EtBr Efflux Research Articles

A novel member of drug/metabolite transporter (DMT) family efflux pump, SA00565, contributes to tetracycline antibiotics resistance in Staphylococcus aureus USA300.

Drug efflux systems have recently been recognized as a significant mechanism responsible for multidrug resistance in bacteria. In this study, we described the identification and characterization of a new chromosomally encoded efflux pump (SA00565) in Staphylococcus aureus. SA00565, which belongs to the drug/metabolite transporter (DMT) superfamily, was predicted to be a 10-transmembrane segment transporter. To evaluate the role of sa00565 in resistance, we generated sa00565 gene deletion mutant (Δsa00565) and assessed its susceptibility to 35 different antibiotic treatments. Our results demonstrated that the Δsa00565 mutant exhibited reduced resistance to tetracycline and doxycycline, with 64-fold and 12-fold decreased MICs, respectively. The mechanism of SA00565-mediated tetracycline resistance was demonstrated that SA00565 possesses the capability to efficiently extrud intracellular tetracycline into the environment. The efflux activity of SA00565 was further validated using EtBr accumulation and efflux assays. In summary, our study uncovered a previously unknown function of a DMT family transporter, which serves as a tetracycline efflux pump, thereby contributing to tetracycline resistance in S. aureus.IMPORTANCEIn this study, we addressed the significance of drug efflux systems in multidrug resistance of Staphylococcus aureus, focusing on the unexplored efflux pump SA00565 in the drug/metabolite transporter (DMT) superfamily. Through phylogenetic analysis, gene knockout, and overexpression experiments, we identified the role of SA00565 in antibiotic resistance. The Δsa00565 mutant showed increased susceptibility to tetracycline and doxycycline in disk diffusion assays, with significantly lower MICs compared to the WT. Remarkably, intracellular tetracycline concentration in the mutant was two- to threefold higher, indicating SA00565 actively eliminates intracellular tetracycline. Our findings emphasize the pivotal contribution of SA00565 to tetracycline antibiotic resistance in S. aureus, shedding light on its functional attributes within the DMT superfamily and providing valuable insights for combating multidrug resistance.

Enhancing bactericidal activities of ciprofloxacin by targeting the trans-translation system that is involved in stress responses in Klebsiella pneumoniae.

Although the trans-translation system is a promising target for antcibiotic development, its antibacterial mechanism in Klebsiella pneumoniae (KP) is unclear. Considering that tmRNA was the core component of trans-translation, this study firstly investigated phenotypic changes caused by various environmental stresses in KP lacking trans-translation activities (tmRNA-deleted), and then aimed to evaluate antibacterial activities of the trans-translation-targeting antibiotic combination (tobramycin/ciprofloxacin) in clinical KP isolates based on inhibition activities of aminoglycosides against trans-translation. We found that the tmRNA-deleted strain P4325/ΔssrA was significantly more susceptible than the wild-type KP strain P4325 under environments with hypertonicity (0.5 and 1M NaCl), hydrogen peroxide (40mM), and UV irradiation. No significant differences in biofilm formation and survivals under human serum were observed between P4325/ΔssrA and P4325. tmRNA deletion caused twofold lower MIC values for aminoglycosides. As for the membrane permeability, tmRNA deletion increased ethidium bromide (EtBr) uptake of KP in the presence or absence of verapamil and carbonyl cyanide-m-chlorophenylhydrazone (CCCP), decreased EtBr uptake in presence of reserpine in P4325/ΔssrA, and reduced EtBr efflux in P4325/ΔssrA in the presence of CCCP. The time-kill curve and in vitro experiments revealed significant bactericidal activities of the tmRNA-targeting aminoglycoside-based antibiotic combination (tobramycin/ciprofloxacin). Thus, the corresponding tmRNA-targeting antibiotic combinations (aminoglycoside-based) might be effective and promising treatment options against multi-drug resistant KP.

Modulatory Effect of Croton heliotropiifolius Kunth Ethanolic Extract on Norfloxacin Resistance in Staphylococcus aureus

The high frequency of infectious diseases has spurred research into effective tactics to combat microorganisms that are resistant to several drugs. The overproduction of the transmembrane efflux pump protein NorA, which may export hydrophilic fluoroquinolones, is a common mechanism of resistance in S. aureus strains. This work evaluated the antimicrobial activity of the ethanolic extract from the leaves of Croton heliotropiifolius (EECH) against different bacterial and fungal strains, as well as investigating its modulating effect on the resistance to norfloxacin in a Staphylococcus aureus SA1199B overproducing the NorA efflux pump. Microdilution assays were used to assess the EECH’s antibacterial efficacy. The MIC of norfloxacin or ethidium bromide (EtBr) against the SA1199B strain was determined in the presence or absence of the EECH in order to assess the modifying influence on drug resistance. The EECH showed no activity against the Gram-positive and Gram-negative bacterial strains tested. The EECH also showed no antifungal activity against Candida albicans ATCC 10231. On the other hand, the extract reduced the MIC values for norfloxacin against SA1199B at subinhibitory concentrations. In addition, the EECH also reduced the MIC values of EtBr at subinhibitory concentrations, suggesting the occurrence of phytochemicals that inhibit efflux pumps. Molecular docking showed that retusin, a flavonoid found in the extract, could compete with norfloxacin at the orthosteric site of the NorA, indicating that it could be a potential efflux pump inhibitor. However, isolated retusin did not enhance the activity of norfloxacin or EtBr and it did not inhibit the EtBr efflux, showing that it is not a NorA inhibitor. Even though C. heliotropiifolius is a source of phytochemicals that function as adjuvants for norfloxacin, isolated retusin cannot be used in conjunction with norfloxacin to treat infections brought on by S. aureus that overproduces NorA.

Inhibition of Staphylococcus pseudintermedius Efflux Pumps by Using Staphylococcus aureus NorA Efflux Pump Inhibitors.

One promising approach in treating antibiotic-resistant bacteria is to "break" resistances connected with antibacterial efflux by co-administering efflux pump inhibitors (EPIs) with antibiotics. Here, ten compounds, previously optimized to restore the susceptibility to ciprofloxacin (CIP) of norA-overexpressing Staphylococcus aureus, were evaluated for their ability to inhibit norA-mediated efflux in Staphylococcus pseudintermedius and synergize with CIP, ethidium bromide (EtBr), gentamycin (GEN), and chlorhexidine digluconate (CHX). We focused efforts on S. pseudintermedius as a pathogenic bacterium of concern within veterinary and human medicine. By combining data from checkerboard assays and EtBr efflux inhibition experiments, the hits 2-arylquinoline 1, dihydropyridine 6, and 2-phenyl-4-carboxy-quinoline 8 were considered the best EPIs for S. pseudintermedius. Overall, most of the compounds, except for 2-arylquinoline compound 2, were able to fully restore the susceptibility of S. pseudintermedius to CIP and synergize with GEN as well, while the synergistic effect with CHX was less significant and often did not show a dose-dependent effect. These are valuable data for medicinal chemistry optimization of EPIs for S. pseudintermedius and lay the foundation for further studies on successful EPIs to treat staphylococcal infections.

Acetylenic spiroketal enol ethers from Artemisia rupestris and their synergistic antibacterial effects on methicillin-resistant Staphylococcus aureus

Synergistic bioassay-guided isolation of the extracts of Artemisia rupestris L, which belongs to the family Asteraceae, afforded two acetylenic spiroketal enol ethers, namely rupesdiynes A (1) and B (2). Their structures were determined based on spectroscopic analysis and experimental and calculated ECD investigations. The two compounds exhibited synergistic activity and were able to reduce the minimum inhibitory concentration (MIC) of oxacillin four-fold, with a fractional inhibitory concentration index (FICI) of 0.5 in combination with oxacillin against the oxacillin-resistant EMRSA-16. Biofilm formation inhibitory and Ethidium bromide (EtBr) efflux assay were further employed to verify the possible mechanism of the synergistic antibacterial effect. Additionally, molecular docking studies were conducted to investigate the binding affinities of the two compounds with penicillin-binding protein 2a (PBP2a) of EMRSA-16. Taken together, rupesdiynes A (1) and rupesdiyne B (2) showed moderate synergistic activity against EMRSA-16 with oxacillin via inhibiting biofilm formation and efflux pump activity, respectively.

Inhibition of efflux pump encoding genes and biofilm formation by sub-lethal photodynamic therapy in methicillin susceptible and resistant Staphylococcus aureus

BackgroundPhotodynamic therapy (PDT) is an effective method to inactivate microorganisms which is based on reactive oxygen species (ROS) generated by photosensitizer and light at certain wavelength. Exposure to sub-lethal dose of PDT (sPDT) could activate the regulatory systems in the surviving bacteria in response to oxidative stress. This study aimed to evaluate the effect of sPDT on efflux pump and biofilm formation in Staphylococcus aureus (S. aureus), which are two important virulence related factors. MethodsDifferent light irradiation time and toluidine blue O (TBO) concentrations were tested to select a sPDT in methicillin-susceptible and methicillin-resistant S. aureus (MSSA and MRSA). Efflux function was evaluated with EtBr efflux experiment. Biofilm formation was evaluated by crystal violet staining. Gene expressions of norA, norB, sepA, mepA and mdeA following sPDT were analyzed with real-time PCR. ResultsSub-lethal PDT was set at 40 J/cm2 associated with 0.5 μM TBO. Efflux function was significantly inhibited in both strains. The average expression levels of mdeA and mepA in MSSA and MRSA were increased by (3.09, 1.77, 1.57) and (3,44, 1.59, 6.29) fold change respectively, norB and sepA were decreased by (3.77, 6.14) and (3.02, 3.47) fold change respectively. Expression level of norA was decreased by 5.44-fold change in MSSA but increased by 2.80-fold change in MRSA. Biofilm formation in both strains was impeded. ConclusionsTBO-mediated sPDT could inhibit efflux pump function, alter efflux pump encoding gene expression levels and retard biofilm formation in MSSA and MRSA. Therefore, sPDT is proposed as a potential adjuvant therapy for infections.

A Novel Tigecycline Adjuvant ML-7 Reverses the Susceptibility of Tigecycline-Resistant Klebsiella pneumoniae.

The increasing incidence of tigecycline resistance undoubtedly constitutes a serious threat to global public health. The combination therapies had become the indispensable strategy against this threat. Herein, 11 clinical tigecycline-resistant Klebsiella pneumoniae which mainly has mutations in ramR, acrR, or macB were collected for tigecycline adjuvant screening. Interestingly, ML-7 hydrochloride (ML-7) dramatically potentiated tigecycline activity. We further picked up five analogs of ML-7 and evaluated their synergistic activities with tigecycline by using checkerboard assay. The results revealed that ML-7 showed certain synergy with tigecycline, while other analogs exerted attenuated synergistic effects among tigecycline-resistant isolates. Thus, ML-7 was selected for further investigation. The results from growth curves showed that ML-7 combined with tigecycline could completely inhibit the growth of bacteria, and the time-kill analysis revealed that the combination exhibited synergistic bactericidal activities for tigecycline-resistant isolates during 24 h. The ethidium bromide (EtBr) efflux assay demonstrated that ML-7 could inhibit the functions of efflux pump. Besides, ML-7 disrupted the proton motive force (PMF) via increasing ΔpH, which in turn lead to the inhibition of the functions of efflux pump, reduction of intracellular ATP levels, as well as accumulation of ROS. All of which promoted the death of bacteria. And further transcriptomic analysis revealed that genes related to the mechanism of ML-7 mainly enriched in ABC transporters. Taken together, these results revealed the potential of ML-7 as a novel tigecycline adjuvant to circumvent tigecycline-resistant Klebsiella pneumoniae.

Role of medicinal plants from North Western Himalayas as an efflux pump inhibitor against MDR AcrAB-TolC Salmonella enterica serovar typhimurium: In vitro and In silico studies

Ethnopharmacological relevanceZingiber officinale Roscoe has been utilized traditionally to cure various diseases like cold, cough, diarrhoea, nausea, asthma, vomiting, toothache, stomach upset, respiratory disorders, joint pain, and throat infection. It is also consumed as spices and ginger tea. Aim of the studyThe current study was aimed to identify the phytocompounds of traditional medicinal plants of North-Western Himalaya that could inhibit the AcrAB-TolC efflux pump activity of Salmonella typhimurium and become sensitive to antibiotic killing at reduced dosage. Material and methodsMedicinal plant extracts were prepared using methanol, aqueous, and ethyl acetate and tested for efflux pump inhibitory activity of Salmonella typhimurium NKS70, NKS174, and NKS773 strains using Ethidium Bromide (EtBr)-agar cartwheel assay. Synergism was assessed by the agar well diffusion method and EPI activity by berberine uptake and EtBr efflux inhibition assays. Microdilution method and checkerboard assays were done to determine the minimum inhibitory concentration (MIC) and fractional inhibitory concentration index (FICI) respectively for a bioactive compound. To validate the phytocompound and efflux pump interaction, molecular docking with 6IE8 (RamA) and 6IE9 (RamR) targets was done using autoDock vina software. Toxicity prediction and drug-likeness were predicted by using ProTox-II and Molinspiration respectively. ResultsMethanolic and ethyl acetate extracts of P. integerrima, O. sanctum, C. asiatica, M. charantia, Z. officinale, and W. somnifera in combination with ciprofloxacin and tetracycline showed synergistic antimicrobial activity with GIIs of 0.61–1.32 and GIIs 0.56–1.35 respectively. Methanolic extract of Z. officinal enhanced the antimicrobial potency of berberine (2 to 4-folds) and increased the EtBr accumulation. Furthermore, bioassay-guided fractionation leads to the identification of lariciresinol in ethyl acetate fraction, which decreased the MIC by 2-to 4-folds. The ΣFIC values varied from 0.30 to 0.55 with tetracycline, that indicated synergistic/additive effects. Lariciresinol also showed a good binding affinity with 6IE8 (−7.4 kcal mol−1) and 6IE9 (−8.2 kcal mol−1), which is comparable to tetracycline and chenodeoxycholic acid. Lariciresinol followed Lipinski's rule of five. ConclusionThe data suggest that lariciresinol from Z. officinale could be a potential efflux pump inhibitor that could lead to effective killing of drug resistant Salmonella typhimurium at lower MIC. Molecular docking confirmed the antibacterial EPI mechanism of lariciresinol in Salmonella typhimurium and confirmed to be safe for future use.

Acidic amino acids as counterions of ciprofloxacin: Effect on growth and pigment production in Staphylococcus aureus NCTC 8325 and Pseudomonas aeruginosa PAO1.

Antimicrobial resistance (AMR) is emerging as a global threat to public health. One of the strategies employed to combat AMR is the use of adjuvants which act to enhance or reinstate antimicrobial activity by inhibiting resistance mechanisms. However, these adjuvants are themselves not immune to selecting resistant phenotypes. Thus, there is a need to utilise mechanisms which are either less likely to or unable to trigger resistance. One commonly employed mechanism of resistance by microorganisms is to prevent antimicrobial uptake or efflux the antibiotic which manages to permeate its membrane. Here we propose amino acids as antimicrobial adjuvants that may be utilizing alternate mechanisms to fight AMR. We used a modified ethidium bromide (EtBr) efflux assay to determine its efflux in the presence of ciprofloxacin within Staphylococcus aureus (NCTC 8325) and Pseudomonas aeruginosa (PAO1). In this study, aspartic acid and glutamic acid were found to inhibit growth of both bacterial species. Moreover, a reduced production of toxic pigments, pyocyanin and pyoverdine by P. aeruginosa was also observed. As evident from similar findings with tetracycline, these adjuvants, may be a way forward towards tackling antimicrobial resistance.

Ethidium bromide exposure unmasks an antibiotic efflux system in Rhodococcus equi.

This study introduces a newly created strain (Rhodococcus equiEtBr25) by exposing R. equi ATCC 33701 to ethidium bromide (EtBr), a substrate for MDR transporters. Such an approach allowed us to investigate the resulting phenotype and genetic mechanisms underlying the efflux-mediated resistance in R. equi. R. equi ATCC 33701 was stimulated with increasing concentrations of EtBr. The antimicrobial susceptibility of the parental strain and R. equiEtBr25 was investigated in the presence/absence of efflux pump inhibitors (EPIs). EtBr efflux was evaluated by EtBr-agar method and flow cytometry. The presence of efflux pump genes was determined by conventional PCR before to quantify the expression of 30 genes coding for membrane transporters by qPCR. The presence of erm(46) and mutations in 23S rRNA, and gyrA/gyrB was assessed by PCR and DNA sequencing to exclude the occurrence of resistance mechanisms other than efflux. R. equi EtBr25 showed an increased EtBr efflux. Against this strain, the activity of EtBr, azithromycin and ciprofloxacin was more affected than that of rifampicin and azithromycin/rifampicin combinations. Resistances were reversed by combining the antimicrobials with EPIs. Gene expression analysis detected a marked up-regulation of REQ_RS13460 encoding for a Major Facilitator Superfamily (MFS) transporter. G→A transition occurred in the transcriptional repressor tetR/acrR adjacent to REQ_RS13460. Exposure of R. equi to EtBr unmasked an efflux-mediated defence against azithromycin and ciprofloxacin, which seemingly correlates with the overexpression of a specific MFS transporter. This genotype may mirror an insidious low-level resistance of clinically important isolates that could be countered by EPI-based therapies.

Neolignans from Piper betle Have Synergistic Activity against Antibiotic-Resistant Staphylococcus aureus

A phytochemical investigation of an extract of the leaves of Piper betle, guided by a synergistic antibacterial screen, led to the isolation and structural elucidation of 10 new neolignans, Pibeneolignan A-J (1-10), together with 11 known compounds. The structures and absolute configurations of the new compounds were elucidated on the basis of spectroscopic data, single-crystal X-ray diffraction analysis, and experimental and calculated ECD investigations. Compounds 1 and 2 are new naturally occurring neolignan skeletons, based on the cyclohept-2-ene-1,4-dione framework. We propose that these natural products are biosynthetically formed from bicyclic [3.2.1] neolignans by oxidative cleavage and ring opening at C-1' and C-2'. Among these compounds, 9, 13, 15, and 16, in combination with norfloxacin against an effluxing S. aureus strain (SA1199B), exhibited significant synergistic activity with fractional inhibitory concentration indices (FICIs) of 0.078, 0.156, 0.125, and 0.25, respectively. Bacterial growth curves, ethidium bromide (EtBr) efflux, and qRt-PCR were further employed to verify their synergistic antibacterial mechanism. Furthermore, computational molecular modeling suggested the binding of compounds 14-17 and 19 to the active site of the modeled structure of the NorA efflux pump, which is the main efflux pump in SA1199B.

Evidence of Calcium Signaling and Modulation of the LmrS Multidrug Resistant Efflux Pump Activity by Ca2 + Ions in S. aureus.

Calcium ions (Ca2+) play a pivotal role in eukaryote cell signaling and regulate many physiological functions. Although a similar role for Ca2+ in prokaryotes has been difficult to demonstrate, there is increasing evidence for Ca2+ as a cell regulator in bacteria. The purpose of this study was to investigate Ca2+ signaling and the effect of Ca2+ on the Staphylococcus aureus multidrug resistant efflux pump LmrS. We hypothesized that antibiotics act by increasing Ca2+ concentrations, which in turn enhance the efflux activity of LmrS. These Ca2+ transients were measured by luminometry in response to various antibiotics by using the photoprotein aequorin reconstituted within live bacterial cells. Efflux associated with LmrS was measured by the increase in fluorescence due to the loss of ethidium bromide (EtBr) from both S. aureus cells and from E. coli cells in which the lmrs gene of S. aureus was expressed. We found that addition of antibiotics to cells generated unique cytosolic Ca2+ transients and that addition of CaCl2 to cells enhanced EtBr efflux whereas addition of Ca2+ chelators or efflux pump inhibitors significantly decreased EtBr efflux from cells. We conclude that antibiotics induce a Ca2+ mediated response through transients in cytosolic Ca2+, which then stimulates LmrS efflux pump.

Modulation of the Drug Resistance by Platonia insignis Mart. Extract, Ethyl Acetate Fraction and Morelloflavone/Volkensiflavone (Biflavonoids) in Staphylococcus aureus Strains Overexpressing Efflux Pump Genes.

Microbial resistance to antibiotics is a global public health problem, which requires urgent attention. Platonia insignis is a native species from the eastern Brazilian Amazon, used in the treatment of burns and wounds. To evaluate the antimicrobial activity of the hydroalcoholic extract of P. insignis (PIHA), the ethyl acetate fraction (PIAE), and its subfraction containing a mixture of biflavonoids (BF). Moreover, the effect of these natural products on the antibiotic activity against S. aureus strains overexpressing efflux pump genes was also evaluated. Minimal inhibitory concentrations were determined against different species of microorganisms. To evaluate the modulatory effect on the Norfloxacin-resistance, the MIC of this antibiotic was determined in the absence and presence of the natural products at subinhibitory concentrations. Inhibition of the EtBr efflux assays were conducted in the absence or presence of natural products. PIHA showed a microbicidal effect against S. aureus and C. albicans, while PIAE was bacteriostatic for S. aureus. PIAE and BF at subinhibitory concentrations were able to reduce the MIC of Norfloxacin acting as modulating agents. BF was able to inhibit the efflux of EtBr efflux in S. aureus strains overexpressing specific efflux pump genes. P. inignisis, a source of efflux pump inhibitors, including volkensiflavone and morelloflavone, which were able to potentiate the Norfloxacin activity by NorA inhibition, being also able to inhibit QacA/B, TetK and MsrA. Volkensiflavone and morelloflavone could be used as an adjuvant in the antibiotic therapy of multidrug resistant S. aureus strains overexpressing efflux pumps.

Synthesis and Evaluation of 2-Aminothiophene Derivatives as Staphylococcus aureus Efflux Pump Inhibitors.

2-aminothiophene derivatives (2AT) in which the thiophene ring is fused with a cycloalkyl or a N-acylated piperidine ring by positions 5 and 6 and carrying a 3-carbethoxy group were synthesized and their bacterial growth and enzyme inhibitory effects against efflux proteins of Staphylococcus aureus leading to resistance to fluoroquinolones and erythromycin (ERY) were investigated. Compounds that most effectively decreases the minimum inhibitory concentrations (MICs) of ciprofloxacin (CIP) were assayed for their dose and time effects on the accumulation and efflux of ethidium bromide (EtBr) in the SA-1 strain. None of the compounds displayed antibacterial activity however, three derivatives carrying 2-amino, 2-aminoacetyl and 2-aminotrifluoroacetyl group enhanced the activity of CIP and ERY by 8- and 16-fold, respectively, and were able to restore the sensitivity of resistant strains, acting as typical efflux pump inhibitors (EPIs). The 2-aminoacetyl and 2-aminotrifluoroacetyl derivatives and two other piperidinyl 2-aminotrifluoroacetyl derivatives increased EtBr accumulation in a dose- and time-dependent manner, and one of them was also able to inhibit the EtBr efflux. Taken together, these results represent an important advance in the development of new EPIs, and demonstrate that 2AT represent a good scaffold for developing new antibiotic adjuvants.

Antimicrobial activity and inhibition of the NorA efflux pump of Staphylococcus aureus by extract and isolated compounds from Arrabidaea brachypoda

Arrabidaea brachypoda is a native shrub of the Brazilian Cerrado widely used in the folk medicine for treatment of renal diseases and articular pains. This study aimed to, first, evaluate the antimicrobial activity of both extracts and isolated molecules Brachydins BR-A and BR-B obtained from the flowers of A. brachypoda against Staphylococcus aureus, Escherchia coli and Candida albicans species. A second objective was to investigate if these natural products were able to potentiate the Norfloxacin activity against the strain Staphylococcus aureus SA1199-B that overexpress the norA gene encoding the NorA efflux pump. Extracts and isolated compounds were analyzed by HPLC-PDA and LC-ESI-MS respectively. Minimal inhibitory concentrations of Norfloxacin or Ethidium Bromide (EtBr) were determined in the presence or absence of ethanolic extract, dichloromethane fraction, as well as BR-A or BR-B by microdilution method. Only BR-B showed activity against Candida albicans. Addition of ethanolic extract, dichloromethane fraction or BR-B to the growth media at sub-inhibitory concentrations enhanced the activity of both Norfloxacin and EtBr against S. aureus SA1199-B, indicating that these natural products and its isolated compound BR-B were able to modulate the fluoroquinolone-resistance possibly by inhibition of NorA. Moreover, BR-B inhibited the EtBr efflux in the SA1199-B strain confirming that it is a NorA inhibitor. Isolated BR-B was able to inhibit an important mechanism of multidrug-resistance very prevalent in S. aureus strains, thus its use in combination with Norfloxacin could be considered as an alternative for the treatment of infections caused by S. aureus strains overexpressing norA.

Baicalin Inhibits Biofilm Formation and the Quorum-Sensing System by Regulating the MsrA Drug Efflux Pump in Staphylococcus saprophyticus.

Staphylococcus saprophyticus (S. saprophyticus) is one of the main pathogens that cause serious infection due to its acquisition of antibiotic resistance. The efflux pump decreases antibiotic abundance, and biofilm compromises the penetration of antibiotics. It has been reported that baicalin is a potential agent to inhibit efflux pumps, biofilm formation, and quorum-sensing systems. The purpose of this study was to investigate whether baicalin can inhibit S. saprophyticus biofilm formation and the quorum-sensing system by inhibiting the MsrA efflux pump. First, the mechanism of baicalin inhibiting efflux was investigated by the ethidium bromide (EtBr) efflux assay, measurement of ATP content, and pyruvate kinase (PK) activities. These results revealed that baicalin significantly reduced the efflux of EtBr, the ATP content, and the activity of PK. Moreover, its role in biofilm formation and the agr system was studied by crystal violet staining, confocal laser scanning microscopy, scanning electron microscopy, and real-time polymerase chain reaction. These results showed that baicalin decreased biofilm formation, inhibited bacterial aggregation, and downregulated mRNA transcription levels of the quorum-sensing system regulators agrA, agrC, RNAIII, and sarA. Correlation analysis indicated that there was a strong positive correlation between the efflux pump and biofilm formation and the agr system. We demonstrate for the first time that baicalin inhibits biofilm formation and the agr quorum-sensing system by inhibiting the efflux pump in S. saprophyticus. Therefore, baicalin is a potential therapeutic agent for S. saprophyticus biofilm-associated infections.

Flavonoids from Artemisia rupestris and their synergistic antibacterial effects on drug-resistant Staphylococcus aureus

This study seeks to discover flavonoids from a traditional Chinese herb, Artemisia rupestris L., with synergistic antibacterial effects against multidrug-resistant Staphylococcus aureus. Five flavonoids, artemetin (1), chrysosplenetin (2), pachypodol (3), penduletin (4) and chrysoeriol (5) were obtained by various column chromatographic methods. Their chemical structures were determined on the basis of comprehensive spectroscopic analysis and comparison with literature data. Three of the compounds (2, 4 and 5) exhibited synergistic activity when combined with norfloxacin against SA1199B, an effluxing fluoroquinolone-resistant strain. The fractional inhibitory concentration indices (FICIs) of 2, 4 and 5 in combination with norfloxacin were 0.375, 0.079 and 0.266 respectively, suggesting synergy. Compound 5 also showed synergistic effects against EMRSA-15 and EMRSA-16 when combined with ciprofloxacin and oxacillin exhibiting FICIs of 0.024 and 0.375 respectively. Real time ethidium bromide (EtBr) efflux assay, qRT-PCR and molecular docking were employed to explore the mechanisms of the synergistic effects.

A structure–activity relationship study of phenyl sesquiterpenoids on efflux inhibition against Staphylococcus aureus

Sixteen natural acetophenone sesquiterpene derivatives were isolated from Ferula ferulioides and 11 synthetic acetophenone sesquiterpene analogues were evaluated to explore the structure–activity relationships (SARs) on their antibacterial activities against a panel of bacteria including drug-resistant Staphylococcus aureus. The minimum inhibitory concentration (MIC) values of the compounds were in the range of 0.5–128 mg/L. Preliminary SAR studies showed that subtle modifications on both the 2’,4’-dihydroxyphenyl moiety and the side chain reduced their activity against drug-resistant S. aureus. All of the compounds that showed no or only weak direct antibacterial activity were tested for their efflux inhibitory effects, among which four compounds showed significant efflux inhibition against drug-resistant strains. Natural product 14 showed significant inhibitory effects for EtBr efflux in strain SA1199B, which has reduced susceptibility to fluoroquinolones by efflux. Compounds 5, 14 and F-3 moderately inhibited EtBr efflux in the macrolide-resistant strain RN4220 and compound 13 moderately inhibited efflux in an MRSA and effluxing tetracycline-resistant strain.

<p>A novel mechanism of action of ketoconazole: inhibition of the <em>NorA</em> efflux pump system and biofilm formation in multidrug-resistant <em>Staphylococcus aureus</em></p>

Background: The rapid emergence of antimicrobial resistance among Gram-positive organisms, especially staphylococci, has become a serious clinical challenge. Efflux machinery and biofilm formation are considered two of the main causes of antimicrobial resistance and therapy failure.Aim: Our study aims to evaluate the antibiofilm and efflux pump inhibitory activity of the antifungal ketoconazole against multidrug-resistant (MDR) Staphylococcus aureus.Methods: Ketoconazole was tested for its effect on the following: minimum inhibitory concentrations (MICs) of ciprofloxacin, norfloxacin, levofloxacin, and ethidium bromide (EtBr) by the broth microdilution method, the efflux of EtBr by NorA-positive MDR S. aureus, and the relative expression of NorA, NorB, and NorC efflux pump genes. Docking studies of ketoconazole were performed using 1PW4 (glycerol-3-phosphate transporter from Escherichia coli which was the representative structure from the major facilitator superfamily).Results: Ketoconazole significantly decreased the MICs of levofloxacin, ciprofloxacin, norfloxacin, and EtBr (a substrate for efflux pump) by 8 to 1024-fold (P<0.01) and decreased the efflux of EtBr. Furthermore, a time-kill assay revealed that combinations of levofloxacin with ketoconazole or carbonyl cyanide m-chlorophenylhydrazone showed no growth for the tested strains after 24 h in comparison to the effect of levofloxacin alone. Docking studies and the ability of ketoconazole to diminish the relative expression of NorA gene in comparison to control (untreated strains) confirmed its action as an efflux pump inhibitor. Conclusion: The findings showed that the antifungal ketoconazole has no antibacterial activity but can potentiate the activity of the fluroquinolones against MDR S. aureus via inhibiting efflux pump and biofilm formation in vitro.

Evaluation of efflux pump activity of multidrug-resistant Salmonella Typhimurium isolated from poultry wet markets in India.

Introduction: Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium) is one of the major cause of foodborne zoonoses in humans. Poultry acts as a reservoir for S. Typhimurium without showing clinical signs and has become a source of infection to humans. Besides, it also became a source of multidrug-resistant (MDR) strains of S. Typhimurium.Methods: In the present study, we have isolated 9 S. Typhimurium from 503 samples from environmental sources of poultry wet markets in the Kadapa District of Andhra Pradesh, India. The role of efflux pump activity in antibiotic resistance was evaluated by ethidium bromide cartwheel test and efflux pump inhibition assay.Results: Eight out of nine isolates were resistant to two or more classes of antibiotics. The efflux pump activity of these isolates by ethidium bromide cartwheel method revealed that 66.6% isolates had shown evidence of pronounced efflux activity. The zone of inhibition (ZOI) of resistant antibiotics for each isolate was estimated in the presence or absence of 25 µg/mL of PAβN. Overall, cephalosporins (cefazolin, cefamandole, and cefaclor), kanamycin, polymyxin-B, piperacillin, and imipenem showed significant increase (≥2 mm) of ZOI, indicating the role of efflux pumps for efflux of these drugs. A maximum of 4 antibiotics among EtBr efflux pump positive isolates and 2 antibiotics among EtBr efflux pump negative isolates showed increased ZOI in the presence of PAβN.Conclusion: The results indicate that efflux pumps of MDR S. Typhimurium may contribute to resistance for at least one antibiotic, even in EtBr cartwheel test negative isolates.