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

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 positive-blood-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.

Fig 1

Overall summary of sample preparation process. Step 1, collect blood culture broth in a test tube; step 2, incubate blood culture broth with lysis buffer for 2 min; step 3, add lysate to the filter membrane for 40 s, making an effort to keep the lysate within a defined area on the membrane; step 4, wash the filter membrane three times with wash buffer and three times with water; step 5, remove microorganisms from the filter membrane using the microswab applicator; step 6, transfer the microorganisms to the MALDI target plate using the microswab applicator; step 7, add matrix on top of the microorganisms to prepare the slide for spectrum acquisition.

Fig 2

Manifold used for processing of blood culture samples. Three separate valves with attached filters allow simultaneous processing of three samples. The manifold is attached to a vacuum source via a vesicle to store waste and is operated under a biological safety hood.