Comparison of multilocus sequence typing and ...

Journal of Microbiological Methods 127 (2016) 214–218

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Comparison of multilocus sequence typing and multilocus typing microarray of Chlamydia trachomatis strains from Argentina and Chile Jenny Isaksson a,1, Lucía Gallo Vaulet b,1, Linus Christerson a, Anke Ruettger c, Konrad Sachse c, Carolina Entrocassi b, Érica Castro d, Marcelo Rodríguez Fermepin b, Björn Herrmann a,⁎ a

Section of Clinical Bacteriology, Department of Medical Sciences, Uppsala University, S-751 85 Uppsala, Sweden Universidad de Buenos Aires, Cátedra de Microbiología Clínica, Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina, Junin 956, CP1113 Ciudad Autónoma de Buenos Aires, Argentina Institute of Molecular Pathogenesis, Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), D-07743 Jena, Germany d Facultad de Medicina, Universidad de San Sebastián, Chile, Lientur 1457, Concepción, Chile b c

a r t i c l e

i n f o

Article history: Received 30 March 2016 Received in revised form 5 June 2016 Accepted 5 June 2016 Available online 07 June 2016 Keywords: Chlamydia trachomatis DNA array Genotyping MLST ompA

a b s t r a c t This study compared conventional ompA genotyping of Chlamydia trachomatis with multilocus sequence typing (MLST) and multilocus typing (MLT) DNA microarray. DNA extracts of 104 C. trachomatis positive specimens were analyzed by ompA sequencing and MLST and of these 76 by MLT array. Obtained MLST sequence types (STs) were compared to sequences in the database http:// mlstdb.uu.se. The resolution obtained for MLST (35 STs) was 2.1 higher than for ompA sequencing (17 variants) and 1.3 higher than MLT array (27 MLT groups). Among the 104 samples the predominant genotype E could be divided into 5 ompA variants and 23 STs of which 16 had not been reported in previous studies. The most common STs, ST3 and ST56, were identified as founders and are common in several countries on a global scale. The MLST and the MLT array provided similar strain discrimination capacity and showed considerably higher resolution than conventional ompA sequencing. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Chlamydia trachomatis is one of the most common sexually transmitted infections worldwide (World Health Organization, 2012). If left untreated, serious sequelae may arise, such as ectopic pregnancy and infertility in women and epididymitis in men. To understand the epidemiology of chlamydia infections and its spread in sexual networks and between populations adequate genotyping tools are necessary. For many years, C. trachomatis typing has been based on the major outer membrane protein (MOMP) and the encoding ompA gene, where 17 genotypes can be distinguished (Pedersen et al., 2009). In most countries almost half of all urogenital chlamydia infections are of serotype E, and within this serotype the ompA E/Bour genotype is predominant Abbreviations: MLST, multilocus sequence typing; MLT, multilocus typing DNA microarray; ST, sequence type. ⁎ Corresponding author. E-mail addresses: [email protected] (J. Isaksson), [email protected] (L. Gallo Vaulet), [email protected] (L. Christerson), anke.ruettger@fli.bund.de (A. Ruettger), [email protected] (K. Sachse), [email protected] (C. Entrocassi), [email protected] (É. Castro), [email protected] (M. Rodríguez Fermepin), [email protected] (B. Herrmann). 1 Both authors contributed equally to the presented work.

http://dx.doi.org/10.1016/j.mimet.2016.06.005 0167-7012/© 2016 Elsevier B.V. All rights reserved.

(Jónsdóttir et al., 2003; Jurstrand et al., 2001; Gravningen et al., 2012; Lysén et al., 2004; Peuchant et al., 2012). Therefore, other typing methods with higher discriminating capacity were developed in recent years, such as multilocus sequence typing (MLST), multilocus typing (MLT) array and multilocus variable number tandem repeats analysis (MLVA). MLST relies on DNA sequencing of several genomic loci. There are three such schemes described for C. trachomatis. Two of them are based on housekeeping genes, have a resolution similar to ompA sequencing and are suitable for evolutionary studies (Dean et al., 2009; Pannekoek et al., 2008). The third scheme was developed by Klint et al. (2007), and is intended for short-term clinical epidemiology and outbreak investigations. MLT array is based on specific hybridization patterns from PCR products and is less laborious than sequence based MLST and MLVA. It has been used to identify chlamydial species (Borel et al., 2008; Sachse et al., 2005) and for genotyping strains of C. trachomatis (Ruettger et al., 2011) and Chlamydia psittaci (Sachse et al., 2008). An MLT array using the MLST scheme designed by Klint et al. was recently published with promising results (Christerson et al., 2011). There is currently no consensus about which high-resolution genotyping method is the best for clinical and epidemiological studies of C.

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trachomatis infections. However, the MLST scheme by Klint et al. is the most frequently used and has generated the largest database (http:// mlstdb.bmc.uu.se). The aim of this study was to type C. trachomatis obtained from clinical samples in two hospitals in Buenos Aires, Argentina and from a C. trachomatis prevalence study conducted at Concepción city, Chile, using three different methods for comparison: ompA gene sequencing, MLT array and MLST. 2. Materials and methods

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analyzed using the MLT Line software, version 2.01., almost as previously described (Christerson et al., 2011).

2.5. Diversity index The diversity index (D) of a typing method refers to the probability that two unrelated strains sampled from the test population will be placed into different typing groups. D was determined using Hunter and Gaston's modification of Simpson's diversity index (Hunter and Gaston, 1988).

2.1. Clinical samples A total of 104 C. trachomatis-positive samples collected from symptomatic adult patients and neonates with neonatal conjunctivitis in two hospitals of Buenos Aires city (the University Hospital “Hospital de Clínicas Jose de San Martin”, Universidad de Buenos Aires and the National Hospital “Prof. A. Posadas”; 34 cervical swabs, 17 male urethral swabs and 33 conjunctiva swabs from neonates) between 2005 and 2007 and from a C. trachomatis prevalence study conducted in Concepción city, Chile, in 2005 (20 cervical swabs), were included in this study. C. trachomatis was detected by ompA nested PCR at the University Hospital laboratory in Buenos Aires as previously described (Gallo Vaulet et al., 2010). A real-time PCR targeting the 23SrRNA gene in Chlamydiaceae spp. (Ehricht et al., 2006) was used after transport to Germany for quality control of DNA. C. trachomatis positive samples with a cycle of threshold value b37 were selected for genotyping. 2.2. DNA extraction QIAamp DNA minikit (Qiagen, Hilden, Germany) was used according to the manufacturer's instructions and DNA was used for typing assays.

2.6. eBURST analysis eBURST version 3 (http://eburst.mlst.net/, Department of Infectious Disease Epidemiology, Imperial College London, The United Kingdom) software was used to identify founders among the sequence types. A founder was defined as the ST with the largest number of single locus variants in a group. An ST that appears to have diversified to produce multiple single locus variants is called a subgroup founder. A list of all STs was inserted into the single dataset function at the eBURST website and the number of loci was set to 5 (ompA not included). The analysis was computed generating groups and predicted founders, and for each larger group a diagram was drawn.

2.7. Minimum spanning tree analysis BioNumerics software (version 7.0; Applied Maths, Sint-MartensLatem, Belgium) was used to construct a minimum spanning tree analysis of all entries in the study (ompA not included). The sequence data was also analyzed together with all other samples in the database and was entered into the BioNumerics software. As the algorithm, we used the predefined template “MST for categorical data” plug-in, which uses the categorical coefficient to calculate the similarity matrix.

2.3. Multilocus sequence typing The typing scheme includes the amplification by PCR and sequencing of five regions of the C. trachomatis genome as previously described (Bom et al., 2011) with modifications for CT058 and pbpB (Bom et al., 2013). These regions included the genes encoding hypothetical proteins CT058, CT144, CT172; and portions of the genes pbpB and hctB. Allele number was assigned by comparing each sequence to all known alleles available in the C. trachomatis MLST database of the Uppsala University, Sweden (http://mlstdb.bmc.uu.se, version 2.0) (n = 2089 entries comprising 415 sequence types (ST)). Only samples in which all five loci were successfully amplified, sequenced, and identified obtained a full MLST ST were included in further analysis. In addition, ompA sequence determination was performed as previously described (Christerson et al., 2012).

2.8. Ethics The study has been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. All samples used in this study were re-coded in order to anonymize patient records/information prior to analysis. Therefore individuals could not be matched with their samples and their epidemiological and clinical data. Samples and epidemiological data were collected for diagnostic purpose under standard of care protocols of sexually transmitted infection in each location. Informed consent, as approved by our institutional Ethics Committee, was not required and only taken for newborn samples from their parents. Neither additional samples nor personal data were requested for this study. Moreover genetic analysis was only performed on bacterial DNA isolates.

2.4. Multilocus typing array 3. Results MLT array was performed on 76 of 104 samples due to scarce sample volume. Amplification of the five MLST regions and ompA was conducted in three separate reactions using the Qiagen multiplex PCR kit (Qiagen, Hilden, Germany) and 5′-biotinylated primers as described previously (Christerson et al., 2011). ArrayStrip™ units consisting of 8 connected plastic vessels in microtiter format, each carrying a microarray chip, were used. The hybridization reactions were performed using the Hybridization Kit (Alere Technologies GmbH, Jena, Germany) following the manufacturer's instructions. Hybridization data was processed using Iconoclust software, version 3.3 (Alere Technologies). Normalized signal intensities were calculated automatically by the software, subsequently transferred into Microsoft Office Excel 2003, and

3.1. ompA typing Complete sequences of the ompA gene were obtained for all 104 study samples. Genotype E was the most frequently detected, 59% (n = 61), of which E/Bour ompA (type 6) was represented by 55 strains. The other genotypes identified were: D 11% (n = 11); Da 1.0% (n = 1), F 11% (n = 11), G 6.7% (n = 7), H 1.9% (n = 2), I 1.0% (n = 1), J 6.7% (n = 7), and K 2.9% (n = 3). Altogether 19 ompA genotype variants were detected: 5 E, 4 G, 3 J, 2 D, and one each of the remaining genotypes Da, F, H, I and K. The ompA type 60 was only observed in Chilean genital samples.

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3.2. MLST Full profiles for the five targets were obtained in 101 of the 104 (97%) samples tested and resulted in 50 STs. A total of 18 new alleles were found (CT058, n = 7; hctB, n = 4; CT144, n = 3; CT172, n = 2; pbpB, n = 2) resulting in 30 new STs. The distribution of alleles and STs for the samples is given in Table S1. The predominating genotype E could be divided into 23 STs, of which 16 had not been reported in previous studies. The most common types among genotype E were ST3

(n = 20) and ST56 (n = 15), while two STs were represented by 2 and 4 samples, respectively and 19 STs were singletons. The predominant ompA type 6, corresponding to genotype E/Bour, was found in 21 different STs. The diversity of the MLST STs was examined by constructing a minimum spanning tree analysis (Fig. 1). Argentinian/Chilean samples were found in the larger circles representing the three founding STs among heterosexuals, especially ST3 and ST56. Three study samples from women had STs highly correlated to men having sex with men

Fig. 1. Minimum spanning tree analysis based on the five MLST target regions of the 101 samples included in this study and the entire database (415 STs from 2089 samples). Sphere sizes indicate the numbers of samples in each sphere. Solid branches show single-locus variants and dashed branches show double-locus variants. The most common STs are indicated by numbers.

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(MSM). Overall, the Argentinian and Chilean samples were spread over most parts of the MST that are linked to heterosexuals (i.e. excluding MSM related and trachoma clusters).

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In 73 of the 76 samples (96%) conclusive results were obtained using the microarray assay resulting in 29 different groups. Genotype E could be divided into 11 groups. Two groups were predominant: Group 4 (corresponding to MLST ST3) was represented by 17 samples and group 6 (corresponding to ST56) by 15 samples. Six groups comprised 2–5 samples and 21 groups were represented by a single sample. Complete analysis of MLT array data is given in Table S2.

i.e. sample 6380 was incorrectly genotyped in the hctB region by the MLT array. The resolution obtained for MLST was 2.1 higher than ompA sequence determination and 1.3 higher than MLT array. The MLT showed 1.7 higher resolution than ompA typing. The resolution of each methodology varied for different ompA genotypes. For example, genotype E was differentiated into 5 ompA types, 9 MLT array types and 16 MLST STs. Thus, compared to ompA typing the resolution of MLT array was 1.8 fold and for MLST it was 3.2 fold higher. Diversity index was calculated for each typing method used in this study and was 0.69 (95% confidence interval [CI], 0.63 to 0.75) for ompA gene sequencing, 0.89 (0.86 to 0.91) using MLT array and 0.94 (0.92 to 0.95) using MLST. When D was calculated for the entire MLST dataset of 101 samples, it was 0.94 (0.92 to 0.95).

3.4. Comparison between ompA sequencing, MLST and MLT array

4. Discussion

Of the 76 samples tested by MLT array, 70 (92%) could be analyzed by the three methodologies used in this study. This resulted in 17 ompA variants, 35 MLST STs and 27 MLT groups (Table 1). The MLT array results were mainly consistent with the MLST analysis. However, there was one inconsistent result between the MLT array and MLST,

The comparison of genotyping methods showed that MLST provided a significantly higher resolution than ompA sequencing and slightly higher than the MLT array. This is in agreement with previous studies where MLST showed up to 5-fold higher resolution than ompA (Gravningen et al., 2012). A D value above 0.95 is considered an “ideal” cutoff value for a molecular typing method (Van Belkum et al., 2007). In the current study MLST was close to that value (0.94) when calculated on the entire dataset of 101 samples, as well as when including only the 70 samples that could be analyzed also by MLT array. This is in agreement with previous studies reporting D between 0.84 and 0.97 (Herrmann et al., 2015). The overall value for the database including N2000 entries was 0.97, indicating that size and epidemiological relatedness have an impact on the assessment in separate studies. The MLT array had a D value similar to MLST while ompA was assessed as considerably lower. The only prior comparison with MLT array was based on samples from Norway, where MLT attained 90% of the resolution of MLST (Christerson et al., 2011). The somewhat lower value in the current study (83%) could be attributed to the fact that in 2009 when the array was designed the number of known sequence variants was lower. Later studies have detected more variants than expected (Herrmann et al., 2015) and this has been clearly confirmed in this study, where 30 new STs were found. The MLT array was earlier shown to be highly specific (Christerson et al., 2011), but needs additional probes to detect all currently known genotypes. MLST analysis also showed that the resolution varies for different ompA genotypes. The most common genotype E, comprising 40% to 60% of all C. trachomatis cases in most studies among heterosexuals, could be differentiated into 23 STs in this study and samples with the most common variant of E (Bour) could be separated into 21 STs. This indicates the limitation of using conventional ompA typing compared to high resolution methods. The predominant MLST STs found in Argentina and Chile, ST3 and ST56, were defined as founders and are common also in several of the other countries included in the database, ST3 in Suriname (16%), Tunisia (36%), China (14%) and the Netherlands (6%) and ST56 in the Netherlands (8%), Sweden (6%) and Norway (13%). This indicates that some STs are widely spread in many countries, while the single locus variants to these founders appear to have limited spread. Notably two women from Chile had ST108, which only has been found in 85 men having sex with men in other studies (Bom et al., 2013; Christerson et al., 2012). Similarly one sample each from one woman and one man in Argentina had STs linked to the MSM associated ST108 and ST109, respectively. These findings may be coincidental, but can also support STs that previously linked to MSM in Europe and USA may be occurring in heterosexuals in other parts of the world. The spread of STs would be more related to epidemiological patterns rather than tissue tropism, as suggested recently (Versteeg et al., 2014). To understand why a few STs predominate, more advanced genetic analysis is needed. Furthermore, the role of the host response to

3.3. MLT array analysis

Table 1 Comparison of genotyping methods by analysis of 70 samples with ompA sequencing, MLT array and MLST. Typing methodology C. trachomatis genotype D

E

F

G

H I J K

(n)

MLST ST

(n)

2

(5)

35

(3)

1

363c 372c (3) 85 186 (50) 3

ompA typea

6

58b 59b 60b 61b 24

(1) (1) (1) (1) (8)

9 10 11 13 35

(1) (1) (1) (1) (2)

36 38 53 12

(1) (1) (2) (2)

n: number of samples. a Type refers to sequence variant of ompA. b New allele. c New ST.

56 59 86 87 153 357c 361c 364c 365c 366c 369c 370c 374c 56 357c 16 356c 12 77 91 148 373c 362c 367c 359c 97 368c 100 91 108 358c 360c

MLT array type

2 14 (1) 12 (1) 2 (2) 21 (1) 19 (13) 1 4 (10) 6 (1) 25 (1) 4 (2) 4 (1) 6 (3) 3 (1) 9 (1) 6 (1) 15 (1) 6 (1) 22 (1) 6 (1) 6 (1) 6 (1) 3 (1) 29 (1) 4 (1) 11 (1) 27 (4) 6 16 (2) 11 (1) 8 (1) 10 (1) 17 (1) 8 (1) 18 (1) 20 (1) 23 (1) 28 (2) 26 (1) 5 (1) 7

(n) (2) (1) (1) (1) (2) (1) (1) (12) (10) (1) (1) (2) (1) (3) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (3) (2) (1) (1) (1) (1) (1) (1) (1) (1) (2) (1) (1)

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different infecting strains is not well understood and may also affect the distribution of STs. In addition, to provide a better perception of evolutionary changes for C. trachomatis and link it to short term variations it would be rewarding to combine our MLST system with data from a housekeeping-based MLST. The size of this study is quite small and the collection of samples is arbitrary rather than unbiased, which in deed is a limitation. A high proportion of the samples were from neonatal conjunctivitis cases. However, the ompA genotype distribution is similar to that in other countries (Pedersen et al., 2009) and indicates that our study reflects strain distribution in most countries where genotyping of C. trachomatis has been performed. This study is the first using high-resolution genotyping of C. trachomatis in South America, except Suriname. Many new MLST STs have been identified but only two predominated. This is in agreement with a more comprehensive MLST analysis study recently published (Herrmann et al., 2015). Moreover, the assessment of the MLT array shows the method to be reliable, but further adaptation is needed before broad-scale use can be considered. Use of whole genome sequencing is rapidly increasing, also in clinical microbiology, and the cost is continuously falling. Nevertheless the cost in terms of money and labor are still too high compared to MLST and the additional information obtained is normally not needed for epidemiological investigations. Furthermore, whole genome sequencing requires a substantial amount of DNA which is not available in a considerable proportion of the clinical chlamydia samples. In conclusion, MLST and MLT DNA array showed higher discrimination capacity than conventional ompA sequencing. A surprisingly high number of new MLST genotypes (STs) were detected, but only a few genetic founders are predominating and appear to be globally spread. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.mimet.2016.06.005. Conflict of interest statement None of the authors has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the present paper. Acknowledgments Susana Di Bartolomeo who cautiously collected samples from the National Hospital Prof. A. Posadas. BH acknowledges support by local funds from Uppsala University Hospital. The study was also financed by a grant of the Universidad de Buenos Aires UBACyT B810 and UBACyT 20020090200460 awarded to MRF. KS acknowledges support by the national research network “Zoonotic chlamydiae – Models of chronic and persistent infections in humans and animals”, which was funded by the Federal Ministry of Education and Research (BMBF) of Germany under grant 01KI0720. References Bom, R.J.M., Christerson, L., Schim van der Loeff, M.F., Coutinho, R.A., Herrmann, B., Bruisten, S.M., 2011. Evaluation of high-resolution typing methods for Chlamydia trachomatis in samples from heterosexual couples. J. Clin. Microbiol. 49, 2844–2853. Bom, R.J.M., van der Helm, J.J., Schim van der Loeff, M.F., van Rooijen, M.S., Heijman, T., Matser, A., de Vries, H.J., Bruisten, S.M., 2013. Distinct transmission networks of Chlamydia trachomatis in men who have sex with men and heterosexual adults in Amsterdam, The Netherlands. PLoS 8 (1), e53869. http://dx.doi.org/10.1371/journal. pone.0053869.

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