Rapid Detection and Discrimination of Chromosome - and MCR-plasmidmediated Resistance To Polymyxins by MALDI-TOF
Rapid Detection and Discrimination of Chromosome - and MCR-plasmidmediated Resistance To Polymyxins by MALDI-TOF
Anais Potron4,6, Patrick Plesiat4,6, Racha Beyrouthy4,7, Frédéric Robin4,7, Richard Bonnet4,7, Thierry Naas2–4,
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, Bicêtre Hospital, Assistance Publique - Hôpitaux de Paris, Le
Kremlin-Bicêtre, 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-Bicêtre, France; 4French National Reference Center for Antibiotic
Resistance, Le Kremlin-Bicêtre, 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
Besançon, Besançon, France; 7Bacteriology Unit, University Hospital of Clermont-Ferrand, Clermont-Ferrand, France
*Corresponding author. Hôpital de Bicêtre, Service de Bactériologie-Hygiène, 78 rue du Général Leclerc, 94270, Le Kremlin-Bicêtre, France.
Tel: !33 (0) 1 45 21 20 19; E-mail: [email protected] 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.
C The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
V
For permissions, please email: [email protected].
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Dortet et al.
increasingly prevalent worldwide, with many producing (disruptions, deletions, mutations) involved are not systematically
carbapenem-hydrolysing enzymes [carbapenemase-producing described or characterized. Regarding plasmid-encoded resist-
Enterobacteriaceae (CPE)].2 This phenomenon has accelerated the ance, five families of mcr genes have already been reported in
use of polymyxins in CPE-endemic countries such as Greece and Enterobacteriaceae.31–35 MCR-2, MCR-3, MCR-4 and MCR-5 share
Italy, and as a result increased resistance to polymyxins is now only 81%, 34%, 33% and 31% amino acid identity, respectively,
being reported.3–7 with MCR-1 (Figure S1, available as Supplementary data at JAC
In Enterobacteriaceae, acquired resistance to polymyxins arises Online). This diversity will inevitably lead to the failure of systemat-
from modifications to the drug target, i.e. LPS.8,9 These modifica- ic detection of polymyxin resistance as the available molecular
tions consist of addition(s) of cationic groups such as phosphoe- biology tools are dedicated to mcr-1 and/or mcr-2 detection
thanolamine (pETN) and/or 4-amino-L-arabinose (L-Ara4N) to the (Figure S2).31,32,34,35
lipid A part of the LPS. This results in decreased binding of the poly- To overcome these issues, we developed a cost-effective assay
myxin to the LPS due to electrostatic repulsion between the added based on MALDI-TOF technology, named the MALDIxin test, which
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Rapid detection of colistin resistance by MALDI-TOF MS JAC
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 CST MIC (mg/L) mcr-1/-2 PCRa PRRE. coli interpretation
CST, colistin.
a
!, positive PCR for the gene indicated in parentheses; #, negative PCR.
b
Strains were isolated from the same patient and have been previously reported by Beyrouthy et al.42 as E. coli WI1 and E. coli WI2, respectively.
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Dortet et al.
SUSCEPTIBLE RESISTANT
O O OH O
H 2N
O P O P O O
HO O
OH OH O H O
pETN NH
O NH OH
O O
O
O OH
O O OH
O
ke
O O H3C
O O OH H3C
-li
O 14 H3 C 14 H 3C
OH
O O OH CR H 3C
14 H3C 14
M
12
O 14
Modified Lipid A
(1821 m/z)
M
CR
O OH O
-li
Ch de
en
H3C O P O O
ke
H 3C HO O
ro d r
co
14 H 3C 14 H 3C OH O H O O O
m es
NH NH2
14 H3C NH O P O P O
os is
H 3C 14 O O
12 O O
om tan
14 OH OH
O OH
e- ce
O O OH pETN
O
Native Lipid A
(1796 m/z)
H3C H 3C
14 H3C 14 H 3C
14 H3C 14
Modified Lipid A
Plasmid-encoded H 3C
12 (1919 m/z)
resistance 14
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.
1821.2) to the intensity of the peak of native lipid A (for E. coli m/z chromosome-encoded (0.1 , PRRE. coli , 0.5) and plasmid-encoded
1796.2) allows accurate distinction between polymyxin-susceptible resistance (PRRE. coli .0.5).
and polymyxin-resistant isolates, but also discrimination between
chromosome-encoded and MCR-related (i.e. plasmid-encoded) re-
sistance to polymyxin. (Figure 2d). The PRR value for all susceptible Performance of the MALDIxin test on colonies grown on
E. coli strains was found to be 0 (Table S1 and Figure 2d). The ratio clinically relevant media
ranged from 0.109 to 0.481 (average of 0.210) for E. coli strains with We assessed the performance of the MALDIxin test using colonies
chromosome-encoded resistance to polymyxin, and from 0.533 to grown on media routinely used to test for antimicrobial susceptibil-
4.844 (average of 2.340) for all MCR producers (Table S1 and ity, i.e. MH agar. In addition, because of the rapid rise of MCR-1-
Figure 2d). The distribution of the PRRE. coli values of polymyxin- producing Enterobacteriaceae since 2016, it has been suggested
susceptible isolates compared with E. coli strains with chromosome- that polymyxin-supplemented media should be used to screen
encoded resistance to polymyxin and MCR-producers were all signifi- organisms isolated from infected patients.41 Accordingly, the
cantly different (P , 0.001, Welch’s t-test). Analysis of receiver oper- MALDIxin test was conducted on three E. coli strains (a WT strain, a
ating characteristic (ROC) curves allowed us to define two cut-off strain with chromosome-encoded resistance and an MCR-1-
values for PRRE. coli (0.1 and 0.5) that discriminate polymyxin- producing strain), grown on LB agar, MH agar and polymyxin
susceptible (PRRE. coli ,0.1) and polymyxin-resistant isolates B-supplemented MH agar at final concentrations of either 1 or
(PRRE. coli.0.1) and also allows further discrimination between 2 mg/L. As shown in Figure S3, similar spectra were obtained
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Rapid detection of colistin resistance by MALDI-TOF MS JAC
(a) (d) P < 0.0001
P < 0.0001
5
1796.2
Polymyxin
susceptible
4.5
Intensity
I1919 + I1821
4 PRRE. coli =
I1796
3.5
PRRE. coli
(chromosome)
2
Intensity
1919.2
1.5
+ 123
+ 25
0.1
+ 123 0
Polymyxin Chromosome- Plasmid-encoded
susceptible encoded (mcr-like)
1600 1720 1840 1960 2080 2200
m/z
Polymyxin resistant
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
40 of 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 (Table S1). 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.
irrespective of the growth medium, with the PRRE. coli being between Validation of the MALDIxin test on a prospective
2.89 and 5.69 for MCR-1-producing strains and between 0.21 and collection of carbapenemase-producing E. coli from the
0.37 for strains harbouring chromosome-encoded resistance. French NRC
To identify any impact of colony age, the MALDIxin test was
performed on fresh colonies (overnight incubation) grown on both In order to further validate the MALDIxin test, all carbapenemase-
LB and MH agar and colonies stored for 48 h, 1 week or 2 weeks producing E. coli received from the French NRC during October and
(agar plates were kept at 4 C). Similar spectra were obtained for November 2016 were blindly analysed in triplicate (three colonies
all tested colonies leading to no significant differences in their selected from the same MH agar plate). This collection of
PRRE. coli (Table S2). 78 carbapenemase-producing E. coli strains included 2 KPC
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Dortet et al.
Research workflow
Litres of 250 ml
Readout
bacterial Lipid Lipid A
culture extraction
MALDI-TOF
Routine workflow +
2h Polymyxin 16-24 h Plasmid
resistant
Readout
isolate – MICs
Polymyxin NP test Colistin-MAC Chromosome
(Dipicolinic acid
Bacterial
identification
– +
MALDI-TOF Chromosome Plasmid
15min
MALDIxin test
Polymyxin susceptibility (R / S)
Readout
AND Lipid A
Plasmid- vs chromosome-
encoded resistance
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.
producers, 11 NDM producers, 2 VIM producers, 63 OXA-48-like become last-resort antimicrobials, largely used for the treatment
producers and 1 isolate producing two different carbapenemases of severe bacterial infections. In Enterobacteriaceae, resistance to
(Table 1). Among these isolates, two NDM-1 producers and one polymyxins results from alterations to lipid A, caused either by
OXA-48 producer were also found to be resistant to colistin, with adaptive chromosomal mutations or by acquired plasmid-
MICs of 4 mg/L.42 PCR analysis revealed that these three isolates encoded MCR enzymes. Irrespective of the resistance mechanism,
were positive for the mcr-1 gene (Table 1). When analysed using the detection of polymyxin resistance remains difficult and time
the MALDIxin test, all polymyxin-susceptible isolates (n " 75) consuming in clinical microbiology laboratories, and relies solely
showed a PRRE. coli of 0, corresponding to polymyxin susceptibility, on the determination of colistin or polymyxin B MICs using BMD.17
whilst for the three MCR-1-producing isolates a PRRE. coli .0.5 was To provide an accurate, reliable, rapid and cost-effective alterna-
obtained (1.73+0.23 and 2.34+0.12 for the two NDM-1-producers tive, we developed a diagnostic test based on MALDI-TOF MS for
and 1.29+0.63 for the OXA-48-producer), accurately classifying the detection of polymyxin resistance in Gram-negative bacteria.
them as resistant to polymyxin by a plasmid-encoded mechanism We call this method the MALDIxin test. After initial validation using
(Table 1). Therefore, in this prospective study, the MALDIxin test re- a well-characterized collection of polymyxin-susceptible and -re-
liably and rapidly identified all MCR-producing strains while con- sistant strains, we performed a prospective evaluation on
ventional methods would have needed 24–48 h with broth dilution carbapenemase-producing E. coli during which the MALDIxin test
method (16–24 h) and subsequent DNA extraction and PCR for accurately detected all MCR-1 producers. This result was obtained
mcr-1 and mcr-2 detection on resistant isolates (4 h). directly from isolated colonies within 15 min, whilst the confirm-
ation of colistin resistance and MCR production using standard
Discussion techniques would have required an additional 24–48 h (Figure 3).
An additional challenge when it comes to polymyxin resistance
Owing to the increasing incidence of carbapenemase-producing is the rapid dissemination of MCR-producing Enterobacteriaceae.
Enterobacteriaceae, polymyxins (colistin and polymyxin B) have Therefore, discrimination between chromosome- and plasmid-
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Rapid detection of colistin resistance by MALDI-TOF MS JAC
encoded resistance is important due to the higher potential for dis- microbiology laboratories (Figure 3). However, we need to note
semination of plasmid-encoded compared with chromosome- that routine use of the MALDIxin test will require switching the
encoded resistance mechanisms. In the future this distinction may MALDI-TOF MS machine to the negative ion mode, which is not
also be crucial in order to facilitate the use of MCR inhibitors, which currently used for bacterial identification; this is necessary due to
are being developed.20,21,43,44 Currently, this discrimination mostly the inherent negative charge of lipid A.
relies on molecular tests dedicated to the detection of mcr genes. Overall, the MALDIxin test is a rapid (15 min) and accurate diag-
These are based on real-time PCR,45–47 loop-mediated isothermal nostic tool (Figure 3), which represents a major advance in the de-
amplification48 and microarray techniques.49 These molecular tection of polymyxin resistance by directly assessing modification
assays are costly and, whilst they detect mcr-1 and/or mcr-2, they of lipid A, the cellular target of the polymyxins, in intact bacteria.
are not able to detect all members of the highly divergent and rap- The test has been validated for E. coli on different media
idly expanding mcr gene family (Figures S1 and S2). In fact, there (Figure S3), with fresh and aged colonies. Of note, polymyxin resist-
are growing numbers of reports describing new MCR families, all of ance is currently a much bigger clinical problem in Klebsiella pneu-
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40 Pieretti G, Cipolletti M, D’Alonzo D et al. A combined fermentative- 47 Dona V, Bernasconi OJ, Kasraian S et al. A SYBRV Green-based real-time
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