptibility testing. Furthermore, we delved into the potential resistance mechanisms of K. pneumoniae via genome sequencing, fluorescence quantitative PCR, and bioinformatics analysis. This study observed that the mutation frequency of K. pneumoniae increased from 10-7 to 1.5 x 10-1 under S-AM stress, and the intracellular reactive oxygen species (ROS) levels of the evolved strains significantly increased compared to that of the control strains. Additionally, the evolved strains exhibited heightened resistance to polymyxin, kanamycin, chloramphenicol, erythromycin, and moxifloxacin. Whole genome sequencing analysis results showed the presence of three non-synonymous single nucleotide polymorphism (SNP) mutation sites, one synonymous SNP mutation site, two intergenic SNPs, and one intergenic insertion site in the evolved strains. These non-synonymous gene mutations include the HAMP domain-containing histidine kinase crrB gene, sensor domain-containing diguanylate cyclase, and type 3 fimbria adhesin subunit mrkD gene. Notably, the mutated crrB gene, in collaboration with the crrA gene, enhanced the modification of the lipid A component of the cell membrane lipopoly-saccharide (LPS) in the evolved strains. This modification increased the positive charge on the bacterial cell membrane surface, hindering polymyxin-cell interactions and contributing to increased polymyxin resistance in K. pneumoniae. Fluorescence quantitative PCR results revealed that exposure to S-AM enhanced the mRNA expressions of two-component regulatory system genes (e.g., crrAB, phoPQ, pmrAB, and pmrDFIK), multidrug efflux pump genes (e.g., emrD and oqxAB), and the intrinsic resistance gene aadA2, which might be responsible for increased bacterial resistance to multiple antibiotics. In summary, this study is the first to demonstrate that S-AM stress triggers multiple antibiotic resistance in K. pneumoniae and elucidates the underlying molecular mechanisms, serving as an early warning for potential health risks associated with long-term medication in hypertensive patients. It lays a foundation for further exploration of antibiotic resistance in diverse intestinal bacteria under prolonged S-AM stress. [ABSTRACT FROM AUTHOR]

Multidrug efflux pumps are the frontline defense mechanisms of Gram-negative bacteria, yet little is known of their relative fitness trade-offs under gut conditions such as low pH and the presence of antimicrobial food molecules. Low pH contributes to the proton-motive force (PMF) that drives most efflux pumps. We show how the PMF-dependent pumps AcrAB-TolC, MdtEF-TolC, and EmrAB-TolC undergo selection at low pH and in the presence of membrane-permeant phytochemicals. Competition assays were performed by flow cytometry of co-cultured Escherichia coli K-12 strains possessing or lacking a given pump complex. All three pumps showed negative selection under conditions that deplete PMF (pH 5.5 with carbonyl cyanide 3-chlorophenylhydrazone or at pH 8.0). At pH 5.5, selection against AcrAB-TolC was increased by aromatic acids, alcohols, and related phytochemicals such as methyl salicylate. The degree of fitness cost for AcrA was correlated with the phytochemical's lipophilicity (logP). Methyl salicylate and salicylamide selected strongly against AcrA, without genetic induction of drug resistance regulons. MdtEF-TolC and EmrAB-TolC each had a fitness cost at pH 5.5, but salicylate or benzoate made the fitness contribution positive. Pump fitness effec ts were not explained by gene expression (measured by digital PCR). Between pH 5.5 and 8.0, acrA and emrA were upregulated in the log phase, whereas mdtE expression was upregulated in the transition-to-stationary phase and at pH 5.5 in the log phase. Methyl salicylate did not affect pump gene expression. Our results suggest that lipophilic non-acidic molecules select against a major efflux pump without inducing antibiotic resistance regulons. [ABSTRACT FROM AUTHOR]

The infections of multidrug-resistant bacteria, particularly the infections caused by Gram-negative bacterial infections, are considered the main global serious health problems. Multidrug-resistant bacteria possess different resistance mechanisms, the most important of which are drug efflux pumps due to their function in recognizing harmful compounds, especially antibiotics, and expelling, them outside the cell, which leads to a decrease in their concentration to sub-toxic levels and losing their effectiveness. One of the most important approaches that can be used to inhibit drug efflux pumps is using small molecule inhibitors targeting specifically pumps to prevent their expelling antibiotics from inside the cell before reaching the required concentration that kills bacteria. The drug efflux pumps inhibition approach is emerging as a new attractive and promising approach in reversing antibiotic resistance in many clinically relevant pathogenic bacteria. This review will discuss the requirements that must be met in inhibitors that target AcrB, which is the main subunit protein responsible for multiple antibiotic resistance in the main pump of AcrAB-TolC which belongs to E. coli, which were specifically chosen due to their various homologs in many Gram-negative bacteria which all of which are implicated in multi-drug resistance (MDR), and the availability of the pump's functions and chemical structures.

Multidrug resistance (MDR) poses a severe threat to human health. The existence of efflux pumps primarily contributes to resistance in Gram-negative bacterial infections. Efflux pump inhibitors (EPIs) can potentially improve the efficacy of antibiotics and reduce bacterial pathogenicity by inhibiting the mechanism of these pumps. Here we report Bac-EPIC, a web interface consisting of AcrA_Ipred, and AcrB_Ipred tools, can aid in the identification of potential EPIs that might bind to AcrAB-TolC pump subunits, alter its structure in vivo, prevent efflux, and enhance antibiotic efficacy in Escherichia coli (E. coli) and other Gram-negative bacteria. The web server is developed by collecting and archiving structural moieties from literature-reported EPIs. The web interface can be utilized in decision-making upon structural similarity-based screening on whether a compound should be examined experimentally or disregarded. It takes the SMILES format of chemical structure as an input. Users can also draw the 2D structure and get the output report of the possible structural similarity profile, similarity percentage, and the active moieties in the compound. The designed web interface is publicly accessible for in silico efflux pump inhibition prediction, at http://14.139.62.46/AcrAB_Ipred/. Overall, the webserver increases the diversity of EPIs, and proposes mechanistic insights to understand and regulate the efflux pump assembly & its function, ultimately aiding in fight against antibiotic resistance.

Introduction: Although carbapenemases are frequently reported in resistant A. baumannii clinical isolates, other chromosomallymediated elements of resistance that are considered essential are frequently underestimated. Having a wide substrate range, multidrug efflux pumps frequently underlie antibiotic treatment failure. Recognizing and exploiting variations in multidrug efflux pumps and penicillin-binding proteins (PBPs) is an essential approach in new antibiotic drug discovery and engineering to meet the growing challenge of multidrug-resistant Gram-negative bacteria. Methods: A total of 980 whole genome sequences of A. baumannii were analyzed. Nucleotide sequences for the genes studied were queried against a custom database of FASTA sequences using the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) system. The correlation between different variants and carbapenem Minimum Inhibitory Concentrations (MICs) was studied. PROVEAN and I-Mutant predictor suites were used to predict the effect of the studied amino acid substitutions on protein function and protein stability. Both PsiPred and FUpred were used for domain and secondary structure prediction. Phylogenetic reconstruction was performed using SANS serif and then visualized using iTOL and Phandango. Results: Exhibiting the highest detection rate, AdeB codes for an important efflux-pump structural protein. T48V, T584I, and P660Q were important variants identified in the AdeB-predicted multidrug efflux transporter pore domains. These can act as probable targets for designing new efflux-pump inhibitors. Each of AdeC Q239L and AdeS D167N can also act as probable targets for restoring carbapenem susceptibility. Membrane proteins appear to have lower predictive potential than efflux pump-related changes. OprB and OprD changes show a greater effect than OmpA, OmpW, Omp33, and CarO changes on carbapenem susceptibility. Functional and statistical evidence make the variants T636A and S382N at PBP1a good markers for imipenem susceptibility and potential important drug targets that can modify imipenem resistance. In addition, PBP3_370, PBP1a_T636A, and PBP1a_S382N may act as potential drug targets that can be exploited to counteract imipenem resistance. Conclusion: The study presents a comprehensive epidemiologic and statistical analysis of potential membrane proteins and efflux-pump variants related to carbapenemsusceptibility in A. baumannii, shedding light on their clinical utility as diagnostic markers and treatment modification targets for more focused studies of candidate elements. [ABSTRACT FROM AUTHOR]

The excessive use of antibiotics has led to the emergence of multidrug-resistant (MDR) pathogens in clinical settings and food-producing animals, posing significant challenges to clinical management and food control. Over the past few decades, the discovery of antimicrobials has slowed down, leading to a lack of treatment options for clinical infectious diseases and foodborne illnesses. Given the increasing prevalence of antibiotic resistance and the limited availability of effective antibiotics, the discovery of novel antibiotic potentiators may prove useful for the treatment of bacterial infections. The application of antibiotics combined with antibiotic potentiators has demonstrated successful outcomes in bench-scale experiments and clinical settings. For instance, the use of efflux pump inhibitors (EPIs) in combination with antibiotics showed effective inhibition of MDR pathogens. Thus, this review aims to enable the possibility of using novel EPIs as potential adjuvants to effectively control MDR pathogens. Specifically, it provides a comprehensive summary of the advances in novel EPI discovery and the underlying mechanisms that restore antimicrobial activity. In addition, we also characterize plant-derived EPIs as novel potentiators. This review provides insights into current challenges and potential strategies for future advancements in fighting antibiotic resistance. [ABSTRACT FROM AUTHOR]

ABSTRACT 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.

IntroductionAlthough carbapenemases are frequently reported in resistant A. baumannii clinical isolates, other chromosomally mediated elements of resistance that are considered essential are frequently underestimated. Having a wide substrate range, multidrug efflux pumps frequently underlie antibiotic treatment failure. Recognizing and exploiting variations in multidrug efflux pumps and penicillin-binding proteins (PBPs) is an essential approach in new antibiotic drug discovery and engineering to meet the growing challenge of multidrug-resistant Gram-negative bacteria.MethodsA total of 980 whole genome sequences of A. baumannii were analyzed. Nucleotide sequences for the genes studied were queried against a custom database of FASTA sequences using the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) system. The correlation between different variants and carbapenem Minimum Inhibitory Concentrations (MICs) was studied. PROVEAN and I-Mutant predictor suites were used to predict the effect of the studied amino acid substitutions on protein function and protein stability. Both PsiPred and FUpred were used for domain and secondary structure prediction. Phylogenetic reconstruction was performed using SANS serif and then visualized using iTOL and Phandango.ResultsExhibiting the highest detection rate, AdeB codes for an important efflux-pump structural protein. T48V, T584I, and P660Q were important variants identified in the AdeB-predicted multidrug efflux transporter pore domains. These can act as probable targets for designing new efflux-pump inhibitors. Each of AdeC Q239L and AdeS D167N can also act as probable targets for restoring carbapenem susceptibility. Membrane proteins appear to have lower predictive potential than efflux pump-related changes. OprB and OprD changes show a greater effect than OmpA, OmpW, Omp33, and CarO changes on carbapenem susceptibility. Functional and statistical evidence make the variants T636A and S382N at PBP1a good markers for imipenem susceptibility and potential important drug targets that can modify imipenem resistance. In addition, PBP3_370, PBP1a_T636A, and PBP1a_S382N may act as potential drug targets that can be exploited to counteract imipenem resistance.ConclusionThe study presents a comprehensive epidemiologic and statistical analysis of potential membrane proteins and efflux-pump variants related to carbapenem susceptibility in A. baumannii, shedding light on their clinical utility as diagnostic markers and treatment modification targets for more focused studies of candidate elements.

Microorganisms need to constantly exchange with their habitat to capture nutrients and expel toxic compounds. The ATP-binding cassette (ABC) transporters, a family of membrane proteins especially abundant in microorganisms, are at the core of these processes. Due to their extraordinary ability to expel structurally unrelated compounds, some transporters play a protective role in different organisms. Yet, the downside of these multidrug transporters is their entanglement in the resistance to therapeutic treatments. Intriguingly, some multidrug ABC transporters show a high level of ATPase activity, even in the absence of transported substrates. Although this basal ATPase activity might seem a waste, we surmise that this inherent capacity allows multidrug transporters to promptly translocate any bound drug before it penetrates into the cell. Many ATP-binding cassette (ABC) transporters display a basal ATPase activity in the absence of transported substrates. Multidrug ABC exporters function according to an alternating access mechanism between inward- and outward-facing conformations. The basal ATPase activity of microbial multidrug ABC exporters is often either not or poorly stimulated by the presence of drugs. The sole binding of drugs to multidrug ABC exporters does not produce a major conformational change. When drugs stimulate the ATPase activity, they likely shift the equilibrium toward the outward-facing conformation of multidrug ABC exporters, a conformation prone to drug release. [ABSTRACT FROM AUTHOR]

Campylobacter (C.) spp. are the most important foodborne, bacterial, and zoonotic pathogens worldwide. Resistance monitoring of foodborne bacterial pathogens is an important tool to control antimicrobial resistance as a part of the "One Health" approach. The detection and functionality of new resistance genes are of paramount importance in applying more effective screening methods based on whole genome sequencing (WGS). Most tetracycline-resistant C. spp. isolates harbor tet(O), a gene that encodes a ribosomal protection protein. Here we describe tet(O)_3, which has been identified in two food isolates of C. jejuni and is very similar to the tet(O) gene in Streptococcus pneumoniae, having a truncated promoter sequence. This gene confers resistance to tetracycline below 1 mg/L, which is the epidemiological cut-off value. We have analyzed the entire genome of these two isolates, together with a C. jejuni isolate found to have high-level resistance to tetracycline. In contrast to the highly resistant isolate, the promoter of tet(O)_3 is highly responsive to tetracycline, as observed by reverse transcription polymerase chain reaction (RT-PCR). In addition, the two isolates possess a CRISPR repeat, fluoroquinolone resistance due to the gyrA point mutation C257T, a β-lactamase resistance gene blaOXA-184, a multidrug efflux pump CmeABC and its repressor CmeR, but no plasmid. Low-level antibiotic resistant C. jejuni might therefore have an advantage for surviving in non-host environments. [ABSTRACT FROM AUTHOR]

ABSTRACT 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.

ABSTRACT The continued challenges of the COVID-19 pandemic combined with the growing problem of antimicrobial-resistant bacterial infections has severely impacted global health. Specifically, the Gram-negative pathogen Klebsiella pneumoniae is one of the most prevalent causes of secondary bacterial infection in COVID-19 patients, with approximately an 83% mortality rate observed among COVID-19 patients with these bacterial coinfections. K. pneumoniae belongs to the ESKAPE group of pathogens, a group that commonly gives rise to severe infections that are often life-threatening. Recently, K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae has drawn wide public attention, as the mortality rate for this infection can be as high as 71%. The most predominant and clinically important multidrug efflux system in K. pneumoniae is the acriflavine resistance B (AcrB) multidrug efflux pump. This pump mediates resistance to different classes of structurally diverse antimicrobial agents, including quinolones, β-lactams, tetracyclines, macrolides, aminoglycosides, and chloramphenicol. We here report single-particle cryo-electron microscopy (cryo-EM) structures of K. pneumoniae AcrB, in both the absence and the presence of the antibiotic erythromycin. These structures allow us to elucidate specific pump-drug interactions and pinpoint exactly how this pump recognizes antibiotics. IMPORTANCE Klebsiella pneumoniae has emerged as one of the most problematic and highly antibiotic-resistant pathogens worldwide. It is the second most common causative agent involved in secondary bacterial infection in COVID-19 patients. K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae is a major concern in global public health because of the high mortality rate of this infection. Its drug resistance is due, in a significant part, to active efflux of these bactericides, a major mechanism that K. pneumoniae uses to resist to the action of multiple classes of antibiotics. Here, we report cryo-electron microscopy (cryo-EM) structures of the prevalent and clinically important K. pneumoniae AcrB multidrug efflux pump, in both the absence and the presence of the erythromycin antibiotic. These structures allow us to understand the action mechanism for drug 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.

Fatty acids salts exert bactericidal and bacteriostatic effects that inhibit bacterial growth and survival. However, bacteria can overcome these effects and adapt to their environment. Bacterial efflux systems are associated with resistance to different toxic compounds. Here, several bacterial efflux systems were examined to determine their influence on fatty acid salt resistance in Escherichia coli. Both acrAB and tolC E. coli deletion strains were susceptible to fatty acid salts, while plasmids carrying acrAB, acrEF, mdtABC, or emrAB conferred drug resistance to the ΔacrAB mutant, which indicated complementary roles for these multidrug efflux pumps. Our data exemplify the importance of bacterial efflux systems in E. coli resistance to fatty acid salts. [ABSTRACT FROM AUTHOR]

There is an urgent need to find novel treatments for combating multidrug-resistant bacteria. Multidrug efflux pumps that expel antibiotics out of cells are major contributors to this problem. Therefore, using efflux pump inhibitors (EPIs) is a promising strategy to increase antibiotic efficacy. However, there are no EPIs currently approved for clinical use especially because of their toxicity. This study investigates sodium malonate, a natural, non-hazardous, small molecule, for its use as a novel EPI of AcrAB-TolC, the main multidrug efflux pump of the Enterobacteriaceae family. Using ethidium bromide accumulation experiments, we found that 25 mM sodium malonate inhibited efflux by the AcrAB-TolC and other MDR pumps of Escherichia coli to a similar degree than 50 μΜ phenylalanine-arginine-β-naphthylamide, a well-known EPI. Using minimum inhibitory concentration assays and molecular docking to study AcrB-ligand interactions, we found that sodium malonate increased the efficacy of ethidium bromide and the antibiotics minocycline, chloramphenicol, and ciprofloxacin, possibly via binding to multiple AcrB locations, including the AcrB proximal binding pocket. In conclusion, sodium malonate is a newly discovered EPI that increases antibiotic efficacy. Our findings support the development of malonic acid/sodium malonate and its derivatives as promising EPIs for augmenting antibiotic efficacy when treating multidrug-resistant bacterial infections. [ABSTRACT FROM AUTHOR]

The excessive use of antibiotics has led to the emergence of multidrug-resistant (MDR) pathogens in clinical settings and food-producing animals, posing significant challenges to clinical management and food control. Over the past few decades, the discovery of antimicrobials has slowed down, leading to a lack of treatment options for clinical infectious diseases and foodborne illnesses. Given the increasing prevalence of antibiotic resistance and the limited availability of effective antibiotics, the discovery of novel antibiotic potentiators may prove useful for the treatment of bacterial infections. The application of antibiotics combined with antibiotic potentiators has demonstrated successful outcomes in bench-scale experiments and clinical settings. For instance, the use of efflux pump inhibitors (EPIs) in combination with antibiotics showed effective inhibition of MDR pathogens. Thus, this review aims to enable the possibility of using novel EPIs as potential adjuvants to effectively control MDR pathogens. Specifically, it provides a comprehensive summary of the advances in novel EPI discovery and the underlying mechanisms that restore antimicrobial activity. In addition, we also characterize plant-derived EPIs as novel potentiators. This review provides insights into current challenges and potential strategies for future advancements in fighting antibiotic resistance.

ABSTRACT 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.

ABSTRACT The transcriptional repressor AcrR is the main regulator of the multidrug efflux pump AcrAB-TolC, which plays a major role in antibiotic resistance and cell physiology in Escherichia coli and other Enterobacteriaceae. However, it remains unknown which ligands control the function of AcrR. To address this gap in knowledge, this study tested whether exogenous and/or endogenous molecules identified as potential AcrR ligands regulate the activity of AcrR. Using electrophoretic mobility shift assays (EMSAs) with purified AcrR and the acrAB promoter and in vivo gene expression experiments, we found that AcrR responds to both exogenous molecules and cellular metabolites produced by E. coli. In total, we identified four functional ligands of AcrR, ethidium bromide (EtBr), an exogenous antimicrobial known to be effluxed by the AcrAB-TolC pump and previously shown to bind to AcrR, and three polyamines produced by E. coli, namely, putrescine, cadaverine, and spermidine. We found that EtBr and polyamines bind to AcrR both in vitro and in vivo, which prevents the binding of AcrR to the acrAB promoter and, ultimately, induces the expression of acrAB. Finally, we also found that AcrR contributes to mitigating the toxicity produced by excess polyamines by directly regulating the expression of AcrAB-TolC and two previously unknown AcrR targets, the MdtJI spermidine efflux pump and the putrescine degradation enzyme PuuA. Overall, these findings significantly expand our understanding of the function of AcrR by revealing that this regulator responds to different exogenous and endogenous ligands to regulate the expression of multiple genes involved in efflux and detoxification. IMPORTANCE Multidrug efflux pumps can remove antibiotics and other toxic molecules from cells and are major contributors to antibiotic resistance and bacterial physiology. Therefore, it is essential to better understand their function and regulation. AcrAB-TolC is the main multidrug efflux pump in the Enterobacteriaceae family, and AcrR is its major transcriptional regulator. However, little is known about which ligands control the function of AcrR or which other genes are controlled by this regulator. This study contributes to addressing these gaps in knowledge by showing that (i) the activity of AcrR is controlled by the antimicrobial ethidium bromide and by polyamines produced by E. coli, and (ii) AcrR directly regulates the expression of AcrAB-TolC and genes involved in detoxification and efflux of excess polyamines. These findings significantly advance our understanding of the biological role of AcrR by identifying four ligands that control its function and two novel targets of this regulator.

[Display omitted] • Strain SH-1 showed a high removal rate of Cr(VI). • Strain SH-1 of Bacillus cereus exhibits resistance to Cr(VI) and antibiotics. • Cr(VI) triggers the expression of the HAE-1 family of efflux pump genes in bacteria. The simultaneous selection of antibiotic resistance genes (ARGs) induced by heavy metals and antibiotics has emerged as a growing environmental problem. This study investigated the combined effects of chromium (Cr(VI)) and antibiotics on the ARGs of Bacillus cereus SH-1. As Cr(VI) concentration increased, it triggered reactive oxygen species oxidative stress in SH-1, increased antioxidant enzyme activity, enhanced plasmid conjugative transfer, and reduced the efficiency of Cr(VI) removal by SH-1. Antibiotic resistance varied with increasing tetracycline and amoxicillin minimum inhibitory concentrations (MICs), whereas azithromycin and chloramphenicol MICs decreased with Cr(VI) induction. The overexpression of eight genes of the HAE-1 family of efflux pumps was detected using metagenomics and proteomics. Co-contamination with Cr(VI) and antibiotics has led to the emergence and spread of antibiotic-resistant bacteria. Therefore, resistance gene contamination resulting from Cr(VI)-polluted environments cannot be overlooked. [ABSTRACT FROM AUTHOR]

Increased expression of efflux pumps is an important mechanism of antibiotic resistance in Pseudomonas aeruginosa , and treatment with inhibitors of active efflux pumps seems an attractive strategy to combat with multidrug resistance. Assays using ethidium bromide (EtBr), which accumulates by binding to nucleic acids, are often employed to assess the efficacy of efflux pump inhibitors (EPIs). However, few studies have reported on assays using other nucleic acid dyes. We used different classes of EPIs for MexAB- or MexXY-OprM to measure the accumulation of various fluorescent dyes, including SYBR Safe, AtlasSight, and GelGreen. Escherichia coli MG1655Δ acrB Δ tolC strain harboring plasmids carrying the mexAB-oprM (pABM) or mexXY-oprM (pXYM) genes of P. aeruginosa were constructed. Then, the accumulation of the above-mentioned nucleic acid dyes and EtBr was measured to assess the efflux ability in the presence and absence of EPIs (MexAB-OprM-specific inhibitor of pyridopyrimidine derivative [ABI-PP], berberine, non-specific inhibitor of phenylalanine-arginine β-naphthylamide [PAβN], and protonophore of carbonyl cyanide m -chlorophenyl hydrazone [CCCP]). Decreased accumulations of nucleic acid dyes were observed in strains with pABM or pXYM compared with the parental strain. ABI-PP or berberine addition significantly increased the accumulation of any nucleic acids in the strains with the specific pumps. PAβN or CCCP addition showed increased accumulation of almost all dye in strains with pABM or pXYM. However, the inhibition patterns of EPIs differed according to the nucleic acid dyes used. Accumulation assays for EPIs were suitable to evaluate EPI candidates using various nucleic acid dyes. [ABSTRACT FROM AUTHOR]

trans‐Anethole (anethole) is a phenylpropanoid; with other drugs, it exhibits synergistic activity against several fungi and is expected to be used in new therapies that cause fewer patient side effects. However, the detailed substructure(s) of the molecule responsible for this synergy has not been fully elucidated. We investigated the structure–activity relationships of phenylpropanoids and related derivatives, with particular attention on the methoxy group and the double bond of the propenyl group in anethole, as well as the length of the p‐alkyl chain in p‐alkylanisoles. Antifungal potency was largely related to p‐alkyl chain length and the methoxy group of anethole, but not to the double bond of its propenyl group. Production of reactive oxygen species also played a role in these fungicidal activities. Inhibition of drug efflux was associated with the length of the p‐alkyl chain and the double bond of the propenyl group in anethole, but not with the methoxy group. Although a desirable synergy was observed between n‐dodecanol and anethole or p‐alkylanisoles with a length of C2–C6 in alkyl chains, it cannot be explained away as being solely due to the inhibition of drug efflux. Similar results were obtained when phenylpropanoid derivatives were combined with fluconazole against Candida albicans. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii has emerged as one of the dominant nosocomial human pathogens associated with high morbidity and mortality globally. Increased incidences of carbapenem-resistant A. baumannii (CRAB) have resulted in an enormous socioeconomic burden on health-care systems. Here, we report the genotypic and phenotypic characterization of novel ST1816 and ST128 variants in A. baumannii strains belonging to International clone II (GC2) with capsule types KL1:OCL8 and KL3:OCL1d from India. Sequence analysis revealed the presence of diverse virulome and resistome in these clinical strains, in addition to islands, prophages, and resistance genes. The oxacillinase bla OXA–23 detected in the genomic island also highlighted the coexistence of bla OXA–66 / bla OXA–98 , bla ADC73 / bla ADC–3 , and bla TEM–1D in their mobile scaffolds, which is alarming. Together with these resistance-determining enzymes, multidrug efflux transporters also harbored substitutions, with increased expression in CRAB strains. The hotspot mutations in colistin resistance-conferring operons, PmrAB, LpxACD, and AdeRS, were additionally confirmed. Phenotype microarray analysis indicated that multidrug-resistant strains A. baumannii DR2 and A. baumannii AB067 preferred a range of antimicrobial compounds as their substrates relative to the other. To our knowledge, this is the first comprehensive report on the characterization of A. baumannii variants ST1816 and ST128, with different genetic makeup and genome organization. The occurrence of CRAB infections worldwide is a severe threat to available limited therapeutic options; hence, continued surveillance to monitor the emergence and dissemination of such novel ST variants in A. baumannii is imperative. [ABSTRACT FROM AUTHOR]

Objective To study the mutation and expression of genes of Salmonella when screened by different concentrations of quinolone and fluoroquinolones during propagation and their relation with antibiotic resistance. Methods Salmonella strain was cultured in broth medium and screened on nutrition plate with different concentration of quinolone and fluoroquinolones. Antimicrobial susceptibility of the screened subculture was tested by broth microdilution method, mutation of genes in quinolone resistance-determining region (QRDR) was detected using polymerase chain reaction (PCR) and DNA sequencing method, and expression level of the encoding genes of multi-drug associated efflux pump AcrAB-TolC was detected by real-time qPCR. Results Antibiotic resistance level of the subcultures screened from LB plate with quinolone and fluoroquinolones inducement increased in different extents. Mutation of Asp87Tyr in gyrA in QRDR was detected from the fifth to the seventh generation of nalidixic acid screened strains. Mutation of Asp87Asn in gyrA in QRDR was detected from the fourth to the seventh generation of ciprofloxacin screened strains. No amino acid mutation was detected from gyrA in the first to the seventh generation of gatifloxacin, levofloxacin and delafloxacin screened strains. Compared to the expression level of the multi-drug efflux pump AcrAB-TolC encoding genes of the original strain, those of the screened strains had significantly (P

Avian pathogenic Escherichia coli (APEC) is a specific group of extraintestinal pathogenic E. coli that causes a variety of extraintestinal diseases in chickens, ducks, pigeons, turkeys, and other avian species. These diseases lead to significant economic losses in the poultry industry worldwide. However, owing to excessive use of antibiotics in the treatment of infectious diseases, bacteria have developed antibiotic resistance. The development of multidrug efflux pumps is one important bacterial antibiotic resistance mechanism. A multidrug efflux pump, MdtH, which belongs to the major facilitator superfamily of transporters, confers resistance to quinolone antibiotics such as norfloxacin and enoxacin. LsrR regulates hundreds of genes that participate in myriad biological processes, including mobility, biofilm formation, and antibiotic susceptibility. However, whether LsrR regulates mdtH transcription and then affects bacterial resistance to various antibiotics in APEC has not been reported. In the present study, the lsrR mutant was constructed from its parent strain APECX40 (WT), and high-throughput sequencing was performed to analyze the transcriptional profile of the WT and mutant XY10 strains. The results showed that lsrR gene deletion upregulated the mdtH transcript level. Furthermore, we also constructed the lsrR- and mdtH-overexpressing strains and performed antimicrobial susceptibility testing, antibacterial activity assays, real-time reverse transcription PCR, and electrophoretic mobility shift assays to investigate the molecular regulatory mechanism of LsrR on the MdtH multidrug efflux pump. The lsrR mutation and the mdtH-overexpressing strain decreased cell susceptibility to norfloxacin, ofloxacin, ciprofloxacin, and tetracycline by upregulating mdtH transcript levels. In addition, the lsrR-overexpressing strain increased cell susceptibility to norfloxacin, ofloxacin, ciprofloxacin, and tetracycline by downregulating mdtH transcript levels. Electrophoretic mobility shift assays indicated that LsrR directly binds to the mdtH promoter. Therefore, this study is the first to demonstrate that LsrR inhibits mdtH transcription by directly binding to its promoter region. This action subsequently increases susceptibility to the aforementioned four antibiotics in APECX40.

ABSTRACT Antibiotic resistance among bacterial pathogens continues to pose a serious global health threat. Multidrug-resistant (MDR) strains of the Gram-negative organism Acinetobacter baumannii utilize a number of resistance determinants to evade current antibiotics. One of the major resistance mechanisms employed by these pathogens is the use of multidrug efflux pumps. These pumps extrude xenobiotics directly out of bacterial cells, resulting in treatment failures when common antibiotics are administered. Here, the structure of the novel tetracycline antibiotic TP-6076, bound to both the Acinetobacter drug efflux pump AdeJ and the ribosome from Acinetobacter baumannii, using single-particle cryo-electron microscopy (cryo-EM), is elucidated. In this work, the structure of the AdeJ–TP-6076 complex is solved, and we show that AdeJ utilizes a network of hydrophobic interactions to recognize this fluorocycline. Concomitant with this, we elucidate three structures of TP-6076 bound to the A. baumannii ribosome and determine that its binding is stabilized largely by electrostatic interactions. We then compare the differences in binding modes between TP-6076 and the related tetracycline antibiotic eravacycline in both targets. These differences suggest that modifications to the tetracycline core may be able to alter AdeJ binding while maintaining interactions with the ribosome. Together, this work highlights how different mechanisms are used to stabilize the binding of tetracycline-based compounds to unique bacterial targets and provides guidance for the future clinical development of tetracycline antibiotics. IMPORTANCE Treatment of antibiotic-resistant organisms such as A. baumannii represents an ongoing issue for modern medicine. The multidrug efflux pump AdeJ serves as a major resistance determinant in A. baumannii through its action of extruding antibiotics from the cell. In this work, we use cryo-EM to show how AdeJ recognizes the experimental tetracycline antibiotic TP-6076 and prevents this drug from interacting with the A. baumannii ribosome. Since AdeJ and the ribosome use different binding modes to stabilize interactions with TP-6076, exploiting these differences may guide future drug development for combating antibiotic-resistant A. baumannii and potentially other strains of MDR bacteria.

Campylobacter (C.) spp. are the most important foodborne, bacterial, and zoonotic pathogens worldwide. Resistance monitoring of foodborne bacterial pathogens is an important tool to control antimicrobial resistance as a part of the “One Health” approach. The detection and functionality of new resistance genes are of paramount importance in applying more effective screening methods based on whole genome sequencing (WGS). Most tetracycline-resistant C. spp. isolates harbor tet(O), a gene that encodes a ribosomal protection protein. Here we describe tet(O)_3, which has been identified in two food isolates of C. jejuni and is very similar to the tet(O) gene in Streptococcus pneumoniae, having a truncated promoter sequence. This gene confers resistance to tetracycline below 1 mg/L, which is the epidemiological cut-off value. We have analyzed the entire genome of these two isolates, together with a C. jejuni isolate found to have high-level resistance to tetracycline. In contrast to the highly resistant isolate, the promoter of tet(O)_3 is highly responsive to tetracycline, as observed by reverse transcription polymerase chain reaction (RT-PCR). In addition, the two isolates possess a CRISPR repeat, fluoroquinolone resistance due to the gyrA point mutation C257T, a β-lactamase resistance gene blaOXA-184, a multidrug efflux pump CmeABC and its repressor CmeR, but no plasmid. Low-level antibiotic resistant C. jejuni might therefore have an advantage for surviving in non-host environments.

Acinetobacter baumannii has emerged as one of the dominant nosocomial human pathogens associated with high morbidity and mortality globally. Increased incidences of carbapenem-resistant A. baumannii (CRAB) have resulted in an enormous socioeconomic burden on health-care systems. Here, we report the genotypic and phenotypic characterization of novel ST1816 and ST128 variants in A. baumannii strains belonging to International clone II (GC2) with capsule types KL1:OCL8 and KL3:OCL1d from India. Sequence analysis revealed the presence of diverse virulome and resistome in these clinical strains, in addition to islands, prophages, and resistance genes. The oxacillinase blaOXA–23detected in the genomic island also highlighted the coexistence of blaOXA–66/blaOXA–98, blaADC73/blaADC–3, and blaTEM–1D in their mobile scaffolds, which is alarming. Together with these resistance-determining enzymes, multidrug efflux transporters also harbored substitutions, with increased expression in CRAB strains. The hotspot mutations in colistin resistance-conferring operons, PmrAB, LpxACD, and AdeRS, were additionally confirmed. Phenotype microarray analysis indicated that multidrug-resistant strains A. baumannii DR2 and A. baumannii AB067 preferred a range of antimicrobial compounds as their substrates relative to the other. To our knowledge, this is the first comprehensive report on the characterization of A. baumannii variants ST1816 and ST128, with different genetic makeup and genome organization. The occurrence of CRAB infections worldwide is a severe threat to available limited therapeutic options; hence, continued surveillance to monitor the emergence and dissemination of such novel ST variants in A. baumannii is imperative.

There is an urgent need to find novel treatments for combating multidrug-resistant bacteria. Multidrug efflux pumps that expel antibiotics out of cells are major contributors to this problem. Therefore, using efflux pump inhibitors (EPIs) is a promising strategy to increase antibiotic efficacy. However, there are no EPIs currently approved for clinical use especially because of their toxicity. This study investigates sodium malonate, a natural, non-hazardous, small molecule, for its use as a novel EPI of AcrAB-TolC, the main multidrug efflux pump of the Enterobacteriaceae family. Using ethidium bromide accumulation experiments, we found that 25 mM sodium malonate inhibited efflux by the AcrAB-TolC and other MDR pumps of Escherichia coli to a similar degree than 50 μΜ phenylalanine-arginine-β-naphthylamide, a well-known EPI. Using minimum inhibitory concentration assays and molecular docking to study AcrB-ligand interactions, we found that sodium malonate increased the efficacy of ethidium bromide and the antibiotics minocycline, chloramphenicol, and ciprofloxacin, possibly via binding to multiple AcrB locations, including the AcrB proximal binding pocket. In conclusion, sodium malonate is a newly discovered EPI that increases antibiotic efficacy. Our findings support the development of malonic acid/sodium malonate and its derivatives as promising EPIs for augmenting antibiotic efficacy when treating multidrug-resistant bacterial infections.

Bacterial genomes encode various multidrug efflux pumps (MDR) whose specific conditions for fitness advantage are unknown. We show that the efflux pump MdtEF-TolC, in Escherichia coli, confers a fitness advantage during exposure to extreme acid (pH 2). Our flow cytometry method revealed pH-dependent fitness trade-offs between bile acids (a major pump substrate) and salicylic acid, a membrane-permeant aromatic acid that induces a drug resistance regulon but depletes proton motive force (PMF). The PMF drives MdtEF-TolC and related pumps such as AcrAB-TolC. Deletion of mdtE (with loss of the pump MdtEF-TolC) increased the strain's relative fitness during growth with or without salicylate or bile acids. However, when the growth cycle included a 2-h incubation at pH 2 (below the pH growth range), MdtEF-TolC conferred a fitness advantage. The fitness advantage required bile salts but was decreased by the presence of salicylate, whose uptake is amplified by acid. For comparison, AcrAB-TolC, the primary efflux pump for bile acids, conferred a PMF-dependent fitness advantage with or without acid exposure in the growth cycle. A different MDR pump, EmrAB-TolC, conferred no selective benefit during growth in the presence of bile acids. Without bile acids, all three MDR pumps incurred a large fitness cost with salicylate when exposed at pH 2. These results are consistent with the increased uptake of salicylate at low pH. Overall, we showed that MdtEF-TolC is an MDR pump adapted for transient extreme-acid exposure and that low pH amplifies the salicylate-dependent fitness cost for drug pumps. [ABSTRACT FROM AUTHOR]

The infections of multidrug-resistant bacteria, particularly the infections caused by Gram-negative bacterial infections, are considered the main global serious health problems. Multidrug-resistant bacteria possess different resistance mechanisms, the most important of which are drug efflux pumps due to their function in recognizing harmful compounds, especially antibiotics, and expelling, them outside the cell, which leads to a decrease in their concentration to sub-toxic levels and losing their effectiveness. One of the most important approaches that can be used to inhibit drug efflux pumps is using small molecule inhibitors targeting specifically pumps to prevent their expelling antibiotics from inside the cell before reaching the required concentration that kills bacteria. The drug efflux pumps inhibition approach is emerging as a new attractive and promising approach in reversing antibiotic resistance in many clinically relevant pathogenic bacteria. This review will discuss the requirements that must be met in inhibitors that target AcrB, which is the main subunit protein responsible for multiple antibiotic resistance in the main pump of AcrAB-TolC which belongs to E. coli , which were specifically chosen due to their various homologs in many Gram-negative bacteria which all of which are implicated in multi-drug resistance (MDR), and the availability of the pump's functions and chemical structures. [ABSTRACT FROM AUTHOR]

Abstract Background OqxAB efflux pump has been found to mediate multidrug resistance (MDR) in various bacteria over the past decades. The updates on the nature and epidemiology of OqxAB efflux pump need to be fully reviewed to broaden our understanding of this MDR determinant. Methods A literature search using the keyword of “oqxAB” was conducted in the online databases of Pubmed and ISI Web of Science with no restriction on the date of publication. The 87 publications were included into this review as references due to their close relevance to the nature and/or epidemiology of OqxAB efflux pump. Results The oqxAB gene generally locates on chromosome and/or plasmids flanked by IS26-like elements in clinical isolates of Enterobacteriaceae and Klebsiella pneumoniae, conferring low to intermediated resistance to quinoxalines, quinolones tigecycline, nitrofurantoin, several detergents and disinfectants (benzalkonium chloride, triclosan and SDS). It could co-spread with other antimicrobial resistance genes (bla CTX-M, rmtB and aac(6′)-Ib etc.), virulence genes and heavy metal resistance genes (pco and sil operons). Both RarA (activator) and OqxR (repressor) play important roles on regulation of the expression of OqxAB. Conclusions The dissemination of oqxAB gene may pose a great risk on food safety and public health. Further investigation and understanding of the natural functions, horizontal transfer, and regulation mechanism of the OqxAB efflux pump will aid in future strategies of antimicrobial usage.

ABSTRACT The substantial rise in multidrug-resistant bacterial infections is a current global imperative. Cumulative efforts to characterize antimicrobial resistance in bacteria has demonstrated the spread of six families of multidrug efflux pumps, of which resistance-nodulation-cell division (RND) is the major mechanism of multidrug resistance in Gram-negative bacteria. RND is composed of a tripartite protein assembly and confers resistance to a range of unrelated compounds. In the major enteric pathogen Campylobacter jejuni, the three protein components of RND are posttranslationally modified with N-linked glycans. The direct role of N-linked glycans in C. jejuni and other bacteria has long been elusive. Here, we present the first detailed account of the role of N-linked glycans and the link between N-glycosylation and antimicrobial resistance in C. jejuni. We demonstrate the multifunctional role of N-linked glycans in enhancing protein thermostability, stabilizing protein complexes and the promotion of protein-protein interaction, thus mediating antimicrobial resistance via enhancing multidrug efflux pump activity. This affirms that glycosylation is critical for multidrug efflux pump assembly. We present a generalized strategy that could be used to investigate general glycosylation system in Campylobacter genus and a potential target to develop antimicrobials against multidrug-resistant pathogens. IMPORTANCE Nearly all bacterial species have at least a single glycosylation system, but the direct effects of these posttranslational protein modifications are unresolved. Glycoproteome-wide analysis of several bacterial pathogens has revealed general glycan modifications of virulence factors and protein assemblies. Using Campylobacter jejuni as a model organism, we have studied the role of general N-linked glycans in the multidrug efflux pump commonly found in Gram-negative bacteria. We show, for the first time, the direct link between N-linked glycans and multidrug efflux pump activity. At the protein level, we demonstrate that N-linked glycans play a role in enhancing protein thermostability and mediating the assembly of the multidrug efflux pump to promote antimicrobial resistance, highlighting the importance of this posttranslational modification in bacterial physiology. Similar roles for glycans are expected to be found in other Gram-negative pathogens that possess general protein glycosylation systems.

Acinetobacter baumannii has emerged as a nosocomial pathogen and considered as a major cause of hospital acquired infections. While A. baumannii in humans have been extensively studied in various situations, the study of A. baumannii in companion animals is still limited. The aim of this study was to examine the presence and antimicrobial resistance profile of A. baumannii isolates from companion rabbits. A total of 17 nasal swab from rabbit carcasses were obtained. A. baumannii was detected in 23.53% (4/17) of the samples and all the isolates exhibited resistance to chloramphenicol, erythromycin, spectinomycin and trimethoprim. Fifty percent of the isolates (2/4) were multidrug resistance (MDR) A. baumannii (ABR601 and ABR604) and produced all the Ade multidrug efflux tested including AdeABC, AdeFGH and AdeIJK. The other 2 isolates, including ABR602 and ABR603, demonstrated different expression profile of the Ade systems i.e. AdeIJK and AdeFGH-AdeIJK, respectively. Of all the isolates, ABR604 and ABR601 were resistant to 12 and 10 out of the 15 antimicrobials tested, respectively. This study indicates the presence of MDR A. baumannii in pets other than dogs and cats and highlights the possible role of these animals as reserviors for the spread of MDR A. baumannii to humans, animals and environment. [ABSTRACT FROM AUTHOR]

ABSTRACT Nontyphoidal salmonellae (NTS) are exposed to reactive oxygen species (ROS) during their residency in the gut. To survive oxidative stress encountered during infection, salmonellae employ several mechanisms. One of these mechanisms involves the multidrug efflux pump MacAB, although the natural substrate of this pump has not been identified. MacAB homologs in pseudomonads secrete products of nonribosomal peptide synthesis (NRPS). In Salmonella enterica serovar Typhimurium, the siderophore enterobactin is produced by NRPS in response to iron starvation and this molecule can be processed into salmochelin and several linear metabolites. We found that Salmonella mutants lacking the key NRPS enzyme EntF are sensitive to peroxide mediated killing and cannot detoxify extracellular H2O2. Moreover, EntF and MacAB function in a common pathway to promote survival of Salmonella during oxidative stress. We further demonstrated that S. Typhimurium secretes siderophores in iron-rich media when peroxide is present and that these MacAB-secreted metabolites participate in protection of bacteria against H2O2. We showed that secretion of anti-H2O2 molecules is independent of the presence of the known siderophore efflux pumps EntS and IroC, well-described efflux systems involved in secretion of enterobactin and salmochelin. Both salmochelin and enterobactin are dispensable for S. Typhimurium protection against ROS; however, linear metabolites of enterobactin produced by esterases IroE and Fes are needed for bacterial survival in peroxide-containing media. We determined that linearized enterobactin trimer protects S. Typhimurium against peroxide-mediated killing in a MacAB-dependent fashion. Thus, we suggest that linearized enterobactin trimer is a natural substrate of MacAB and that its purpose is to detoxify extracellular reactive oxygen species. IMPORTANCE Nontyphoidal Salmonella bacteria induce a classic inflammatory diarrhea by eliciting a large influx of neutrophils, producing a robust oxidative burst. Despite substantial progress understanding the benefits to the host of the inflammatory response to Salmonella, little is known regarding how Salmonella can simultaneously resist the damaging effects of the oxidative burst. The multidrug efflux pump MacAB is important for survival of oxidative stress both in vitro and during infection. We describe a new pathway used by Salmonella Typhimurium to detoxify extracellular reactive oxygen species using a multidrug efflux pump (MacAB) to secrete a linear siderophore, a metabolite of enterobactin. The natural substrates of many multidrug efflux pumps are unknown, and functional roles of the linear metabolites of enterobactin are unknown. We bring two novel discoveries together to highlight an important mechanism used by Salmonella to survive under the oxidative stress conditions that this organism encounters during the classic inflammatory diarrhea that it also induces.

Infectious diseases caused by bacterial species of the Vibrio genus have had considerable significance upon human health for centuries. V. cholerae is the causative microbial agent of cholera, a severe ailment characterized by profuse watery diarrhea, a condition associated with epidemics, and seven great historical pandemics. V. parahaemolyticus causes wound infection and watery diarrhea, while V. vulnificus can cause wound infections and septicemia. Species of the Vibrio genus with resistance to multiple antimicrobials have been a significant health concern for several decades. Mechanisms of antimicrobial resistance machinery in Vibrio spp. include biofilm formation, drug inactivation, target protection, antimicrobial permeability reduction, and active antimicrobial efflux. Integral membrane-bound active antimicrobial efflux pump systems include primary and secondary transporters, members of which belong to closely related protein superfamilies. The RND (resistance-nodulation-division) pumps, the MFS (major facilitator superfamily) transporters, and the ABC superfamily of efflux pumps constitute significant drug transporters for investigation. In this review, we explore these antimicrobial transport systems in the context of Vibrio spp. pathogenesis and virulence.

Abstract Background The plasmid-encoded multidrug efflux pump oqxAB confers bacterial resistance primarily to olaquindox, quinolones, and chloramphenicol. The aims of this study were to investigate the prevalence of oqxAB among Escherichia coli isolates from dogs, cats, and humans in Henan, China and the susceptibilities of E. coli isolates to common antibiotics. Methods From 2012 to 2014, a total of 600 samples which included 400 rectal samples and 200 clinical human specimens were tested for the presence of E. coli. All isolates were screened for oqxAB genes by PCR and sequencing. The MICs of 11 antimicrobial agents were determined by the broth microdilution method. A total of 30 representative oqxAB-positive isolates were subjected to ERIC-PCR and MLST. Additionally, conjugation experiments and southern hybridizations were performed. Results Of 270 isolates, 58.5% (62/106) of the isolates from dogs, 56.25% (36/64) of the isolates from cats, and 42.0% (42/100) of the isolates from humans were positive for the oqxAB. Olaquindox resistance was found for 85.7%-100% of oqxAB-positive isolates. Of oqxAB-positive isolates from dogs, cats, and humans, ciprofloxacin resistance was inspected for 85.8%, 59.1%, and 93.8%, respectively. Several oqxAB-positive isolates were demonstrated by ERIC-PCR and MLST, and have high similarity. Phylogenetic analysis showed that oqxAB-positive isolates could be divided into 7 major clusters. OqxAB-positive conjugants were obtained, southern hybridization verified that the oqxAB gene complex was primarily located on plasmids. Conclusion In conclusion, oqxAB-positive isolates were widespread in animals and humans in Henan, China. Carriage of oqxAB on plasmids of E. coli isolates may facilitate the emergence of multidrug resistant and its transmission via horizontal transfer, and might pose a potential threat to public health.

Due to excessive use of antimicrobial agents in the treatment of infectious diseases, bacteria have developed resistance to antibacterial drugs and toxic compounds. The development of multidrug efflux pumps is one of the important mechanisms of bacterial drug resistance. A multidrug efflux pump, EmrD, belonging to the major facilitator superfamily of transporters, confers resistance to many antimicrobial agents. BasSR, a typical two-component signal transduction system (TCS), regulates susceptibility to the cationic antimicrobial peptide, polymyxin B, and the anionic bile detergent, deoxycholic acid, in Escherichia coli. However, whether or not the BasSR TCS affects susceptibility or resistance to other antimicrobial agents and transcription of emrD has not been reported in E. coli. In the present study, we constructed the basSR mutants of wild-type MG1655 and clinical strain APECX40 and performed antimicrobial susceptibility testing, antibacterial activity assays, real-time reverse transcription–PCR experiments and electrophoretic mobility shift assays (EMSA) to investigate the molecular mechanism by which BasSR regulates the EmrD multidrug efflux pump. Results showed that the basSR mutation increased cell susceptibility to eight antimicrobial agents, including ciprofloxacin, norfloxacin, doxycycline, tetracycline, clindamycin, lincomycin, erythromycin, and sodium dodecyl sulfate, by downregulating the transcriptional levels of emrD. Furthermore, EMSA indicated that BasR could directly bind to the emrD promoter. Therefore, this study was the first to demonstrate that BasSR activates transcription of emrD by binding directly to its promoter region, and then decreases susceptibility to various antimicrobial agents in E. coli strains, APECX40 and MG1655. [ABSTRACT FROM AUTHOR]

Polluted waters are an important reservoir for antibiotic resistance genes and multidrug-resistant bacteria. This report describes the microbial community, antibiotic resistance genes, and the genetic profile of extended spectrum β-lactamase strains isolated from rivers at, Pune, India. ESBL-producing bacteria isolated from diverse river water catchments running through Pune City were characterized for their antibiotic resistance. The microbial community and types of genes which confer antibiotic resistance were identified followed by the isolation of antibiotic-resistant bacteria on selective media and their genome analysis. Four representative isolates were sequenced using next generation sequencing for genomic analysis. They were identified as Pseudomonas aeruginosa, Escherichia coli, and two isolates were Enterobacter cloacae. The genes associated with the multidrug efflux pumps, such as tolC, macA, macB, adeL, and rosB, were detected in the isolates. As MacAB-TolC is an ABC type efflux pump responsible for conferring resistance in bacteria to several antibiotics, potential efflux pump inhibitors were identified by molecular docking. The homology model of their MacB protein with that from Escherichia coli K12 demonstrated structural changes in different motifs of MacB. Molecular docking of reported efflux pump inhibitors revealed the highest binding affinity of compound MC207-110 against MacB. It also details the potential efflux pump inhibitors that can serve as possible drug targets in drug development and discovery. [ABSTRACT FROM AUTHOR]

Campylobacter jejuni is a prevalent gastrointestinal pathogen associated with increasing rates of antimicrobial resistance development. It was also the first bacterium demonstrated to possess a general N-linked protein glycosylation pathway capable of modifying > 80 different proteins, including the primary C ampylobacter m ultidrug e fflux pump, CmeABC. Here we demonstrate that N-glycosylation is necessary for the function of the efflux pump and may, in part, explain the evolutionary pressure to maintain this protein modification system. Mutants of cmeA in two common wildtype (WT) strains are highly susceptible to erythromycin (EM), ciprofloxacin and bile salts when compared to the isogenic parental strains. Complementation of the cmeA mutants with the native cmeA allele restores the WT phenotype, whereas expression of a cmeA allele with point mutations in both N-glycosylation sites is comparable to the cmeA mutants. Moreover, loss of CmeA glycosylation leads to reduced chicken colonization levels similar to the cmeA knock-out strain, while complementation fully restores colonization. Reconstitution of C. jejuni CmeABC into Escherichia coli together with the C. jejuni N-glycosylation pathway increases the EM minimum inhibitory concentration and decreases ethidium bromide accumulation when compared to cells lacking the pathway. Molecular dynamics simulations reveal that the protein structures of the glycosylated and non-glycosylated CmeA models do not vary from one another, and in vitro studies show no change in CmeA multimerization or peptidoglycan association. Therefore, we conclude that N-glycosylation has a broader influence on CmeABC function most likely playing a role in complex stability. [ABSTRACT FROM AUTHOR]

ABSTRACT Resistance-nodulation-cell division multidrug efflux pumps are membrane proteins that catalyze the export of drugs and toxic compounds out of bacterial cells. Within the hydrophobe-amphiphile subfamily, these multidrug-resistant proteins form trimeric efflux pumps. The drug efflux process is energized by the influx of protons. Here, we use single-particle cryo-electron microscopy to elucidate the structure of the Acinetobacter baumannii AdeB multidrug efflux pump embedded in lipidic nanodiscs to a resolution of 2.98 Å. We found that each AdeB molecule within the trimer preferentially takes the resting conformational state in the absence of substrates. We propose that proton influx and drug efflux are synchronized and coordinated within the transport cycle. IMPORTANCE Acinetobacter baumannii is a successful human pathogen which has emerged as one of the most problematic and highly antibiotic-resistant Gram-negative bacteria worldwide. Multidrug efflux is a major mechanism that A. baumannii uses to counteract the action of multiple classes of antibiotics, such as β-lactams, tetracyclines, fluoroquinolones, and aminoglycosides. Here, we report a cryo-electron microscopy (cryo-EM) structure of the prevalent A. baumannii AdeB multidrug efflux pump, which indicates a plausible pathway for multidrug extrusion. Overall, our data suggest a mechanism for energy coupling that powers up this membrane protein to export antibiotics from bacterial cells. Our studies will ultimately inform an era in structure-guided drug design to combat multidrug resistance in these Gram-negative pathogens.

Multidrug efflux pumps are the frontline defense mechanisms of Gram-negative bacteria, yet little is known of their relative fitness trade-offs under gut conditions such as low pH and the presence of antimicrobial food molecules. Low pH contributes to the proton-motive force (PMF) that drives most efflux pumps. We show how the PMF-dependent pumps AcrAB-TolC, MdtEF-TolC, and EmrAB-TolC undergo selection at low pH and in the presence of membrane-permeant phytochemicals. Competition assays were performed by flow cytometry of co-cultured Escherichia coli K-12 strains possessing or lacking a given pump complex. All three pumps showed negative selection under conditions that deplete PMF (pH 5.5 with carbonyl cyanide 3-chlorophenylhydrazone or at pH 8.0). At pH 5.5, selection against AcrAB-TolC was increased by aromatic acids, alcohols, and related phytochemicals such as methyl salicylate. The degree of fitness cost for AcrA was correlated with the phytochemical's lipophilicity (logP). Methyl salicylate and salicylamide selected strongly against AcrA, without genetic induction of drug resistance regulons. MdtEF-TolC and EmrAB-TolC each had a fitness cost at pH 5.5, but salicylate or benzoate made the fitness contribution positive. Pump fitness effects were not explained by gene expression (measured by digital PCR). Between pH 5.5 and 8.0, acrA and emrA were upregulated in the log phase, whereas mdtE expression was upregulated in the transition-to-stationary phase and at pH 5.5 in the log phase. Methyl salicylate did not affect pump gene expression. Our results suggest that lipophilic non-acidic molecules select against a major efflux pump without inducing antibiotic resistance regulons.IMPORTANCEFor drugs that are administered orally, we need to understand how ingested phytochemicals modulate drug resistance in our gut microbiome. Bacteria maintain low-level resistance by proton-motive force (PMF)-driven pumps that efflux many different antibiotics and cell waste products. These pumps play a key role in bacterial defense by conferring resistance to antimicrobial agents at first exposure while providing time for a pathogen to evolve resistance to higher levels of the antibiotic exposed. Nevertheless, efflux pumps confer energetic costs due to gene expression and pump energy expense. The bacterial PMF includes the transmembrane pH difference (ΔpH), which may be depleted by permeant acids and membrane disruptors. Understanding the fitness costs of efflux pumps may enable us to develop resistance breakers, that is, molecules that work together with antibiotics to potentiate their effect. Non-acidic aromatic molecules have the advantage that they avoid the Mar-dependent induction of regulons conferring other forms of drug resistance. We show that different pumps have distinct selection criteria, and we identified non-acidic aromatic molecules as promising candidates for drug resistance breakers., Competing Interests: The authors declare no conflict of interest.

Nagilactone E, an antifungal agent derived from the root bark of Podocarpus nagi, inhibits 1,3-β glucan synthesis; however, its inhibitory activity is weak. Anethole, the principal component of anise oil, enhances the antifungal activity of nagilactone E. We aimed to determine the combinatorial effect and underlying mechanisms of action of nagilactone E and anethole against the budding yeast Saccharomyces cerevisiae. Analyses using gene-deficient strains showed that the multidrug efflux pump PDR5 is associated with nagilactone E resistance; its transcription was gradually restricted in cells treated with the drug combination for a prolonged duration but not in nagilactone-E-treated cells. Green-fluorescent-protein-tagged Pdr5p was intensively expressed and localized on the plasma membrane of nagilactone-E-treated cells but not in drug-combination-treated cells. Quick-freeze deep-etch electron microscopy revealed the smoothening of intertwined fiber structures on the cell surface of drug-combination-treated cells and spheroplasts, indicating a decline in cell wall components and loss of cell wall strength. Anethole enhanced the antifungal activity of nagilactone E by enabling its retention within cells, thereby accelerating cell wall damage. The combination of nagilactone E and anethole can be employed in clinical settings as an antifungal, as well as a food preservative to restrict food spoilage.