scholarly journals Synthesis of Omeprazole Analogues and Evaluation of These as Potential Inhibitors of the Multidrug Efflux Pump NorA of Staphylococcus aureus

2006 ◽  
Vol 51 (3) ◽  
pp. 831-838 ◽  
Author(s):  
Céline Vidaillac ◽  
Jean Guillon ◽  
Corinne Arpin ◽  
Isabelle Forfar-Bares ◽  
Boubakar B. Ba ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
High Performance ◽  
Efflux Pump ◽  
Critical Role ◽  
Inhibitory Effect ◽  
Multidrug Efflux Pump ◽  
Structural Element ◽  
Efflux Pump Inhibitor ◽  
Antiulcer Agent ◽  
The Stability

ABSTRACT A series of 11 pyrrolo[1,2-a]quinoxaline derivatives, 1a to 1k, sharing structural analogies with omeprazole, a eukaryotic efflux pump inhibitor (EPI) used as an antiulcer agent, was synthesized. Their inhibitory effect was evaluated using Staphylococcus aureus strain SA-1199B overexpressing NorA. By determinations of the MIC of norfloxacin in the presence of these EPIs devoid of intrinsic antibacterial activity and used at 128 μg/ml, and by the checkerboard method, compound 1e (MIC decrease, 16-fold; fractional inhibitory concentration index [ΣFIC], 0.18) appeared to be more active than compounds 1b to 1d, reserpine, and omeprazole (MIC decrease, eightfold; ΣFIC, 0.31), followed by compounds 1a and 1f (MIC decrease, fourfold; ΣFIC, 0.37) and 1g to 1k (MIC decrease, twofold; ΣFIC, 0.50 to 0.56). By time-kill curves combining norfloxacin (1/4 MIC) and the most efficient EPIs (128 μg/ml), compound 1e persistently restored the bactericidal activity of norfloxacin (inoculum reduction, 3 log10 CFU/ml at 8 and 24 h), compound 1f led to a delayed but progressive decrease in the number of viable cells, and compounds 1b to 1d and omeprazole acted synergistically (inoculum reduction, 3 log10 CFU/ml at 8 h but further regrowth), while compound 1a and reserpine slightly enhanced norfloxacin activity. The bacterial uptake of norfloxacin monitored by high-performance liquid chromatography confirmed that compounds 1a to 1f increased antibiotic accumulation, as did reserpine and omeprazole. Since these EPIs did not disturb the Δψ and ΔpH, they might directly interact with the pump. A structure-activity relationships study identified the benzimidazole nucleus of omeprazole as the main structural element involved in efflux pump inhibition and highlighted the critical role of the chlorine substituents in the stability and efficiency of compounds 1e to 1f. However, further pharmacomodulation is required to obtain therapeutically applicable derivatives.

scholarly journals The Phenolic Diterpene Totarol Inhibits Multidrug Efflux Pump Activity in Staphylococcus aureus

2007 ◽  
Vol 51 (12) ◽  
pp. 4480-4483 ◽  
Author(s):  
Eileen C. J. Smith ◽  
Glenn W. Kaatz ◽  
Susan M. Seo ◽  
Neale Wareham ◽  
Elizabeth M. Williamson ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Antimicrobial Activity ◽  
Antimicrobial Agent ◽  
Efflux Pump ◽  
Resistant Mutant ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Efflux Pump Inhibitor ◽  
Pump Activity ◽  
Combination Studies

ABSTRACT The phenolic diterpene totarol had good antimicrobial activity against effluxing strains of Staphylococcus aureus. Subinhibitory concentrations reduced the MICs of selected antibiotics, suggesting that it may also be an efflux pump inhibitor (EPI). A totarol-resistant mutant that overexpressed norA was created to separate antimicrobial from efflux inhibitory activity. Totarol reduced ethidium efflux from this strain by 50% at 15 μM (1/4× MIC), and combination studies revealed marked reductions in ethidium MICs. These data suggest that totarol is a NorA EPI as well as an antistaphylococcal antimicrobial agent.

scholarly journals Staphylococcus aureus Mutants Isolated via Exposure to Nonfluorinated Quinolones: Detection of Known and Unique Mutations

2001 ◽  
Vol 45 (12) ◽  
pp. 3422-3426 ◽  
Author(s):  
Siddhartha Roychoudhury ◽  
Tracy L. Twinem ◽  
Kelly M. Makin ◽  
Mark A. Nienaber ◽  
Chuiying Li ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Sequence Analysis ◽  
Efflux Pump ◽  
Serial Passage ◽  
Efflux Pump Inhibitor ◽  
In Vitro Development ◽  
Development Of Resistance ◽  
High Level ◽  
Vitro Development

ABSTRACT The in vitro development of resistance to the new nonfluorinated quinolones (NFQs; PGE 9262932, PGE 4175997, and PGE 9509924) was investigated in Staphylococcus aureus. At concentrations two times the MIC, step 1 mutants were isolated more frequently with ciprofloxacin and trovafloxacin (9.1 × 10−8 and 5.7 × 10−9, respectively) than with the NFQs, gatifloxacin, or clinafloxacin (<5.7 × 10−10). Step 2 and step 3 mutants were selected via exposure of a step 1 mutant (selected with trovafloxacin) to four times the MICs of trovafloxacin and PGE 9262932. The step 1 mutant contained the known Ser80-Phe mutation in GrlA, and the step 2 and step 3 mutants contained the known Ser80-Phe and Ser84-Leu mutations in GrlA and GyrA, respectively. Compared to ciprofloxacin, the NFQs were 8-fold more potent against the parent and 16- to 128-fold more potent against the step 3 mutants. Mutants with high-level NFQ resistance (MIC, 32 μg/ml) were isolated by the spiral plater-based serial passage technique. DNA sequence analysis of three such mutants revealed the following mutations: (i) Ser84-Leu in GyrA and Glu84-Lys and His103-Tyr in GrlA; (ii) Ser-84Leu in GyrA, Ser52-Arg in GrlA, and Glu472-Val in GrlB; and (iii) Ser84-Leu in GyrA, Glu477-Val in GyrB, and Glu84-Lys and His103-Tyr in GrlA. Addition of the efflux pump inhibitor reserpine (10 μg/ml) resulted in 4- to 16-fold increases in the potencies of the NFQs against these mutants, whereas it resulted in 2-fold increases in the potencies of the NFQs against the parent.

scholarly journals Efflux-Related Resistance to Norfloxacin, Dyes, and Biocides in Bloodstream Isolates of Staphylococcus aureus

2007 ◽  
Vol 51 (9) ◽  
pp. 3235-3239 ◽  
Author(s):  
Carmen E. DeMarco ◽  
Laurel A. Cushing ◽  
Emmanuel Frempong-Manso ◽  
Susan M. Seo ◽  
Tinevimbo A. A. Jaravaza ◽  
...  
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Efflux Pump ◽  
Resistance Mechanism ◽  
Efflux Pump Inhibitor ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Environmental Surfaces ◽  
Genes Encoding ◽  
High Level

ABSTRACT Efflux is an important resistance mechanism in Staphylococcus aureus, but its frequency in patients with bacteremia is unknown. Nonreplicate bloodstream isolates were collected over an 8-month period, and MICs of four common efflux pump substrates, with and without the broad-spectrum efflux pump inhibitor reserpine, were determined (n = 232). A reserpine-associated fourfold decrease in MIC was considered indicative of efflux. Strains exhibiting efflux of at least two of the four substrates were identified (“effluxing strains” [n = 114]). For these strains, MICs with or without reserpine for an array of typical substrates and the expression of mepA, mdeA, norA, norB, norC, and qacA/B were determined using quantitative real-time reverse transcription-PCR (qRT-PCR). A fourfold or greater increase in gene expression was considered significant. The most commonly effluxed substrates were ethidium bromide and chlorhexidine (100 and 96% of effluxing strains, respectively). qRT-PCR identified strains overexpressing mepA (5 [4.4%]), mdeA (13 [11.4%]), norA (26 [22.8%]), norB (29 [25.4%]), and norC (19 [16.7%]); 23 strains overexpressed two or more genes. Mutations probably associated with increased gene expression included a MepR-inactivating substitution and norA promoter region insertions or deletions. Mutations possibly associated with increased expression of the other analyzed genes were also observed. Effluxing strains comprised 49% of all strains studied (114/232 strains), with nearly half of these overexpressing genes encoding MepA, MdeA, and/or NorABC (54/114 strains). Reduced susceptibility to biocides may contribute to persistence on environmental surfaces, and efflux of drugs such as fluoroquinolones may predispose strains to high-level target-based resistance.

scholarly journals Response of Staphylococcus aureus to Salicylate Challenge

2006 ◽  
Vol 189 (1) ◽  
pp. 220-227 ◽  
Author(s):  
James T. Riordan ◽  
Arunachalam Muthaiyan ◽  
Wayne Van Voorhies ◽  
Christopher T. Price ◽  
James E. Graham ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Antimicrobial Resistance ◽  
Transcriptome Analysis ◽  
Target Genes ◽  
Efflux Pump ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Antibiotic Target ◽  
Unique Mechanism ◽  
Formate Metabolism

ABSTRACT Growth of Staphylococcus aureus with the nonsteroidal anti-inflammatory salicylate reduces susceptibility of the organism to multiple antimicrobials. Transcriptome analysis revealed that growth of S. aureus with salicylate leads to the induction of genes involved with gluconate and formate metabolism and represses genes required for gluconeogenesis and glycolysis. In addition, salicylate induction upregulates two antibiotic target genes and downregulates a multidrug efflux pump gene repressor (mgrA) and sarR, which represses a gene (sarA) important for intrinsic antimicrobial resistance. We hypothesize that these salicylate-induced alterations jointly represent a unique mechanism that allows S. aureus to resist antimicrobial stress and toxicity.

scholarly journals Molecular Mechanism of MBX2319 Inhibition of Escherichia coli AcrB Multidrug Efflux Pump and Comparison with Other Inhibitors

2014 ◽  
Vol 58 (10) ◽  
pp. 6224-6234 ◽  
Author(s):  
Attilio V. Vargiu ◽  
Paolo Ruggerone ◽  
Timothy J. Opperman ◽  
Son T. Nguyen ◽  
Hiroshi Nikaido
Keyword(s):  
Efflux Pump ◽  
Efflux Pumps ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Efflux Pump Inhibitor ◽  
Content Type ◽  
X Ray Crystallography ◽  
Resistance Nodulation Division ◽  
Hydrophobic Portion ◽  
Dynamics Simulations

ABSTRACTEfflux pumps of the resistance nodulation division (RND) superfamily, such as AcrB, make a major contribution to multidrug resistance in Gram-negative bacteria. The development of inhibitors of the RND pumps would improve the efficacy of current and next-generation antibiotics. To date, however, only one inhibitor has been cocrystallized with AcrB. Thus,in silicostructure-based analysis is essential for elucidating the interaction between other inhibitors and the efflux pumps. In this work, we used computer docking and molecular dynamics simulations to study the interaction between AcrB and the compound MBX2319, a novel pyranopyridine efflux pump inhibitor with potent activity against RND efflux pumps ofEnterobacteriaceaespecies, as well as other known inhibitors (D13-9001, 1-[1-naphthylmethyl]-piperazine, and phenylalanylarginine-β-naphthylamide) and the binding of doxorubicin to the efflux-defective F610A variant of AcrB. We also analyzed the binding of a substrate, minocycline, for comparison. Our results show that MBX2319 binds very tightly to the lower part of the distal pocket in the B protomer of AcrB, strongly interacting with the phenylalanines lining the hydrophobic trap, where the hydrophobic portion of D13-9001 was found to bind by X-ray crystallography. Additionally, MBX2319 binds to AcrB in a manner that is similar to the way in which doxorubicin binds to the F610A variant of AcrB. In contrast, 1-(1-naphthylmethyl)-piperazine and phenylalanylarginine-β-naphthylamide appear to bind to somewhat different areas of the distal pocket in the B protomer of AcrB than does MBX2319. However, all inhibitors (except D13-9001) appear to distort the structure of the distal pocket, impairing the proper binding of substrates.

scholarly journals MepR, a Repressor of the Staphylococcus aureus MATE Family Multidrug Efflux Pump MepA, Is a Substrate-Responsive Regulatory Protein

2006 ◽  
Vol 50 (4) ◽  
pp. 1276-1281 ◽  
Author(s):  
Glenn W. Kaatz ◽  
Carmen E. DeMarco ◽  
Susan M. Seo
Keyword(s):  
Staphylococcus Aureus ◽  
Efflux Pump ◽  
Regulatory Protein ◽  
Inducible Promoter ◽  
Multidrug Efflux ◽  
Recognition Sequence ◽  
Multidrug Efflux Pump ◽  
Mobility Shift ◽  
Repressive Effect ◽  
Operator Interaction

ABSTRACT The mepRAB gene cluster of Staphylococcus aureus encodes a MarR family repressor (MepR; known to repress mepA expression), a MATE family multidrug efflux pump (MepA), and a protein of unknown function (MepB). In this report, we show that MepR also is autoregulatory, repressing the expression of its own gene. Exposure of strains containing a mepR::lacZ fusion with mepR provided in trans under the control of an inducible promoter, or a mepA::lacZ fusion alone, to subinhibitory concentrations of MepA substrates resulted in variably increased expression mainly of mepA. Mobility shift assays revealed that MepR binds upstream of mepR and mepA, with an apparently higher affinity for the mepA binding site. MepA substrates abrogated MepR binding to each site in a differential manner, with the greatest effect observed on the MepR-mepA operator interaction. DNase I footprinting identified precise binding sites which included promoter motifs, inverted repeats, and transcription start sites for mepR and mepA, as well as a conserved GTTAG motif, which may be a signature recognition sequence for MepR. Analogous to other multidrug efflux pump regulatory proteins such as QacR, the substrate-MepR interaction likely results in its dissociation from its mepA, and in a more limited fashion its mepR, operator sites and relief of its repressive effect. The enhanced effect of substrates on mepA compared to mepR expression, and on the MepR-mepA operator interaction, results in significant relief of mepA and relative maintenance of mepR repression, leading to increased MepA protein unimpeded by MepR when the need for detoxification exists.

scholarly journals Signal Synthase-Type versus Catabolic Monooxygenases: Retracing 3-Hydroxylation of 2-Alkylquinolones and Their N-Oxides by Pseudomonas aeruginosa and Other Pulmonary Pathogens

2021 ◽  
Vol 87 (6) ◽  
Author(s):  
Niklas H. Ritzmann ◽  
Steffen L. Drees ◽  
Susanne Fetzner
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Efflux Pump ◽  
Biological Activities ◽  
Multidrug Efflux Pump ◽  
Therapeutic Success ◽  
Signal Molecule ◽  
Physiological Level ◽  
Content Type

ABSTRACT The multiple biological activities of 2-alkylquinolones (AQs) are crucial for virulence of Pseudomonas aeruginosa, conferring advantages during infection and in polymicrobial communities. Whereas 2-heptyl-3-hydroxyquinolin-4(1H)-one (the “Pseudomonas quinolone signal” [PQS]) is an important quorum sensing signal molecule, 2-alkyl-1-hydroxyquinolin-4(1H)-ones (also known as 2-alkyl-4-hydroxyquinoline N-oxides [AQNOs]) are antibiotics inhibiting respiration. Hydroxylation of the PQS precursor 2-heptylquinolin-4(1H)-one (HHQ) by the signal synthase PqsH boosts AQ quorum sensing. Remarkably, the same reaction, catalyzed by the ortholog AqdB, is used by Mycobacteroides abscessus to initiate degradation of AQs. The antibiotic 2-heptyl-1-hydroxyquinolin-4(1H)-one (HQNO) is hydroxylated by Staphylococcus aureus to the less toxic derivative PQS-N-oxide (PQS-NO), a reaction probably also catalyzed by a PqsH/AqdB ortholog. In this study, we provide a comparative analysis of four AQ 3-monooxygenases of different organisms. Due to the major impact of AQ/AQNO 3-hydroxylation on the biological activities of the compounds, we surmised adaptations on the enzymatic and/or physiological level to serve either the producer or target organisms. Our results indicate that all enzymes share similar features and are incapable of discriminating between AQs and AQNOs. PQS-NO, hence, occurs as a native metabolite of P. aeruginosa although the unfavorable AQNO 3-hydroxylation is minimized by export as shown for HQNO, involving at least one multidrug efflux pump. Moreover, M. abscessus is capable of degrading the AQNO heterocycle by concerted action of AqdB and dioxygenase AqdC. However, S. aureus and M. abscessus orthologs disfavor AQNOs despite their higher toxicity, suggesting that catalytic constraints restrict evolutionary adaptation and lead to the preference of non-N-oxide substrates by AQ 3-monooxygenases. IMPORTANCE Pseudomonas aeruginosa, Staphylococcus aureus, and Mycobacteroides abscessus are major players in bacterial chronic infections and particularly common colonizers of cystic fibrosis (CF) lung tissue. Whereas S. aureus is an early onset pathogen in CF, P. aeruginosa establishes at later stages. M. abscessus occurs at all stages but has a lower epidemiological incidence. The dynamics of how these pathogens interact can affect survival and therapeutic success. 2-Alkylquinolone (AQ) and 2-alkylhydroxyquinoline N-oxide (AQNO) production is a major factor of P. aeruginosa virulence. The 3-position of the AQ scaffold is critical, both for attenuation of AQ toxicity or degradation by competitors, as well as for full unfolding of quorum sensing. Despite lacking signaling functionality, AQNOs have the strongest impact on suppression of Gram-positives. Because evidence for 3-hydroxylation of AQNOs has been reported, it is desirable to understand the extent by which AQ 3-monooxygenases contribute to manipulation of AQ/AQNO equilibrium, resistance, and degradation.

scholarly journals Effect of azithromycin and phenylalanine-arginine beta-naphthylamide on quorum sensing and virulence factors in clinical isolates of Pseudomonas aeruginosa

2021 ◽  
Author(s):  
Amel Elsheredy ◽  
Ingy El-Soudany ◽  
Eglal Elsherbini ◽  
Dalia Metwally ◽  
Abeer Ghazal
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Virulence Factors ◽  
Efflux Pump ◽  
Cell Communication ◽  
Inhibitory Effect ◽  
Resistance Rate ◽  
Clinical Isolates ◽  
Efflux Pump Inhibitor ◽  
Bacteria Growth

Background and Objectives: Pseudomonas aeruginosa is a problematic opportunistic pathogen causing several types of nosocomial infections with a high resistance rate to antibiotics. Production of many virulence factors in P. aeruginosa is regulated by quorum sensing (QS), a cell-to-cell communication mechanism. In this study, we aimed to assess and compare the inhibitory effect of azithromycin (AZM) and EPI- PAβN (efflux pump inhibitor- Phenylalanine-Arginine Beta-Naphthylamide) on QS system and QS-dependent virulence factors in P. aeruginosa clinical isolates. Materials and Methods: A total of 50 P. aeruginosa isolates were obtained from different types of clinical specimens. Isolates were investigated for detection of QS system molecules by AHL cross-feeding bioassay and QS-dependent virulence factors; this was also confirmed by detection of QS genes (lasR, lasI, rhlR, and rhlI) using PCR assay. The inhibitory effect of sub-MIC AZM and EPI PAβN on these virulence factors was assessed. Results: All the P. aeruginosa, producing QS signals C4 HSL, failed to produce C4 HSL in the presence of sub-MIC AZM, In the presence of EPI PAβN (20 µg/ml) only 14 isolates were affected, there was a significant reduction in QS-dependent virulence factors production (protease, biofilm, rhamnolipid and pyocyanin) in the presence of either 20 µg/ml EPI or subMIC of AZM with the inhibitory effect of AZM was more observed than PAβN. Conclusion: Anti-QS agents like AZM and EPI (PAβN) are useful therapeutic options for P. aeruginosa due to its inhibitory effect on QS-dependent virulence factors production without selective pressure on bacteria growth, so resistance to these agents is less likely to develop.

scholarly journals CmeABC Functions as a Multidrug Efflux System in Campylobacter jejuni

2002 ◽  
Vol 46 (7) ◽  
pp. 2124-2131 ◽  
Author(s):  
Jun Lin ◽  
Linda Overbye Michel ◽  
Qijing Zhang
Keyword(s):  
Campylobacter Jejuni ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Intrinsic Resistance ◽  
Multidrug Efflux Pump ◽  
Efflux Pump Inhibitor ◽  
Gram Negative ◽  
Multidrug Efflux Pumps

ABSTRACT Campylobacter jejuni, a gram-negative organism causing gastroenteritis in humans, is increasingly resistant to antibiotics. However, little is known about the drug efflux mechanisms in this pathogen. Here we characterized an efflux pump encoded by a three-gene operon (designated cmeABC) that contributes to multidrug resistance in C. jejuni 81-176. CmeABC shares significant sequence and structural homology with known tripartite multidrug efflux pumps in other gram-negative bacteria, and it consists of a periplasmic fusion protein (CmeA), an inner membrane efflux transporter belonging to the resistance-nodulation-cell division superfamily (CmeB), and an outer membrane protein (CmeC). Immunoblotting using CmeABC-specific antibodies demonstrated that cmeABC was expressed in wild-type 81-176; however, an isogenic mutant (9B6) with a transposon insertion in the cmeB gene showed impaired production of CmeB and CmeC. Compared to wild-type 81-176, 9B6 showed a 2- to 4,000-fold decrease in resistance to a range of antibiotics, heavy metals, bile salts, and other antimicrobial agents. Accumulation assays demonstrated that significantly more ethidium bromide and ciprofloxacin accumulated in mutant 9B6 than in wild-type 81-176. Addition of carbonyl cyanide m-chlorophenylhydrazone, an efflux pump inhibitor, increased the accumulation of ciprofloxacin in wild-type 81-176 to the level of mutant 9B6. PCR and immunoblotting analysis also showed that cmeABC was broadly distributed in various C. jejuni isolates and constitutively expressed in wild-type strains. Together, these findings formally establish that CmeABC functions as a tripartite multidrug efflux pump that contributes to the intrinsic resistance of C. jejuni to a broad range of structurally unrelated antimicrobial agents.

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