Skip to main content
Premium Trial:

Request an Annual Quote

Imperial College Team Develops MALDI Assay for Detecting Polymyxin Resistance


NEW YORK (GenomeWeb) – Researchers from Imperial College London have developed a MALDI mass spec-based method for detecting polymyxin resistance in Enterobacteriaceae.

In a study presented this week at the European Congress of Clinical Microbiology and Infectious Diseases annual meeting in Vienna, the scientists used MALDI to test for polymyxin resistance in 53 Escherichia coli and 81 Klebsiella pneumoniae strains, finding that they were able to identify resistant strains with 100 percent accuracy.

They were also able to distinguish between strains whose resistance was mediated by chromosomal changes and those with resistance mediated by a plasmid, which has important implications for patient management, said Laurent Dortet, an Imperial College researcher and first author on the study.

MALDI mass spec has made significant inroads in the research and clinical microbiology markets for microorganism identification, with Bruker and BioMérieux leading the way with, respectively, their MALDI Biotyper and Vitek MS platforms. More recently, these companies, as well as outside researchers, have taken an interest in applying the technology to the detection of antibiotic resistance, where MALDI-based methods could offer a less expensive and faster way to assess an infection's susceptibility to different agents.

In the case of the Imperial College polymyxin work, the researchers were able to identify mass spec features corresponding to modifications to bacterial lipopolysaccharides (LPS) that confer resistance to this class of antibiotics.

Specifically, these modifications consist of the addition of cationic groups like phosphoethanolamine (pETN) or 4-amino-L-arabinose (L-Ara4N) to the lipid A portion of the LPS in Enterobacteriaceae.

These additions lead to a change in the mass of lipid A that can be detected by mass spec as a peak at m/z 1919, which corresponds to the addition of pETN to the m/z 1796 of lipid A in its unmodified form. In an analysis of a collection of 53 E. coli and 81 K. pneumoniae strains that included nine polymyxin-resistant varieties of the former and 49 polymyxin-resistant varieties of the latter as confirmed by conventional colistin susceptibility testing, the researchers detected the m/z 1919 peak in all resistant isolates and in none of the susceptible ones.

Dortet said that the lipid A modification and its role in causing polymyxin resistance has been well known for many years, as was the fact that it would cause the observed mass shift. He noted that the issue of polymyxin resistance has gained new currency in recent years as areas with high prevalence of carbapenem-resistant infections have begun using more polymyxins.

"It has started to become an issue in countries where polymyxin has become the first treatment for infections, meaning in countries where there is a high rate of [carbapenem-resistance]," Dortet said, citing Greece and Italy as European Union countries where this was a particular problem. "If someone is very ill with sepsis, for example, the physician just starts colistin therapy before having the results of the susceptibility testing," he said.

Traditional susceptibility testing can take three or four days, Dortet said, while MALDI-based methods require around a day, most of which is taken up growing colonies to be run on the mass spec. The mass spec analysis itself takes around 15 minutes, he said.

In addition to identifying polymyxin-resistant strains, the researchers were also able to distinguish between organisms whose resistance stemmed from alterations in their own DNA versus those whose resistance was derived from a plasmid carrying the gene mcr-1, which confers polymyxin resistance. In the latter cases, an additional peak is present at 1821 m/z.

Dortet said that he and his colleagues have not yet characterized this peak, but that it appeared to be linked to the production of the pETN transferase encoded by mcr-1 as it was observed only in MCR-1 producing isolates.

Distinguishing between resistance due to chromosomal alterations and plasmids is very important to patient management Dortet said, given the ability of plasmids to move between bacteria.

"If the resistance is due to a plasmid, there can be a lot of dissemination of this [resistance]," he said, adding that clinicians dealing with a patient with plasmid-mediated polymyxin resistance may want to confine the patient in their own room or assign dedicated staff to their care.

In the ECCMID study, Dortet and his colleagues looked at E. coli and 81 K. pneumoniae, but he said that they had shown the approach worked in Acinobacter, as well.

He added that they hoped to demonstrate the ability to detect the resistance marker directly from patient samples, which would eliminate the need for culturing samples prior to mass spec analysis, significantly speeding the identification and resistance detection process.

Dortet said he believed direct detection of the polymyxin markers would be possible, though the field has struggled to develop such methods thus far, limited by factors including the complexity of samples like patient blood and the sensitivity constraints of the MALDI instruments typically used in such workflows.

MALDI-based approaches similar to those used in the ECCMID study have also been applied to other forms of antibiotic resistance, most notably the detection of carbapenem-resistant organisms. For instance, researchers from institutions including the National Institutes of Health and the University of Bologna have used MALDI to detect a peak specific to the pKpQIL_p019 protein, which is often present on the same plasmid as the bkaKPC gene. The bkaKPC gene produces the protein carbapenemase KPC, which can confer resistance to carbapenems.

Other researchers, including scientists at Bruker, are developing methods using MALDI mass spec to identify resistant strains by detecting the hydrolysis products generated by KPC. Bruker has also developed a research-use-only method on the MALDI Biotyper for detection of carbapenem-resistant strains of Bacteroides fragilis and Staphylococcus aureus via detection of the presence of, respectively, the cfiA gene and the mecA cassette expressing the protein PSM.

Dortet said that he and his colleagues aimed to work with Bruker or BioMérieux to incorporate their polymyxin assay as part of those vendors' MALDI microbiology platforms.