Dodecanoic acid & palmitic acid disarms rifampicin resistance by putatively targeting mycobacterial efflux pump Rv1218c

Background & objectives: Drug-resistant tuberculosis (TB) jeopardizes the treatment process with poor outcomes. Efflux pumps (EPs) belonging to the ABC transporter family in Mycobacterium tuberculosis confer resistance to rifampicin (RMP) besides genetic mutations thus serving as a target for a potential adjunct therapeutic inhibitory molecule. Rv1218c is one such pump that was previously reported to be active in multidrug-resistant TB clinical isolates. Methods: In this study, the inhibition potential of Rv1218c-EP was tested on 8 molecules that were shortlisted by in silico methods. These molecules were subjected to the minimum inhibitory concentration (MIC) determination, checkerboard drug combination assay, ethidium bromide-DNA binding assay, and in vitro and ex vivo cytotoxicity assay. Results: Based on the outcome of the study, two molecules dodecanoic acid (DA) and palmitic acid (PA) were found to be potential enough to decrease the MIC of RMP by 8 to 1000 folds against multidrug-resistant clinical isolates and Rv1218c expressing recombinant Mycobacterium smegmatis. Interpretation & conclusions: These molecules were also found to reduce the time taken by RMP to kill these drug-resistant Mycobacteria to 48 h, unlike control isolates that survived more than 240 h of RMP exposure. The functional concentration of both molecules was non-toxic to the epithelial and blood mononuclear cells. With further comprehensive scientific validation, PA and DA could be recommended as adjunct therapeutic molecules with first-line anti-TB drugs to treat drug-resistant TB.

Development of the novel ACLY inhibitor 326E as a promising treatment for hypercholesterolemia

Hepatic cholesterol accumulation is an important contributor to hypercholesterolemia, which results in atherosclerosis and cardiovascular disease (CVD). ATP-citrate lyase (ACLY) is a key lipogenic enzyme that converts cytosolic citrate derived from tricarboxylic acid cycle (TCA cycle) to acetyl-CoA in the cytoplasm. Therefore, ACLY represents a link between mitochondria oxidative phosphorylation and cytosolic de novo lipogenesis. In this study, we developed the small molecule 326E with an enedioic acid structural moiety as a novel ACLY inhibitor, and its CoA-conjugated form 326E-CoA inhibited ACLY activity with an IC50 = 5.31 ± 1.2 μmol/L in vitro. 326E treatment reduced de novo lipogenesis, and increased cholesterol efflux in vitro and in vivo. 326E was rapidly absorbed after oral administration, exhibited a higher blood exposure than that of the approved ACLY inhibitor bempedoic acid (BA) used for hypercholesterolemia. Chronic 326E treatment in hamsters and rhesus monkeys resulted in remarkable improvement of hyperlipidemia. Once daily oral administration of 326E for 24 weeks prevented the occurrence of atherosclerosis in ApoE-/- mice to a greater extent than that of BA treatment. Taken together, our data suggest that inhibition of ACLY by 326E represents a promising strategy for the treatment of hypercholesterolemia.

The host-targeting compound peruvoside has a broad-spectrum antiviral activity against positive-sense RNA viruses

Positive-sense RNA viruses modify intracellular calcium stores, endoplasmic reticulum and Golgi apparatus (Golgi) to generate membranous replication organelles known as viral factories. Viral factories provide a conducive and substantial enclave for essential virus replication via concentrating necessary cellular factors and viral proteins in proximity. Here, we identified the vital role of a broad-spectrum antiviral, peruvoside in limiting the formation of viral factories. Mechanistically, we revealed the pleiotropic cellular effect of Src and PLC kinase signaling via cyclin-dependent kinase 1 signaling leads to Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1) phosphorylation and Golgi vesiculation by peruvoside treatment. The ramification of GBF1 phosphorylation fosters GBF1 deprivation consequentially activating downstream antiviral signaling by dampening viral factories formation. Further investigation showed signaling of ERK1/2 pathway via cyclin-dependent kinase 1 activation leading to GBF1 phosphorylation at Threonine 1337 (T1337). We also showed 100% of protection in peruvoside-treated mouse model with a significant reduction in viral titre and without measurable cytotoxicity in serum. These findings highlight the importance of dissecting the broad-spectrum antiviral therapeutics mechanism and pave the way for consideration of peruvoside, host-directed antivirals for positive-sense RNA virus-mediated disease, in the interim where no vaccine is available.

Advances in abscisic acid biosynthesis / 生物工程学报

Abscisic acid, a plant hormone that inhibits growth, is a key factor in balancing plant endogenous hormones and regulating growth and metabolism. Abscisic acid can improve the drought resistance and salt tolerance of crops, reduce fruit browning, reduce the incidence rate of malaria and stimulate insulin secretion, so it has a broad application potential in agriculture and medicine. Compared with traditional plant extraction and chemical synthesis, abscisic acid synthesis by microorganisms is an economic and sustainable route. At present, a lot of progress has been made in the synthesis of abscisic acid by natural microorganisms such as Botrytis cinerea and Cercospora rosea, while the research on the synthesis of abscisic acid by engineered microorganisms is rarely reported. Saccharomyces cerevisiae, Yarrowia lipolytica and Escherichia coli are common hosts for heterologous synthesis of natural products due to their advantages of clear genetic background, easy operation and friendliness for industrial production. Therefore, the heterologous synthesis of abscisic acid by microorganisms is a more promising production method. The author reviews the research on the heterologous synthesis of abscisic acid by microorganisms from five aspects: selection of chassis cells, screening and expression enhancement of key enzymes, regulation of cofactors, enhancement of precursor supply and promotion of abscisic acid efflux. Finally, the future development direction of this field is prospected.

Research progress on efflux pump inhibitors of Mycobacterium tuberculosis in recent years / 药学学报

Currently, the resistance of first-line anti-tuberculosis drugs has made the prevention and treatment of tuberculosis increasingly difficult, posing a serious threat to global public health. Several studies have shown that efflux pumps are one of the important causes for bacteria to develop multi-drug resistance and extremely-drug resistance, and efflux pump inhibitors can inhibit the efflux of antibacterial drugs, thereby reducing bacterial drug resistance. Numerous natural products and synthetic compounds have been reported to possess efflux pump inhibitory activity, but they have not been applied in clinical settings because of their toxicity, pharmacokinetic properties, etc. Therefore, we summarized the efflux pump inhibitory activity, antimicrobial activity, and structure-activity relationships of reported efflux pump inhibitors against Mycobacterium tuberculosis in recent years, providing references for the development of new efflux pump inhibitors with better activity and lower toxicity.

Recent advances in the development of antibacterial potentiators / 中华检验医学杂志

Multidrug-resistant bacteria that can′t be treated with any common antibacterial drugs have become a global medical crisis. Therefore, there is an urgent need for new antibacterial potentiators to restore the sensitivity of bacteria to the antibacterial drug. This review elaborates on the novel antibacterial synergistic methods and their underlying mechanisms, clinical experimental data and efficacy, and the progress of drug research and development. This review aims to raise awareness about antibacterial potentiators among the public.

Antimicrobial resistance in Pseudomonas aeruginosa

ABSTRACT@#In recent years, antimicrobial resistance (AMR) has become a global public health concern. The growth of resistant bacteria is increasing dramatically, while the number of new antibiotics accessible is decreasing. This is especially true in the case of Pseudomonas aeruginosa, an important causative agent of healthcare-associated infections. The ability of P. aeruginosa to survive in different environments and on medical devices has made it more resistant to antibiotics. This causes bacteremia in hospitalized patients, ventilator-associated pneumonia, catheter-associated urinary tract infections and wound infections, particularly in patients with severe burns, bed ulcers and immunocompromised individuals. The rise in the AMR rate in both developed and developing countries may be attributed to a number of factors such as variations in the standard health care, large population, awareness about antibiotic resistance, inadequate training on rationale antibiotic usage and inadequate infection control facilities in many hospitals. The emergence of Extensive Drug Resistance (XDR) and Pan Drug Resistance (PDR) among organisms that cause various infections leads to increased treatment costs, morbidity and mortality, leaving no therapeutic options. This review highlights the different mechanisms of antibiotic resistance, including intrinsic and acquired resistance, which are frequently observed in P. aeruginosa.

Study on the susceptibility genes that mediate the resistance of Helicobacter pylori to clarithromycin and levofloxacin based on comprehensive antibiotic research database comparison / 中华消化杂志

Objective:Taking clinical strains of Helicobacter pylori ( H. pylori) with different antimicrobial resistance as the research object, to explore the new genes related to the resistance of H. pylori to clarithromycin (CLA) and levofloxacin (LVX) based on whole-genome sequencing. Methods:From September 1st, 2016 to August 31st, 2019, 1 749 patients with upper gastrointestinal symptoms and positive 13C urea breath test who visited the Department of Gastroenterology and Hepatology, the University of Hong Kong-Shenzhen Hospital were enrolled. After gastric mucosal biopsy, H. pylori was isolated and cultured from gastric mucosa. Ninety H. pylori strains were successfully preserved. According to the results of in vitro drug sensitivity test, a total of 40 strains including 10 strains with single-drug resistance to CLA (CLA group), 10 strains with single-drug resistance to LVX (LVX group), 10 strains with dual-resistance to CLA and LVX (dual resistance group) and 10 strains sensitive to CLA, LVX, amoxicillin, furazolidone, tetracycline and metronidazole (all sensitive group) were screened out. By whole-genome sequencing and comparison to the comprehensive antibiotic research database (CARD), single nucleotide variations (SNV) and indels were analyzed, genes related to H. pylori resistance to CLA and LVX were screened out and the differences of new genes among 4 groups were analyzed. Independent sample t test, one-way analysis of variance, least significant difference method and chi-square test were used for statistical analysis. Results:Among the 4 groups there were no statistically significant differences in the number of SNV (74 952.00±8 755.21, 77 128.10±3 191.35, 78 639.90±601.23 and 77 474.60±2 421.05) and Indels (2 582.20±265.45, 2 653.60±108.37, 2 667.10±43.82 and 2 641.10±80.25) (all P>0.05). Compared to CARD, a total of 223 drug resistance-related genes were detected, of which 19 genes related to CLA mono-resistance in CLA group, 24 genes related to LVX mono-resistance in LVX group, 16 genes related to CLA mono-resistance, 14 genes related to LVX mono-resistance, and 12 dual resistance-related genes in dual resistance group, and 11 genes related to CLA mono-resistance, 17 genes related to LVX mono-resistance, and 13 dual resistance-related genes in all sensitive group. Among the genes related to CLA mono-resistance, the detection rates of erythromycin esterase gene ( ere)B in CLA group, LVX group, dual resistance group and all sensitive group were 0/10, 0/10, 3/10, 0/10, respectively, and the difference was statistically significant( χ2=5.79, P=0.049). The detection rate of erythromycin ribosomal methylase gene ( erm) family in CLA group and dual resistance group was higher than that in LVX group and all sensitive group (45.0%, 9/20 vs. 10.0%, 2/20), and the difference was statistically significant ( χ2=6.14, P=0.013). The detection rates of free methionine-(R)-sulfoxide reductase gene ( msrC) in CLA group, LVX group, dual resistance group and all sensitive group were 10/10, 7/10, 6/10, 4/10, respectively, and the difference was statistically significant ( χ2=8.97, P=0.030). Among the genes related to LVX mono-resistance, the detection rate of quinolone resistance pentapeptide repeat protein gene ( qnr) family in LVX group and dual resistance group was higher than that in CLA group and all sensitive group (60.0%, 12/20 vs. 25.0%, 5/20), and the difference was statistically significant ( χ2=5.01, P=0.025). The detection rates of qnrB4 in CLA group, LVX group, dual resistance group and all sensitive group were 1/10, 3/10, 7/10, 1/10, respectively, and the difference was statistically significant ( χ2=10.17, P=0.010). The number of efflux transporter genes related to CLA mono-resistance in 4 groups were less than those of LVX mono-resistance and dual drug resistance (11 vs. 29 and 11 vs. 23), and the differences were statistically significant ( χ2=11.87, 5.80; P=0.001, 0.016). The detected numbers of LVX resistance-related efflux transport genes in CLA group, LVX group, dual resistance group and all sensitive group were 28, 40, 24 and 27, respectively, and the difference was statistically significant ( χ2=10.26, P=0.016). Conclusions:Erm family and msrC may be important genes that mediate the resistance of H. pylori to CLA, and qnr family is related to mediating the resistance of H. pylori to LVX. Efflux transport genes may play a synergistic role in the process of drug efflux, and are more likely to mediate H. pylori resistance to LVX.

Arsenic on cholesterol efflux and the expression of ABCA1, ABCG1 and SRBI / 预防医学

Objective@#To explore the impact of arsenic on cholesterol efflux and the expression of ATP-binding cassette, sub-family A, member 1 ( ABCA1 ), ATP-binding cassette transporter G1 ( ABCG1 ), and scavenger receptor class B member I ( SRBI ) in macrophages, so as to provide the evidence for the mechanism of arsenic induced atherosclerosis.@*Methods@#The human myeloid leukemia mononuclear cells ( THP-1 ), induced by phorbol myristate acetate, and mouse primary macrophages were treated with 0, 0.625, 1.25, 2.5 and 5 μmol/L NaAsO2 for 48 hours. Then the cells treated with 2.5 μmol/L NaAsO2 were changed to arsenic free mediums for 48 hours and collected every 12 hours to analyze the time effect of arsenic. The expression levels of ABCA1, ABCG1 and SRBI were determined by quantitative polymerase chain reaction and western blotting. Cholesterol efflux rates were measured by 3H isotope tracer. @*Results@#Arsenic significantly down-regulated the expression levels of ABCA1 and ABCG1, and cholesterol efflux in a dose-dependent manner. The levels of ABCA1 mRNA decreased by 69% and 72%, the levels of ABCG1 mRNA decreased by 42% and 34%, and the rate of cholesterol efflux decreased by 55% and 59% in THP-1 and mouse primary macrophages cells treated with 5 μmol/L NaAsO2 ( all P<0.05 ). Arsenic had no significant effect on SRBI expression ( all P>0.05 ). Arsenic inhibited ABCA1 expression and cholesterol efflux in THP-1 in a time-dependent manner. Compared with cells before the exposure of arsenic, the level of ABCA1 mRNA and the rate of cholesterol efflux in THP-1 bottomed at 48 hours by 43% and 42%, and gradually recovered when arsenic was removed. @*Conclusions@#Arsenic inhibits cholesterol efflux by down-regulating the expression of ABCA1 and ABCG1 in macrophages.

2,3,5,4'-Tetrahydroxystibane-2-O-β-D-glucoside induces liver injury by disrupting bile acid homeostasis and phospholipids efflux / 中国中药杂志

Polygonum multiflorum is a traditional Chinese herbal medicine and has many biological activities such as hair-blacking, anti-atherosclerosis, anti-inflammatory and anti-aging. However, the liver injury induced by P. multiflorum has aroused wide attention in recent years. 2,3,5,4'-tetrahydroxystibane-2-O-β-D-glucoside(TSG) is a main component of P. multiflorum, but the role of TSG in inducing liver injury is unclear. The aim of present study was to evaluate TSG's potential liver injury and effects on bile acid homeostasis and phospholipids efflux. C57 BL/6 J mice received intraperitoneal administration of 400 mg·kg~(-1) of TSG daily for 15 days, and then biochemical indexes of liver injury and changes of phospholipid content were detected. The changes of bile acid compositions were detected by LC-MS/MS. The results showed TSG 400 mg·kg~(-1) significantly increased the content of serum total bile acid(TBA) and alkaline phosphatase(ALP). Elevated free bile acid levels were observed in TSG-treated groups, including β-muricholic acid(β-MCA), ursodeoxycholic acid(UDCA), hyodeoxycholic acid(HDCA), chenodeoxycholic acid(CDCA), deoxcholic acid(DCA) in serum and β-MCA, CDCA in liver. TSG inhibited the protein expression of farnesoid X receptor(FXR) and down stream bile salt export pump(BSEP), which may result in the accumulation of bile acid. TSG also inhibited the expression of 25-hydroxycholesterol-7 alpha-hydroxylase(CYP7 B1), which may disturb the alternative pathway for bile acid synthesis. In addition, intraperitoneal injection of TSG 400 mg·kg~(-1) significantly decreased the content of phospholipids in bile. The research showed that TSG significantly inhibited the expression of multidrug resistance protein 2(MDR2) and destroyed the regular distribution of MDR2 on the bile duct membrane of liver. In vitro results showed that the IC_(50) of TSG on HepG2 cells was about 1 500 μmol·L~(-1) and TSG at 500 μmol·L~(-1)(for 24 h) could destroy the distribution of MDR2 on the bile duct membrane of liver. In conclusion, TSG induced liver injury by disrupting bile acid homeostasis and phospholipids efflux.

Antibacterial activity and inhibition against Staphylococcus aureus NorA efflux pump by ferulic acid and its esterified derivatives

Objective: To evaluate the inhibitory activity of ferulic acid and four of its esterified derivatives (methyl, ethyl, propyl, and butyl) against resistance mechanisms in Staphylococcus aureus strains. Methods: Ferulic acid derivatives were obtained by esterification with methanol, ethanol, propanol, and butanol, and then characterized by hydrogen and carbon-13 nuclear magnetic resonance analysis. The minimum inhibitory concentrations (MIC) of ferulic acid and its esterified derivatives, ethidium bromide, and norfloxacin were obtained using the microdilution test, while the efflux pump inhibition test was conducted by examining reduction in the MICs of norfloxacin and ethidium bromide. Molecular docking was also carried out using the Schrodinger Suite 2015 molecular modeling software. A three-dimensional model of NorA efflux pump was generated using I-TASSER. The best scoring model was used as a receptor for ligand-receptor docking. Results: The methyl and butyl ester derivatives did not demonstrate significant antimicrobial activity. However, a significant synergic effect was evidenced when norfloxacin was combined with the ethyl and propyl esterified derivatives. The docking study demonstrated favorable energy of interaction between ferulate derivatives and NorA, and amino acid residues TYR57, TYR58, and LEU255 were present commonly in stabilizing all complexes. The PCA analysis corroborated the docking hypothesis that the lipophilic character and hydrogen bond interactions were the most relevant characteristics involved with NorA inhibitors. The pharmacokinetic parameters of ferulic acid derivatives showed good ADMET properties, demonstrating that they can be easily absorbed and have no effect or inhibit the cytochrome P450 enzyme complex, revealing their potential as drug candidates. Conclusions: This study provides strong evidence that the molecular basis for this activity is potentially due to the NorA efflux pump.

Nordihydroguaiaretic acid reverses efflux pump system MexCD-OprJ mediated ceftazidime resistance of pseudomonas aeruginosa and its related mechanisms / 中草药

Objective: To evaluate the effect of nordihydroguaiaretic acid (NDGA) on ceftazidime resistance of Pseudomonas aeruginosa mediated by efflux pump system MexCD-OprJ and explore its mechanism. Methods: The bacterial solution with a concentration of 0.5 mcburney was diluted and inoculated in a 96-well plate, and NDGA and ceftazidime were added by the checkerboard dilution method. At the same time, the untreated control group, NDGA control group and ceftazidime control group were set; After being cultured for 24 h, the absorbance was measured by an enzyme micro-plate reader, the minimum inhibitory concentration (MIC) of each drug was recorded and the bacteriostatic rate and fractional inhibitory concentration (FIC) index were calculated. Bacteria were inoculated with the bacterial liquid coating method in the 96-well plates, and the bacterial colony number was counted after 24 h of culture. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the gene expressions of efflux pump membrane protein MexC, MexD, OprJ and nfxB. Results: Compared with ceftazidime or NDGA alone, combination of ceftazidime and NDGA significantly inhibited the growth of efflux pump system MexCD-OprJ-mediated ceftazidime-resistant P. aeruginosa (P < 0.05); The pharmacological effects of ceftazidime and NDGA showed synergistic or additive effects; After combined administration, the MIC values of ceftazidime and NDGA were significantly decreased, and the MIC value of some ceftazidime had no significant difference from that of ceftazidime-sensitive P. aeruginosa; Compared with ceftazidime alone, the gene expressions of efflux pump membrane proteins MexC, MexD and OprJ were significantly decreased after combined application of ceftazidime and NDGA (P < 0.05), while the expression of nfxB was significantly increased (P < 0.05). Conclusion: The mechanism of NDGA on ceftazidime resistance of P. aeruginosa mediated by efflux pump system MexCD-OprJ is related to its ability to down-regulate the gene expression of efflux pump membrane proteins MexC, MexD and OprJ, and up-regulate the gene expression of the negative regulatory gene nfxB of the above three proteins.

Molecular characteristics of Acinetobacter baumannii and the changes of efflux pump expression under tigecycline pressure / 医学研究生学报

Objective　Acinetobacter baumannii (A. baumannii) is a commonly infective bacterium in the hospital. This study aims to analyze its molecular epidemiological characteristics, detect the carrying rate of efflux pump and regulatory protein genes, and investigate the effects of tigecycline on the efflux pump and expression of regulatory protein genes.　Methods　A total of 183 A. baumannii strains were collected from inpatients of the affiliated hospital of Jiangsu University from May 2017 to March 2019. They were divided into an antimicrobial-resistant group (one or more antimicrobial-resistant strains, 139 strains) and a sensitive group (the drugs in the drug sensitivity test were all non-resistant strains, 44 strains). Repeated sequence PCR was used for homology analysis of the strains, and pulse-field gel electrophoresis (PFGE) was used as the gold standard for homology analysis to verify and compare some strains. PCR was used to detect the occurrence of drug resistance-related genes. Based on homology analysis, efflux pump carrying rate detection and antibiotics sensitivity test results, 6 clinical strains carrying all efflux pump genes but different resistance phenotypes were selected as experimental strains, including sensitive strains (SAB), the multidrug resistance strain (MDRAB) and the extensively drug-resistant strain (XDRAB). All strains were induced in vitro with the minimum inhibitory concentration (MIC) of tigecycline. The induced strains were categorized as induction group, and the same strains cultured in LB agar without tigecycline was used as a control group. MIC was used to analyze the tigecycline susceptibility, and RT-qPCR was used to detect the gene expression of efflux pumps, such as TetB, AbaQ and regulatory proteins (AdeS and BaeS), in drug-resistant strains.　Results　Homology analysis showed that there were 45 clonal groups in the detected clinical isolates, with no obvious outbreak of epidemic clonal groups. Efflux pumps and regulatory proteins were widely distributed in the clinical isolates, and the expression of AdeB, TetB, AbeS, AdeS in MDRAB and XDRAB is significantly higher than that insensitive group SAB. Continuous in vitro induction with tigecycline could increase the antimicrobial resistance of some clinical strains and even significantly increase the expression levels of efflux pumps and regulatory proteins.　Conclusion　A. baumannii is widely distributed in the clinic, and efflux pumps and regulatory proteins might play an important role in drug resistance process. The unreasonable use of tigecycline could enhance the tolerance of A. baumannii by up-regulating the expression of some bacterial efflux pumps.

Role of Efflux Pump Gene adeIJK to Multidrug Resistance in Acinetobacter baumannii Clinical Isolates / 대한임상미생물학회지

BACKGROUND: The emergence of multidrug-resistant Acinetobacter baumannii as a nosocomial pathogen is one of the major public health problems. The aim of this study was to evaluate the role of an efflux pump gene adeJ for the multidrug resistance of A. baumannii clinical isolates.METHODS: Two groups (MDRAB and SAB) of A. baumannii clinical isolates were studied. The SAB group consisted of strains that did not meet the criteria of MDRAB and were susceptible to more categories of antibiotics than MDRAB. Antimicrobial susceptibility results obtained by VITEKII system were used in data analysis and bacterial group allocation. We performed real-time reverse transcription PCR to determine relative expression of adeJ. We compared relative expression of adeJ in comparison groups by considering two viewpoints: i) MDRAB and SAB groups and ii) susceptible and non-susceptible groups for each antibiotic used in this study.RESULTS: The mean value of relative expression of adeJ of MDRAB and SAB groups was 1.4 and 0.92, respectively, and showed significant difference (P=0.002). The mean values of relative expression of adeJ of susceptible and non-susceptible groups to the antibiotics cefepime, ceftazidime, ciprofloxacin, imipenem, meropenem, tigecycline, piperacillin/tazobactam, ticarcillin/clavulanic acid, trimethoprim/sulfamethoxazole, piperacillin, and gentamicin showed statistically significant differences.CONCLUSION: The overexpression of adeIJK might contribute to the multi-drug resistance in A. baumannii clinical isolates. Further, the overexpression of adeIJK might be one of the factors contributing to the resistance to numerous antibiotics.

Plant-derived secondary metabolites as the main source of efflux pump inhibitors and methods for identification / 药物分析学报

The upsurge of multiple drug resistance (MDR) bacteria substantially diminishes the effectiveness of antibiotic arsenal and therefore intensifies the rate of therapeutic failure. The major factor in MDR is efflux pump-mediated resistance. A unique pump can make bacteria withstand a wide range of struc-turally diverse compounds. Therefore, their inhibition is a promising route to eliminate resistance phenomenon in bacteria. Phytochemicals are excellent alternatives as resistance-modifying agents. They can directly kill bacteria or interact with the crucial events of pathogenicity, thereby decreasing the ability of bacteria to develop resistance. Numerous botanicals display noteworthy efflux pumps inhibi-tory activities. Edible plants are of growing interest. Likewise, some plant families would be excellent sources of efflux pump inhibitors (EPIs) including Apocynaceae, Berberidaceae, Convolvulaceae, Cucur-bitaceae, Fabaceae, Lamiaceae, and Zingiberaceae. Easily applicable methods for screening plant-derived EPIs include checkerboard synergy test, berberine uptake assay and ethidium bromide test. In silico high-throughput virtual detection can be evaluated as a criterion of excluding compounds with efflux substrate-like characteristics, thereby improving the selection process and extending the identification of EPIs. To ascertain the efflux activity inhibition, real-time PCR and quantitative mass spectrometry can be applied. This review emphasizes on efflux pumps and their roles in transmitting bacterial resistance and an update plant-derived EPIs and strategies for identification.

Amino acid exporters and metabolic modification of Corynebacterium glutamicum - a review / 生物工程学报

Amino acids are important compounds with a wide range of applications in the food, medicine and chemical industries. Corynebacterium glutamicum is a powerful workhorse commonly used in industrial amino acid production, with the scale of more than one million tons. In addition to its efficient anabolism, the effective exporters also ensure the high amino acid production by C. glutamicum. In this review, the research progress of amino acid exporter of C. glutamicum is summarized, to provide the foundation for further improving amino acid production by C. glutamicum via metabolic engineering.

Is the efflux pump inhibitor Verapamil a potential booster for isoniazid against Mycobacterium tuberculosis?

The membrane-based efflux pump systems are recognized to have an important role in pathogenicity and drug resistance in Mycobacterium tuberculosis by the extrusion of toxic substrates and drugs from the inner bacillus. This study aimed to investigate the in vitro interaction of Verapamil (VP), an efflux pump inhibitor, with the classical first-line anti-tuberculosis drug isoniazid (INH) in resistant and susceptible M. tuberculosis clinical isolates. Seven multidrug-resistant (MDR), three INH monoresistant and four susceptible M. tuberculosis clinical isolates were tested for the INH and VP combination by modified Resazurin Microtiter Assay Plate (REMA). Fractional Inhibitory Concentration (FIC) and Modulation Factor (MF) were determined. The INH plus VP combination showed no significant change in the Minimum inhibitory concentration (MIC) values of INH (FIC≥ 0.5; MF=1 or 2).The use of VP in tuberculosis therapy should be managed carefully, considering the resistance caused by specific mutation in katG and inhA genes, in which the use of these EPIs may have no success. The use of EPIs as an adjunctive drug in the anti-tuberculosis therapy should be further investigated on a larger number of M. tuberculosis clinical isolates with different resistant profile.

Perfil de tolerancia al triclosán y detección de los genes MexA, MexC, AcrB y oqxA relacionados con la expresión de bombas de expulsión en aislados clínicos del género Enterobacter aerogenes y Enterobacter cloacae / Triclosan tolerance profile and detection of MexA, MexC, AcrB and oqxA genes related to the expression of expulsion pumps in clinical isolates of Enterobacter aero- genes and Enterobacter cloacae / Perfil de tolerância ao triclosan e detecção dos genes mexA, mexC, acrB e oqxA relacionados à expressão de bombas de expulsão em isolados clínicos do gênero Enterobacter aerogenes e Enterobacter cloacae

Introducción. La resistencia a los antimicrobianos y la tolerancia a biocidas está dada por mecanismos comunes, generados por su uso en diferentes ambientes; mecanismos como la expresión de bombas de expulsión presentes en bacterias del género Enterobacter circulantes amenaza la eficacia de los antimicrobianos limitando las opciones de terapia antibiótica. Objetivos: Determinar el perfil de tolerancia al triclosán y detección de genes asociados a bombas de expulsión en aislados clínicos de Enterobacter aerogenes y Enterobacter cloacae. Materiales y Métodos: Se realizó un estudio observacional, descriptivo y de corte transversal, se determinaron perfiles de tolerancia al triclosán por microdilución, de susceptibilidad antimicrobiana, confirmación fenotípica de mecanismos de resistencia, por reacción en cadena de la polimerasa, se identificó la presencia de genes que codifican para bombas de expulsión. Resultados: El 17% correspondió a Enterobacter cloacae y el 6% Enterobacter aerogenes. El 93,7% de los aislados clínicos del género Enterobacter presentó el fenotipo de resistencia BLEE y AmpC. En el 81,3% de los aislamientos se obtuvo la presencia de al menos un gen relacionado con las expresión de bombas de expulsión, siendo frecuentes MexC y AcrB; no identificó presencia del gen oqxA. Conclusiones: La resistencia a diferentes grupos de antibióticos se identifica en especies de Enterobacter circulante, así la presencia de enzimas BLEE y AmpC, la presencia de genes relacionados con bombas de expulsión y la alta tolerancia al triclosán. Palabras clave: Triclosán, Resistencia, Bombas de expulsión, Genes, Biocida

Introduction. Antimicrobial resistance and tolerance to biocides is given by common mechanisms, generated by the use of antimicrobial and biocidal substances in different environments, these me- chanisms such as the expression of expulsion pumps present in bacteria of the Enterobacter genus circulating threatens the efficacy of antimicrobials by limiting antibiotic therapy options. Objective: to determine the triclosan tolerance profile and detection of genes associated with expul- sion pumps in clinical isolates of Enterobacter aerogenes and Enterobacter cloacae. Materials and Methods: An observational, descriptive and the cross-sectional study was performed, triclosan tolerance profiles were determined by microdilution, antimicrobial susceptibility, phenotypic confirmation of resistance mechanisms, by the presence of polymerase chain reaction, the presence of genes that code for expulsion pumps. Results: The 17% corresponded to Enterobacter cloacae and 6% Enterobacter aerogenes. 93.7% of the clinical isolates of the genus Enterobacter presented the ESBL and AmpC resistance phenotype. In 81.3% of the isolates, the presence of at least one gene related to the expression of ejection pumps was obtained, with MexC and AcrB being frequent; did not identify the presence of the oqxA gene. conclusions: The resistance to different groups of antibiotics is identified in circulating Enterobacter species, as well as the presence of ESBL and AmpC enzymes, the presence of genes related to ejection pumps, and high tolerance to triclosan.

Introdução.A resistência antimicrobiana e a tolerância a biocidas esta dada pelos mecanismos comuns gerados pelo uso em diferentes ambientes; mecanismos como a expressão de bombas de expulsão presentes em bactérias do gênero Enterobacter circulantes ameaza a eficácia das antimicrobiana limitando as opções de terapia antibiótica. Objetivos: Determinar o perfil de tolerância ao triclosan e detecção dos genes asociados a bombas de expulsão em isolados clínicos Enterobacter aerogenes e Enterobacter cloacae. Materiais e Métodos: Realizou-se um estudo observacional, descritivo e de corte transversal, deter- minaram-se perfiles de tolerância ao triclosan por microdiluição, de susceptibilidade antimicrobiana, confirmação de mecanismos de resistência fenotípica por reação em cadeia da polimerase, identifi- cou-se a presença de genes que codificam para bombas de expulsão. Resultados: 17% correspondeu ao Enterobacter cloacae e 6% ao Enterobacter aerogenes. 93,7% em isolados clínicos do gênero Enterobacter presentou o fenótipo de resistência BLEE e AmpC. No 81% dos isolamentos se obteve a presença de pelo menos um gen relacionado à expressão de bombas de expulsão, sindo frequentes mexC e acrB; não se identificou a presença do gen oqxA. Conclusões: A resistência de diferentes grupos de antibióticos se identificou em espécies de Entero- bacter circulante, assim a presença de enzimas BLEE e AmpC, a presença de genes relacionados com bombas de expulsão e a alta tolerância ao triclosan.

Effect of Tanreqing Injection and Antibiotics on Multi-drug Resistant Pseudomonas aeruginosa Efflux Pump / 中国实验方剂学杂志

Objective:To study the interaction between Tanreqing injection and commonly used antibiotics against multi-drug resistant Pseudomonas aeruginosa (MDR-PA) and the effect on bacterial efflux pump. Method:Antibiotic susceptibility test was performed with bacteria. Paper diffusion method (Kirby-Bauer, KB) combined with efflux pump inhibitor (50 mg·L-1) was used to measure the diameter of the inhibition zone, and real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) was used to detect gene expression of efflux pump Positive efflux pump strain. KB method was used to observe the changes of Tanreqing (final concentration 3 g·L-1) and antibiotics on the diameter of the zone of inhibition. Strains were co-cultured with Tanreqing and antibiotic sub-inhibitory concentrations for Real-time PCR detection. KB method was used to observe the effect of Tanreqing on the diameter of bacteriostatic ring after the continuous use of efflux pump-positive bacteria. Result:Two MDR-PA efflux pump-positive strains were identified and screened. Tanreqing has synergistic antibacterial effect with aloxicillin, aztreonam, meropenem, ceftazidime, cefoperazone, and Shupushen. In inhibiting the expression levels of bacterial efflux pump genes, the four drugs were compared by the effect: cefoperazone>Tanreqing>ceftazidime>Shupushen. After Tanreqing continued to act on efflux pump-positive strains, it could have a better effect in combination with ceftazidime, cefoperazone, and Shupushen. Conclusion:Tanreqing, ceftazidime, cefoperazone, and Shupushen can reduce the drug resistance of bacteria by down-regulating the expressions of bacterial efflux pump genes, and reducing the clinical dose of antibiotics, and thus play a bacteriostatic effect.

Detection of efflux pump CmeABC in enrofloxacin resistant Campylobacter spp. strains isolated from broiler chickens (Gallus gallus domesticus) in the state of Rio de Janeiro, Brazil / Detecção da bomba de efluxo CmeABC em cepas de Campylobacter spp. resistentes à enrofloxacina isoladas de frangos de corte (Gallus gallus domesticus) no Estado do Rio de Janeiro, Brasil

Fowls are the main reservoirs of the highly important food-originating pathogen called Campylobacter spp. and broilers' meat and byproducts are the main vehicles of this microorganism. Increasing of Campylobacter spp. resistant strains to fluorquinolones, an antimicrobial class often employed in poultry farming and in human medicine has become a great concern to poultry breeders. In fact, several studies evaluated increasing bacterial resistance against these antimicrobial agents. The role of CmeABC efflux system has been underscored among the resistance mechanisms in Campylobacter spp. to fluorquinolones. This study investigated the occurrence of CmeABC efflux pump in 81 and 78 enrofloxacin resistant strains of Campylobacter jejuni and C. coli respectively, isolated from broilers collected from six abattoirs situated at São José do Vale do Rio Preto/RJ poultry center and from two commercial abattoirs situated at Metropolitan Region of Rio de Janeiro, from 2013 to 2016. The resistance to enrofloxacin was assessed by agar dilution to determine minimum inhibitory concentration (MIC). The CmeABC efflux system was investigated through the detection of genes genes cmeA, cmeB and cmeC by PCR. The activity of CmeABC efflux pump was investigated in 20 strains by using the efflux pump inhibitor Phenylalanine-Arginine ß-Naphthylamide (PAßN). The three genes cmeA, cmeB and cmeC were detected in 94.3% of the strains (C. jejuni = 80 and C. coli = 70), whereas the system was absent or incomplete in 5.7% of strains (C. jejuni = 1 and C. coli = 8). MIC varied between 0.5µg/ml and 64µg/ml, and 88.7% of strains were enrofloxacin resistant and 11.3% featuring intermediate resistance. The inhibition of the efflux pump by PAßN reduced the MIC to enrofloxacin up to eight times in fifteen strains (75%). These results indicate that this system is frequent and active in Campylobacter spp. Resistant strains in the presence of enrofloxacin.(AU)

As aves são os principais reservatórios de Campylobacter spp., importante patógeno de origem alimentar e a carne de frango e produtos derivados são os principais veículos desse microrganismo. O aumento de cepas de Campylobacter spp. resistentes às fluorquinolonas, uma classe antimicrobiana frequentemente empregada na avicultura e na medicina humana, tornou-se uma grande preocupação para os produtores de aves e vários estudos avaliaram o aumento da resistência bacteriana a esses antimicrobianos. O papel do sistema de efluxo CmeABC tem sido enfatizado entre os mecanismos de resistência em Campylobacter spp. à fluorquinolonas. O presente estudo investigou a ocorrência da bomba de efluxo CmeABC em 81 cepas de Campylobacter jejuni e 78 cepas de Campylobacter coli resistentes à enrofloxacina, isoladas de frangos de corte coletados em seis abatedouros situados no polo avícola de São José do Rio Preto/RJ e de dois abatedouros comerciais situados na Região Metropolitana do Rio de Janeiro, de 2013 a 2016. A resistência à enrofloxacina foi avaliada pelo método de diluição em ágar para determinar a concentração inibitória mínima (CIM). O sistema de efluxo CmeABC foi investigado através da detecção dos genes cmeA, cmeB e cmeC por PCR. A atividade da bomba de efluxo CmeABC foi investigada em 20 cepas utilizando o inibidor da bomba de efluxo Phenylalanine-Arginine ß-Naftilamida (PAßN). Os três genes cmeA, cmeB e cmeC foram detectados em 94,3% das cepas (C. jejuni = 80 e C. coli = 70), enquanto o sistema estava ausente ou incompleto em 5,7% das cepas (C. jejuni = 1 e C coli = 8). A CIM variou entre 0,5µg/ml e 64µg/ml e 88,7% das cepas foram resistentes à enrofloxacina, enquanto 11,3% apresentaram resistência intermediária. A inibição da bomba de efluxo pelo PAßN reduziu a CIM da enrofloxacina até oito vezes em quinze cepas (75%). Estes resultados indicam que este sistema é frequente e ativo em cepas resistentes de Campylobacter spp. na presença de enrofloxacina.(AU)