Monday, November 19, 2012

Ion/Molecule Reaction Mass Spectrometry Identifies Gram-Negative Bacteria

MALDI-TOF mass spectrometry is now being used to identify microorganisms, and this is based on an accurate molecular weight measurement and characterization of the organism's biomolecules, including proteins, peptides, polysaccharides and nucleic acids. Following microbial growth, approximately 105 cells are added to a target plate (usually stainless steel), and following the addition of an energy absorbing matrix, the mixture is ionized, and the ionized particles are accelerated in an electric field. The molecules are separated according to their mass to charge ratio, and the resulting mass spectrum is compared with an internal identification library or database.

Ion/molecule reaction mass spectrometry examines volatile or gas-phase compounds, and scientists in Germany and the UK are now using this technique for the rapid identification of Gram negative bacteria. The following excerpt from is an overview of their work.


Critically ill patients in hospitals can be severely affected by infections, which slow down their recovery or, in extreme cases, cause death. A recent international study found that the mortality rate in intensive care units rose from 11% in non-infected patients to 25% in those who had been infected. The majority of the infections (64%) were of respiratory origin and 62% of these were with Gram-negative bacteria.

It goes without saying that rapid identification of infection could be a lifesaver, helping the clinician to prescribe the appropriate antibiotics. However, the conventional microbiological tests can take several hours or days to complete, by which time any infection will have taken a stronger hold.

One alternative route being explored in recent years involves the volatile metabolites that are emitted from bacteria. There have been numerous reports outlining the use of GC/MS to distinguish between various microorganisms and these have led to direct mass spectrometry techniques like proton transfer reaction mass spectrometry which can produce results much quicker than GC/MS.

Now, a team of scientists in Europe has developed a method to discriminate between bacteria using ion/molecule reaction mass spectrometry (IMR MS) as part of a broader programme to investigate the use of volatile compounds for the diagnosis of infections in vivo. Their method has the advantage of providing results within three minutes and is suitable for automation, a big plus for clinical testing labs.

Michael Dolch and colleagues from the Ludwig-Maximilians-University of Munich, Germany, V&F Medical Development GmbH, Absam, Austria, and Avacta/Oxford Medical Diagnostics Ltd, Oxford, UK, published details of their new procedure in Journal of Applied Microbiology.

Ion/molecule reactions

The team concentrated on seven Gram-negative bacteria that were prevalent in patients with nocosomial pneumonia. They were grown on blood agar plates and their volatile metabolites were measured at various times by IMR MS using headspace analysis. The ion/molecule reactions were initiated using mercury ions which were allowed to react with the volatiles to produce positive ions for detection.

A set of 65 m/z values were selected for monitoring based on preliminary work. The identities of some of the peaks were confirmed by comparison with the IMR mass spectra of authentic compounds, including ammonia, hydrogen sulphide, propene, acetyl group, acetaldehyde, ethanol, methanethiol, acetone, propanal, toluene and indole.

The raw data signals were normalised against that of isoprene as a reference gas. Then peaks were marked as originating from the microorganisms if they exceeded a threshold intensity that was 100% different from the background noise of the broth solutions. These peaks were processed by principal components analysis to see if the bacterial species could be distinguished.

Positive identification of Gram-negative bacteria

No peaks reached the threshold value after 4 hours of incubation but all species showed significant increases for some masses after 8 hours and the peaks at m/z values of 105, 106 and 107 showed decreased abundances. These are likely to be benzyl derivatives but their structures were not elucidated.

After 24 hours of culture, the IMR mass spectra were sufficiently developed to allow the seven bacteria to be distinguished. Any combination of the first three components in the PCA of the signals allowed discrimination equally between Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris and Serratia marcescens.

The clusters in the PCA plots for Enterobacter cloacae and Klebsiella oxytoca overlapped significantly. However, the introduction of minor spectral differences, namely the presence of peaks at m/z 29, 60 and 79 from K. oxytoca and their absence from E. cloacae, allowed their differentiation in a separate PCA analysis.

So, seven Gram-negative bacterial species can be detected after only 8 hours of growth and distinguished from each other after 24 hours of growth. Their early identification will allow timely intervention with a treatment program to combat their growth and protect the patients.

The rapid test is complete within three minutes using a process that is amenable to automation. “Currently no such diagnostic tool exists, but we certainly hope it will in the future allowing for species differentiation even outside the usual working hours, and thus shorten the time required for adaptions of antibiotic treatment in response to microbial testing results,” said Dolch.


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