Amodiaquine resistance in Plasmodium berghei is associated with PbCRT His95Pro mutation, loss of chloroquine, artemisinin and primaquine sensitivity, and high transcript levels of key transporters

Background: The human malaria parasite Plasmodium falciparum has evolved complex drug evasion mechanisms to all available antimalarials. To date, the combination of amodiaquine-artesunate is among the drug of choice for treatment of uncomplicated malaria. In this combination, a short acting, artesunate is partnered with long acting, amodiaquine for which resistance may emerge rapidly especially in high transmission settings. Here, we used a rodent malaria parasite Plasmodium berghei ANKA as a surrogate of P. falciparum to investigate the mechanisms of amodiaquine resistance. Methods: We used serial technique to select amodiaquine resistance by submitting the parasites to continuous amodiaquine pressure. We then employed the 4-Day Suppressive Test to monitor emergence of resistance and determine the cross-resistance profiles. Finally, we genotyped the resistant parasite by PCR amplification, sequencing and relative quantitation of mRNA transcript of targeted genes. Results: Submission of P. berghei ANKA to amodiaquine pressure yielded resistant parasite within thirty-six passages. The effective dosage that reduced 90% of parasitaemia (ED90) of sensitive line and resistant line were 4.29mg/kg and 19.13mg/kg, respectively. After freezing at -80ºC for one month, the resistant parasite remained stable with an ED90 of 18.22mg/kg. Amodiaquine resistant parasites are also resistant to chloroquine (6fold), artemether (10fold), primaquine (5fold), piperaquine (2fold) and lumefantrine (3fold). Sequence analysis of Plasmodium berghei chloroquine resistant transporter revealed His95Pro mutation. No variation was identified in Plasmodium berghei multidrug resistance gene-1 (Pbmdr1), Plasmodium berghei deubiquitinating enzyme-1 or Plasmodium berghei Kelch13 domain nucleotide sequences. Amodiaquine resistance is also accompanied by high mRNA transcripts of key transporters; Pbmdr1, V-type/H+ pumping pyrophosphatase-2 and sodium hydrogen ion exchanger-1 and Ca2+/H+ antiporter. Conclusions: Selection of amodiaquine resistance yielded stable “multidrug-resistant’’ parasites and thus may be used to study common resistance mechanisms associated with other antimalarial drugs. Genome wide studies may elucidate other functionally important genes controlling AQ resistance in P. berghei.

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Amodiaquine resistance in Plasmodium berghei is associated with PbCRT His95Pro mutation, loss of chloroquine, artemisinin and primaquine sensitivity, and high transcript levels of key transporters

Wellcome Open Research ◽

10.12688/wellcomeopenres.11768.2 ◽

2018 ◽

Vol 2 ◽

pp. 44 ◽

Cited By ~ 1

Author(s):

Loise Ndung'u ◽

Benard Langat ◽

Esther Magiri ◽

Joseph Ng'ang'a ◽

Beatrice Irungu ◽

...

Keyword(s):

Plasmodium Berghei ◽

Malaria Parasite ◽

Pcr Amplification ◽

Multidrug Resistant ◽

Resistance Mechanisms ◽

Cross Resistance ◽

Multidrug Resistance Gene ◽

Resistance Level ◽

Drug Of Choice ◽

Long Acting

Background: The human malaria parasite Plasmodium falciparum has evolved drug evasion mechanisms to all available antimalarials. The combination of amodiaquine-artesunate is among the drug of choice for treatment of uncomplicated malaria. In this combination, a short-acting, artesunate is partnered with long-acting, amodiaquine for which resistance may emerge rapidly especially in high transmission settings. Here, we used a rodent malaria parasite Plasmodium berghei ANKA as a surrogate of P. falciparum to investigate the mechanisms of amodiaquine resistance. Methods: We used the ramp up approach to select amodiaquine resistance. We then employed the 4-Day Suppressive Test to measure the resistance level and determine the cross-resistance profiles. Finally, we genotyped the resistant parasite by PCR amplification, sequencing and relative quantitation of mRNA transcript of targeted genes. Results: Submission of the parasite to amodiaquine pressure yielded resistant line within thirty-six passages. The effective doses that reduced 90% of parasitaemia (ED90) of the sensitive and resistant lines were 4.29mg/kg and 19.13mg/kg respectively. The selected parasite retained resistance after ten passage cycles in the absence of the drug and freezing at -80ºC for one month with ED90 of 20.34mg/kg and 18.22mg/kg. The parasite lost susceptibility to chloroquine by (6-fold), artemether (10-fold), primaquine (5-fold), piperaquine (2-fold) and lumefantrine (3-fold). Sequence analysis of Plasmodium berghei chloroquine-resistant transporter revealed His95Pro mutation. We found no variation in the nucleotide sequences of Plasmodium berghei multidrug resistance gene-1 (Pbmdr1), Plasmodium berghei deubiquitinating enzyme-1 or Plasmodium berghei Kelch13 domain. However, high mRNA transcripts of essential transporters; Pbmdr1, V-type/H+ pumping pyrophosphatase-2 and sodium hydrogen ion exchanger-1 and Ca2+/H+ antiporter accompanies amodiaquine resistance. Conclusions: The selection of amodiaquine resistance yielded stable “multidrug-resistant’’ parasites and thus may be used to study shared resistance mechanisms associated with other antimalarial drugs. Genome-wide analysis of the parasite may elucidate other functionally relevant genes controlling AQ resistance in P. berghei.

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Isolation and characterization of Drosophila multidrug resistance gene homologs

Molecular and Cellular Biology ◽

10.1128/mcb.11.8.3940-3948.1991 ◽

1991 ◽

Vol 11 (8) ◽

pp. 3940-3948

Author(s):

C T Wu ◽

M Budding ◽

M S Griffin ◽

J M Croop

Keyword(s):

Amino Acid ◽

Cell Lines ◽

Multidrug Resistant ◽

Amino Acid Identity ◽

Resistant Cell ◽

Cross Resistance ◽

Multidrug Resistance Gene ◽

Isolation And Characterization ◽

P Glycoprotein

Mammalian multidrug-resistant cell lines, selected for resistance to a single cytotoxic agent, display cross-resistance to a broad spectrum of structurally and functionally unrelated compounds. These cell lines overproduce a membrane protein, the P-glycoprotein, which is encoded by a member(s) of a multigene family, termed mdr or pgp. The amino acid sequence of the P-glycoprotein predicts an energy-dependent transport protein with homology to a large superfamily of proteins which transport a wide variety of substances. This report describes the isolation and characterization of two Drosophila homologs of the mammalian mdr gene. These homologs, located in chromosomal sections 49EF and 65A, encode proteins that share over 40% amino acid identity to the human and murine mdr P-glycoproteins. Fly strains bearing disruptions in the homolog in section 49EF have been constructed and implicate this gene in conferring colchicine resistance to the organism. This work sets the foundation for the molecular and genetic analysis of mdr homologs in Drosophila melanogaster.

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Activity of a New Antipseudomonal Cephalosporin, CXA-101 (FR264205), against Carbapenem-Resistant and Multidrug-Resistant Pseudomonas aeruginosa Clinical Strains

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00834-09 ◽

2009 ◽

Vol 54 (2) ◽

pp. 846-851 ◽

Cited By ~ 64

Author(s):

Carlos Juan ◽

Laura Zamorano ◽

José L. Pérez ◽

Yigong Ge ◽

Antonio Oliver

Keyword(s):

Pcr Amplification ◽

Multidrug Resistant ◽

Cross Resistance ◽

Cloning And Sequencing ◽

Complete Collection ◽

Highly Active ◽

Carbapenem Resistant ◽

Extended Spectrum ◽

Almost All ◽

Hospital Study

ABSTRACT The activity of the new cephalosporin CXA-101 (CXA), previously designated FR264205, was evaluated against a collection of 236 carbapenem-resistant P. aeruginosa isolates, including 165 different clonal types, from a Spanish multicenter (127-hospital) study. The MICs of CXA were compared to the susceptibility results for antipseudomonal penicillins, cephalosporins, carbapenems, aminoglycosides, and fluoroquinolones. The MIC of CXA in combination with tazobactam (4 and 8 μg/ml) was determined for strains with high CXA MICs. The presence of acquired β-lactamases was investigated by isoelectric focusing and PCR amplification followed by sequencing. Additional β-lactamase genes were identified by cloning and sequencing. The CXA MIC50/MIC90 for the complete collection of carbapenem-resistant P. aeruginosa isolates was 1/4 μg/ml, with 95.3% of the isolates showing an MIC of ≤8 μg/ml. Cross-resistance with any of the antibiotics tested was not observed; the MIC50/MIC90 of CXA-101 was still 1/4 when multidrug-resistant (MDR) strains (42% of all tested isolates) or AmpC-hyperproducing clones (53%) were analyzed. Almost all (10/11) of the strains showing a CXA MIC of >8 μg/ml produced a horizontally acquired β-lactamase, including the metallo-β-lactamase (MBL) VIM-2 (one strain), the extended-spectrum β-lactamase (ESBL) PER-1 (one strain), several extended-spectrum OXA enzymes (OXA-101 [one strain], OXA-17 [two strains], and a newly described OXA-2 derivative [W159R] designated OXA-144 [four strains]), and a new BEL variant (BEL-3) ESBL (one strain), as identified by cloning and sequencing. Synergy with tazobactam in these 11 strains was limited, although 8 μg/ml reduced the mean CXA MIC by 2-fold. CXA is highly active against carbapenem-resistant P. aeruginosa isolates, including MDR strains. Resistance was restricted to still-uncommon strains producing an acquired MBL or ESBL.

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The Building Blocks of Antimicrobial Resistance in Pseudomonas aeruginosa: Implications for Current Resistance-Breaking Therapies

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.665759 ◽

2021 ◽

Vol 11 ◽

Author(s):

R. Frèdi Langendonk ◽

Daniel R. Neill ◽

Joanne L. Fothergill

Keyword(s):

Adjuvant Therapy ◽

Antimicrobial Stewardship ◽

Acquired Resistance ◽

High Stress ◽

Building Blocks ◽

Multidrug Resistant ◽

Resistance Mechanisms ◽

World Health ◽

Cross Resistance ◽

Great Promise

P. aeruginosa is classified as a priority one pathogen by the World Health Organisation, and new drugs are urgently needed, due to the emergence of multidrug-resistant (MDR) strains. Antimicrobial-resistant nosocomial pathogens such as P. aeruginosa pose unwavering and increasing threats. Antimicrobial stewardship has been a challenge during the COVID-19 pandemic, with a majority of those hospitalized with SARS-CoV2 infection given antibiotics as a safeguard against secondary bacterial infection. This increased usage, along with increased handling of sanitizers and disinfectants globally, may further accelerate the development and spread of cross-resistance to antibiotics. In addition, P. aeruginosa is the primary causative agent of morbidity and mortality in people with the life-shortening genetic disease cystic fibrosis (CF). Prolonged periods of selective pressure, associated with extended antibiotic treatment and the actions of host immune effectors, results in widespread adaptive and acquired resistance in P. aeruginosa found colonizing the lungs of people with CF. This review discusses the arsenal of resistance mechanisms utilized by P. aeruginosa, how these operate under high-stress environments such as the CF lung and how their interconnectedness can result in resistance to multiple antibiotic classes. Intrinsic, adaptive and acquired resistance mechanisms will be described, with a focus on how each layer of resistance can serve as a building block, contributing to multi-tiered resistance to antimicrobial activity. Recent progress in the development of anti-resistance adjuvant therapies, targeting one or more of these building blocks, should lead to novel strategies for combatting multidrug resistant P. aeruginosa. Anti-resistance adjuvant therapy holds great promise, not least because resistance against such therapeutics is predicted to be rare. The non-bactericidal nature of anti-resistance adjuvants reduce the selective pressures that drive resistance. Anti-resistance adjuvant therapy may also be advantageous in facilitating efficacious use of traditional antimicrobials, through enhanced penetration of the antibiotic into the bacterial cell. Promising anti-resistance adjuvant therapeutics and targets will be described, and key remaining challenges highlighted. As antimicrobial stewardship becomes more challenging in an era of emerging and re-emerging infectious diseases and global conflict, innovation in antibiotic adjuvant therapy can play an important role in extending the shelf-life of our existing antimicrobial therapeutic agents.

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Isolation and characterization of Drosophila multidrug resistance gene homologs.

Molecular and Cellular Biology ◽

10.1128/mcb.11.8.3940 ◽

1991 ◽

Vol 11 (8) ◽

pp. 3940-3948 ◽

Cited By ~ 81

Author(s):

C T Wu ◽

M Budding ◽

M S Griffin ◽

J M Croop

Keyword(s):

Amino Acid ◽

Cell Lines ◽

Multidrug Resistant ◽

Amino Acid Identity ◽

Resistant Cell ◽

Cross Resistance ◽

Multidrug Resistance Gene ◽

Isolation And Characterization ◽

P Glycoprotein

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Art-175 Is a Highly Efficient Antibacterial against Multidrug-Resistant Strains and Persisters of Pseudomonas aeruginosa

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02668-14 ◽

2014 ◽

Vol 58 (7) ◽

pp. 3774-3784 ◽

Cited By ~ 90

Author(s):

Yves Briers ◽

Maarten Walmagh ◽

Barbara Grymonprez ◽

Manfred Biebl ◽

Jean-Paul Pirnay ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Outer Membrane ◽

Time Lapse ◽

Bactericidal Effect ◽

Multidrug Resistant ◽

Resistance Mechanisms ◽

Cross Resistance ◽

Chronic Infections ◽

Content Type ◽

Resistant Strains

ABSTRACTArtilysins constitute a novel class of efficient enzyme-based antibacterials. Specifically, they covalently combine a bacteriophage-encoded endolysin, which degrades the peptidoglycan, with a targeting peptide that transports the endolysin through the outer membrane of Gram-negative bacteria. Art-085, as well as Art-175, its optimized homolog with increased thermostability, are each composed of the sheep myeloid 29-amino acid (SMAP-29) peptide fused to the KZ144 endolysin. In contrast to KZ144, Art-085 and Art-175 pass the outer membrane and killPseudomonas aeruginosa, including multidrug-resistant strains, in a rapid and efficient (∼5 log units) manner. Time-lapse microscopy confirms that Art-175 punctures the peptidoglycan layer within 1 min, inducing a bulging membrane and complete lysis. Art-175 is highly refractory to resistance development by naturally occurring mutations. In addition, the resistance mechanisms against 21 therapeutically used antibiotics do not show cross-resistance to Art-175. Since Art-175 does not require an active metabolism for its activity, it has a superior bactericidal effect againstP. aeruginosapersisters (up to >4 log units compared to that of the untreated controls). In summary, Art-175 is a novel antibacterial that is well suited for a broad range of applications in hygiene and veterinary and human medicine, with a unique potential to target persister-driven chronic infections.

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Efficacy of Artilysin Art-175 against Resistant and Persistent Acinetobacter baumannii

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00285-16 ◽

2016 ◽

Vol 60 (6) ◽

pp. 3480-3488 ◽

Cited By ~ 52

Author(s):

Valerie Defraine ◽

Joris Schuermans ◽

Barbara Grymonprez ◽

Sander K. Govers ◽

Abram Aertsen ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Outer Membrane ◽

Multidrug Resistant ◽

Resistance Mechanisms ◽

Cross Resistance ◽

Content Type ◽

Highly Active ◽

Development Of Resistance ◽

Microfluidic Flow ◽

Dividing Cells

Bacteriophage-encoded endolysins have shown promise as a novel class of antibacterials with a unique mode of action, i.e., peptidoglycan degradation. However, Gram-negative pathogens are generally not susceptible due to their protective outer membrane. Artilysins overcome this barrier. Artilysins are optimized, engineered fusions of selected endolysins with specific outer membrane-destabilizing peptides. Artilysin Art-175 comprises a modified variant of endolysin KZ144 with an N-terminal fusion to SMAP-29. Previously, we have shown the high susceptibility ofPseudomonas aeruginosato Art-175. Here, we report that Art-175 is highly bactericidal against stationary-phase cells of multidrug-resistantAcinetobacter baumannii, even resulting in a complete elimination of large inocula (≥108CFU/ml). Besides actively dividing cells, Art-175 also kills persisters. Instantaneous killing ofA. baumanniiupon contact with Art-175 could be visualized after immobilization of the bacteria in a microfluidic flow cell. Effective killing of a cell takes place through osmotic lysis after peptidoglycan degradation. The killing rate is enhanced by the addition of 0.5 mM EDTA. No development of resistance to Art-175 under selection pressure and no cross-resistance with existing resistance mechanisms could be observed. In conclusion, Art-175 represents a highly active Artilysin against bothA. baumanniiandP. aeruginosa, two of the most life-threatening pathogens of the orderPseudomonadales.

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Varied-shaped gold nanoparticles with nanogram killing efficiency as potential antimicrobial surface coatings for the medical devices

Scientific Reports ◽

10.1038/s41598-021-91847-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ewelina Piktel ◽

Łukasz Suprewicz ◽

Joanna Depciuch ◽

Sylwia Chmielewska ◽

Karol Skłodowski ◽

...

Keyword(s):

Gold Nanoparticles ◽

Medical Devices ◽

Multidrug Resistant ◽

Resistance Mechanisms ◽

Antimicrobial Spectrum ◽

Antimicrobial Coatings ◽

Low Toxicity ◽

Spherical Gold Nanoparticles ◽

Cost Efficient ◽

Bactericidal Efficiency

AbstractMedical device-associated infections are a serious medical threat, particularly for patients with impaired mobility and/or advanced age. Despite a variety of antimicrobial coatings for medical devices being explored to date, only a limited number have been introduced for clinical use. Research into new bactericidal agents with the ability to eradicate pathogens, limit biofilm formation, and exhibit satisfactory biocompatibility, is therefore necessary and urgent. In this study, a series of varied-morphology gold nanoparticles in shapes of rods, peanuts, stars and spherical-like, porous ones with potent antibacterial activity were synthesized and thoroughly tested against spectrum of Candida albicans, Pseudomonas aeruginosa, Staphylococcus aureus clinical strains, as well as spectrum of uropathogenic Escherichia coli isolates. The optimization of gold nanoparticles synthesis allowed to develop nanomaterials, which are proved to be significantly more potent against tested microbes compared with the gold nanoformulations reported to date. Notably, their antimicrobial spectrum includes strains with different drug resistance mechanisms. Facile and cost-efficient synthesis of gold nanoparticles, remarkable bactericidal efficiency at nanogram doses, and low toxicity, underline their potential for development as a new coatings, as indicated by the example of urological catheters. The presented research fills a gap in microbial studies of non-spherical gold nanoparticles for the development of antimicrobial coatings targeting multidrug-resistant pathogens responsible for device-associated nosocomial infections.

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1280. In-Vitro Activity of Cefiderocol, Imipenem/Relebactam, & Ceftazidime/Avibactam in Ceftolozane/Tazobactam Resistant Strains of Multidrug Resistant Pseudomonas aeruginosa

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.1463 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S655-S655

Author(s):

Daniel Navas ◽

Angela Charles ◽

Amy Carr ◽

Jose Alexander

Keyword(s):

Multidrug Resistant ◽

Resistance Mechanisms ◽

Vitro Activity ◽

Clinical Isolates ◽

Resistance Testing ◽

Clinical Samples ◽

In Vitro Activity ◽

Carbapenemase Gene ◽

Resistant Strains

Abstract Background The activity of imipenem/relebactam (I/R), ceftazidime/avibactam (CZA) and cefiderocol (FDC) were evaluated against clinical isolates of multidrug resistant (MDR) strains of P. aeruginosa which was resistant to ceftolozane/tazobactam (C/T). The recent increase of MDR P. aeruginosa strains isolated from clinical samples has prompted research and development of new antimicrobials that can withstand its multiple resistance mechanisms. C/T is an effective option for treatment of MDR P. aeruginosa in our facility with only 10% of resistance in MDR strains, but the emergence of resistance may occur due to the presence of a carbapenemase gene or an ampC mutation. Methods Antimicrobial susceptibility testing for C/T Etest® (bioMérieux, Inc.) were performed on all MDR strains initially screened by the VITEK2® (bioMérieux, Inc.). 10% (n=20) of all MDR isolates were resistant to C/T by the CLSI 2019 breakpoints. These resistant isolates were tested for presence of a carbapenemase gene using the GeneXpert CARBA-R (Cepheid®) PCR and against CZA Etest® (bioMérieux, Inc.) I/R gradient strips (Liofilchem®) and FDC broth microdilution (Thermo Scientific™ Sensititre™). Results A total of 20 clinical isolates of MDR P. aeruginosa resistant to C/T were tested following standardized CLSI protocols and techniques. All 20 isolates were screened for the presence of a carbapenemase gene (blaVIM, blaNDM, blaKPC, blaOXA-48, blaIMP). A blaVIM gene was detected in 6 (30%) out of 20 isolates. FDC demonstrated the greatest activity with 85% (n=17) of susceptible isolates (CLSI MIC <4µg/dL). CZA (CLSI MIC <8µg/dL) and I/R (FDA MIC <2µg/dL) showed 15% (n=3) and 10% (n=2) of susceptible isolates respectively. FDC was active against all 6 blaVIM isolates, where all 6 strains were resistant to CZA and I/R as expected. 3 isolates tested non-susceptible against FDC; additional characterization was not performed at this time. Conclusion Based on these results, FDC demonstrated the greatest in-vitro activity against C/T resistant strains of MDR P. aeruginosa. FDC also demonstrated activity against all 6 MDR P. aeruginosa carrying blaVIM gene. FDC is a strong option to consider on MDR P. aeruginosa strains based on a resistance testing algorithm and a cost/effective protocol. Disclosures All Authors: No reported disclosures

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