NEW YORK – With the release earlier this year of its MALDI Biotyper Sirius system, Bruker is moving beyond the platform's traditional focus on proteins and into analysis of other analytes that could boost its capabilities in areas like antibiotic resistance testing and microbial identification.
Specifically, the instrument's ability to run in negative-ion mode enables more effective analysis of lipids, which researchers are exploring for applications like detecting antibiotic-resistant organisms and identifying bacteria directly from tissue.
To date, researchers and clinicians have primarily used the MALDI Biotyper to look at bacterial proteins because these molecules were more amenable to analysis by the instrument's positive ion mode, said Gerald Larrouy-Maumus, group leader at the MRC Centre for Molecular Bacteriology & Infection at Imperial College London.
While lipids are also helpful in identifying organisms and assessing antibiotic resistance, the Biotyper was not as good at measuring these molecules, which benefit from analysis in negative ion mode, said Larrouy-Maumus, who is working with Bruker to develop and commercialize a Biotyper-based test for colistin-resistant bacteria.
The addition of negative ion mode to the Sirius system allows researchers to analyze both proteins and lipids effectively.
"You can do bacterial typing based on the protein level like before, but on top of that you have a new range of opportunities thanks to the negative ion mode," Larrouy-Maumus said.
"As the lipid composition of microbial cell walls and membranes can be quite characteristic, the MALDI Biotyper Sirius opens up new fields of analyses," said Wolfgang Pusch, executive VP microbiology & diagnostics at Bruker.
Perhaps the most notable application of the lipid analysis enabled by the instrument is improved antibiotic susceptibility testing, which is a major area of focus for Bruker and MALDI-based microbiology research in general.
In a study published last year in Nature Scientific Reports, Larrouy-Maumus and his colleagues demonstrated the effectiveness of a MALDI-based assay, which they called the MALDIxin test, to identify colistin-resistant Acinetobacter baumannii isolates in around 15 minutes by detecting modifications to the organism's lipid A. Traditional phenotypic minimum inhibitory concentration testing can take as long as 24 hours, the authors wrote.
As antibiotic resistance has grown, colistin has become an antibiotic of last resort, particularly in European Union countries like Italy and Greece. More frequent use of this drug has, in turn, led to increased resistance, making a more rapid assay like the MALDIxin test potentially valuable.
In 2017, the researchers presented data from the assay in which they were able to test for colistin resistance in 53 Escherichia coli and 81 Klebsiella pneumoniae strains, identifying the resistant strains with 100 percent accuracy.
Larrouy-Maumus said that the assay has also been able to detect colistin resistance missed by phenotypic testing, which he noted retains a measure of subjectivity since growth is measured by eye.
"With MALDI, either you have a peak [indicating resistance] or you don't," he said.
Additionally, he noted that the test is able to distinguish between resistance mediated by an alteration in the bacteria's chromosome, which cannot be transferred between organisms, and resistance mediated by a plasmid, which can be transferred. Being able to make this distinction is important to controlling the spread of resistant bacteria.
Led by Bruker and BioMérieux, which offers the competing Vitek MS system, MALDI mass spec has become an established technology in the clinical microbiology lab for microbial ID. As that market has matured, attention has increasingly turned to using MALDI for antibiotic resistance assays, where it offers the potential for a cheaper, faster, and more accurate alternative to existing tests.
Bruker currently offers CE-IVD-marked Biotyper tests for cephalosporin and carbapenem resistance. It offers both tests in the US as research-use-only versions.
Nathan Ledeboer, professor of pathology and medical director of microbiology at the Medical College of Wisconsin, suggested that whether MALDI was able to make significant inroads into antibiotic susceptibility testing would depend substantially on how highly automated the process can be made.
"When you think about the number of different dilutions and drugs you test [in a susceptibility assay], you may be testing up to 135 'drug-bug' combinations," he said. "To try to do that manually would be incredibly difficult, so I think you really have to develop a system that can handle inoculation and incubation and then transfer whatever the end product is to the MALDI target."
"Is this going to be kind of a boutique assay that you use for one or two high-value drugs at one or two high-value dilutions, which is a bit more manageable in a manual fashion, or do you really intend it to be a replacement for traditional phenotypic testing, in which case it needs to be highly automated," he said.
Pusch said that Bruker's strategy was to focus on "antibiotic resistance and susceptibility testing for urgent cases, like sepsis patients," adding that "the sample throughput for this application is much lower than for broad [antibiotic susceptibility testing]."
"Rapid resistance and susceptibility testing in blood culture workflows are especially in our focus," Pusch said. "In these cases, guided by rapid MALDI Biotyper identification and rapid functional resistance testing, therapies can potentially be adjusted the same day by the clinician."
He cited as an example the company's Bologna Workflow, which it developed in collaboration with researchers at the University Hospital, Policlinico Sant'Orsola-Malpighi, Bologna, Italy, from which it takes its name. The workflow combines microbial identification from positive blood cultures with testing for resistance to cephalosporin and carbapenem antibiotics and, according to clinicians at the hospital, has reduced the time to make an ID and determine susceptibility from as many as 48 hours to between 30 minutes and two hours.
In addition to antibiotic resistance testing, the lipid analysis enabled by the Sirius system could also prove useful for microbial identification, filling in gaps where a conclusive identification can't be made by protein data alone, Larrouy-Maumus said.
He noted, for instance, that it was difficult using only protein information to distinguish between Escherichia coli and Shigella but that the two could be distinguished using lipid analysis.
"This is still academic work, and we still have to improve [the method], but this is what we are doing in our lab, using positive ion mode for [identification] as usual, and if that is not enough we switch to negative ion mode to get more information to discriminate," he said.
While it is perhaps unlikely, given the broad adoption of MALDI-based proteomic profiling for microbial ID, that Bruker would move to a primarily lipid-based approach, lipid-level analyses do offer some potential advantages, Larrouy-Maumus noted.
For instance, sample preparation for lipid-based assays can be more straightforward than that for protein assays, which, depending on the target organism, can require time-consuming steps to extract the proteins for analysis.
"It is complicated to get access to [bacterial] proteins, which are cytosolic, but lipids are exposed on the surface," Larrouy-Maumus said.
Lipids have been an area of focus for other mass spec platforms applied to microbial identification. For instance, much of the microbiology work done using the rapid evaporative ionization mass spectrometry (REIMS) technology developed by Larrouy-Maumus' Imperial College colleague Zoltan Takats has focused on lipid analysis. Waters acquired the REIMS technology, which uses electromagnetic current to ionize samples, in 2014.
"REIMS is a beautiful technology," Larrouy-Maumus said, but he noted that MALDI systems have an advantage in terms of throughput and instrument cost as well as the fact that they are already entrenched in the clinical marketplace.