scholarly journals Antioxidant Enzyme Expression in Clinical Isolates of Pseudomonas aeruginosa: Identification of an Atypical Form of Manganese Superoxide Dismutase

2001 ◽  
Vol 69 (12) ◽  
pp. 7396-7401 ◽  
Author(s):  
Bradley E. Britigan ◽  
Rachel A. Miller ◽  
Daniel J. Hassett ◽  
Michael A. Pfaller ◽  
Michael L. McCormick ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antioxidant Enzyme ◽  
Catalase Activity ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Growth Conditions ◽  
Clinical Isolates ◽  
Missense Mutations ◽  
Enzyme Expression

ABSTRACT Expression of superoxide dismutases (FeSOD and MnSOD) and catalases by laboratory strains of Pseudomonas aeruginosa is modulated by exogenous factors. Whether clinical isolates behave similarly and whether antioxidant enzyme expression influencesP. aeruginosa virulence remain unclear. Fifty-sevenP. aeruginosa blood culture isolates, plus seven pairs of blood and local-site isolates, were examined for FeSOD, MnSOD, and catalase production in vitro. Under iron-replete growth conditions FeSOD and catalase activities were maximized. MnSOD was not detected. FeSOD and catalase activity decreased under iron-limited growth conditions, whereas MnSOD activity appeared. SOD and catalase activity did not change with site of isolation or by patient. MnSOD could not be expressed by one isolate due to a missense mutation insodA that produced a premature stop codon. Eleven percent of the isolates expressed a novel, rapidly migrating MnSOD that was associated with missense mutations in the normal stop codon ofsodA. We conclude that clinical P. aeruginosa isolates vary little in FeSOD and catalase expression. Some strains produce a newly described MnSOD variant, whereas one is deficient in MnSOD production. The absence of MnSOD expression in a P. aeruginosa strain causing invasive human disease indicates that MnSOD is probably not essential forP. aeruginosa virulence.

scholarly journals In VitroActivities of the Novel Investigational Tetrazoles VT-1161 and VT-1598 Compared to the Triazole Antifungals against Azole-Resistant Strains and Clinical Isolates ofCandida albicans

2019 ◽  
Vol 63 (6) ◽  
Author(s):  
Andrew T. Nishimoto ◽  
Nathan P. Wiederhold ◽  
Stephanie A. Flowers ◽  
Qing Zhang ◽  
Steven L. Kelly ◽  
...  
Keyword(s):  
Fungal Infections ◽  
Stop Codon ◽  
Geometric Mean ◽  
Premature Stop Codon ◽  
Resistance Mechanisms ◽  
Clinical Isolates ◽  
Azole Resistance ◽  
Content Type ◽  
Mutant Strains

ABSTRACTThe fungal Cyp51-specific inhibitors VT-1161 and VT-1598 have emerged as promising new therapies to combat fungal infections, includingCandidaspp. To evaluate theirin vitroactivities compared to other azoles, MICs were determined by Clinical and Laboratory Standards Institute (CLSI) method for VT-1161, VT-1598, fluconazole, voriconazole, itraconazole, and posaconazole against 68 C. albicansclinical isolates well characterized for azole resistance mechanisms and mutant strains representing individual azole resistance mechanisms. VT-1161 and VT-1598 demonstrated potent activity (geometric mean MICs ≤0.15 μg/ml) against predominantly fluconazole-resistant (≥8 μg/ml) isolates. However, five of 68 isolates exhibited MICs greater than six dilutions (>2 μg/ml) to both tetrazoles compared to fluconazole-susceptible isolates. Four of these isolates likewise exhibited high MICs beyond the upper limit of the assay for all triazoles tested. A premature stop codon inERG3likely explained the high-level resistance in one isolate. VT-1598 was effective against strains with hyperactive Tac1, Mrr1, and Upc2 transcription factors and against mostERG11mutant strains. VT-1161 MICs were elevated compared to the control strain SC5314 for hyperactive Tac1 strains and two strains with Erg11 substitutions (Y132F and Y132F&K143R) but showed activity against hyperactive Mrr1 and Upc2 strains. While mutations affecting Erg3 activity appear to greatly reduce susceptibility to VT-1161 and VT-1598, the elevated MICs of both tetrazoles for four isolates could not be explained by known azole resistance mechanisms, suggesting the presence of undescribed resistance mechanisms to triazole- and tetrazole-based sterol demethylase inhibitors.

scholarly journals Interplay of Efflux System, ampC, and oprD Expression in Carbapenem Resistance of Pseudomonas aeruginosa Clinical Isolates

2006 ◽  
Vol 50 (5) ◽  
pp. 1633-1641 ◽  
Author(s):  
John Quale ◽  
Simona Bratu ◽  
Jyoti Gupta ◽  
David Landman
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Target Genes ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Carbapenem Resistance ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Carbapenem Resistant ◽  
High Level ◽  
Increased Activity

ABSTRACT Carbapenems are important agents for the therapy of infections due to multidrug-resistant Pseudomonas aeruginosa; the development of carbapenem resistance hampers effective therapeutic options. To assess the mechanisms leading to resistance, 33 clinical isolates with differing degrees of carbapenem susceptibility were analyzed for the expression of the chromosomal β-lactamase (ampC), the porin that is important for the entry of carbapenems (oprD), and the proteins involved in four efflux systems (mexA, mexC, mexE, and mexX). Real-time reverse transcriptase PCR was performed using primers and fluorescent probes for each of the target genes. The sequencing of regulatory genes (ampR, mexR, nalC, nalD, mexT, and mexZ) was also performed. Diminished expression of oprD was present in all imipenem- and meropenem-resistant isolates but was not required for ertapenem resistance. Increased expression of ampC was not observed in several isolates that were overtly resistant to carbapenems. Increased expression of several efflux systems was observed in many of the carbapenem-resistant isolates. Increased efflux activity correlated with high-level ertapenem resistance and reduced susceptibility to meropenem and aztreonam. Most isolates with increased expression of mexA had mutations affecting nalC and/or nalD. Two isolates with mutations leading to a premature stop codon in mexZ had markedly elevated mexX expressions, although mutations in mexZ were not a prerequisite for overexpression. β-Lactam resistance in clinical isolates of P. aeruginosa is a result of the interplay between diminished production of oprD, increased activity of ampC, and several efflux systems.

scholarly journals Cephalosporin nitric oxide-donor prodrug DEA-C3D disperses biofilms formed by clinical cystic fibrosis isolates of Pseudomonas aeruginosa

2019 ◽  
Vol 75 (1) ◽  
pp. 117-125 ◽  
Author(s):  
Odel Soren ◽  
Ardeshir Rineh ◽  
Diogo G Silva ◽  
Yuming Cai ◽  
Robert P Howlin ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Clinical Isolates ◽  
Nitric Oxide Donor ◽  
Confocal Laser ◽  
Broth Microdilution Method

Abstract Objectives The cephalosporin nitric oxide (NO)-donor prodrug DEA-C3D (‘DiEthylAmin-Cephalosporin-3′-Diazeniumdiolate’) has been shown to initiate the dispersal of biofilms formed by the Pseudomonas aeruginosa laboratory strain PAO1. In this study, we investigated whether DEA-C3D disperses biofilms formed by clinical cystic fibrosis (CF) isolates of P. aeruginosa and its effect in combination with two antipseudomonal antibiotics, tobramycin and colistin, in vitro. Methods β-Lactamase-triggered release of NO from DEA-C3D was confirmed using a gas-phase chemiluminescence detector. MICs for P. aeruginosa clinical isolates were determined using the broth microdilution method. A crystal violet staining technique and confocal laser scanning microscopy were used to evaluate the effects of DEA-C3D on P. aeruginosa biofilms alone and in combination with tobramycin and colistin. Results DEA-C3D was confirmed to selectively release NO in response to contact with bacterial β-lactamase. Despite lacking direct, cephalosporin/β-lactam-based antibacterial activity, DEA-C3D was able to disperse biofilms formed by three P. aeruginosa clinical isolates. Confocal microscopy revealed that DEA-C3D in combination with tobramycin produces similar reductions in biofilm to DEA-C3D alone, whereas the combination with colistin causes near complete eradication of P. aeruginosa biofilms in vitro. Conclusions DEA-C3D is effective in dispersing biofilms formed by multiple clinical isolates of P. aeruginosa and could hold promise as a new adjunctive therapy to patients with CF.

Effect of sub-inhibitory concentrations of cefepime on biofilm formation by Pseudomonas aeruginosa

2021 ◽  
pp. 1-8
Author(s):  
Soheir A.A. Hagras ◽  
Alaa El-Dien M.S. Hosny ◽  
Omneya M. Helmy ◽  
Mounir M. Salem-Bekhit ◽  
Faiyaz Shakeel ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Clinical Isolates ◽  
Polymeric Chain ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Protein Encoding ◽  
Encoding Gene ◽  
Fimbrial Protein

This study investigated the effect of cefepime at sub-minimum inhibitory concentrations (sub-MICs) on in vitro biofilm formation (BF) by clinical isolates of Pseudomonas aeruginosa. The effect of cefepime at sub-MIC levels (½–1/256 MIC) on in vitro BF by six clinical isolates of P. aeruginosa was phenotypically assessed following 24 and 48 h of challenge using the tissue culture plate (TCP) assay. Quantitative real-time polymeric chain reaction (qRT-PCR) was employed to observe the change in expression of three biofilm-related genes, namely, a protease-encoding gene (lasA), fimbrial protein-encoding gene (cupA1), and alginate-encoding gene (algC), in a weak biofilm-producing strain of P. aeruginosa following 24 and 48 h of challenge with sub-MICs of cefepime. The BF morphology in response to cefepime was imaged using scanning electron microscopy (SEM). The TCP assay showed strain-, time-, and concentration-dependent changes in in vitro BF in P. aeruginosa following challenge with sub-MICs of cefepime, with a profound increase in strains with inherently no or weak biofilm-producing ability. RT-PCR revealed time-dependent upregulation in the expression of the investigated genes following challenge with ½ and ¼ MIC levels, as confirmed by SEM. Cefepime at sub-MICs could upregulate the expression of BF-related genes and enhance BF by P. aeruginosa clinical isolates.

scholarly journals Membrane blebbing as an assessment of functional rescue of dysferlin-deficient human myotubes via nonsense suppression

2010 ◽  
Vol 109 (3) ◽  
pp. 901-905 ◽  
Author(s):  
Bingjing Wang ◽  
Zhaohui Yang ◽  
Becky K. Brisson ◽  
Huisheng Feng ◽  
Zhiqian Zhang ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Nonsense Suppression ◽  
Muscle Membrane ◽  
Membrane Repair ◽  
Vitro Assay ◽  
Membrane Blebbing ◽  
Human Myotubes

Mutations that result in the loss of the protein dysferlin result in defective muscle membrane repair and cause either a form of limb girdle muscular dystrophy (type 2B) or Miyoshi myopathy. Most patients are compound heterozygotes, often carrying one allele with a nonsense mutation. Using dysferlin-deficient mouse and human myocytes, we demonstrated that membrane blebbing in skeletal muscle myotubes in response to hypotonic shock requires dysferlin. Based on this, we developed an in vitro assay to assess rescue of dysferlin function in skeletal muscle myotubes. This blebbing assay may be useful for drug discovery/validation for dysferlin deficiency. With this assay, we demonstrate that the nonsense suppression drug, ataluren (PTC124), is able to induce read-through of the premature stop codon in a patient with a R1905X mutation in dysferlin and produce sufficient functional dysferlin (∼15% of normal levels) to rescue myotube membrane blebbing. Thus ataluren is a potential therapeutic for dysferlin-deficient patients harboring nonsense mutations.

scholarly journals In Vitro Activity of Ceftolozane-Tazobactam vs. Antimicrobial Non-Susceptible Pseudomonas aeruginosa Clinical Isolates Obtained from Across Canada as Part of the CANWARD Study, 2008–2016

2017 ◽  
Vol 4 (suppl_1) ◽  
pp. S372-S372
Author(s):  
Andrew Walkty ◽  
Heather J Adam ◽  
Melanie Baxter ◽  
Philippe Lagace-Wiens ◽  
James Karlowsky ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Multidrug Resistant ◽  
Vitro Activity ◽  
Clinical Isolates ◽  
In Vitro Activity ◽  
Research Support ◽  
Research Relationship ◽  
Inhibitor Combination ◽  
Patient Infection

Abstract Background Ceftolozane-tazobactam (C/T) is a novel β-lactam β-lactamase inhibitor combination with a broad spectrum of activity that includes Pseudomonas aeruginosa. The purpose of this study was to evaluate the in vitro activity of C/T and relevant comparators vs. a large collection of antimicrobial non-susceptible (NS) P. aeruginosa clinical isolates obtained from patients across Canada (CANWARD, 2008–2016). Methods From January 2008 to December 2016, inclusive, 12 to 15 sentinel hospitals across Canada submitted clinical isolates from patients attending ERs, medical and surgical wards, hospital clinics, and ICUs (CANWARD). Each center was asked to annually submit clinical isolates (consecutive, one per patient/infection site) from blood, respiratory, urine, and wound infections. Susceptibility testing was performed using broth microdilution as described by CLSI. Multidrug-resistant (MDR) P. aeruginosa were defined as isolates that tested NS to at least one antimicrobial from ≥3 classes. Extensively drug-resistant (XDR) P. aeruginosa were defined as isolates that tested NS to at least one antimicrobial from ≥5 classes. Results 3229 P. aeruginosa isolates were obtained as a part of CANWARD. The in vitro activity of C/T and relevant comparators is presented below. Conclusion C/T demonstrated excellent in vitro activity vs. antimicrobial NS P. aeruginosa clinical isolates, including MDR, XDR, and meropenem NS subsets. It may prove useful in the treatment of infections caused by these organisms. Disclosures D. Hoban, Abbott: Research relationship, Research support Achaogen: Research relationship, Research support Astellas: Research relationship, Research support Merck Canada: Research relationship, Research support Merck USA: Research relationship, Research support Paratek Pharma: Research relationship, Research support Pharmascience: Research relationship, Research support Sunovion: Research relationship, Research support Tetraphase: Research relationship, Research support The Medicines Co.: Research relationship, Research support Zoetis: Research relationship, Research support; G. Zhanel, Achaogen: Research relationship, Research support Astellas: Research relationship, Research support Merck Canada: Research relationship, Research support Merck USA: Research relationship, Research support Paratek Pharma: Research relationship, Research support Pharmascience: Research relationship, Research support Sunovion: Research relationship, Research support Tetraphase: Research relationship, Research support The Medicines Co.: Research relationship, Research support Zoetis: Research relationship, Research support

scholarly journals Results from the China Antimicrobial Surveillance Network (CHINET) in 2017 of the In Vitro Activities of Ceftazidime-Avibactam and Ceftolozane-Tazobactam against Clinical Isolates of Enterobacteriaceae and Pseudomonas aeruginosa

2019 ◽  
Vol 63 (4) ◽  
Author(s):  
Dandan Yin ◽  
Shi Wu ◽  
Yang Yang ◽  
Qingyu Shi ◽  
Dong Dong ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Clinical Isolates ◽  
Surveillance Network ◽  
Content Type ◽  
Highly Active ◽  
Microdilution Method ◽  
Carbapenem Resistant ◽  
Antimicrobial Surveillance ◽  
Broth Microdilution Method

ABSTRACT The in vitro activities of ceftazidime-avibactam (CZA), ceftolozane-tazobactam (C-T), and comparators were determined for 1,774 isolates of Enterobacteriaceae and 524 isolates of Pseudomonas aeruginosa collected by 30 medical centers from the China Antimicrobial Surveillance Network (CHINET) in 2017. Antimicrobial susceptibility testing was performed by the CLSI broth microdilution method, and blaKPC and blaNDM were detected by PCR for all carbapenem-resistant Enterobacteriaceae (CRE). Ceftazidime-avibactam demonstrated potent activity against almost all Enterobacteriaceae (94.6% susceptibility; MIC50, ≤0.25 mg/liter; MIC90, ≤0.25 to >32 mg/liter) and good activity against P. aeruginosa (86.5% susceptibility; MIC50/90, 2/16 mg/liter). Among the CRE, 50.8% (189/372 isolates) were positive for blaKPC-2, which mainly existed in ceftazidime-avibactam-susceptible Klebsiella pneumoniae isolates (92.1%, 174/189). Among the CRE, 17.7% (66/372 isolates) were positive for blaNDM, which mainly existed in strains resistant to ceftazidime-avibactam (71.7%, 66/92). Ceftolozane-tazobactam showed good in vitro activity against Escherichia coli and Proteus mirabilis (MIC50/90, ≤0.5/2 mg/liter; 90.5 and 93.8% susceptibility, respectively), and the rates of susceptibility of K. pneumoniae (MIC50/90, 2/>64 mg/liter) and P. aeruginosa (MIC50/90, 1/8 mg/liter) were 52.7% and 88.5%, respectively. Among the CRE strains, 28.6% of E. coli isolates and 85% of K. pneumoniae isolates were still susceptible to ceftazidime-avibactam, but only 7.1% and 1.9% of them, respectively, were susceptible to ceftolozane-tazobactam. The rates of susceptibility of the carbapenem-resistant P. aeruginosa isolates to ceftazidime-avibactam (65.7%) and ceftolozane-tazobactam (68%) were similar. Overall, both ceftazidime-avibactam and ceftolozane-tazobactam were highly active against clinical isolates of Enterobacteriaceae and P. aeruginosa recently collected across China, and ceftazidime-avibactam showed activity superior to that of ceftolozane-tazobactam against Enterobacteriaceae, whereas ceftolozane-tazobactam showed a better effect against P. aeruginosa.

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