MALDI ToF with negative ion mode: A new approach for rapid detection of colistin resistance

Antimicrobial resistance (AMR) is one of the biggest public health concerns of our time. Currently, resistance has been reported for almost all available antibiotics, from first-line to last-resort drugs. AMR leads to increased morbidity and mortality of hospitalized patients and imposes a substantial financial burden on healthcare systems worldwide. The limited pipeline of new antimicrobials and the continuous emergence of multidrug-resistant (MDR) organisms needs to be countered by novel antibacterial strategies coupled with rapid diagnostics to detect resistance, particularly in the case of gram-negative bacteria.

In gram-negative bacteria, intrinsic resistance to many antibiotics limits therapeutic options. Thus, polymyxins (polymyxin B or colistin) are used as last-resort antibiotics. Unfortunately, resistance to colistin is now emerging due to acquired resistance, which results from modifications of the drug target, i.e., the lipopolysaccharide (LPS). Although colistin-resistant strains are still rare, their detection is one of the key issues to improving the treatment of the patient. Unfortunately, the methods currently available in routine laboratories for the detection of resistance to colistin still rely on bacterial growth in the presence of colistin. These procedures require 16–20 h in culture, whether determining susceptibility to colistin minimal inhibitory concentration (MIC) using the broth microdilution method (BMD; reference method).

Considering the need to develop, a fast and robust assay for the detection of colistin resistance in gram-negative bacteria, Bruker came up with the latest advanced version of MALDI ToF system that is capable of analyzing lipid molecules. MALDI biotyper Sirius system has negative ion mode in addition to positive ion mode, which broadens the applications beyond routine microbial identification. The analytes that are acidic in nature, such as those containing phosphate or carboxylate groups, are more efficiently ionized by the generation of anions. As such, detection of Lipid A, which contains both long-chain fatty acid and phosphate groups (at 208 carbon 1 and 4ˊ), is superior when anions are generated using the negative ion mode. Therefore, the newly introduced negative ion mode of the MALDI Biotyper Sirius allows efficient detection of both native Lipid A and its modified forms. The new MBT Lipid Xtract™ kit can be combined with MALDI Biotyper Sirius for fast and easy extraction of lipids and analysis.

As discussed earlier, the modification of Lipid A is a common mechanism of colistin resistance in organisms beyond E. coli. The structure of Lipid A from a range of bacterial species (including Klebsiella pneumonia, Shigella spp., and Pseudomonas aeruginosa) can be determined by MALDI-TOF mass spectrometry, this technique provides a broadly applicable basis for the development of new diagnostics in many species of Gram-negative bacteria. Indeed, the Lipid A of Salmonella spp., which has been reported to carry MCR-enzymes (19) is similar to that of E. coli and can be detected using the negative-ion mode of the MALDI Biotyper Sirius as a peak at m/z 1796.2. Similarly, Lipid A from Acinetobacter baumannii can be directly detected using MALDI-TOF mass spectrometry. Colistin resistance in this organism, primarily resulting from the overexpression of the chromosomally-encoded pETN transferase PmrC, can be detected as a +123 m/z addition to the peak corresponding to native bis-phosphorylated hepta236 acyl Lipid A.

The strength of these approaches is that they could serve as a first-line strategy to rapidly, reliably, and cost-effectively identify colistin resistance with minimal changes to existing clinical equipment and workflows. It is expected that MALDI-TOF mass spectrometers with negative-ion mode capabilities approved for routine clinical use will make their way into hospital laboratories as part of the regular equipment upgrade cycle, allowing the implementation of this approach alongside existing positive-ion mode applications. If required for epidemiological purposes, in-depth characterization of the isolated strains could be pursued at a second stage using culture-, PCR- or sequencing-based methods. This type of pipeline would ensure that treatment of patients with challenging MDR gram-negative infections happens as efficiently as possible, improving patient outcomes without compromising the necessary epidemiological component of clinical microbiology.