scholarly journals Mutations in PA2491 (mexS) Promote MexT-Dependent mexEF-oprN Expression and Multidrug Resistance in a Clinical Strain of Pseudomonas aeruginosa

2005 ◽  
Vol 187 (4) ◽  
pp. 1246-1253 ◽  
Author(s):  
Mara L. Sobel ◽  
Shadi Neshat ◽  
Keith Poole
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Multidrug Resistance ◽  
Clinical Isolate ◽  
Multidrug Resistant ◽  
Clinical Strain ◽  
Insertion Mutant ◽  
Multidrug Efflux ◽  
Probable Significance

ABSTRACT Disruption of the PA2491 gene in a mini-Tn5-tet insertion mutant of a clinical isolate of Pseudomonas aeruginosa increased expression of the mexEF-oprN multidrug efflux genes and decreased production of outer membrane protein OprD, concomitant with enhanced resistance to chloramphenicol, quinolones, and imipenem, which was reminiscent of previously described nfxC mutants. PA2491 encodes a probable oxidoreductase previously shown to be positively regulated by the MexT positive regulator of mexEF-oprN expression (T. Köhler, S. F. Epp, L. K. Curty, and J. C. Pechére, J. Bacteriol. 181:6300-6305, 1999). Spontaneous multidrug-resistant mutants of the P. aeruginosa clinical isolate hyperexpressing mexEF-oprN and showing reduced production of OprD were readily selected in vitro, and all of them were shown to carry mutations in PA2491, highlighting the probable significance of such mutations as determinants of MexEF-OprN-mediated multidrug resistance in vivo.

scholarly journals Protein Modulator of Multidrug Efflux Gene Expression in Pseudomonas aeruginosa

2007 ◽  
Vol 189 (15) ◽  
pp. 5441-5451 ◽  
Author(s):  
Denis M. Daigle ◽  
Lily Cao ◽  
Sebastien Fraud ◽  
Mark S. Wilke ◽  
Angela Pacey ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Multidrug Resistant ◽  
In Vitro Transcription ◽  
Direct Result ◽  
Titration Calorimetry ◽  
Multidrug Efflux ◽  
Transcription Assay ◽  
Repressor Activity

ABSTRACT nalC multidrug-resistant mutants of Pseudomonas aeruginosa show enhanced expression of the mexAB-oprM multidrug efflux system as a direct result of the production of a ca. 6,100-Da protein, PA3719, in these mutants. Using a bacterial two-hybrid system, PA3719 was shown to interact in vivo with MexR, a repressor of mexAB-oprM expression. Isothermal titration calorimetry (ITC) studies confirmed a high-affinity interaction (equilibrium dissociation constant [KD ], 158.0 ± 18.1 nM) of PA3719 with MexR in vitro. PA3719 binding to and formation of a complex with MexR obviated repressor binding to its operator, which overlaps the efflux operon promoter, suggesting that mexAB-oprM hyperexpression in nalC mutants results from PA3719 modulation of MexR repressor activity. Consistent with this, MexR repression of mexA transcription in an in vitro transcription assay was alleviated by PA3719. Mutations in MexR compromising its interaction with PA3719 in vivo were isolated and shown to be located internally and distributed throughout the protein, suggesting that they impacted PA3719 binding by altering MexR structure or conformation rather than by having residues interacting specifically with PA3719. Four of six mutant MexR proteins studied retained repressor activity even in a nalC strain producing PA3719. Again, this is consistent with a PA3719 interaction with MexR being necessary to obviate MexR repressor activity. The gene encoding PA3719 has thus been renamed armR (antirepressor for MexR). A representative “noninteracting” mutant MexR protein, MexRI104F, was purified, and ITC confirmed that it bound PA3719 with reduced affinity (5.4-fold reduced; KD , 853.2 ± 151.1 nM). Consistent with this, MexRI104F repressor activity, as assessed using the in vitro transcription assay, was only weakly compromised by PA3719. Finally, two mutations (L36P and W45A) in ArmR compromising its interaction with MexR have been isolated and mapped to a putative C-terminal α-helix of the protein that alone is sufficient for interaction with MexR.

scholarly journals Overcoming multidrug-resistance in vitro and in vivo using the novel P-glycoprotein inhibitor 1416

2012 ◽  
Vol 32 (6) ◽  
pp. 559-566 ◽  
Author(s):  
Yan Xu ◽  
Feng Zhi ◽  
Guangming Xu ◽  
Xiaolei Tang ◽  
Sheng Lu ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Cancer Chemotherapy ◽  
Antitumour Activity ◽  
Multidrug Resistant ◽  
The Novel ◽  
Mice Model ◽  
P Glycoprotein ◽  
Channel Currents

MDR (multidrug-resistance) represents a major obstacle to successful cancer chemotherapy and is usually accomplished by overexpression of P-gp (P-glycoprotein). Much effort has been devoted to developing P-gp inhibitors to modulate MDR. However, none of the inhibitors on the market have been successful. 1416 [1-(2,6-dimethylphenoxy)-2-(3,4-dimethoxyphenylethylamino)propane hydrochloride (phenoprolamine hydrochloride)] is a new VER (verapamil) analogue with a higher IC50 for blocking calcium channel currents than VER. In the present paper, we examined the inhibition effect of 1416 on P-gp both in vitro and in vivo. 1416 significantly enhanced cytotoxicity of VBL (vinblastine) in P-gp-overexpressed human multidrug-resistant K562/ADM (adriamycin) and KBV cells, but had no such effect on the parent K562 and KB cells. The MDR-modulating function of 1416 was further confirmed by increasing intracellular Rh123 (rhodanmine123) content in MDR cells. Human K562/ADM xenograft-nude mice model verified that 1416 potentiates the antitumour activity of VBL in vivo. RT-PCR (reverse transcriptase-PCR) and FACS analysis demonstrated that the expression of MDR1/P-gp was not affected by 1416 treatment. All these observations suggest that 1416 could be a promising agent for overcoming MDR in cancer chemotherapy.

scholarly journals Design of Epitope-Based Peptide Vaccine against Pseudomonas aeruginosa Fructose Bisphosphate Aldolase Protein Using Immunoinformatics

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Mustafa Elhag ◽  
Ruaa Mohamed Alaagib ◽  
Nagla Mohamed Ahmed ◽  
Mustafa Abubaker ◽  
Esraa Musa Haroun ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Peptide Vaccine ◽  
Multidrug Resistant ◽  
Fructose Bisphosphate ◽  
Mhc I ◽  
Mhc Ii ◽  
Fructose Bisphosphate Aldolase ◽  
Hospital Acquired

Pseudomonas aeruginosa is a common pathogen that is responsible for serious hospital-acquired infections, ventilator-associated pneumonia, and various sepsis syndromes. Also, it is a multidrug-resistant pathogen recognized for its ubiquity and its intrinsically advanced antibiotic-resistant mechanisms. It usually affects immunocompromised individuals but can also infect immunocompetent individuals. There is no vaccine against it available till now. This study predicts an effective epitope-based vaccine against fructose bisphosphate aldolase (FBA) of Pseudomonas aeruginosa using immunoinformatics tools. The protein sequences were obtained from NCBI, and prediction tests were undertaken to analyze possible epitopes for B and T cells. Three B cell epitopes passed the antigenicity, accessibility, and hydrophilicity tests. Six MHC I epitopes were found to be promising, while four MHC II epitopes were found promising from the result set. Nineteen epitopes were shared between MHC I and II results. For the population coverage, the epitopes covered 95.62% worldwide excluding certain MHC II alleles. We recommend in vivo and in vitro studies to prove its effectiveness.

Peptide polymer displaying potent activity against clinically isolated multidrug resistant Pseudomonas aeruginosa in vitro and in vivo

2020 ◽  
Vol 8 (2) ◽  
pp. 739-745 ◽  
Author(s):  
Weinan Jiang ◽  
Ximian Xiao ◽  
Yueming Wu ◽  
Weiwei Zhang ◽  
Zihao Cong ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antimicrobial Activity ◽  
Host Defense ◽  
Multidrug Resistant ◽  
Host Defense Peptide

Host defense peptide mimicking peptide polymer displayed potent in vitro and in vivo antimicrobial activity against clinically isolated multidrug resistant Pseudomonas aeruginosa.

scholarly journals Expression of the multidrug resistance operon mexA-mexB-oprM in Pseudomonas aeruginosa: mexR encodes a regulator of operon expression.

1996 ◽  
Vol 40 (9) ◽  
pp. 2021-2028 ◽  
Author(s):  
K Poole ◽  
K Tetro ◽  
Q Zhao ◽  
S Neshat ◽  
D E Heinrichs ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Multidrug Resistance ◽  
Gene Product ◽  
Antimicrobial Agents ◽  
Resistant Mutant ◽  
Multidrug Resistant ◽  
Lacz Fusion ◽  
Base Substitution ◽  
Knockout Mutant

The region upstream of the multiple antibiotic resistance efflux operon mexA-mexB-oprM in Pseudomonas aeruginosa was sequenced, and a gene, mexR, was identified. The predicted MexR product contains 147 amino acids with a molecular mass of 16,964 Da, which is consistent with the observed size of the overexpressed mexR gene product. MexR was homologous to MarR, the repressor of MarA-dependent multidrug resistance in Escherichia coli, and other repressors of the MarR family. A mexR knockout mutant showed a twofold increase in expression of both plasmid-borne and chromosomal mexA-reporter gene fusions compared with the MexR+ parent strain, indicating that the mexR gene product negatively regulates expression of the mexA-mexB-oprM operon. Furthermore, the cloned mexR gene product reduced expression of a plasmid-borne mexA-lacZ fusion in E. coli, indicating that MexR represses mexA-mexB-oprM expression directly. Consistent with the increased expression of the efflux operon in the mexR mutant, the mutant showed an increase (relative to its MexR+ parent) in resistance to several antimicrobial agents. Expression of a mexR-lacZ fusion increased threefold in a mexR knockout mutant, indicating that mexR is negatively autoregulated. OCR1, a nalB multidrug-resistant mutant which overproduces OprM, exhibited a greater than sevenfold increase in expression of a chromosomal mexA-phoA fusion compared with its parent. Introduction of a mexR knockout mutation in strain OCR1 eliminated this increase in efflux gene expression and, as expected, increased the susceptibility of the strain to a variety of antibiotics. The nucleotide sequences of the mexR genes of OCR1 and its parental strain revealed a single base substitution in the former which would cause a predicted substitution of Trp for Arg at position 69 of its mexR product. These data suggest that MexR possesses both repressor and activator function in vivo, the activator form being favored in nalB multidrug-resistant strains.

scholarly journals Mutations in PA3574 (nalD) Lead to Increased MexAB-OprM Expression and Multidrug Resistance in Laboratory and Clinical Isolates of Pseudomonas aeruginosa

2005 ◽  
Vol 49 (5) ◽  
pp. 1782-1786 ◽  
Author(s):  
Mara L. Sobel ◽  
Didier Hocquet ◽  
Lily Cao ◽  
Patrick Plesiat ◽  
Keith Poole
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Multidrug Resistance ◽  
Resistant Mutant ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Multidrug Efflux ◽  
Efflux System ◽  
Clinical Strains ◽  
Transposon Insertion ◽  
Multidrug Efflux System

ABSTRACT Mutations in genes mexR and nalC have previously been shown to drive overexpression of the MexAB-OprM multidrug efflux system in Pseudomonas aeruginosa. A transposon insertion multidrug-resistant mutant of P. aeruginosa overproducing MexAB-OprM was disrupted in yet a third gene, PA3574, encoding a probable repressor of the TetR/AcrR family that we have dubbed NalD. Clinical strains overexpressing MexAB-OprM but lacking mutations in mexR or nalC were also shown to carry mutations in nalD. Moreover, the cloned nalD gene reduced the multidrug resistance and MexAB-OprM expression of the transposon mutant and clinical isolates, highlighting the significance of the nalD mutations vis-à-vis MexAB-OprM overexpression in these isolates.

scholarly journals Synergistic Efficacy of Aedes aegypti Antimicrobial Peptide Cecropin A2 and Tetracycline against Pseudomonas aeruginosa

2017 ◽  
Vol 61 (7) ◽  
Author(s):  
Zhaojun Zheng ◽  
Nagendran Tharmalingam ◽  
Qingzhong Liu ◽  
Elamparithi Jayamani ◽  
Wooseong Kim ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Aedes Aegypti ◽  
Mammalian Cells ◽  
Galleria Mellonella ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Synergistic Activity ◽  
Content Type

ABSTRACT The increasing prevalence of antibiotic resistance has created an urgent need for alternative drugs with new mechanisms of action. Antimicrobial peptides (AMPs) are promising candidates that could address the spread of multidrug-resistant bacteria, either alone or in combination with conventional antibiotics. We studied the antimicrobial efficacy and bactericidal mechanism of cecropin A2, a 36-residue α-helical cationic peptide derived from Aedes aegypti cecropin A, focusing on the common pathogen Pseudomonas aeruginosa. The peptide showed little hemolytic activity and toxicity toward mammalian cells, and the MICs against most clinical P. aeruginosa isolates were 32 to 64 μg/ml, and its MICs versus other Gram-negative bacteria were 2 to 32 μg/ml. Importantly, cecropin A2 demonstrated synergistic activity against P. aeruginosa when combined with tetracycline, reducing the MICs of both agents by 8-fold. The combination was also effective in vivo in the P. aeruginosa/Galleria mellonella model (P < 0.001). We found that cecropin A2 bound to P. aeruginosa lipopolysaccharides, permeabilized the membrane, and interacted with the bacterial genomic DNA, thus facilitating the translocation of tetracycline into the cytoplasm. In summary, the combination of cecropin A2 and tetracycline demonstrated synergistic antibacterial activity against P. aeruginosa in vitro and in vivo, offering an alternative approach for the treatment of P. aeruginosa infections.

Baicalin-induced Cytotoxicity and Apoptosis in Multidrug-resistant MC3/5FU Mucoepidermoid Carcinoma Cell Line

2019 ◽  
Vol 16 (12) ◽  
pp. 1339-1347 ◽  
Author(s):  
Xiaofang Xu ◽  
Qihong Li ◽  
Kaitao Yu ◽  
Ghulam Murtaza ◽  
Bin Liu
Keyword(s):  
Flow Cytometry ◽  
Multidrug Resistance ◽  
Nude Mice ◽  
Multidrug Resistant ◽  
Clinical Findings ◽  
Hoechst 33342 ◽  
Protein Levels ◽  
Transmission Electron

Background: Multidrug Resistance (MDR) is a serious hindrance to cancer chemotherapy and profoundly influences the clinical findings. Many Traditional Chinese Medicines (TCM) have been tested with the aim of developing effective resistance modulators or anticancer drugs to overcome the MDR of human cancers. Methods: The anticancer effect of baicalin on multidrug-resistant MC3/5FU (5-fluorouracil) cells was investigated by MTT test and xenografts in nude mice. Cell apoptosis was studied by transmission electron microscopy, Hoechst-33342 staining, DNA fragmentation detection, and flow cytometry. RT-PCR and Rhodamine 123 efflux assay was also used to detect its effect on ABC drug transporter proteins, ABCB1 (P-glycoprotein, P-gp) and ABCC1 (multidrug resistance protein 1, MRP1). Results: The results indicate that there was no significant effect of baicalin on ABC transporters expression or efflux function, although it induced potent growth inhibition in MC3/5FU cells. Flow cytometry, Hoechst 33342 staining and transmission electron microscope revealed that baicalin caused MC3/5FU cell death through the induction of apoptosis. It is demonstrated that baicalininduced apoptosis could be mediated by up-regulation of Bax and caspase-3 protein levels and downregulation of Bcl-2 protein levels. In addition, daily intraperitoneal injection of baicalin (100 and 200 mg/kg) for 2 weeks significantly inhibited the growth of MC3/5FU cells xenografts in nude mice. Conclusion: Our results suggest that baicalin possesses considerable cytotoxic activity in multidrug resistance MC3/5FU cells in vitro and in vivo.

scholarly journals A Galleria mellonella infection model reveals double and triple antibiotic combination therapies with enhanced efficacy versus a multidrug-resistant strain of Pseudomonas aeruginosa

2014 ◽  
Vol 63 (7) ◽  
pp. 945-955 ◽  
Author(s):  
Jessica Krezdorn ◽  
Sophie Adams ◽  
Peter J. Coote
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Resistant Strain ◽  
Galleria Mellonella ◽  
Multidrug Resistant ◽  
Therapeutic Benefit ◽  
Infection Model ◽  
Combination Therapies ◽  
Multidrug Resistant Strain

The aim of this study was to compare the inhibitory effect of antibiotic combinations in vitro with efficacy in Galleria mellonella larvae in vivo to identify efficacious combinations that target Pseudomonas aeruginosa. P. aeruginosa NCTC 13437, a multidrug-resistant strain resistant to β-lactams and aminoglycosides, was used. Susceptibility to cefotaxime, piperacillin, meropenem, amikacin, levofloxacin and colistin alone, or in dual or triple combinations, was measured in vitro via a 24 h time-kill assay. In vitro results were then compared with the efficacy of the same dual or triple antibiotic combinations versus G. mellonella larvae infected with P. aeruginosa. G. mellonella haemolymph burden of P. aeruginosa was determined over 96 h post-infection and treatment with the most potent combination therapies. Many dual and triple combinations of antibiotics displayed synergistic inhibition of multidrug-resistant P. aeruginosa in vitro. There was little correlation between combinations that were synergistic in vitro and those that showed enhanced efficacy in vivo versus infected G. mellonella larvae. The most potent dual and triple combinations in vivo were cefotaxime plus piperacillin, and meropenem plus piperacillin and amikacin, respectively. Fewer combinations were found to offer enhanced therapeutic benefit in vivo compared with in vitro. The therapeutic benefit arising from treatment with antibiotic combinations in vivo correlated with reduced larval burden of P. aeruginosa. This study has identified antibiotic combinations that merit further investigation for their clinical potential and has demonstrated the utility of using G. mellonella to screen for novel antibiotic treatments that demonstrate efficacy in vivo.

scholarly journals Multicenter Evaluation of Ceftazidime-Avibactam and Ceftolozane-Tazobactam Inhibitory Activity against Meropenem-Nonsusceptible Pseudomonas aeruginosa from Blood, Respiratory Tract, and Wounds

2017 ◽  
Vol 61 (10) ◽  
Author(s):  
Mordechai Grupper ◽  
Christina Sutherland ◽  
David P. Nicolau
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Inhibitory Activity ◽  
Clinical Utility ◽  
Multidrug Resistant ◽  
Resistance Mechanisms ◽  
Broth Microdilution ◽  
Content Type ◽  
Carbapenem Resistant

ABSTRACT The recent escalation of occurrences of carbapenem-resistant Pseudomonas aeruginosa has been recognized globally and threatens to erode the widespread clinical utility of the carbapenem class of compounds for this prevalent health care-associated pathogen. Here, we compared the in vitro inhibitory activity of ceftazidime-avibactam and ceftolozane-tazobactam against 290 meropenem-nonsusceptible Pseudomonas aeruginosa nonduplicate clinical isolates from 34 U.S. hospitals using reference broth microdilution methods. Ceftazidime-avibactam and ceftolozane-tazobactam were active, with ceftolozane-tazobactam having significantly higher inhibitory activity than ceftazidime-avibactam. The heightened inhibitory activity of ceftolozane-tazobactam was sustained when the site of origin (respiratory, blood, or wound) and nonsusceptibility to other β-lactam antimicrobials was considered. An extensive genotypic search for enzymatically driven β-lactam resistance mechanisms revealed the exclusive presence of the VIM metallo-β-lactamase among only 4% of the subset of isolates nonsusceptible to ceftazidime-avibactam, ceftolozane-tazobactam, or both. These findings suggest an important role for both ceftazidime-avibactam and ceftolozane-tazobactam against carbapenem-nonsusceptible Pseudomonas aeruginosa. Further in vitro and in vivo studies are needed to better define the clinical utility of these novel therapies against the increasingly prevalent threat of multidrug-resistant Pseudomonas aeruginosa.

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