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Tetracycline Resistance Genes in Treponema spp.- an Analysis of 4355 Spirochaetales Genomes

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08 March 2024

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11 March 2024

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Abstract

Background: The resurgence of syphilis, primarily in MSM populations, has necessitated novel prophylactic strategies, such as the use of doxycycline post-exposure prophylaxis (doxy-PEP). However, the potential for increased doxycycline use to select for tetracycline resistance represents significant challenges in managing this sexually transmitted infection. This study aims to identify chromosomal mutations associated with tetracycline resistance in Spirochaetales to inform treatment strategies and surveillance measures.Methods: Whole genome sequences (WGS) from the Spirochaetales order, including 4,355 genomes, were analyzed for the presence of mutations in 16S rRNA and non-synonymous mutations in the rpsC and rpsJ genes. The study utilized WGS from GenBank® and sequence data from the PubMLST Treponema pallidum isolate collection. Genetic resistance to tetracycline was detected using a combination of BLASTN searches and gene-gene analysis. Results: A transition mutation TGA to TGG at positions 965-967 in the 16S rRNA gene was detected in 5.6% of Treponema spp. and 3.97% of Spirochaeta spp. genomes. The rpsJ gene exhibited a V57G amino acid substitution across a significant subset of Treponema spp. Notably, the V57K mutation was common in Spirochaeta spp. The rpsC gene had the H178Q mutation and was found to be present in the Spirochaetales bacterium.Conclusion: The identification of mutations associated with tetracycline resistance in Spirochaetales provides a foundation for the development of rapid molecular diagnostics and guides the clinical selection of antibiotics for syphilis treatment. This study underscores the complexity of antibiotic resistance mechanisms and the critical importance of surveillance in the era of antibiotic prophylaxis for STI management.

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Subject: Biology and Life Sciences - Life Sciences

Introduction

The incidence of bacterial STIs in general, but syphilis in particular, has recently increased dramatically in a number of countries, including Belgium [1]. These increases have led to calls for novel measures to reverse this trend. One of the most promising interventions is the ingestion of doxycycline after condomless sex (Doxycycline post-exposure prophylaxis or doxy-PEP), which has been shown to reduce the incidence of syphilis, chlamydia and possibly gonorrhoea in men who have sex with men (MSM) [2,3,4]. These findings have led a number of organizations, such as the Centers for Disease Control and Prevention (CDC) and the European AIDS Clinicians Society (EACS), to promote the use of doxy-PEP in sub-populations of MSM [5]. We have calculated that the introduction of doxy-PEP in Belgian HIV-pre-exposure prophylaxis (PrEP) cohorts would increase the consumption of doxycycline by up to 88-fold [6]. This intense consumption of doxycycline could induce and select for antimicrobial resistance (AMR) to tetracyclines and other antimicrobials in a range of bacterial species [7,8,9]. Doxycycline is a vital backup medication for individuals who cannot receive intramuscular penicillin due to frequent stock-outs, silicone implants or penicillin allergy [10]. If doxy-PEP were to induce tetracycline resistance in Treponema pallidum subsp. pallidum, the causative agent of syphilis, this would have far-reaching consequences.

Previous genotypic assessments of T. pallidum have not found mutations that have been predicted to cause tetracycline resistance [11, 12]. One exception is a study from China that detected the tetB gene directly from 15/171 syphilitic lesions via PCR amplification [13]. Concerns have, however, been raised that the tetB genes detected represented contamination from other bacteria present in these clinical samples [14]. Because T. pallidum is not known to contain any mobile elements, point mutations in specific regions of the gene encoding the 16S rRNA or specific ribosomal proteins would be the most plausible pathway to tetracycline resistance [12, 15]. Furthermore, A2058G and A2059G mutations in T. pallidum's 23S rRNA that confer resistance to macrolides have arisen independently in multiple lineages of T. pallidum and spread to attain prevalences close to 100% [16, 17]. The 16S and 23S rRNA are part of the same single-copy operon in T. pallidum, and thus, point mutations are possible in the 16S rRNA gene.

T. pallidum is challenging to culture in vitro, and thus far, a single study has established that the doxycycline MICs of the four strains assessed were between 0.06mg/L to 0.1mg/L [18]. No study has attempted to induce tetracycline resistance in vitro or in vivo. For such studies and for the evaluation of suspected tetracycline resistance in human infections, it would be useful to know which resistance mutations are possible based on what resistance-associated mutations occur in related bacterial species. This study assesses the occurrence and prevalence of tetracycline resistance associated with chromosomal mutations in Spirochetales. The Spirochetales order contains three families: Spirochaetaceae, Brachyspiraceae, and Leptospiraceae. Previous studies have found that mutations in 16S, rpsC and rpsJ have been shown to be associated with tetracycline susceptibility in several other spirochaetes, such as Borrelia spp., and Leptospira spp. [19,20,21].

Materials and Methods

Dataset for Analysis

The whole genome sequence (WGS) used in this study was acquired from GenBank®. This collection included 4,355 genomes from the order Spirochaetales (NCBI Taxonomy ID: 136). Additionally, the study incorporated 16S sequence data from PubMLST Treponema pallidum isolate collection. This collection included 613 sequences.

Detection of genetic resistance to tetracycline

WGS were screened for tetracycline resistance, and the analysis was carried out as described [22]. In brief, WGS (n = 4355) was analyzed using chewBBACA version 2.8.5 [23]. Firstly, a training file was created from the complete genome of T. pallidum (AE000520.1) using Prodigal and used in subsequent steps [24]. Secondly, a study-specific T. pallidum schema was created, and a FASTA file for each coding sequence (CDS) was generated. Thirdly, a BLAST database was created using the study-specific schema, herein referred to as the schema database. This was followed by a BLASTN search against the schema database using the rpsC and rpsJ genes from T. pallidum (AE000520.1) as queries. The multiple sequence alignment files were imported into CLC Genomics Workbench (v20), and the CDSs were translated. The presence of non-synonymous substitutions in the following proteins was further evaluated: rpsC (Lys4; His175) and rpsJ (Val57).

For 16S analysis, a BLASTN search using the 16S rRNA T. pallidum (AE000520.1) sequence was queried against the BLAST database that was created using all 4,355 genomes. A BLASTN search was also performed against T. pallidum isolate collection in PubMLST. Thus, for the 16S analysis, the dataset included the genomes and sequences from Genbank and PubMLST, respectively.

Results

Mutations in the 16S rRNA gene

Our analyses revealed a transition mutation from TGA to TGG at positions 965 to 967 (Escherichia coli 16S rRNA numbering) observed among Treponema (5.6% occurrence) and Spirochaeta spp. (3.97% occurrence) genomes. Additional mutations at the same positions, including CGC, GGT, CGA, TGR(A/G), TGC and AGC, were detected less frequently (Table 1).

Figure 1. Flowchart describing genomes and sequences used in the analyses.

Mutations in the rpsJ gene

Analysis of the rpsJ gene, which encodes the 30S ribosomal subunit protein S10, showed the presence of the V57G substitution that was observed in both the Treponema porcinum genomes and 20% of Treponema sp. genomes (Table 1). The presence of additional variants, V57I and V57K, was also observed at a lower incidence (Table 1). The substitution V57K (169 GTG-AAG 171) frequently appeared in the Spirochaeta spp. (1.69% of genomes) and other Treponema spp. (1.12% of genomes) (Table 1).

Mutations in the rpsC gene

An H178Q substitution in the rpsC gene, associated with the 30S ribosomal subunit protein S3, was found in 80% of the genomes of the Spirochaetales bacterium (Table 1).

Discussion

The emergence and dissemination of antimicrobial resistance (AMR) are of utmost concern in infectious disease management, particularly in the context of sexually transmitted infections (STIs) such as syphilis. The resurgence of syphilis incidence in various countries, including Belgium, necessitates innovative interventions to curb this trend [1]. One such intervention is the administration of doxycycline as post-exposure prophylaxis (doxy-PEP), which has demonstrated efficacy in reducing the incidence of syphilis and other STIs among men who have sex with men (MSM) [5]. However, the extensive use of doxycycline raises concerns about the potential induction of AMR, especially considering its critical role as an alternative to penicillin, the first-line treatment [10].

Our study focused on the potential chromosomal mutations associated with tetracycline resistance within the Spirochetales order. The genomic data from this order, including 4,355 whole genome sequences and an additional 613 16S rRNA sequences, were systematically analyzed for the presence of mutations in 16S rRNA and non-synonymous mutations in the rpsC and rpsJ genes, which encode the 30S ribosomal subunit proteins S3 and S10, respectively.

Our analysis revealed the transition mutation TGA to TGG at positions 965 to 967 (Escherichia coli 16S rRNA numbering) in the 16S rRNA gene of Treponema and Spirochaeta spp. genomes. The identification of this mutation across these species suggests a potential mechanism of resistance that might become more prevalent under selective pressure from increased doxycycline use [25]. Although less frequent, other mutations in this gene point to the potential for diverse resistance mechanisms within these genera.

In the rpsJ gene, the V57G amino acid substitution was present in both the genomes of Treponema porcinum and a significant subset of Treponema spp., and this mutation could confer a fitness advantage in the presence of tetracycline. The V57I and V57K substitutions were also observed but at lower frequencies. Notably, the GTG-AAG (V57K) mutation at nucleotide positions 169-171 was relatively common in Spirochaeta spp. and other Treponema spp.. The minimal inhibitory concentration (MIC) of E. coli to tetracycline with V57K substitution was found to be 0.75 μg/ml, and Escherichia coli with V57I substitution showed a slightly increased MICs to tigecycline and tetracycline [26]. Substitutions in V57 amino acid positions that confer resistance to tetracycline or tigecycline have been observed in both Gram-negative and Gram-positive bacteria, namely E. coli, Neisseria gonorrhoeae, Neisseria meningitidis, Klebsiella pneumoniae, Bacillus subtilis, Enterococcus faecium, Enterococcus faecalis and Staphyloccus aureus [26,27,28,29].

The rpsC gene analysis uncovered an H178Q (corresponding to the H175 position in Streptococcus pneumoniae) substitution in a substantial majority of the Spirochaetales bacterium genomes, highlighting the critical nature of this mutation within this specific bacterial group [30].

In light of the widespread use of doxy-PEP, the putative mutations associated with tetracycline resistance reported in this study provide a prerequisite for developing molecular methods for the rapid detection and surveillance of antibiotic-resistant T. pallidum in clinical specimens [31]. Our study contributes to the foundational knowledge necessary for guiding future studies and public health strategies to mitigate the spread of AMR in the context of STI management.

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Table 1. List of the tetracycline resistance associated mutations.

Ribosomal genes	Source of genomes	Organism	Total no of genomes	Mutations	No of genomes with mutations	No of genomes with 2 copies	No of genomes with >2 copies	Copy number
16S rRNA	Genbank	Treponema parvum	5	TGA 965-967 TGG	2	2		1-2 copies
		Treponema parvum	5	TGA 965-967 GGT	1	0		1 copy
		Treponema brennaborense	4	TGA 965-967 TGG	1	1		4 copies
		Treponema peruense	4	TGA 965-967 TGG	1	1		4 copies
		Treponema bryantii	5	TGA 965-967 TGG	2	0	3	1-4 copies
		Treponema sp.	60	TGA 965-967 CGC	1	0		1 copy
		Other Treponema spp.	1341	TGA 965-967 TGG	71	24	9	1-3 copies
		Spirochaeta spp.	1006	TGA 965-967 CGA	8	0		1 copy
		Spirochaeta spp.	1006	TGA 965-967 TGG	40	18		1 to 5 copies
	PubMLST	Treponema pallidum	544	TGA 965-967 TGG	1	-	-	-
				TGA 965-967 TGR(A/G)	2	-	-	-
				TGA 965-967 TGC	1	-	-	-
				TGA 965-967 AGC	1	-	-	-
rpsJ (30S ribosomal subunit protein S10)	Genbank	Candidatus Borreliella tachyglossi	1	V57A	1	-	-	-
		Treponema porcinum	2	V57G	2	-	-	-
		Treponema sp.	60	V57G	12	-	-	-
				V57I	1	-	-	-
				V57K	3	-	-	-
		Borrelia persica	1	V57I	1	-	-	-
		Spirochaeta spp.	1006	V57G	1	-	-	-
				V57I	4	-	-	-
				V57K	17	-	-	-
		Other Treponema spp.	1341	V57K	15	-	-	-
rpsC (30S ribosomal subunit protein S3)	Genbank	Spirochaetales bacterium	5	H178Q	4	-	-	-

- Not available or not determined.

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