Single Nucleotide Polymorphism Typing of Global Salmonella enterica

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Single Nucleotide Polymorphism Typing of Global Salmonella enterica

     JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2010, p. 35043509 Vol. 48, No. 10 0095-1137/10/$12.00 doi:10.1128/JCM.00709-10 Copyright ? 2010, American Society for Microbiology. All Rights Reserved.

    Single Nucleotide Polymorphism Typing of Global Salmonella enterica

    Serovar Typhi Isolates by Use of a Hairpin Primer

    Real-Time PCR Assay

    Sophie Octavia and Ruiting Lan*

    School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia

    Received 6 April 2010/Returned for modication 19 June 2010/Accepted 20 July 2010

    Salmonella enterica serovar Typhi is highly homogeneous. Single nucleotide polymorphisms (SNPs) have

    been shown to be valuable markers for molecular typing of S. enterica serovar Typhi. Here, we used a hairpin

    primer real-time PCR assay for SNP typing of S. enterica serovar Typhi isolates. Forty-two SNPs were selected from a comparison of 19 published S. enterica serovar Typhi genomes and sequences from other studies. The

    SNPs were used to type 71 global S. enterica serovar Typhi isolates and differentiated these S. enterica serovar

    Typhi isolates and the 19 genome sequenced strains into 25 SNP pro;les. Phylogenetic analysis revealed that these SNP pro;les were grouped into six major clusters. These clusters can be identi;ed by using ;ve SNPs,

    while the full differentiation of the 25 SNP pro;les requires a minimum of 24 SNPs. This real-time PCR-based

    SNP typing method will be useful for global epidemiological analysis.

     Salmonella enterica serovar Typhi is highly homogeneous netic bias which revealed the full path of the last common ancestors connecting strains CT18 and Ty2 but only the node (10, 17, 18). The lack of genetic diversity is a major challenge locations for the other SNP proles (14). to the development of suitable typing methods to differentiate Advances in technology, such as high-throughput sequenc- S. enterica serovar Typhi isolates for both phylogenetic and ing, allow SNPs to be discovered to obtain a full resolution of epidemiological purposes. Single nucleotide polymorphisms the phylogenetic relationships of isolates. A recent study by (SNPs) are considered the most valuable markers, particularly Holt et al. (8) utilized 454 and/or Solexa technologies to se- for studying the evolutionary relationships of isolates of homo- geneous pathogenic clones, such as Bacillus anthracis (16), quence 19 S. enterica serovar Typhi isolates selected from the Mycobacterium tuberculosis (4), and Yersinia pestis (1). ve major clusters found by Roumagnac et al. (17). There were SNPs have been used as markers for molecular typing of S. more than 1,700 SNPs found, and these gave a fully resolved enterica serovar Typhi in a large study by Roumagnac et al. phylogenetic tree of these isolates. These SNPs are invaluable (17). A total of 88 SNPs, found from analysis of 200 gene resources for investigation of the evolutionary history of global fragments from 105 diverse S. enterica serovar Typhi isolates, S. enterica serovar Typhi isolates. This study aimed to select a could differentiate 481 global S. enterica serovar Typhi isolates better set of SNPs on the basis of the genome tree and the

    into 85 haplotypes (SNP proles) and ve major clusters (17). previous SNP studies by Roumagnac et al. (17) to differentiate

    However, despite the large number of SNPs used, each of the and establish the phylogenetic relationships of global S. en-

     ve clusters was supported by only a single SNP and there was terica serovar Typhi isolates, using real-time (R-T) PCR assays

     little resolution of the relationships of the haplotypes within a based on hairpin (HP) primers (6).

     cluster. Eighty of the SNPs have also been used to differentiate 140 Indonesian S. enterica serovar Typhi isolates into nine MATERIALS AND METHODS haplotypes (2). Bacterial isolates. The 71 global S. enterica serovar Typhi isolates used were We have also shown that genome-wide SNPs are useful for the same set of isolates from a previous study of Octavia and Lan (14). Genome sequenced strains CT18 and Ty2 were also used as controls. The SNP data for the molecular typing and determining the relationships of global S. remaining 17 genome sequenced strains were obtained from GenBank and were enterica serovar Typhi isolates (14). Thirty-seven SNPs selected included in the analysis. from a comparison of the genomes of S. enterica serovar Typhi SNP selection and primer design. A total of 42 SNPs were selected from a strains CT18 (15) and Ty2 (3) were typed using restriction comparison of 19 S. enterica serovar Typhi genomes (11) and are listed in Table enzyme digestion to differentiate 73 global S. enterica serovar S1 in the supplemental material. Locus tags were used as SNP names, as there was only one SNP per gene selected. The SNP location in the gene is shown in Typhi isolates into 23 SNP proles and four distinct genetic Table S1 in the supplemental material. The primers for HP real-time PCR assay groups. As the SNPs were selected by comparison of only two were designed to produce small amplicons (about 100 bp) and to optimally S. enterica serovar Typhi genomes, this resulted in a phyloge- anneal at 60?C (see Table S1 in the supplemental material). Hairpin primers were designed on the basis of the principles described by Hazbon and Alland (6). A secondary mismatch was introduced in some of the primers (noted in Table S1 in the supplemental material) to further decrease the afnity of the mismatched * Corresponding author. Mailing address: School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, primers, therefore permitting a larger threshold cycle (C) value difference TNSW 2052, Australia. Phone: 61-2 9385 2095. Fax: 61-2 9385 1591. between the matched and mismatched HP primers. E-mail: HP R-T PCR assay. Each PCR mixture contained 10 ng of chromosomal Supplemental material for this article may be found at http://jcm DNA, 3 l of SYBR green (Quantace), 0.5 l each of 10 M forward and reverse primers (see Table S1 in the supplemental material), and Milli-Q water to a nal Published ahead of print on 28 July 2010. volume of 10 l. All reactions were performed in a Rotor-gene 6000 sequence



    detector system (Corbett Life Science, Australia). Thermal cycling conditions Reliability of HP R-T PCR for SNP typing. We used two sets were as follows: stage 1, 95?C for 10 min and then hold; stage 2, 10 cycles of of SNPs to test the reliability of HP R-T PCR SNP typing. We 72?C for 30 s, 95?C for 15 s, and 69?C for 30 s, with the temperature being rst typed the four cluster-dividing SNPs (STY0778, STY1397, lowered 1?C in the last step for every cycle; and stage 3, 72?C for 30 s, 95?C STY1583, and STY4105) from Octavia and Lan (14) in the 73 for 20 s, and 60?C for 30 s, repeated 40 times. Data for analysis were collected isolates, including the 71 global S. enterica serovar Typhi iso- at the last step of stage 3. Bioinformatic analyses. The PAUP program (19) was used to construct a lates and 2 genome sequenced strains, CT18 and Ty2, which maximum-parsimony tree from the SNP data and calculate the homoplasy index. have previously been typed using restriction enzyme digestion. SNPT software (4) was used to determine the minimum number of SNPs re- The typing results for these SNPs were consistent with previous quired for typing. values were 5.36, results. The average differences in the CT 12.53, 8.22, and 5.41 for STY0778, STY1397, STY1583, and STY4105, respectively. RESULTS AND DISCUSSION We then tested the four cluster-dividing SNPs (STY2513, Selection of SNPs. A total of 42 SNPs were selected for STY2629, STY3196, and STY3622) from Roumagnac et al. typing on the basis of the data from genome sequencing of 19 (17). Twenty-nine of the 73 isolates have previously been in- S. enterica serovar Typhi isolates (8) and the SNP typing results dependently typed for these four SNPs by Roumagnac et al. of Octavia and Lan (14) and Roumagnac et al. (17). We pre- (17). All except three nucleotides were consistent with previ- viously mapped the SNPs from these three studies onto the ous data. The three discrepancies observed were in the assign- genome tree of the 19 strains whose genomes were sequenced ment of nucleotides for two of the four SNPs. The isolate of (11), and SNP selection was based on this mapping. SNPs ST60 was typed as H50 by Roumagnac et al. (17), SNP showing reverse/parallel changes were avoided. We rst se- STY2629 should be a G nucleotide for this isolate but was lected SNPs that have been used in previous studies to divide typed as nucleotide A in the present study. Similarly, isolates S. enterica serovar Typhi isolates into clusters, including four R1962 and T189 were both typed as H42 by Roumagnac et al. SNPs (STY0778, STY1397, STY1583, and STY4105) by us (17) due to allele T in SNP STY3196, but both had nucleotide (14) and four (STY2513, STY2629, STY3196, and STY3622) C in the present study. To determine whether our HP R-T by Roumagnac et al. (17). We then selected SNPs to cover PCR results were correct, we sequenced the SNPs from these branches with at least one SNP for branches with 40 or more isolates, which conrmed our HP R-T PCR results. SNPs. The SNPs typed by Roumagnac et al. (17) were selected For the remaining 34 SNPs selected, we had only strains as the rst preference. In total, 15 and 22 SNPs were selected CT18 and Ty2 as controls. The HP R-T PCR typing results for from the internal nodes and terminal branches of the genome these two strains matched the genome data, which further tree, respectively (see Fig. S1 in the supplemental material). conrmed the reliability of the HP R-T PCR for SNP typing. Finally, we used the haplotype frequency data from Roumag- We typed the remaining SNPs for the 71 isolates using the HP nac et al. (17) to select SNPs so that common haplotypes can R-T PCR assay. The average Cvalue difference for the SNPs Tbe separated. There were 16 most common haplotypes, with typed was 7.92 (see Table S1 in the supplemental material), each haplotype having from 3 to 156 isolates. SNPs separating and the Cvalues between the two sets of primers were dis- T11 haplotypes (haplotype 1 [H1], H8, H42, H45, H46, H50, tinctive, which correlates with the matched and mismatched H52, H55, H58, H59, and H85) have already been selected on primer pairs for each nucleotide. the basis of the criteria described above. Five additional SNPs Polymorphisms among the S. enterica serovar Typhi iso- (STY1503, STY1919, STY2389, STY3876, and STY4562) were lates. The SNP data for the 71 isolates, 2 fully sequenced selected to separate the remaining ve common haplotypes genome strains (CT18 and Ty2), and 17 partially sequenced (H39, H81, H15, H56, and H84), respectively (17). The 42 genome strains (8) were combined for comparison (Table 1). SNPs selected resolved the 19 genomes into 13 groups, and the Twenty-three of the 42 SNPs were informative. Four pairs of terminal branches between strains E03-4983 and 404ty col- SNPs, STY0539 and STY3196, STY2513 and STY4095, lapsed into a single branch. As these two branches represent STY336 and STY717, and STY144 and STY2443, gave the the distinctive isolates with the z66 agellar antigen, the col- same patterns of polymorphisms among the SNP proles. The

    lapse of divisions between these two lineages has no impact on 90 isolates were distinguished into 25 SNP proles. The same

    their identication. level of differentiation can also be achieved by typing 24 SNPs Out of the 42 SNPs, 25 were synonymous SNPs (sSNPs), 14 (indicated by asterisks in Table 1). Twelve proles were unique were nonsynonymous SNPs (nsSNPs), and 3 were nonsense to a single isolate, while 13 others were shared by more than mutations. The SNPs selected were mostly from genes of one isolate. SNP proles 6 and 25 contained more than 10 known functions. Only 4 of the 42 SNPs (STY0321, STY0336, isolates, and SNP prole 25 was the largest SNP prole, con-

    STY1583, and STY1720) are in genes that might be related to taining 19 isolates.

    virulence, and these SNPs can potentially be under selection The 29 isolates also typed by Roumagnac et al. (17) were pressure, giving rise to reverse or parallel changes in different previously differentiated into 14 haplotypes using 88 biallelic lineages. These SNPs were selected, as they were from previ- polymorphisms. In the present study, the 29 isolates were dis- ous studies and are key SNPs required for resolving the lin- tinguished into 13 SNP proles. Some haplotypes previously

    eages involved (11, 17). However, the SNP data (see below) dened by Roumagnac et al. (17) were further differentiated showed no reverse or parallel changes in these SNPs, and such using our set of 42 SNPs. H8, H50, H52, and H59 were each changes overall are rare, suggesting that reverse or parallel further divided into two or three proles. However, some other

    changes are not an issue in inferring relationships on the basis haplotypes collapsed into the same SNP prole in the present

    of the SNPs selected. study; for example, SNP prole 6 contained four different


A G STY4562 cluster .. .. .. .. .. .. .. A. G STY4188 arranged for nucleotides. ... G G G G G G G G A STY4105 were .... .... base . . T C STY3969 STY3622 proles . . T T C STY3820 and SNP consensus ...... ...... ............ ...... .. A A G IV, STY3725 the The .... .... .... C to T T T T T T STY3622 cluster SNPs. . T T T C STY3499 for the identical ...... . . . A G of STY3082

... . STY3196 A A A A G STY2629 ........... ........... locationsIII, .......... ........ ........ .......... . . . . T T nucleotides C STY2513,STY4095 the ........................ uster T . C C C C C C C C C C STY2331 for cl indicate ........ ...... ............ for T C C STY1720 Dots material . . . T C b STY1693 STY2331 SNP ... . A A A G STY1583 column. II, ................................ ................ ........... . T C STY1503 each supplemental cluster .... T G G G G G for STY1397 the ****************** for in ...... T C STY1143 S1 ................... ...... . A G nucleotide STY0962 STY4105 Table I, ... ... ... A A A G STY0961 proles See .... .... A A A G STY0778 cluster SNP predominant .......... for 1. T T * genome. C STY0717,STY0336 the ................ ........................ ........ ................ .. .. .. . . A G STY0563 CT18 STY2629 TABLE C T T T T T T T T T T T STY0539, STY3196 Typhi represent ............. . A G STY0321 data ............................ boldface), ....... serovar . . T C STY0221 in base . . . A A A G STY0144,STY2443 enterica proles. ................................................ ................................ ............ ................ ........ ***** ..... .... .... .... .... .... .... .... .... .... . . . . . S. A G STY0029 Consensus SNP the (highlighted to 3125 parentheses. 417Ty (H59) 26T51 SNPs (H58), 26T49, (H14), (H59), (H52), (H58), clusters. 26T32 E02- (H42), CC6 in individual (H50) (H8) (H76), T202 26T56, ve 425Ty,701Ty, 420Ty 26T9, according In24 ST1 are In20 804N 26T50 (H59), of into 374 26T38, (H42), (H58), 26T30, E03-4983 In15 R1962 ST60 a (17) respective 26T6, ST309, 26T40, name, 419Ty,423Ty,446Ty, (H39), 26T37, 416Ty R1637, (H58), TYT1668 ISP-04-06979 T189 al. AG3 IP.E88 (H85) ST24B, Isolate 26T17, the (H81),(H50), (H11), (H42), PL27566 (H59), et minimum (H58) Tp2 locus ferentiation (H52), (H6) (H45)(H46) (H50) 26T19, (H55) (H42) 418Ty,421Ty,445Ty, (H52) A into 3126, 353, J185 CC7, TYT1669 (H59), 25T-40, 26T24, PL73203, (H8) the (H16), (H58), ST1106, (H63) 404ty 1. dif (H8) (H1) (H39), (H10) full ISP-03-07467 TYT1677 withFig. E03-9804 26T12, 444Ty,702Ty, 414Ty IP.E88 ST1002 (H50),(H50), (H59),(H59), 3123, (H52) 2759 proles Roumagnac E02E00-1180-7866 E98-3139 E98-0664 E98-2068 CDC3434-73, E01-6750 422Mar92CDC1707CDC9032 -81- 85 CDC1196-74 CDC3137-73 25T-36, 25T-44, CDC382-82, 415Ty, in Tp1 R1167,CT18 ST145, Ty2 M223 ST24A 15098S PNG32 for to SNP tree 4 2 3 1 9 8 5 6 7 6 the 14 10 13 11 12 17 18 24 25 21 20 15 19 22 23 designated 1 no. SNP required prole are according divide parsimony SNPs to typed the base to used haplotypes cluster (minimum) SNPs be minimum TheThe ferentiating SNP can the SNP a b , according Consensus III II Dif I IV VI V, V


FIG. 1. A maximum-parsimony tree to illustrate the evolutionary relationships of the SNP proles (SP). For SNP proles containing genome

    strains, the strain names are in parentheses after the SNP prole numbers. H58 genome strains include AG3, E02-2759, ISP-04-06979, ISP-03- 07467, 804N, and E03-9804. The roman numerals correspond to the cluster number. Clusters dened by Roumagnac et al. (17) were marked RI to RV. SNPs are labeled on the branch. Five cluster-dividing SNPs are highlighted in boldface.

    haplotypes (H50, H11, H42, and H76), with the last three being prole 24 contained three isolates, including 2 of the genome

    sequenced strains, 404ty and E03-4983, while SNP prole 25 less frequent haplotypes (17). SNP prole 6 also contained

    contained 19 isolates, including 1 genome sequenced strain, multiple divergent types, determined by multilocus variable-

    J185. Two strains from SNP prole 25, including J185, were number tandem-repeat (VNTR) analysis (MLVA) (13), sug-

    gesting that there is more diversity within this SNP prole. A z66 negative (2, 8, 17) and ST1106.

    genome from this lineage should be sequenced for better phy- Evolutionary relationships of the SNP pro;les. A maxi-

    logenetic coverage of its diversity. mum-parsimony phylogenetic tree was constructed to deter- Two of our isolates, isolates 25T-44 and 26T24, were mine the relationships of the 25 SNP proles. There was only

    grouped together with six genome sequenced strains of H58. one most parsimonious tree found, and the homoplasy index According to Roumagnac et al. (17), H58 was mostly isolated was very low (0.003) due to a T nucleotide rather than a C from Southeast Asia (17), and recent H58 isolates, particularly nucleotide for SNP STY2331 in SNP prole 14 (Table 1). As

    those from Vietnam, are associated with resistance to nalidixic the prole was represented by the genome strain M223 only, acid (12). Both of our isolates were from Canada. There was the change was potentially a sequencing error and remained to no information on whether these two isolates were imported be conrmed. The observation of a low homoplasy index was cases, but multidrug-resistant isolates from travelers to South- concordant with observations from previous two SNP studies east Asia have been reported in Canada (5). by Roumagnac et al. (17) and Holt et al. (8). Eighteen isolates with the z66 agellar antigen were differ- The SNP proles could be divided into six clusters (clusters entiated into two SNP proles, SNP proles 24 and 25. SNP I to VI), with at least one SNP supporting each cluster (Fig. 1).


    Cluster I consisted of four SNP proles (proles 1 to 4) sup- lance of S. enterica serovar Typhi, and a combination of SNP ported by STY2629; cluster II contained four SNP proles typing and VNTR typing can be used for local epidemiological (proles 8, 9, 10, and 14) supported by STY4105; cluster III analysis.

    contained SNP proles 5 to 7 supported by STY2331; cluster Concluding comments. Previous studies by Roumagnac et

    IV had three SNP proles (proles 11 to 13) supported by al. (17) and Octavia and Lan (14) have shown that SNPs are STY0539, STY3196, and STY0961; and cluster V had ve SNP very useful for typing and provide better insights into the proles (proles 16 to 18, 24, and 25) supported by STY3622 evolutionary relationships of S. enterica serovar Typhi isolates. and STY1397, which also separate cluster V from cluster VI, SNP typing in these two studies was achieved using restriction which contained six SNP proles (proles 15, 19, and 20 to 23). enzyme digestion (14) or dHPLC (17). We have shown in the A minimum of ve SNPs (STY2629, STY4105, STY2331, present study that the HP R-T PCR assay is an alternative STY3196, and STY3622) is required to differentiate these six method for SNP typing and was applied to type 42 SNPs. The clusters. Note that three of the SNPs (STY2629, STY3196, and HP R-T PCR assay is not gel based, unlike the two aforemen- STY3622) were also cluster-dividing SNPs from Roumagnac et tioned methods, and the results could be obtained directly al. (17). The ve SNPs act like dichotomous keys sequentially after completion of the PCRs. Therefore, the use of this differentiating the clusters. method greatly reduced the time for SNP typing. Two recent Comparison of evolutionary relationships established using studies by Kariuki et al. (9) and Holt et al. (7) employed the different molecular markers. SNPs have also been used as GoldenGate bead array high-throughput platform (Illumina) molecular markers for S. enterica serovar Typhi in a similar to type 1,500 SNPs in 94 Kenyan and 62 Nepalese S. enterica study by Roumagnac et al. (17). A total of 88 SNPs (referred serovar Typhi isolates, respectively. However, the method is far to as biallelic polymorphisms), found upon analysis of 200 gene less cost efcient than the HP R-T PCR assay used in the fragments from 105 diverse S. enterica serovar Typhi isolates present study and is thus less suitable for routine use in labo- using denaturing high-performance liquid chromatography ratories, particularly in developing countries where typhoid (dHPLC), could differentiate 481 global S. enterica serovar fever is endemic. In addition, the 1,500 SNPs used by Kariuki Typhi isolates into 85 SNP haplotypes (17). Four SNPs et al. (9) and Holt et al. (7) were highly redundant, as only (STY2513, STY2629, STY3196, and STY3622) divided their eight and six haplotypes were found among the 94 and 62 haplotypes into ve major clusters (17). We have also included isolates studied, respectively. these four SNPs for typing in the present study. We can allo- In the present study, we selected 42 SNPs on the basis of the cate our SNP proles according to the Roumagnac et al. (17) phylogenetic distribution of the genome-wide SNPs from 19 clustering scheme, and we designated them RI to RV (Fig. 1). diverse S. enterica serovar Typhi strains (8), SNPs found by The clustering was different to some extent between the two Roumagnac et al. (17), and SNPs used by Octavia and Lan studies. Clusters I and V correspond to RI and RIII, respec- (14). We have shown that this set of SNPs can distinguish many tively. Clusters II, III, and VI were grouped together as RII, of the major SNP proles, although they may not be able to suggesting that our selected SNPs have a better resolution to fully resolve the less common SNP proles found by Roumag- further differentiate cluster RII. Cluster VI was divided into nac et al. (17). A minimum of 5 and 24 SNPs differentiated the RIV and RV. We can divide cluster VI into two subclusters isolates used in the present study into six major clusters and 25 corresponding to RIV and RV using either STY1583 or SNP proles, respectively. Nevertheless, the full set of 42 SNPs STY2513/ST4095. Therefore, despite the difference in cluster may offer a higher level of differentiation in other S. enterica division, the evolutionary relationships of the SNP proles serovar Typhi populations. With the exibility of the HP R-T were consistent, and there was no conict at the cluster level. PCR assay, large-scale typing of S. enterica serovar Typhi iso- These 73 S. enterica serovar Typhi isolates have also been lates can be done progressively using the ve cluster-dividing typed using MLVA with nine VNTRs (13). For SNP proles SNPs rst, followed by the other 19 minimum SNPs and then containing multiple isolates, all were divided further into mul- the remaining SNPs for full resolution. In the studies of Kari- tiple MLVA proles (see Fig. S2 in the supplemental mate- uki et al. (9) and Holt et al. (7), H58 (SNP prole 23 in the rial). In addition, MLVA proles with the same SNP proles present study) was subdivided into two sublineages and several were not always grouped together, as was also found in our subtypes. Division into sublineages requires one SNP and can previous study (13). The number of VNTR differences was be easily added to our scheme. smaller between the isolates within SNP proles 7 and 25, and We have recently shown that SNPs are well suited for their corresponding MLVA proles belonged to the same global and long-term epidemiological analyses, while MLVA cluster. Moreover, the SNP clusters dened in the MLVA is suitable only for short-term epidemiological anal- present study were not correlated with the clusters dened yses (13). SNP typing has also been shown to be valuable to using VNTR markers. The SNP clusters were no longer visible study the local population structure and epidemiology of S. and were distributed among different MLVA clusters. Highly enterica serovar Typhi in Indonesia, Kenya, and Nepal (2, 7, polymorphic VNTRs appeared to evolve too fast to have re- 9). The SNPs and strains typed in the present study form the tained sufcient phylogenetic information. Evolutionary rela-

    basis of a future database for international comparison. tionships based on VNTRs would best be viewed within SNP

    Further studies should include testing of an expanded col- proles (see Fig. S2 in the supplemental material). SNPs are

    lection of isolates. In conclusion, the present study provided necessary to resolve the genetic relationships of S. enterica

    a simple and cost-efcient alternative SNP typing method serovar Typhi isolates, and VNTRs should be used only to

    and a better selected set of SNPs for epidemiological studies achieve a further resolution of closely related isolates. There-

    of S. enterica serovar Typhi. fore, SNP typing will be more appropriate for global surveil-


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