Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Is a Superior Diagnostic Tool for the Identification and Differentiation of Mycoplasmas Isolated from Animals.

In veterinary diagnostic laboratories, identification of mycoplasmas is achieved by demanding, cost-intensive, and time-consuming methods that rely on antigenic or genetic identification. Since matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) seems to represent a promising alternative to the currently practiced cumbersome diagnostics, we assessed its applicability for the identification of almost all mycoplasma species isolated from vertebrate animals so far. For generating main spectrum profiles (MSPs), the type strains of 98 Mycoplasma, 11 Acholeplasma, and 5 Ureaplasma species and, in the case of 69 species, 1 to 7 clinical isolates were used. To complete the database, 3 to 7 representatives of 23 undescribed Mycoplasma species isolated from livestock, companion animals, and wildlife were also analyzed. A large in-house library containing 530 MSPs was generated, and the diversity of spectra within a species was assessed by constructing dendrograms based on a similarity matrix. All strains of a given species formed cohesive clusters clearly distinct from all other species. In addition, phylogenetically closely related species also clustered closely but were separated accurately, indicating that the established database was highly robust, reproducible, and reliable. Further validation of the in-house mycoplasma library using 335 independent clinical isolates of 32 mycoplasma species confirmed the robustness of the established database by achieving reliable species identification with log scores of ≥1.80. In summary, MALDI-TOF MS proved to be an excellent method for the identification and differentiation of animal mycoplasmas, combining convenience, ease, speed, precision, and low running costs. Furthermore, this method is a powerful and supportive tool for the taxonomic resolution of animal mycoplasmas.

Membrane properties of a plant-pathogenic mycoplasmalike organism.

In terms of biosystematics, the plant-pathogenic mycoplasmalike organisms (MLOs) have been tentatively placed into the class Mollicutes. Certain physiological tests have been used to distinguish families within this class: the sterol-nonrequiring Acholeplasmataceae differ from the sterol-requiring Mycoplasmataceae in that the former are more resistant to lysis by digitonin and more sensitive to lysis in hypotonic salt solutions. To test MLOs for these membrane properties and thus assist in their definitive classification, a dot-blot microassay procedure was used to detect nucleic acids released from lysed cells. The results show that MLOs resemble acholeplasmas grown in the absence of sterols in that they are resistant to digitonin and sensitive to hypotonic salt solutions. The MLOs can be differentiated from acholeplasmas grown without sterols by their greater resistance to lysis in hypotonic sucrose solutions.