AcrAB-TolC Efflux Pump Research Articles

The Role of AcrAB-TolC Efflux Pumps on Quinolone Resistance of E. coli ST131.

Escherichia coli ST131 is a cause for global concern because of its high multidrug resistance and several virulence factors. In this study, the contribution of acrAB-TolC efflux system of E. coli ST131 to fluoroquinolone resistance was evaluated. A total of nonrepetitive 111 ciprofloxacin-resistant E. coli isolates were included in the study. Multilocus sequence typing was used for genotyping. Expressions of acrA, acrB, and TolC efflux pump genes were measured by RT-PCR. Mutations in marA, gyrA, parC, and aac(6')-lb-cr positivity were studied by Sanger sequencing. Sixty-four (57.7%) of the isolates were classified as ST131, and 52 (81.3%) of the ST131 isolates belonged to H30-Rx subclone. In ST131, CTX-M 15 positivity (73%) and aac(6')-lb-cr carriage (75%) were significantly higher than those in non-ST131 (12.8% and 51%, respectively) (P < 0.05). The ampicillin-sulbactam (83%) resistance was higher, and gentamicin resistance (20%) was lower in ST131 than that in non-ST131 (64% and 55%, respectively) (P = 0.001 and P = 0.0002). Numbers of the isolates with MDR or XDR profiles did not differ in both groups. Multiple in-dels (up to 16) were recorded in all quinolone-resistant isolates. However, marA gene was more overexpressed in ST131 compared to that in non-ST131 (median 5.98 vs. 3.99; P = 0.0007). Belonging to H30-Rx subclone, isolation site, ciprofloxacin MIC values did not correlate with efflux pump expressions. In conclusion, the marA regulatory gene of AcrAB-TolC efflux pump system has a significant impact on quinolone resistance and progression to MDR profile in ST131 clone. Efflux pump inhibitors might be alternative drugs for the treatment of infections caused by E. coli ST131 if used synergistically in combination with antibiotics.

Effect of aminoacyl-tRNA synthetase mutations on susceptibility to ciprofloxacin in Escherichia coli.

Chromosomal mutations that reduce ciprofloxacin susceptibility in Escherichia coli characteristically map to drug target genes (gyrAB and parCE), and genes encoding regulators of the AcrAB-TolC efflux pump. Mutations in RNA polymerase can also reduce susceptibility, by up-regulating the MdtK efflux pump. We asked whether mutations in additional chromosomal gene classes could reduce susceptibility to ciprofloxacin. Experimental evolution, complemented by WGS analysis, was used to select and identify mutations that reduce susceptibility to ciprofloxacin. Transcriptome analysis, genetic reconstructions, susceptibility measurements and competition assays were used to identify significant genes and explore the mechanism of resistance. Mutations in three different aminoacyl-tRNA synthetase genes (leuS, aspS and thrS) were shown to reduce susceptibility to ciprofloxacin. For two of the genes (leuS and aspS) the mechanism was partially dependent on RelA activity. Two independently selected mutations in leuS (Asp162Asn and Ser496Pro) were studied in most detail, revealing that they induce transcriptome changes similar to a stringent response, including up-regulation of three efflux-associated loci (mdtK, acrZ and ydhIJK). Genetic analysis showed that reduced susceptibility depended on the activity of these loci. Broader antimicrobial susceptibility testing showed that the leuS mutations also reduce susceptibility to additional classes of antibiotics (chloramphenicol, rifampicin, mecillinam, ampicillin and trimethoprim). The identification of mutations in multiple tRNA synthetase genes that reduce susceptibility to ciprofloxacin and other antibiotics reveals the existence of a large mutational target that could contribute to resistance development by up-regulation of an array of efflux pumps.

DNA Damage Repair and Drug Efflux as Potential Targets for Reversing Low or Intermediate Ciprofloxacin Resistance in E. coli K-12.

Ciprofloxacin is a potent antibacterial drug that is widely used in human clinical applications. As a consequence of its extensive use, resistance has emerged in almost all clinically relevant bacterial species. A mean to combat the observed ciprofloxacin resistance is by reversing it via co-administration of a potentiating compound, also known as a helper drug. Here, we report on the current advances in identifying ciprofloxacin helper drugs, and put them into perspective of our own findings. We searched for potential helper drug targets in Escherichia coli strains with different levels of ciprofloxacin resistance using transcriptomics i.e., RNAseq and by deletion of genes associated with hyper-susceptibility to ciprofloxacin. Differential gene expression analysis of the highly ciprofloxacin resistant uropathogenic E. coli strain, ST131 UR40, treated with a clinically relevant concentration of ciprofloxacin (2 μg/mL), showed that the transcriptome was unaffected. Conversely, genetic screening of 23 single gene deletions in the high-level ciprofloxacin resistant laboratory derived E. coli strain, LM693, led to a significant decrease in the minimal inhibitory concentration for several genes, including genes encoding the AcrAB-TolC efflux pump, SOS-response proteins and the global regulator Fis. In addition, deletion of acrA, tolC, recA, or recC rendered two E. coli strains with intermediate susceptibility to ciprofloxacin fully susceptible according to the CLSI recommended breakpoint. Our results corroborate the AcrAB-TolC efflux pump and the SOS response proteins, RecA and RecC, as potential targets for ciprofloxacin helper drugs in treatment of human bacterial infections caused by E. coli strains with intermediate sensitivity to ciprofloxacin.

Characteristics of Resistance Mechanisms and Molecular Epidemiology of Fluoroquinolone-Nonsusceptible Salmonella enterica Serovar Typhi and Paratyphi A Isolates from a Tertiary Hospital in Dhaka, Bangladesh.

The aim of this study was to characterize the fluoroquinolone (FQ) resistance mechanism of Salmonella enterica serovar Typhi and Paratyphi A in Bangladesh. Salmonella Typhi isolates were classified into sequence type (ST) 1, ST2, and ST2209 and Salmonella Paratyphi A isolates were classified into ST85 and ST129. The most common STs of the FQ-nonsusceptible strain were ST1 (44.4%) and ST129 (66.6%). Thirty-nine percent of Salmonella Typhi isolates were multidrug resistant, and these were all ST1, which is the type prevalent in the Indian subcontinent. Although plasmid-mediated quinolone resistance genes were not detected in any of the tested strains, single and double mutations were identified in the quinolone resistance-determining region (QRDR). The most common QRDR mutation was GyrA_Ser83Phe (66.7% for Salmonella Typhi and 100% for Salmonella Paratyphi A). Treatment with an efflux pump inhibitor resulted in susceptibility of the strains to levofloxacin. All isolates demonstrated 100% susceptibility to ceftriaxone, azithromycin, and carbapenem. Our results suggest that mutations in gyrase A and enhancement of efflux pump activity are responsible for the resistance to FQs; in particular, the AcrAB-TolC efflux pump may be an important resistance factor for levofloxacin. To control the spread of FQ-nonsusceptible Salmonella Typhi, intensive surveillance in endemic areas, including Bangladesh, and effective infection control are necessary.

Antibacterial Efficacy of Polysaccharide Capped Silver Nanoparticles Is Not Compromised by AcrAB-TolC Efflux Pump.

Antibacterial therapy is of paramount importance in treatment of several acute and chronic infectious diseases caused by pathogens. Over the years extensive use and misuse of antimicrobial agents has led to emergence of multidrug resistant (MDR) and extensive drug resistant (XDR) pathogens. This drastic escalation in resistant phenotype has limited the efficacy of available therapeutic options. Thus, the need of the hour is to look for alternative therapeutic approaches to mitigate healthcare concerns caused due to MDR bacterial infections. Nanoparticles have gathered much attention as potential candidates for antibacterial therapy. Equipped with advantages of, wide spectrum bactericidal activity at very low dosage, inhibitor of biofilm formation and ease of permeability, nanoparticles have been considered as leading therapeutic candidates to curtail infections resulting from MDR bacteria. However, substrate non-specificity of efflux pumps, particularly those belonging to resistance nodulation division super family, have been reported to reduce efficacy of many potent antibacterial therapeutic drugs. Previously, we had reported antibacterial activity of polysaccharide-capped silver nanoparticles (AgNPs) toward MDR bacteria. We showed that AgNPs inhibits biofilm formation and alters expression of cytoskeletal proteins FtsZ and FtsA, with minimal cytotoxicity toward mammalian cells. In the present study, we report no reduction in antibacterial efficacy of silver nanoparticles in presence of AcrAB-TolC efflux pump proteins. Antibacterial tests were performed according to CLSI macrobroth dilution method, which revealed that both silver nanoparticles exhibited bactericidal activity at very low concentrations. Further, immunoblotting results indicated that both the nanoparticles modulate the transporter AcrB protein expression. However, expression of the membrane fusion protein AcrA did show a significant increase after exposure to AgNPs. Our results indicate that both silver nanoparticles are effective in eliminating MDR Enterobacter cloacae isolates and their action was not inhibited by AcrAB-TolC efflux protein expression. As such, the above nanoparticles have strong potential to be used as effective and alternate therapeutic candidates to combat MDR gram-negative Enterobacterial pathogens.

High Salt Cross-Protects Escherichia coli from Antibiotic Treatment through Increasing Efflux Pump Expression.

Environmental stresses often co-occur when bacteria encounter antibiotic treatment inside the human body. The cellular response to environmental stressors can alter the global gene expression pattern of bacteria. However, the relationship between the cellular stress response and antibiotic susceptibility remains poorly understood. Here we studied the effect of high salt, an important environmental stress condition inside the human body, on bacterial susceptibility to antibiotics. We found that high salt reduces the susceptibility of Escherichia coli to tetracycline and chloramphenicol, leading to a cross-protection effect. The cross-protection effect originates from the increased AcrAB-TolC efflux pump expression level under high-salt conditions. Our study demonstrates that stress-induced gene expression alterations can cross-protect bacteria from antibiotic treatment and should thus be considered when investigating antibiotic susceptibility and applying antimicrobial treatment.IMPORTANCE Environmental stresses often co-occur when bacteria confront antibiotic treatment. We provide a clear example that a natural stress condition (high salt) can cross-protect bacteria from antibiotic treatment by triggering the bacterial stress response program (elevated AcrAB-TolC efflux pump expression). Our study highlights the importance of taking the co-occurrence of bacterial environmental stresses into consideration when investigating antibiotic susceptibility and applying antimicrobial treatment.

Bioactive compounds from the African medicinal plant Cleistochlamys kirkii as resistance modifiers in bacteria.

Cleistochlamys kirkii (Benth) Oliv. (Annonaceae) is a medicinal plant traditionally used in Mozambique to treat infectious diseases. The aim of this study was to find resistance modifiers in C.kirkii for Gram-positive and Gram-negative model bacterial strains. One of the most important resistance mechanisms in bacteria is the efflux pump-related multidrug resistance. Therefore, polycarpol (1), three C-benzylated flavanones (2-4), and acetylmelodorinol (5) were evaluated for their multidrug resistance-reverting activity on methicillin-susceptible and methicillin-resistant Staphylococcus aureus and Escherichia coli AG100 and AG100 A strains overexpressing and lacking the AcrAB-TolC efflux pump system. The combined effects of antibiotics and compounds (2 and 4) were also assessed by using the checkerboard microdilution method in both S.aureus strains. The relative gene expression of the efflux pump genes was determined by real-time reverse transcriptase quantitative polymerase chain reaction. The inhibition of quorum sensing was also investigated. The combined effect of the antibiotics and compound 2 or 4 on the methicillin-sensitive S.aureus resulted in synergism. The most active compounds 2 and 4 increased the expression of the efflux pump genes. These results suggested that C.kirkii constituents could be effective adjuvants in the antibiotic treatment of infections.

Antimicrobial Resistance, Biofilm Formation, and Phylogenetic Grouping of Uropathogenic Escherichia coli Isolates in Egypt: The Role of Efflux Pump-Mediated Resistance

Background: Urinary tract infections are the most commonly encountered infections in clinics and outpatient settings and are mainly caused by Uropathogenic Escherichia coli (UPEC). Multidrug-resistant bacteria (MDR) have become a major public health threat, worldwide. Objectives: This study aimed to investigate the prevalence of the MDR phenotype, efflux pump-mediated resistance, and the ability for in vitro biofilm formation among UPEC clinical isolates from Egypt. Methods: Uropathogenic E. coli isolates were collected from two Egyptian governorates, identified, and classified to their corresponding phylogenetic group by the polymerase chain reaction (PCR). Antimicrobial susceptibility testing was done using the Kirby-Bauer disk diffusion method. AcrAB-TolC efflux pump major genes were detected by PCR; efflux pump-mediated resistance was determined by the efflux pump inhibitor microplate-based assay. The ability for in-vitro biofilm formation was also tested. Results: The phylogenetic analysis of the UPEC isolates revealed that most of the isolates belonged to groups B2 and D. The MDR phenotype was detected in 90.8% of UPEC isolates; efflux pump-mediated resistance was detected in all MDR isolates. The acrA, acrB, and tolC were detected in 74.84% of MDR isolates. The ability for in-vitro biofilm formation was recorded in 76.5% of the UPEC isolates. Conclusions: The MDR phenotype and the ability for in-vitro biofilm formation were predominant among UPEC in Egypt. The high prevalence of MDR efflux pumps necessitates the application of new treatment strategies to inhibit this phenomenon.

Transcriptional Modulation of Penicillin-Binding Protein 1b, Outer Membrane Protein P2 and Efflux Pump (AcrAB-TolC) during Heat Stress Is Correlated to Enhanced Bactericidal Action of Imipenem on Non-typeable Haemophilus influenzae.

Objective: The purpose of the present study was to investigate the penicillin binding proteins (PBPs), drug influx and efflux modulations during heat stress and their effects on the bactericidal action of imipenem on non-typeable Haemophilus influenzae (NTHi).Methods: The two NTHi clinical isolates (GE47 and GE88, imipenem MICs by E-test > 32 μg/mL) examined in this study were collected at Geneva University Hospitals. The imipenem killing activity was assessed after incubation of the NTHi strains at either 37 or 42°C for 3 h with increasing concentrations of imipenem. The detection of PBPs was carried out by Bocillin-FL. Global transcriptional changes were monitored by RNA-seq after pre-incubation of bacterial cells at either 37 or 42°C, and the expression levels of relevant target genes were confirmed by qRT-PCR.Results: Quantitation of NTHi viable cells after incubation with 0.25 μg/mL of imipenem for 3 h revealed more than a twofold decrease in GE47 and GE88 viable cells at 42°C as compared to 37°C. Transcriptome analysis showed that under heat stress conditions, there were 141 differentially expressed genes with a | log2(fold change)| > 1, including 67 up-regulated and 74 down-regulated genes. The expression levels of ponB (encoding PBP1b) and acrR (regulator of AcrAB-TolC efflux pump) were significantly increased at 42°C. In contrast, the transcript levels of ompP2 (encoding the outer membrane protein P2) and acrB gene (encoding AcrB) were significantly lower under heat stress condition.Conclusion: This study shows that the transcriptional modulation of ponB, ompP2, acrR, and acrB in the heat stress response is correlated to enhanced antimicrobial effects of imipenem on non-typeable H. influenzae.

Stress Introduction Rate Alters the Benefit of AcrAB-TolC Efflux Pumps.

Stress tolerance studies are typically conducted in an all-or-none fashion. However, in realistic settings-such as in clinical or metabolic engineering applications-cells may encounter stresses at different rates. Therefore, how cells tolerate stress may depend on its rate of appearance. To address this, we studied how the rate of stress introduction affects bacterial stress tolerance by focusing on a key stress response mechanism. Efflux pumps, such as AcrAB-TolC of Escherichia coli, are membrane transporters well known for the ability to export a wide variety of substrates, including antibiotics, signaling molecules, and biofuels. Although efflux pumps improve stress tolerance, pump overexpression can result in a substantial fitness cost to the cells. We hypothesized that the ideal pump expression level would involve a rate-dependent trade-off between the benefit of pumps and the cost of their expression. To test this, we evaluated the benefit of the AcrAB-TolC pump under different rates of stress introduction, including a step, a fast ramp, and a gradual ramp. Using two chemically diverse stresses, the antibiotic chloramphenicol and the jet biofuel precursor pinene, we assessed the benefit provided by the pumps. A mathematical model describing these effects predicted the benefit as a function of the rate of stress introduction. Our findings demonstrate that as the rate of introduction is lowered, stress response mechanisms provide a disproportionate benefit to pump-containing strains, allowing cells to survive beyond the original inhibitory concentrations.IMPORTANCE Efflux pumps are ubiquitous in nature and provide stress tolerance in the cells of species ranging from bacteria to mammals. Understanding how pumps provide tolerance has far-reaching implications for diverse fields, from medicine to biotechnology. Here, we investigated how the rate of stressor appearance impacts tolerance. We focused on two distinct substrates of AcrAB-TolC efflux pumps, the antibiotic chloramphenicol and the biofuel precursor pinene. Interestingly, tolerance is highly dependent on the rate of stress introduction. Therefore, it is important to consider not only the total quantity of a stressor but also the rate at which it is applied. The implications of this work are significant because environments are rarely static; antibiotic concentrations change during dosing, and metabolic engineering processes change with time.

Regulation of the AcrAB-TolC efflux pump in Enterobacteriaceae

Bacterial multidrug efflux systems are a major mechanism of antimicrobial resistance and are fundamental to the physiology of Gram-negative bacteria. The resistance-nodulation-division (RND) family of efflux pumps is the most clinically significant, as it is associated with multidrug resistance. Expression of efflux systems is subject to multiple levels of regulation, involving local and global transcriptional regulation as well as post-transcriptional and post-translational regulation. The best-characterised RND system is AcrAB-TolC, which is present in Enterobacteriaceae. This review describes the current knowledge and new data about the regulation of the acrAB and tolC genes in Escherichia coli and Salmonella enterica.

Vaborbactam: Spectrum of Beta-Lactamase Inhibition and Impact of Resistance Mechanisms on Activity in Enterobacteriaceae.

ABSTRACTVaborbactam (formerly RPX7009) is a new beta-lactamase inhibitor based on a cyclic boronic acid pharmacophore. The spectrum of beta-lactamase inhibition by vaborbactam and the impact of bacterial efflux and permeability on its activity were determined using a panel of strains with beta-lactamases cloned from various classes and a panel of Klebsiella pneumoniae carbapenemase 3 (KPC-3)-producing isogenic strains with various combinations of efflux and porin mutations. Vaborbactam is a potent inhibitor of class A carbapenemases, such as KPC, as well as an inhibitor of other class A (CTX-M, SHV, TEM) and class C (P99, MIR, FOX) beta-lactamases. Vaborbactam does not inhibit class D or class B carbapenemases. When combined with meropenem, vaborbactam had the highest potency compared to the potencies of vaborbactam in combination with other antibiotics against strains producing the KPC beta-lactamase. Consistent with broad-spectrum beta-lactamase inhibition, vaborbactam reduced the meropenem MICs for engineered isogenic strains of K. pneumoniae with increased meropenem MICs due to a combination of extended-spectrum beta-lactamase production, class C beta-lactamase production, and reduced permeability due to porin mutations. Vaborbactam crosses the outer membrane of K. pneumoniae using both OmpK35 and OmpK36, but OmpK36 is the preferred porin. Efflux by the multidrug resistance efflux pump AcrAB-TolC had a minimal impact on vaborbactam activity. Investigation of the vaborbactam concentration necessary for restoration of meropenem potency showed that vaborbactam at 8 μg/ml results in meropenem MICs of ≤2 μg/ml in the most resistant engineered strains containing multiple mutations. Vaborbactam is a highly active beta-lactamase inhibitor that restores the activity of meropenem and other beta-lactam antibiotics in beta-lactamase-producing bacteria, particularly KPC-producing carbapenem-resistant Enterobacteriaceae.

Effect of total alkaloid extract of local Sophora alopecuroides on minimum inhibitory concentration and intracellular accumulation of ciprofloxacin, and acrA expression in highly resistant Escherichia coli clones.

Multidrug-resistant phenotypes in Gram-negative bacteria such as Escherichia coli mainly result from overexpression of the AcrAB-TolC efflux pump. Efflux pump inhibitors may be obtained from plant extracts, such as alkaloid extract of Sophora spp. The aims of this study were to evaluate the antibacterial and anti-pump potential of local Sophora alopecuroides total alkaloid extract and commercial matrine in combination with ciprofloxacin in highly resistant E. coli clones. Alkaloid extract of S. alopecuroides seeds was prepared by acid-base solution and chloroform extraction. The percentage of matrine in this extract was measured by high-performance liquid chromatography (HPLC). Minimum inhibitory concentrations (MICs) of the extract and matrine in combination with ciprofloxacin were determined by the microbroth dilution method. The anti-pump activity of these combined materials was measured by ciprofloxacin accumulation assay and real-time PCR. The alkaloid extract contained 31.2% matrine. A synergistic interaction [fractional inhibitory concentration index (FICI)=0.13] was seen between the extract (1.56mg/mL) and ciprofloxacin (1μg/mL) in highly resistant E. coli clones. A significant decrease (P<0.05) in the expression level of acrA was seen in the presence of ciprofloxacin in clones pre-treated with plant extract or matrine for 36h. Accumulation of ciprofloxacin enhanced 2.8- and 2-fold following addition of matrine and plant extract, respectively, in these clones. Alkaloid extract of local S. alopecuroides and matrine have anti-pump activity; further investigations are necessary to elucidate their exact mechanisms of action.

The Role of AcrAB-TolC Efflux Pump in Mediating Fluoroquinolone Resistance in Naturally Occurring Salmonella Isolates from China

The involvement of AcrAB-TolC efflux pump in regulating fluoroquinolone resistance of naturally occurring Salmonella isolates is insufficiently investigated. In this study, the regulatory genes, acrR, ramR, marRAB, and soxRS of AcrAB-TolC efflux pump, of 27 naturally occurring fluoroquinolone-resistant Salmonella isolates collected in China were sequenced. The expression levels of acrB, ramA, marA, and soxS were also examined using quantitative real-time polymerase chain reaction. Gene alterations were mainly observed for acrR (three mutation types) and ramR (four mutation types), not for marRAB (no mutation) or soxRS (one mutaton type). Overexpressions were also mainly observed for acrB and ramA, not for marA or soxS. Some mutations/deletions in ramR caused highly elevated expression of ramA. Complementation with wild-type ramR gene reduced mRNA levels of acrB and ramA by 1.7- to 2.2-fold and 10.5- to 30.1-fold, respectively, and lowered fluoroquinolones (FQ) minimum inhibitory concentrations by 2- to 8-fold. Neither MarA nor SoxS was found to be associated with increased FQ resistance. This study shows that the AcrAB efflux pump is playing a role in mediating fluoroquinolone resistance, and RamA may be the major global regulator of AcrAB-TolC-mediated fluoroquinolone resistance in Salmonella.

Network-Based Identification of Adaptive Pathways in Evolved Ethanol-Tolerant Bacterial Populations.

Efficient production of ethanol for use as a renewable fuel requires organisms with a high level of ethanol tolerance. However, this trait is complex and increased tolerance therefore requires mutations in multiple genes and pathways. Here, we use experimental evolution for a system-level analysis of adaptation of Escherichia coli to high ethanol stress. As adaptation to extreme stress often results in complex mutational data sets consisting of both causal and noncausal passenger mutations, identifying the true adaptive mutations in these settings is not trivial. Therefore, we developed a novel method named IAMBEE (Identification of Adaptive Mutations in Bacterial Evolution Experiments). IAMBEE exploits the temporal profile of the acquisition of mutations during evolution in combination with the functional implications of each mutation at the protein level. These data are mapped to a genome-wide interaction network to search for adaptive mutations at the level of pathways. The 16 evolved populations in our data set together harbored 2,286 mutated genes with 4,470 unique mutations. Analysis by IAMBEE significantly reduced this number and resulted in identification of 90 mutated genes and 345 unique mutations that are most likely to be adaptive. Moreover, IAMBEE not only enabled the identification of previously known pathways involved in ethanol tolerance, but also identified novel systems such as the AcrAB-TolC efflux pump and fatty acids biosynthesis and even allowed to gain insight into the temporal profile of adaptation to ethanol stress. Furthermore, this method offers a solid framework for identifying the molecular underpinnings of other complex traits as well.

Fitness cost constrains the spectrum of marR mutations in ciprofloxacin-resistant Escherichia coli.

ObjectivesTo determine whether the spectrum of mutations in marR in ciprofloxacin-resistant clinical isolates of Escherichia coli shows evidence of selection bias, either to reduce fitness costs, or to increase drug resistance. MarR is a repressor protein that regulates, via MarA, expression of the Mar regulon, including the multidrug efflux pump AcrAB-TolC.MethodsIsogenic strains carrying 36 different marR alleles identified in resistant clinical isolates, or selected for resistance in vitro, were constructed. Drug susceptibility and relative fitness in growth competition assays were measured for all strains. The expression level of marA, and of various efflux pump components, as a function of specific mutations in marR, was measured by qPCR.ResultsThe spectrum of genetic alterations in marR in clinical isolates is strongly biased against inactivating mutations. In general, the alleles found in clinical isolates conferred a lower level of resistance and imposed a lower growth fitness cost than mutations selected in vitro. The level of expression of MarA correlated well with the MIC of ciprofloxacin. This supports the functional connection between mutations in marR and reduced susceptibility to ciprofloxacin.ConclusionsMutations in marR selected in ciprofloxacin-resistant clinical isolates are strongly biased against inactivating mutations. Selection favours mutant alleles that have the lowest fitness costs, even though these cause only modest reductions in drug susceptibility. This suggests that selection for high relative fitness is more important than selection for increased resistance in determining which alleles of marR will be selected in resistant clinical isolates.

SoxRS induces colistin hetero-resistance in Enterobacter asburiae and Enterobacter cloacae by regulating the acrAB-tolC efflux pump.

Colistin is the last drug option for the treatment of MDR Gram-negative bacterial infections. Several types of resistance to colistin have been identified, including hetero-resistance, which has been observed in several Gram-negative pathogens. During a routine surveillance project on antimicrobial resistance, we found abnormal colistin-resistant Enterobacter asburiae and Enterobacter cloacae isolates. E. cloacae is an intestinal commensal bacterium and a well-known opportunistic nosocomial pathogen. To characterize the molecular mechanism of colistin hetero-resistance in Enterobacter spp. Several approaches (WGS, transposome mutagenesis and RT-PCR analysis) were used to discover the molecular mechanism of colistin hetero-resistance. Genomic analysis of mutant clones generated by transposome mutagenesis suggests that hetero-resistance is linked with overexpression of the acrAB-tolC efflux pump. Transcriptional analysis further found that naturally elevated soxRS triggers the induction of the acrAB-tolC efflux pump proteins followed by the development of colistin hetero-resistance in E. asburiae and E. cloacae. Transcriptional analysis results were further verified as demonstrating the development of hetero-resistance in colistin-susceptible strains by plasmid-based overexpression of soxRS. Our observations highlight the importance of such findings, which previously were only superficially described because of the challenges associated with their detection, in the context of common modes of colistin resistance in Gram-negative bacteria. This study constitutes a unique demonstration of efflux-based high-level colistin hetero-resistance, controlled by a soxRS regulator in Gram-negative bacteria.

Potentiation of Antibiotic Activity by a Novel Cationic Peptide: Potency and Spectrum of Activity of SPR741.

Novel approaches for the treatment of multidrug-resistant Gram-negative bacterial infections are urgently required. One approach is to potentiate the efficacy of existing antibiotics whose spectrum of activity is limited by the permeability barrier presented by the Gram-negative outer membrane. Cationic peptides derived from polymyxin B have been used to permeabilize the outer membrane, granting antibiotics that would otherwise be excluded access to their targets. We assessed the in vitro efficacies of combinations of SPR741 with conventional antibiotics against Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii Of 35 antibiotics tested, the MICs of 8 of them were reduced 32- to 8,000-fold against E. coli and K. pneumoniae in the presence of SPR741. The eight antibiotics, azithromycin, clarithromycin, erythromycin, fusidic acid, mupirocin, retapamulin, rifampin, and telithromycin, had diverse targets and mechanisms of action. Against A. baumannii, similar potentiation was achieved with clarithromycin, erythromycin, fusidic acid, retapamulin, and rifampin. Susceptibility testing of the most effective antibiotic-SPR741 combinations was extended to 25 additional multidrug-resistant or clinical isolates of E. coli and K. pneumoniae and 17 additional A. baumannii isolates in order to rank the potentiated antibiotics. SPR741 was also able to potentiate antibiotics that are substrates of the AcrAB-TolC efflux pump in E. coli, effectively circumventing the contribution of this pump to intrinsic antibiotic resistance. These studies support the further development of SPR741 in combination with conventional antibiotics for the treatment of Gram-negative bacterial infections.

Biophysical characterization of E. coli TolC interaction with the known blocker hexaamminecobalt

BackgroundThe tripartite efflux pump AcrAB-TolC in E. coli is involved in drug resistance by transporting antibiotics out of the cell. The outer membrane protein TolC can be blocked by various cations, including hexaamminecobalt, thereby TolC represents a potential target for reducing antimicrobial resistance as its blockage may improve efficacy of antibiotics. MethodsWe utilized single channel electrophysiology measurements for studying TolC conductance in the absence and presence of the known TolC blocker hexaamminecobalt. Association and dissociation constants of hexaamminecobalt were determined using surface plasmon resonance measurements. Minimum inhibitory concentration (MIC) assays in the absence and presence of antibiotics were carried out for investigating the antibacterial effect of hexaamminecobalt and its potential to reduce MICs. ResultsTolC gating in the absence of any ligand is voltage dependent and asymmetric at high applied voltages. Hexaamminecobalt binds to TolC with high affinity and kinetic data revealed fast association and dissociation rates. Despite potent binding to TolC, hexaamminecobalt does not possess an intrinsic antimicrobial activity against E. coli nor does it reduce MIC values of antibiotics erythromycin and fusidic acid. ConclusionsTolC opening can be effectively blocked by small molecules. More potent channel blockers are needed in order to investigate the eligibility of TolC as drug target. General significanceTolC, a potentially interesting pharmaceutical target can be addressed by small molecules, blocking the channel. Biophysical characterization of the binding processes will support future identification and optimisation of more potent TolC blockers in order to validate TolC as a pharmaceutical target.

Identification and Structure-Activity Relationships of Novel Compounds that Potentiate the Activities of Antibiotics in Escherichia coli.

In Gram-negative bacteria, efflux pumps are able to prevent effective cellular concentrations from being achieved for a number of antibiotics. Small molecule adjuvants that act as efflux pump inhibitors (EPIs) have the potential to reinvigorate existing antibiotics that are currently ineffective due to efflux mechanisms. Through a combination of rigorous experimental screening and in silico virtual screening, we recently identified novel classes of EPIs that interact with the membrane fusion protein AcrA, a critical component of the AcrAB-TolC efflux pump in Escherichia coli. Herein, we present initial optimization efforts and structure-activity relationships around one of those previously described hits, NSC 60339 (1). From these efforts we identified two compounds, SLUPP-225 (17h) and SLUPP-417 (17o), which demonstrate favorable properties as potential EPIs in E. coli cells including the ability to penetrate the outer membrane, improved inhibition of efflux relative to 1, and potentiation of the activity of novobiocin and erythromycin.