Hcca matrix solution

Bacterial strains were analyzed by MALDI-TOF MS in an MBT Smart MALDI Biotyper (Bruker Daltonics, Bremen, Germany) using the updated database containing 9957 mass spectra profiles (MSPs). A few bacterial colonies of each enterococcal isolate were spotted onto the MALDI target plate. On-plate protein extraction was performed by applying 1 µL of formic acid on each target spot and letting it dry at room temperature before adding 1 µL of HCCA matrix solution (Bruker Daltonics, Bremen, Germany), following the manufacturer’s instructions. Spectra were acquired in positive mode in the range of 2000 to 20,000 Da, applying default settings [19 (link)].

Mass spectra were generated using a MALDI Biotyper 3.0 Ultraflex platform (Bruker Daltonics, Massachusetts, USA). For individual suspected S. argenteus isolates, with white colonies, 1 ml of crude protein extract or one colony was deposited on a 96‐spot polished steel target plate (Bruker Daltonics), air‐dried and covered with 1 ml of HCCA matrix solution (Bruker Daltonics) (Kolecka et al., 2013). As a positive control and calibration reference, 1 ml of Bacterial Test Standard (Bruker Daltonics) was used. The main spectrum was acquired using the MALDI Biotyper Automated Flex Control software v.3.0 (Bruker Daltonics). The identification of isolates was performed using the Bruker database and in‐house databases from Chantratita et al. (2016) and Moradigaravand et al. (2017).

Bacterial isolates were identified by MALDI-TOF mass spectrometry (MALDI-TOF MS) with ethanol-formic acid extraction procedure with HCCA matrix solution according to the manufacturer’s instructions (Bruker Daltonik GmbH, Bremen, Germany) with the usage of an extended custom database in Biotyper software (Bruker) for identification of bifidobacteria [50 (link)].
Based on species variability, the selected isolates were then identified by 16S rRNA gene amplicon sequencing. DNA was isolated using PrepMan Ultra™ (Applied Biosystems, Waltham, MA, USA) according to the manufacturer’s instructions. For PCR amplifications of the 16S rRNA gene, the universal pair of primers fD1/rP2 [51 (link)] was used, except for pair 285F/261R [52 (link)] for the identification of bifidobacteria. PCR products were purified by the EZNA Cycle Pure Kit (Omega Bio-Tek, Norcross, GA, USA) and sequenced by Eurofins Genomics (Ebersberg, Germany). The obtained sequences were processed in Chromas Lite 2.5.1 (Technelysium Pty Ltd., Tewantin, Australia), BioEdit [53 ] with ClustalW algorithm [54 (link)], and compared with 16S rRNA gene sequences in BLAST rRNA/ITS (https://blast.ncbi.nlm.nih.gov/https://www.ezbiocloud.net/, accessed on 4 November 2022) and EZBioCloud databases (https://www.ezbiocloud.net/, accessed on 4 November 2022).

At both institutes, samples were independently prepared for mass spectrometry by ethanol-formic acid extraction according to manufacturer’s instructions before being spotted on a 96-spot steel plate target and covered with alpha-cyano-4-hydroxy-cinnamic acid (HCCA) matrix solution (Bruker Daltonik GmbH, Bremen, Germany). Mass spectra were measured using a microflex LT MALDI-TOF MS system (Bruker Daltonik) operated by the Biotyper automation software flexControl (v3.4.135.0, Bruker Daltonik). To increase data robustness, twelve technical replicate spectra were acquired from four different target spots using the recommended instrument settings for bacterial identification (linear positive ion detection mode, 60 Hz laser frequency, 20 kV acceleration voltage, 18.1–18.2 kV IS2 voltage). Spectra were initially analyzed at BfR using the Bruker Biotyper software (v3.1) with MSP library version MBT_7311 (7311 entries), the Security-Relevant (SR) Database (104 entries) and a customized in-house database to confirm identification as B. melitensis.

Information about the different strains used in this study is provided in Data Set S1 in the supplemental material. Samples were either collected in the framework of routine diagnostic procedures or as part of investigations on a permit issued by the accredited Institutional Animal Care and Use Committee of the International Livestock Research Institute in Nairobi, Kenya (approval no. IACUC 2014.8). The Staphylococcaceae strains were isolated using standard methods (37 ) without any enrichment to select for high MICs and stored for subsequent use at −80°C. Species designation of the various strains was first performed via MALDI-TOF MS analysis and then, once the draft genomes were obtained (see below), confirmed by genomic sequence analysis using the type strain genome server (TYGS) (18 (link)). For MALDI-TOF MS analysis, strains were streaked onto trypticase soy agar with 5% sheep blood (TSA-B; Becton, Dickinson and Co.) and incubated at 37°C overnight. A small amount of colony material was transferred onto a steel target plate, 1 μL of 70% formic acid was added to the colony material, and it was allowed to air dry before 1 μL of HCCA matrix solution (α-cyano-4-hydroxycinnamic acid; Bruker Daltonics) was added. MALDI-TOF MS measurements to identify the species were performed using a Microflex LT instrument (Bruker Daltonics) and the MBT 7854 MSP Library.

One of the above-mentioned bacterial suspensions was pelleted after centrifugation (10,000 × g for 2 min) and the supernatant was discarded. The remaining bacterial cell pellet was dissolved in 50 μL of 70% formic acid thorough vortexing, and 50 μL of pure acetonitrile was added to the solution, vortexed, and centrifuged (10,000 × g for 2 min). One microliter of the supernatant was spotted onto a steel target plate and air-dried, which was immediately overlaid with 1 μL of HCCA matrix solution (Bruker Daltonics) (Chen et al., 2013 (link)). Identifications were performed by the Bruker microflex MALDI-TOF MS system using the MALDI Biotyper 3.0 RTC database (Bruker Daltonics) as described above.