Multilocus sequence-typing scheme for Borrelia miyamotoi — the erythema-free ixodid tick-borne borreliosis pathogens

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Abstract

Introduction. Borrelia miyamotoi is a pathogen of erythema-free ixodid tick-borne borreliosis (ITBB), a disease widespread in Russia. To date, there are no generally accepted methods for B. miyamotoi genotyping. The multilocus sequencing typing (MLST) scheme of Borrelia was originally developed for B. burgdorferi, and does not have the required discrimination power for monitoring the ITBB pathogens.

The objective of this study is to develop the MLST scheme for B. miyamotoi.

Materials and Methods. The whole genome sequences of 10 reference strains (GenBank) were analyzed for the selection of the house-keeping loci. The MLST scheme development was based on principles published by the authors of the method. For this experiment, 81 B. miyamotoi strains and positive clinical samples were used to test the MLST scheme.

Results. After analyzing the genomic data, 8 house-keeping loci were chosen for MLST, for which the PCR and sequencing primers were designed. Each MLST loci was represented by several alleles (from 4 to 7) which form 15 sequence types. The genetic diversity of pathogens isolated from ITBB patients and ticks were characterized.

Discussion. Based on pairwise distances between allelic profiles, the sequence types can be classified into four groups. The first two groups are clonal complexes; the other two groups are formed by once identified sequence types. The first clonal complex unites 11 sequence types (80 or 88% of the characterized B. miyamotoi), the second consists of 2 sequence types (9 or 9.8%). The genetic differences between B. miyamotoi are associated with the sources of strains and biological isolates. The MLST based classification confirms the previously described genetic heterogeneity of B. miyamotoi populations associated with ecologically unrelated vectors of ITBB pathogens.

Conclusion. The proposed MLST scheme is an appropriate tool for ITBB pathogen classification and evolutionary change characterization within clonal complexes.

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Introduction

Borrelia miyamotoi is the pathogen of the erythematous form of ixodid tick-borreliosis (ITBB) which belongs to the group of relapsing fever pathogens, but is transmitted by ticks of the Ixodes genus. The history of discovery, taxonomy, biological properties of pathogens, aspects of pathogenesis, clinic and epidemiology of ITBB were published in a monograph by A.E. Platonov in 2017 [1]. A relevant area of B. miyamotoi research is the development of intraspecies pathogen classification methods. This is due to the necessity to study the clinical features of ITBB caused by different genetic variants of the pathogen, including those associated with the phenomenon of immune evasion [1], as well as the necessity to solve classical epidemiological problems related to monitoring pathogens. Because of the severely limited use of mass parallel sequencing, which is primarily due to the difficulty of culturing B. miyamotoi, as well as the low concentration of pathogens in the biological material sample, there is a necessity to develop affordable polymerase chain reaction (PCR)-based and fragment sequencing-based methods for antigenic and genetic characterization of ITBB pathogens.

To characterize the antigenic diversity of B. miyamotoi, we have proposed a method of determining the main surface proteins [2, 3], which allows simultaneous detection of several antigenic variants and identification of the most clinically and epidemiologically significant variants of ITBB pathogens circulating in Russia. However, antigen detection cannot be used for the study of evolutionary processes occurring in the bacterial population to identify pathogens with increased virulent or pathogenic properties.

The triple-locus typing scheme based on the sequencing of p66, glpQ, and 16S gene fragments [4], proposed earlier by domestic researchers could be used to characterize representatives of the Borrelia genus, but this scheme does not have the necessary discrimination power for B. miyamotoi.

A convenient tool for studying the genetic properties of pathogens that are not under immune system pressure, allowing identification of individual strains and characterization of their genetic relationships, is the method of multilocus sequencing typing (MLST), which has been successfully used in epidemiological practice since 1998 [5, 6]. In addition to the previously demonstrated advantages of using multiple genetic loci in analyzing genetic relationships of pathogens, an important advantage of MLST is the absolute interlaboratory reproducibility of the results, which allows combining genetic and epidemiologic information into a single database [7]. Currently, the PubMLST Internet resource contains information on MLST schemes for more than 130 species of microorganisms; for many species, published MLST schemes are the golden standard for their intraspecific characterization. At the same time, the developed MLST scheme for bacteria of the Borrelia genus and its modifications do not have sufficient discrimination power to differentiate B. miyamotoi. This is due to the fact that the MLST scheme was initially developed for B. burgdorferi [8], which does not allow its use for monitoring Russian ITBB pathogens. Since the B. miyamotoi genome has significant differences from bacteria of the Borrelia burgdorferi sensu lato complex, additional analysis of available whole-genome sequences is necessary in order to develop and validate the B. miyamotoi MLST methodology.

Materials and methods

Nucleotide sequences

When selecting sequences for MLST, we used whole genome sequences of 6 Russian strains — Izh-4 (GenBank identification number CP024390.2, number in the State Collection of Pathogenic Microorganisms and Cell Cultures "SCPM-Obolensk" B-8324), Izh-5 (CP024205. 2, B-8325), Izh-14 (CP024371.2, B-8326), Izh-16 (CP024351.2, B-8327), Yekat-1 (CP024333.2, B-8328), Yekat-6 (CP024316.2, B-8329) [1, 9] as well as 4 foreign strains – FR64b (CP004217. 2), HT-31 (CP114703.1), LB-2001 (CP114690.1) and CA17-2241 (CP021872.1) [1, 10, 11], known since the beginning of the study.

Strains and samples of biological material

During development, the MLST scheme was tested on 7 strains, NL-IR-1 (CP044783.1), Yekat-18 (CP037471.1, B-8810), Yekat-76 (CP037058.1, B-8814), Yekat-19 (CP036557.1, B-8811), Yekat-17 (CP037215. 2, B-8330), Yekat-21 (CP036914.2, B-8812), Yekat-31 (CP036726.1, B-8813) and 81 B. miyamotoi DNA samples isolated in 2012-2022 from 50 tick suspensions and the blood of 31 ITBB patients. The suspensions were obtained from Ixodes vector ticks collected in Western Europe and European regions of Russia (I. ricinus), as well as central and eastern regions of Russia (I. persulcatus). Most of the biological samples were obtained and characterized during the development of a methodology for determining the B. miyamotoi main surface protein antigenic properties [2]. The study was conducted with voluntary informed consent of patients. The study protocol was approved by the Ethical Committee of the Central Research Institute of Epidemiology of Rospotrebnadzor (protocol No. 83 of 26.06.2018).

Furthermore, 9 strains whose whole-genome sequences were used to select MLST loci were studied. The characteristics of the studied strains and biological material samples are given in Table 1.

 

Table 1. The B. miyamotoi DNA samples sources and their genetic characteristics (sequence types and clonal complex)

DNA source

Sequence type

Сlonal complex

Territory

Years

Number of samples

Blood of patients with ixodid tick-borne borreliosis caused by B. miyamotoi (n = 37)

ST-1*

I

Sverdlovsk Region

Udmurt Republic

2016–2018

2016

19

1

ST-2*

I

Sverdlovsk Region

Udmurt Republic

2017

2016

2

1

ST-3

I

Udmurt Republic

2016

1

ST-4

I

Sverdlovsk Region

2016

1

ST-5*

I

Udmurt Republic

2016

1

ST-12*

I

Sverdlovsk Region

Krasnoyarsk Territory

2017

2017

1

1

ST-13

I

Sverdlovsk region

2017

1

ST-14*

I

Novosibirsk Region

Khabarovsk Territory

Sverdlovsk Region

Krasnoyarsk Territory

2012

2012

2017

2017, 2019

1

2

2

3

Suspensions of ticks of Ixodes genus (n = 54)

ST-1*

I

Novosibirsk Region

Sverdlovsk Region

Samara Region

Omsk Region

2014, 2017

2013–2014

2019

2022

4

3

1

5

ST-2*

I

Altai Republic

Samara Region

2016

2019

1

1

ST-5*

I

Omsk Region

2022

1

ST-6

I

Japan

1992

1

ST-7

I

Japan

1990

1

ST-8

–

Connecticut, USA

2001

1

ST-9

–

California, USA

2015

1

ST-10

II

Netherlands

Czech Republic

Kabardino-Balkarian Republic

2018

2019

2021

1

6

1

ST-11

II

Kabardino-Balkarian Republic

2021

1

ST-12*

I

Novosibirsk Region

Omsk Region

2017

2022

4

3

ST-14*

I

Altai Republic

Amur Region

Novosibirsk Region

Omsk Region

2016

2016

2017

2022

6

1

9

1

ST-15

I

Tomsk Region

2014

1

Note. *Sequence types found both in patient samples and ticks.

 

PCR and sequencing

DNA extraction and PCR were performed using reagents produced by the Central Research Institute of Epidemiology of Rospotrebnadzor. Amplification and sequencing of gene fragments were performed with primers (Table 2) according to the following procedure: 95ºC — 15 min (1 cycle), 95ºC — 10 s, 60ºC — 20 s, 72ºC — 20 s (40 cycles). The PCR and sequencing methods carried out with the help of Applied Biosystems equipment and reagents were similar to those used in previous studies [2, 12].

 

Table 2. Fragments of genes for MLST, PCR and sequencing primers

Fragment name

Protein*

5’-3’ primer sequences

The length of the PCR products, bp

The length of the MLST loci, bp

acpS

holo-ACP synthase

gACgAAATCAATAggATgTgATATAATAAAggT

CTATTACAAATgCAATAgAgTACTCCCTTTCA

365

192

nusB

Transcription antitermination factor NusB

ggATTTAAgACATAAggCTAgAgTTTTAgCTTTTC

gAgCTCTCCATATTTTTTAACAAAgCATCAAg

417

380

motB

Flagellar motor protein MotB

CCTgAATATATgTTgACATATggAgACATggTT

CCTgCAAATCCAgATACCTCAAATTTACTC

623

413

dnaX

DNA polymerase III subunit gamma/tau

CTgCTATTAAgAAgCgTCCCAgAgAT

CTgATCAAAAAgAgTATAAgCATCCCTTACAC

653

560

rplP

50S ribosomal protein L16

gTATAgAAAgAAgCAAAgAggAAgACTgTCA

CACCTCAAATCTCgCCTTATCACAAATATAg

393

269

сdd

Cytidine deaminase

GAAGCTGCAAGAAATAATGCATATTCACCAT

GCTGCATAATCCTATTATTTGTAAAGATAGC

620

233

lysM

LysM peptidoglycan-binding domain-containing protein

gACTTgATCCTggTgCTATTgTTAAAgCTAg

CCCgATCTCTAAgCTTAgAAgATCTAATTgC

624

522

miaA

tRNA (adenosine(37)-N6)-dimethylallyltransferase MiaA

TCCTACgggTgTAggTAAAAgTgACATT

CCTCTTCAAgCAATCCACAATCAATC

626

555

Note. *The designation of the protein from NCBI (www.ncbi.nlm.nih.gov). **The nucleotide positions of MLST loci in the reference strains were presented in the Table 3.

 

Analysis of MLST data

Sequencing results with allele designations and sequence types (ST) were processed in accordance with the method developer requirements [5–7]. ST analysis using BURST and UPGMA algorithms and genetic tree construction were carried out using the "START2 v. 1.0.5" software [13].

Results

In accordance with the MLST principles, the selection of DNA fragments for allele designation (MLST loci) was subject to the following conditions: the fragments should be located in unlinked genes within the chromosome and represented by several alleles; furthermore, the combinations of alleles should allow for intraspecies pathogen classification [5, 6]. Whole-genome sequence analysis of 6 strains allowed us to identify several dozen groups of unlinked genetic loci, 8 of which were sequentially selected (Table 2), allowing us to differentiate epidemiologically unrelated strains which were isolated at different times, in different territories, and from different sources. Table 3 presents our proposed allele designations and profiles that determine ST strains and B. miyamotoi DNA in biological samples. For allele designations, the MLST loci coordinates in the whole-genome nucleotide sequences of the reference strains are given, except for the nusB-6 and lysM-7 alleles, for which identification numbers in the GenBank database are given. Differences in the MLST loci are represented by single-nucleotide substitutions, except for the nusB fragment, for which 2 alleles (nusB-4 and nusB-6 in ST-10 and ST-11 allele profiles) containing a 6-nucleotide insertion were identified.

 

Table 3. Designations of alleles and sequence types

DNA source

Fragments (lenght, bp)***

Sequence type

acpS (192)

nusB (380, 386)

motB (413)

dnaX (560)

rplP (269)

сdd (233)

lysM (522)

miaA (555)

Strains**

Yekat-1*

1

896361–896170

1

800974–801353

1

613232–613644

1

423437–423996

1

404829–405097

1

265228–265460

1

253627–254148

1

37083–37637

ST-1

Izh-14

1

1

1

1

2

404724–404992

1

1

1

ST-2

Izh-4

1

1

1

1

1

1

2

253612–254133

2

37068–37622

ST-3

Yekat-6

1

2

800953–801332

2

613211–613623

1

1

1

1

1

ST-4

Izh-5

2

896194–896385

1

1

2

423437–423996

1

2

265228–265460

1

1

ST-5

FR64b

2

1

1

2

1

1

3

650921–651442

1

ST-6

HT31

2

1

1

2

1

1

1

3

37098–37652

ST-7

LB-2001

3

897013–897204

3

801812–802191

3

613265–613677

3

423552–424111

3

404960–405228

3

265566–265798

4

253956–254477

4

37153–37707

ST-8

CA17-2241

5

10054–10245

5

105075–105454

5

292933–293345

6

482530–483089

5

501393–501661

5

640132–640364

6

651447–651968

6

868175–868729

ST-9

NL-IR-1

4

893710–893901

4

798494–798879

4

611883–612295

4

422636–423195

4

404082–404350

4

265475–265707

5

253875–254396

5

37104–37658

ST-10

DNA samples

136_KBR21

4

6

4

4

4

4

5

5

ST-11

Bal_Y17

2

1

1

1

1

1

1

1

ST-12

Sha_Y17

1

1

1

1

1

1

1

2

ST-13

4426_Y17

2

1

1

2

1

1

1

1

ST-14

2154_T14

2

1

1

1

1

1

7

1

ST-15

Note. *The Izh-16 strain is assigned to ST-1. **Alleles and sequence types of the first nine strains were designated based on the whole genome sequences, allelic profiles of the NL-IR-1 strain and isolates were obtained by testing the MLST scheme. ***For the unique alleles the nucleotide positions of MLST loci in the reference strains sequences described in Material and Methods section are indicated. The alleles nusB-6 and lysM-7 have identification numbers in the GenBank database OR192576 and OR134830, respectively.

 

When developing the in silico MLST scheme, 9 STs were found in 10 strains. Sequencing of gene fragments included in the MLST scheme was performed for 9 strains, and no discordant results were found in comparison with the data of whole-genome sequencing presented in GenBank. Further analysis of nucleotide sequences from DNA samples revealed 6 more STs, of which 2 were formed by two new alleles and 4 by new combinations of previously identified alleles. ST-1 (33; 36%) and ST-14 (25; 27.4%) were the most frequent in the studied sample, ST-12 (9; 10%), ST-10 (8; 9%), ST-2 (5; 5.5%) and ST-5 (2; 2.2%) were less frequent. ST-3, ST-4, ST-6, ST-7, ST-8, ST-9, ST-11, ST-13 and ST-15 occurred once (1.1% each).

The figure presents a dendrogram illustrating the genetic relationships of ST. The dendrogram and the results presented in Table 1 combine data on 91 representatives of the B. miyamotoi species from different sources.

 

Genetic relationships of sequence types within the B. miyamotoi species based on allelic profiles differences (the UPGMA algorithm). The number in parentheses is the strain or isolates name for the sequence types found once. The Roman numeral is the designation of clonal complexes.

 

Discussion

The genetic relationships of B. miyamotoi presented in the figure, the determination of which was based on the analysis of the number of mismatches in allelic profiles, allow us to classify the designated STs into 4 groups. The first 2 groups correspond to the clonal complexes labeled I and II in the figure, the other 2 groups are formed by ST-8 and ST-9, which have differences in all 8 loci from each other and from the other STs.

The first clonal complex (I) brings together 11 STs which are found in 80 (88%) of the characterized strains and DNA samples. ST-14 can be designated as the central one, because the allelic profile corresponding to it has the maximum number of mismatches at 1 MLST locus from the allelic profiles of other STs of this clonal complex, equal to 4. The second clonal complex (II) is formed by ST-10 and ST-11 which are found in 8 (8.7%) DNA samples and 1 (1.1%) strain. The allelic profiles of ST-10 and ST-11 have mismatches at 1 MLST locus and differ from the allelic profiles of the first clonal complex STs at all 8 MLST loci.

Table 1 presents STs and characterization of B. miyamotoi DNA sources. Most of the characterized DNA samples have STs found both in samples from ITBB patients and in tick suspensions. ST-3, ST-4, and ST-13 found once in ITBB pathogens and ST-6, ST-7, ST-11, and ST-15 found in tick suspensions do not allow the determination of specific genotypes associated with ITBB cases. Presumably, all representatives of the first clonal complex found in vectors can cause ITBB.

The pronounced genetic differences of characterized B. miyamotoi are related to the B. miyamotoi DNA sources. All the pathogens included in the first clonal complex were isolated from I. persulcatus tick suspensions obtained in Russia. The first clonal complex also contains strains FR64b and HT31 isolated in Japan from the same vector species. STs included in the second clonal complex were found in I. ricinus tick suspensions obtained from European countries and in the Kabardino-Balkar Republic. ST-8 and ST-9 B. miyamotoi were isolated from ticks of I. scapularis and I. pacificus species, respectively, distributed in North America.

Thus, the proposed classification based on MLST confirms the previously demonstrated genetic heterogeneity of B. miyamotoi populations corresponding to Asian (clonal complex I), European (clonal complex II) and American (ST not included in clonal complexes I and II) genotypes [1, 14, 15], circulating in different continents, which is associated with ecologically unrelated vectors of the pathogens.

Conclusion

In this study, based on the available whole-genome data, an MLST scheme was developed to differentiate B. miyamotoi pathogens collected in different territories. By the example of the first ST clonal complex distribution, it is possible to draw a conclusion about the most common variants of B. miyamotoi circulating in Russia and some foreign countries. This approach may become a convenient tool for monitoring the pathogens of ITBB and characterizing evolutionary changes in the described clonal complexes. The use of MLST involves the examination of biological material samples after qualitative detection of B. miyamotoi DNA or obtaining a culture of the pathogen. Due to the complexity of B. miyamotoi cultivation, the bulk of B. miyamotoi pathogens can be characterized using MLST and the rest are characterized with the help of whole-genome sequencing, which at the present stage is necessary for a thorough analysis of evolutionary changes occurring in bacterial populations.

Further studies should be focused on expanding the selection of characterized isolates, including validation of the developed in silico MLST scheme using nucleotide sequence information published in GenBank, as well as searching for possible relationships between the described B. miyamotoi ST and antigenic variants determined with the help of the main surface protein typing data [2]. After extended testing of the proposed MLST scheme, the possibility of combining all the obtained data into a common database, similar to existing standards for intraspecific characterization of other pathogens, may be considered [7].

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About the authors

Konstantin O. Mironov

Central Research Institute for Epidemiology

Author for correspondence.
Email: mironov@pcr.ru
ORCID iD: 0000-0001-8207-9215

D. Sci. (Med.), Head, Laboratory of molecular methods for genetic polymorphisms research, Central Research Institute for Epidemiology

Russian Federation, Moscow

Anton V. Titkov

Central Research Institute for Epidemiology

Email: mironov@pcr.ru
ORCID iD: 0000-0001-7548-9267

researcher, Laboratory of zoonoses, Central Research Institute for Epidemiology

Russian Federation, Moscow

Konstantin V. Kuleshov

Central Research Institute for Epidemiology

Email: mironov@pcr.ru
ORCID iD: 0000-0002-5238-7900

Cand. Sci. (Biol.), Head, Laboratory of molecular diagnostics and epidemiology of intestinal infections, Central Research Institute for Epidemiology

Russian Federation, Moscow

Alexander E. Platonov

Central Research Institute for Epidemiology

Email: mironov@pcr.ru
ORCID iD: 0000-0001-7450-0081

D. Sci. (Biol.), Prof., chief researcher, Laboratory of zoonoses, Central Research Institute for Epidemiology

Russian Federation, Moscow

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