Rapid Identification of Bacteria and Yeasts from Positive-Blood-Culture Bottles by Using a Lysis-Filtration Method and Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrum Analysis with the SARAMIS Database

Abstract

c Rapid identification of microorganisms causing bloodstream infections directly from a positive blood culture would decrease the time to directed antimicrobial therapy and greatly improve patient care. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is a fast and reliable method for identifying microorganisms from positive culture. This study evaluates the performance of a novel filtration-based method for processing positive-blood-culture broth for immediate identification of microorganisms by MALDI-TOF with a Vitek MS research-use-only system (VMS). BacT/Alert non-charcoal-based blood culture bottles that were flagged positive by the BacT/Alert 3D system were included. An aliquot of positiveblood-culture broth was incubated with lysis buffer for 2 to 4 min at room temperature, the resulting lysate was filtered through a membrane, and harvested microorganisms were identified by VMS. Of the 259 bottles included in the study, VMS identified the organisms in 189 (73%) cultures to the species level and 51 (19.7%) gave no identification (ID), while 6 (2.3%) gave identifications that were considered incorrect. Among 131 monomicrobic isolates from positive-blood-culture bottles with one spot having a score of 99.9%, the IDs for 131 (100%) were correct to the species level. In 202 bottles where VMS was able to generate an ID, the IDs for 189 (93.6%) were correct to the species level, whereas the IDs provided for 7 isolates (3.5%) were incorrect. In conclusion, this method does not require centrifugation and produces a clean spectrum for VMS analysis in less than 15 min. This study demonstrates the effectiveness of the new lysis-filtration method for identifying microorganisms directly from positive-blood-culture bottles in a clinical setting. R ecently, new mass spectrometry (MS) technology has been introduced as a way to quickly and accurately identify bacteria. Compared to standard phenotypic identification, this technology is rapid, requires reagents that are inexpensive (after initial purchase of the instrument), and could provide accurate results comparable to those provided by 16S rRNA sequencing (1). Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS has been shown to accurately identify bacteria grown on solid medium to the species level (2-4) and has the potential to serve as a fast and reliable method for identifying microorganisms directly from blood.Rapid identification of organisms causing bloodstream infections after a blood culture turns positive has the potential to improve patient care, and protocols have been developed for use with MALDI-TOF instrumentation. Previously published studies have explored a variety of approaches to accomplish this task, generally using a series of washes, centrifugations, protein extraction, and analysis using dedicated databases (e.g., BioTyper and SARAMIS) (1,3,(5)(6)(7)(8)(9)(10)(11).The Vitek MS research-use-only (RUO) system (VMS) with the SARAMIS database by bioMérieux (Durham, NC) is a research-us...

Optimized Use of the MALDI BioTyper System and the FilmArray BCID Panel for Direct Identification of Microbial Pathogens from Positive Blood Cultures

“…However, the method requires manual processing, such as centrifugation or filtration concentration, prior to analysis, and the MALDI-TOF MS procedures for BC are still being standardized (13,29,30). Low microbial loads, especially low fungal loads (Ͻ10 4 CFU/ml), can be a relevant cause of ineffective direct ID using the MALDI BioTyper system (11,28).…”

Enzymatic Digestion for Improved Bacteria Separation from Leafy Green Vegetables

“…The aqueous food matrix passes through a filter and target bacteria are retained on the filter while the liquid food sample is discarded. The solid that retented on the filter depends upon the size of the microorganisms and pore of the filter (21).…”

Rapid Identification of Positive Blood Cultures by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Using Prewarmed Agar Plates

“…To decrease the time to identification, attempts have been made to identify bacteria directly from positive blood cultures using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), but the broth medium and blood interfere with mass spectrometry (4,5). Various extraction protocols have been developed to isolate bacteria from culture vials, but these are laborious and result in decreased accuracy (4)(5)(6)(7)(8)(9)(10). An easier and cheaper method has been described; a sample from a positive blood culture bottle is spotted onto a prewarmed agar plate and identified by MALDI-TOF MS following 3 to 5 h of incubation without the need for ethanol-formic acid protein extraction (11).…”