Influence of High Mutation Rates on the Mechanisms and Dynamics of In Vitro and In Vivo Resistance Development to Single or Combined Antipseudomonal Agents

ABSTRACT We studied the mechanisms and dynamics of the development of resistance to ceftazidime (CAZ) alone or combined with tobramycin (TOB) or ciprofloxacin (CIP) in vitro and in vivo (using a mouse model of lung infection with human antibiotic regimens). Pseudomonas aeruginosa strain PAO1 and its hypermutable derivative PAOΔmutS were used, and the results were compared with those previously obtained with CIP, TOB, and CIP plus TOB (CIP-TOB) under the same conditions. An important (200-fold) amplification of the number of resistant mutant cells was documented for PAOΔmutS-infected mice that were under CAZ treatment compared to the number for mice that received placebo, whereas the median number of resistant mutant cells was below the detection limits for mice infected by PAO1. These results were intermediate between the high amplification with CIP (50,000-fold) and the low amplification with TOB (10-fold). All CAZ-resistant single mutant cells selected in vitro or in vivo hyperproduced AmpC. On the other hand, the three combinations studied were found to be highly effective in the prevention of in vivo resistance development in mice infected with PAOΔmutS, although the highest therapeutic efficacy (in terms of mortality and total bacterial load reduction) compared to those of the individual regimens was obtained with CIP-TOB and the lowest was with CAZ-CIP. Nevertheless, mutant cells that were resistant to the three combinations tested were readily selected in vitro for PAOΔmutS (mutation rates from 1.2 × 10−9 to 5.8 × 10−11) but not for PAO1, highlighting the potential risk for antimicrobial resistance development associated with the presence of hypermutable strains, even when combined therapy was used. All five independent CAZ-TOB-resistant PAOΔmutS double mutants studied presented the same resistance mechanism (AmpC hyperproduction plus an aminoglycoside resistance mechanism not related to MexXY), whereas four different combinations of resistance mechanisms were documented for the five CAZ-CIP-resistant double mutants.

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Oxidative Stress Induction of the MexXY Multidrug Efflux Genes and Promotion of Aminoglycoside Resistance Development inPseudomonas aeruginosa

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01495-10 ◽

2010 ◽

Vol 55 (3) ◽

pp. 1068-1074 ◽

Cited By ~ 65

Author(s):

Sebastien Fraud ◽

Keith Poole

Keyword(s):

Multidrug Efflux ◽

Efflux System ◽

Aminoglycoside Resistance ◽

Resistance Development ◽

Enhanced Resistance ◽

Resistant Mutants ◽

Multidrug Efflux System

ABSTRACTExposure to reactive oxygen species (ROS) (e.g., peroxide) was shown to induce expression of the PA5471 gene, which was previously shown to be required for antimicrobial induction of the MexXY components of the MexXY-OprM multidrug efflux system and aminoglycoside resistance determinant inPseudomonas aeruginosa. mexXYwas also induced by peroxide exposure, and this too was PA5471 dependent. The prospect of ROS promotingmexXYexpression and aminoglycoside resistance recallsP. aeruginosainfection of the chronically inflamed lungs of cystic fibrosis (CF) patients, where the organism is exposed to ROS and where MexXY-OprM predominates as the mechanism of aminoglycoside resistance. While ROS did not enhance aminoglycoside resistancein vitro, long-term (8-day) exposure ofP. aeruginosato peroxide (mimicking chronicin vivoROS exposure) increased aminoglycoside resistance frequency, dependent upon PA5471 andmexXY. This enhanced resistance frequency was also seen in a mutant strain overexpressing PA5471, in the absence of peroxide, suggesting that induction of PA5471 by peroxide was key to peroxide enhancement of aminoglycoside resistance frequency. Resistant mutants selected following peroxide exposure were typically pan-aminoglycoside-resistant, withmexXYgenerally required for this resistance. Moreover, PA5471 was required formexXYexpression and aminoglycoside resistance in these as well as several CF isolates examined.

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Honokiol Restores Polymyxin Susceptibility to MCR-1-Positive Pathogens both In Vitro and In Vivo

Applied and Environmental Microbiology ◽

10.1128/aem.02346-19 ◽

2019 ◽

Vol 86 (5) ◽

Cited By ~ 1

Author(s):

Yan Guo ◽

Xiaohong Lv ◽

Yanling Wang ◽

Yonglin Zhou ◽

Na Lu ◽

...

Keyword(s):

Combination Therapy ◽

Molecular Simulation ◽

Natural Compound ◽

Combined Therapy ◽

Bacterial Load ◽

Promising Alternative ◽

Content Type ◽

Time Kill

ABSTRACT The emergence of the plasmid-mediated colistin resistance gene mcr-1 has led to serious multidrug-resistant (MDR) Enterobacteriaceae infections, which are a great threat to the clinic. This study aims to find an inhibitor of MCR-1 to reestablish the use of polymyxins against MDR Enterobacteriaceae infections. Here, we determined that the natural compound honokiol could enhance the efficacy of polymyxins against MDR Enterobacteriaceae infections by a checkerboard MIC assay, a time-kill assay, a combined disk test, Western blotting, molecular simulation dynamics, and mouse infection models. The MIC results indicated that honokiol can recover the sensitivity of polymyxins against MCR-1-positive Klebsiella pneumoniae and Escherichia coli (with a fractional inhibitory concentration index ranging from 0.09 ± 0.00 to 0.27 ± 0.06). Based on time-kill curve analysis, all of the tested bacteria were killed within 1 h following the combined therapy with honokiol and polymyxins. Molecular simulation dynamics results suggested that honokiol directly binds to the MCR-1 active region, reducing the biological activity of MCR-1. The combination of honokiol and polymyxins could increase the 40% protection rate and reduce the bacterial load on the thigh muscles of mice. Our study indicates that honokiol is a predominant natural compound whose combination therapy with polymyxins is very promising in future treatment options for MCR-1-positive Enterobacteriaceae infections. IMPORTANCE In the present study, honokiol could effectively inhibit the activity of MCR-1 and showed almost no cytotoxicity to MH-S cells. According to our results, the combination of honokiol and polymyxin had a clear synergistic effect against MCR-1-positive Enterobacteriaceae in vitro. Combination therapy also showed a powerful therapeutic effect in vivo, which can significantly improve mouse livability, reduced the load of bacteria, and reduced pathological change. This combined therapy of small molecule compounds and antibiotics may not continue to induce new bacterial resistance, due to the fact that MCR-1 targeted by honokiol is not indispensable for the bacterial viability; on the other hand, it can reduce the dosage of combined antibiotics, and it is also a promising alternative therapy for the treatment of drug-resistant infections in the future.

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Evolution of the Pseudomonas aeruginosa Aminoglycoside Mutational Resistome In Vitro and in the Cystic Fibrosis Setting

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02583-17 ◽

2018 ◽

Vol 62 (4) ◽

Cited By ~ 20

Author(s):

Carla López-Causapé ◽

Rosa Rubio ◽

Gabriel Cabot ◽

Antonio Oliver

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Respiratory Infections ◽

Aminoglycoside Resistance ◽

Resistance Development ◽

Content Type ◽

High Doses ◽

High Level

ABSTRACT Inhaled administration of high doses of aminoglycosides is a key maintenance treatment of Pseudomonas aeruginosa chronic respiratory infections in cystic fibrosis (CF). We analyzed the dynamics and mechanisms of stepwise high-level tobramycin resistance development in vitro and compared the results with those of isogenic pairs of susceptible and resistant clinical isolates. Resistance development correlated with fusA1 mutations in vitro and in vivo. pmrB mutations, conferring polymyxin resistance, were also frequently selected in vitro . In contrast, mutational overexpression of MexXY, a hallmark of aminoglycoside resistance in CF, was not observed in in vitro evolution experiments.

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Targeting IDH1/2 mutant cancers with combinations of ATR and PARP inhibitors

NAR Cancer ◽

10.1093/narcan/zcab018 ◽

2021 ◽

Vol 3 (2) ◽

Author(s):

Amrita Sule ◽

Jinny Van Doorn ◽

Ranjini K Sundaram ◽

Sachita Ganesa ◽

Juan C Vasquez ◽

...

Keyword(s):

Kinase Inhibitors ◽

Parp Inhibitor ◽

Parp Inhibitors ◽

Protein Kinase Inhibitors ◽

Isocitrate Dehydrogenase 1 ◽

Normal Product ◽

Synthetic Lethal Interactions ◽

Mutant Cells

Abstract Mutations in the isocitrate dehydrogenase-1 and -2 (IDH1/2) genes were first identified in glioma and acute myeloid leukemia (AML), and subsequently found in multiple other tumor types. These neomorphic mutations convert the normal product of enzyme, α-ketoglutarate (αKG), to the oncometabolite 2-hydroxyglutarate (2HG). Our group recently demonstrated that 2HG suppresses the high-fidelity homologous recombination (HR) DNA repair pathway, resulting in a state referred to as ‘BRCAness’, which confers exquisite sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. In this study, we sought to elucidate sensitivity of IDH1/2-mutant cells to DNA damage response (DDR) inhibitors and, whether combination therapies could enhance described synthetic lethal interactions. Here, we report that ATR (ataxia telangiectasia and Rad3-related protein kinase) inhibitors are active against IDH1/2-mutant cells, and that this activity is further potentiated in combination with PARP inhibitors. We demonstrate this interaction across multiple cell line models with engineered and endogenous IDH1/2 mutations, with robust anti-tumor activity in vitro and in vivo. Mechanistically, we found ATR and PARP inhibitor treatment induces premature mitotic entry, which is significantly elevated in the setting of IDH1/2-mutations. These data highlight the potential efficacy of targeting HR defects in IDH1/2-mutant cancers and support the development of this combination in future clinical trials.

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The Antitumor Activity of Sodium Selenite Alone and in Combination with Gemcitabine in Pancreatic Cancer: An In Vitro and In Vivo Study

Cancers ◽

10.3390/cancers13133169 ◽

2021 ◽

Vol 13 (13) ◽

pp. 3169

Author(s):

Kevin Doello ◽

Cristina Mesas ◽

Francisco Quiñonero ◽

Gloria Perazzoli ◽

Laura Cabeza ◽

...

Keyword(s):

Pancreatic Cancer ◽

Sodium Selenite ◽

Molecular Mechanisms ◽

Combined Therapy ◽

Significant Inhibition ◽

In Vivo Studies ◽

C57bl Mice ◽

Migration Capacity

Sodium selenite acts by depleting enzymes that protect against cellular oxidative stress. To determine its effect alone or in combination with gemcitabine (GMZ) in pancreatic cancer, we used PANC-1 and Pan02 cell lines and C57BL mice bearing a Pan02-generated tumor. Our results demonstrated a significant inhibition of pancreatic cancer cell viability with the use of sodium selenite alone and a synergistic effect when associated with GMZ. The molecular mechanisms of the antitumor effect of sodium selenite alone involved apoptosis-inducing factor (AIF) and the expression of phospho-p38 in the combined therapy. In addition, sodium selenite alone and in association with GMZ significantly decreased the migration capacity and colony-forming ability, reduced tumor activity in multicellular tumor spheroids (MTS) and decreased sphere formation of cancer stem cells. In vivo studies demonstrated that combined therapy not only inhibited tumor growth (65%) compared to the untreated group but also relative to sodium selenite or GMZ used as monotherapy (up to 40%), increasing mice survival. These results were supported by the analysis of C57BL/6 albino mice bearing a Pan02-generated tumor, using the IVIS system. In conclusion, our results showed that sodium selenite is a potential agent for the improvement in the treatment of pancreatic cancer and should be considered for future human clinical trials.

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DNA methylation specifies chromosomal localization of MeCP2.

Molecular and Cellular Biology ◽

10.1128/mcb.16.1.414 ◽

1996 ◽

Vol 16 (1) ◽

pp. 414-421 ◽

Cited By ~ 223

Author(s):

X Nan ◽

P Tate ◽

E Li ◽

A Bird

Keyword(s):

Dna Methylation ◽

Cultured Cells ◽

Centromeric Heterochromatin ◽

Intact Protein ◽

Chromosomal Protein ◽

Mouse Cells ◽

Low Levels ◽

Mutant Cells

MeCP2 is a chromosomal protein that is concentrated in the centromeric heterochromatin of mouse cells. In vitro, the protein binds preferentially to DNA containing a single symmetrically methylated CpG. To find out whether the heterochromatic localization of MeCP2 depended on DNA methylation, we transiently expressed MeCP2-LacZ fusion proteins in cultured cells. Intact protein was targeted to heterochromatin in wild-type cells but was inefficiently localized in mutant cells with low levels of genomic DNA methylation. Deletions within MeCP2 showed that localization to heterochromatin required the 85-amino-acid methyl-CpG binding domain but not the remainder of the protein. Thus MeCP2 is a methyl-CpG-binding protein in vivo and is likely to be a major mediator of downstream consequences of DNA methylation.

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MBX-500, a Hybrid Antibiotic withIn VitroandIn VivoEfficacy against Toxigenic Clostridium difficile

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00508-12 ◽

2012 ◽

Vol 56 (9) ◽

pp. 4786-4792 ◽

Cited By ~ 16

Author(s):

Michelle M. Butler ◽

Dean L. Shinabarger ◽

Diane M. Citron ◽

Ciarán P. Kelly ◽

Sofya Dvoskin ◽

...

Keyword(s):

Weight Gain ◽

Clostridium Difficile ◽

Severe Disease ◽

Normal Weight ◽

New Drugs ◽

Hamster Model ◽

Resistance Development ◽

Content Type

ABSTRACTClostridium difficileinfection (CDI) causes moderate to severe disease, resulting in diarrhea and pseudomembranous colitis. CDI is difficult to treat due to production of inflammation-inducing toxins, resistance development, and high probability of recurrence. Only two antibiotics are approved for the treatment of CDI, and the pipeline for therapeutic agents contains few new drugs. MBX-500 is a hybrid antibacterial, composed of an anilinouracil DNA polymerase inhibitor linked to a fluoroquinolone DNA gyrase/topoisomerase inhibitor, with potential as a new therapeutic for CDI treatment. Since MBX-500 inhibits three bacterial targets, it has been previously shown to be minimally susceptible to resistance development. In the present study, thein vitroandin vivoefficacies of MBX-500 were explored against the Gram-positive anaerobe,C. difficile. MBX-500 displayed potency across nearly 50 isolates, including those of the fluoroquinolone-resistant, toxin-overproducing NAP1/027 ribotype, performing as well as comparator antibiotics vancomycin and metronidazole. Furthermore, MBX-500 was a narrow-spectrum agent, displaying poor activity against many other gut anaerobes. MBX-500 was active in acute and recurrent infections in a toxigenic hamster model of CDI, exhibiting full protection against acute infections and prevention of recurrence in 70% of the animals. Hamsters treated with MBX-500 displayed significantly greater weight gain than did those treated with vancomycin. Finally, MBX-500 was efficacious in a murine model of CDI, again demonstrating a fully protective effect and permitting near-normal weight gain in the treated animals. These selective anti-CDI features support the further development of MBX 500 for the treatment of CDI.

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TERT Promoter Revertant Mutation Inhibits Melanoma Growth through Intrinsic Apoptosis

Biology ◽

10.3390/biology11010141 ◽

2022 ◽

Vol 11 (1) ◽

pp. 141

Author(s):

Yanbing Wang ◽

Yiwu Chen ◽

Chang Li ◽

Zhiwei Xiao ◽

Hongming Yuan ◽

...

Keyword(s):

Therapeutic Option ◽

B Cell Lymphoma ◽

Inhibitory Effect ◽

Migration And Invasion ◽

Catalytic Core ◽

Tert Promoter ◽

Human Telomerase ◽

Mutant Cells

Human telomerase is a specialized DNA polymerase whose catalytic core includes both TERT and human telomerase RNA (hTR). Telomerase in humans, which is silent in most somatic cells, is activated to maintain the telomere length (TEL) in various types of cancer cells, including melanoma. In the vast majority of tumor cells, the TERT promoter is mutated to promote proliferation and inhibit apoptosis. Here, we exploited NG-ABEmax to revert TERT -146 T to -146 C in melanoma, and successfully obtained TERT promoter revertant mutant cells. These TERT revertant mutant cells exhibited significant growth inhibition both in vitro and in vivo. Moreover, A375−146C/C cells exhibited telomere shortening and the downregulation of TERT at both the transcription and protein levels, and migration and invasion were inhibited. In addition, TERT promoter revertant mutation abrogated the inhibitory effect of mutant TERT on apoptosis via B-cell lymphoma 2 (Blc-2), ultimately leading to cell death. Collectively, the results of our work demonstrate that reverting mutations in the TERT promoter is a potential therapeutic option for melanoma.

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Evidence that the metabolite repair enzyme NAD(P)HX epimerase has a moonlighting function

Bioscience Reports ◽

10.1042/bsr20180223 ◽

2018 ◽

Vol 38 (3) ◽

Cited By ~ 10

Author(s):

Thomas D. Niehaus ◽

Mona Elbadawi-Sidhu ◽

Lili Huang ◽

Laurence Prunetti ◽

Jesse F. Gregory ◽

...

Keyword(s):

Vitamin B6 ◽

Weakly Bound ◽

E Coli ◽

Essentially Normal ◽

Metabolite Repair ◽

Mutant Cells ◽

Dehydratase Activity ◽

Related Compounds

NAD(P)H-hydrate epimerase (EC 5.1.99.6) is known to help repair NAD(P)H hydrates (NAD(P)HX), which are damage products existing as R and S epimers. The S epimer is reconverted to NAD(P)H by a dehydratase; the epimerase facilitates epimer interconversion. Epimerase deficiency in humans causes a lethal disorder attributed to NADHX accumulation. However, bioinformatic evidence suggest caution about this attribution by predicting that the epimerase has a second function connected to vitamin B6 (pyridoxal 5′-phosphate and related compounds). Specifically, (i) the epimerase is fused to a B6 salvage enzyme in plants, (ii) epimerase genes cluster on the chromosome with B6-related genes in bacteria, and (iii) epimerase and B6-related genes are coexpressed in yeast and Arabidopsis. The predicted second function was explored in Escherichia coli, whose epimerase and dehydratase are fused and encoded by yjeF. The putative NAD(P)HX epimerase active site has a conserved lysine residue (K192 in E. coli YjeF). Changing this residue to alanine cut in vitro epimerase activity by ≥95% but did not affect dehydratase activity. Mutant cells carrying the K192A mutation had essentially normal NAD(P)HX dehydratase activity and NAD(P)HX levels, showing that the mutation had little impact on NAD(P)HX repair in vivo. However, these cells showed metabolome changes, particularly in amino acids, which exceeded those in cells lacking the entire yjeF gene. The K192A mutant cells also had reduced levels of ‘free’ (i.e. weakly bound or unbound) pyridoxal 5'-phosphate. These results provide circumstantial evidence that the epimerase has a metabolic function beyond NAD(P)HX repair and that this function involves vitamin B6.

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