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<pre>
Annals of Clinical Microbiology and
Antimicrobials
BioMed Central
Open Access
Research
Multilocus sequence typing method for identification and genotypic
classification of pathogenic Leptospira species
Niyaz Ahmed*†1,2, S Manjulata Devi†1, M de los Á Valverde3, P Vijayachari4,
Robert S Machang'u5, William A Ellis6 and Rudy A Hartskeerl2,7
Address: 1Pathogen Evolution Group, Centre for DNA Fingerprinting and Diagnostics (CDFD), Hyderabad 500076, India, 2ISOGEM working
group on Spirochetes, The International Society for Genomic and Evolutionary Microbiology (ISOGEM), Sassari, Italy, 3National Reference Center
Leptospirosis. INCIENSA (Costarrican Institute for Research in Nutrition and Health), Costa Rica, 4Regional Medical Research Centre (RMRC),
Port Blair, India, 5Department of Veterinary Microbiology and Parasitology, Sokoine University of Agriculture, P. O. Box 3019, Morogoro,
Tanzania, 6Veterinary Sciences Division (VSD), The Queen's University of Belfast, Stoney Road, Stormont, Belfast, Northern Ireland, BT4 3SD, UK
and 7WHO/FAO/OIE and National Collaborating Centre for Reference and Research on Leptospirosis, KIT Biomedical Research, KIT (Koninklijk
Instituut voor de Tropen/Royal Tropical Institute) Meibergdreef 39, 1105 AZ Amsterdam, The Netherlands
Email: Niyaz Ahmed* - niyaz.cdfd@gmail.com; S Manjulata Devi - manju@cdfd.org.in; M de los Á Valverde - mvalverde@inciensa.sa.cr;
P Vijayachari - vijayacharip@yahoo.com; Robert S Machang'u - machangu2001@yahoo.com; William A Ellis - bill.ellis@dardni.gov.uk;
Rudy A Hartskeerl - r.hartskeerl@kit.nl
* Corresponding author †Equal contributors
Published: 23 November 2006
Annals of Clinical Microbiology and Antimicrobials 2006, 5:28
28
doi:10.1186/1476-0711-5-
Received: 12 October 2006
Accepted: 23 November 2006
This article is available from: http://www.ann-clinmicrob.com/content/5/1/28
© 2006 Ahmed et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: Leptospira are the parasitic bacterial organisms associated with a broad range of mammalian hosts
and are responsible for severe cases of human Leptospirosis. The epidemiology of leptospirosis is complex and
dynamic. Multiple serovars have been identified, each adapted to one or more animal hosts. Adaptation is a
dynamic process that changes the spatial and temporal distribution of serovars and clinical manifestations in
different hosts. Serotyping based on repertoire of surface antigens is an ambiguous and artificial system of
classification of leptospiral agents. Molecular typing methods for the identification of pathogenic leptospires up to
individual genome species level have been highly sought after since the decipherment of whole genome sequences.
Only a few resources exist for microbial genotypic data based on individual techniques such as Multiple Locus
Sequence Typing (MLST), but unfortunately no such databases are existent for leptospires.
Results: We for the first time report development of a robust MLST method for genotyping of Leptospira.
Genotyping based on DNA sequence identity of 4 housekeeping genes and 2 candidate genes was analyzed in a
set of 120 strains including 41 reference strains representing different geographical areas and from different
sources. Of the six selected genes, adk, icdA and secY were significantly more variable whereas the LipL32 and
LipL41 coding genes and the rrs2 gene were moderately variable. The phylogenetic tree clustered the isolates
according to the genome-based species.
Conclusion: The main advantages of MLST over other typing methods for leptospires include reproducibility,
robustness, consistency and portability. The genetic relatedness of the leptospires can be better studied by the
MLST approach and can be used for molecular epidemiological and evolutionary studies and population genetics.
Page 1 of 10
(page number not for citation purposes)
Annals of Clinical Microbiology and Antimicrobials 2006, 5:28
Background
Leptospirosis is a zoonotic and an emerging infectious
disease caused by the pathogenic Leptospira species and is
identified in the recent years as a global public health
problem because of its increased mortality and morbidity
in different countries. Leptospirosis is frequently misdiagnosed as a result of its protean and non-specific presentation resembling many other febrile diseases, notably viral
haemorrhagic fevers such as dengue [1]. There is, for certain, an underestimation of the leptospirosis problem due
to lack of awareness and under-recognition through a lack
of proper use of diagnostic tools.
The common mode of transmission of the infection in
humans is either by direct or indirect contact with the
urine of infected animals and may lead to potential lethal
disease. A unique feature of this organism is to parasitize
in a wide variety of wild and domestic animals [2]. Traditionally, two species have been identified, i.e. Leptospira
interrogans and L. biflexa for pathogenic and non-pathogenic leptospires, respectively. The serovar is the basic
identifier, characterized on the basis of serological criteria.
To date nearly 300 serovars have been identified under the
species L. interrogans alone that have been distributed
among 25 different serogroups of antigenically similar
serovars [3].
http://www.ann-clinmicrob.com/content/5/1/28
MLST is a simple PCR based technique, which makes use
of automated DNA sequencers to assign and characterize
the alleles present in different target genes. The method
allows one to generate sequence data in a low to highthroughput scale, which is unambiguous and suitable for
epidemiological and population studies. The selected loci
are generally the housekeeping genes, which evolve very
slowly over an evolutionary time-scale [16] and hence
qualify as highly robust markers of ancient and modern
ancestry. The sequencing of multiple loci provides a balance between technical feasibility and resolution. MLST
has been applied to the study of many other bacterial species like Neisseria meningitides [17], Streptococcus pneumoniae [18], Yersinia species [19], Campylobacter jejuni [20]
and Helicobacter pylori [21].
Our present study is the first attempt to use the MLST,
which currently differentiates the species and examines
the intra and interspecies relationships of Leptospira. This
method in future could be developed as a highly sophisticated genotyping system based on integrated genome
analysis approaches to correctly identify and track leptospiral strains and is expected to greatly facilitate epidemiology of leptospirosis apart from deciphering the origins
and evolution of leptospires in a global sense.
Methods
Previously a classification system based on DNA-DNA
hybridization studies has been introduced, which now
comprises 17 Leptospira species [4-7]. Among these, 7 species: L. interrogans, L. borgpetersenii, L. santarosai, L.
noguchii, L. weilli, L. kirschneri and L. alexanderi are considered as the main agents of leptospirosis [5,6]. The enormous inventory of serovars, based mainly on an everchanging surface antigen repertoire, throws an artificial
and unreliable scenario of strain diversity. It is therefore
difficult to track strains whose molecular identity keeps
changing according to the host and the environmental
niches they inhabit and cross through.
Other than the serological methods, molecular tools that
have been employed so far for sub-classification and cataloguing of leptospiral agents include restriction endonuclease assay (REA) [8,9], pulsed field gel electrophoresis
(PFGE) [10,11], restriction fragment length polymorphism (RFLP) [12], arbitrarily primed PCR [13], Variable
Number of Tandem Repeats (VNTR) analysis [14] and fluorescent amplified fragment length polymorphism
(FAFLP) [15]. All these techniques however, suffer from
certain disadvantages that include requirement of large
quantity of pure and high quality DNA, low discriminatory power, low reproducibility, ambiguous interpretation of data and problems associated with transfer of data
between different laboratories [14].
Bacterial strains
Bacterial strains (Table 1) were cultured by the WHO reference laboratory at the KIT Biomedical Research Centre
at The Royal Tropical Institute, Amsterdam, The Netherlands (all isolates and reference strains labelled RK3) and
at the Veterinary Sciences Division (VSD), The Queen's
University of Belfast, United Kingdom (reference strains
labelled RB3) and the WHO reference centre at Port Blair
India (labelled isol 15). A total of 120 strains consisting of
79 isolates and 41 reference strains from different sources
and geographical regions were analyzed by MLST. The 41
reference strains included in the study belonged to six
Leptospira species (L. interrogans; L. kirschneri; L. noguchii;
L. borgpetersenii; L. santarosai and L. alexanderi).
Selection and validation of target genes for MLST
The candidate loci sequences were obtained from the
strains L. interrogans Fiocruz L1-130 and L. interrogans Lai
56601 strains from the Leptolist server. Six genes, namely
adk (Adenylate Kinase), icdA (Isocitrate dehydrogenase),
LipL32 (outer membrane lipoprotein LipL32), rrs2 (16S
rRNA), secY (pre-protein translocase SecY protein), and
LipL41 (outer membrane Lipoprotein LipL41) (Table 2)
were selected for MLST analysis. Many sequences of the
rrs2, LipL32 and LipL41 are available in the GenBank [2].
PCR primers were designed for these genes based on GenBank records in the conserved regions flanking the variable internal fragments of the target regions. PCR primers
Page 2 of 10
(page number not for citation purposes)
Annals of Clinical Microbiology and Antimicrobials 2006, 5:28
http://www.ann-clinmicrob.com/content/5/1/28
Table 1: Details of leptospiral strains and isolates used for MLST based
Labels
Genome Species
Serogroup
Serovar
Strain
Geographical area
Source
INT 01
L. interrogans
Canicola
Sumneri
Sumner
Malaysia
RB3
INT 02
L. interrogans
Canicola
Portlandvere
MY 1039
Jamaica
RB3
INT 03
L. interrogans
Pomona
Pomona
Pomona
Australia
RB3
INT 04
L. interrogans
Pomona
Proechimys
1161 U
Panama
RB3
INT 05
L. interrogans
Pomona
Kenniwicki
LT 1026
USA
RB3
INT 06
L. interrogans
Grippotyphosa
Grippotyphosa
Moskva V
Unknown
RB4
INT 07
L. interrogans
Grippotyphosa
Muelleri
RM 2
Malaysia
RB3
INT 08
L. interrogans
Sejroe
Roumanica
LM 294
Roumania
RB3
INT 09
L. interrogans
Sejroe
Saxkoebing
Mus 24
Denmark
RB3
INT 10
L. interrogans
Sejroe
Hardjoprajitno
Hardjoprajitno
Indonesia
RB3
INT 11
L. interrogans
Icterohaemorrhagiae
Lai
Lai
China
RB3
INT 12
L. interrogans
Icterohaemorrhagiae
Copenhageni
M 20
Denmark
RB3
INT 13
L. interrogans
Grippotyphosa
Valbuzzi
Valbuzzi
Australia
RB3
INT 14
L. interrogans
Pyrogenes
Manilae
LT 398
Phillipins
RB3
INT 15
L. interrogans
Australis
Australis
Ballico
Ballico
RK3
INT 16
L. interrogans
Icterohaemorrhagiae
Icterohaemorrhagiae
RGA
Germany
RK3
INT 17
L. interrogans
Grippotyphosa
Ratnapura
Field Isolate 1
South Andaman
Isol 15
INT 18
L. interrogans
Icterohaemorrhagiae
Copenhageni
Field Isolate 2
South Andaman
Isol 15
INT 19
L. interrogans
Grippotyphosa
Ratnapura
Field Isolate 3
South Andaman
Isol 15
INT 20
L. interrogans
Grippotyphosa
Ratnapura
Field Isolate 4
South Andaman
Isol 15
INT 21
L. interrogans
Grippotyphosa
Valbuzzi
Field Isolate 5
South Andaman
Isol 15
INT 22
L. interrogans
Icterohaemorrhagiae
Copenhageni
Field Isolate 6
South Andaman
Isol 15
INT 23
L. interrogans
Grippotyphosa
Valbuzzi
Field Isolate 7
North Andaman
Isol 15
INT 24
L. interrogans
Grippotyphosa
Valbuzzi
Field Isolate 8
North Andaman
Isol 15
INT 25
L. interrogans
Grippotyphosa
Ratnapura
Field Isolate 9
South Andaman
Isol 15
INT 26
L. interrogans
Grippotyphosa
Ratnapura
Field Isolate 10
South Andaman
Isol 15
INT 27
L. interrogans
Grippotyphosa
Ratnapura
Field Isolate 11
South Andaman
Isol 15
INT 28
L. interrogans
Grippotyphosa
Unknown
Field Isolate 12
South Andaman
Isol 15
INT 29
L. interrogans
Grippotyphosa
Unknown
Field Isolate 13
South Andaman
Isol 15
INT 30
L. interrogans
Sejroe
Sejroe
Field Isolate 14
South Andaman
Isol 15
INT 31
L. interrogans
Pomona
Unknown
Field Isolate 15
South Andaman
Isol 15
INT 32
L. interrogans
Grippotyphosa
Ratnapura
Field Isolate 16
South Andaman
Isol 15
INT 33
L. interrogans
Australis
Ramisi
Field Isolate 17
South Andaman
Isol 15
INT 34
L. interrogans
Grippotyphosa
Unknown
Field Isolate 18
South Andaman
Isol 15
INT 35
L. interrogans
Grippotyphosa
Valbuzzi
Field Isolate 19
South Andaman
Isol 15
INT 36
L. interrogans
Grippotyphosa
Valbuzzi
Field Isolate 20
South Andaman
Isol 15
INT 37
L. interrogans
Hebdomadis
Goiano
Bovino 131
Brazil
RB3
INT 38
L. interrogans
Canicola*
Canicola*
M12/90
Brazil
Isol
INT 39
L. interrogans
Icterohaemorrhagiae*
Copenhageni*
M9/99
Brazil
Isol
INT 40
L. interrogans
Australis*
Rushan*
L01
Brazil
Isol
INT 41
L. interrogans
Canicola*
Canicola*
L02
Brazil
Isol
Page 3 of 10
(page number not for citation purposes)
Annals of Clinical Microbiology and Antimicrobials 2006, 5:28
http://www.ann-clinmicrob.com/content/5/1/28
Table 1: Details of leptospiral strains and isolates used for MLST based (Continued)
INT 42
L. interrogans
Canicola*
Canicola*
L03
Brazil
Isol
INT 43
L. interrogans
Canicola*
Canicola*
L09
Brazil
Isol
INT 44
L. interrogans
Icterohaemorrhagiae*
Copenhageni*
L10
Brazil
Isol
INT 45
L. interrogans
Canicola*
Canicola*
L14
Brazil
Isol
INT 46
L. interrogans
Lyme*
Lyme*
K30B
UK
Isol
INT 47
L. interrogans
Australis*
Australis*
K9H
UK
Isol
INT 48
L. interrogans
Icterohaemorrhagiae*
Copenhageni*
Isolate 9
Costa Rica
Isol
INT 49
L. interrogans
Unknown*
Unknown*
Isolate 10
Costa Rica
Isol
INT 50
L. interrogans
Australis*
Lora*
1992
Tanzania
Isol
INT 51
L. interrogans
Australis*
Lora*
2324
Tanzania
Isol
INT 52
L. interrogans
Australis*
Lora*
2364
Tanzania
Isol
INT 53
L. interrogans
Australis*
Lora*
2366
Tanzania
Isol
INT 54
L. interrogans
Ballum*
Kenya*
4885
Tanzania
Isol
INT 55
L. interrogans
Ballum*
Kenya*
4883
Tanzania
Isol
KIR 01
L. kirschneri
Canicola
Kuwait
136/2/2
Kuwait
RB3
KIR 02
L. kirschneri
Canicola
Schueffneri
Vleermuis 90 C
Indonesia
RB3
KIR 03
L. kirschneri
Pomona
Mozdok
5621
Soviet Union (Russia)
RB3