Rapid extended-spectrum b-lactamases detection using DNA chips
Abstract number: O367
Leinberger D., Grimm V., Rubtsova M., Henn A., Egorov A., Schroeppel K., Wichelhaus T., Knabbe C., Schmid R., Bachmann T.
Objectives: Conventional culture based bacterial identification and antimicrobial susceptibility testing methods are too slow to enable prospective therapy and provide limited information depth and there is a high demand for improved alternatives. Molecular methods such as DNA chips open the way for rapid testing and prospective therapy. A continuously emerging problem of microbial resistance is related to Extended Spectrum Beta-Lactamases (ESBL). The majority of clinically relevant ESBL variants derive from the genes blaTEM-1 or blaSHV-1 by mutations that alter the amino acid configuration and increasingly from the acquisition and mutation of genes from the CTX-M gene family. In the past decade further enzymes like the OXA lactamases and plasmid-mediated AmpC lactamases have emerged in the clinics as a new severe problem as they are likely to hydrolyse modern cephalosporins. The presentation will demonstrate developments made under the German PathoGenoMik programme on the example of the ESBL chips as well as future routes for rapid microbial diagnostics in the clinic with special focus on novel technologies for faster assay times and microfluidic integration.
Methods: Here we describe the development and application of fluorescence based diagnostic oligonucleotide microarrays for the genotyping of TEM, SHV, and CTX-M b-lactamase variants based on allele specific hybridisation. To increase the coverage, we developed chip modules to genotype plasmid-mediated AmpC and OXA-type beta lactamases. To enable a single all-in-one b-lactam resistance test for the clinic, we fully integrated individual chip modules (TEM, SHV, CTX-M) to a single ESBL chip platform.
Results: The ESBL chip was validated using 60 clinical samples taken over one year in the clinical routine of different Gram-negative organisms (phenotypically characterised as ESBL). The detected variants included TEM-1, SHV-1, SHV-5, SHV-12, CTX-M3, CTX-M9, CTX-M14 and CTX-M15. Analytically difficult samples were correctly identified such as mixed resistances of various genotypes. All ESBL phenotypes could be ascribed to the presence of a CTX-M variant (78%) or SHV variant (22%), whereas no ESBL TEM variant was found. As the total assay could be performed in 4h.
Conclusion: The ESBL chip presents a promising example in our development of a panel of diagnostic microbial chips. They can now be further integrated with the whole analytical chain to provide clinically meaningful and cost effective tools.
|Session name:||18th European Congress of Clinical Microbiology and Infectious Diseases|
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