title: Identification of bovine-associated coagulase-negative staphylococci by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a direct transfer protocol.
abstract: This study evaluated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) for the identification of bovine-associated coagulase-negative staphylococci (CNS), a heterogeneous group of different species. Additionally, we aimed to expand the MALDI-ToF MS database with new reference spectra as required to fill the gaps within the existing commercial spectral library. A total of 258 isolates of CNS were used in the study, covering 16 different CNS species. The majority of the isolates were previously identified by rpoB gene sequencing (n = 219), and the remainder were identified by sequencing of 16S rRNA, hsp60, or both rpoB and hsp60. The genotypic identification was considered the gold standard identification. All MALDI-ToF MS identifications were carried out using the direct transfer method. In a preliminary evaluation (n = 32 isolates; 2 of each species) with the existing commercial database, MALDI-ToF MS showed a typeability of 81% (26/32) and an accuracy of 96% (25/26). In the main evaluation (n = 226 isolates), MALDI-ToF MS with the existing commercial Biotyper (Bruker Daltonics Inc., Billerica, MA) database achieved a typeability of 92.0% (208/226) and an accuracy of 99.5% (207/208). Based on the assessment of the existing commercial database and prior knowledge of the species, a total of 13 custom reference spectra, covering 8 species, were created and added to the commercial database. Using the custom reference spectra expanded database, isolates were identified by MALDI-ToF MS with 100% typeability and 100% accuracy. Whereas the MALDI-ToF MS manufacturer's cutoff for species-level identification is 2.000, the reduction of the species level cutpoint to ≥1.700 improved the species-level identification rates (from 64 to 92% for the existing commercial database) when classifying CNS isolates. Overall, MALDI-ToF MS using the direct transfer method was shown to be a highly reliable tool for the identification of bovine-associated CNS.
authors.affiliation: Department of Health Management, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island, Canada C1A 4P3. Electronic address: mcameron@upei.ca.
authors.forename: M
authors.initials: M
authors.lastname: Cameron
authors.affiliation: Department of Production Animal Health, Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada T2N 1N4.
authors.forename: H W
authors.initials: HW
authors.lastname: Barkema
authors.affiliation: Department of Production Animal Health, Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada T2N 1N4.
authors.forename: J
authors.initials: J
authors.lastname: De Buck
authors.affiliation: M-team and Mastitis and Milk Quality Research Unit, Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium.
authors.forename: S
authors.initials: S
authors.lastname: De Vliegher
authors.affiliation: Department of Health Management, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island, Canada C1A 4P3.
authors.forename: M
authors.initials: M
authors.lastname: Chaffer
authors.affiliation: Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island, Canada C1A 4P3.
authors.forename: J
authors.initials: J
authors.lastname: Lewis
authors.affiliation: Department of Health Management, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island, Canada C1A 4P3.
authors.forename: G P
authors.initials: GP
authors.lastname: Keefe
mesh_terms: D000818:Animals; D002417:Cattle; D003030:Coagulase; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013210:Staphylococcus / Q000235:genetics
chemical_list: D003030:Coagulase; D012336:RNA, Ribosomal, 16S
keywords: bovine coagulase-negative staphylococci; matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; species identification