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      • P 39 : Reliable and Reproducible Method for Rapid Identification of Nocardia Species by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

        ( Kazushi Kashio ),( Masahiro Toyokawa ),( Nagasawa Mitsuaki ) 대한임상병리사협회 2015 임상미생물검사학회 발표자료집 Vol.2015 No.-

        Background Recently, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been challenged for identification of Nocardia species. However, there is no reliable and reproducible method for Nocardia species. Methods Our modified extraction procedure is consisted of heat-inactivated bacteria after dissociation in 0.5% Tween-20, mechanical breaking of the cell wall with acid-washed glass beads and protein extraction with formic acid and acetonitrile. In this study, a panel of 25 clinical strains were characterized by a microflex LT (Bruker Daltonik). As reference methods for species identification, full-length 16S rRNA gene sequencing and some phenotypical tests were used. Results Of the 25 acquired spectra aligned with the MALDI BioTyper database, only 4 isolates (16%) were correctly identified to the genus level. In a next step, we made a own Nocardia database by the analysis of 13 strains (13 different species including N. elegans, N. otitidiscaviarum, N. asiatica, N. abscessus, N. brasiliensis, N. thailandica, N. farcinica, N. nova, N. mikamii, N. cyriacigeorgica, N. asteroids, Nocardiopsis alba, Micromonospora sp.) and registered to the MALDI BioTyper database. Then we established our database (MALDI BioTyper database + own Nocardia database) by the analysis of 12 challenge strains. Correct identification was achieved for 12 of the 12 isolates (100%), including 8 strains identified to the species level and 4 strains identified to the genus level according to the manufacture’s log score specifications. The N. elegans spectra clustered quite closely with the N. nova cluster therefore some strains of those species can’t identify species level by MALDI-TOF MS. In the estimation of reproducibility of our method intended for 4 strains, both of within-run and between-run reproducibility were excellent. Conclusion(s) These data indicate that our method is reliable, reproducible and rapid method for identification of Nocardia species without any substantial costs for consumables.

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