Maldi biotyper system

Plasma samples were analyzed with the MALDI Biotyper System (Bruker Daltonik GmbH, Bremen, Germany). Total number of plasma samples analyzed per group were n = 29 for the LPS group, n = 22 for the IS group and n = 25 for the CTL group. Plasma samples were analyzed with the standard method for microbial biotyping. Briefly, 1 μl of plasma was loaded onto each spot in duplicate and 1 μl of the matrix (alpha-cyano-4-hydroxycinnamic acid matrix in 50% acetonitrile and 2.5% trifluoroacetic acid) was added to each dried spot.
Continuous mass spectra were obtained with a Microflex LT/SH MALDI-TOF mass spectrometer using the flexControl software version 3.4.135.0. Acquisition conditions were ionization mode: LD + , acquisition method: MBT_FC.par, acquisition mode: qsim, tof Mode: linear, acquisition Operator Mode: linear, and digitizerType: Bruker BD0G5, within a mass range of 2000–20,000 Da. Spectra were obtained in the manual mode, using 60% of laser intensity with 40 laser repetition in each shot and reaching between 400–500 spectra by acquisition. Internal calibration was performed every day following manufacturer’s instructions (bacterial test standard; Bruker Daltonik GmbH).

Representative isolates (n=38) from samples collected at 26 fish farms in 14 counties in Sweden were included in the study. Three isolates from fish farms in Finland were also included for reference. Included isolates were verified as F. psychrophilum by MALDI-TOF MS performed on a MALDI Biotyper System (Bruker) by use of main spectra projections prepared and validated for identification of F. psychrophilum and F. columnare according to Bruker’s instructions. Fish farm locations were aggregated to county level to protect the anonymity of study participants. The isolates were collected in connection with disease outbreaks between 1988 and 2016 and originated from rainbow trout (n=36), Arctic charr (n=2), brown trout (n=1) and fish farm water samples (n=2). Of the fish isolates, 33 were from kidneys, one each was from skin, fin, gill and abscess samples while the remaining were from unspecified tissues (see Table S1 for details).

A total of 20 mL of whole blood (10 mL for the aerobic and 10 mL for the anaerobic bottle) was drawn for one set of routine blood culture testing, as previously described [22 (link)]. BCs were incubated in the BACTEC FX system (Becton-Dickinson, Franklin Lakes, New Jersey, United States) for a maximum of 5 days. Positive BCs were further examined by gram-staining and microscopy. Sub-cultivation of retrieved pathogens on agar plates was done according to standard techniques [22 (link)]. Identification of the pathogens was performed by Matrix-Assisted Laser-Desorption Ionization–Time-Of-Flight (MALDI-TOF) using MALDI Biotyper^® system, MBT Compass Software IVD V4.2 (Bruker Daltonik, Bremen, Germany).

Results of the molecular tests were compared against conventional BC and subsequent analysis in the MALDI Biotyper System (Bruker, Billerica, Massachusetts, United States) for identification of pathogens in positive blood cultures. This served as the gold standard of diagnosing blood stream infections. The results of the individual tests were compiled and concordant or discrepant results were used for the calculation of sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV), and overall diagnostic accuracy, as described elsewhere [30 (link)]. In the case of uninterpretable PCR results (e.g., due to inhibition of PCR amplification), the samples were excluded from the further analysis.

Two hundred and three clinical isolates of Candida spp. obtained from urine samples processed in the Laboratory of Microbiology of the Virgen de las Nieves University Hospital (Granada, Spain) were selected. CHROMagar Orientation medium (Becton Dickinson, Franklin Lakes, NJ, USA) was used for the growth of isolates. All colonies with yeast compatible morphology were subcultured using a CHROMagar Candida medium (Becton Dickinson). Species were identified using filamentation test and ASM Vitek system (bioMérieux, Madrid, Spain) or MALDI Biotyper system (Bruker Daltonics, Billerica, MA, USA). All isolates were stored at −40° C until the susceptibility study.

Positive BCs of patients admitted in emergency and internal medicine departments, deemed representative of a single BSI event, and showing Gram-negative bacilli at Gram staining were subjected to recovery of bacterial pellet using the MBT Sepsityper IVD Kit (Bruker DALTONIK GmbH, Bremen, Germany). MALDI-TOF MS analysis of the bacterial pellets was then performed using the MALDI BioTyper system in accordance with the manufacturer’s instructions (Bruker DALTONIK GmbH, Bremen, Germany). Bacterial identification was considered reliable with a score of > 1.80. In case of EB identification, NG-Test CTX-M MULTI and NG-Test Carba 5 assays were performed adding ten drops of lysis buffer to the remaining bacterial pellet; after vortexing for 5 s, 100 μL of each suspension was added to the sample well of the respective test cassette, as previously described [12 (link), 23 (link)]. According to LFIA results, all BCs included in the study were divided in three phenotypic group: (1) CTX-M-p (LFIAs tested CTX-M positive and main carbapenemases negative), (2) main-carbapenemases- or CTX-M-and-main-carbapenemases-producer (CA-p, LFIAs tested CTX-M negative and main carbapenemases positive or CTX-M positive and main carbapenemases positive), and (3.) CTX-M-and-main-carbapenemases-non-producer (CTX-M-CA-np, LFIAs tested CTX-M and main carbapenemases negative).