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NIH Researchers Develop LC-MS/MS Method for Strain-Level ID of Acinetobacter


NEW YORK (GenomeWeb) – Researchers at the National Institutes of Health have developed a tandem mass spec approach to strain typing Acinetobacter baumannii isolates.

The method, which was detailed in a paper published last month in Clinical Chemistry, uses LC-MS/MS to detect strain-specific peptides from clinical A. baumannii isolates, which could allow clinicians to identify and track specific organism strains in, for instance, cases of hospital infections or outbreaks.

The paper follows recent similar work by researchers at the Public Health Agency of Canada's National Microbiology Laboratory who used LC-MS/MS for H-typing of Escherichia coli. Together, the studies suggest a way forward for LC-MS/MS in clinical microbiology, which, in terms of mass spec, has to date been dominated by MALDI-TOF platforms like Bruker's MALDI Biotyper and BioMériuex's Vitek MS.

MALDI-based microbial IDs have typically used intact protein profiling to generate proteomic fingerprints specific to a given organism. The recently described LC-MS/MS approaches, on the other hand, use a conventional bottom-up proteomics workflow wherein the target proteins are digested into peptides and then identified via mass spec.

Because it does not require protein digestion or LC separation, MALDI offers advantages in terms of throughput and ease of use. The recent Clinical Chemistry papers suggest, however, that LC-MS/MS applied to a peptide-based approach might prove a more powerful tool for strain-level detection.

"There are a variety of approaches that are being used, each of which has its merits and domain of application," said John Dekker, an NIH scientist and author on the A. baumannii paper. "One thing that has become apparent is that fragmentation-based methods [like conventional LC-MS/MS] may have more power to identify strain-specific signatures than intact protein profiling methods [like MALDI]."

In fact, the NIH initially set out to develop a MALDI-based method for distinguishing between A. baumannii strains, looking at 22 isolates from a larger collection of samples taken during an outbreak at the NIH Clinical Center. However, they were unable to separate all 22 isolates into strain groups based on their MALDI data and so moved to LC-MS/MS in hopes that the higher resolution data provided by that method would improve upon the MALDI approach.

Using 15 isolates with available whole genome sequences the researchers created a database from which they identified peptides specific to particular A. baumannii strains that they could use for strain-level identification. They then developed both multiple-reaction monitoring assays on an Agilent Chip-Cube 6495 triple quadrupole and high-resolution assays on an Agilent QTOF 6450 to detect these peptide markers.

The work was a proof-of-principle study, Dekker said, adding that he and his colleagues are not currently using it in clinical practice but are considering it for epidemiological applications.

As the authors noted, several challenges remain in terms of optimizing the approach. A primary issue is obtaining enough high-quality strain-specific genomes from which to select strain-specific peptides for making identifications. The larger the set of isolates they are able to obtain, they wrote, the higher the method's specificity.

Generally speaking, Dekker said, the work indicates that finding enough strain-specific peptides to confidently make strain-level identifications should not be a problem.

"Tryptic digest offers an extraordinary number of peptides from which to choose genome-specific markers," he said. "While we do not yet have a general answer to the question of exactly how many peptides are required on average to be confident of a particular strain-level identification from a statistical point of view, our work suggests that unique marker peptides are quite abundant and discoverable by these methods."

The substantial number of markers to choose from also means that researchers are able to select for high-abundance peptides or peptides that are otherwise amenable to mass spec detection.

Currently, pulsed-field gel electrophoresis or genome sequencing are commonly used for strain-level identification, but, Dekker said, a mass spec approach could have advantages compared to these methods.

"Once marker peptides have been identified and validated, tryptic digestion followed by mass spectrometry using a triple quad is a fast and easy procedure for classifying isolates, provided that expertise and instruments are available," he said. "The technique also has potentially higher resolution than traditional MLST [multilocus sequence typing] sequencing and is significantly faster and cheaper than whole-genome sequencing."

One possible challenge in terms of implementation is the fact that, while MALDI has made significant inroads into clinical microbiology labs, LC-MS/MS instruments are less common, Dekker said.

"These instruments are more likely to be found in the chemistry section of a clinical lab rather than the diagnostic microbiology section," he said.

However, Nathan Ledeboer, medical director for the clinical microbiology and molecular diagnostics laboratories at the Medical College of Wisconsin, suggested that the familiarity of clinical chemistry departments with LC-MS/MS instruments could prove an advantage.

"There are obviously a lot of MS/MS instruments that are in the clinical chemistry sections of these laboratories, and this could be an additional application that could be used on those," he told GenomeWeb.

Ledeboer, who was not involved in either of the Clinical Chemistry studies, noted that the idea of use LC-MS/MS for microbial ID is not especially new, but that researchers and clinicians have struggled with the question of throughput. Specifically, he noted, there are challenges surrounding the time required for extraction and tryptic digestion of the target proteins and with the LC-MS/MS cycle times.

With regard to the former, the NIH team presented a 15-minute extraction and digestion protocol that, at least in the case of their A. baumannii assay, brings sample prep down to a time competitive with MALDI.

There still remains the issue of liquid chromatography, however. For the A. baumannii MRM assay, the total LC-MS/MS cycle time was 11 minutes per sample. The Public Health Agency's E. coli assay, on the other hand, required around four hours.

"So I think that the question then becomes how scalable is [the LC-MS/MS approach]," Ledeboer said. "The advantage of a MALDI-based approach is that it is exceedingly scalable. If you think about the average clinical laboratory that is bringing through anywhere from 50 to 300 to 1,000 samples a day, can the analysis time be fast enough to get that done in a reasonable time frame?"

A leader in MALDI-based clinical microbiology, BioMérieux is also pursuing LC-MS/MS approaches. In 2015 the company presented research at the Mass Spectrometry Applications to the Clinical Laboratory annual meeting using MRM to quantify expression levels of efflux pumps and β-lactamase, bacterial proteins both linked to resistance to a variety of antibiotics. The year before that it presented on similar work.

Bruker, meanwhile, is working on MALDI-based assays for strain-level identification and antibiotic resistance and, in fact, last month released new research-use-only features for the MALDI Biotyper enabling some strain-level identification and detection of carbapenem-resistant organisms.

Ledeboer said, though, that it was unclear whether MALDI would ultimately prove the best technology for such work. "I certainly think Bruker and BioMérieux are trying to push it, but I'm not certain that ultimately MALDI is going to give us the level of strain resolution that we are going to need," he said.

MALDI-based platforms might seem to have advantages from a regulatory perspective, as both the Bruker and BioMérieux platforms have received clearances from a variety of regulatory agencies, including the US Food and Drug Administration. However, Ledeboer noted, strain-level identification is not commonly used in clinical practice but rather for purposes like identifying and tracking the organisms behind an outbreak, which is not regulated by the FDA.

"So the importance of having an FDA approved solution is a little bit less acute," he said.