α cyano 4 hydroxycinnamic acid hcca matrix

The Bacillus sp. NRRL B-14911 and B. infantis JCM 13438 T cells grown in LB broth at 37^o C were plated onto tryptic soy agar with 5 % sheep blood agar plates. Following overnight incubation, individual colonies were picked and spotted onto the MALDI-TOF target. The spots were overlaid with 1 μl of α-cyano-4-hydroxycinnamic acid (HCCA) matrix (Bruker), and the mass spectra were acquired using MALDI-TOF MS, Microflex LT system in a linear positive mode (Bruker Daltonik, Billerica, MA). Instrument calibration was performed using standard reference BTS Escherichia coli (Bruker). For bacterial identification, MALDI Biotyper 3.0, Reference Library 1.0 Version 3.1.2 was used [22 (link), 23 (link)]. The cut-off scores used for species identification were: 2.300 to 3.000–highly probable species identification; 2.000 to 2.299–secure genus identification and probable species identification; 1.700 to 1.999–probable genus identification; and 0.000 to 1.699–not reliable for species identification.

On 20 November 2020, NYTJ35 was isolated at Xiangya Hospital of Central South University (Changsha, China) from the urine sample of a healthy male individual who was living in the community. This isolate was identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; Bruker Daltonics GmbH, Bremen, Germany). Briefly, one colony from an overnight culture was taken with a disposable loop and spotted onto a metal plate and the spots were then covered with 1 μL of a α-cyano-4-hydroxy-cinnamic acid (HCCA) matrix (Bruker Daltonik GmbH, Bremen, Germany). Then, bacterial samples on the microplate were analyzed with MALDI-TOF MS. Finally, MALDI Biotyper® (Bruker Daltonik GmbH, Bremen, Germany) software was used to classify the isolate at the genus and species level. The quality control strain was Escherichia coli ATCC 25922 (National Center for Clinical Laboratories, Beijing, China).

All isolates were initially screened using conventional methods including microscopy (Gram film), catalase and coagulase testing. Prolex™ staph latex kits (ProLab Diagnostics, Neston, UK) were used to identify CNS and S. aureus. All potential staphylococcal isolates were then fully identified using a Matrix-assisted laser desorption ionization time flight mass-spectroscopy (MALDI-TOF-MS, Microflex LT, Bruker Daltonics, Coventry, UK) in a positive linear mode (2000–20,000 m/z range). Samples were prepared as described previously (Mkrtchyan et al., 2013 (link)). In brief, 3–5 colonies of fresh cultures were added into 300 μl distilled water and mixed with 900 μl absolute ethanol. The suspension was centrifuged for 2 min at 13,000 × g and the pellets were re-suspended in 25 μl of 70% formic acid and then mixed with 25 μl pure acetonitrile. The mixture was centrifuge for 2 min at 13,000 × g. One microliter aliquots of the obtained supernatant were spotted on the MALDI target plate and overlaid with 1 μl of α-Cyano-4-hydroxycinnamic acid (HCCA) matrix (Bruker Daltonics, Coventry, UK). The resulting spectra for each isolate was analyzed by MALDI-Biotyper 3.0 software (Bruker Daltonics, Coventry, UK). Escherichia coli DH5α (Bruker Daltonics, Coventry, UK) was used as a standard for calibration and quality control.

For direct MALDI-TOF MS analysis (using Sepsityper™ kit and IH protocol), duplicate 1 μL of extracted protein supernatant from the last centrifugation were applied to the polished steel target plate and once dried, were immediately overlaid with 1 μL α-cyano-4-hydroxycinnamic acid (HCCA) matrix (Bruker Daltonics, Bremen, Germany) solution before MALDI-TOF MS analysis. The final score used for interpretation for each sample was the higher one from two measurements. For MALDI-TOF MS analysis after BC subculture (standard method), a small portion of a single colony (after 24 or 48 h of incubation) was smeared onto the ground steel target plate using a wooden cocktail stick, and covered with 1 μL HCCA matrix solution immediately. Measurements were performed with the Bruker Biotyper MALDI-TOF MS system using FlexControl 3.3 and MALDI Biotyper V.3.3.1.2 software (Bruker Daltonics) as previously described (Zhou et al., 2016 (link)).

Initially, the positive blood cultures were subjected to Gram staining to determine the presence of gram-positive or gram-negative bacteria. Based on these results, appropriate agar plates, including blood, Maconkey, chocolate, and anaerobic blood agar, were used for further culturing. The plates were grown in an incubator (Thermo Fisher Scientific, USA) at 35°C in a 5% CO₂ or anaerobic atmosphere until visible colonies appeared. Identification was then performed with the Bruker microflex MALDI-TOF MS system using the MALDI Biotyper 3.0 Realtime classification (RTC) database (Bruker Daltonics, Bremen, Germany) as described previously (Chen et al., 2013 (link); Tian et al., 2016 (link); Zhou et al., 2017 (link)). In brief, a pure bacterial colony was smeared onto a steel target plate with a wood toothpick, and 70% formic acid solution was added to lyse the bacterial cells. Once the formic acid solution dried, 1 μL of α-cyano-4-hydroxycinnamic acid (HCCA) matrix (Bruker Daltonics) solution was added for subsequent MALDI-TOF MS (Bruker Daltonics) identification. The calibration and validation of MALDI-TOF MS was carried out once a week with a bacterial test standard (BTS) according to the manufacturer's instructions (Bruker Daltonics).