High-resolution Multilocus Sequence Typing Research Articles

A core-genome multilocus sequence typing scheme for the detection of genetically related Streptococcus pyogenes clusters.

The recently observed increase in invasive Streptococcus pyogenes infections causes concern in Europe. However, conventional molecular typing methods lack discriminatory power to aid investigations of outbreaks caused by S. pyogenes. Therefore, there is an urgent need for high-resolution molecular typing methods to assess genetic relatedness between S. pyogenes isolates. In the current study, we aimed to develop a novel high-resolution core-genome multilocus sequence typing (cgMLST) scheme for S. pyogenes and compared its discriminatory power to conventional molecular typing methods. The cgMLST scheme was designed with the commercial Ridom SeqSphere+ software package. To define a cluster threshold, the scheme was evaluated using publicly available data from nine defined S. pyogenes outbreaks in the United Kingdom. The cgMLST scheme was then applied to 23 isolates from a suspected S. pyogenes outbreak and 117 S. pyogenes surveillance isolates both from the Netherlands. MLST and emm-typing results were used for comparison to cgMLST results. The allelic differences between isolates from defined outbreaks ranged between 6 and 31 for isolates with the same emm-type, resulting in a proposed cluster threshold of <5 allelic differences out of 1,095 target loci. Seven out of twenty-three (30%) isolates from the suspected outbreak had an allelic difference of <2, thereby identifying a potential cluster that could not be linked to other isolates. The proposed cgMLST scheme shows a higher discriminatory ability when compared to conventional typing methods. The rapid and simple analysis workflow allows for extended detection of clusters of potential outbreak isolates and surveillance and may facilitate the sharing of sequencing results between (inter)national laboratories.

Delineating Mycobacterium abscessus population structure and transmission employing high-resolution core genome multilocus sequence typing

Mycobacterium abscessus is an emerging multidrug-resistant non-tuberculous mycobacterium that causes a wide spectrum of infections and has caused several local outbreaks worldwide. To facilitate standardized prospective molecular surveillance, we established a novel core genome multilocus sequence typing (cgMLST) scheme. Whole genome sequencing data of 1991 isolates were employed to validate the scheme, re-analyze global population structure and set genetic distance thresholds for cluster detection and taxonomic identification. We confirmed and amended the nomenclature of the main dominant circulating clones and found that these also correlate well with traditional 7-loci MLST. Dominant circulating clones could be linked to a corresponding reference genome with less than 250 alleles while 99% of pairwise comparisons between epidemiologically linked isolates were below 25 alleles and 90% below 10 alleles. These thresholds can be used to guide further epidemiological investigations. Overall, the scheme will help to unravel the apparent global spread of certain clonal complexes and as yet undiscovered transmission routes.

High-resolution multilocus sequence typing for Chlamydia trachomatis: improved results for clinical samples with low amounts of C. trachomatis DNA

BackgroundSeveral Multilocus Sequence Typing (MLST) schemes have been developed for Chlamydia trachomatis. Bom’s MLST scheme for MLST is based on nested PCR amplification and sequencing of five hypervariable genes and ompA. In contrast to other Chlamydia MLST schemes, Bom’s MLST scheme gives higher resolution and phylogenetic trees that are comparable to those from whole genome sequencing. However, poor results have been obtained with Bom’s MLST scheme in clinical samples with low concentrations of Chlamydia DNA.ResultsIn this work, we present an improved version of the scheme that is based on the same genes and MLST database as Bom’s MLST scheme, but with newly designed primers for nested-1 and nested-2 steps under stringent conditions. Furthermore, we introduce a third primer set for the sequencing step, which considerably improves the performance of the assay. The improved primers were tested in-silico using a dataset of 141 Whole Genome Sequences (WGS) and in a comparative analysis of 32 clinical samples. Based on cycle threshold and melting curve analysis values obtained during Real-Time PCR of nested-1 & 2 steps, we developed a simple scoring scheme and flow chart that allow identification of reaction inhibitors as well as to predict with high accuracy amplification success. The improved MLST version was used to obtain a genovars distribution in patients attending an STI clinic in Tel Aviv.ConclusionsThe newly developed MLST version showed great improvement of assay results for samples with very low concentrations of Chlamydia DNA. A similar concept could be applicable to other MLST schemes.

Genetic diversity of urogenital Chlamydia trachomatis before and after mass drug administration for trachoma

Background The WHO recommends treatment of trachoma with community-wide mass drug administration (MDA) with a single dose of azithromycin as part of the SAFE strategy (surgery, antibiotics, facial cleanliness and environmental improvement). In the Solomon Islands, this programme had demonstrable collateral benefit by reducing the prevalence of urogenital Chlamydia trachomatis(Ct) infections. We evaluated the impact of this treatment on population genetics of urogenital Ct. Methods Two vaginal swabs were collected from consecutive women attending antenatal clinics during cross-sectional surveys before and after MDA. For every swab positive for Ctinfection, DNA was extracted from the second swab, enriched and sequenced using paired-end sequencing. Whole-genome sequences were aligned against selected references. Diversity was assessed using genome-wide pairwise diversity and a high-resolution multi-locus sequence typing (hr-MLST-6) scheme. ARIBA software was used to test for evidence of antimicrobial resistance to macrolides. Results Whole-genome sequence data was obtained from 23/49 (47%) pre-MDA and 32/41 (78%) post-MDA Ct-positive samples. Most strains were serotype E and F, and tissue tropism genes were consistent with their urogenital nature. Genetic diversity of Ctwas lower by both pairwise and hr-MLST-6 diversity metrics in the post-MDA sample than the pre-MDA sample. There was no evidence of mutations known to confer resistance to macrolides in any of the samples collected. Conclusions Reduced diversity after MDA may represent selection pressure from mass antibiotic delivery. The absence of antimicrobial resistance is encouraging. The collateral impact (both positive and negative) of large-scale preventative chemotherapy programmes should be considered when deciding whether should be implemented.

Molecular characterization of Treponema pallidum subsp. pallidum in Switzerland and France with a new multilocus sequence typing scheme.

Syphilis is an important public health problem and an increasing incidence has been noted in recent years. Characterization of strain diversity through molecular data plays a critical role in the epidemiological understanding of this re-emergence. We here propose a new high-resolution multilocus sequence typing (MLST) scheme for Treponema pallidum subsp. pallidum (TPA). We analyzed 30 complete and draft TPA genomes obtained directly from clinical samples or from rabbit propagated strains to identify suitable typing loci and tested the new scheme on 120 clinical samples collected in Switzerland and France. Our analyses yielded three loci with high discriminatory power: TP0136, TP0548, and TP0705. Together with analysis of the 23S rRNA gene mutations for macrolide resistance, we propose these loci as MLST for TPA. Among clinical samples, 23 allelic profiles as well as a high percentage (80% samples) of macrolide resistance were revealed. The new MLST has higher discriminatory power compared to previous typing schemes, enabling distinction of TPA from other treponemal bacteria, distinction between the two main TPA clades (Nichols and SS14), and differentiation of strains within these clades.

A novel high-resolution multilocus sequence typing of Giardia intestinalis Assemblage A isolates reveals zoonotic transmission, clonal outbreaks and recombination

Molecular epidemiology and genotyping studies of the parasitic protozoan Giardia intestinalis have proven difficult due to multiple factors, such as low discriminatory power in the commonly used genotyping loci, which has hampered molecular analyses of outbreak sources, zoonotic transmission and virulence types. Here we have focused on assemblage A Giardia and developed a high-resolution assemblage-specific multilocus sequence typing (MLST) method. Analyses of sequenced G. intestinalis assemblage A genomes from different sub-assemblages identified a set of six genetic loci with high genetic variability. DNA samples from both humans (n = 44) and animals (n = 18) that harbored Giardia assemblage A infections, were PCR amplified (557–700 bp products) and sequenced at the six novel genetic loci. Bioinformatic analyses showed five to ten-fold higher levels of polymorphic sites than what was previously found among assemblage A samples using the classic genotyping loci. Phylogenetically, a division of two major clusters in assemblage A became apparent, separating samples of human and animal origin. A subset of human samples (n = 9) from a documented Giardia outbreak in a Swedish day-care center, showed full complementarity at nine genetic loci (the six new and the standard BG, TPI and GDH loci), strongly suggesting one source of infection. Furthermore, three samples of human origin displayed MLST profiles that were phylogenetically more closely related to MLST profiles from animal derived samples, suggesting zoonotic transmission. These new genotyping loci enabled us to detect events of recombination between different assemblage A isolates but also between assemblage A and E isolates. In summary, we present a novel and expanded MLST strategy with significantly improved sensitivity for molecular analyses of virulence types, zoonotic potential and source tracking for assemblage A Giardia.

Lymphogranuloma venereum rates increased and Chlamydia trachomatis genotypes changed among men who have sex with men in Sweden 2004–2016

This study aimed to determine the incidence of lymphogranuloma venereum (LGV) in Sweden since 2004 and to study in detail a consecutive number of Chlamydia trachomatis cases in men who have sex with men (MSM) during a 10 month period (September 2014 to July 2015). LGV increased from sporadic import cases in 2004 to comprise a spread within Sweden in 2016. Initially, only the L2b ompA genotype was detected, but in 2015 half of the genotyped LGV cases were L2 genotype. The changing genotype distribution in Sweden is linked to increased LGV spread in Europe. High-resolution multilocus sequence typing of 168 C. trachomatis cases from MSM in 2015 resulted in 29 sequence types, of which 3 accounted for 49 % of cases. The increased rates and different genotypes of LGV indicate that more concern for high-risk taking MSM is needed to avoid further spread of this invasive infection.

Monitoring therapy success of urogenital Chlamydia trachomatis infections in women: A prospective observational cohort study

IntroductionThe use of a nucleic acid amplification test (NAAT) as a test of cure after treatment is subject to discussion, as the presence of C. trachomatis nucleic acids after treatment may be prolonged and intermittent without presence of infectious bacteria. We used cell culture to assess if a positive RNA- or DNA-based NAAT after treatment indicates the presence of viable C. trachomatis.MethodsWe included women with asymptomatic urogenital C. trachomatis infection visiting the Amsterdam STI clinic from September 2015 through June 2016. Endocervical swabs were collected prior to treatment with azithromycin, and during three follow-up visits 7, 21 and 49 days after treatment. Collected swabs were subjected to C. trachomatis culture and a RNA- and DNA-based NAAT. High-resolution multilocus sequence typing (hr-MLST) was used to further differentiate potential re-infections.ResultsWe included 90 women with a positive RNA-test prior to receiving treatment of whom 81 (90%) were also DNA-positive, and 69 (76.7%) culture-positive. Prolonged and intermittent positive RNA and DNA results over time were observed. Three women had culture positive results at the second visit, but all were negative at the third visit. Five women had NAAT-positive results at the fourth visit of whom three women were also culture-positive indicating a viable infection. All five women reported unprotected sexual contact since the first visit. From 2, hr-MLST sequence types were obtained. One had a different sequence type indicating a new infection the other was identical to the previously found indicating a potentially persisting infection.ConclusionMost RNA- or DNA-positive results after treatment of urogenital C. trachomatis may be caused by non-viable molecular remnants since they cannot be confirmed by culture. In a minority viable C. trachomatis was found in culture at the second visit, indicating that patients may remain infectious at least 7 days after treatment.

A snapshot of Chlamydia trachomatis genetic diversity using multilocus sequence type analysis in an Australian metropolitan setting.

High-resolution screening methodologies which enable the differentiation of Chlamydia trachomatis at the strain level, directly from clinical samples, can provide the detailed information required for epidemiological questions such as the dynamics of treatment failure. In addition, they give a detailed snapshot of circulating C. trachomatis genetic variation, data which are currently lacking for the Australian population. In the context of two Australian clinical trials, we assessed the genetic diversity of C. trachomatis and compared these to strains circulating globally. We used high-resolution multilocus sequence typing (MLST) of five highly variable genetic regions of C. trachomatis to examine variation in Australia. Samples with established genovars were drawn from a pool of 880 C. trachomatis-positive samples from two clinical studies, whereby 76 sample pairs which remained C. trachomatis-positive for the same genovar after treatment underwent MLST analysis to distinguish between treatment failure and reinfection. MLST analysis revealed a total of 25 sequence types (STs), six new allele variants and seven new STs not described anywhere else in the world, when compared to those in the international C. trachomatis MLST database. Of the eight most common global STs, seven were found in Australia (four derived from men who have sex with men (MSM) and three from heterosexuals). Newly identified STs were predominantly found in samples from the MSM population. In conclusion, MLST provided a diverse C. trachomatis strain profile, with novel circulating STs, and could be used to identify local sexual networks to focus on interventions such as testing and partner notification to prevent reinfection.

High-resolution multilocus sequence typing of urogenital Chlamydia trachomatis among STD clinic outpatients in Guangxi Zhuang Autonomous Region

Objective To investigate the type distribution of urogenital Chlamydia trachomatis (Ct) among STD clinic outpatients in Guangxi Zhuang Autonomous Region, and to estimate the prevalence of Ct infection among the patients during posttreatment follow-up. Methods Urethral and cervical swabs were collected from male and female outpatients with confirmed urogenital Ct infection, respectively, in Institute of Dermatology of Guangxi Zhuang Autonomous Region. The patients with positive results in preliminary screening tests were followed up after treatment, and specimens were collected at follow-up visits. General and clinical information was also obtained from these patients. DNA was extracted from these samples by using the QIAxtractor instrument. Nested PCR was performed to amplify the major outer membrane protein A (ompA) gene for ompA typing, and to amplify CT046 (hctB), CT058, CT144, CT172 and CT682 (pbpB) genes for high-resolution multilocus sequence typing (hr-MLST). Then, PCR products were sequenced, and ompA and MLST types of Ct were determined by sequence alignment and MLST analysis, respectively. The obtained MLST sequence types (STs) were compared with those from an Italian population by using the BioNumerics7 software, and a minimum spanning tree (MST) was generated. Results Totally, 44 and 6 Ct-positive specimens were collected at first visits and follow-up visits respectively. Among the 50 specimens, 42 underwent successful ompA typing and hr-MLST, and 7 ompA genotypes and 15 hr-MLST STs were identified, including 3 first reported STs. The distribution of STs of Ct isolates from Guangxi Zhuang Autonomous Region was significantly different from that from the Italian population. Among the 6 followed patients with posttreatment Ct infection, 3 were confirmed to be reinfected with Ct, and the other 3 failed to be diagnosed because of unsuccessful genotyping. Conclusion The genotypes of Ct strains isolated from STD clinic outpatients in Guangxi Autonomous Region were characteristic, and Ct reinfection occurred in some patients during follow-up. Key words: Chlamydia trachomatis; Multilocus sequence typing; Follow-up studies; Cluster analysis

Does typing of Chlamydia trachomatis using housekeeping multilocus sequence typing reveal different sexual networks among heterosexuals and men who have sex with men?

BackgroundChlamydia trachomatis infections remain the most common bacterial sexually transmitted infection worldwide. To gain more insight into the epidemiology and transmission of C. trachomatis, several schemes of multilocus sequence typing (MLST) have been developed. We investigated the clustering of C. trachomatis strains derived from men who have sex with men (MSM) and heterosexuals using the MLST scheme based on 7 housekeeping genes (MLST-7) adapted for clinical specimens and a high-resolution MLST scheme based on 6 polymorphic genes, including ompA (hr-MLST-6).MethodsSpecimens from 100 C. trachomatis infected men who have sex with men (MSM) and 100 heterosexual women were randomly selected from previous studies and sequenced. We adapted the MLST-7 scheme to a nested assay to be suitable for direct typing of clinical specimens. All selected specimens were typed using both the adapted MLST-7 scheme and the hr-MLST-6 scheme. Clustering of C. trachomatis strains derived from MSM and heterosexuals was assessed using minimum spanning tree analysis.ResultsSufficient chlamydial DNA was present in 188 of the 200 (94 %) selected samples. Using the adapted MLST-7 scheme, full MLST profiles were obtained for 187 of 188 tested specimens resulting in a high success rate of 99.5 %. Of these 187 specimens, 91 (48.7 %) were from MSM and 96 (51.3 %) from heterosexuals. We detected 21 sequence types (STs) using the adapted MLST-7 and 79 STs using the hr-MLST-6 scheme. Minimum spanning tree analyses was used to examine the clustering of MLST-7 data, which showed no reflection of separate transmission in MSM and heterosexual hosts. Moreover, typing using the hr-MLST-6 scheme identified genetically related clusters within each of clusters that were identified by using the MLST-7 scheme.ConclusionNo distinct transmission of C. trachomatis could be observed in MSM and heterosexuals using the adapted MLST-7 scheme in contrast to using the hr-MLST-6. In addition, we compared clustering of both MLST schemes and demonstrated that typing using the hr-MLST-6 scheme is able to identify genetically related clusters of C. trachomatis strains within each of the clusters that were identified by using the MLST-7 scheme.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-016-1486-2) contains supplementary material, which is available to authorized users.

P08.35 Cluster analysis ofchlamydia trachomatisstrains using two multilocus sequence typing schemes shows differences in discrimination of msm strains versus those of heterosexuals

Introduction Despite intensive efforts Chlamydia trachomatis (Ct) remains the most common bacterial sexually transmitted infection worldwide. Therefore, to gain more insight into the epidemiology and transmission of Ct, multilocus sequence typing (MLST) schemes have been developed. However, there is no consensus regarding the use of an MLST scheme for epidemiological studies. Therefore, the objective of this study was to investigate the clustering of Ct strains using two MLST schemes with differing discriminatory capacities. Methods We selected and tested samples from Ct infected men having sex with man (MSM) and heterosexual women using two existing MLST schemes. One MLST scheme was based on 6 highly variable targets in the chlamydial genome, also referred to as high-resolution MLST (hr-MLST-6) and the other scheme was based on 7 housekeeping genes (MLST-7). For this study, the existing MLST-7 scheme was modified to a nested PCR enabling to test clinical samples. Sequences obtained from both MLST schemes were analysed for cluster analysis and phylogenetic relationships. Results Full profiles were obtained for 175 samples; 84 (48%) were from MSM and 91 (52%) from heterosexuals. We detected 32 sequence types (STs) using the MLST-7scheme and 78 STs using the hr-MLST-6scheme. Cluster analysis using hr-MLST-6data showed segregation between isolates from MSM and heterosexuals. However, cluster analysis using the MLST-7 data did not show this segregation between isolates from MSM and heterosexuals. Conclusion The MLST-7 scheme was not able to discern closely related Ct strains over a small time-frame confirming that it is more suitable for evolutionary studies. For short-term cluster analysis the hr-MLST-6scheme is considered more suitable to study the epidemiology and transmission of Ct in various populations. Disclosure of interest statement This work was funded by the Public Health Service of Amsterdam, the Netherlands. None of the authors report a conflict of interest.

High-resolution multilocus sequence typing reveals novel urogenital Chlamydia trachomatis strains in women in Mopani district, South Africa

ObjectivesRecently, we reported a high prevalence (16%) of urogenital Chlamydia trachomatis infections among women in a rural setting in South Africa. Molecular epidemiological studies on C. trachomatis infections could provide...

Urogenital Chlamydia trachomatis strain types, defined by high-resolution multilocus sequence typing, in relation to ethnicity and urogenital symptoms among a young screening population in Amsterdam, The Netherlands

IntroductionPrevious studies found conflicting results regarding associations between urogenital Chlamydia trachomatis infections and ethnicity or urogenital symptoms among at-risk populations using either ompA-based genotyping or high-resolution multilocus sequence typing (MLST)....

No indication for tissue tropism in urogenital and anorectal Chlamydia trachomatis infections using high-resolution multilocus sequence typing.

BackgroundPrevious studies showed that C. trachomatis strains found in MSM are different from those in heterosexuals. This study investigates whether the differences in strain distribution between MSM and heterosexuals are due to tissue tropism.MethodsC. trachomatis positive samples were collected from MSM (anorectal) and women (anorectal, cervical, vaginal, pharyngeal) visiting the STI outpatient clinic of Amsterdam between 2008 and 2013. All samples were typed using multilocus sequence typing (MLST). Epidemiological data were derived from electronic patient records.ResultsWe obtained full MLST data for C. trachomatis from 207 MSM and 185 women, all with anorectal infections. Six large clusters were identified of which 3 consisted predominantly of samples from women (89%-100%), whereas the other 3 consisted predominantly of samples from MSM (97%-100%). Furthermore, we obtained full MLST data from 434 samples of 206 women with concurrent infections at multiple anatomical locations. No association was observed between C. trachomatis cluster and the anatomical location of infection.ConclusionWe found no indication for tissue tropism in urogenital, pharyngeal and anorectal C. trachomatis infections. Combined with results from previously conducted studies, we hypothesize that MSM and heterosexuals have different distributions of C. trachomatis strains due to their separate sexual networks.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2334-14-464) contains supplementary material, which is available to authorized users.

Use of Chlamydia trachomatis high-resolution typing: an extended case study to distinguish recurrent or persistent infection from new infection

ObjectivesRepeated infections of Chlamydia trachomatis may be new infections or persistent infections due to treatment failure or due to unresolved infections in sexual partners. We aimed to establish the value...

P3.257 Distinct But Also Highly Similar Chlamydia Trachomatis Strains in Nanjing, China and in Amsterdam, the Netherlands

In China, Chlamydia trachomatis infections are endemic in the general population, but most infections are found in specific risk groups, such as female sex workers. The most prevalent C. trachomatis...

Multilocus Sequence Typing of Genital Chlamydia trachomatis in Norway Reveals Multiple New Sequence Types and a Large Genetic Diversity

BackgroundThe Chlamydia trachomatis incidence rate in Finnmark, the most northern and sparsely populated county in Norway, has been twice the national average. This population based cross-sectional study among Finnmark high school students had the following aims: i) to examine distribution of multilocus sequence types (STs) of C. trachomatis in a previously unmapped area, ii) to compare chlamydia genetic diversity in Finnmark with that of two urban regions, and iii) to compare discriminatory capacity of multilocus sequence typing (MLST) with conventional ompA sequencing in a large number of chlamydia specimens.Methodology ompA sequencing and a high-resolution MLST system based on PCR amplification and DNA sequencing of five highly variable genetic regions were used. Eighty chlamydia specimens from adolescents aged 15–20 years in Finnmark were collected in five high schools (n = 60) and from routine clinical samples in the laboratory (n = 20). These were compared to routine clinical samples from adolescents in Tromsø (n = 80) and Trondheim (n = 88), capitals of North and Central Norway, respectively.Principal Findings ompA sequencing detected 11 genotypes in 248 specimens from all three areas. MLST displayed 50 STs providing a five-fold higher resolution. Two-thirds of all STs were novel. The common ompA E/Bour genotype comprised 46% and resolved into 24 different STs. MLST identified the Swedish new variant of C. trachomatis not discriminated by ompA sequencing. Simpson's discriminatory index (D) was 0.93 for MLST, while a corrected Dc was 0.97. There were no statistically significant differences in ST genetic diversity between geographic areas. Finnmark had an atypical genovar distribution with G being predominant. This was mainly due to expansion of specific STs of which the novel ST161 was unique for Finnmark.Conclusions/SignificanceMLST revealed multiple new STs and a larger genetic diversity in comparison to ompA sequencing and proved to be a useful tool in molecular epidemiology of chlamydia infections.

P4-S1.06 High-resolution Multilocus Sequence Typing (MLST) of Chlamydia trachomatis from three catchment areas in North and Central Norway

BackgroundThe C trachomatis incidence rate in the county of Finnmark in North Norway is twice as high as in the rest of the country. This study genetically characterises the C...

O4-S1.01 Rapid array-based multilocus genotyping of Chlamydia trachomatis: the easy and economical alternative

BackgroundGenotyping ofChlamydia trachomatis (C.) is an important technique to understand its epidemiology. Methods such as DNA sequencing of the ompA gene or multilocus sequence typing (MLST), either offer limited epidemiological...