Multi Locus Sequence Analysis MLSA as an alternative to Whole DNADNA Hybridization WDDH in Borrelia

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Multi Locus Sequence Analysis (MLSA) as an
alternative to Whole DNA/DNA Hybridization (WDDH)
in Borrelia burgdorferi sensu lato Taxonomy.

• Guy BARANTON and Daniele POSTIC
• INSTITUT PASTEUR Paris, FRANCE

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B. burgdorferi sensu lato, a complex of species
 Non pathogenic  species
Pathogenic Species
B.valaisiana B. lusitaniae B. bissettii B.
sinica B. andersonii B. japonica B. turdi B.
tanukii B. spp.
B. burgdorferi sensu stricto
B. afzelii
B. garinii
Preferential Tropism
articular
cutaneous
neurologic
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Geographic Distribution of B. burgdorferi sensu
lato
I. ricinus B. garinii B. afzelii B. burgdorferi
ss B. valaisiana B. lusitaniae Borrelia spp
I. persulcatus B. garinii B. afzelii
B. japonica I. ovatus B. turdi I.
turdus B. tanukii I. tanuki
I. pacificus
B. burgdorferi ss
I. scapularis
I. dentatus B. andersonii
I. spinipalpis B. bissettii Borrelia spp
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WDDH is considered as the gold standard in
taxonomy

• The species criteria have been formalized in
  1987
• 70 or more whole DNA relatedness within a
  species
• - DTm lower than 5C within a species
• Both criteria should be taken in consideration
• (Wayne L. G., Brenner D. J.et al. IJSB 1987
  37463-464)

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WDDH advantages and inconvenients

• - WDDH indeed reflects the true evolutive
  distances between two genomes, however
• - Time consuming and restricted to experienced
  labs
• - Delineation of a new species requires
  experiments with all previously known species -gt
  increasing work and large collections of bacteria
• - Not quite reproducible from one experiment to
  another
• - High cost

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• MLST has been used to replace MLEE
• It uses a cluster analysis procedure which
  supposes some constraints
• - to target housekeeping genes (both because they
  were previously used in MLEE and they are
  chronometric genes
• - To use sequences of about 400bp from /_ seven
  loci which warrant optimal allelic diversity.

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• Gevers, D. al. (2005). Nature Rev Microbiol 3,
  733-739.
• Suggest to use MLSA adapted from MLST in
  prokaryotic taxonomy.
• Our proposal is to sequence diverse fragments of
  loci representative of the large genetic
  diversity of the whole genome.
• It would realize a sort of  abstract  of the
  concerned bacterial genome

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• MLSA constraints are different from MLST ones
• (distances method instead of cluster analysis)
• - Large diversity of loci conserved/variable
  chronometric/adaptative, chromosomal/plasmidic,
  coding/non coding
• - The size of the loci is not criticable, a high
  number is to be preferred. Whole size about
  1/1000 of the genome
• - To avoid loci often laterally transferred and
  then to prefer informational genes instead of
  operational ones.

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• Borrelia spielmanii
• This species has been published by D. Richter in
  2004 (Appl Environ Microbiol 70, 6414-6419) but
  could not be validated because of the lack of
  DNA/DNA relatedness values.
• These isolates have been isolated in France from
  I. ricinus collected on dormice. However half of
  the sequences of rrf-rrl intergenic spacer
  typical for B. spielmanii in databank were from
  human.

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The newness of the method implies a 3 steps
strategy

• Step 1 To assess the method
• Sequencing of the 7 loci from several strains
  of each known species in Europe. Comparaison of
  the distances deduced from the concatenated
  sequences with the DNA/DNA relatedness values
  previously obtained for the concerned strains

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The newness of the method implies a 3 steps
strategy

• Step 2 Same strategy on the species known in
  North America including atypical strains which,
  studied by WDDH lead to border-line results and
  could not be classified this way.
• Delineation of potential new genomospecies

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The newness of the method implies a 3 steps
strategy

• Step 3 Both Borrelia sp. and B. valaisiana
  strains (considered as non monophyletic) from
  Europe wich have not be submitted to WDDH will be
  studied by MLSA.
• Finally, the simultaneous phylogenetic
  treatment of the concatenated sequences from all
  the studied isolates, whatever the continent they
  originated from, should allow to suggest
  relationships between species and genomospecies.

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• Material and Methods
• - 42 isolates 19 for step 1, 16 for step2 and
  all 42 for step3
• - seven loci sequenced in both directions
• - Alignment with Clustal W, concatenation with
  Word, phylogenetic analysis with Mega 3.

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Table 2. Characteristics of the amplified
fragments and corresponding primer sequences.
Numbering derives from Borrelia burgdorferi s.s.
strain B31T.

• Locus Length Primers Sequence
  analysed    
• rrs 522 F AGAGTTTGATCCTGGCTTAG (10-29)
  30-551
• R
  CTTTACGCCCAATAATCCCGA (572-552)
• fla 457 F AACACACCAGCATCACTTTCAGG (475-497)
  497-844
• R
  GATTWGCRTGCGCAATCATTGCC (976-954)
• groEL 268 F TACGATTTCTTATGTTGAGGG (552-572)
  573-840
• R
  CATTGCTTTTCGTCTATCACC (861-841)
• hbb 327 F GCGAAGAATTCATAAAAATAAGGCTGC (-79 -53
  ) 1-327
• R
  TATAAGAATTCACGATATTAACTGGC (End 26)
• recA 156 F GTGGATCTATTGTATTAGATGAAGCTCTTG
  (170-199) 206-361
• R
  GCCAAAGTTCTGAAACATTAACTCCCAAAG (391-362)
• ospA 261 F AATAGGTCTAATATTAGCCTTAATAGC (21-47)
  48-308
• R
  TTGATACTAATGTTTTGCCATCTTCTT (334-308)
• rrl-rrf 197 F CTGCGAGTTCGCGGGAGAG (3' rrf)
  197
• R
  AAGCTCCTAGGCATTCACCATA (5' rrl)    

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Table 1. Characteristics of the Borrelia
burgdorferi s.l. isolates used in step1.

• Isolate Species Geographic origin
  Biologic origin Reference
• B31T B. burgdorferi  s.s. USA I.
  scapularis Johnson et al., 1984
• IP1 B. burgdorferi  s.s. France Human
  CSF Boerlin et al., 1992
• 212 B. burgdorferi  s.s. France I.
  ricinus Postic et al., 1994
• VS461T B. afzelii Switzerland I.
  ricinus Boerlin et al., 1992
• PGau B. afzelii Germany Human skin Wilske
  et al., 1988
• BO23 B. afzelii Sweden Human skin Postic
  et al., 1994
• 20047T B. garinii France I.
  ricinus Valsangiacomo 1997
• PBi B. garinii Germany Human
  CSF Preac-Mursic 1986
• NT29 B. garinii Japan I.
  persulcatus Postic et al., 1994
• VS116T B. valaisiana Switzerland I.
  ricinus Wang et al., 1997
• UK B. valaisiana U. Kingdom I.
  ricinus Wang et al., 1997
• PotiB2T B. lusitaniae Portugal I.
  ricinus Le Fleche et al., 1997
• PotiB1 B. lusitaniae Portugal I.
  ricinus Le Fleche et al., 1997
• PC-Eq17T B. spielmanii France I.
  ricinus Richter et al., 2004
• PC-Eq2/1 B. spielmanii France I.
  ricinus Richter et al., 2004
• PC-Eq2r B. spielmanii France I.
  ricinus Richter et al., 2004
• A14S B. spielmanii Netherlands Human
  skin Wang et al., 1999

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• Delineation of Borrelia burgdorferi sensu lato
  species
• by multilocus sequence analysis
• and confirmation of the delineation of Borrelia
  spielmanii
• Dania Richter, Danièle Postic, Natacha Sertour,
  Ian Livey,
• Franz-Rainer Matuschka, and Guy Baranton
• IJSEM Papers in Press, published online 9
  December 2005
• lthttp//ijs.sgmjournals.org/misc/pip.shtml

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Table 3 Characteristics of the isolates used in
step 2

• Isolate Species Geo. origin Biologic
  origin Reference
• B31T B. bor.  ss USA I. scapularis Johnson
  IJSB 1984
• 297 B. bor.  ss USA Human CSF Steere NEJM 1983
• Sh2-82 B. bor.  ss USA I. scapularis Schwann
  II 1988
• 21123 B. and. New York I. dentatus Postic IJSB
  1994
• 21133 B. and. New York I. dentatus Postic IJSB
  1
• 19952 B. and. New York I. dentatus Postic IJSB
  1994
• DN127 B. bis. California I. pacificus Bissett
  JCM 1987
• CA55 B. bis. California I. neotomae Postic
  IJSB 1994
• CA128 B. bis. California I. neotomae Brown
  Science 1992
• CA2 B. sp. California I. neotomae Brown Lane
  Science 1992
• CA8 B. sp. California I. pacificus Brown Lane
  Science 1992
• CA13 B. sp. California I. neotomae Schwann JCM
  1993
• CA28 B. sp. California I. pacificus Brown Lane
  Science 1992
• CA29 B. sp. California I. pacificus Brown Lane
  Science 1992

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Table 4 Characteristics of the additional
isolates used in step 3

• Isolate Species Geographic origin Biologic
  origin Reference
• M7 B. val. Netherlands I. ricinus Wang
  Res Mic 2000
• Am501 B. val. Japan I. persulcatus
  Masuzawa 1996
• NE49 B. sp. Switzerland I. ricinus
  Postic JCM 1994
• 5LM218 B. sp. France I. ricinus
  unpublished
• Z41493 B. sp. Germany I. ricinus Postic
  JCM 1994
• Z41293 B. sp. Germany I. ricinus Postic
  JCM 1994
• Z51094 B. sp. Germany I. ricinus Postic
  JCM 1994
• Z52794 B. sp. Germany I. ricinus Postic
  JCM 1994
• Z57394 B. sp. Germany I. ricinus Postic
  JCM 1994

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Conclusion 1

• Validation of both MLSA method in taxonomy and
  B. spielmanii (IJSEM in press).
• Delineation of 3 additional genomospecies in the
  USA.
• Confirmation of 2 geographical clusters of B.
  burgdorferi s. l. in Old and New Worlds

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Conclusion 2

• European B. valaisiana does constitute a
  monophyletic species.
• Delineation of a 4th genomospecies mainly
  comprising european isolates clustering as does
  B. burgdorferi s. s. with North American species.
• Outgroup geographical position of B. lusitaniae.