Diversity of Orientia tsutsugamushi clinical isolates in Cambodia reveals active selection and recombination process

Infection, Genetics and Evolution 15 (2013) 25–34

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Diversity of Orientia tsutsugamushi clinical isolates in Cambodia reveals active selection and recombination process Veasna Duong a,1, Kim Blassdell a,1, Thinh Thi Xuan May b, Lay Sreyrath a, Laurent Gavotte c, Serge Morand c, Roger Frutos d,⇑, Philippe Buchy a,⇑⇑ a

Virology Unit, Institut Pasteur in Cambodia, 5 Monivong blvd, PO Box 983, Phnom Penh, Cambodia Virology Department, Institut Pasteur in Nha Trang, 10 Tran Phu Street, Nha Trang, Viet Nam UM2, ISEM, UMR 5554, CNRS-UM2-IRD, Université Montpellier 2 CC065, Place E. Bataillon, 34095 Montpellier Cedex 5, France d Cirad, UMR 17, Cirad-Ird, TA-A17/G, Campus International de Baillarguet, 34398 Montpellier Cedex 5, France b c

a r t i c l e

i n f o

Article history: Available online 18 September 2010 Keywords: Orientia tsutsugamushi Scrub typhus MLST Phylogeny Recombination Selective pressure Polymorphism Genetic diversity

a b s t r a c t Orientia tsutsugamushi, the causative agent of scrub typhus in South East Asia and Pacific, is an obligate intracellular bacterium closely related to the Rickettsia. The pathogen is transmitted to humans through the bites of infected larvae of trombiculid mites of the genus Leptotrombidium in which is maintained trough vertical transmission mechanism. The infection in rodents has been described in over 20 species. Scrub typhus is commonly confused with other tropical fevers and late diagnosis and treatment can lead to severe organ failures and a strain-dependent mortality rate of up to 50%. A MLST scheme associating seven core function genes: adk, lepB, lipA, lipB, secY, sodB and sucA was developed and validated on seven Cambodian strains detected in patients and two complete reference genomes from Korea and Japan. Sequence data were analyzed both with respect to sequence type (ST) diversity and DNA polymorphism. Differing trends were revealed. DNA polymorphism and phylogeny of individual gene loci indicated a significant level of recombination and genetic diversity. However, the ST distribution is clearly clonal and the clinical situation can be summarized by the formula: one patient, one strain, one ST. This contradiction is only apparent and is most likely the consequence of the unique life cycle of O. tsutsugamushi. The quasi exclusive vertical transmission mode in mites generates repeated bottlenecks and small-size populations and strongly limits genetic diversity. O. tsutsugamushi has developed specific mechanisms for generating genetic diversity which include recombination, duplication and conjugation. Recombination and other mechanisms for increasing genetic diversity are likely to occur in rodents which can act as maintenance hosts, although occurrence in mites cannot be excluded. Consequences for the epidemiology of scrub typhus are discussed. Ó 2010 Elsevier B.V. All rights reserved.

1. Introduction Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular bacterium closely related to the Rickettsia (a-proteobacteria). The pathogen is transmitted to humans through the bites of infected larvae of trombiculid mites, or chiggers, of the genus. Leptotrombidium in which it is vertically maintained (Watt and Parola, 2003). Scrub typhus is a major cause of febrile illness in South-East Asia and the Pacific (Kelly et al., 2009; Suttinont et al., 2006) but the real burden of the disease is probably underes⇑ Corresponding author. Tel.: +855 12 80 29 82; fax: +33 4 67 59 37 98. ⇑⇑Corresponding author. E-mail addresses: [email protected] (R. Frutos), [email protected] (P. Buchy). 1 Co-first authors. 1567-1348/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.meegid.2010.08.015

timated as scrub typhus can easily be mistaken for other infections. Late diagnosis and treatment can lead to severe organ failure and a mortality rate of up to 50% (Chattopadhyay and Richards, 2007). An estimated one million cases occur annually and this figure is increasing; one billion people are considered as at risk for scrub typhus (Watt and Parola, 2003). Patients are mostly rural individuals in contact with recently modified habitats, i.e. farmers and people living in newly established urban zones. The development of the ecotourism industry in many South-East Asian countries may lead to an increase in the incidence of the disease. The severity of the disease depends on geographic location (Kelly et al., 2009), strain (Chattopadhyay and Richards, 2007), bacterial load (Sonthayanon et al., 2009) and other undetermined causes such as host response to infection and immuno-genetic background. This diversity in severity might be related to the high genetic diversity recorded but also to the high plasticity of the

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genome. Cho et al. (2007) and Nakayama et al. (2008) showed that O. tsutsugamushi is characterized by an unusually high amount of repeats, transposons and conjugative elements. These unique features are likely to play a major role in host-driven selection, the capacity for adaptation to a novel environment (i.e. the host) and the capacity for evading host-defences (Cho et al., 2007; Nakayama et al., 2008). O. tsutsugamushi thus displays a high rate of genetic and genomic plasticity which could make it capable of adapting quickly to novel hosts or increasing in virulence. O. tsutsugamushi is characterized by an extremely high number of duplicated gene sequences which account for 46.7% of the whole 2.1 megabase genome. Furthermore, it bears more than 400 transposases, 60 phage integrases, 70 reverse transcriptases, 10 types of transposable elements, and 7 types of short repeats of unknown origin (Cho et al., 2007; Nakayama et al., 2008). Although the O. tsutsugamushi genome is the largest chromosome of all Rickettsiales, the core genome is more reduced than that of the other Rickettsiales, suggesting a higher dependency on host cells. This high genomic plasticity displayed by O. tsutsugamushi may explain the difficulties encountered with diagnosis and phylogenetic analysis. The current lack of accessible, inexpensive and specific diagnostic techniques for scrub typhus and also of a vaccine, often leads to late treatment and increased mortality. The use of the 56-kDa type-specific antigen gene has allowed for a better understanding of the structure of the populations of O. tsutsugamushi in Thailand (Blacksell et al., 2008). The 56-kDa antigen is exposed at the surface of the outer membrane and is involved in the stimulation of neutralizing antibodies (Stover et al., 1990; Seong et al., 1997, 2000). Although powerful, this typing system is a single locus approach targeting what is likely a positive selection pressure, which might not reflect the true diversity of O. tsutsugamushi. A very recent report described an efficient MLST marker set for deciphering the diversity of O. tsutsugamushi and underscored the major role of recombination in this diversity (Sonthayanon et al., 2010). We report on the analysis of clinical samples of O. tsutsugamushi isolated from hospitalized patients originating from different regions in Cambodia. These isolates were subjected to a multilocus sequence typing (MLST) associated to DNA polymorphism analysis. We report here that the clinical samples display a highly clonal organization whereas DNA polymorphism is significant with a low level of linkage disequilibrium and occurrence of intragenomic recombination. Finally, we report another validated MLST scheme for O. tsutsugamushi. 2. Materials and methods 2.1. Sample collection O. tsutsugamushi isolates used in this study were detected from febrile patients suspected for scrub typhus disease and admitted at Calmette Hospital, Phnom Penh, Cambodia in 2008. Whole blood was collected in EDTA tubes and biopsies of eschars, when present, were sampled for diagnostic purposes. Blood was processed to obtain peripheral blood mononuclear cells (PBMC). Results of the diagnostic PCR (Paris et al., 2009) on PBMC and skin biopsies were sent to the physician in a timely manner to allow optimal clinical management. Remaining PBMCs and tissues samples were stored at 80 °C prior to MLST studies and the database was anonymized. The project was approved by the Cambodia Ethics Committee for Health Research and the Institutional Review Board of the Institut Pasteur, Paris, France. 2.2. Reference strains Two full-length reference strains currently available were used in the phylogenetic analysis alongside in the Cambodian isolates.

These strains are the O. tsutsugamushi strains Boryong (NC 009488) isolated in Korea (Cho et al., 2007) and O. tsutsugamushi strain Ikeda (NC 010793) from Japan (Nakayama et al., 2008). 2.3. Detection of O. tsutsugamushi Genomic DNA was extracted from PBMC and/or eschars using QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. A SYBGreen real time PCR using targeting the groEL gene was adapted from Paris et al. (2009) and performed on extracted DNA for the accurate identification of O. tsutsugamushi. PCR reactions were performed on a LightCycler 480 II (Roche, Rotkreuz, Switzerland). The melting curve of the amplicon (160 bp) was ranging between 83 and 84 °C. Amplicons were directly sequenced (Macrogen, Korea) to confirm the identification. 2.4. Analysis of the 56-kDa gene The 56-kDa gene was amplified as described by Blacksell et al. (2008) and sequences were aligned with Muscle and Clustal W in SeaView 4.2.5 (Gouy et al., 2010). Neighbor-Joining distribution trees were designed with 100 bootstrap replicates in SeaView 4.2.5. The reference sequences for the genotypes Karp (AY956315), Kato (M63382) and Gilliam (DQ485289) were used for comparison. 2.5. Target loci Selection of MLST loci, i.e. adk, lepB, lipA, lipB, secY, sodB and sucA (Supplementary Table 1) was based both on comparative genomic analysis of full-length O. tsutsugamushi genomes (Cho et al., 2007; Nakayama et al., 2008) and reports of orthologous genes used for MLST analysis on other pathogens (Adakal et al., 2009, 2010; Allsopp et al., 2003; Allsopp and Allsopp, 2007; Harbottle et al., 2006; Inokuma et al., 2001; McCombie et al., 2006; Ahmed et al., 2006). Orthologous genes from Rickettsia typhi strain Wilmington (McLeod et al., 2004), accession number NC 006142, were used for outgroup rooting. 2.6. Primers and PCR reactions Primers were designed using Primer 3 (Rozen and Skaletsky, 2000). Primers sequences are given in Supplementary Table 1. The bacterial titer in blood samples from infected patients was high enough to allow for direct-amplification without nested PCR. PCR amplifications were performed in 25 ll GoTaq Flexi reaction buffer (Promega) (MgCl2 at 1.7 lM final with 0.167 mM dATP, 0.167 mM dGTP 0.167 mM dCTP and 0.167 mM dTTP, 0.167 lM of each of the corresponding forward and reverse primers, 2.5U Taq polymerase (Promega). The PCR program consisted of an initial denaturation step for 2 min at 94 °C followed by 15 cycles of 1 min denaturation at 94 °C, 1 min annealing at the relevant temperature according to each gene (Supplementary Table 1) and 2 min elongation at 72 °C followed by a final extension step of 7 min at 72 °C. 25 cycles were performed for the second round under same conditions. The PCR products were sequenced by Macrogen Company, South Korea. 2.7. Sequence alignment and phylogenetic trees Consensus sequences were obtained using Nucmer (Kurtz et al., 2004) and Contig Aligner (http://nbc11.biologie.uni-kl.de/framed/ left/menu/auto/right/contig_aligner/) and multiple sequence alignments were conducted with ClustalX2.0.3 (Thompson et al., 1997). Whenever relevant, gap regions were eliminated to perform

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alignments for phylogenetic purpose. Verification of reading frames was performed with Expasy Translate tool (http:// www.expasy.ch/tools/dna.html). Maximum Likelihood trees were built from combined data from three independent runs with 75% majority rule consensus for inference relatedness (program was run with 50,000 burn-in followed by 50,000 subsequent iterations). Similarity and difference matrices were constructed from ClustalX2 alignments using BioEdit 7.0.9.0 (Hall, 1999). For individual genes phylogenies, alignments were conducted with ClustalX2.0.3. Phylogenetic analyses were performed using maximum likelihood (ML) method for each locus separately. JModelTest (Posada, 2008) was used to select the optimal evolution model by evaluating the selected parameters using the Akaike Information Criterion (AIC). This approach suggested the model HKY for adk, lipA, lipB, sucB and sucA; HKY + G for lepB and GTR + G for secY and sodB. Under the selected models, the parameters were optimized and ML analyses were performed with phyML (version 2.4.4) (Guindon and Gascuel, 2003). Nodes robustness was assessed with 500 bootstrap replicates. 2.8. MLST analyses Identical DNA sequences at a given locus between strains were arbitrarily coded with the same allele number (i.e. each allele or haplotype was given a unique identifier), following MLST conventions (Urwin and Maiden, 2003). Each unique allelic profile was assigned a Sequence Type or ST (a unique series of allele, or haplotype, identifiers). Allele sequences have been deposited in Genbank. 2.9. Genetic and DNA polymorphism analyses Genetic similarity and difference matrices were constructed from ClustalX2 alignments (Thompson et al., 1997) using BioEdit 7.0.9.0 (Hall, 1999). DNA sequence polymorphism and all subsequent tests were investigated using several functions from the DnaSP 5.00.02 package (Librado and Rozas, 2009). Haplotypes (alleles) were calculated according to Nei (1987). Nucleotide diversity, Pi (p), the average number of nucleotide differences per site between two sequences was calculated according to Nei (1987), using the Jukes and Cantor (1969) correction. Theta (Watterson’s mutation parameter) was calculated for the whole sequence from S (Watterson, 1975). Eta (g) is the total number of mutations, and S is the number of segregating (polymorphic) sites. Ka (the number of non-synonymous substitutions per non-synonymous site), and Ks (the number of synonymous substitutions per synonymous site) for any pair of sequences were calculated according to Nei and Gojobori (1986). Tajima’s D test (Tajima, 1989) was used for testing the hypothesis that all mutations are selectively neutral (Kimura, 1983). Other tests of neutrality are Fu and Li’s tests D* and F* (Fu and Li, 1993) and Fu’s Fs statistic (Fu, 1997). ZnS statistics (Kelly, 1997) is the average of R2 (Hill and Robertson, 1968) over all pairwise comparisons. It reflects the excess of linkage disequilibrium compared with that expected under neutrality. Wall’s B and Q statistics (Wall, 1999) were also considered. Significant pairwise associations were assessed by Fisher exact test and Bonferroni procedure. 3. Results 3.1. Identification of haplotypes (alleles) and sequence types (ST) The strains described in this work were identified as O. tsutsugamushi by real time PCR (Paris et al., 2009) confirmed by amplicon sequencing (data not shown). O. tsutsugamushi strain 005 was

Table 1 Summary of STs for the selected O. tsutsugamushi strains. Strains

MLST loci adk

lepB

lipA

lipB

secY

sodB

sucA

Strain 005 1 1 1 1 1 1 1 Strain 009 2 2 2 2 2 2 2 Strain 072 3 3 3 3 2 3 3 Strain 210 4 4 4 4 3 4 4 Strain 257 5 5 5 5 2 5 5 Strain 358 6 6 2 6 2 2 4 Strain New 7 7 2 7 3 6 4 Strain Otb 8 8 6 8 4 7 6 Strain Oti 9 9 7 9 5 8 7 N haplotypes 9 9 7 9 5 8 7 S 15 29 21 10 28 13 22 Polymorphism (%) 3.61 6.81 4.32 2.70 7.19 2.70 4.99 Size (bp) 415 426 486 371 389 481 441 NH: number of haplotypes; S: polymorphic sites.

selected as the reference strain for ST determination. As a consequence, strain 005 carried the haplotype (allele) 1 for all the loci considered. STs and loci polymorphism are presented in Table 1. Haplotype sequences were deposited in GenBank. The overall number of haplotypes identified among the nine strains analyzed were 9 for adk, 9 for lepB, 7 for lipA, 9 for lipB, 5 for secY, 8 for sodB and 7 for sucA (Table 1). Polymorphic sites or S (i.e. number of mutated sites per locus) varied from 10 to 28 (Table 1). When weighting these data considering the size of the amplified fragment, the resulting percentage of site polymorphism varied from 2.70% to 7.19% (Table 1). Interestingly, secY, the locus displaying the higher level of polymorphism (7.19%), is also the locus with the lowest number of haplotypes (5). With the exception of strains 358 and New, each strain displays a unique sequence type (ST), with each locus showing a different haplotype (Table 1). When considering the sequence identity matrix and the sequence difference matrix (Supplementary Table 2), most of the polymorphism is borne by the two reference strains, O. tsutsugamushi Boryong (Otb) from Korea and O. tsutsugamushi Ikeda (Oti) from Japan. The Cambodian strains were more homogeneous in terms of polymorphism (Supplementary Table 2). 3.2. Tree topology of concatenated sequences The tree topology obtained when considering all concatenated sequences using orthologous sequences from R. typhi as an outgroup or using midpoint rooting were similar. As a consequence only midpoint rooting is shown in Fig. 1. The two reference strains branched apart directly at the root from all the other strains. Only the strain New clearly differentiated itself from the other Cambodian strains by branching separately with a bootstrap value of 100. All the other strains made a weak structure characterized by weak bootstraps (Fig. 1). 3.3. Phylogeny of individual MLST loci A similar phylogenetic analysis was conducted on each locus considered separately. For each locus, trees were constructed with both midpoint rooting and outgroup rooting using the relevant orthologous genes from R. typhi. As before with concatenated sequences, identical results were obtained for each locus with both midpoint and outgroup rooting. Therefore, only midpoint rooting distribution is shown in Fig. 2. Each locus yielded a different tree topology (Fig. 2). Interestingly, lipA and lipB, two functionally associated genes coding for the lipoyl syntase and lipoyl transferase, respectively, yielded trees with different topologies. Two pairs of loci at contiguous locations on the genome and thus physically

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Fig. 1. Maximum-likelihood tree of the concatenated MLST loci sequences. ML tree was drawn using a midpoint rooting. Bootstrap values were calculated for nodes.

linked, i.e. secY-adk and sodB-sucA also provided trees with different topology. adk haplotypes were distributed into two blocks associated with strong bootstraps (Fig. 2a) whereas secY haplotypes were individualized with high bootstrap values (Fig. 2e). Similarly, the physically linked sodB and sucA loci displayed differing tree topologies (Fig. 2f and g). With respect to adk, lepB, secY, sodB and sucA, the two reference strains (i.e. O. tsutsugamushi Ideka and O. tsutsugamushi Boryong) both reference strains branch separately. However, when considering lipA and lipB these two reference strains are differentially associated with the other strains (Fig. 2c and d). 3.4. Strain distribution based on the 56-kDa gene This distribution is displayed in Fig. 3. The clinical samples were compared to the Boryong and the Ikeda strain, and to the reference strains for the genotypes Karp, Kato and Gilliam. The Cambodian clinical samples were distributed into three different groups. S05 and New made a distinct group with the Kato type strain whereas S09, S210 and S358 were associated with the Karp strain and Otb. S210 and S358 displayed the same 56-kDa gene sequence. S072, S257 and Oti were grouped together in a separate subcluster, not associated with any of the reference genotype.

nificant non-random pairs was brought down to zero for all loci (Table 2).

3.6. DNA polymorphism The occurrence of recombination and low level of linkage disequilibrium were expected to translate into DNA polymorphism. A DNA polymorphism analysis was therefore conducted using the DnaSp 5.00.02 package. Polymorphism was found to be relatively high with h (theta) values ranging from 3.679 to 11.038 (Table 2). Polymorphism data differs significantly from that observed in Ehrlichia ruminantium, another Rickettsiale, using a set of loci sharing five out of seven members with the current set of loci, i.e. lipA, lipB, secY, sodB and sucA (Adakal et al., 2010). Two populations of E. ruminantium were characterized by Adakal et al. (2010): population 1 in genomic stasis and population 2 in expansion following clonal emergence. h ranged from 0.322 to 5.478 and from 0.265 to 2.913 for population 1 and population 2, respectively. Values observed for h in O. tsutsugamushi were much higher. g and S were also higher in O. tsutsugamushi than in both populations of E. ruminantium (Adakal et al., 2010).

3.5. Recombination and linkage disequilibrium

3.7. Selective pressure

Recombination events were detected using the four gametes tests on three loci, i.e. lepB, lipB, secY and sodB (Table 2) suggesting that such events have indeed occurred. In addition, linkage disequilibrium appeared to be weak with low values for Kelly’s ZnS statistics and Wall’s B and Q statistics (Table 2). Furthermore, although a rather high number of pairs were considered for pairwise comparisons, from 45 to 378 depending on the locus, only a few associations, i.e. from 0 to 13, were considered significantly non-random when using an exact Fisher test (Table 2). When using a Bonferroni procedure on the exact Fisher test, the number of sig-

The presence of a relatively high level of DNA polymorphism, haplotype diversity and recombination conflicts somewhat with the observed clonal organization of the strains as shown by the ST distribution (Table 3). One can thus expect the presence of selective pressure to explain this apparent discrepancy and selection pressure analysis was therefore conducted. Both Tajima’s D test and Fu and Li’s D* and F* were not significant and failed to reject the null hypothesis of neutrality (Table 3). However, Fu’s Fs test yielded strongly negative values usually associated with populations in expansion or genetic hitchhiking (Table 3).

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Fig. 2. Maximum-likelihood trees of the individual MLST loci sequences. a: adk; b: lepB; c: lipA; d: lipB; e: secY; f: sodB; g: sucA. All trees are midpoint rooted. Midpoint and outgroup rooting using Rickettsia typhi orthologous sequences yielded the exact same trees. Bootstrap values were calculated for nodes. R. typhi accession number is NC 006142.

3.8. Distribution of strains and clinical picture All strains have been isolated from hospitalized patients. However, all patients originated from and were infected in South-Eastern provinces (Supplementary Fig. 1). The patient infected by strain S09 came from Kratie, whereas the patient from whom the ST reference strain S05 was isolated from was from the southern province of Prey Veng but sojourned in Kratie prior to experiencing symptoms. Strains New and S358 were detected in patients living in Kandal province (Southern Cambodia), whereas the last three patients from whom strains 072, 210

and 257 were isolated from came from the Central-Eastern province of Kampong Cham. With the exception of the patient infected by strain S05, for whom doubt exists regarding the place of infection, all other patients were most likely infected in their province of origin. No correlation could be found between the origin of the patients and the genotype of the strains. Even patients living in the same province were infected by strains displaying totally unique ST. The only two strains, displaying some common haplotypes with other strains (i.e. New and S358) were isolated from patients coming from the same province (Prey Veng).

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Fig. 3. Neighbor-Joining distribution tree of the 56-kDa protein gene. The Neighbor-Joining distribution tree was designed with 100 bootstrap replicates in SeaView 4.2.5. Accession numbers for the reference sequences for the genotypes Karp, Kato and Gilliam are AY956315, M63382 and DQ485289, respectively.

Table 2 Assessment of recombination events and linkage disequilibrium for the selected O. tsutsugamushi strains.

adk lepB lipA lipB secY sodB sucA

Hp

Pa

S

g

g(s)

h

p

Rm

ZnS

B

Q

PW

F

BP

9 9 7 9 5 8 7

4 11 3 4 8 9 2

15 29 21 10 13 28 21

15 30 22 10 14 28 21

11 17 18 6 5 19 19

5519 11,038 8095 3679 5151 10,302 8094

0.01084 0.02263 0.01097 0.00854 0.01011 0.02128 0.01184

0 5 0 1 1 3 0

0.1330 0.1533 0.5997 0.1385 0.4117 0.1483 0.3397

0.0000 0.1111 0.5789 0.1111 0.4545 0.0741 0.4286

0.0000 0.1786 0.6000 0.2000 0.5833 0.1429 0.5455

105 378 190 45 66 378 231

3** 2* 0 0 13*** 4* 1*

0 0 0 0 0 0 0

HP: haplotypes; Pa: parsimony informative sites; S: polymorphic sites; g: total number of mutations; g(s): number of singletons; h: Watterson’s mutation parameter (calculated from Eta); p: nucleotide diversity; Rm: minimal recombination events; ZnS: ZnS statistic; B: Wall’s B statistics; Q: Wall’s Q statistics; PW: number of pairwise comparisons; v2: number of significant associations tested by chi-square test; F: number of significant associations tested by a Fisher exact test. BP: number of significant associations tested by a Fisher exact test after Bonferroni procedure. * Significant at 0.01 < P < 0.05. ** Significant at 0.001 < P < 0.01. *** Mixed test results (i.e. some sites are significant at 0.01 < P < 0.05 and others are significant at 0.001 < P < 0.01).

Table 3 DNA polymorphism and neutrality tests of the selected MLST target loci from O. tsutsugamushi. Hp adk lepB lipA lipB secY sodB sucA

9 9 7 9 5 8 7

S 15 29 21 10 13 28 21

g 15 30 22 10 14 28 21

g(s) 11 17 18 6 5 19 19

Pa 4 11 3 4 8 9 2

h 5.519 11.038 8.095 3.679 5.151 10.302 8.094

p 0.01084 0.02263 0.01097 0.00854 0.01011 0.02128 0.01184

Na 7 6 7 7 5 14 10

Ns 7 24 15 3 9 13 11

ka

ks

0.00579 0.00400 0.00466 0.00711 0.00490 0.01409 0.00645

0.02621 0.08999 0.03096 0.01328 0.02624 0.04853 0.02801

ka/ks 0.218 0.042 0.148 0.533 0.184 0.284 0.227

Tajima’s D 0.8909NS 0.6357NS 1.6864NS 0.6486NS 0.2702NS 0.9827NS 1.75432*

D*

F* NS

1.1838 0.7202NS 1.7127NS 0.6625NS 0.0539NS 1.0211NS 1.8859NS

Fu’s Fs NS

1.2460 0.7850NS 1.9126NS 0.7365NS 0.1187NS 1.1349NS 2.0781NS

5.264 2.904 1.080 6.683 1.140 1.454 1.129

NH: number of haplotypes; PA: parsimony informative sites; S: polymorphic sites; g(s): number of singletons; g: total number of mutations; h: Watterson’s mutation parameter (per sequence calculated from Eta); p: nucleotide diversity; Na: number of non-synonymous substitutions; Ns: number of synonymous substitutions; ka: rate of non-synonymous substitutions; ks: rate of synonymous substitutions; NS: not significant. * Significant at P < 0.05.

4. Discussion The main objective in this work was to analyze an apparent contradiction in the population structure of O. tsutsugamushi in order to identify ways of efficiently addressing the molecular epidemiology of scrub typhus. The components of this apparent

discrepancy are a clonal organization and mode of transmission on one hand and DNA polymorphism and high genomic plasticity on the other hand. The first trait to emphasize is the high clonality of the human clinical samples analyzed during this study. They are all unique, even with patients coming from the same region. The ST of each

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patient is unique, generally with a specific allele (haplotype) for each locus, for each strain. This could be summarized by the formula: one patient/one strain/one ST, a shortcut in good agreement with the currently recognized mode of transmission. O. tsutsugamushi is transmitted to humans following the bite of infected trombiculid mites, also known as chigger mites. It also circulates in wild reservoirs, such as rodents, through the bites of infected chigger mites. The transmission in chiggers is vertical (transovarial transmission) and only the larval stage is infective (Coleman et al., 2003). A controversy still remains with respect to the role of rodents, and perhaps other small mammals, in the circulation, transmission and maintenance of scrub typhus in the wild. Trombiculid larvae were reported to only feed upon the host on which they attach (Traub et al., 1975), thus limiting the opportunities for transmission. Trombiculid mites might thus be regarded as the true hosts, with small mammals being maintenance hosts (Coleman et al., 2003) and human accidental dead-end hosts. Owing to the life cycle of trombiculid mites, no host-to-host movement of attached larvae occurs and only newly hatched infected larvae can infect humans and small mammals, thus transmitting the strain of O. tsutsugamushi they received from their mother. However, horizontal transmission has been demonstrated (Traub et al., 1975) as well as effective transmission to mites co-feeding on infected rodents (Frances et al., 2000) and infection of naive mites through feeding on infected rodents (Takahashi et al., 1990). Co-infection in humans was reported recently in 25% of Thai patients analyzed (Sonthayanon et al., 2010). This was not observed in this work but it might simply be a consequence of the small sample size, although differences between the situation Thailand and Cambodia with respect to scrub typhus cannot be excluded. Several species of chigger mites can transmit scrub typhus (Coleman et al., 2003) with perhaps differential preferences in respect to host, habitat or behaviour. This could further complicate the patch and clonal structure of the population and thus the genotype distribution in clinical cases. This very specific mode of transmission is expected to generate clonal populations and O. tsutsugamushi could be regarded as patches or islands of genotypes determined by the filiation of the chigger mites and the movement of rodent populations. It is therefore expected to find different strains in each patient, i.e. clones. An additional parameter could increase the clonal effect as the human susceptibility to the infection (e.g., previous immunization against a strain, genetic susceptibility, etc.). However, the high genetic diversity and genomic plasticity of O. tsutsugamushi, is apparently conflicting with this conclusion. Indeed, the data reported here indicates the presence of a significant level of DNA polymorphism and a low level of linkage disequilibrium. This is expected from a global open population but not from clonal, vertically transmitted populations of an intracellular pathogen. In this case, the paradigm of genomic stasis which characterizes obligate intracellular bacteria is expected to apply. Indeed, following genome size reduction and massive loss of genes when adapting to the intracellular lifestyle (Moran and Mira, 2001; Moran and Plague, 2004; Sallstrom and Andersson, 2005), the genome of cell parasites remain highly stable over long periods of time (Tamas et al., 2002; Klasson and Andersson, 2004). Such genomic stasis was described on E. ruminantium, another Rickettsiale (Adakal et al., 2009, 2010). Polymorphism is supposed to be eroded over time with only positively selected mutations that bring a selective advantage being maintained, with the possible exception of deleterious mutations due to Muller’s ratchet. However, the data reported here suggests a situation somewhat different, where genetic diversity is more important and not limited to mutations but also involves rearrangements. Five loci out of the seven analyzed in this work were studied in a similar way for E. ruminantium, the causative agent of heartwater in ruminants (Adakal et al., 2009, 2010).

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DNA polymorphism at these loci in O. tsutsugamushi is significantly higher than in E. ruminantium, although the latter, in which genetic exchange and recombination have been described, is neither clonally organized nor vertically transmitted (Allsopp and Allsopp, 2007; Adakal et al., 2009, 2010). Furthermore, no linkage disequilibrium could be shown for the O. tsutsugamushi strains studied. This could be due to the small sample size, however owing to the high clonality observed, linkage disequilibrium should have been detectable even with these few samples. Furthermore, recombination events have been detected using a four gametes test even with the small sample size. A larger sample size would be necessary to strengthen the statistical analysis, however the phylogenetic analysis of the target loci and more specifically those physically linked, i.e. adk-secY and sodB-sucA, brought essential information for determining the occurrence of recombination in O. tsutsugamushi. The divergence observed in the topology of the phylogenetic trees and the differential evolutionary models fitting these loci are not in agreement either with a strict vertical transmission route or a limited genetic variability based on accumulation of substitutions in a context of positive selection. Positive selective pressure was not detected in the way expected. Instead Fu’s Fs data was indicative of a population in expansion or genetic hitchhiking. This is in agreement with the occurrence of recombination and the diverging phylogeny of target loci and must also be considered in the light of genomic data. O. tsutsugamushi is an exceptional bacterium displaying a set of unique genomic features, the first one being a very high genomic plasticity. Almost 50% of the genome is made of repetitive sequences derived from integrative and conjugative elements and the repeat density is 200 times higher than that of Rickettsia prowazekii (Cho et al., 2007; Nakayama et al., 2008). Among other features are more than 400 transposases, 60 phage integrases, 70 reverse transcriptases, 10 types of transposable elements, and 7 types of short repeats of unknown origins (Cho et al., 2007; Nakayama et al., 2008). Perhaps the most striking feature is the massive duplication of components of conjugative type IV secretion system. A total of 359 tra genes were identified in 79 different locations throughout the genome (Cho et al., 2007). Diversity is most likely linked for a large part to the proliferation of conjugative transfer system to which intragenomic recombination is associated (Cho et al., 2007). Furthermore, comparative genomic analysis of the two full-length genomes currently available has shown extensive genome shuffling (Nakayama et al., 2008). All together these genomic data and the results reported in this work indicate that recombination, duplication and rearrangement are major drives for the genomic diversity and complexity of O. tsutsugamushi. Using a set clinical sample strains from Thailand, Sonthayanon et al. (2010) also demonstrated very recently the extensive occurrence of recombination in O. tsutsugamushi. Horizontal transfer is also a marked trait since a large number of foreign genes have been described in the genome of O. tsutsugamushi (Nakayama et al., 2008). Since O. tsutsugamushi is an obligate intracellular parasite, recombination requires a coinfection which could potentially occur either in the vector or in the mammalian host. Recombination could be horizontal, for instance involving multiple infection of the same mammalian host by larvae from different lineages or multiple mite infection by feeding or co-feeding on a host bearing different strains. These mechanisms are not exclusive and can both occur. Owing to the vertical transmission of O. tsutsugamushi, coinfection might also involve strains transmitted to rodents by different generations of mites adding thus more potential for diversity and plasticity. There is at this stage a remaining apparent contradiction that must be investigated. Chigger mites, which feed on a single host throughout the larval stage, were reported to be unable to transmit acquired O. tsutsugamushi strains to the

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offspring through the transovarial route (Takahashi et al., 1994). However, since recombination was found in strains isolated from human patients, both in this work and in Sonthayanon et al. (2010), the recombined strains must have been taken at one stage by a chigger mite and transmitted to the off-spring which further infected the human patients. Vertical transmission of acquired strains must thus exist. However, at this stage one can only speculate on the mechanisms involved. Several hypotheses can be considered to explain this contradiction. Takahashi et al. (1994) reported a lack of transovarial transmission on Leptotrombidium pallidum. However, the efficiency of vertical transmission seems to be species or population dependent. Different species of naturally infected chigger mites were shown to vertically transmit O. tsutsugamushi with differing efficacy. Leptotrombidium chiangraiensis was shown to vertically transmit the pathogen 100% over two generations whereas Leptotrombidium imphalum displayed a transmission efficiency of only 62.3% after two generations (Phasomkusolsil et al., 2009). Frances et al. (2001) reported that two naturally infected populations of the same species, Leptotrombidium deliense, displayed totally different capacity for vertical transmission. Mites originating from adult V3M were capable to transmit O. tsutsugamushi at 100% over two generations and at 86.6% at the third generation whereas mites originating from adult V3F transmitted at 100% at F1 and 0% at F2 (Frances et al., 2001). Other works on the three same species, i.e. L. deliense, L. chiangraiensis and L. imphalum, showed transmission rates ranging from 7% to 80% depending on the colonies (Lerdthusnee et al., 2002). A populationdependent competence for vertical transmission might explain the contradiction if the work reported by Takahashi et al. (1994) was conducted on a population poorly efficient for vertical transmission. Furthermore, transmission of O. tsutsugamushi to mites other than Leptotrombidium, i.e. Blankaartia acuscutellaris, was shown when cofeeding with L. deliense (Frances et al., 2000). The transmission by genera other than Leptotrombidium might thus be another possible explanation. On another hand, since O. tsutsugamushi acts as a symbiont of the chigger mites, the presence of an incompatibility mechanism as shown in the related Wolbachia symbionts (Engelstäder and Telshow, 2009) might also explain the population driven differential competence for vertical transmission. Finally, one cannot exclude the occurrence of recombination within the mite between the newly acquired strain and the resident strain which is normally transmitted. The remaining question is whether the strong clonality observed in strains isolated from human patients is truly in contradiction with the occurrence of recombination and the high diversity and plasticity of O. tsutsugamushi. Cho et al. (2007) have developed the complex genome hypothesis in which they suggest that the proliferation of conjugative secretion systems and associated genes provide a basis for further positive or diversifying selection. The data reported in this work fully support and complement this hypothesis. O. tsutsugamushi is unique both by its vertical transmission route and by its very high genomic plasticity and massive duplication of conjugative elements. O. tsutsugamushi displays two opposing evolutionary processes (Fuxelius et al., 2007; Darby et al., 2007). The first process is gene losses due to adaptation to intracellular parasitism and O. tsutsugamushi displays the smallest set of metabolic core function genes among the Rickettsiales and the highest level of dependency to the host cell. On the other hand, it displays the largest and most repeated genome among the same Rickettsiales (Fuxelius et al., 2007). These unique and sometimes apparently conflicting genomic features should be regarded as an adaptation to the very unique mode of transmission and life cycle of O. tsutsugamushi. Mammals perhaps not only play a role in the maintenance of scrub typhus (Coleman et al., 2003) but may also be ‘‘diversity-boosting’’ hosts.

As vertical transmission is its primary route of transmission in the vector, O. tsutsugamushi has little opportunity for genetic exchange. However, this vertical transmission is not absolute and co-infections occur but probably remain relatively rare events. There might thus be a very high selective pressure for conjugative systems, recombination and duplication to optimize and take advantage of any co-infection event to generate an increased genetic diversity by insertion and recombination allowing for an increased genetic basis for further selection (Cho et al., 2007). In the chigger mite, O. tsutsugamushi is exposed to repeated bottlenecks because of vertical transmission and small population size, to which must be added purifying selection driven by rodent host. The trombiculid ‘‘vector’’ is most likely the true host, to which O. tsutsugamushi has adapted, perhaps as symbiont rather than a pathogen as suggested by the vertical mode of transmission. This could explain the massive loss of metabolic genes. However, the repeated bottlenecks and small population sizes consecutive to this predominant vertical transmission mode requires an alternative process for the creation of diversity. This alternative process involving recombination, rearrangements, duplications and conjugations might have been selected to occur in the maintenance of host, i.e. small mammals. Owing to the significant rate of plasticity and polymorphism observed, the combinations of MLST alleles they bear in patients are only driven by chance and unlikely to be repeated. They correspond to unique individual stochastic events. The expected outcome of these rare events is an apparent clonal distribution which does not reflect the true diversity and plasticity of O. tsutsugamushi. This work not only provides an explanation for apparently contradictory features in O. tsutsugamushi (Cho et al., 2007; Fuxelius et al., 2007), but also raises the question of the epidemiological survey of scrub typhus. Human clinical isolates are most likely to be clones selected on an unknown basis from already highly monophyletic small populations, and therefore cannot provide a good base for molecular epidemiology analysis and understanding of the dynamic of scrub typhus. An efficient genotyping approach was developed with the 56 kDa protein gene (Blacksell et al., 2008). However this approach is monolocus-based and therefore cannot bring an insight on the relative dynamics of the target genes. Owing to the high genetic plasticity and unique features and mechanisms displayed of O. tsutsugamushi, monolocus markers are not adapted for typing and phylogenetic studies. Furthermore, phylogeny and typing themselves might not be relevant if addressed as with other open-population bacteria. A separate MLST analysis, based on different target loci, was very recently published (Sonthayanon et al., 2010) and underscored also the potential of multilocus typing for analysing the diversity and discriminating strains of O. tsutsugamushi. This work indicates that the epidemiology of scrub typhus must be addressed at a larger scale with multilocus markers by comparing strains circulating in rodents and more importantly in chigger mites. It also provides another validated MLST scheme and a set of tools for tackling the population analysis of this re-emerging and expanding disease.

Acknowledgments This study was supported by a grant from the Institut Pasteur, Paris (Actions Concertées Inter-Pasteuriennes). RF was supported by the Franco-Thai Hubert Curien project PHC 20624VK and by the ANR project CERoPath. A very similar and excellent work conducted in parallel was published by Sonthayanon et al. (2010) during the review process of this manuscript. Reference to this work was thus included in the revised version of the manuscript.

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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.meegid.2010.08.015.

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