2018_High-resolution melting PCR analysis for rapid genotyping of Burkholderia mallei

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Infection, Genetics and Evolution
journal homepage: www.elsevier.com/locate/meegid
Short communication
High-resolution melting PCR analysis for rapid genotyping of Burkholderia
mallei
Girault G.a, Wattiau P.b, Saqib M.c, Martin B.a, Vorimore F.a, Singha H.d, Engelsma M.e,
Roest H.J.e, Spicic S.f, Grunow R.g, Vicari N.h, De Keersmaecker S.C.J.i, Roosens N.H.C.i,
Fabbi M.h, Tripathi B.N.d, Zientara S.a, Madani N.a, Laroucau K.a,⁎
a Paris-Est University, ANSES, Laboratory for Animal Health, Bacterial Zoonosis Unit, European Union Reference Laboratory for Equine Diseases/Glanders, Maisons-
Alfort, France
bDepartment of Bacterial Diseases, CODA-CERVA (Veterinary and Agrochemical Research Centre), Brussels, Belgium
cUniversity of agriculture, Faisalabad, Pakistan
d ICAR-National Research Centre on Equines, Hisar, India
eWageningenBioveterinary Research, Lelystad, The Netherlands
f Croatian Veterinary Institute, Department for Bacteriology and Parasitology, Laboratory for Bacterial Zoonoses and Molecular Diagnosis of Bacterial Diseases, Zagreb,
Croatia
g Centre for Biological Threats and Special Pathogens, Robert Koch Institute, Berlin, Germany
h IstitutoZooprofilatticoSperimentaledellaLombardia e dell'EmiliaRomagna "Bruno Ubertini", Pavia, Italy
i Platform Biotechnology and Molecular Biology, Scientific Institute of Public Health, Brussels, Belgium
A R T I C L E I N F O
Keywords:
Burkholderia mallei
Genotyping
HRM
A B S T R A C T
Burkholderia (B.) mallei is the causative agent of glanders. A previous work conducted on single-nucleotide
polymorphisms (SNP) extracted from the whole genome sequences of 45 B. mallei isolates identified 3 lineages
for this species. In this study, we designed a high-resolution melting (HRM) method for the screening of 15
phylogenetically informative SNPs within the genome of B. mallei that subtype the species into 3 lineages and 12
branches/sub-branches/groups. The present results demonstrate that SNP-based genotyping represent an in-
teresting approach for the molecular epidemiology analysis of B. mallei.
1. Short communication
Burkholderia (B.) mallei is the causative agent of glanders in equids
and camels, a disease recently qualified as a re-emergent due to the
increased number of cases reported in several parts of the world during
the last 20 years (Khan et al., 2013). B. mallei is a genetically homo-
genous species that is very closely related to the much more diverse
species B. pseudomallei from which it recently evolved. Its genome is
thought to be continuously evolving through random insertion se-
quence-mediated recombination events (Losada et al., 2010). Due to a
lack of diversity, only molecular characterization techniques with high
discrimination power could enhance genetic differentiation at strain
level. Most of the genotyping methods applied to B. mallei were initially
developed for B. pseudomallei. Whereas multilocus sequence typing
based on 7 housekeeping genes failed to differentiate B. mallei strains
which match the ST40 sequence type for nearly all of them (Godoy
et al., 2003; Losada et al., 2010), a 23-loci Multiple Locus Variable
Number of Tandem repeats Analysis (MLVA) method derived from the
32-loci MLVA method for B. pseudomallei (U'Ren et al., 2007) succeeded
to distinguish B. mallei outbreak isolates for example in Pakistan and
Emirates (Hornstra et al., 2009; Scholz et al., 2014). However, this
method requires the analysis of 23 loci which is technically demanding,
expensive and time-consuming. Given that variable number of tandem
repeats are inappropriate for determining deep levels of evolutionary
relatedness (Hornstra et al., 2009) and given the increasing availability
of whole genome sequences (WGS), it is now possible to interrogate
nearly every base of the genome and to identify specific single nu-
cleotide polymorphisms (SNPs) able to discriminate B. mallei isolates at
strain level. The post- real-time PCR high resolution melting (HRM)
analysis offers the possibility to discriminate different amplicons based
on their melting temperature (Tm) and allows the detection of genetic
variations such as SNPs (Tamburro and Ripabelli, 2017).
In a previous work, a minimum spanning tree based on 2296 B.
mallei specific SNPs extracted from the WGS of 45 strains identified 3
lineages for B. mallei (Laroucau et al., 2018). In this study, inside these
3 lineages (L1 to L3), subdivisions into branches (Br, up to 3), sub-
https://doi.org/10.1016/j.meegid.2018.05.004
Received 12 January 2018; Received in revised form 2 May 2018; Accepted 4 May 2018
⁎ Corresponding author.
E-mail address: karine.laroucau@anses.fr (K. Laroucau).
Infection, Genetics and Evolution 63 (2018) 1–4
Available online 08 May 2018
1567-1348/ © 2018 Elsevier B.V. All rights reserved.
T
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https://doi.org/10.1016/j.meegid.2018.05.004
https://doi.org/10.1016/j.meegid.2018.05.004
mailto:karine.laroucau@anses.fr
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branches (sB, up to 3) and even groups (Gp) could be identified, as
illustrated in Fig. 1. A first set of 15 SNPs specific for each of these
lineages, branches, sub-branches and groups was identified in silico
using BioNumerics 7.6.1 (Applied Maths) and PCR primers targeting
these SNPs were designed using Primer 3web version 4.0.0 (http://
www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi)
(Table 1). Singleplex PCR amplifications were conducted on a ViiA7™
Real-Time PCR instrument (Life Technologies) using the LightCycler®
480 High Resolution Melting Master Mix (Roche Diagnostics). Reaction
mixtures consisted of 10 ng DNA, 0.2 μM of each primer, 10 μL Light-
Cycler® 480 HRM master mix and 2.5 mM MgCl2 in 20 μL final volume.
The following amplification parameters were used: 10min at 95 °C
followed by 40 cycles consisting in 30 s at 94 °C, 30 s at 55 °C and 30 s at
72 °C. Samples were next heated to 95 °C for 30 s, cooled down to 65 °C
for 1min and heated from 65 °C to 95 °C at a rate of 0.025 °C/s with 25
fluorescence acquisitions/°C. HRM data were analyzed by the ViiA7™
Software version 1.2.1. For each of the SNPs, synthetic DNA oligonu-
cleotides carrying either the C/G or A/T substitutions (Eurofins,
Germany) were included as controls (Table 1, Supplementary material
1). All yielded amplicons produced a single melting peak. Each peak
was characterized by a melting curve, with Tm values depending on the
SNP carried by the amplicon. On average, differences in Tm values of
about 0.4 to 1.1 °C were observed between the two allelic states
(Table 1).
This panel of 15 SNPs was first validated on 8 DNA preparations
from the 45 fully sequenced strains used for the phylogenetic clustering
(Laroucau et al., 2018). As determined from WGS data, all strains
clustered within their predicted lineage, branch, sub-branch and/or
group, namely L1 for NCTC120/2002734306, L2B2sB1Gp1 for
ATCC23344, L2B2sB1Gp2 for NCTC10245/ATCC10399 and China 5,
L3B2 for 16-2438_BM#8, and L3B3sB3 for NCTC10229, Ivan and
NCTC10247 (Table 2).
The developed PCR-HRM method was further applied to 33 DNA
preparations from either ancient or contemporary B. mallei strains
isolated in Croatia (Croatia_1957), Hungary (64.12 and NCTC10230),
India (Mukteswar, NCTC3708, NCTC3709, 3711, 3712, 3851, 3855,
NCTC10248(TR)
16-2438_BM#816-2438_BM#816-2438_BM#816-2438_BM#816-2438_BM#816-2438_BM#816-2438_BM#816-2438_BM#8-
102102102102102102102102102
11, strain_1111, strain_1111, strain_1111, strain_1111, strain_1111, strain_1111, strain_1111, strain_1111, strain_11
200272127620027212762002721276200272127620027212762002721276200272127620027212762002721276
200272128020027212802002721280200272128020027212802002721280200272128020027212802002721280
20027343062002734306200273430620027343062002734306200273430620027343062002734306
307630763076307630763076307630763076371237123712371237123712371237123712
6, strain_66, strain_66, strain_66, strain_66, strain_66, strain_66, strain_66, strain_66, strain_6
A188, BURK080A188, BURK080A188, BURK080A188, BURK080A188, BURK080A188, BURK080A188, BURK080A188, BURK080A188, BURK080
A193, BURK081A193, BURK081A193, BURK081A193, BURK081A193, BURK081A193, BURK081A193, BURK081A193, BURK081A193, BURK081
ATCC 10399ATCC 10399ATCC 10399ATCC 10399ATCC 10399ATCC 10399ATCC 10399ATCC 10399
BMQBMQBMQBMQBMQBMQBMQBMQBMQ
Bahrain1Bahrain1Bahrain1Bahrain1Bahrain1Bahrain1Bahrain1Bahrain1Bahrain1
BudapestBudapestBudapestBudapestBudapestBudapestBudapestBudapestBudapest
China5China5China5China5China5China5China5China5
FMH 23344FMH 23344FMH 23344FMH 23344FMH 23344FMH 23344FMH 23344FMH 23344FMH 23344
FMHFMHFMHFMHFMHFMHFMHFMHFMH
GB8 horse 4GB8 horse 4GB8 horse 4GB8 horse 4GB8 horse 4GB8 horse 4GB8 horse 4GB8 horse 4GB8 horse 4
India86-567-2India86-567-2India86-567-2India86-567-2India86-567-2India86-567-2India86-567-2India86-567-2India86-567-2
JHUJHUJHUJHUJHUJHUJHUJHUJHU
KC_1092KC_1092KC_1092KC_1092KC_1092KC_1092KC_1092KC_1092KC_1092
Kweiyang#4Kweiyang#4Kweiyang#4Kweiyang#4Kweiyang#4Kweiyang#4Kweiyang#4Kweiyang#4Kweiyang#4
NCTC 10229NCTC 10229NCTC 10229NCTC 10229NCTC 10229NCTC 10229NCTC 10229NCTC 10229
NCTC 10247_v2NCTC 10247_v2NCTC 10247_v2NCTC 10247_v2NCTC 10247_v2NCTC 10247_v2NCTC 10247_v2NCTC 10247_v2
PRL-20PRL-20PRL-20PRL-20PRL-20PRL-20PRL-20PRL-20PRL-20
SR092700ISR092700ISR092700ISR092700ISR092700ISR092700ISR092700ISR092700ISR092700I
Turkey10Turkey10Turkey10Turkey10Turkey10Turkey10Turkey10Turkey10Turkey10
Turkey1Turkey1Turkey1Turkey1Turkey1Turkey1Turkey1Turkey1Turkey1
Turkey2Turkey2Turkey2Turkey2Turkey2Turkey2Turkey2Turkey2Turkey2
Turkey3Turkey3Turkey3Turkey3Turkey3Turkey3Turkey3Turkey3Turkey3
Turkey4Turkey4Turkey4Turkey4Turkey4Turkey4Turkey4Turkey4Turkey4
Turkey5Turkey5Turkey5Turkey5Turkey5Turkey5Turkey5Turkey5Turkey5
Turkey7Turkey7Turkey7Turkey7Turkey7Turkey7Turkey7Turkey7Turkey7
Turkey8Turkey8Turkey8Turkey8Turkey8Turkey8Turkey8Turkey8Turkey8
Turkey9Turkey9Turkey9Turkey9Turkey9Turkey9Turkey9Turkey9Turkey9
V-120V-120V-120V-120V-120V-120V-120V-120V-120
ATCC 23344ATCC 23344ATCC 23344ATCC 23344ATCC 23344ATCC 23344ATCC 23344ATCC 23344
20000310632000031063200003106320000310632000031063200003106320000310632000031063
NCTC 10247_v1NCTC 10247_v1NCTC 10247_v1NCTC 10247_v1NCTC 10247_v1NCTC 10247_v1NCTC 10247_v1NCTC 10247_v1
SAVP1SAVP1SAVP1SAVP1SAVP1SAVP1SAVP1SAVP1SAVP1
2002734299, Ivan2002734299, Ivan2002734299, Ivan2002734299, Ivan2002734299, Ivan2002734299, Ivan2002734299, Ivan2002734299, Ivan
Turkey6Turkey6Turkey6Turkey6Turkey6Turkey6Turkey6Turkey6Turkey6
2000031281, China_72000031281, China_72000031281, China_72000031281, China_72000031281, China_72000031281, China_72000031281, China_72000031281, China_72000031281, China_7
Bahrain
Brazil
Burma
China
France
Hungary
India
Iran
NR
Pakistan
Turkey
United Kingdom
United States
L3
L1
L2
B1
B2
B1
B2
B3
sB1
sB2
sB3
sB2
sB1 Grp2
Grp1
NCTC 10229
16 2438_BM#8
ATCC 10399
China5
2000031063
ATCC 23344
2002734299, Ivan
A198, A199(IR)
A200(IR)
52.236(IR)
2007_2, 2017_1, 56, KH1, 
KH2(PK)
3711, 3712, 3851, 3855, 
3880, 3881, 3893, 3897, 
3912, 3932, 4295(IN)
NCTC 10247_v2
NCTC 10247_v1
A187
2002734306
IZLSER1411(IT)
RKI 03-0444
NCTC3708(IN)
NCTC3709(IN)
NCTC10260(TR)
Bogor (ID)
Mukteswar (IN)
Zagreb (YU)
Croatia_1957(HR)
A
B
64.12, NCTC10230(HU)
Fig. 1. A. SNP-based tree determined from 45 B. mallei publicly available whole genome sequences. Whole-genome sequences of 45 B. mallei strains present in public
databases were aligned and mapped against the reference sequence ATCC 23344 using the BWA algorithm implemented in BioNumerics with 90% parameter
identity. Strain-specific SNPs were identified using the BioNumericswgSNP module and then filtered using fixedconditions (minimum 30× coverage, removal of
repeated elements, contiguous SNPs, ambiguous and non-informative bases, removal of gaps)(Laroucau et al., 2018). A tree was generatedin BioNumerics using the
filtered SNP matrix as input and using the maximum parsimony algorithm (BioNumerics 7.6.1) (Applied Maths). DNA preparations from8 fully sequenced strains
initially tested with the panel of SNPs markers are highlighted in yellow.PCR-HRM clustering results for the B. mallei DNAs included in this study, without pre-
liminary WGS information, are shown in blue. B. Normalized melting curves obtained for three SNP markers (L1, L2 and L3). (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
G. Girault et al. Infection, Genetics and Evolution 63 (2018) 1–4
2
http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi
http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi
3880, 3881, 3893, 3897, 3912, 3932, 4295), Indonesia (Bogor), Italy
(IZSLER1411), Iran (A198, A199, A200, 52.236), Pakistan (2007_2,
2017_1, KH1, KH2 and 56), Turkey (NCTC10260 and NCTC10248),
former Yougoslavia (Zagreb), or from unknown origin (A187,
RKI_03–04444). Results are summarized in Table 2. The Croatian strain
Croatia_1957, the Hungarian strains 64.12 and NCTC10230, the Italian
strain IZSLER 1411 and three of the Iranian isolates (A198, A199 and
52.236) clustered within the L3B3sB3 branch. Two of the 14 Indian
strains (NCTC3708, NCTC3709) as well as one Iranian isolate (A200)
clustered within the L3B2 lineage which also includes the Indian BMQ
isolate. Among the Turkish strains, NCTC10260 grouped in L3B3sB3
together with the fully sequenced NCTC10247 strain also isolated in
Turkey at the same period, whereas the NCTC10248 strain clustered in
L1, a lineage which already includes a Turkish strain 6. The RKI
03–04444 strain of unknown origin clustered in L2B1, along with
Bogor, Mukteswar and Zagreb strains. Strain A187 clustered in L1. Five
strains collected between 2007 and 2017 in Pakistan and 11 of the
recent Indian strains (3711, 3712, 3851, 3855, 3880, 3881, 3893, 3897,
3912, 3932, 4295) grouped in L2B2sB2, together with strains fully se-
quenced and recently isolated in these two countries. PCR-HRM data
suggest that recent B. mallei isolates fall in three groups with strains
from Dubai, UAE, Bahrain clustering in L2B1, strains from India and
Pakistan clustering in L2B2sB2 and strains from Brazil clustering in
L3B2. This method should be now applied to a larger number of recent
strains to confirm this hypothesis.
The in silico analysis of the 45 publicly available WGS allowed the
design of a first panel of 15 PCR-HRM markers. The SNP clustering
determined by the new proposed PCR-HRM method was validated in
comparison with publicly available WGS as well as with unpublished
WGS data from 29 of the 33 DNA preparations of ancient or con-
temporary B. mallei strains included in this study (A187, NCTC10248,
IZSLER1411, A198, A199, 64.12, NCTC10230, NCTC10260, 52.236,
A200, NCTC3708, NCTC3709, 56, RKI 03–0444, 3711, 3712, 3851,
3855, 3880, 3881, 3893, 3897, 3912, 3932, 4295, Bogor, Mukteswar
and Zagreb). Comparative whole-genome sequencing offers now a
powerful way for in-depth characterization of pathogens, including the
identification of informative SNPs. In the future, with the increase in
availability of WGS, new SNP positions will emerge and could be added
to the currently PCR-HRM panel used in this study. This new PCR-HRM
tool is rapid, reliable and could be used in field investigations in
countries where the disease is endemic. Such studies are required to
trace back the origin and spread of strains circulating during outbreaks
of this important and re-emergent zoonosis.
Table 1
List of primers used for this study and melting temperature (Tm) values determined for the respective controls tested 6 times.
Subtype name Genomic
position
Forward primer (5′-3′) Reverse primer (5′-3′) Amplicon size
(pb)
SNP for the respective
subtype
SNP for the other subtype
Allele Tm values (°C) Allele Tm values (°C)
L1 330,697TCGAGGCAATCAGTTAATATCCG CGCGCGGAACAACAATGA 60 C 79,00 ± 0,03 T 78,41 ± 0,06
L2 2,621,027 CGCAGTGAAAGATCGGTGAG CCTGCTGTTCTTCATGGTCG 69 A 83,73 ± 0,03 G 84,17 ± 0,02
L2B1 354,181 TCCCGATCTTCTGGATGG GAAGAGCGCGGACGAATA 68 A 84,39 ± 0,02 G 84,97 ± 0,02
L2B2 1,408,904 ACCCTTACACGATCGAAAGGT GGCCGCTACCCCTAAGATAG 96 C 85,18 ± 0,02 T 84,43 ± 0,02
L2B2sB1 1,853,849 CACCGGCTTCTCGAACTT GGTCGAGCTTCACGATGTC 62 T 83,87 ± 0,03 C 84,34 ± 0,02
L2B2sB1Gp1 1,163,826 CGAACTCTCATCTTCAAGGCA CGTACCTTGCCGCAAAATTG 76 T 79,11 ± 0,02 C 79,71 ± 0,02
L2B2sB1Gp2 559,637 GAAGATCACGACCGTTCAGC TATTACGCCTTGACGTTCGC 60 G 85,01 ± 0,02 A 83,95 ± 0,03
L2B2sB2 707,292 CGAGCCGTTCCGTTTGATG TATCTCAAAACATCGGCCGC 60 T 82,52 ± 0,03 C 83,31 ± 0,04
L3 2,557,840 GTGCCCGTCTTCTTGTACG AAGTGGAACCAGTGGCTGTT 65 T 82,91 ± 0,02 C 83,59 ± 0,03
L3B1 309,945 TTTCTCGTATCTGCCGCTGT TGGAACAGCAGATAGATCACGT 75 T 84,49 ± 0,02 C 85,14 ± 0,01
L3B2 1,767,871 CTTCTCGATCTGCACCGC GACCTGTACATCCGCGACT 66 A 83,66 ± 0,03 G 84,10 ± 0,02
L3B3 135,971 CGCTCGACATGATGAAGAAG CGTCGCGAGATCGTTCAT 77 T 85,08 ± 0,02 C 85,82 ± 0,02
L3B3sB1 155,657 GCGCTCGGGATGAATTTCTT CTTCCTGCGCGTTGTACATG 73 T 81,50 ± 0,03 C 82,18 ± 0,03
L3B3sB2 1,560,255 AGATCGTCGACTCGGTGGT CAGCACGAATTTGTTCGAGA 89 A 85,29 ± 0,04 G 85,96 ± 0,03
L3B3sB3 922,706 CTGCTCGATGCAGCCTTC ATGCCGCTCTACCTGTCG 72 T 85,86 ± 0,02 C 86,38 ± 0,01
Table 2
List of B. mallei DNA used for this study and PCR-HRM clustering results.
Name Country Host Year of
isolation
SNP clustering
found by HRM
analysis
16-2438_BM#8 Brazil Mule 2016 L3B2
ATCC23344 Burma Human 1944 L2B2sB1Gp1
NCTC10245/
ATCC10399
China Horse 1956,
1942?
L2B2sB1Gp2
China 5 China Horse 1956 L2B2sB1Gp2
Croatia_1957 Croatia – 1957 L3B3sB3
64.12 Hungary Horse 1961 L3B3sB3
NCTC10229 Hungary unknown 1961 L3B3sB3
NCTC10230 Hungary Horse 1961 L3B3sB3
Ivan Hungary Horse 1961 L3B3sB3
NCTC3708 India unknown 1932 L3B2
NCTC3709 India Horse 1932 L3B2
Mukteswar India Horse – L2B1
3711 India Mule 2015 L2B2sB2
3712 India Horse 2015 L2B2sB2
3851 India Horse 2016 L2B2sB2
3855 India Horse 2016 L2B2sB2
3880 India Mule 2016 L2B2sB2
3881 India Horse 2016 L2B2sB2
3893 India Mule 2016 L2B2sB2
3897 India Horse 2016 L2B2sB2
3912 India Mule 2016 L2B2sB2
3932 India Mule 2016 L2B2sB2
4295 India Horse 2018 L2B2sB2
Bogor Indonesia Horse – L2B1
IZSLER 1411 Italy Horse 1959 L3B3sB3
A198 Iran Horse 1948 L3B3sB3
A200 Iran Horse 1948 L3B2
52.236 Iran Mule 1952 L3B3sB3
A199/strain 324 Iran Horse 1954 L3B3sB3
2007_2 Pakistan Gelding 2007 L2B2sB2
2017_1 Pakistan Donkey 2017 L2B2sB2
KH1 Pakistan Gelding 2013 L2B2sB2
KH2 Pakistan Gelding 2013 L2B2sB2
56 Pakistan Horse 2015 L2B2sB2
NCTC10247 Turkey unknown 1960 L3B3sB3
NCTC10260 Turkey Human 1949 L3B3sB3
NCTC10248 Turkey Human 1950 L1
NCTC120/
2002734306
UK Unknown 1920 L1
Zagreb Yugoslavia Horse – L2B1
A187/strain A – – 1954 L1
RKI 03-04444 – – – L2B1
G. Girault et al. Infection, Genetics and Evolution 63 (2018) 1–4
3
Acknowledgments
This project was supported by the European Commission's
Directorate-General for Health and Consumers (EC n°180/2008). We
would like to acknowledge Christophe Cordevant from the Anses sci-
entific direction for his precious support as well as the fruitful colla-
boration of the CRBIP - Biological Resource Center of Institut Pasteur,
Paris, France, for providing some of the bacterial strains used in this
study.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.meegid.2018.05.004.
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	High-resolution melting PCR analysis for rapid genotyping of Burkholderia mallei
	Short communication
	Acknowledgments
	Supplementary data
	References