Accepted Manuscript Real- time quantitative PCR assay with Taqman* probe for rapid detection of MCR-1 plasmid-mediated colistin resistance Selma Chabou, Thongpan Leangapichart, Liliane Okdah, Stephanie Le Page, Linda Hadjadj, Jean-Marc Rolain PII:
S2052-2975(16)30071-3
DOI:
10.1016/j.nmni.2016.06.017
Reference:
NMNI 202
To appear in:
New Microbes and New Infections
Received Date: 4 May 2016 Revised Date:
29 June 2016
Accepted Date: 30 June 2016
Please cite this article as: Chabou S, Leangapichart T, Okdah L, Le Page S, Hadjadj L, Rolain J-M, Real- time quantitative PCR assay with Taqman* probe for rapid detection of MCR-1 plasmid-mediated colistin resistance, New Microbes and New Infections (2016), doi: 10.1016/j.nmni.2016.06.017. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Full-length title: Real- Time quantitative PCR assay with Taqman* probe for rapid
2
detection of MCR-1 plasmid-mediated colistin resistance.
3
Category: New Technologies for Infectious and Tropical Diseases
4
Short title (for the running head): mcr-1 colistin resistance qPCR assay
RI PT
1
5
Author list: Selma Chabou1, Thongpan Leangapichart1, Liliane Okdah1, Stephanie Le Page1,
7
Linda Hadjadj 1, and Jean-Marc Rolain1*
8
Affiliations: 1 URMITE UM 63 CNRS 7278 IRD 198 INSERM U1905, IHU Méditerranée
9
Infection, Faculté de Médecine et de Pharmacie, Aix-Marseille Université, 27 boulevard Jean
M AN U
SC
6
Moulin, 13385 Marseille CEDEX 05, France
11
* Corresponding author:
[email protected]
TE D
10
12
Key words: colistin resistance; mcr-1 gene; antibiotic resistance surveillance;
14
Number of text words: 1006
15
Number of figures: 1
16
Number of tables: 2
17
Number of references: 18
18
Abstract word count: 100
19 20
AC C
EP
13
ACCEPTED MANUSCRIPT
ABSTRACT
22
Here we report the development of two rapid real-time quantitative PCR assays with TaqMan®
23
probes to detect MCR-1 plasmid-mediated colistin resistance gene from bacterial isolates and
24
fecal samples from chickens. Specificity and sensitivity of the assay were at 100% on bacterial
25
isolates including 18 colistin-resistant isolates carrying the mcr-1 gene (6 K. pneumoniae and 12
26
E. coli) with a calibration curve linear from 101 to 108 DNA copies. Five out of 833 fecal samples
27
from chickens from Algeria were positive from which 3 E. coli strains were isolated and
28
confirmed to harbor the mcr-1 gene by standard PCR and sequencing.
M AN U
SC
RI PT
21
29 30 31
EP AC C
33
TE D
32
ACCEPTED MANUSCRIPT
INTRODUCTION
35
The increasing prevalence of infections caused by multidrug-resistant (MDR) Gram-negative
36
bacteria combined with few antimicrobial agents being in development has led to a resurgence in
37
interest in colistin as a last-line therapy with the inevitable risk of emerging resistance (1-3).
38
MCR-1 plasmid-mediated colistin resistance is a member of the phosphoethanolamine transferase
39
enzyme family, with expression in Escherichia coli resulting in the addition of
40
phosphoethanolamine to lipid A and resistance to colistin (4). This plasmid-mediated colistin
41
resistance is an emerging concern that has already spread worldwide (5) in E. coli and K.
42
pneumoniae from pigs, chicken, retail meat, pork and humans (4). In animal health, colistin is
43
used to prevent infections from E. coli isolates that are known to cause serious side effects such
44
as diarrhea, sepsis and colibacillosis, which result in huge economic losses (6) . The extensive
45
use of antibiotics in food-animal production has been shown to increase the risk of transferring
46
resistant bacteria to humans (7).
47
There is a need to screen for colistin resistance in patients even without prior history of colistin
48
usage for the timely detection and isolation of patients harboring such resistant strains to prevent
49
clonal transmission (8). For this reason the aim of this study was to develop rapid real-time
50
quantitative PCR to detect the MCR-1 plasmid-mediated colistin resistance and to evaluate its
51
sensitivity and specificity both from strains and stool samples.
52
MATERIALS AND METHODS
53
Specific primers and probes designer
54
We design specific primers and probes to develop two real-time quantitative PCR assays (PE1
55
and PE2) for detection of MCR-1 encoding gene (Table 1). Specificity of the primers and probes
AC C
EP
TE D
M AN U
SC
RI PT
34
ACCEPTED MANUSCRIPT
were verified in silico by blastN analysis on the National Center for Biotechnology Information
57
(NCBI) database.
58
Sample collection
59
Bacterial strains
60
A total of 100 strains from humans and animals were used in this study including 18 colistin-
61
resistant isolates carrying the mcr-1 gene (6 K. pneumoniae and 12 E. coli). Phenotypic and
62
genotypic features of these strains are summarized in Table 2.
63
Chicken stool collection
64
A total of 833 feces of broilers were collected between August and February 2015 from 8
65
regions in Algeria (El Tarf, Souk Ahras, Skikda, Setif, Jijel, Algiers, Biskra, and Ourgla; n = 503)
66
and in three slaughterhouses in Marseille (n = 330). All extracted DNA from the 833 feces of
67
broilers were tested using our qPCR assay and positive samples were inoculated on agar for
68
isolation of positive mcr-1 isolates.
69
Molecular analysis
70
Strategy for PCR amplification and sequencing
71
Standard PCR amplification and sequencing of the MCR-1-encoding gene was used as the gold
72
standard and performed as previously described (4). Quantification of the MCR-1 encoding gene
73
using our two sets of primers and probes was performed using a quantitative CFX96Tm Real Time
74
system C1000Tm Touch thermal cycler (Bio- Rad, Sangapour). The qPCR conditions were as
75
follows: the reaction mixtures were kept at 95°C for 15 min and subsequently put through 35
76
cycles of 95°C for 30s and 60°C for 1min.
77
Specificity and reproducibility of the new system of Real time PCR
AC C
EP
TE D
M AN U
SC
RI PT
56
ACCEPTED MANUSCRIPT
The specificity of the primers and probes were verified in vitro using our local collection of 100
79
strains (Table 2). The sensitivity of our assays were determined using tenfold serial dilutions
80
(between 108 to 101 DNA copies) of E. coli strain P10 by triplicate amplification, the number of
81
MCR-1 in each sample was calculated based on the DNA copy numbers. The obtained Ct values
82
were used to generate the calibration curves as compared to the number of bacteria quantified by
83
standard bacterial count on agar plates. The standard curve was constructed on the basis of the
84
concordance between Ct values and number of log UFC/mL. The limit of detection was based on
85
the final dilution detected by PCR. Efficacy of qPCR was calculated from a standard curve
86
according to Rutledge and Cote (17).
87
RESULTS
88
Specificity and Technical sensitivity of the quantitative PCR
89
BlastN analysis of the primers and probe designed for development of the real-time PCR assay
90
showed in silico a 100% homology with the MCR-1-encoding gene only. The sensitivity of the
91
real time PCR using the 18 mcr-1 positive strains using serial ten-fold dilutions of a calibrated
92
inoculum was excellent with a calibration curve linear from 101–108 DNA copies corresponding
93
to 35–21 Ct (Fig. 1). Regressions formula and PCR efficiency of the two real-time PCR assays
94
are shown in Figure 1. The reproducibility of the two qPCR assays was excellent, with a positive
95
PCR at 21.4 ± 0.4 Ct for PE1 and 20.8+/- 0.4 Ct for PE2 when testing one colony re-suspended in
96
200µL of sterile water (Table 2). The specificity of the two qPCR assays in vitro against a panel
97
of 82 clinically relevant bacteria negative for mcr-1 gene was at 100% (all real-time PCR were
98
negative, Table 2).
99
Screening of feces from broilers
AC C
EP
TE D
M AN U
SC
RI PT
78
ACCEPTED MANUSCRIPT
Five out of 503 fecal samples from chickens from Algeria were positive from which 3 E. coli
101
strains were isolated and confirmed to harbor the mcr-1 gene by standard PCR and sequencing.
102
None of the 330 samples from France were positive.
103
DISCUSSION
104
The recent description and emergence of mcr-1 plasmid-mediated resistance to colistin in humans
105
and animals is a major concern worldwide (5). In this study, two new qPCR assays using
106
Taqman* probes were developed that demonstrate high sensitivity and specificity for
107
confirmation of the presence of this gene in colistin-resistant bacterial isolates as well as for
108
screening directly from stool samples. Indeed both systems have the same performance to screen
109
for the presence of mcr-1 containing isolates and in stools. We recommend to use PE1 as a first
110
set of primers for the rapid screening of mcr-1 and PE2 system to confirm the positive results.
111
Recently, Bontron et al have reported a real-time PCR assay using SYBR green as fluorescent
112
marker with similar sensitivity (18). However it is well known that Taqman* probes enhance
113
specificity that is a critical point when testing directly from biological samples. Our real time
114
PCR assays had advantages including sensitivity, specificity and the possibility to detect MCR-1
115
plasmid-mediated colistin resistance very quickly (256
None
0
0
Algeria
unpublished data
S. marcescens (n=2)
-
>256
None
0
0
Algeria
unpublished data
AC C
EP
TE D
M AN U
SC
* ; Mutation, +; Positive, -; Negative.
RI PT
P. mirabilis (n=2)
RI PT
PE1
107 8
10 10 7 6
10 5 10 4
21.39
7.50E+05
26.42 29.1
7.50E+04
105
31.43
7.50E+03
104
34.21
7.50E+02
PE1
log CFU/Reaction 8
R² = 0.9742
4
y = -3.065x + 40.11
2
slope = -3.065 20
25
30
35
PCR efficiency = [(10-1/-3.065)-1] x 100% = 111.97%
AC C
0
EP
Limit of detection 75 CFU/ml
6
TE
10
106
7.50E+06
Con.
SC
108
Ct Avg.
M AN U
CFU/reaction
D
Con.
PE2
108 107
106
105 104
CFU/reaction
Ct Avg.
108
7.50E+06
20.84
107
7.50E+05
27.195
106
7.50E+04
28.795
105
7.50E+03
31.525
7.50E+02
35.825
104
log CFU/Reaction
PE2
8
Limit of detection 75 CFU/ml
6
R² = 0.9588
4
y = -3.43x + 42.11
2
slope = -3.43
0 20
25
30
35
40
PCR efficiency = [(10-1/-3.43)-1] x 100%= 95.68%
Fig 1. Real-time polymerase chain reaction (PCR) sensitivity test to detect -encoding plasmid-mediated colistin resistance (MCR-1) encoding gene from E.coli strain P10. PE1 and PE2 are two qPCR assays developed.
