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ETH Zurich Team Uses MALDI for Single-Cell Analysis of Microorganisms

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NEW YORK (GenomeWeb) – Researchers from the Swiss Federal Institute of Technology Zurich have developed a method for single-cell analysis of microorganisms using MALDI mass spectrometry.

Detailed in a paper published last week in Applied and Environmental Microbiology, the technique could allow researchers to better study heterogeneity within microbial populations and might also have applications for clinical microbiology work, Jasmin Krismer, an ETH Zurich researcher and first author on the study, told GenomeWeb. 

Microbes, Krismer said, are often studied using bulk analyses looking at groups of organisms, but heterogeneity within populations can have important implications for phenomena like antibiotic resistance or the colonization of new habitats.

Approaches like next-generation sequencing and fluorescent tagging have allowed researchers to characterize microbial populations at the single-cell level. However, a mass spec-based approach has potential advantages in the case of some analytes. For instance, in the case of proteins, mass spec-based methods allow for a much more comprehensive analysis of a cell's contents than tagging-based methods. And, in the case of small molecules — the focus of the AEM study — mass spec could allow researchers to look at analytes too small to be labeled without potentially changing their biological activity.

In what Krismer described as essentially a proof of concept, she and her colleagues applied their technique to the alga Chlamydomonas reinhardtii, using it to distinguish between two different strains of the organism.

Chlamydomonas reinhardtii "is a microbe, but it's not as small as bacteria or some of the relevant bacteria that are studied," she said. "I think it's a step in that direction," she added, noting that algae are some 1,000 times larger by volume than, for instance, a small bacteria."

Key to the approach was use of a piezo-driven contactless spotting instrument for spotting single cells with a minimum of cross-contamination. For mass spec analysis the researchers used both a Bruker SolariX 9.4T FT-ICR instrument attached to a MALDI source and an AB Sciex 5800 MALDI machine.

The researchers focused on small molecules, which Krismer said were more amenable to a single-cell analysis due to their higher copy numbers. Proteins, she noted, would be more of a challenge for the method — in part due to their typically lower abundance and also due to the need for trypsin digestion, which complicates sample prep.

"I think proteins will be a really big challenge to detect on the single-cell level [using the technique]," she said. "However, maybe some abundant proteins will be detectable."

She added that small molecules like lipids could provide information useful for identifying microbes at the single-cell level. Indeed, at the Mass Spectrometry Applications to the Clinical Laboratory 2015 annual meeting in April, there was considerable discussion of the potential of lipid profiles for typing organisms in clinical microbiology.

For instance, work presented at MSACL by Imperial College London researcher Nicole Strittmatter demonstrated the use of lipid profiling for detection of bacteria directly from human colorectal tissue samples. Strittmatter and her colleagues were able to identify lipidomic markers for a number of bacteria at taxa levels ranging from phylum down to species.

This research used the REIMS technology developed by Zoltan Takats, the leader of Strittmatter's group.

One significant reason for the interest at the conference surrounding the REIMS lipidomics work was its apparent ability to identify bacteria directly from samples without the need for culturing, which is required by the two currently available mass spec-based clinical microbiology systems, Bruker's MALDI Biotyper and BioMérieux's Vitek MS.

Krismer suggested that, given its high sensitivity, her group's single-cell MALDI approach might similarly be capable of direct identifications.

She noted, however, that she and her colleagues have not yet investigated the method's suitability for biotyping microorganisms. More immediately, she said, they hope to use the method to study actual environmental systems, as opposed to the model organisms on which they tested it.

"I'm very fond of evolutionary experiments as well as studying outdoor samples," she said. "From my side, it's really going to be a challenge to work under real-life conditions instead of the artificial conditions we have been [investigating] until now."

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