Use of PCR coupled with electrospray ionization mass spectrometry for rapid identification of bacterial and yeast bloodstream pathogens from blood culture bottles

Abstract

Sepsis is among the top 10 causes of mortality in the United States. Rapid administration of antibiotics is one of the most important contributors to patient survival, yet only a limited number of methods exist for rapid identification of microbes cultivated from bloodstream infections, which can lead to sepsis. While traditional single-target molecular methods have been shown to greatly improve survival for septic patients by enabling rapid deescalation of broad-spectrum antibiotics, multiplex methods offer even greater possibilities. A novel multiplex method, PCR coupled to electrospray ionization mass spectrometry (PCR/ESI-MS), was used to identify the genus and species of microorganisms found to cause human bloodstream infections. DNA was directly extracted from 234 BacT-Alert blood culture bottles, and results were compared to those obtained by clinical reference standard methods. The study results demonstrated 98.7% and 96.6% concordance at the genus and species levels, respectively. Mixtures of microbes were identified in 29 blood culture bottles, including mixed species of the same genus, as well as mixtures containing Gram-positive and Gram-negative organisms, exemplifying the PCR/ESI-MS capability to identify multiple organisms simultaneously without the need for cultivation. This study demonstrates high analytical accuracy in comparison to routine subculture of blood culture bottles and phenotypic identification of microbes. Without foreknowledge of the microorganisms potentially present, the PCR/ESI-MS methods can deliver accurate results in as little as 5 to 6 h after a positive alarm from the automated blood culture system; however, current batch mode testing limits the method's clinical utility at this time.

FIG. 1.

Determining the base composition from mass spectral data: example using BCB115. (A) The spectra are collected and displayed as a 96-well plate format. Mass spectra can be viewed independently for each of the 96 wells. (B) Electrospray-generated mass spectra. This sample contains the PCR amplicons from two different organisms: Bacteroides thetaiotaomicron (top) and Staphylococcus aureus (bottom). (C) Deconvoluted mass spectra showing molecular masses of the forward and reverse strands of each PCR amplicon. An algorithm is used to calculate the base composition from the molecular mass and was calculated to be 30A-27G-23C-19T (top) and 27A-30G-21C-21T (bottom).

FIG. 2.

Work flow for this study.

FIG. 3.

Base composition data obtained from sample BCB115, which contained a mixture of the Gram-negative anaerobe Bacteroides thetaiotaomicron and the Gram-positive methicillin-resistant Staphylococcus aureus. The composition of the PCR amplicon produced by each primer is shown.