Maldi tof ms

One hundred thirteeen clinical Klebsiella species isolates recovered in the Barnes-Jewish Hospital microbiology laboratory (St. Louis, MO) from 2016 to 2017 were evaluated in this study. Of these, 56 were consecutively collected isolates identified by Bruker Biotyper MALDI-TOF MS as K. variicola (research-use-only database v6). This identification was confirmed using a PCR-restriction fragment length polymorphism (RFLP) assay targeting the yggE gene (F: 5′-TGTTACTTAAATCGCCCTTACGGG-3′; R: 5′-CAGCGATCTGCAAAACGTCTACT-3′; restriction enzyme: BciVI) that was designed to distinguish K. variicola from K. pneumoniae. A 94.6% proportion (53/56) of isolates were confirmed as K. variicola using the yggE PCR-RFLP assay.
The remaining 58 isolates were randomly selected from a banked collection of K. pneumoniae strains historically recovered from clinical specimens (29 from urine, 25 from blood, and 1 each from abdominal wound, tracheal aspirate, bronchial washing, and bile). Each of these isolates underwent Bruker MALDI-TOF MS and yggE PCR-RFLP to confirm their identification. Five percent (3/58) were confirmed as K. variicola using MALDI-TOF MS and the yggE PCR-RFLP assay.

Gram stains were done directly from positive blood culture bottles. According to the results of the staining, specimens from the positive bottles were subcultured onto relevant agar plates. The microorganisms grown on the agar plates were identified by Vitek2 XL (bioMérieux, Marcy l’Etoile, France), by Bruker MALDI-TOF MS (Bruker Daltonics, Bremen, Germany), by growth characteristics on selective agar plates and by a panel of validated desktop spot tests, including catalase, and indole spot. The isolates (n = 100) included in the study constituted of Bacteroides fragilis (n = 35), Clostridium perfringens (13), Bacteroides thetaiotaomicron (12), Bacteroides ovatus (4), Fusobacterium nucleatum (4), Eggerthella lenta (4), Bacteroides vulgates (3), Clostridium ramosum (3), Veillonella parvula (2), Clostridium cadaveris (2), Bifdobacterum breve (2), Propinibacterium acnes (2), Clostridium septicum (2), Prevotella buccae (2), Veillonella atypical (1), Alistipes fingoldii (1), Fusobacterium mortiferum (1), Clostridium tertium (1), Clostridium clostridioforme (1), Clostridium hathewayi (1), Clostridium innocuum (1), Parabacteroides goldsteinii (1), Propionibacterium species (1) and Lactobacillus species (1). All bacterial species included in the study were present in the Bruker Biotyper 3.1 software and library (version 4613, Bruker Daltonics) used for spectrum analysis.

A full loop of colonies was selected and suspended in 300 μL of deionized water containing 900 μL of absolute ethanol. Following centrifugation, the pellet was then dissolved in 50 μL of 70% formic acid and mixed in a vortex with 50 μL of acetonitrile. The samples were centrifuged, and 1 μL of the clear supernatant was spotted onto the MALDI target plate and air-dried at room temperature. Each spot was then overlaid with 1 μL of CHCA and completely air-dried before the Bruker MALDI-TOF MS measurement. The microorganism was identified, and data analyses were performed using the Bruker LT microflex MALDI-TOF MS (Bruker Daltonics, Bremen, Germany) with Bruker BioTyper 3.0 system software (Bruker Daltonics) [24 (link),25 (link)]. A MALDI Biotyper log(score) value >2.0 represents reliable identification at the genus level and a score <1.7 represents ambiguous identification [4 (link),5 (link)].

Immediately after explantation, tubes containing the implants in 1.5 ml of thioglycollate medium were labelled with a code number and transferred to the microbiology laboratory. Within 4 h, tubes were vortexed for 90 s with a vortex mixer (VELP Scientifica) and then sonicated at a frequency of 40 kHz at 22 °C for 5–7 min (BANDELIN Electronic GmbH & Co. KG, Berlin, Germany) and finally vortexed again for 90 s to obtain a biofilm disintegration. The sonication fluid was centrifuged at 3200 rpm for 15 min, the supernatant was carefully removed, and the sediment was resuspended in 100 μl of medium. A 10-μl volume of medium was added onto aerobic Columbia sheep blood agar plates and incubated at 37 °C for 5 days. The same volume was placed onto anaerobic Schaedler sheep blood agar and incubated for 10 days in an anaerobic jar with CO₂ generating system GasPak™ (Becton, Dickinson and Company). The semiquantitative estimate was made by counting on plates and expressed in colony-forming units (CFU/ml). The minimum detection level was 5 CFU/ml.
Microbial identification was performed by Bruker MALDI-TOF MS (Bruker Daltonics, Billerica, MA, USA), and for each implant, species found on plates were identified for multiple comparisons.

Microbiological analysis of samples was carried out by the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; Bruker Daltonics, Bremen, Germany) based on protein fingerprints. Single colonies of fresh overnight cultures were used for the ethanol-formic acid extraction. Each sample spot was covered with 2 µL of matrix solution (saturated solution of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile with 2.5% trifluoroacetic acid; Bruker Daltonics) and air-dried for 15 min. Raw data of protein spectrum of each isolate was imported into the Biotyper software, version 2.0 (Bruker Daltonics) and analyzed [97 (link)].

To rapidly and presumptively identify extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales, oral and stool swabs stored in default liquid cultures were spread directly onto CHROMagar ESBL medium plates (Kanto Chemical) [14 (link)]. The plates were incubated at 37 °C for 24 h. A single bacterial colony from each swab sample was grown on CHROM agar ESBL plates and used for subsequent testing. Colonies were identified using MALDI-TOF MS (Bruker Daltonics). Among the bacterial isolates from the stool samples of the group with a history of antimicrobial treatment, we randomly selected three Escherichia coli isolates from swab samples of three patients, extracted their DNA and conducted genome sequencing using an Illumina NovaSeq 6000 in a 2×150 bp paired-end run protocol.