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Genomic approach to high resolution typing of Mycoplasma bovis

Shukla, Ankit; Kokotovic, Branko

Publication date:2015

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Citation (APA):Shukla, A., & Kokotovic, B. (2015). Genomic approach to high resolution typing of Mycoplasma bovis. Postersession presented at Progress in human and animal mycoplasmology, Pendik/Istanbul, Turkey.

Genomic approach to high resolution typing of

Mycoplasma bovis

Ankit Shukla* and Branko Kokotovic

OMB_030-87 OMB_018-87 OMB_016-87 OMB_022-87 OMB_014-87 OMB_028-87 OMB_033-87 OMB_011-87

2.0E-5

OMB_408-98 OMB_405-88

OMB_009-84 OMB_006-84 OMB_008-84

OMB_003-81

OMB_130-95 OMB_430-98

OMB_167-00 OMB_001-01

OMB_003-99

OMB_393-98

OMB_004-81

PG45

OMB_344-97

AMB_230-11 AMB_231-11

AMB_224-11

BMB_021-12

AMB_023-11

AMB_025-11

AMB_024-11

AMB_071-11

AMB_072-11

AMB_037-11

AMB_045-11

AMB_101-11

AMB_094-11

BMB_023-12

BMB_001-12

CMB_430-13

BMB_118-12

BMB_004-12

BMB_002-12

CMB_531-13

DMB_059-14

BMB_003-12

DMB_028-14

BMB_027-12

BMB_028-12

AMB_068-11

AMB_066-11

CMB_149-13

CMB_141-13

CMB_150-13

AMB_123-11

AMB_103-11

AMB_104-11

AMB_124-11

AMB_121-11

CMB_063-13

AMB_228-11

AMB_164-11

CMB_111-13

BMB_009-12

BMB_008-12

BMB_094-12

AMB_105-11

AMB_102-11

AMB_122-11

AMB_038-11

AMB_120-11

AMB_128-11

DMB_284-14

AMB_078-11

AMB_063-11

AMB_029-11

AMB_030-11

AMB_040-11

AMB_042-11

AMB_039-11

AMB_048-11

CMB_076-13

BMB_030-12

BMB_085-12

AMB_059-11

AMB_058-11

AMB_044-11

AMB_083-11

AMB_116-11

AMB_223-11

CMB_033-13

BMB_164-12

BMB_010-12

BMB_011-12

CMB_096-13

CMB_103-13

CMB_105-13

CMB_094-13

CMB_098-13

CMB_023-13

CMB_110-13

CMB_109-13

CMB_574-13

DMB_228-14

DMB_288-14

3.0E-4

6553 SNPs

1709 SNPs

3rd M.bovis outbreak

1st M.bovis isolate reported in DK

1st M.bovis outbreak

2nd M.bovis outbreak

1981 2

1984 3

8 1987

1

3 1998

1 1999

1 2000

1 2001

41 2011

18 2013

6 2014

№ isolates Year

Sporadic M.bovis isolates

1 1997

1 1995

1 1988

16 2011

Background

Conventional MLST (cMLST) approach relying on variations within portions of few housekeeping genes has been extensively used to study structure and dynamics of bacterial populations. Although highly useful in identifying global population structure, its resolution power does not permit analysis of genetic variations within a single clonal line of descent over short time frames.

Objective

To examine genetic diversity within clonal lineages of Mycoplasma bovis during outbreak periods and within short geographical distances by using MLST based on partial (cMLST), full length sequences (eMLST) of housekeeping genes, a complete set of coding regions (wgMLST) in the M. bovis chromosome and genome wide single nucleotide polymorphism (SNP) analysis.

Materials & Methods The examination was carried out by analysis of 104 Danish M.bovis isolates collected throughout Denmark (Fig. 1) over a period of 34 years. For cMLST and eMLST sequences of the housekeeping genes included in a recently published MLST scheme (1) were extracted from reference genome (PG45), and pre-processed sequencing reads were mapped on them using Burrows-Wheeler algorithm (Fig. 2).

Results & Discussion Classical MLST based on partial gene sequences (cMLST) divided the 104 Danish M.bovis isolates into two distinct types (Fig. 3a), which differed only differed at tkt locus. Including full length housekeeping gene sequences (eMLST) of the used MLST scheme resulted in differentiation of the test population in 7 types (Fig. 3b) based on additional 4 loci (atpA, dnaA, tufA and rpoD). Substantial number of variations was detected in the regions flanking partial sequences used in cMLST scheme, some of which contributed to further discrimination within the group (Fig. 4). wgMLST based on analysis of 365 full length coding sequences spanning more than 400 kb, which were common for all strains in the test population revealed three distinct groups comprising 80 different types. Genome wide SNP based on analysis of coding and non-coding regions spanning more than 600 kb clearly identified outbreak strains and separated them from sporadic isolates in contrast to cMLST, eMLST and wgMLST .

Fig 4.: atpA gene sequence with bars indicating polymorphisms in

the test population. Partial gene sequence used in classical MLST

scheme is indicated by red rectangle. Additional mutations

facilitating further differentiation of the strains were found in the

flanking region and are indicated by asterisk.

Partial sequence used in cMLST scheme

*

References 1. Rosales et al. 2015. J. Clin. Microbiol., 53, 789-94

Fig 5.: Genome wide SNP tree representing 3 distinct groups

based on coding and non-coding regions

Fig. 1.: Spatiotemporal distribution of Danish Isolates

Fig. 2.: Bioinformatics Worklfow

Conclusions

Both cMLST and eMLST methods were able to divide the test population in two distinct types based on variations in partial and full sequences of housekeeping genes used in the cMLST scheme (1). Extending the analysis to include full length sequences (eMLST) of the housekeeping genes have only marginally improved the actual resolution of the cMLST method. wgMLST improved the resolution and revealed three distinct groups, however it did not completely resolve outbreak strains from 1984. The highest level of resolution was attained by genome wide SNP analysis which clearly distguished outbreak strains including outbreak strains from 1984, which allowed for detailed analysis of outbreaks and microevolution of a given clonal line.

Group 3

Group 2

Group 1

DMB_318-14

(c) wgMLST

(b) eMLST (a) cMLST

1

Group 1+2

Group 3

Group 1+2

Group 3

Group 1

1

Group 2

Group 3

Fig. 3.: Minimum spanning tree based on (a) Classical MLST gene

fragments (b) eMLST (c) whole genome MLST

*Ankit Shukla, Research group for Bacteriology and Pathology, National Veterinary Institute, Technical University of Denmark, Bülowsvej 27, 1970 Copenhagen V, Denmark. Phone: +45 93911187 Fax: +45 3588 6100 e-mail: anshu@vet.dtu.dk