J Infect Chemother 20 (2014) 656e659
Contents lists available at ScienceDirect
Journal of Infection and Chemotherapy journal homepage: http://www.elsevier.com/locate/jic
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Multilocus VNTR analysis-ompA typing of venereal isolates of Chlamydia trachomatis in Japan Masaaki Satoh, Motohiko Ogawa, Masayuki Saijo, Shuji Ando* Department of Virology 1, National Institute of Infectious Diseases, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 May 2014 Received in revised form 13 June 2014 Accepted 23 June 2014 Available online 25 July 2014
In this study, we investigated the prevalence of genital Chlamydia trachomatis isolated in Japan using a high-resolution genotyping method, the multilocus VNTR analysis (MLVA)-ompA typing method. Seventeen serotypes of C. trachomatis standard strain (A-L3) and 44 clinical isolates were obtained from clinical settings. Genotyping of the ompA gene allowed clinical isolates to be divided into nine serotypes: B (6.8%), D (15.9%), E (25%), F (20.5%), G (18.1%), H (6.8%), Ia (2.3%), J (2.3%), and K (2.3%). These isolates were further divided into 28 types after combining ompA genotyping data with MLVA data (Huntere Gaston discriminatory index D, 0.949). Thus, our results demonstrated that MLVA could identify clinical isolates that could not be distinguished by ompA typing. © 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Keywords: Chlamydia trachomatis VNTR MLVA-ompA Clinical isolates
Chlamydia trachomatis is an obligate intracellular pathogen infecting eukaryotic host cells and is one of the major causes of sexually transmitted infectious diseases worldwide. Eighteen serotypes of C. trachomatis have been identified and can be divided, according to pathogenicity, into three biovars: AeC and Ba (trachoma); DeK, Da, and Ia (venereal infection); and L1eL3 and L2a (LGV). Genotyping of C. trachomatis has been effectively used as an epidemiological tool to determine the prevalence of the pathogen. Methods used for genotyping of C. trachomatis have advanced from conventional typing using serum or monoclonal antibodies to molecular genotyping. In the polymerase chain reaction (PCR)based method, the ompA gene has become the primary target for genotyping of C. trachomatis because the nucleotide sequence of this gene reflects the serotype [1]. Furthermore, because of the difficulties in diagnosing persistent or recurrent infections using only the ompA sequence due to the high conservation of this gene, high-resolution typing methods have been developed, including multilocus-sequencing typing (MLST) and multilocus variable number of tandem repeat (VNTR) analysis (MLVA), which accompanied the publication of the whole genome of several C. trachomatis serotypes [2e4]. MLST analysis is based on several housekeeping genes and analyzes evolutional changes [2], while
* Corresponding author. Department of Virology 1, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. Tel.: þ81 3 5285 1111; fax: þ81 3 5285 1208. E-mail address: [email protected] (S. Ando).
MLVA is based on analysis of combined sequencing of the ompA gene plus three VNTR loci, as proposed by Pederson et al. [3] and further modified by Wang et al. [4]. This combined analysis method is termed MLVA-ompA typing. The advantages of MLVA include a higher capacity of discrimination among microbial strains and the potential for additional applications, such as tracing bacterial isolates [5]. However, these advantages remain controversial because of complicated patterns of gene expression and variations in gene sequences obtained by the typing methods in these organisms. In this study, we used MLVA-ompA analysis to evaluate the diversity among isolates from venereal samples collected from clinical settings in the 1980s and stored in our laboratory. Elementary bodies of standard strains of C. trachomatis (kindly provided by Dr. Kuo and Dr. Wang of Washington University and propagated previously; see Table 1) were used for a pilot study. Forty-four clinical isolates were obtained from 35 patients with sexually transmitted infections in Tokyo (25 men and 10 women) and from gynecological samples of pregnant women in the Saitama prefecture (N ¼ 2) and the Nagano prefecture (N ¼ 7). These isolates were passaged using HeLa-229 cells and stored in our laboratory. DNA extraction was performed from standard strains and clinical isolates using a Qiagen DNA mini kit (Qiagen, Venlo, the Netherlands) according to the manufacturer's instructions. PCR was used to amplify regions of the ompA gene and three VNTR loci (CT1335, CT1299, and CT1291, as defined in previous reports [3,4]) of C. trachomatis using previously described methods [4]. Cycle sequence reaction was performed using a BigDye Terminator (version 3.1) Cycle Sequencing Kit (Life Technologies, Carlsbad, CA,
http://dx.doi.org/10.1016/j.jiac.2014.06.010 1341-321X/© 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
M. Satoh et al. / J Infect Chemother 20 (2014) 656e659
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Table 1 MLVA-ompA typing of standard strains. Serotype
A B Ba C D Da E F G I Ia J K L1 L2 L2a L3
Strain name
G-17/OT TW-5/OT AP-2/OT TW-3/OT UW-3/CX TW-448/OT UW-5/CX UW-6/CX UW-57/CX UW-12/Ur UW-202/Br UW-36/CX UW-31/CX 440/Bubo 434/Bubo UW-396/Nr 404/Bubo
ompA gene similarity
MLVA code
Length (bp)
Assigned in this study
Identical/total (reference strain: GenBank accession number)
1028 1022 1022 1031 1019 1019 1019 1025 1025 1028 1031 1031 1031 1019 1022 1022 1031
AB915579 AB915580 AB915581 AB915582 AB915583 AB915584 AB915585 AB915586 AB915587 AB915588 AB915589 AB915590 AB915591 AB915592 AB915593 AB915594 AB915595
1027/1028 (A/HAR13: CP000051) 1022/1022 (B/HAR36: DQ064297) 1021/1022 (Ba/Apache-2: DQ064282) 1031/1031 (C/TW3/OT: AF352789) 1019/1019 (D/EC: CP002054) 1017/1019 (Da/TW-448: X62921) 1019/1019 (E/Bour: HE601870) 1024/1025 (F/IC-CAL3: DQ064287) 1024/1025 (G/UW-57: DQ064299) 1028/1028 (I/UW-12: DQ064290) 1031/1031 (Ia/SotonIa 1: HE601808) 1030/1031 (J/UW-36: DQ064292) 1030/1031 (K/UW-31: DQ064293) 1019/1019 (L1/440/LN: HE601950) 1022/1022 (L2/434/Bu: CP003963) N. R. 1031/1031 (L3/404/LN: HE601955)
3. 5. 3 3. 5. 3 N. I.a N. I.a 3. 6a. 4 8. 7. 2 8. 5. 1 3. 5. 2 12. 4a. 5 14b. 4a. 3 13. 5. 9b 3. 4a. 5 3. 6a 4 5. 9. 3bc 5. 9 .3bc 1. 9. 8bc 5. 9. 8bc
Bold font : 100% identical. Sequences of serotype H were not determined because of insufficient materials. Underline: the same MLVA pattern as the reference strain. N. R. : Not Registered. a N. I. : Not identified due to unreadable chromatogram of PCR product. b New variant code. c Modified variant code according to Wang's rule (2011).
Fig. 1. Phylogenetic trees showing the relationships among clinical isolates, standard strains, and reference sequences using the ompA gene. This tree was constructed by the neighbor-joining method using MEGA 6 software. Red letters (B, DeH, Ia, J, and K) indicate identified serotypes of clinical isolates in this study, and parenthetical percentages indicate the ratio of each serotype for all isolates. Clinical isolates are indicated as “No” or ”S”, and standard strains are indicated as “-niid”. Previously reported sequences were used as references because they were identical or showed high similarity with the sequences of clinical isolates, followed by those of the strains B-AB695151, D-AB695166, D-AB695172, K-AB695161, and Ia-AB695145, which were isolated in Sapporo, North Japan, and the strains DeICeCAL8, D-DB-185, E-Bour, F-SW4, G9301, G11222, G-UW57, H-VR-879, H-UW-4, and J-UW36. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Table 2 MLVA-ompA typing of 44 clinical isolates. Serotype (number of sample)
B (3) D (7)
E (11)
ompA gene similarity Strain name
Genbank accession number
(Number of samples: Identical/total bp)
B1 B2 D1 D2 D3 D4 D5 E1 E2
e e e e D/ICeCAL8 D/B-185 D/B-185 E/Bour E/Bour F/ICeCAL3
AB695151 a AB695151 a AB695172 a AB695166 a DQ064285 X62919 X62919 HE601870 HE601870 DQ064287
(1: 1016/1022) (2: 1017/1022) (1: 1019/1019) (3: 1019/1019) (1: 1019/1019) (1: 1019/1019) (1: 1018/1019) (10: 1019/1019) (1: 1018/1019) (9: 1025/1025)
G1 G2 G3 H1 H2
G/9301 G/UW-57 G/11222 H/VR-879 H/UW-4 e J/UW-36 e
CP001930 DQ064299 CP001888 JX564246 AF304857 AB695145 a DQ064292 AB695161 a
(1: 1025/1025) (2: 1025/1025) (5: 1025/1025) (2: 1031/1031) (1: 1030/1031) (1: 1031/1031) (1: 1031/1031) (1: 1031/1031)
F (9) G (8)
H (3) Ia (1) J (1) K (1)
MLVA code (number of samples)
Group
3. 4. 4 (1) 3. 4a. 2 (2) 3. 4a. 3 (1) 3. 4a. 3 (1) 3. 4a. 4 (1) 3. 4. 4 (1) 6. 5. 1 (1) 13. 4a. 4 (1) 3. 4a. 5 (1) 8. 5. 1 (9) 8. 6. 1 (1) 8. 6. 1 (1) 8. 5. 1 (2) 8. 5. 2 (1) 8. 6. 2 (1) 8. 3. 1 (1) 8. 4. 1 (3) nb. 5. 1 (1) 3. 4a. 2 (1) 3. 4a. 2 (1) 3. 4a. 4 (1) 12. 4a. 2 (4) 12. 4a. 4 (1) 3. 1. 3 (2) 3. 4a. 4 (1) 3. 6a. 3 (1) 3. 4. 3 (1) 3. 4a. 2 (1)
Bold font: 100% identical to reference strain at ompA gene sequence. Group was defined based on the analysis of ompA sequence. Underline: previously reported MLVA code. a Isolate derived from Sapporo, North Japan (Yamazaki et al. BMC Infect Dis. 2012). b ‘n’ indicates no PCR fragment.
USA). Subsequently, the samples were analyzed using an ABI 3130 genetic analyzer (Life Technologies). In addition to comparison of the obtained nucleotide sequences of ompA with the GenBank registered sequences, each sequence of three VNTR loci was assigned a variant code according to the rules outlined by Wang et al. [4]. Consequently, the genotype of each strain was defined as the combination of the ompA gene and the three variant codes. We determined the MLVA-ompA sequences of 17 standard strains and 44 clinical isolates. Of the 17 standard strains, the ompA sequences of serotypes B, C, D, E, I, Ia, L1, L2, and L3 were identical to those of reference strains registered in the GenBank. However, those of serotypes A, Ba, Da, F, G, J, and K exhibited 1-2 bp differences. The DNA sequences of the ompA gene of the 17 standard strains reported here were registered in the DDBJ and GenBank nucleotide databases (Table 1). Considering the previous report of observation of the amino acid mutation in MOMP in the same strain [6], 1e2 bp nucleotide change in ompA gene in the same strain might be frequent, or importantly, even standard strains might exhibit quasispecies. Further analysis for elucidation of the cause of this 1e2 bp change will be required in the future. The MLVAs of serotypes D, E, L1, and L2 were identical to those of each serotype reported previously (D-3-6a-4 and E-8-5-1) [4] or registered in the GenBank (L1-5-9-3b and L2c-5-9-3b), while those of the other strains were identified as having unique MLVA-ompA patterns. Interestingly, new variant codes of VNTR (bold font) were identified in serotypes I (CT1335, code 14: GAAAAAGG-6T7A-GCTTTTGT) and Ia (CT1291, code 9: AAAATGGTCT-14C-TATTG), and modified variant codes (bold, underlined font) were identified in serotypes L1 and L2 (CT1291, code 3b: AAAATAGTCTA-9C-TATTG) and L2a and L3 (CT1291, code 8b: AAAATAGTCTA-7C-TATTG) when using Wang's rules of assignment [4]. Because these results suggested that this method may be effective as a high-resolution genotyping method in C. trachomatis isolates, we applied this method to 44 clinical isolates. In our isolates, nine serotypes were identified by ompA sequence analysis (Fig. 1). Furthermore, genetic variants were observed in serotypes B, D, E, G, and H in the ompA sequence (Table 2). Several isolates, which were indistinguishable by ompA gene sequencing, were
identified by MLVA code; these included serotypes E (E1: 8-5-1 and 8-6-1), F (8-3-1, 8-4-1, 8-5-1, 8-5-2, 8-6-2, and n-5-1), G (G1: 3-10a3 and 3-4a-2, G3: 12-4a-4 and 12-4a-2), and D (D5: 3-4a-3, 3-4a-4, and 3-4-4), as shown in Table 2. Therefore, our identification of several new MLVA codes or sequences in standard strains A and B (trachoma) and L1, L2, L2a, and L3 (LGV) suggested that this method may be applicable for genetic survey of the prevalence of Chlamydia-related diseases, as previously reported [7]. Interestingly, in our clinical isolates, the discriminatory power was calculated as 0.949 using the HuntereGaston discriminatory index D; that obtained using ompA genotyping alone was 0.897. As compared with data from previous reports [8e10], these data supported that the present method may be suitable for evaluation of the diversity of C. trachomatis. Clinical isolates with serotypes E, F, G, H, and J and some with serotype D exhibited the same or similar genetic patterns as foreign reference strains (e.g., E/Bour, F/ IC-CAL3, D/IC-CAL8, G/9301, G/11222, G/UW-57, H/VR-879, and J/ UW-36) by ompA analysis. On the other hand, those with serotypes B, Ia, and K and some with serotype D exhibited the same or similar genetic patterns as isolates in Sapporo, Japan (Table 2). Furthermore, when combined with observations of several patterns of MLVA within the same serotype (e.g., serotypes E, F, D, and G, see Table 2) and those described in a previous report [11], these data suggested that populations within each serotype and the evolutional rate of each serotype may be divergent in Japanese populations. Therefore, further MLVA-ompA analysis of clinical isolates of C. trachomatis is expected to provide a strong tool for elucidating the genetic diversity and geographical and/or sequence evolution of this pathogen in isolates from Japan and throughout the globe. Conflicts of interest None. References [1] Pedersen LN, Herrmann B, Moller JK. Typing Chlamydia trachomatis: from egg yolk to nanotechnology. FEMS Immunol Med Microbiol 2009;55:120e30.
M. Satoh et al. / J Infect Chemother 20 (2014) 656e659 [2] Klint M, Fuxelius HH, Goldkuhl RR, Skarin H, Rutemark C, Andersson SG, et al. High-resolution genotyping of Chlamydia trachomatis strains by multilocus sequence analysis. J Clin Microbiol 2007;45:1410e4. [3] Pederson LN, Podenphant L, Moller J. High discriminative genotyping of Chlamydia trachomatis using omp1 and a set of variable number tandem repeats. Clin Microbiol Infect 2008;14:644e52. [4] Wang Y, Skilton RJ, Cutcliffe LT, Andrew E, Clark IN, Marsh P. Evaluation of a high resolution genotyping method for Chlamydia trachomatis using routine clinical samples. PLoS One 2011;6. http://dx.doi.org/10.1371/journal. pone.0016971. [5] Belkum AV. Tracing isolates of bacterial species by multilocus variable number of tandem repeat analysis (MLVA). FEMS Immunol Med Microbiol 2007;49: 22e7. [6] Hayes LJ, Picket MA, Conlan JW, Ferris S, Everson JS, Ward ME, et al. The major outer-memberane proteins of Chlamydia trachomatis serovars A and B: intraserovar amino acid changes do not alter specificities of serovar- and C subspecies-reactive antibody-binding domains. J Gen Microbiol 1990;136: 1559e66.
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[7] Mejuto P, Boga JA, Junquera M, Torreblanca A, Leiva PS. Genotyping Chlamydia trachomatis strains among men who have sex with men from a Northern Spain region: a cohort study. BMJ Open 2013;3. http://dx.doi.org/10.1136/ bmjopen-2012-002330. [8] Ikryannikova LN, Shkarupeta MM, Shitikov EA, Il’ina E, Govorun VM. Comparative evaluation of new typing schemes for urogenital Chlamydia trachomatis isolates. FEMS Immunol Med Microbiol 2010;59:188e96. [9] Belkum AV, Tassios PT, Dijkshoorn L, Haeggman S, Cookson B, Fry N, et al. Guidelines for the validation and application of typing methods for use in bacterial epidemiology. Clin Mibrobiol Infect 2007;13:1e46. [10] Labiran C, Clarke IN, Cutcliffe LT, Wang Y, Skilton RJ, Persson K, et al. Genotyping markers used for multi locus VNTR analysis with ompA (MLVA-ompA) and multi sequence typing (MST) retain stability in Chlamydia trachomatis. Front Cell Infect Microbiol 2012;2. http://dx.doi.org/10.3389/fcimb.2012.00068. [11] Yamazaki T, Matsumoto M, Matsuo J, Abe K, Minami K, Yamaguchi H. Frequency of Chlamydia trachomatis in Ureaplasma-positive healthy women attending their first prenatal visit in a community hospital in Sapporo, Japan. BMC Infect Dis 2012;12. http://dx.doi.org/10.1186/1471-2334-12-82.
Contents lists available at ScienceDirect
Journal of Infection and Chemotherapy journal homepage: http://www.elsevier.com/locate/jic
Note
Multilocus VNTR analysis-ompA typing of venereal isolates of Chlamydia trachomatis in Japan Masaaki Satoh, Motohiko Ogawa, Masayuki Saijo, Shuji Ando* Department of Virology 1, National Institute of Infectious Diseases, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 May 2014 Received in revised form 13 June 2014 Accepted 23 June 2014 Available online 25 July 2014
In this study, we investigated the prevalence of genital Chlamydia trachomatis isolated in Japan using a high-resolution genotyping method, the multilocus VNTR analysis (MLVA)-ompA typing method. Seventeen serotypes of C. trachomatis standard strain (A-L3) and 44 clinical isolates were obtained from clinical settings. Genotyping of the ompA gene allowed clinical isolates to be divided into nine serotypes: B (6.8%), D (15.9%), E (25%), F (20.5%), G (18.1%), H (6.8%), Ia (2.3%), J (2.3%), and K (2.3%). These isolates were further divided into 28 types after combining ompA genotyping data with MLVA data (Huntere Gaston discriminatory index D, 0.949). Thus, our results demonstrated that MLVA could identify clinical isolates that could not be distinguished by ompA typing. © 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Keywords: Chlamydia trachomatis VNTR MLVA-ompA Clinical isolates
Chlamydia trachomatis is an obligate intracellular pathogen infecting eukaryotic host cells and is one of the major causes of sexually transmitted infectious diseases worldwide. Eighteen serotypes of C. trachomatis have been identified and can be divided, according to pathogenicity, into three biovars: AeC and Ba (trachoma); DeK, Da, and Ia (venereal infection); and L1eL3 and L2a (LGV). Genotyping of C. trachomatis has been effectively used as an epidemiological tool to determine the prevalence of the pathogen. Methods used for genotyping of C. trachomatis have advanced from conventional typing using serum or monoclonal antibodies to molecular genotyping. In the polymerase chain reaction (PCR)based method, the ompA gene has become the primary target for genotyping of C. trachomatis because the nucleotide sequence of this gene reflects the serotype [1]. Furthermore, because of the difficulties in diagnosing persistent or recurrent infections using only the ompA sequence due to the high conservation of this gene, high-resolution typing methods have been developed, including multilocus-sequencing typing (MLST) and multilocus variable number of tandem repeat (VNTR) analysis (MLVA), which accompanied the publication of the whole genome of several C. trachomatis serotypes [2e4]. MLST analysis is based on several housekeeping genes and analyzes evolutional changes [2], while
* Corresponding author. Department of Virology 1, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. Tel.: þ81 3 5285 1111; fax: þ81 3 5285 1208. E-mail address: [email protected] (S. Ando).
MLVA is based on analysis of combined sequencing of the ompA gene plus three VNTR loci, as proposed by Pederson et al. [3] and further modified by Wang et al. [4]. This combined analysis method is termed MLVA-ompA typing. The advantages of MLVA include a higher capacity of discrimination among microbial strains and the potential for additional applications, such as tracing bacterial isolates [5]. However, these advantages remain controversial because of complicated patterns of gene expression and variations in gene sequences obtained by the typing methods in these organisms. In this study, we used MLVA-ompA analysis to evaluate the diversity among isolates from venereal samples collected from clinical settings in the 1980s and stored in our laboratory. Elementary bodies of standard strains of C. trachomatis (kindly provided by Dr. Kuo and Dr. Wang of Washington University and propagated previously; see Table 1) were used for a pilot study. Forty-four clinical isolates were obtained from 35 patients with sexually transmitted infections in Tokyo (25 men and 10 women) and from gynecological samples of pregnant women in the Saitama prefecture (N ¼ 2) and the Nagano prefecture (N ¼ 7). These isolates were passaged using HeLa-229 cells and stored in our laboratory. DNA extraction was performed from standard strains and clinical isolates using a Qiagen DNA mini kit (Qiagen, Venlo, the Netherlands) according to the manufacturer's instructions. PCR was used to amplify regions of the ompA gene and three VNTR loci (CT1335, CT1299, and CT1291, as defined in previous reports [3,4]) of C. trachomatis using previously described methods [4]. Cycle sequence reaction was performed using a BigDye Terminator (version 3.1) Cycle Sequencing Kit (Life Technologies, Carlsbad, CA,
http://dx.doi.org/10.1016/j.jiac.2014.06.010 1341-321X/© 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
M. Satoh et al. / J Infect Chemother 20 (2014) 656e659
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Table 1 MLVA-ompA typing of standard strains. Serotype
A B Ba C D Da E F G I Ia J K L1 L2 L2a L3
Strain name
G-17/OT TW-5/OT AP-2/OT TW-3/OT UW-3/CX TW-448/OT UW-5/CX UW-6/CX UW-57/CX UW-12/Ur UW-202/Br UW-36/CX UW-31/CX 440/Bubo 434/Bubo UW-396/Nr 404/Bubo
ompA gene similarity
MLVA code
Length (bp)
Assigned in this study
Identical/total (reference strain: GenBank accession number)
1028 1022 1022 1031 1019 1019 1019 1025 1025 1028 1031 1031 1031 1019 1022 1022 1031
AB915579 AB915580 AB915581 AB915582 AB915583 AB915584 AB915585 AB915586 AB915587 AB915588 AB915589 AB915590 AB915591 AB915592 AB915593 AB915594 AB915595
1027/1028 (A/HAR13: CP000051) 1022/1022 (B/HAR36: DQ064297) 1021/1022 (Ba/Apache-2: DQ064282) 1031/1031 (C/TW3/OT: AF352789) 1019/1019 (D/EC: CP002054) 1017/1019 (Da/TW-448: X62921) 1019/1019 (E/Bour: HE601870) 1024/1025 (F/IC-CAL3: DQ064287) 1024/1025 (G/UW-57: DQ064299) 1028/1028 (I/UW-12: DQ064290) 1031/1031 (Ia/SotonIa 1: HE601808) 1030/1031 (J/UW-36: DQ064292) 1030/1031 (K/UW-31: DQ064293) 1019/1019 (L1/440/LN: HE601950) 1022/1022 (L2/434/Bu: CP003963) N. R. 1031/1031 (L3/404/LN: HE601955)
3. 5. 3 3. 5. 3 N. I.a N. I.a 3. 6a. 4 8. 7. 2 8. 5. 1 3. 5. 2 12. 4a. 5 14b. 4a. 3 13. 5. 9b 3. 4a. 5 3. 6a 4 5. 9. 3bc 5. 9 .3bc 1. 9. 8bc 5. 9. 8bc
Bold font : 100% identical. Sequences of serotype H were not determined because of insufficient materials. Underline: the same MLVA pattern as the reference strain. N. R. : Not Registered. a N. I. : Not identified due to unreadable chromatogram of PCR product. b New variant code. c Modified variant code according to Wang's rule (2011).
Fig. 1. Phylogenetic trees showing the relationships among clinical isolates, standard strains, and reference sequences using the ompA gene. This tree was constructed by the neighbor-joining method using MEGA 6 software. Red letters (B, DeH, Ia, J, and K) indicate identified serotypes of clinical isolates in this study, and parenthetical percentages indicate the ratio of each serotype for all isolates. Clinical isolates are indicated as “No” or ”S”, and standard strains are indicated as “-niid”. Previously reported sequences were used as references because they were identical or showed high similarity with the sequences of clinical isolates, followed by those of the strains B-AB695151, D-AB695166, D-AB695172, K-AB695161, and Ia-AB695145, which were isolated in Sapporo, North Japan, and the strains DeICeCAL8, D-DB-185, E-Bour, F-SW4, G9301, G11222, G-UW57, H-VR-879, H-UW-4, and J-UW36. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Table 2 MLVA-ompA typing of 44 clinical isolates. Serotype (number of sample)
B (3) D (7)
E (11)
ompA gene similarity Strain name
Genbank accession number
(Number of samples: Identical/total bp)
B1 B2 D1 D2 D3 D4 D5 E1 E2
e e e e D/ICeCAL8 D/B-185 D/B-185 E/Bour E/Bour F/ICeCAL3
AB695151 a AB695151 a AB695172 a AB695166 a DQ064285 X62919 X62919 HE601870 HE601870 DQ064287
(1: 1016/1022) (2: 1017/1022) (1: 1019/1019) (3: 1019/1019) (1: 1019/1019) (1: 1019/1019) (1: 1018/1019) (10: 1019/1019) (1: 1018/1019) (9: 1025/1025)
G1 G2 G3 H1 H2
G/9301 G/UW-57 G/11222 H/VR-879 H/UW-4 e J/UW-36 e
CP001930 DQ064299 CP001888 JX564246 AF304857 AB695145 a DQ064292 AB695161 a
(1: 1025/1025) (2: 1025/1025) (5: 1025/1025) (2: 1031/1031) (1: 1030/1031) (1: 1031/1031) (1: 1031/1031) (1: 1031/1031)
F (9) G (8)
H (3) Ia (1) J (1) K (1)
MLVA code (number of samples)
Group
3. 4. 4 (1) 3. 4a. 2 (2) 3. 4a. 3 (1) 3. 4a. 3 (1) 3. 4a. 4 (1) 3. 4. 4 (1) 6. 5. 1 (1) 13. 4a. 4 (1) 3. 4a. 5 (1) 8. 5. 1 (9) 8. 6. 1 (1) 8. 6. 1 (1) 8. 5. 1 (2) 8. 5. 2 (1) 8. 6. 2 (1) 8. 3. 1 (1) 8. 4. 1 (3) nb. 5. 1 (1) 3. 4a. 2 (1) 3. 4a. 2 (1) 3. 4a. 4 (1) 12. 4a. 2 (4) 12. 4a. 4 (1) 3. 1. 3 (2) 3. 4a. 4 (1) 3. 6a. 3 (1) 3. 4. 3 (1) 3. 4a. 2 (1)
Bold font: 100% identical to reference strain at ompA gene sequence. Group was defined based on the analysis of ompA sequence. Underline: previously reported MLVA code. a Isolate derived from Sapporo, North Japan (Yamazaki et al. BMC Infect Dis. 2012). b ‘n’ indicates no PCR fragment.
USA). Subsequently, the samples were analyzed using an ABI 3130 genetic analyzer (Life Technologies). In addition to comparison of the obtained nucleotide sequences of ompA with the GenBank registered sequences, each sequence of three VNTR loci was assigned a variant code according to the rules outlined by Wang et al. [4]. Consequently, the genotype of each strain was defined as the combination of the ompA gene and the three variant codes. We determined the MLVA-ompA sequences of 17 standard strains and 44 clinical isolates. Of the 17 standard strains, the ompA sequences of serotypes B, C, D, E, I, Ia, L1, L2, and L3 were identical to those of reference strains registered in the GenBank. However, those of serotypes A, Ba, Da, F, G, J, and K exhibited 1-2 bp differences. The DNA sequences of the ompA gene of the 17 standard strains reported here were registered in the DDBJ and GenBank nucleotide databases (Table 1). Considering the previous report of observation of the amino acid mutation in MOMP in the same strain [6], 1e2 bp nucleotide change in ompA gene in the same strain might be frequent, or importantly, even standard strains might exhibit quasispecies. Further analysis for elucidation of the cause of this 1e2 bp change will be required in the future. The MLVAs of serotypes D, E, L1, and L2 were identical to those of each serotype reported previously (D-3-6a-4 and E-8-5-1) [4] or registered in the GenBank (L1-5-9-3b and L2c-5-9-3b), while those of the other strains were identified as having unique MLVA-ompA patterns. Interestingly, new variant codes of VNTR (bold font) were identified in serotypes I (CT1335, code 14: GAAAAAGG-6T7A-GCTTTTGT) and Ia (CT1291, code 9: AAAATGGTCT-14C-TATTG), and modified variant codes (bold, underlined font) were identified in serotypes L1 and L2 (CT1291, code 3b: AAAATAGTCTA-9C-TATTG) and L2a and L3 (CT1291, code 8b: AAAATAGTCTA-7C-TATTG) when using Wang's rules of assignment [4]. Because these results suggested that this method may be effective as a high-resolution genotyping method in C. trachomatis isolates, we applied this method to 44 clinical isolates. In our isolates, nine serotypes were identified by ompA sequence analysis (Fig. 1). Furthermore, genetic variants were observed in serotypes B, D, E, G, and H in the ompA sequence (Table 2). Several isolates, which were indistinguishable by ompA gene sequencing, were
identified by MLVA code; these included serotypes E (E1: 8-5-1 and 8-6-1), F (8-3-1, 8-4-1, 8-5-1, 8-5-2, 8-6-2, and n-5-1), G (G1: 3-10a3 and 3-4a-2, G3: 12-4a-4 and 12-4a-2), and D (D5: 3-4a-3, 3-4a-4, and 3-4-4), as shown in Table 2. Therefore, our identification of several new MLVA codes or sequences in standard strains A and B (trachoma) and L1, L2, L2a, and L3 (LGV) suggested that this method may be applicable for genetic survey of the prevalence of Chlamydia-related diseases, as previously reported [7]. Interestingly, in our clinical isolates, the discriminatory power was calculated as 0.949 using the HuntereGaston discriminatory index D; that obtained using ompA genotyping alone was 0.897. As compared with data from previous reports [8e10], these data supported that the present method may be suitable for evaluation of the diversity of C. trachomatis. Clinical isolates with serotypes E, F, G, H, and J and some with serotype D exhibited the same or similar genetic patterns as foreign reference strains (e.g., E/Bour, F/ IC-CAL3, D/IC-CAL8, G/9301, G/11222, G/UW-57, H/VR-879, and J/ UW-36) by ompA analysis. On the other hand, those with serotypes B, Ia, and K and some with serotype D exhibited the same or similar genetic patterns as isolates in Sapporo, Japan (Table 2). Furthermore, when combined with observations of several patterns of MLVA within the same serotype (e.g., serotypes E, F, D, and G, see Table 2) and those described in a previous report [11], these data suggested that populations within each serotype and the evolutional rate of each serotype may be divergent in Japanese populations. Therefore, further MLVA-ompA analysis of clinical isolates of C. trachomatis is expected to provide a strong tool for elucidating the genetic diversity and geographical and/or sequence evolution of this pathogen in isolates from Japan and throughout the globe. Conflicts of interest None. References [1] Pedersen LN, Herrmann B, Moller JK. Typing Chlamydia trachomatis: from egg yolk to nanotechnology. FEMS Immunol Med Microbiol 2009;55:120e30.
M. Satoh et al. / J Infect Chemother 20 (2014) 656e659 [2] Klint M, Fuxelius HH, Goldkuhl RR, Skarin H, Rutemark C, Andersson SG, et al. High-resolution genotyping of Chlamydia trachomatis strains by multilocus sequence analysis. J Clin Microbiol 2007;45:1410e4. [3] Pederson LN, Podenphant L, Moller J. High discriminative genotyping of Chlamydia trachomatis using omp1 and a set of variable number tandem repeats. Clin Microbiol Infect 2008;14:644e52. [4] Wang Y, Skilton RJ, Cutcliffe LT, Andrew E, Clark IN, Marsh P. Evaluation of a high resolution genotyping method for Chlamydia trachomatis using routine clinical samples. PLoS One 2011;6. http://dx.doi.org/10.1371/journal. pone.0016971. [5] Belkum AV. Tracing isolates of bacterial species by multilocus variable number of tandem repeat analysis (MLVA). FEMS Immunol Med Microbiol 2007;49: 22e7. [6] Hayes LJ, Picket MA, Conlan JW, Ferris S, Everson JS, Ward ME, et al. The major outer-memberane proteins of Chlamydia trachomatis serovars A and B: intraserovar amino acid changes do not alter specificities of serovar- and C subspecies-reactive antibody-binding domains. J Gen Microbiol 1990;136: 1559e66.
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[7] Mejuto P, Boga JA, Junquera M, Torreblanca A, Leiva PS. Genotyping Chlamydia trachomatis strains among men who have sex with men from a Northern Spain region: a cohort study. BMJ Open 2013;3. http://dx.doi.org/10.1136/ bmjopen-2012-002330. [8] Ikryannikova LN, Shkarupeta MM, Shitikov EA, Il’ina E, Govorun VM. Comparative evaluation of new typing schemes for urogenital Chlamydia trachomatis isolates. FEMS Immunol Med Microbiol 2010;59:188e96. [9] Belkum AV, Tassios PT, Dijkshoorn L, Haeggman S, Cookson B, Fry N, et al. Guidelines for the validation and application of typing methods for use in bacterial epidemiology. Clin Mibrobiol Infect 2007;13:1e46. [10] Labiran C, Clarke IN, Cutcliffe LT, Wang Y, Skilton RJ, Persson K, et al. Genotyping markers used for multi locus VNTR analysis with ompA (MLVA-ompA) and multi sequence typing (MST) retain stability in Chlamydia trachomatis. Front Cell Infect Microbiol 2012;2. http://dx.doi.org/10.3389/fcimb.2012.00068. [11] Yamazaki T, Matsumoto M, Matsuo J, Abe K, Minami K, Yamaguchi H. Frequency of Chlamydia trachomatis in Ureaplasma-positive healthy women attending their first prenatal visit in a community hospital in Sapporo, Japan. BMC Infect Dis 2012;12. http://dx.doi.org/10.1186/1471-2334-12-82.
