Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

Summary

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

Abstract

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

Introduction

Patients with bloodstream infection (BSI) due to bacteria continue to have high in-hospital mortality, ranging from 6-48%¹. The delivery of appropriate empiric antibiotic promotes survival and in the subset of patients with severe sepsis, each hour delay to appropriate therapy correlates to decreased survival^2,3. Accordingly, a key objective of the clinical laboratory is to rapidly detect, identify and communicate the presence of bacteria in blood cultures to inform clinical decisions. It has been demonstrated that the microbiology laboratory has the greatest influence on antimicrobial therapy at the time of reporting the Gram stain⁴ a....

Protocol

1. Blood Culture Broths Flag as “Positive”

1. Remove the signaled blood culture bottle from the continuous monitoring incubation cabinet and place it into a biological safety cabinet. Note: Bottles can contain hazardous microorganisms and universal precautions need to be followed. Due to the risk of infectious aerosols in sampling, all sampling procedures must be performed in a Biosafety Class II laminar flow cabinet.

2. Gram Stain is Prepared

1. Prepare a Gram stain from the signaled blood culture broth as per local institutional protocols. Note: When Gram negative organisms are identified on microscopy....

Results

The generated MALDI-TOF MS spectra are compared to the integrated reference database of spectra. A logarithmic score is assigned for the confidence of the match between the test isolate and the reference database isolates, with the recommendation of a score ≥1.7 required for probable identification to genera level (Figure 1) and ≥2.0 for probable identification to species (Figure 2). Report to species level when the score ≥1.7 and the first 5 identifications matched by the Typing softwar.......

Discussion

It is important when applying MALDI-TOF MS to blood culture broth that the post centrifugation steps are performed with sufficient care not to remix the separated components. It is particularly important to remove the blood culture constituents and human cellular proteins, including hemoglobin, which may produce spikes interfering with the MALDI-TOF spectra.

Although MS manufactures recommend cut of score of ≥2.0 for species and ≥1.7 for genus identification, other reports have sug.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BACTEC Plus Aerobic/F Medium	Becton Dickinson; BD, Franklin Lakes, NJ, USA	442192
BACTEC Lytic/10 Anaerobic/F Medium	Becton Dickinson; BD, Franklin Lakes, NJ, USA	442265
BACTEC Peds Plus Medium	Becton Dickinson; BD, Franklin Lakes, NJ, USA	442194
Vacutainer - Blood transfer device	Becton Dickinson; BD, Franklin Lakes, NJ, USA	364880	Single use sampling device reducing the risk of needlestick injury
Vacutainer SST 5.0 ml, Advance plus	Becton Dickinson; BD, Franklin Lakes, NJ, USA	367954	Serum separating tube
Syringe 10 ml	Becton Dickinson; BD, Franklin Lakes, NJ, USA	302143
Transfer pipette	Samco, USA	222-20S
Transfer pipette (fine tipped)	Samco, USA	232-20S
Microcentrifuge tube (2.0 ml)	Eppendorf, Hamburg, Germany	0030.120.094
Sterile water (DNAse and RNAse free)	Life Technologies, Carlsbad, California, USA	10977-015
Formic acid	Sigma-Aldrich, St. Louis, Missouri, USA	F0507
Matrix solution	Bruker Daltonics, Bremen Germany	285074	10 mg/ml α-cyano-4-hydroxycinnamic acid, 50% acetonitrile, 2.5% trifluoroacetic acid
Benchtop microflex LT MALDI-TOF MS	Bruker Daltonics, Bremen Germany		Utilizing BioTyper 3.1 (Build 65) and FlexControl 3.3 (Build 99) software