Agreement with nucleic acid sequencing for species present in Mycobacterium Library v1.0 was 97.6% (81/83) for isolates cultured on solid medium and 97.5% (79/81) for those cultured in broth.

ABSTRACTCastellaniella defragransis aBetaproteobacteriumcapable of coupling the oxidation of monoterpenes with denitrification. Geraniol dehydrogenase (GeDH) activity was induced during growth with limonene in comparison to growth with acetate. The N-terminal sequence of the purified enzyme directed the cloning of the corresponding open reading frame (ORF), the first bacterial gene for a GeDH (geoA, forgeranioloxidation pathway). TheC. defragransgeraniol dehydrogenase is a homodimeric enzyme that affiliates with the zinc-containing benzyl alcohol dehydrogenases in the superfamily of medium-chain-length dehydrogenases/reductases (MDR). The purified enzyme most efficiently catalyzes the oxidation of perillyl alcohol (kcat/Km= 2.02 × 106M−1s−1), followed by geraniol (kcat/Km= 1.57 × 106M−1s−1). ApparentKmvalues of <10 μM are consistent with anin vivotoxicity of geraniol above 5 μM. In the genetic vicinity ofgeoAis a putative aldehyde dehydrogenase that was namedgeoBand identified as a highly abundant protein during growth with phellandrene. Extracts ofEscherichia coliexpressinggeoBdemonstratedin vitroa geranial dehydrogenase (GaDH) activity. GaDH activity was independent of coenzyme A. The irreversible formation of geranic acid allows for a metabolic flux from β-myrcene via linalool, geraniol, and geranial to geranic acid.

e Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has demonstrated its ability to promptly identify nontuberculous mycobacteria using the Mycobacteria Library v2.0. However, some species are particularly difficult to identify reliably using this database, providing a low log(score). In this study, the identification power of an updated Mycobacteria Library (v3.0) has been evaluated. Overall, 109 NTM isolates were analyzed with both databases. The v3.0 database allowed a high-level confidence in the identification [log(score) value, >1.8] of 91.7% of the isolates versus 83.5% with the v2.0 version (P < 0.01). Rapid identification of nontuberculous mycobacteria (NTM) with matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) has outperformed molecular techniques, such as GenoType (Hain Lifescience GmbH, Nehren, Germany), and provides accurate identification that correlates well with 16S rRNA gene sequencing when applied to the most common species of NTM (1-3). The Mycobacteria Library database (Bruker Daltonik GmbH, Bremen, Germany) available so far (v2.0) provided low scores, particularly for NTM belonging to the slow-growing groups (3-5). In the present study, we assessed the power of a new database to identify NTM (i.e., Mycobacteria Library v3.0) using 109 isolates from 26 NTM species (Table 1) and compared the identification scores of version v2.0 and version v3.0.Ninety-nine nonselected NTM isolates from clinical samples and 10 reference strains (Table 1) were collected in the clinical microbiology laboratory from the Hospital Gregorio Marañón (Madrid, Spain) between January 2011 and May 2015. These isolates were routinely identified by 16S rRNA hsp65 sequencing and, in parallel, by MALDI-TOF MS using a Microflex LT benchtop mass spectrometer (Bruker Daltonik) and the Mycobacteria Library v2.0, containing 313 Mycobacterium isolates from 131 species (Bruker Daltonik) (4). Sample preparation was described elsewhere (6). Briefly, colonies of NTM isolates grown on Lowenstein-Jensen medium were harvested into a 1.5-ml Eppendorf tube with 300 l of deionized water and inactivated for 30 min at 95°C under biosafety level 3 conditions. Then, they were centrifuged at maximal speed and subsequently resuspended in 300 l of water and 900 l of absolute ethanol and centrifuged again at 13,000 rpm. The supernatant was discarded, and the pellet was taken to biosafety level 2 conditions in order to disrupt the mycobacteria cell aggregates with silica bead vortexing and extract the bacterial proteins using formic acid and acetonitrile. In the end, 1 l of supernatant was placed onto a steel plate for MALDI-TOF MS analysis. Samples were analyzed in duplicates; the species identification, using the Mycobacteria Library v2.0, and the higher log(score) value result were recorded. For comparison reasons, all of the protein spectra from the 109 NTM isolates were reanalyzed using the new Mycobacteria Library v3.0, containing 853 references from 149 Mycob...