Beeckman, Delphine Sylvie Anne

Dickx, Veerle

Pannekoek, Yvonne

van der Ende, Arie

Vanrompay, Daisy

Vretou, Evangelia

English

Ghent University Academic Bibliography

MULTI LOCUS SEQUENCE TYPING OF CHLAMYDIALES: CLONAL GROUPINGS WITHIN CHLAMYDOPHILA ABORTUS AND HOST ASSOCIATED GENOTYPES OF CHLAMYDOPHILA PSITTACI.  Y. Pannekoek1, D.S.A. Beeckman2, V. Dickx2, E. Vretou3, D. Vanrompay2 and A. van der Ende1*  1 Academic Medical Center, Center for Infection and Immunity Amsterdam (CINIMA), Department of Medical Microbiology, Amsterdam, the Netherlands; 2 Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium; 3 Laboratory of Biotechnology, Department of Microbiology, Hellenic Pasteur Institute, Athens, Greece  Introduction The obligate intracellular growing bacterium Chlamydia trachomatis causes diseases like trachoma, urogenital infection and lymphogranuloma venereum with severe morbidity. Several serovars and genotypes have been identified, but these could not be linked to clinical disease or outcome. The related Chlamydophila pneumoniae, of which no subtypes are recognized, causes respiratory infections worldwide. Chlamydophila psittaci which can cause zoonotic pneumonia in humans are usually hosted by birds, while Chlamydophila abortus causes abortion and fetal death in mammals, including humans and is hosted by goats, sheeps and their relatives. We developed a multi locus sequence typing (MLST) scheme based on the partial sequences of seven housekeeping genes to understand the population genetic structure of Chlamydiales and the diversity of these species and to evaluate the association between genotype and disease. Methods Strains A collection of 26 strains of C. trachomatis of different serovars and clinical presentation and 18 strains of C. pneumoniae were included in the study. In addition, 29 C. psittaci strains isolated from different bird species and mammals from different geographic locations and 16 C. abortus isolated from goats and sheep from different geographic locations were included. For comparison, sequences of C. caviae, C. felis, C. pecorum (Chlamydophila), and C. muridarum (Chlamydia) were also included. DNA, genes, PCR products and sequences DNA extraction, PCR protocols and DNA sequencing were performed as previously described (1). Phylogenetic and other analyses  Sequences of fragments from seven housekeeping genes (enoA, fumC, gatA, gidA, hemN, hlfX, oppA) were analysed as described before (1). Allel numbers and genotypes were identified at http://pubmlst.org/chlamydiales/. A distance matrix in Nexus format was generated from the set of concatenated allele sequences using MEGA version 4.0.2 (2), which was then used for phylogenetic analyses in SplitsTree 4.0 (3), generating an UPGMA tree. Results Recently, multilocus sequence analysis (MLSA) was introduced to study relatedness of closely related species (4). In this analysis the sequences of multi locus housekeeping fragments are concatenated and used in cluster analysis. Phylogenetic analysis of 38 genotypes among 89 strains of Chlamydiales by Neighbour-Joining method of the aligned concatenated sequences of the housekeeping gene fragments resulted in a tree (Fig. 1). comparable to that obtained with 16S rRNA gene and 23S rRNA gene sequences (5). Of note, the distance between C. abortus and C. psittaci is closer than the distance between the other species, indicating that C. psittaci and C. abortus are more related than the other species to each other. Also, the group of C. abortus contains one C. psittaci strain 84/2334 (ST36) isolated from a Yellow-crowned amazon.  C. pneumoniaeC. trachomatisC. psitacciC. abortusC. caviaeC. pecorumC. felisC. muridarum Figure 1. Phylogenetic analyses of concatenated sequences of 7 housekeeping gene fragments of Chlamydiales strains. Concatenated sequences of seven housekeeping gene fragments were aligned and analysed in MEGA 3.1. Phylogenetic tree was constructed using the Neighbour-Joining algorithm with Kimura-2 parameter.  A tree produced from the allelic profiles of the C. psittaci and C. abortus strains showed groupings among C. psittaci which were associated with host species with some exceptions (Fig 2, Table 1).   Figure 2. Phylogenetic analyses of concatenated sequences of 7 housekeeping gene fragments of C. psittaci and C. abortus strains.  Concatenated sequences were aligned and analysed in MEGA 3.1. Phylogenetic tree was constructed using the Neighbour-Joining algorithm with Kimura-2 parameter. Bootstrap test was for 1000 repetitions. Bold numbers indicate bootstrap values over 50%. Colours correspond with those in Table 1.  The group of C. psittaci strains (ST34, ST35, ST27, and ST26), mainly isolated from pigeons, contains one strain isolated from a ferret and human (ST35). Also, the strains isolated from ducks (all with genotype ST28) are closely related to strains isolated from parrots and  ST-34 ST-35 ST-27 ST-26 ST-24 ST-28 ST-32 ST-37 ST-41 ST-31 ST-36 ST-30 ST-25 ST-19 ST-2962868210074977854797010097parakeets (all of genotype ST24). Strain WC, divided from these strains with a bootstrap value of 54%.  Table 1. C. psittaci and C. abortus strains according to genotype (ST). Colours correspond to those in Figure 1.  Strain ST species Animal country   96/1867/30 34 C. psittaci urban Pigeon Italy   CPMN 35 C. psittaci ferret/human USA   99/3759/2 35 C. psittaci urban Pigeon Italy   96/3218 26 C. psittaci urban Pigeon Italy   98/6098 26 C. psittaci urban Pigeon Italy   CP3 27 C. psittaci Pigeon     84/55 24 C. psittaci Budgerigar Germany   89/1291 24 C. psittaci Budgerigar Belgium   91/154 24 C. psittaci Budgerigar Belgium   2000/2675 24 C. psittaci Eastern Rosella Italy   99/3005 24 C. psittaci Elegant Parrot Italy   C_psittaci 6BC 24 C. psittaci Parakeet     VS1 24 C. psittaci Parrot USA   95/99 24 C. psittaci Peach-faced Lovebird Italy   2000/332 24 C. psittaci Scarlet Macaw Italy   91/237 24 C. psittaci Senegal Parakeet Belgium   90/1551 24 C. psittaci       91/0137 24 C. psittaci       CR9 28 C. psittaci Duck Europe   humaan E 28 C. psittaci human     18/290800 28 C. psittaci Pekin Duck Germany   3/20901 28 C. psittaci Pekin Duck Germany   4/20901 28 C. psittaci Pekin Duck Germany   5/20901 28 C. psittaci Pekin Duck Germany   WC 32 C. psittaci Bovine     NJ1  37 C. psittaci Turkey USA   VS225 41 C. psittaci Parakeet USA   M56 31 C. psittaci Muskrat     84/2334 36 C. psittaci Yellow-crowned amazon Germany   LLG 30 C. abortus goat Greece   POS 30 C. abortus sheep Greece   1B 25 C. abortus sheep France   FAG 19 C. abortus goat Greece   MB 19 C. abortus goat Greece   VPIG 19 C. abortus goat Greece   B577 19 C. abortus sheep USA (ATCC)   A22 19 C. abortus sheep UK   AB7 19 C. abortus sheep France   FAS 19 C. abortus sheep Greece   MA 19 C. abortus sheep Greece   MD 19 C. abortus sheep greece   ME 19 C. abortus sheep Greece   C_abortus 19 C. abortus       MF 19 C. abortus   Greece   Krauss-15 29 C. abortus caprine Tunisia Clustering is not associated with the geographic origin of the species. Two C. psittaci strains are more distantly related to the other C. psittaci strains; one isolated from a parakeet and one isolated from a muskrat. Finally, the C. psittaci strain 84/2334 isolated from a parrot is closely related to the C. abortus strains forming one group. Discussion The in this study used MLST scheme was previously applied to analyze clonal groupings among C. trachomatis and C. pneumonia strains (1). We showed that an UPGMA tree produced from the allelic profiles and from concatenated allel sequences resulted in three groups of sequence types. The urogenital strains were distributed over two separated groups; one consisted solely of strains with frequent occurring serovars (E, D and F). The LGV strains grouped in a single cluster, which also included C. trachomatis B/TW5. Recently, another MLST scheme was described showing clonal groupings among C. trachomatis strains with a group consisting exclusively of LGV strains (6). Of note, B/TW5 shares IncA polymorphisms with LGV strains, which were not found among other serovars (7). Phylogenetic analyses of the concatenated allel sequenes of C. psittaci and C. abortus strains indicated an association between C. psittaci genotype and host species, but not with geographic origin. However, there are exceptions like C. psittaci CPMN from a patient found in a cluster with strains isolated from pigeons and C.psittaci human E from a patient found in the group with strains from ducks, suggesting host species-crossing by C. psittaci. Host species-crossing of C. abortus may be indicated by strain 84/2334. Although this strain is annotated as being a C. psittaci strain, it groups with all C. abortus strains in the MLSA analyses. Previously, this strain was identified as the missing link between C. psittaci and C. abortus (8). It was shown that 84/2334 has DNA sequences identical to an extrachromosomal plasmid in duck C. psittaci strain N352, to rnpB in strain R54 from a brown skua and to the rrn intergenic spacer in parakeet strain Prk/Daruma. Analysis of ompA and the rrn spacer revealed progressive diversification of the strains, with 84/2334 resembling what might have been a recent ancestor of C. abortus. Our analyses of concatenated sequences of 7 housekeeping alleles which are not under selective pressure of the host show that 84/2334 branches off before all C. abortus strains, indicating that this strain differs from C. abortus but is more closely related to C. abortus than to C. psittaci and should therefore be classified as C. abortus. Acknowledgements This research was partly funded by the Sixth Framework Program of the European Commission, Proposal/Contract no.: 512061 (Network of Excellence 'European Virtual Institute for Functional Genomics of Bacterial Pathogens' [http://www.noe-epg.uni-wuerzburg.de]. This publication made use of the Chlamydiales MLST website (http://pubmlst.org/ chlamydiales/) developed by Keith Jolley and sited at the University of Oxford (9). The development of this site has been funded by the Wellcome Trust. D.S.A. Beeckman is a Post-doctoral fellow of the Research Foundation - Flanders (FWO - Vlaanderen). References 1. Pannekoek Y, Morelli G, Kusecek B, Morre SA, Ossewaarde JM, Langerak AA, van der Ende A. Multi locus sequence typing of Chlamydiales: clonal groupings within the obligate intracellular bacteria Chlamydia trachomatis. BMC Microbiol 2008, 8:42. 2. Tamura K, Dudley J, Nei M & Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24:1596-1599. 3. Rozas J, Sanches-DelBarrio JC, Messeguer X, Rozas R: DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 2003, 19:4. Bishop CJ, Aanensen DM, Jordan GE, Kilian M, Hanage WP, Spratt BG. Assigning strains to bacterial species via the internet. 2496-2497.  5. Everett KDE, Bush RM, Andersen AA: Emended description of the order Chlamydiales, proposal of ParaChlamydiaceae fam. nov. and Simkaniaceae fam. nov., each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new genus and five new species, and standards for the identification of organisms. Int J Syst Bacteriol 1999, 49:415-440 BMC Biol. 2009;7:3. 6. Dean D, Bruno WJ, Wan R, Gomes JP, Devignot S, Mehari T, de Vries HJ, Morre SA, Myers G, Read TD, Spratt BG. Predicting phenotype and emerging strains among Chlamydia trachomatis infections. Emerg Infect Dis 2009, 15:1385-94. 7. Pannekoek Y, van der Ende A, Eijk PP, van Marle J, de Witte MA, Ossewaarde JM, van den Brule AJ, Morré SA, Dankert J. Normal IncA expression and fusogenicity of inclusions in Chlamydia trachomatis isolates with the incA I47T mutation. Infect Immun. 2001;69:4654-6. 8. Van Loock M, Vanrompay D, Herrmann B, Vander Stappen J, Volckaert G, Goddeeris BM, Everett KD. Missing links in the divergence of Chlamydophila abortus from Chlamydophila psittaci. Int J Syst Evol Microbiol. 2003;53:761-70. 9. Jolley KA, Chan MS, Maiden MC. mlstdbNet - distributed multi-locus sequence typing (MLST) databases. BMC Bioinformatics. 2004;5:86. 

Multi locus sequence typing of Chlamydiales: clonal groupings within Chlamydophila abortus and host associated genotypes of Chlamydophila psittaci

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Multi locus sequence typing of Chlamydiales: clonal groupings within Chlamydophila abortus and host associated genotypes of Chlamydophila psittaci

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