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Swedish Researchers Use MALDI-TOF to Type Clostridium Difficile

NEW YORK (GenomeWeb) – A pair of researchers from the Public Health Agency of Sweden has developed a MALDI-TOF-based approach for typing Clostridium difficile based on the patterns of high-molecular weight proteins the bacterium contains.

As the duo reported in PLOS One today, this MALDI-TOF-based high-molecular weight typing approach identifies specific proteins on the bacterium surface, and uses the pattern of those proteins to identify the clonal type to which it belongs.

Sweden's Kristina Rizzardi and Thomas Åkerlund tested some 500 isolates, corresponding to 59 PCR ribotypes, and were able to resolve 35 high-molecular weight types.

"The method developed in this study, HMW typing, is in our mind an important complement to other typing methods, specifically for screening purposes in hospitals, environment, or in animal settings," Rizzardi and Åkerlund wrote in their paper.

As MALDI-TOF has become a standard clinical laboratory tool, the researchers added that their high-molecular weight typing
 "method has the potential to rapidly analyze a large number of strains and improve local surveillance at a low running cost."

Quick and early detection of C. difficile transmissions between patients can enable hospitals to move to stem the spread of an outbreak, they noted, while also guiding antibiotic use.

In this study, the researchers developed a MALDI-TOF-based method to examine the high-molecular weight — between 30,000 daltons and 50,000 daltons — protein profiles from whole cells, corresponding to the size of the C. difficile S-layer proteins.

To check that those peaks actually corresponded to the S-layer proteins, the researched extracted S-layer proteins from strains from the European Centre for Disease Prevention and Control Cardiff collection, using two different methods, and compared those profiles to the ones generated by whole-cell mass spec.

The major peaks from the whole-cell mass spec and the extractions were, the researchers found, of the same size, indicating they did correspond to the S-layer proteins. However, the researchers noted that there were additional peaks in the whole-cell mass spec analysis that weren't in the S-layer protein extraction samples, suggesting that other proteins are also detected by whole-cell mass spec.

The researchers constructed a MALDI-TOF database of 170 isolates, representing some 23 PCR ribotypes, by drawing on the Cardiff reference strains as well as on clinical isolates commonly found in Sweden.

At the same time, they also developed a PCR ribotyping database based on 1,179 C. difficile isolates collected through the Swedish national surveillance program between 2009 and 2012.

They validated the MALDI-TOF database using a cohort of 222 additional isolates amassed from more than two dozen Swedish clinical labs that they also typed by PCR ribotyping. The additional PCR ribotypes and high-molecular weight profiles found in the validation cohort that were lacking in the initial database were further confirmed through the additional analysis of 108 isolates belonging to the same PCR ribotypes.

Rizzardi and Åkerlund then analyzed 500 isolates using both methods. Through the MALDI-TOF whole-cell mass spec approach, they were able to generate 24 high-molecular weight protein profiles, 14 of which correlated to 13 PCR ribotypes, The researchers noted that some PCR ribotypes could have two or more HMW profiles.

At the same time, researchers were able to uncover 59 PCR ribotypes using that approach.

By tweaking their extraction approach, the researchers were able to improve the resolution of the high-molecular weight profiles, increasing the number of types they could resolve to 35.

While this approach is less discriminatory than PCR ribotyping, Rizzardi and Åkerlund noted that it is fast and simple and represents a cost-effective way to screen isolates from an outbreak.