scholarly journals Effect of Vitamin K3 Inhibiting the Function of NorA Efflux Pump and Its Gene Expression on Staphylococcus aureus

Membranes ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 130 ◽  
Author(s):  
Saulo R. Tintino ◽  
Veruska C. A. de Souza ◽  
Julia M. A. da Silva ◽  
Cícera Datiane de M. Oliveira-Tintino ◽  
Pedro S. Pereira ◽  
...  
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Structural Changes ◽  
Efflux Pump ◽  
Direct Interaction ◽  
Vitamin K3 ◽  
Efflux Pump Inhibitor ◽  
Microdilution Method ◽  
Dual Mechanism ◽  
Hospital Acquired

Resistance to antibiotics has made diseases that previously healed easily become more difficult to treat. Staphylococcus aureus is an important cause of hospital-acquired infections and multi-drug resistant. NorA efflux pump, present in bacteria S. aureus, is synthesized by the expression of the norA gene. Menadione, also known as vitamin K3, is one of the synthetic forms of vitamin K. Therefore, the aim of this study is to verify the menadione effect on efflux inhibition through NorA pump gene expression inhibition and assess the effects of menadione in bacterial membrane. The effect of menadione as an efflux pump inhibitor (EPI) was evaluated by the microdilution method, fluorimetry, electron microscopy, and by RT-qPCR to evaluate gene expression. In the molecular docking, association with menadione induces increased fluorescence intensity. Menadione was observed (100% of the clusters) interacting with residues ILE12, ILE15, PHE16, ILE19, PHE47, GLN51, ALA105, and MET109 from NorA. The results showed the norA gene had its expression significantly diminished in the presence of menadione. The simulation showed that several menadione molecules were able to go through the bilayer and allow the entry of water molecules into the hydrophobic regions of the bilayer. When present within membranes, menadione may have caused membrane structural changes resulting in a decline of the signaling pathways involved in norA expression. Menadione demonstrated to be an efflux pump inhibitor with dual mechanism: affecting the efflux pump by direct interaction with protein NorA and indirectly inhibiting the norA gene expression, possibly by affecting regulators present in the membrane altered by menadione.

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 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 Characterization of NorR Protein, a Multifunctional Regulator of norA Expression in Staphylococcus aureus

2003 ◽  
Vol 185 (10) ◽  
pp. 3127-3138 ◽  
Author(s):  
Que Chi Truong-Bolduc ◽  
Xiamei Zhang ◽  
David C. Hooper
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Efflux Pump ◽  
Protein A ◽  
Protein Overexpression ◽  
Wild Type ◽  
Liquid Media ◽  
Cell Surface Properties ◽  
Regulatory Systems

ABSTRACT We characterized a Staphylococcus aureus norA gene expression regulator, NorR, initially identified from its binding to the norA promoter. The norR gene was 444 bp in length, located ∼7 kb upstream from the norA gene, and encoded a predicted 17.6-kDa protein. Overexpression of norR in wild-type S. aureus strain ISP794 led to a fourfold decrease in sensitivity to quinolones and ethidium bromide and an increase in the level of norA transcripts, suggesting that NorR acts as a positive regulator of norA expression. Overexpression of norR in sarA and agr mutants did not alter quinolone sensitivity or levels of norA transcription, indicating that the presence of these two global regulatory systems is necessary for NorR to affect the expression of norA. Insertion and disruption of norR in ISP794 increased resistance to quinolones by 4- to 16-fold but had no effect on norA transcription, suggesting that NorR acts as a repressor for another unidentified efflux pump or pumps. These mutants also exhibited an exaggerated clumping phenotype in liquid media, which was complemented fully by a plasmid-encoded norR gene. Collectively, these results indicate that NorR is a multifunctional regulator, affecting cell surface properties as well as the expression of NorA and likely other multidrug resistance efflux pumps.

The Expression of Efflux Pump Genes in Methicillin-Resistant Staphylococcus aureus (MRSA) Strains Isolated from Blood Cultures in Iran

2020 ◽  
Vol 15 (5) ◽  
Author(s):  
Arezoo Bostanmaneshrad ◽  
Jamileh Norouzi ◽  
Gita Eslami ◽  
Ali Hashemi
Keyword(s):  
Staphylococcus Aureus ◽  
Efflux Pump ◽  
Resistance Mechanism ◽  
Expression Level ◽  
Resistance Pattern ◽  
Dilution Method ◽  
University Hospitals ◽  
Efflux Pump Inhibitor ◽  
Disk Diffusion Test ◽  
Methicillin Resistant

Background: Efflux pump is a significant resistance mechanism in Staphylococcus aureus. A total of 100 patients with bacteremia from Shahid Beheshti University Hospitals of Tehran in Iran were tested for the expression of efflux pump genes, contributing to S. aureus antimicrobial resistance. Objectives: this study was conducted to identify resistance pattern, and to evaluate the inhibitory effect of efflux pump, MIC of ciprofloxacin, and expression levels of norA, norB, and norC efflux pump genes in the presence of an efflux pump inhibitor against MDR S. aureus. Methods: A total of 100 MRSA isolates were investigated in different hospitals of Shahid Beheshti University of Medical Sciences from April 2017-2018. Owing to new consensus guidelines from the Clinical and Laboratory Standards Institute (CLSI), both the Kirby-Bauer disk diffusion test and micro-dilution method were used to evaluate antimicrobial susceptibility. Efflux pump activity using carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was identified as a chemical efflux pump inhibitor. E-test was used to determine Vancomycin-resistant antibiotic. Broth micro-dilution method for S. aureus isolates resistant to ciprofloxacin has been developed for minimum inhibitory concentration (MIC) of ciprofloxacin and CCCP and their composition. Quantitative Real-time Polymerase Chain Reaction (qRT-PCR) was used to investigate the expression level of norA, norB, and norC efflux pump genes. Results: A total of 38 of 45 MRSA isolates (84.4%) showed resistance to ciprofloxacin. Moreover, 100% of isolates had the norA and norB genes. Further, 95% of S. aureus isolates had the norC gene. According to this study, ciprofloxacin MIC has decreased by CCCP compared to ciprofloxacin. There was an increase in the expression level of norA, norB, and norC efflux pump genes in methicillin-resistant and ciprofloxacin-resistant S. aureus strains based on RT- PCR. In this study, four different spA types were obtained as the most prevalent type of spA by t037and t790 (23.3%) and t030 (14.1%) and t044 (12.2%). Conclusions: This study indicates that the prevalence of ciprofloxacin-resistant S. aureus strains has a rising trend among MRSA clinical isolates. The ability of S. aureus isolates to be converted into drug-resistant strains using efflux pump mechanism has become a widespread concern.

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 NorB, an Efflux Pump in Staphylococcus aureus Strain MW2, Contributes to Bacterial Fitness in Abscesses

2008 ◽  
Vol 190 (21) ◽  
pp. 7123-7129 ◽  
Author(s):  
Yanpeng Ding ◽  
Yoshikuni Onodera ◽  
Jean C. Lee ◽  
David C. Hooper
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Single Copy ◽  
Aureus Strain ◽  
Bacterial Survival ◽  
Bacterial Fitness

ABSTRACT While remaining a major problem in hospitals, Staphylococcus aureus is now spreading in communities. Strain MW2 (USA400 lineage) and other community methicillin-resistant S. aureus strains most commonly cause skin infections with abscess formation. Multidrug resistance (MDR) efflux pumps contribute to antimicrobial resistance but may also contribute to bacterial survival by removal of environmental toxins. In S. aureus, NorA, NorB, NorC, and Tet38 are chromosomally encoded efflux pumps whose overexpression can confer MDR to quinolones and other compounds (Nor pumps) or tetracyclines alone (Tet38), but the natural substrates of these pumps are not known. To determine the role of these efflux pumps in a natural environment in the absence of antibiotics, we used strain MW2 in a mouse subcutaneous abscess model and compared pump gene expression as determined by reverse transcription-PCR in the abscesses and in vitro. norB and tet38 were selectively upregulated in vivo more than 171- and 24-fold, respectively, whereas norA and norC were downregulated. These changes were associated with an increase in expression of mgrA, which encodes a transcriptional regulator known to affect pump gene expression. In competition experiments using equal inocula of a norB or tet38 mutant and parent strain MW2, each mutant exhibited growth defects of about two- to threefold in vivo. In complementation experiments, a single-copy insertion of norB (but not a single-copy insertion of tet38) in the attB site within geh restored the growth fitness of the norB mutant in vivo. Our findings indicate that some MDR pumps, like NorB, can facilitate bacterial survival when they are overexpressed in a staphylococcal abscess and may contribute to the relative resistance of abscesses to antimicrobial therapy, thus linking bacterial fitness and resistance in vivo.

scholarly journals 1-benzyl-1,4-diazepane reduces the efflux of resistance-nodulation-cell division pumps in Escherichia coli

2020 ◽  
Vol 15 (11) ◽  
pp. 987-999
Author(s):  
Enrico Casalone ◽  
Tiziano Vignolini ◽  
Laura Braconi ◽  
Lucia Gardini ◽  
Marco Capitanio ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ethidium Bromide ◽  
Inhibitory Concentration ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Efflux Pump Inhibitor ◽  
E Coli ◽  
Microdilution Method ◽  
Photoactivated Localization Microscopy ◽  
Mixed Mechanism

Aim: To investigate the action mechanism of 1-benzyl-1,4-diazepane (1-BD) as efflux pump inhibitor (EPI) in Escherichia coli mutants: Δ acrAB or overexpressing AcrAB and AcrEF efflux pumps. Materials & methods: Effect of 1-BD on: antibiotic potentiation, by microdilution method; membrane functionality, by fluorimetric assays; ethidium bromide accumulation, by fluorometric real-time efflux assay; AcrB expression, by quantitative photoactivated localization microscopy. Results: 1-BD decreases the minimal inhibitory concentration of levofloxacin and other antibiotics and increase ethidium bromide accumulation in E. coli overexpressing efflux pumps but not in the Δ acrAB strain. 1-BD increases membranes permeability, without sensibly affecting inner membrane polarity and decreases acrAB transcription. Conclusion: 1-BD acts as an EPI in E. coli with a mixed mechanism, different from that of major reference EPIs.

scholarly journals Activity of imipenem/relebactam on Klebsiella pneumoniae with different mechanisms of imipenem non-susceptibility

2021 ◽  
Author(s):  
Mervat El-Sayed Mashaly ◽  
Ghada El-Saeed Mashaly
Keyword(s):  
Gene Expression ◽  
Klebsiella Pneumoniae ◽  
Efflux Pump ◽  
Molecular Profile ◽  
Efflux Pump Inhibitor ◽  
Extended Spectrum Beta Lactamase ◽  
Reverse Transcription Pcr ◽  
Treat Ment ◽  
Encoding Genes

Background and Objectives: Imipenem/relebactam (IMP/R) is a newly FDA approved β-lactam/β-lactamase inhibitor combination. Relebactam ability to restore IMP activity could differ according to the cause of imipenem non-susceptibility. Therefore, we investigated the in-vitro activity of IMP/R against Klebsiella pneumoniae with different mechanisms of imi- penem non-susceptibility. Materials and Methods: Imipenem-nonsusceptible (IMP-NS) K. pneumoniae isolates were collected and characterized for β-lactamase encoding genes by multiplex PCR. For IMP-NS carbapenemase-negative isolates, study of Ompk35 & Ompk36 gene expression was performed by reverse transcription-PCR while efflux pump activity was studied by minimum inhibitory concentration (MIC) reduction assay using efflux pump inhibitor. Susceptibility testing of K. pneumoniae to IMP and IMP/R were achieved by broth microdilution (BMD) method. Results: During the study period, 140 isolates of IMP-NS K. pneumoniae were collected. BMD method showed that relebac- tam restored IMP susceptibility in 100%, 60% and 49% of isolates that only harbor AmpC, extended spectrum beta lactamase (ESBL) and carbapenemases, respectively. IMP/R was most potent against all bla KPC and 50% of bla _producing isolates. No demonstrable activity of IMP/R against K. pneumoniae harboring metallo-β-lactamases (MBLs). Out of 18 isolates with IMP non-suceptibility due to porins loss with overproduction of ESBL and/or AmpC, 14 (77.7%) isolates were IMP/R sus- ceptible. IMP/R showed no activity against isolates with only efflux pump hyperactivity. Conclusion: Relebactam could restore IPM activity in KPC or AmpC-producing IMP/NS K. pneumoniae but with no ac- tivity against MBL- producing isolates. Relebactam activity against isolates harbouring-bla OXA-48 or with altered Ompk35 & Ompk36 gene expression and efflux pump hyperactivity need further studies. Therefore, using IMP/R antibiotic in the treat- ment of infections caused by IMP/NS K. pneumoniae should be based on its molecular profile of IMP resistance to optimize the utility of IMP/R.

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