Rapid identification of microorganisms from positive blood cultures by MALDI-TOF mass spectrometry subsequent to very short-term incubation on solid medium

Abstract

Rapid identification of the causative microorganism is important for appropriate antimicrobial therapy of bloodstream infections. Bacteria from positive blood culture (BC) bottles are not readily available for identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Lysis and centrifugation procedures suggested for direct MALDI-TOF MS from positive BCs without previous culture are associated with additional hands-on processing time and costs. Here, we describe an alternative approach applying MALDI-TOF MS from bacterial cultures incubated very briefly on solid medium. After plating of positive BC broth on Columbia blood agar (n = 165), MALDI-TOF MS was performed after 1.5, 2, 3, 4, 5, 6, 7, 8, 12 and (for control) 24 h of incubation until reliable identification to the species level was achieved (score ≥2.0). Mean incubation time needed to achieve species-level identification was 5.9 and 2.0 h for Gram-positive aerobic cocci (GPC, n = 86) and Gram-negative aerobic rods (GNR, n = 42), respectively. Short agar cultures with incubation times ≤2, ≤4, ≤6, ≤8 and ≤12 h yielded species identification in 1.2%, 18.6%, 64.0%, 96.5%, 98.8% of GPC, and in 76.2%, 95.2%, 97.6%, 97.6%, 97.6% of GNR, respectively. Control species identification at 24 h was achieved in 100% of GPC and 97.6% of GNR. Ethanol/formic acid protein extraction performed for an additional 34 GPC isolates cultivated from positive BCs showed further reduction in time to species identification (3.1 h). MALDI-TOF MS using biomass subsequent to very short-term incubation on solid medium allows very early and reliable bacterial identification from positive BCs without additional time and cost expenditure.

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.…”

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

“…Identification of microorganisms from positive blood culture using of MALDI Sepsity per processing kit requires approximately 20 minutes and it does independent of species (6, 10,13,18,20,25,26,32,33). Batch processing added approximately 1 min per blood culture analyzed.Our estimated time to identify bacteria from the positive blood culture, of fixed automated system BD BACTEC Fx signal time to MALDI-TOF mass spectrometry identification time, is not an objective, it includes the blood smear preparation time by Gram staining, …”

“…But in comparison with other studies our estimated time correspond to with foreign laboratories (10).Some studies even describe MALDI-TOF mass spectrometry advantages compared to other direct methods to identify the bacteria from the blood, for example, using polymerase chain reaction and the latter method is limited by the high cost, not every species of microorganisms is determined in one study, in addition, it is enough difficult to realize in practic (28)(29)(30)(31).…”

Utilización de espectrometría de masas para la identificación precoz de hemocultivos positivos: Validación de un algoritmo local

“…Al momento de la redacción de este artículo, sólo hemos encontrado un trabajo que aborda este enfoque metodológico (identificación de hemocultivo positivo mediante EM luego de un corto período de incubación en medio sólido), con resultados comparables a los obtenidos por nosotros 10 .…”