Classifying MRSA: Biomarker peptides of different strains by rapid mass spectrometric profiling

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  • Published: Jul 1, 2012
  • Author: Steve Down
  • Channels: Proteomics & Genomics / Proteomics
thumbnail image: Classifying MRSA: Biomarker peptides of different strains by rapid mass spectrometric profiling

MRSA variants

Three strains of antibiotic-resistant Staphylococcus aureus can be distinguished by the mass spectrometric detection of specific peptides, providing the opportunity for developing a diagnostic test.

Image: Janice Carr / CDC

The common bacterium Staphylococcus aureus, often referred to as Staph in the medical profession, has been treated effectively with antibiotics since their introduction in the 1940s. That is, until the evolution of various super strains that developed resistance to the antibiotics. These variant strains are generally no more aggressive than other infections, simply more resistant to treatment.

The one we all think of is MRSA but there are a number of other types too. MRSA is now commonly used to describe any strain of S. aureus that is resistant to one or more of the powerful penicillin-type antibiotics but, strictly speaking, it refers to the strain that is resistant to methicillin (methicillin-resistant Staphylococcus aureus). However, there are now at least 17 strains of MRSA, with different degrees of immunity to various antibiotics and varying degrees of infection.

Most people associate MRSA with hospitals after the incidence of infections rose sharply over recent years but there is also a separate and distinct strain that spreads throughout the general community. Other strains have also emerged that are resistant to different antibiotics like heterogeneous vancomycin-intermediate SA (hVISA) which is resistant to intermediate levels of the antibiotic, and vancomycin-resistant SA (VISA).

It is important to identify these infections rapidly so that the correct infection control and patient treatment protocols can be instigated. The possibility of using mass spectrometry profiling has emerged recently as an alternative to the classical polymerase chain reaction (PCR) procedure. Many organisms produce peptides or proteins that are unique to them, so spotting them is proof that the organism is present.

This approach has been adopted by Chao-Jung Chen and colleagues from the China Medical University Hospital, Taichung, and the Chang-Gung Memorial Hospital, Linkou, Taoyuan, Taiwan. They investigated the protein profiles of several MRSA strains: hospital-associated MRSA (HA-MRSA), community-associated MRSA (CA-MRSA), hVISA and VISA and used methicillin-sensitive SA (MSSA) to aid method optimisation.

Extracting MRSA isolates

Various bacterial isolates that had been isolated from different body fluids (such as blood, pus and sputum) and catheter tips were grown on two different media before extraction. The extracts were analysed by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI MS) on a time of flight instrument in linear and reflectron modes.

Bacteria grown on brain heart infusion agar gel produced far stronger mass spectral signals than those grown on blood agar plates, so that medium was adopted for all experiments. When the cells were washed with 70% ethanol before extraction, the background noise signals in the mass spectra were sharply reduced.

Subsequent extraction with 70% formic acid followed by an equal volume of acetonitrile gave good ion profiles for the proteins but the use of 2.5% formic acid was better for peptides. The researchers thought that the acid breaks the cell walls first, allowing easier access for extraction by acetonitrile.

Peptide biomarkers

The mass spectral profiles did not allow MSSA and MRSA to be distinguished but they did discriminate between other MRSA types. The researchers defined the different strains in terms of their genetic footprints which are known as SCCmec types and were determined by PCR.

Strains SSCmec I, II and III, which are HA-MRSA types, could be distinguished from strains IV and V, which are CA-MRSA strains. This is an important distinction to be able to make because CA-MRSA can cause nasty infections which destroy the tissue, so rapid treatment is essential.

The peaks at m/z 1774.1 and 1792.1 were present in 95% of the IV and V isolates but only 8% of the I-III isolates. They were attributed to the so-called PSM-alpha peptides which mobilise neutrophils to increase the virulence of the bacterium. The two sets were separated on a 2D cluster plot of these mass spectrometric signal intensities.

For the vancomycin-resistant strains, the peptides at m/z 1835.0 and 1863.0 were present in 88% of the VISA isolates and 50% of the hVISA isolates, allowing these to be identified with some accuracy. Unfortunately, the same two peptides were observed in the mass spectra of 16% of HA-MRSA, 20% of CA-MRSA and 10% of MSSA samples, so this biomarker test would be less accurate than that for CA-MRSA.

The marker peptides in this case were derived from the acyl carrier protein which is involved in fatty acid transport and bacterial cell wall synthesis. VISA cells have thicker walls than MRSA cells, which contributes to vancomycin resistance, so the observation of acyl carrier protein in the isolates could be related to cell wall thickening.

The results suggest that the MALDI mass spectrometric method has the potential to be developed into a test for the rapid identification of the CA-MRSA, HA-MRSA and hVISA/VISA strains of MRSA, although more work will be required to confirm the uniqueness of the biomarker peptides to these particular strains.

Related Links

Analytical Chemistry 2012 (Article in Press): "Peptide biomarker discovery for identification of methicillin-resistant and vancomycin-intermediate Staphylococcus aureus strains by MALDI-TOF"

Article by Steve Down

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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