Two-site study on performances of a commercially available MALDI-TOF MS-based assay for the detection of colistin resistance in Escherichia coli

Colistin is a last resort drug for the treatment of multiple drug-resistant (MDR) Gram-negative bacterial infections. Rapid methods to detect resistance are highly desirable. Here, we evaluated the performance of a commercially available MALDI-TOF MS-based assay for colistin resistance testing in Escherichia coli at two different sites. Ninety clinical E. coli isolates were provided by France and tested in Germany and UK using a MALDI-TOF MS-based colistin resistance assay. Lipid A molecules of the bacterial cell membrane were extracted using the MBT Lipid Xtract Kit™ (RUO; Bruker Daltonics, Germany). Spectra acquisition and evaluation were performed by the MBT HT LipidART Module of MBT Compass HT (RUO; Bruker Daltonics) on a MALDI Biotyper® sirius system (Bruker Daltonics) in negative ion mode. Phenotypic colistin resistance was determined by broth microdilution (MICRONAUT MIC-Strip Colistin, Bruker Daltonics) and used as a reference. Comparing the results of the MALDI-TOF MS-based colistin resistance assay with the data of the phenotypic reference method for the UK, sensitivity and specificity for the detection of colistin resistance were 97.1% (33/34) and 96.4% (53/55), respectively. Germany showed 97.1% (33/34) sensitivity and 100% (55/55) specificity for the detection of colistin resistance by MALDI-TOF MS. Applying the MBT Lipid Xtract™ Kit in combination with MALDI-TOF MS and dedicated software showed excellent performances for E. coli. Analytical and clinical validation studies must be performed to demonstrate the performance of the method as a diagnostic tool.


Introduction
Antimicrobial resistance is an issue of global importance and one of the defining public health concerns of our time [1]. The limited pipeline of novel antimicrobials and the spread of multidrug-resistant (MDR) organisms have increased our reliance on a few last-line antibiotics for the treatment of MDR Gram-negative bacteria. Crucial last-resort agents are the polymyxin antibiotics, such as polymyxin B and colistin [2,3].
In Gram-negative bacteria like Escherichia coli, polymyxin resistance mostly occurs as a consequence of lipopolysaccharide (LPS) modifications, with the addition of phosphoethanolamine (pEtN) and/or 4-amino-L-arabinose (L-Ara4N) cationic groups on the lipid A portion of LPS [4,5]. These lipid A modifications often arise due to (i) alterations of the PmrAB and PhoPQ two-component systems and mutations of the negative regulator of PhoPQ and MgrB that are both chromosome-encoded or (ii) because of the activity of a plasmid-borne pEtN transferase called mobile colistin resistance (MCR) enzyme [6]. The first MCR enzyme, mcr-1, was reported in 2016 [7], and this discovery was followed by the rapid identification of other mobile polymyxin resistance genes [8][9][10][11].
We have previously reported on the development of the MALDIxin test, a diagnostic tool based on matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) that can be used to detect colistin resistance using whole bacteria [12][13][14][15][16][17][18]. Although fast and effective, this test was not optimized for routine use in diagnostic microbiology laboratories. The main limitations were that such an approach was not developed for the MALDI-TOF mass spectrometers widely used for bacterial identification in clinical microbiology laboratories, as well as not having a robust and easy standardized kit and software module to perform automated data analysis and interpretation. In addition, the standard workflow of the MALDIxin test required centrifugation steps [12,13,[16][17][18], the use of either super-2,5-Dihydroxybenzoic acid or Norharmane matrix, which led to the non-homogenous co-crystallization of the biological sample with the matrix on the MALDI target plate, precluding any automation of the data acquisition and processing.
The aim of this study was to determine the performances and reproducibility of a standardized methodology designed (and commercialized) for the low-resolution linear MALDI-TOF MS instrument in negative ion mode employed by the MALDI Biotyper® sirius system (Bruker Daltonics) to accurately identify colistin resistance in clinical E. coli isolates by detecting addition of both pEtN and L-Ara4N moieties to lipid A and reporting the results using an automated software module.

Bacterial strains
A collection of 90 E. coli clinical isolates (Table 1) was used in this study which was provided by the Centre National de Reference associé Résistance aux Antibiotiques "Entérobactéries productrices de carbapénèmases." The same sets of strains were sent as heat-inactivated isolates to Imperial College London, UK, and Bruker Daltonics GmbH & Co KG, Bremen, Germany, for the evaluation of the MBT Lipid Xtract™ Kit (RUO, Bruker Daltonics) in combination with the MBT HT LipidART Module (RUO, Bruker Daltonics). Evaluation centers in the UK and Germany were blinded of the colistin resistance phenotype and of the colistin resistance mechanisms. Preliminary experiments showed comparable results of lipid A extracts of heat-inactivated isolates and viable strains using the MBT Lipid Xtract™ Kit in conjunction with the MBT HT LipidART Module (data not shown).

Genotype determination
Identification of commonly encountered mcr genes (mcr-1 to mcr-10) was performed by PCR and sequencing of the amplicon in case of a positive signal (Table 1) [19]. For colistin-resistant isolates that were negative for the tested mcr genes, mutations in mgrB, pmrA, pmrB, phoP, and phoQ have been checked by PCR and sequencing. The β-lactamase encoding genes were identified as previously described [20].

Susceptibility testing
Broth microdilution was performed as a phenotypic reference method using MICRONAUT MIC-Strip Colistin (Bruker Daltonics), following the manufacturer's instructions. MICRONAUT MIC-Strip Colistin is a validated IVD product according to DIN EN ISO 20776-2 [21] and was tested by EUCAST showing 99% essential agreement [22]. Classification of colistin susceptibility or resistance was done according to EUCAST breakpoints (version 12.0) [23]. In case of discrepancies between the MALDI-TOF MS-based result and classification result of the reference method, broth microdilution was repeated in triplicate, and the median result was used as the final reference result.

Extraction of lipid A
Lipid A from E. coli was extracted using the MBT Lipid Xtract™ Kit, following the manufacturer's instructions. Briefly, bacteria were grown overnight in the Mueller-Hinton agar, the equivalent of a 1-μL inoculation loop placed into a 1.5-mL low-binding microtube, and mixed in 50 μL of MBT Lipid Xtract Hydrolysis buffer. A total of 44 μL of the cell suspension was discarded, and the remaining 6 μL was submitted with the lid of the tube closed to a heating process at 90 °C for 10 min. The tubes were left for 2 min with the lid open to completely evaporate the buffer. The dried pellets were washed with 50 μL of MBT Lipid Xtract washing buffer for a few seconds without dissolving the pellet. The total volume of the washing buffer was discarded by pipetting. Finally, 5 μL of the matrix was pipetted up and down for 15-20 s to resuspend the dried pellet, and 2 μL of the suspension was spotted onto either an MSP 96 polished steel target (Bruker Daltonics, Part-No. 8280800) or an MBT Biotarget 96 (Bruker Daltonics, Part-No. 1840375).

Acquisition of MALDI-TOF mass spectra and data analysis
The spectra were recorded in the linear negative-ion mode (laser intensity 45%, ion source 1 = 15.00 kV) of the MALDI

Results and discussion
The MBT Lipid Xtract™ Kit was evaluated using a panel of 90 E. coli clinical isolates (Table 1). Using broth microdilution as the phenotypic reference method, 34 strains were tested as colistin resistant and 55 strains as colistin susceptible, respectively. One strain showed inconsistent MIC results and was defined as not applicable.
For all colistin-susceptible E. coli clinical isolates in this panel, a single peak at m/z 1796.2 was detected, assigned to hexa-acyl diphosphoryl lipid A containing four C14:0 3-OH, one C14:0, and one C12:0, confirming that the lipid A in these strains was unmodified (Fig. 1). For colistin-resistant clinical isolate strains, both the native lipid A peak and the additional pEtN peak at m/z 1919.2 were observed, corresponding to the addition of one residue of pEtN (+ 123 mass unit) (Fig. 1) or the additional peak at m/z 1927.2 corresponding of the addition of one  (Fig. 1). Evaluation of the performance of the MALDI-TOF MSbased colistin resistance assay was done according to valid guidelines [21], and broth microdilution was used as the phenotypic reference method. The mass spectra of the 90 E. Coli clinical isolates were acquired in the negative ion mode on the MALDI Biotyper® sirius system. Using the MBT HT LipidART Module, the sensitivity and specificity of the method were 97.1 and 96.4%, respectively, for the UK site and 97.1 and 100%, respectively, for the German site (Table 2).
For the German site, one false negative sample was observed (Ecoli_S04). The sample showed an MIC of 8 µg/ mL but was identified as susceptible by the MALDI-TOF MS-based method. This discrepancy was not observed for the viable strain (data not shown).
At the UK site, the same sample was detected as false negative (Ecoli_S04), and two other samples were observed as false positive (E.coli_R59 and Ecoli_S20). The reference method showed a susceptible result, and the MALDI-TOF MS-based method revealed modified lipid A, which means colistin resistance due to calculated (R)-values that were close to the algorithm-based cutoff value.
Regarding the results, the strains, Ecoli_R59 and Ecoli_ S20, show the discrepancy between the two sites. That could be due to the low presence of a non-specific peak at the  m/z assigned to the L-Ara4N range and that the automated software pickup that background as positive or negative depending on the site, regarding the discrepancy between the two sites and whole genome sequencing data, which are Ecoli_R03, Ecoli_R10, Ecoli_R14, Ecoli_R39, Ecoli_ R43, Ecoli_R51, Ecoli_R56, Ecoli_R59, Ecoli_R60, and Ecoli_R61. For strains like Ecoli R_14, one possibility is that mcr-1 expression could be induced by the presence of colistin [24]. However, as no shift in MIC above 2 μg/mL was observed, it is likely that plasmid has been lost due to the long storage of the clinical isolates in the − 80 °C freezer and by freeze/thawing the stocks. We, therefore, recommend performing the assay as soon as possible after the isolation of the strains. For strains like Ecoli_R03, even if carrying mcr-1 and having a MIC at 2 μg/mL, which is at the breakpoint, no modification of the lipid A could be reported. However, the MIC and our assays are performed in vitro, and we cannot rule out that exposure to host factors could stimulate the expression of mcr-1, rendering the bacteria resistant to colistin. In another topic, for example, it has been reported that human serum induces antibiotic tolerance in Staphylococcus aureus by triggering the staphylococcal GraRS two-component system, resulting in increased peptidoglycan accumulation [25]. We cannot rule out that in the case of colistin resistance, host factors could possibly trigger the resistance in strains carrying mcr plasmids. That is why, in some cases, one could recommend taking into consideration strains that have an MIC of 2 μg/mL as resistant [26].
Overall, this study showed a high reproducibility of the MALDI-TOF MS-based colistin resistance assay revealing comparable results for both test sites (Table 2).
Previous studies suggested that the MALDIxin test was promising for the rapid identification of colistin resistance in Gram-negatives [12][13][14][15][16][17][18]. Unfortunately, this homemade version of the test was less suitable for use in routine diagnostics due to the requirement of the high amount of biological material and high laser power at the limit of the MALDI system (> 90%). The MALDI-TOF MS-based assay presented here requires very less biological material, no centrifugation step, and only short handson time. Additionally, the measurement can be performed with the same low-resolution settings used for other clinical applications on the MALDI Biotyper® sirius system. Furthermore, the development of a software module able to automatically interpret the MALDI-TOF spectrum is far more accurate for diagnostic use (Fig. 2). Compared to the MALDIxin test, the assay presented here is, therefore, more suitable for use in routine diagnostics (Fig. 3). Of note, with this new commercial kit, very homogeneous crystallization on the target surface can be achieved with After the appropriate development of in vitro diagnostic (IVD) consumables and software, following further clinical and analytical studies, it will be a very rapid and easy way to diagnose colistin resistance in clinical laboratories in the near future. Time-to-result will be significantly shortened compared to standard susceptibility testing methods in routine diagnostics. Preliminary studies showed that the method might be suitable for testing directly from positive blood cultures (data not shown) and would accelerate the diagnostic by at least one further day providing a same-day result.
This study has some limitations as it focused on E. coli colistin-resistant clinical isolates. Indeed, colistin resistance has also been reported in other Gram-negative bacteria such as Klebsiella spp., Pseudomonas spp., and Acinetobacter baumannii. Accordingly, this new approach should be expanded to those Gram-negative pathogens, too.
Overall, this study represents a major step towards the routine application of MALDI-TOF-based detection of colistin resistance and lays the foundations for a rapid diagnostic test for colistin resistance that will be readily accessible to many clinical microbiology laboratories. As such, it will facilitate improved management and treatment of patients with challenging MDR Gram-negative infections, particularly in countries where carbapenemase production is highly prevalent, leading to the increasing use of polymyxins, for which resistance emerged and started to disseminate.   . 3 Workflow of the MBT Lipid Xtract™ Kit. One colony is transferred in a low-binding 1.5 mL microtube, 50 μL of hydrolysis buffer is added, the colony is suspended, and then, 44 μL are removed. The remaining 6 μL is placed into a thermo block set at 90 °C for 10 min. This hydrolysate is then dried by letting the tube open for 2 min. The microtube is subsequently placed on the bench at room temperature, 50 μL of washing buffer is added, and the solution is vortexed for 1 s.
That washing step allows the removal of molecules that could interfere with the ionization of lipid A. Following that step, 50 μL of the washing buffer is removed. To the remaining material, 5 μL of the matrix is added, and the sample is resuspended. A total of 2 μL is immediately deposited onto the target. Once dried, the MALDI target plate is then placed into MALDI Biotyper sirius® system, and data are analyzed in the negative ion mode Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.