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Rapid detection and discrimination of chromosome- and MCR-plasmid-mediated resistance to polymyxins by MALDI-TOF MS in Escherichia coli: the MALDIxin test

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Rapid detection and discrimination of chromosome- and

MCR-plasmid-mediated resistance to polymyxins by MALDI-TOF MS in

Escherichia coli: the MALDIxin test

Laurent Dortet

1–4

*†, Remy A. Bonnin

3,4

, Ivana Pennisi

1

, Lauraine Gauthier

2–4

, Agne`s B. Jousset

2–4

,

Laura Dabos

3

, R. Christopher D. Furniss

1

, Despoina A. I. Mavridou

1

, Pierre Bogaerts

5

, Youri Glupczynski

5

,

Anais Potron

4,6

, Patrick Plesiat

4,6

, Racha Beyrouthy

4,7

, Fre´de´ric Robin

4,7

, Richard Bonnet

4,7

, Thierry Naas

2–4

,

Alain Filloux

1

and Gerald Larrouy-Maumus

1

1

MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London SW7 2AZ, UK;2Department of Bacteriology-Hygiene, Biceˆtre Hospital, Assistance Publique - Hoˆpitaux de Paris, Le Kremlin-Biceˆtre, France;3EA7361 ‘Structure, dynamic, function and expression of broad spectrum b-lactamases’, Paris-Sud University,

Paris Saclay University, LabEx Lermit, Faculty of Medicine, Le Kremlin-Biceˆtre, France;4French National Reference Center for Antibiotic Resistance, Le Kremlin-Biceˆtre, France;5Laboratory of Clinical Microbiology, Belgian National Reference Center for Monitoring Antimicrobial Resistance in Gram-negative Bacteria, CHU UCL Namur, Yvoir, Belgium;6Bacteriology Unit, University Hospital of

Besanc¸on, Besanc¸on, France;7Bacteriology Unit, University Hospital of Clermont-Ferrand, Clermont-Ferrand, France

*Corresponding author. Hoˆpital de Biceˆtre, Service de Bacte´riologie-Hygie`ne, 78 rue du Ge´ne´ral Leclerc, 94270, Le Kremlin-Biceˆtre, France. Tel: !33 (0) 1 45 21 20 19; E-mail: laurent.dortet@aphp.fr orcid.org/0000-0001-6596-7384

†These authors made an equal contribution to the article.

Received 21 February 2018; returned 10 April 2018; revised 28 June 2018; accepted 26 July 2018 Background: Polymyxins are currently considered a last-resort treatment for infections caused by MDR Gram-negative bacteria. Recently, the emergence of carbapenemase-producing Enterobacteriaceae has accelerated the use of polymyxins in the clinic, resulting in an increase in polymyxin-resistant bacteria. Polymyxin resistance arises through modification of lipid A, such as the addition of phosphoethanolamine (pETN). The underlying mechanisms involve numerous chromosome-encoded genes or, more worryingly, a plasmid-encoded pETN transferase named MCR. Currently, detection of polymyxin resistance is difficult and time consuming.

Objectives: To develop a rapid diagnostic test that can identify polymyxin resistance and at the same time dif-ferentiate between chromosome- and plasmid-encoded resistances.

Methods: We developed a MALDI-TOF MS-based method, named the MALDIxin test, which allows the detection of polymyxin resistance-related modifications to lipid A (i.e. pETN addition), on intact bacteria, in,15 min. Results: Using a characterized collection of polymyxin-susceptible and -resistant Escherichia coli, we demon-strated that our method is able to identify polymyxin-resistant isolates in 15 min whilst simultaneously discrimi-nating between chromosome- and plasmid-encoded resistance. We validated the MALDIxin test on different media, using fresh and aged colonies and show that it successfully detects all MCR-1 producers in a blindly ana-lysed set of carbapenemase-producing E. coli strains.

Conclusions: The MALDIxin test is an accurate, rapid, cost-effective and scalable method that represents a major advance in the diagnosis of polymyxin resistance by directly assessing lipid A modifications in intact bacteria.

Introduction

The increasing prevalence of antibiotic resistance is a threat to glo-bal human health whilst the pipeline of new antimicrobials is ex-tremely limited. One of the most concerning developments is the

rapid dissemination of MDR Gram-negative bacteria. In this con-text, and because of the paucity of alternative therapeutic options, polymyxins (polymyxin B and colistin) have become the last-resort therapy, especially for treating infections caused by carbapenem-resistant Enterobacteriaceae.1 These organisms are becoming

VC The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.

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increasingly prevalent worldwide, with many producing carbapenem-hydrolysing enzymes [carbapenemase-producing Enterobacteriaceae (CPE)].2This phenomenon has accelerated the use of polymyxins in CPE-endemic countries such as Greece and Italy, and as a result increased resistance to polymyxins is now being reported.3–7

In Enterobacteriaceae, acquired resistance to polymyxins arises from modifications to the drug target, i.e. LPS.8,9These

modifica-tions consist of addition(s) of cationic groups such as phosphoe-thanolamine (pETN) and/or 4-amino-L-arabinose (L-Ara4N) to the

lipid A part of the LPS. This results in decreased binding of the poly-myxin to the LPS due to electrostatic repulsion between the added cationic groups and the positively charged antibiotic. Addition of pETN andL-Ara4N is associated with chromosome-encoded

resist-ance mechanisms, such as mutations in the PmrA/PmrB or PhoP/PhoQ two-component systems, or through alterations to the master regulator MgrB.8,9More than 10 different genes have been implicated in chromosome-encoded resistance to polymyxin. However, these chromosomal mutations have a high fitness cost, and cannot be transferred between organisms.9,10Recently, it has

been reported that addition of pETN can also occur through the ex-pression of a plasmid-encoded pETN transferase, named MCR-1.11

Since its initial description, the mcr-1 gene has been identified worldwide in Enterobacteriaceae (mostly Escherichia coli), recov-ered from both animal and human samples.8,12The mcr-1 poly-myxin resistance gene is thus a major threat due to its lack of (or very low) fitness cost and its ability to be transferred between strains and species.13,14In order to contain the spread of

poly-myxin resistance in Enterobacteriaceae, a test that enables both rapid and reliable detection of polymyxin resistance as well as dis-crimination between plasmid- and chromosome-encoded resist-ance is urgently needed.

Currently, broth microdilution (BMD) is the gold standard tech-nique for polymyxin susceptibility testing,12,15generating results

up to 24 h after bacterial isolation.16This method has been recent-ly chosen as the unique reference by the CLSI and by the EUCAST,17which rules out methods classically used for determin-ation of antimicrobial susceptibility, such as agar dilution, disc dif-fusion and gradient difdif-fusion (Etest). Additionally, because of high rates of false susceptibility observed when using automated sys-tems (MicroScanVR

, VitekVR

2 and BD PhoenixTM), results obtained with these approaches would need to be confirmed by BMD.15,18

Recently, a biochemical test, the Rapid Polymyxin NP test, which detects bacterial growth in the presence of a defined concentra-tion of a polymyxin, has been developed.19This test is claimed to be rapid (within 2 h) and is easy to perform, but is not able to dis-criminate between plasmid- and chromosome-encoded resist-ance to polymyxin; the latter requires an additional step, usually molecular detection (e.g. PCR) of mcr-like genes. However, fast de-tection of mcr-positive isolates is one of the key issues in curbing the dissemination of this plasmid-encoded resistance and facilitat-ing the use of any future MCR inhibitors,20,21which may become

essential for the treatment of patients infected with MDR and MCR-producing Enterobacteriaceae.22–30

Molecular biology (e.g. PCR or WGS) is widely used for the detec-tion of many antimicrobial resistance mechanisms. Nonetheless, this may not be the best option for the detection of polymyxin re-sistance. The chromosome-encoded genes associated with poly-myxin resistance are numerous, and the gene modifications

(disruptions, deletions, mutations) involved are not systematically described or characterized. Regarding plasmid-encoded resist-ance, five families of mcr genes have already been reported in Enterobacteriaceae.31–35MCR-2, MCR-3, MCR-4 and MCR-5 share

only 81%, 34%, 33% and 31% amino acid identity, respectively, with MCR-1 (FigureS1, available as Supplementary dataat JAC Online). This diversity will inevitably lead to the failure of systemat-ic detection of polymyxin resistance as the available molecular biology tools are dedicated to mcr-1 and/or mcr-2 detection (FigureS2).31,32,34,35

To overcome these issues, we developed a cost-effective assay based on MALDI-TOF technology, named the MALDIxin test, which aims to detect polymyxin resistance using a single bacterial colony in,15 min. By using a large panel of E. coli strains, we also demon-strate that this technique can, at the same time, efficiently dis-criminate between chromosome- and plasmid-encoded (mcr-like genes) resistance mechanisms.

Materials and methods

Bacterial strains and plasmids

We used a collection of 87 E. coli strains including 41 polymyxin-resistant isolates, of which 29 were MCR producers (18 MCR-1, 2 MCR-1.5, 3 MCR-2, 2 MCR-3 and 4 MCR-5). The 46 polymyxin-susceptible E. coli strains were of various phenotypes, from WT to carbapenemase producers (TableS1). The MALDIxin test was also prospectively evaluated using a collection of 78 iso-lates of carbapenemase-producing E. coli received during October and November 2016, from the French National Reference Centre (NRC) for Antimicrobial Resistance (Table1).

In addition to the above clinical strains, functional mcr-like genes were cloned into pDM1, an isopropyl b-D-thiogalactoside (IPTG)-inducible TetRderivative of pACYC184.36mcr-1, mcr-2 and mcr-5 were cloned into the SacI/XmaI sites of the vector, while for mcr-3 the NdeI/XmaI sites were used. The resulting plasmids were transformed into E. coli DH5a and MC1000.37Whenever these strains were used (during susceptibility test-ing or for the MALDIxin test), 0.5 mM IPTG was added to the growth medium; the pDM1 vector was used as a control.

Susceptibility testing

MICs were determined by BMD according to the guidelines of the CLSI and EUCAST joint subcommittee.17 Results were interpreted using EUCAST breakpoints as updated in 2017.38

MALDIxin test

Bacterial strains were cultured on lysogeny broth (LB) agar and Mueller– Hinton (MH) agar. A single colony was resuspended in 200 lL of distilled water in a 1.5 mL microtube. The bacteria were then washed three times with 200 lL of distilled water and resuspended in 100 lL of double-distilled water. To perform MS analysis, a patented 2,5-dihydroxybenzoic acid (DHB) matrix was used at a final concentration of 10 mg/mL in chloro-form/methanol 90:10 v/v. We loaded 0.4 lL of the bacterial solution onto the target and immediately overlaid it with 0.8 lL of the matrix solution. Bacterial solution and matrix were mixed directly on the target by pipetting and the mix was dried gently under a stream of air (,1 min). MALDI-TOF MS analysis was performed on a 4800 Proteomics Analyzer (Applied Biosystems) using the reflectron mode. Samples were analysed by operat-ing at 20 kV in the negative ion mode usoperat-ing an extraction delay time set at 20 ns. Typically, spectra from 500 to 2000 laser shots were summed to ob-tain the final spectrum. MS data were analysed using Data Explorer version 4.9 (Applied Biosystems).

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Statistical analysis

All experiments were carried out on three independent bacterial cultures (on three different days). Data were compared two-by-two using the un-paired Welch’s t-test. P values,0.05 were considered statistically different.

Results

We have previously reported a method for the direct detection of lipid A from intact and untreated Gram-negative bacteria.39In Enterobacteriaceae, polymyxin resistance is associated with the addition of, at least, pETN on the phosphate group at position 40or

1 of lipid A (Figure1). Accordingly, this modification should be observed on MALDI-TOF spectra obtained from polymyxin-resistant isolates as it corresponds to an increase of 123 mass units compared with the mass assigned to native lipid A (at m/z 1796 in E. coli) (Figure1).

Determination of polymyxin resistance in E. coli using

the MALDIxin test

In polymyxin-susceptible E. coli strains the negative mass spec-trum scanned between m/z 1600 and 2200 was dominated by a set of peaks assigned to bis-phosphorylated hexa-acyl lipid A (Figure2a). The major peak at m/z 1796.2 is known to correspond to the hexa-acyl diphosphoryl lipid A containing four C14:0 3-OH, one C14:0 and one C12:0, and referred to as native lipid A (Figure1, left panel).

In all polymyxin-resistant E. coli strains, an additional peak at m/z 1919.2 was observed (Figure2b and c) independent of the resistance mechanism involved (chromosome- or plasmid-encoded). This peak corresponds to the addition of one pETN moi-ety to the phosphate group at position 1 of native lipid A (Figure1, right bottom panel), leading to an increase of 123 mass units com-pared with the mass assigned to native lipid A (Figure2b and c).

In the case of plasmid-encoded resistance (mcr-like genes in Enterobacteriaceae), a third peak at m/z 1821.2 was routinely observed in addition to the peaks corresponding to the native lipid A (m/z 1796.2) and the pETN-modified lipid A (m/z 1919.2) (Figure2c). This m/z 1821.2 peak was assigned to the addition of a pETN moiety onto the phosphate group at position 40of native lipid

A with concomitant loss of the phosphate group at position 1 (Figure1, right upper panel), and appears to be a specific marker of MCR-like enzymes. This observation is in line with the fact that the dephosphorylation can only occur on the phosphate group at pos-ition 1.40

To further support this observation, we analysed 73 E. coli isolates including 46 polymyxin-susceptible strains with various antimicrobial resistance phenotypes. Of these strains, four possess chromosome-encoded resistance to polymyxin and 23 are MCR producers (Table S1). The intensity of the peaks corresponding to the native lipid A (m/ z 1796.2), the pETN-modified lipid A (m/z 1919.2) and the specific marker of MCR resistance (m/z 1821.2) were recorded from three in-dependent experiments. The ratio [termed polymyxin resistance ratio (PRR) hereafter] of the sum of the intensities of the peaks asso-ciated with modified lipid A (for E. coli peaks at m/z 1919.2 and m/z

Table 1. Detection and characterization of polymyxin resistance using conventional techniques (MICs and PCR) and the MALDIxin test on 78 carbape-nemase-producing E. coli strains received by the French NRC during October and November 2016

Carbapenemase No. of isolates

Polymyxin resistance MALDI-TOF results

CST MIC (mg/L) mcr-1/-2 PCRa PRR E. coli interpretation KPC-2 1 0.25 # 0 susceptible KPC-3b 1 0.25 # 0 susceptible NDM-1 3 0.25 # 0 susceptible NDM-1 1 0.5 # 0 susceptible NDM-1 1 4 !(mcr-1) 1.73+0.23 plasmid-encoded resistance NDM-1 1 4 !(mcr-1) 2.34+0.12 plasmid-encoded resistance NDM-5 2 0.25 # 0 susceptible NDM-5 3 0.5 # 0 susceptible VIM-1 1 0.5 # 0 susceptible VIM-4 1 0.25 # 0 susceptible OXA-48 27 0.25 # 0 susceptible OXA-48 26 0.5 # 0 susceptible OXA-48 1 1 # 0 susceptible

OXA-48b 1 4 !(mcr-1) 1.29+0.63 plasmid-encoded resistance

OXA-181 4 0.25 # 0 susceptible OXA-204 1 0.25 # 0 susceptible OXA-244 1 0.25 # 0 susceptible OXA-244 1 0.5 # 0 susceptible OXA-181!NDM-5 1 0.25 # 0 susceptible CST, colistin.

a!, positive PCR for the gene indicated in parentheses; #, negative PCR.

bStrains were isolated from the same patient and have been previously reported by Beyrouthy et al.42as E. coli WI1 and E. coli WI2, respectively.

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1821.2) to the intensity of the peak of native lipid A (for E. coli m/z 1796.2) allows accurate distinction between polymyxin-susceptible and polymyxin-resistant isolates, but also discrimination between chromosome-encoded and MCR-related (i.e. plasmid-encoded) re-sistance to polymyxin. (Figure2d). The PRR value for all susceptible E. coli strains was found to be 0 (TableS1and Figure2d). The ratio ranged from 0.109 to 0.481 (average of 0.210) for E. coli strains with chromosome-encoded resistance to polymyxin, and from 0.533 to 4.844 (average of 2.340) for all MCR producers (Table S1 and Figure 2d). The distribution of the PRRE. coli values of polymyxin-susceptible isolates compared with E. coli strains with chromosome-encoded resistance to polymyxin and MCR-producers were all signifi-cantly different (P,0.001, Welch’s t-test). Analysis of receiver oper-ating characteristic (ROC) curves allowed us to define two cut-off values for PRRE. coli (0.1 and 0.5) that discriminate polymyxin-susceptible (PRRE. coli ,0.1) and polymyxin-resistant isolates (PRRE. coli.0.1) and also allows further discrimination between

chromosome-encoded (0.1,PRRE. coli,0.5) and plasmid-encoded resistance (PRRE. coli.0.5).

Performance of the MALDIxin test on colonies grown on

clinically relevant media

We assessed the performance of the MALDIxin test using colonies grown on media routinely used to test for antimicrobial susceptibil-ity, i.e. MH agar. In addition, because of the rapid rise of MCR-1-producing Enterobacteriaceae since 2016, it has been suggested that polymyxin-supplemented media should be used to screen organisms isolated from infected patients.41 Accordingly, the

MALDIxin test was conducted on three E. coli strains (a WT strain, a strain with chromosome-encoded resistance and an MCR-1-producing strain), grown on LB agar, MH agar and polymyxin B-supplemented MH agar at final concentrations of either 1 or 2 mg/L. As shown in Figure S3, similar spectra were obtained SUSCEPTIBLE MCR-like MCR-like Chromosome-encoded resistance RESISTANT Plasmid-encoded resistance O O P O O O O O HO H NH O O O O O O H3C H3C H3C 14 14 12 14 14 14 H3C H3C H3C O O O P OH OH OH OH OH OH O O O O O O NH NH HO O H O O O O O O P O O NH2 OH P OH OH OH O O O O O P O OH OH H HO O O OH OH OH NH NH O O O O O O O O O O O O O O O H2N H3C 14 H3C H3C H3C H3C 14 12 14 14 H3C 14 H3C 14 H3C H3C H3C H3C H3C 14 14 12 14 14 P P O OH O O OH OH Modified Lipid A (1919 m/z) Modified Lipid A (1821 m/z) NH pETN pETN Native Lipid A (1796 m/z)

Figure 1. Lipid A modifications caused by chromosome-encoded determinants or MCR enzymes that can be detected by the MALDIxin test. The structure of the native E. coli lipid A is depicted on the left. Phosphate groups are circled in blue, dephosphorylation at position 1 is circled in green, phosphoethanolamine (pETN) groups are circled in orange. Numbers at the end of each fatty acid chain indicate the number of carbon atoms. The positions of the peaks of interest in the mass spectrum for each structure are also given. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

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irrespective of the growth medium, with the PRRE. colibeing between 2.89 and 5.69 for MCR-1-producing strains and between 0.21 and 0.37 for strains harbouring chromosome-encoded resistance.

To identify any impact of colony age, the MALDIxin test was performed on fresh colonies (overnight incubation) grown on both LB and MH agar and colonies stored for 48 h, 1 week or 2 weeks (agar plates were kept at 4C). Similar spectra were obtained for

all tested colonies leading to no significant differences in their PRRE. coli(TableS2).

Validation of the MALDIxin test on a prospective

collection of carbapenemase-producing E. coli from the

French NRC

In order to further validate the MALDIxin test, all carbapenemase-producing E. coli received from the French NRC during October and November 2016 were blindly analysed in triplicate (three colonies selected from the same MH agar plate). This collection of 78 carbapenemase-producing E. coli strains included 2 KPC

1600 1720 1840 1960 2080 2200 Intensity 1796.2 m/z m/z m/z 1600 1720 1840 1960 2080 2200 Intensity + 123 1796.2 1919.2 1600 1720 1840 1960 2080 2200 Intensity 1821.2 1796.2 1919.2 + 123 + 25 Polymyxin susceptible Polymyxin resistant (chromosome) Polymyxin resistant (mcr-1) Polymyxin susceptible Chromosome-encoded (mcr-like) Polymyxin resistant 2.5 3 3.5 4 4.5 5 0.5 1 1.5 PRR E. coli PRRE. coli I1919 + I1821 I1796 2 0 0.4 0.3 0.2 0.1 P < 0.0001 P < 0.0001 P < 0.0001

=

(a) (d) (b) (c) Plasmid-encoded

Figure 2. Results of the MALDIxin test on E. coli. Representative spectra of (a) a polymyxin-susceptible E. coli isolate; (b) a chromosome-encoded polymyxin-resistant E. coli isolate; (c) a polymyxin-resistant E. coli isolate producing MCR-1. Peaks of interest are indicated. The peak at m/z 1796.2 (blue) corresponds to the native E. coli lipid A, the peak at m/z 1821.2 (green) corresponds to the addition of pETN on the phosphate group at position 40of the native lipid A with concomitant loss of the phosphate group at position 1, and the peak at m/z 1919.2 (red) corresponds to the addition of

one pETN on the phosphate group at position 1 of the native lipid A. (d) Distribution of the polymyxin resistance ratios (PPRs) for the 79 tested E. coli strains (TableS1). Three independent experiments were performed for each strain. Cut-off values for discrimination between polymyxin resistance and polymyxin susceptibility (0.1) and for discrimination between chromosome- and MCR-encoded resistance to polymyxin (0.5) are indicated by a red and a black dotted line, respectively. P values (unpaired Welch’s t-test) are indicated. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

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producers, 11 NDM producers, 2 VIM producers, 63 OXA-48-like producers and 1 isolate producing two different carbapenemases (Table1). Among these isolates, two NDM-1 producers and one OXA-48 producer were also found to be resistant to colistin, with MICs of 4 mg/L.42PCR analysis revealed that these three isolates

were positive for the mcr-1 gene (Table1). When analysed using the MALDIxin test, all polymyxin-susceptible isolates (n " 75) showed a PRRE. coliof 0, corresponding to polymyxin susceptibility, whilst for the three MCR-1-producing isolates a PRRE. coli.0.5 was obtained (1.73+0.23 and 2.34+0.12 for the two NDM-1-producers and 1.29+0.63 for the OXA-48-producer), accurately classifying them as resistant to polymyxin by a plasmid-encoded mechanism (Table1). Therefore, in this prospective study, the MALDIxin test re-liably and rapidly identified all MCR-producing strains while con-ventional methods would have needed 24–48 h with broth dilution method (16–24 h) and subsequent DNA extraction and PCR for mcr-1 and mcr-2 detection on resistant isolates (4 h).

Discussion

Owing to the increasing incidence of carbapenemase-producing Enterobacteriaceae, polymyxins (colistin and polymyxin B) have

become last-resort antimicrobials, largely used for the treatment of severe bacterial infections. In Enterobacteriaceae, resistance to polymyxins results from alterations to lipid A, caused either by adaptive chromosomal mutations or by acquired plasmid-encoded MCR enzymes. Irrespective of the resistance mechanism, the detection of polymyxin resistance remains difficult and time consuming in clinical microbiology laboratories, and relies solely on the determination of colistin or polymyxin B MICs using BMD.17 To provide an accurate, reliable, rapid and cost-effective alterna-tive, we developed a diagnostic test based on MALDI-TOF MS for the detection of polymyxin resistance in Gram-negative bacteria. We call this method the MALDIxin test. After initial validation using a well-characterized collection of polymyxin-susceptible and -re-sistant strains, we performed a prospective evaluation on carbapenemase-producing E. coli during which the MALDIxin test accurately detected all MCR-1 producers. This result was obtained directly from isolated colonies within 15 min, whilst the confirm-ation of colistin resistance and MCR production using standard techniques would have required an additional 24–48 h (Figure3).

An additional challenge when it comes to polymyxin resistance is the rapid dissemination of MCR-producing Enterobacteriaceae. Therefore, discrimination between chromosome- and plasmid-Day 0 Litres of bacterial culture Research workflow Routine workflow 2 h 3-5 h 16-24 h 16-24 h Lipid extraction Polymyxin NP test Patient samples Colonies Bacterial identification MALDI-TOF MALDIxin test Polymyxin susceptibility (R / S) AND Plasmid- vs chromosome-encoded resistance 15min Plasmid Plasmid Readout Lipid A Readout MICs Readout Gene(s) Readout Lipid A Chromosome Chromosome

+

+

Standard susceptibility testing Polymyxin MICs DNA extraction + PCR (mcr-1/–2)

Day 1 Day 2 Day 3 Day 4 1-3 weeks

MALDI-TOF Polymyxin resistant isolate Colistin-MAC (Dipicolinic acid inhibition) 250 ml

Figure 3. Comparison of research (purple outline), routine clinical (green outline) and MALDIxin (blue outline) workflows for the detection of chromo-some- and plasmid (MCR)-encoded resistance to polymyxin in Enterobacteriaceae. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

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encoded resistance is important due to the higher potential for dis-semination of plasmid-encoded compared with chromosome-encoded resistance mechanisms. In the future this distinction may also be crucial in order to facilitate the use of MCR inhibitors, which are being developed.20,21,43,44Currently, this discrimination mostly relies on molecular tests dedicated to the detection of mcr genes. These are based on real-time PCR,45–47loop-mediated isothermal amplification48 and microarray techniques.49 These molecular

assays are costly and, whilst they detect mcr-1 and/or mcr-2, they are not able to detect all members of the highly divergent and rap-idly expanding mcr gene family (Figures S1 and S2). In fact, there are growing numbers of reports describing new MCR families, all of which were discovered using a combination of conventional sus-ceptibility testing methods coupled with WGS.31,32,34,35

Despite their divergent ancestral origins and their low identity (FigureS1), all MCR enzymes possess a common enzymatic activity, the add-ition of a pETN moiety to the lipid A of Gram-negative bacteria. The MALDIxin test directly detects this signature and can therefore be used to identify any MCR-encoding strain. Most specifically, our method identifies a specific signature (peak at m/z 1821) associ-ated with MCR production, leading to the accurate discrimination of chromosome- and plasmid-encoded resistance to polymyxins in E. coli (Figures1and2).

Recently, a simple phenotypic method, named Colistin-MAC, has been described for screening for MCR-1-mediated colistin re-sistance.50This method is based on colistin MIC reduction (8-fold

reduction) in the presence of dipicolinic acid, when the polymyxin resistance is caused by MCR-1 production. Although this method is relatively cheap and easy to perform in a clinical microbiology la-boratory, it requires the determination of additional MICs on polymyxin-resistant isolates using the BMD method, which again results in an additional delay of at least 24 h compared with the original molecular biology approach (Figure3). The MALDIxin test is more efficient because of its ability to simultaneously detect polymyxin resistance and its genetic basis, in,15 min (Figure3).

Compared with plasmid-borne mcr-1, the occurrence of chro-mosomally encoded mcr-1 is extremely rare. Of note, despite their abilities to detect MCR producers, neither the MALDIxin test nor the currently available techniques (such as molecular assays dedi-cated to mcr detection or the Colistin-MAC) are able to distinguish between plasmid- and chromosome-encoded mcr. Accordingly, a chromosome-encoded resistance is usually assumed if the test result is negative independently of the technique used.

Another advantage of the MALDIxin test is its amenability to high-throughput use, which may prove invaluable for screening large collections of strains such as those collected by the NRCs. As this technique does not require specific sample preparation, a large number of bacterial isolates can be loaded onto the same MALDI-TOF target (between 48 and 96 positions are available on the standard reusable MALDI target plates) and tested in the same run. Thus, consumable costs and technical handling time are drastically reduced compared with other techniques described for the detection of polymyxin resistance. From a One-Health per-spective, the high-throughput potential of our test will be useful for screening for MCR producers in the livestock industry, where large numbers of mcr-positive isolates are being identified. Finally, the MALDIxin test could easily be incorporated into routine work-flows, because a MALDI-TOF MS-based approach for bacterial identification is now carried out routinely in many clinical

microbiology laboratories (Figure3). However, we need to note that routine use of the MALDIxin test will require switching the MALDI-TOF MS machine to the negative ion mode, which is not currently used for bacterial identification; this is necessary due to the inherent negative charge of lipid A.

Overall, the MALDIxin test is a rapid (15 min) and accurate diag-nostic tool (Figure3), which represents a major advance in the de-tection of polymyxin resistance by directly assessing modification of lipid A, the cellular target of the polymyxins, in intact bacteria. The test has been validated for E. coli on different media (FigureS3), with fresh and aged colonies. Of note, polymyxin resist-ance is currently a much bigger clinical problem in Klebsiella pneu-moniae than in E. coli and MCR production is still a public health issue that has not yet impaired human health. However, prelimin-ary data indicate that it may be also applicable to other Gram-negative bacteria for which pETN addition is involved in polymyxin resistance. These include the clinically important organisms Salmonella spp. and K. pneumoniae (which together with E. coli make up.99.9% of MCR producers),12as well as Acinetobacter baumannii, another emerging pathogen for which resistance to polymyxins is becoming critical.8,51The combination of excellent performance, cost-effectiveness and high-throughput scalability are all desirable attributes that will allow further commercializa-tion of the MALDIxin test. Finally, the test uses a technology that is already available in many clinical microbiology laboratories, thus allowing no-cost and hassle-free implementation.

Funding

This work was partially funded by the University Paris-Sud, France. L. D., R. A. B., L. G., A. B. J. and T. N. are members of the Laboratory of Excellence in Research on Medication and Innovative Therapeutics (LERMIT) supported by a grant from the French National Research Agency (ANR-10-LABX-33). L. D. was supported by funding from ‘the People Programme (Marie Skłodowska-Curie Actions) of the European Union’s Horizon 2020 under REA grant agreement number [654909]’ and from ‘the University Paris Sud’. D. A. I. M. was supported by an ‘MRC Career Development Award (MR/M009505/1)’. G. L.-M. was supported by an ‘MRC Confidence in Concept Award (RSPO_P54990)’.

Transparency declarations

L. Dortet, A. F. and G. L.-M. are co-inventors of the MALDIxin test for which a patent has been filed by Imperial Innovations. The remaining authors have none to declare.

Author contributions

L. Dortet and G. L.-M. had full access to all of the data in the study, and take re-sponsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: L. Dortet and G. L.-M. Acquisition, analysis, or inter-pretation of data: all authors. Drafting of the manuscript: L. Dortet, G. L.-M. and A. F. Critical revision of the manuscript for important intellectual content: L. Dortet, R. C. D. F., D. A. I. M., P. B., Y. G., P. P., R. A. B., T. N., A. F. and G. L.-M.

Supplementary data

Figures S1 to S3 and TablesS1andS2appear asSupplementary dataat JAC Online.

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Figure

Figure 1. Lipid A modifications caused by chromosome-encoded determinants or MCR enzymes that can be detected by the MALDIxin test.
Figure 2. Results of the MALDIxin test on E. coli. Representative spectra of (a) a polymyxin-susceptible E
Figure 3. Comparison of research (purple outline), routine clinical (green outline) and MALDIxin (blue outline) workflows for the detection of chromo- chromo-some- and plasmid (MCR)-encoded resistance to polymyxin in Enterobacteriaceae

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