Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Direct Bacterial Identification from Positive Blood Culture Pellets

Abstract

An ammonium chloride erythrocyte-lysing procedure was used to prepare a bacterial pellet from positive blood cultures for direct matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry analysis. Identification was obtained for 78.7% of the pellets tested. Moreover, 99% of the MALDI-TOF identifications were congruent at the species level when considering valid scores. This fast and accurate method is promising.Blood cultures are the best approach to establishing the etiology of bloodstream infections and infectious endocarditis. Moreover, rapid identification of the etiological agents of such severe infections is pivotal to guiding antimicrobial therapy. Thus, the impact of timely microbiology laboratory reporting is maximal at the notification of positive blood cultures (5). Matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) allows identification of both gram-positive (2, 7) and gram-negative bacteria (1, 4) to the species level in a few minutes by measuring the molecular masses of proteins and other bacterial components obtained from whole bacterial extracts. A relatively low crude bacterial load of about 5 ϫ 10 3 CFU is necessary for reliable MALDI-TOF analysis (3), suggesting that bacterial identification might be done directly with blood culture pellets. We applied a simple procedure for lysing erythrocytes from positive blood cultures and prepared a bacterial pellet for MALDI-TOF MS analysis.Pellets from positive blood culture vials (Plus aerobic/F, Lytic anaerobic/F, and Peds/F; Becton Dickinson) detected by the Bactec 9240 automated blood culture system (Becton Dickinson) were prepared as follows shortly after the automated system flagged a positive vial. Five milliliters of positive medium was added to 40 ml of sterile H 2 O. The sample was mixed and centrifuged at 1,000 ϫ g for 10 min. H 2 O and blood cells were removed (Fig. 1A). The pellet was then suspended in 1 ml of a home-made ammonium chloride lysing solution (0.15 M NH 4 Cl, 1 mM KHCO 3 ) and centrifuged at 140 ϫ g for 10 min (Fig. 1B and C). When the pellet remained hemorrhagic, the supernatant was discarded and the pellet was washed again with 2 ml of H 2 O. MALDI-TOF MS analysis was then directly performed on the bacterial pellet or after an additional extraction step. To extract proteins, 20 l of the pellet was mixed with 1 ml of 70% ethanol. After a further centrifugation at 13,000 ϫ g for 2 min, the pellet was mixed with 25 l of 70% formic acid and 25 l of pure acetonitrile. After centrifugation at 13,000 ϫ g for 2 min, 1 l of the supernatant containing the bacterial extract was transferred onto the MALDI target plate and dried. Subsequently, both unextracted and extracted samples were overlaid with 1 l of MALDI matrix (a saturated solution of ␣-cyano-4-hydroxycinnamic acid in 50% acetonitrile-2.5% trifluoroacetic acid) and dried in air.Mass spectra were then acquired by Microflex MALDI-TOF MS (Bruker Daltonics, Bremen, Germany). MALDI BioTyper 2.0 software was used ...

Rapid Identification of Bacteria Directly from Positive Blood Cultures by Use of a Serum Separator Tube, Smudge Plate Preparation, and Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

“…Several studies have shown that MALDI-TOF MS analysis of samples obtained directly from positive blood cultures can correctly identify 66% to 97% of aerobic and anaerobic organisms causing monomicrobial bacteremia (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). As in the current study, the best results have been obtained with Gram-negative organisms.…”

“…Implementation of MALDI-TOF MS in the microbiology laboratory in conjunction with antibiotic stewardship has been associated with earlier initiation of effective antimicrobial therapy and lower 30-day mortality in patients with bloodstream infections (3). This technology has also been applied to reduce turnaround times for identification of blood culture isolates directly from positive blood culture broths (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). Various techniques have been assessed, generally involving lysis/centrifugation of blood culture pellets in preparation for analysis by MALDI-TOF MS.…”

Improved Identification of Yeast Species Directly from Positive Blood Culture Media by Combining Sepsityper Specimen Processing and Microflex Analysis with the Matrix-Assisted Laser Desorption Ionization Biotyper System

“…One such system, the MALDI Biotyper system (Bruker Daltonics Inc., Billerica, MA), has been successfully used to rapidly identify yeast-like fungal pathogens after they grow on solid medium as pure colonies (2,5,17,28,32). Although direct identification from blood culture media has been successfully applied to bacterial pathogens (8,9,15,22,27), published studies are limited and have yielded variable results for direct identification of yeast-like fungal pathogens directly from positive blood culture medium (8,9,16).…”

An Evaluation Of Direct Identification Of Pathogens From Blood Cultures By Maldi-Tof Mass Spectrometry

“…With a special set of MALDI Sepsityper is possible to identify bacteria directly from the positive blood culture (17)(18)(19)(20)(21)(22)(23)(24)(25). Most reports describe the blood culture must be grown in automated BACTEC system vials (26,27).…”