Abstract
A chemical (ethanol; formic acid; acetonitrile) protein extraction method for the preparation of bacterial samples for matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) identification was evaluated for its ability to inactivate bacterial species. Initial viability tests (with and without double filtration of the extract through 0.2 μM filters), indicated that the method could inactivate Escherichia coli MRE 162 and Klebsiella pneumoniae ATCC 35657, with or without filtration, but that filtration was required to exclude viable, avirulent, Bacillus anthracis UM23CL2 from extracts. Multiple, high stringency, viability experiments were then carried out on entire filtered extracts prepared from virulent B. anthracis Vollum vegetative cells and spores ranging in concentration from 106-108cfu per extract. B. anthracis was recovered in 3/18 vegetative cell extracts and 10/18 spore extracts. From vegetative cell extracts B. anthracis was only recovered from extracts that had undergone prolonged Luria (L)-broth (7 day) and L-agar plate (a further 7 days) incubations. We hypothesise that the recovery of B. anthracis in vegetative cell extracts is due to the escape of individual sub-lethally injured cells. We discuss our results in view of working practises in clinical laboratories and in the context of recent inadvertent releases of viable B. anthracis.
Highlights
Matrix-assisted laser desorption / ionisation time-of-flight mass spectrometry (MALDI-TOF MS) is used in clinical microbiology for the identification of bacterial or fungal strains isolated from positive blood cultures [1]
In this paper we report on the inactivation efficacy of a MALDI-TOF chemical extraction method, on the viability of high concentrations of Bacillus anthracis vegetative cells and spores
Bacillus anthracis Vollum was chosen for the high stringency inactivation experiments based on the results from the initial inactivation experiments
Summary
Matrix-assisted laser desorption / ionisation time-of-flight mass spectrometry (MALDI-TOF MS) is used in clinical microbiology for the identification of bacterial or fungal strains isolated from positive blood cultures [1]. There is a danger that an operative may inadvertently apply an ACDP HG3 bacterial agent directly to a target plate, especially in a geographic region where ACDP HG3 organisms are endemic. In this paper we report on the inactivation efficacy of a MALDI-TOF chemical extraction method (using ethanol, formic acid, acetonitrile, and filtration), on the viability of high concentrations of Bacillus anthracis vegetative cells and spores. This method was designed to be able to be used in a clinical laboratory, within a small microbiological safety cabinet or isolator, with the MALDI-TOF MS system being situated on the bench, outside of primary biocontainment. The method was required to allow generation of mass spectra which could be compared with databases supplied with the Bruker Biotyper MALDI-TOF MS system, and allow routine identification of common clinical strains
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