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Molecular recognition of live methicillin-resistant staphylococcus aureus cells using DNA aptamers.

ABSTRACT: AIM:To generate DNA-aptamers binding to Methicillin-resistant Staphylococcus aureus (MRSA). METHODS:The Cell-Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology was used to run the selection against MRSA bacteria and develop target-specific aptamers. MRSA bacteria were targeted while Enterococcus faecalis bacteria were used for counter selection during that process. Binding assays to determine the right aptamer candidates as well as binding assays on clinical samples were performed through flow cytometry and analyzed using the FlowJo software. The characterization of the aptamers was done by determination of their Kd values and determined by analysis of flow data at different aptamer concentration using SigmaPlot. Finally, the recognition of the complex Gold-nanoparticle-aptamer to the bacteria cells was observed using transmission electron microscopy (TEM). RESULTS:During the cell-SELEX selection process, 17 rounds were necessary to generate enrichment of the pool. While the selection was run using fixed cells, it was shown that the binding of the pools with live cells was giving similar results. After sequencing and analysis of the two last pools, four sequences were identified to be aptamer candidates. The characterization of those aptamers showed that based on their Kd values, DTMRSA4 presented the best binding with a Kd value of 94.61 ± 18.82 nmol/L. A total of ten clinical samples of MRSA , S. aureus and Enterococcus faecalis were obtained to test those aptamers and determine their binding on a panel of samples. DTMRSA1 and DTMRSA3 showed the best results regarding their specificity to MRSA , DTMRSA1 being the most specific of all. Finally, those aptamers were coupled with gold-nanoparticle and their binding to MRSA cells was visualized through TEM showing that adduction of nanoparticles on the aptamers did not change their binding property. CONCLUSION:A total of four aptamers that bind to MRSA were obtained with Kd values ranking from 94 to 200 nmol/L.

SUBMITTER: Turek D

PROVIDER: S-EPMC4244701 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Molecular recognition of live <i>methicillin-resistant staphylococcus aureus</i> cells using DNA aptamers.

Turek Diane D Van Simaeys Dimitri D Johnson Judith J Ocsoy Ismail I Tan Weihong W

World journal of translational medicine 20130101 3

<h4>Aim</h4>To generate DNA-aptamers binding to <i>Methicillin-resistant Staphylococcus aureus (MRSA)</i>.<h4>Methods</h4>The Cell-Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology was used to run the selection against <i>MRSA</i> bacteria and develop target-specific aptamers. <i>MRSA</i> bacteria were targeted while <i>Enterococcus faecalis</i> bacteria were used for counter selection during that process. Binding assays to determine the right aptamer candidates as wel ...[more]

PMID: 25436184

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Project description:Methicillin-resistant Staphylococcus aureus (MRSA) is a major human health problem and recently, domestic animals are described as carriers and possible reservoirs. Twenty seven S. aureus isolates from five turkey farms (n = 18) and two broiler farms (n = 9) were obtained by culturing of choana and skin swabs from apparently healthy birds, identified by Taqman-based real-time duplex nuc-mecA-PCR and characterized by spa typing as well as by a DNA microarray based assay which covered, amongst others, a considerable number of antibiotic resistance genes, species controls, and virulence markers. The antimicrobial susceptibility profiles were tested by agar diffusion assays and genotypically confirmed by the microarray. Five different spa types (3 in turkeys and 2 in broilers) were detected. The majority of MRSA isolates (24/27) belonged to clonal complex 398-MRSA-V. The most frequently occurring spa types were accordingly t011, t034, and t899. A single CC5-MRSA-III isolated from turkey and CC398-MRSA with an unidentified/truncated SCCmec element in turkey and broiler were additionally detected. The phenotypic antimicrobial resistance profiles of S. aureus isolated from both turkeys and broilers against 14 different antimicrobials showed that all isolates were resistant to ampicillin, cefoxitin, oxacillin, doxycycline, and tetracycline. Moreover, all S. aureus isolated from broilers were resistant to erythromycin and azithromycin. All isolates were susceptible to gentamicin, chloramphenicol, sulphonamides, and fusidic acid. The resistance rate against ciprofloxacin was 55.6% in broiler isolates and 42.1% in turkey isolates. All tetracycline resistant isolates possessed genes tetK/M. All erythromycin-resistant broiler isolates carried ermA. Only one broiler isolate (11.1%) carried genes ermA, ermB, and ermC, while 55.6% of turkey isolates possessed ermA and ermB genes. Neither PVL genes (lukF/S-PV), animal-associated leukocidin (lukM and luk-P83) nor the gene encoding the toxic shock syndrome toxin (tst1) were found in turkey and broiler isolates. In conclusion, the detection of MRSA in healthy turkeys and broilers with even additional antibiotic resistance markers is of major public health concern. The difference in antibiotic resistance and virulence markers between MRSA isolates from turkeys and broilers was addressed.

Dataset Information

Molecular recognition of live methicillin-resistant staphylococcus aureus cells using DNA aptamers.

Publications

Molecular recognition of live <i>methicillin-resistant staphylococcus aureus</i> cells using DNA aptamers.

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