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Study of the multidrug membrane transporter of single living Pseudomonas aeruginosa cells using size-dependent plasmonic nanoparticle optical probes.

ABSTRACT: Multidrug membrane transporters (efflux pumps) in both prokaryotes and eukaryotes are responsible for impossible treatments of a wide variety of diseases, including infections and cancer, underscoring the importance of better understanding of their structures and functions for the design of effective therapies. In this study, we designed and synthesized two silver nanoparticles (Ag NPs) with average diameters of 13.1 +/- 2.5 nm (8.1-38.6 nm) and 91.0 +/- 9.3 nm (56-120 nm) and used the size-dependent plasmonic spectra of single NPs to probe the size-dependent transport kinetics of MexAB-OprM (multidrug transporter) in Pseudomonas aeruginosa in real time at nanometer resolution. We found that the level of accumulation of intracellular NPs in wild-type (WT) cells was higher than in nalB1 (overexpression of MexAB-OprM) but lower than in DeltaABM (deletion of MexAB-OprM). In the presence of proton ionophores (CCCP, inhibitor of proton motive force), we found that intracellular NPs in nalB1 were nearly doubled. These results suggest that MexAB-OprM is responsible for the extrusion of NPs out of cells and NPs (orders of magnitude larger than conventional antibiotics) are the substrates of the transporter, which indicates that the substrates may trigger the assembly of the efflux pump optimized for the extrusion of the encountered substrates. We found that the smaller NPs stayed inside the cells longer than larger NPs, suggesting the size-dependent efflux kinetics of the cells. This study shows that multisized NPs can be used to mimic various sizes of antibiotics for probing the size-dependent efflux kinetics of multidrug membrane transporters in single living cells.

SUBMITTER: Nallathamby PD

PROVIDER: S-EPMC2912454 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Study of the multidrug membrane transporter of single living Pseudomonas aeruginosa cells using size-dependent plasmonic nanoparticle optical probes.

Nallathamby Prakash D PD Lee Kerry J KJ Desai Tanvi T Xu Xiao-Hong Nancy XH

Biochemistry 20100701 28

Multidrug membrane transporters (efflux pumps) in both prokaryotes and eukaryotes are responsible for impossible treatments of a wide variety of diseases, including infections and cancer, underscoring the importance of better understanding of their structures and functions for the design of effective therapies. In this study, we designed and synthesized two silver nanoparticles (Ag NPs) with average diameters of 13.1 +/- 2.5 nm (8.1-38.6 nm) and 91.0 +/- 9.3 nm (56-120 nm) and used the size-depe ...[more]

PMID: 20540528

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Project description:BACKGROUND:We aimed to identify the main spreading clones, describe the resistance mechanisms associated with carbapenem- and/or multidrug-resistant P. aeruginosa and characterize patients at risk of acquiring these strains in Estonian hospitals. METHODS:Ninety-two non-duplicated carbapenem- and/or multidrug-resistant P. aeruginosa strains were collected between 27th March 2012 and 30th April 2013. Clinical data of the patients was obtained retrospectively from the medical charts. Clonal relationships of the strains were determined by whole genome sequencing and analyzed by multi-locus sequence typing. The presence of resistance genes and beta-lactamases and their origin was determined. Combined-disk method and PCR was used to evaluate carbapenemase and metallo-beta-lactamase production. RESULTS:Forty-three strains were carbapenem-resistant, 11 were multidrug-resistant and 38 were both carbapenem- and multidrug-resistant. Most strains (54%) were isolated from respiratory secretions and caused an infection (74%). Over half of the patients (57%) were???65 years old and 85% had ?1 co-morbidity; 96% had contacts with healthcare and/or had received antimicrobial treatment in the previous 90 days. Clinically relevant beta-lactamases (OXA-101, OXA-2 and GES-5) were found in 12% of strains, 27% of which were located in plasmids. No Ambler class B beta-lactamases were detected. Aminoglycoside modifying enzymes were found in 15% of the strains. OprD was defective in 13% of the strains (all with CR phenotype); carbapenem resistance triggering mutations (F170 L, W277X, S403P) were present in 29% of the strains. Ciprofloxacin resistance correlated well with mutations in topoisomerase genes gyrA (T83I, D87N) and parC (S87 L). Almost all strains (97%) with these mutations showed ciprofloxacin-resistant phenotype. Multi-locus sequence type analysis indicated high diversity at the strain level - 36 different sequence types being detected. Two sequence types (ST108 (n =?23) and ST260 (n =?18)) predominated. Whereas ST108 was associated with localized spread in one hospital and mostly carbapenem-resistant phenotype, ST260 strains occurred in all hospitals, mostly with multi-resistant phenotype and carried different resistance genotype/machinery. CONCLUSIONS:Diverse spread of local rather than international P. aeruginosa strains harboring multiple chromosomal mutations, but not plasmid-mediated Ambler class B beta-lactamases, were found in Estonian hospitals. TRIAL REGISTRATION:This trial was registered retrospectively in ClinicalTrials.gov ( NCT03343119 ).

Dataset Information

Study of the multidrug membrane transporter of single living Pseudomonas aeruginosa cells using size-dependent plasmonic nanoparticle optical probes.

Publications

Study of the multidrug membrane transporter of single living Pseudomonas aeruginosa cells using size-dependent plasmonic nanoparticle optical probes.

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