Determination of molecular types and resistance to macrolides in Treponema pallidum isolates isolated in the Russian Federation

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Background

Syphilis caused by Treponema pallidum subspecies pallidum (T. pallidum) is a systemic sexually transmitted infectious disease. According to official state statistical surveillance, the incidence rate of all the syphilis forms in the Russian Federation in 2019 was 15.0 cases per 100,000 population; in 2020, 10.5; in 2021, 14.5; and in 2022, 18.9 cases. Hence, a significant increase in the incidence of syphilis was observed throughout the country in 2021–2022 [1].

Molecular typing is an important tool for determining the diversity and antibiotic resistance of circulating T. pallidum isolates. In 1998, the US Centers for Disease Control and Prevention (CDC) first introduced a typing scheme based on determining the number of 60-bp repeats in the arp (acidic repeat protein) gene and sequence differences in the tprII subfamily genes [2], which was subsequently supplemented by sequencing of the tp0548 gene region [3]. Results of molecular genetic typing of an individual clinical isolate are presented using triple digital and alphabetic designation (e.g., 14a/a) characterizing the variants of the arp, tprII and tp0548 genes found in it.

According to research results, the T. pallidum subtypes endemic to the Russian Federation belong to the Street Strain-14 (SS14) line [4], which holds a leading position in the world [5, 6]. Eight molecular subtypes of T. pallidum have been identified in the Russian Federation in 2014–2021: 14d/f, 14d/g, 14b/f, 14c/f, 14i/f, 9d/f, 14b/g, and 14e/f, with stable dominance of subtype 14d/f [7].

In accordance with Russian clinical guidelines, penicillins are first-line medications for treating syphilis [8]. However, despite their proven effectiveness, treatment of syphilitic infection with these drugs is limited by the possible development of allergic reactions [9–11]. In the 1990s, azithromycin, a macrolide antibiotic that was initially considered to be a convenient alternative to the therapeutic option benzathine benzylpenicillin G, started to be used to treat patients with early forms of syphilis.

Azithromycin is easy to use, requires no invasive procedures, has few side effects and can be used in accelerated partner therapy for syphilis [12]. In 1993–2003, azithromycin was included in the list of reserve drugs for treating early syphilis in the Russian Federation in cases when patients are intolerant to penicillin and other reserve drugs, doxycycline and cephalosporin [13–15]. Because of the increasing resistance of T. pallidum to macrolides [16, 17], azithromycin was removed from the subsequent Russian syphilis treatment guidelines. Some foreign guidelines, in particular the US Centers for Disease Control and Prevention (CDC), currently include this antibiotic as a second-line drug for treating early forms of syphilis [18].

A close association was shown to exist between macrolide resistance and the A2058G and A2059G mutations in the 23S rRNA of T. pallidum [19–21]. A feature of T. pallidum is that laboratory testing of its antibiotic sensitivity cannot be performed because of the nonculturability of the syphilis causative agent, which necessitates the use of molecular genetic research methods that allow one to comprehensively study the genetic determinants of resistance. Over the period between 2014 and 2021, three subtypes carrying the A2058G mutation associated with azithromycin resistance have been identified in the Russian Federation: 14d/g, 14b/g and 14b/f [7, 22].

The objective of this study is to analyze the molecular types and resistance to macrolides in T. pallidum isolates designated in the Russian Federation in 2022.

Methods

In 2022, 49 T. pallidum isolates were obtained from patients of dermatovenereological healthcare institutions in three federal districts of the Russian Federation: 24, from the Siberian Federal District; 23, from the Central Federal District, and two, from the North Caucasian Federal District. The diagnosis of syphilis was made based on clinical data and laboratory tests, including rapid plasma reagin (RPR) test, passive hemagglutination (PHA) test, and enzyme-linked immunosorbent assay (ELISA) [23].

Among syphilis patients from whom clinical isolates (separate from erosive and ulcerative elements) containing Treponema pallidum were derived, there were 35 males and 14 females aged 16–76 years. The patients were distributed as follows according to their diagnosis: 14 patients were diagnosed with primary syphilis (A51.1 according to ICD-10); 34 patients, with secondary syphilis (A.51.3); and one patient, with syphilis of other localizations (A51.2).

DNA was extracted from clinical samples using a Proba-NK set of reagents (DNA-Technology, Russia) according to the manufacturer’s instructions. The presence of genetic material of T. pallidum in clinical samples was confirmed by PCR with primers for the species-specific polA gene encoding DNA polymerase I of this microorganism (Table 1) [24].

 

Table 1. Primer sequences used for amplification of T. pallidum target genes

Таблица 1. Последовательности праймеров, использованных для амплификации целевых генов T. pallidum

Gene	Primer name	Nucleotide sequence
arp	ARP-1	5’-ATCTTTGCCGTCCCGTGTGC-3’
	ARP-2	5’-CCGAGTGGGATGGCTGCTTC-3’
tprII	A-1	5’-ACTGGCTCTGCCACACTTGA-3’
	B-2	5’-CTACCAGGAGAGGGTGACGC-3’
	IP-6	5’-CAGGTTTTGCCGTTAAGC-3’
	IP-7	5’-AATCAAGGGAGAATACCGTC-3’
tp0548	tp0548 sense	5’-GGTCCCTATGATATCGTGTTCG-3’
	tp0548 antisense	5’-GTCATGGATCTGCGAGTGG-3’
23 S	23 Sf	5’-GTCTCCCACCTATACTACACAT -3’
	23 Sr	5’-GGAGAGGTTCGTGGTAACACA -3’

 

Molecular typing of the samples with confirmed presence of T. pallidum genetic material was conducted following the algorithm recommended by the Centers for Disease Control and Prevention (CDC) in Druid Hills, Atlanta, Georgia. The procedure and assessment of the method results were described previously [25]. Amplification of T. pallidum genes was performed using specific primers [24] on a DNA amplification T100 Thermal Cycler (Bio-Rad, USA). After the first amplification stage, the PCR product was purified using the QIAquick PCR Purification Kit (Qiagen, Germany). The purified PCR product was utilized for the second amplification stage using labeled terminating nucleotides from the Big Dye Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems, USA). Precipitated PCR products were used for sequencing a variable fragment of the 23S rRNA gene on a 3130 Genetic Analyzer (Applied Biosystems, USA) with the 3130 Data Collection software v. 3.0 [7]. Primary nucleotide sequence decoding was performed using the Sequencing Analysis 5.3.1 software. The analyzed fragments of the target genes were aligned to the reference sequences of T. pallidum using the Mega 5 software.

Results

In 49 clinical samples, the presence of T. pallidum DNA was confirmed using PCR with primers targeting the polA gene. Molecular typing of isolates based on the arp, tpr, and tp0548 genes allowed one to identify the complete molecular subtype of each isolate. One variant of the arp gene (variant 14) and tprII gene (variant d) and three variants of the tp0548 gene (variants d, f, and g) were detected. Therefore, three molecular subtypes were identified in the analyzed population: 14d/f (32 strains, 65%), 14d/d (10 strains, 20%), and 14d/g (7 strains, 15%); the 14d/d subtype was detected for the first time (Figure 1).

 

Fig. 1. Nucleotide sequence variants in the tp0548 gene of T. pallidum

Рис. 1. Варианты нуклеотидных последовательностей в гене tp0548 T. pallidum

 

The A2058G transition, with a proven role in providing high-level resistance to macrolide antibiotics, was associated with 14d/d and 14d/g subtypes (a total of 17 strains, 35%). Table 2 shows the distribution of isolates by federal districts.

 

Table 2. Distribution of T. pallidum subtypes by federal districts

Таблица 2. Распределение субтипов T. pallidum по федеральным округам

Federal District	Territorial entity of the Russian Federation	Number of isolates for each subtype
		14d/f	14d/g*	14d/d*
North Caucasian	Stavropol	—	—	2
Siberian	Kyzyl	24	—	—
Central	Moscow	8	6	8
	Kaluga Oblast	—	1	—

*Subtypes containing a macrolide resistance mutation.

*Субтипы, содержащие мутацию устойчивости к макролидам.

 

Discussion

Because Treponema pallidum is a non-culturable pathogen that does not grow on culture media, antibiotic resistance of this microorganism is studied using molecular genetic methods by determining the known and potential genetic determinants of resistance. Of particular interest is the search for determinants of T. pallidum resistance to broad-spectrum antibacterial drugs used as an alternative treatment regimen in cases of penicillin intolerance, which is observed in 8–12% of the population. Two mutations identified in the 23S rRNA gene are mentioned in the literature. The first mutation, A2058G, confers resistance to macrolides with a 14-membered (erythromycin, roxithromycin, clarithromycin) and 15-membered (azithromycin) lactone ring in T. pallidum. The second mutation, A2059G, confers resistance to 14-, 15-, and 16-membered macrolides (spiramycin and tylosin). A limitation of the current study is that clinical manifestations of the identified resistance cannot be investigated since azithromycin is excluded from federal clinical recommendations for managing syphilis. However, the data on macrolide resistance based on the conducted molecular typing of clinical isolates allowed one to analyze the Russian population of T. pallidum.

In 2022, three subtypes of T. pallidum were identified in the represented federal districts. It is worth noting the ongoing dominance of the endemic subtype 14d/f in Russia, which remains sensitive to macrolides. The macrolide-resistant subtype 14d/g was previously detected in the Siberian and Central Federal Districts. In 2022, a new macrolide-resistant subtype 14d/d emerged in the Central (predominantly) and North Caucasian Federal Districts. This particular subtype of T. pallidum has never been predominant in any country but is among the most common ones in Argentina, also being occasionally found in the Czech Republic, France, and Australia. Isolates of this subtype with the A2058G macrolide resistance mutation have been identified in Brazil.

These findings indicate the continuous expansion of the T. pallidum population in Russia, including the emergence of azithromycin-resistant strains. Thus, in 2013, T. pallidum isolates carrying the A2058G mutation associated with azithromycin resistance belonged to subtypes 14d/g and 14b/f; in 2016, to subtype 14b/g; and in 2022, to subtype 14d/d. Additionally, isolates 14b/g and 14e/f without the macrolide resistance mutation were identified in the Siberian Federal District in 2016 and 2017. According to the Federal State Statistics Service, in 2022, in Moscow, there were twice as many new syphilis cases diagnosed compared to the previous year. A rise in migration flows is one of the reasons for the increase in the incidence rate. A less obvious reason could be the uncontrolled use of antibiotics, which may mask the symptoms of the disease.

Ethical review

Based on our research, patients are not involved, and there is no intervention in the course of treatment. The study exclusively uses biomaterial specimens from individuals in the Russian Federation. The information obtained does not allow either direct or indirect identification of patients and cannot lead to any risks of criminal or civil responsibility for patients, nor jeopardize their financial status, employment, or reputation.

Conclusion

The results of molecular typing of clinical isolates of T. pallidum conducted in 2022 confirmed the existing trend: in 2022, the molecular type 14d/f dominated among the samples of T. pallidum circulating in the Russian Federation, while other subtypes were less common; all the samples with the subtype g of the tp 0548 gene carried the dominant A2058G macrolide resistance marker.

Since Russia is considered a geographic region with low prevalence of macrolide resistance, the emergence of isolates carrying markers of resistance to azithromycin can be attributed to cross-border transmission associated with labor migration or tourism from a specific geographic region. Therefore, continuous monitoring of the Russian population of T. pallidum can facilitate understanding the geographical distribution of the infection and antibiotic resistance of T. pallidum strains.

About the authors

Olga A. Obraztsova

State Research Center of Dermatovenereology and Cosmetology

Author for correspondence.
Email: valeeva19@gmail.com
ORCID iD: 0000-0002-5728-2139
SPIN-code: 6355-4699

Cand. Sci. (Biol.)

Россия, Moscow

Kseniya M. Lagun

State Research Center of Dermatovenereology and Cosmetology

Email: xobanaa@mail.ru
ORCID iD: 0009-0004-9700-2455
SPIN-code: 4770-8904
Россия, Moscow

Georgii L. Katunin

State Research Center of Dermatovenereology and Cosmetology

Email: g.katunin@rambler.ru
ORCID iD: 0000-0003-0599-6305
SPIN-code: 1598-8595

MD, Cand. Sci. (Med.)

Россия, Moscow

Marina V. Shpilevaya

State Research Center of Dermatovenereology and Cosmetology

Email: aniram1970@list.ru
ORCID iD: 0000-0002-9957-4009
SPIN-code: 6600-3311

Cand. Sci. (Biol.)

Россия, Moscow

Nikita Y. Nosov

State Research Center of Dermatovenereology and Cosmetology

Email: nnosov@cnikvi.ru
ORCID iD: 0000-0002-3967-8359
SPIN-code: 8806-8539

Cand. Sci. (Biol.)

Россия, Moscow

References

Supplementary files