Mycoplasma genitalium molecular typing in men with non-gonococcal urethritis discriminates between phylogenetic clusters based on sexual preference and antibiotic resistance

Abstract

Introduction.Mycoplasma genitalium is a sexually transmitted bacterium associated with non-gonococcal urethritis (NGU) in men. The rising macrolide and fluoroquinolone resistance in M. genitalium has become a public health concern, requiring close surveillance.

Gap statement.MgpB/MG309 typing is commonly used to study genotype distribution and resistance patterns of M. genitalium in men who have sex with men (MSM); however, data for men who have sex with women (MSW) are limited.

Aim. The aim of this study was to explore the epidemiology of M. genitalium based on mgpB/MG309 molecular typing in isolates from men diagnosed with NGU, comparing MSM and MSW. Additionally, antibiotic resistance was evaluated to assess associations between the mgpB/MG309 genotypes, antimicrobial resistance profiles, and epidemiological determinants.

Methodology. A subset of previously collected M. genitalium isolates from men diagnosed with NGU in Amsterdam, the Netherlands, between May 2018 and November 2019 was analysed. Molecular typing was performed by sequencing relevant regions of the mgpB and MG309 loci. Macrolide resistance was assessed by detecting mutations in the 23S rRNA gene via quantitative polymerase chain reaction, while fluoroquinolone resistance was determined through sequencing parC and gyrA.

Results. A total of 62 M. genitalium samples were analysed from 33 MSM and 29 MSW. The overall macrolide and fluoroquinolone resistance was 75.8% and 24.2 %, respectively. At the mgpB locus, 24 sequence types (STs) were identified, with ST4 most prevalent in MSM and ST2 in MSW. The MG309 locus revealed 12 distinct short tandem repeat numbers, with repeat 10 being most common in both groups. Phylogenetic analysis based on mgpB sequences revealed two clusters: cluster A included significantly more MSW, whereas cluster B predominantly comprised MSM (P<0.001). Macrolide and fluoroquinolone resistance was significantly higher in cluster B compared with cluster A (P<0.01 and P<0.05, respectively).

Conclusion. Molecular typing of M. genitalium revealed two clusters that differed by sexual preference and antibiotic resistance, highlighting the importance of surveillance of resistance across genotypes. The findings suggest multiclonal spread of resistance through independent mutations. Future studies using next-generation sequencing are needed to further explore the links between sexual transmission and genetic diversity in M. genitalium.

Data Availability

Data are available upon reasonable request. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Introduction

Mycoplasma genitalium is a sexually transmitted bacterium implicated in both acute and persistent non-gonococcal urethritis (NGU) in men [1]. The prevalence of M. genitalium infection among men diagnosed with NGU is ~10–35% [2], making it the second most common cause after Chlamydia trachomatis infection [3]. Due to its minimalistic genome and lack of a cell wall, M. genitalium exhibits intrinsic resistance to several antibiotics, limiting the options for treatment [1]. For symptomatic M. genitalium infections in men, current treatment guidelines in Europe recommend macrolides as the first-line antibiotic, followed by fluoroquinolones as the preferred alternative in case of initial therapy failure [4]. However, the extensive use of these antibiotics has led to widespread resistance in Europe, with the prevalence of macrolide and fluoroquinolone resistance mutations ranging from 8% to 89% and 4% to 34%, respectively [5].

Macrolide treatment failure is attributed to single nucleotide polymorphisms (SNPs) in domain V of the 23S rRNA gene at positions A2058 and A2059, referred to as macrolide resistance-associated mutations (MRAMs) [5,6]. Similarly, fluoroquinolone treatment failure is linked to mutations in the parC and gyrA genes, collectively known as the quinolone resistance-associated mutations (QRAMs). Clinically significant mutations include S83I, S83R, D87N and D87Y in parC, and M95I and D99Y in gyrA.5

To further explore the transmission dynamics of M. genitalium within populations, molecular typing methods are frequently employed. These include SNP analysis of the mgpB gene, which encodes the hypervariable adhesin protein MgPa [7], and the examination of the MG309 gene, which involves quantifying the number of short tandem repeats (STRs), specifically the AGT/AAT motifs [8,9]. Combined, these analyses possess sufficient discriminatory power to effectively distinguish between closely related M. genitalium isolates [10]. While the mgpB/MG309 molecular typing method has been commonly used to investigate M. genitalium transmission in sexual networks of men who have sex with men (MSM) [11,12] and women [11], only limited molecular typing data are available for M. genitalium isolates infecting men who have sex with women (MSW).

A comprehensive understanding of the distribution of M. genitalium genotypes across various populations and their potential association with resistance patterns is essential to combat the growing antimicrobial resistance observed. The aim of this study was to explore the epidemiology based on mgpB/MG309 molecular typing among M. genitalium isolates from men diagnosed with NGU, comparing MSM and MSW. Additionally, antibiotic resistance was evaluated to assess potential associations between the mgpB/MG309 genotypes, antimicrobial resistance profiles, and relevant epidemiological determinants.

Methods

Study patient population and sample selection

A subset of samples from a previously conducted study at the sexually transmitted infection (STI) clinic in Amsterdam, the Netherlands, was used [13]. From May 2018 to November 2019, men diagnosed with urethritis visiting the STI clinic were included. First-void urines were tested for Neisseria gonorrhoeae, C. trachomatis and M. genitalium using transcription-mediated amplification assays (Aptima, Hologic Inc, San Diego, CA, USA). M. genitalium infection was re-evaluated by MgPa quantitative polymerase chain reaction (qPCR) and genotyped for MRAM SNPs by single-target qPCR assays [14,15]. The subset of samples included in the current study consisted of randomly selected urine specimens that tested positive for M. genitalium, with a Ct-value <32.

DNA sequencing and molecular typing

Sanger sequencing of relevant regions of parC (319 bp) and gyrA (228 bp) was performed on remnant DNA using primer combinations as previously described by Deguchi et al. [16]. The cycling conditions for parC and gyrA were as follows: activation step of 95 °C for 2 min; 35 cycles of 95 °C (15 s), 65 °C (30 s) and 72 °C (30 s); final elongation step of 72 °C for 6 min. Sanger sequencing of relevant regions of mgpB (281 bp) and MG309 (~380 bp) was performed using primer combinations as previously described [7,8, 17]. Cycling conditions for mgpB were as follows: activation step of 98 °C for 30 s; second activation step of 95 °C for 2 min; 35 cycles of 95 °C (30 s), 60 °C (30 s) and 72 °C (60 s). For MG309, cycling conditions included: activation step of 95 °C for 15 min; 35 cycles of 95 °C (45 s), 56.8 °C (60 s) and 72 °C (60 s); final elongation step of 72 °C for 10 min.

PCR products were purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany). Subsequently, samples were sent for high-throughput Sanger sequencing to Eurofins Genomics (Ebersberg, Germany). Sequence analysis was performed using Bionumerics Version 7.6.3. (Applied Maths, BioMérieux, Marcy-l'Étoile, France). Obtained sequences were compared to the M. genitalium G37 reference strain (accession number NC_000908.2). For MG309, typing was determined by the number of STRs, independent of the specific repeat motifs, as is outlined in previously conducted studies [9,18]. Sequence typing of mgpB was based on SNPs, using the Public databases for molecular typing and microbial genome diversity (PubMLST) [19]. Newly found sequences were deposited to the PubMLST database, and consecutive sequence type (ST) numbering was given for isolates differing from the previously known genotypes. For mgpB, a maximum-likelihood phylogenetic tree using the best-fitting TPM2u+F+I substitution model and 1,000 bootstrap values was generated by IQ-TREE (version 2.3.4). Tree annotation was performed using iTol (version 6.9). The individual and combined discriminatory index (DI) for the mgpB and MG309 typing methods was calculated by applying Simpson’s index of diversity [20].

Statistical analysis

Patient information was extracted from electronic patient files which were blinded for the investigators. The variable ‘educational level’ was categorized into four groups as follows: low (no education, primary school, lower secondary vocational education, and intermediate secondary general education), middle (higher secondary general education, senior secondary vocational education, and pre-university secondary education), high (higher professional or university education) and unknown. Sexual preference was self-reported by the patients. Bisexual men were excluded from this study. The Chi-square test, Fisher’s exact test and Mann–Whitney U test were used to evaluate significant differences in epidemiological factors between MSM and MSW (P-value < 0.05). Statistical analyses were performed in SPSS Statistics (version 26.0, IBM, Armonk, NY, USA), and GraphPad Prism (version 8, GraphPad Software, La Jolla California, USA) was used to create figures.

Results

Patient characteristics

Sequencing was performed on M. genitalium-positive specimens originating from 126 patients diagnosed with NGU, successfully achieving sequencing for all four targets in isolates from 66 (52.4%) patients. Four of the patients were excluded since no sociodemographic and/or clinical data were available, leaving 62 (49.2%) patients for further analyses. Of these, 33 (53.2%) self-identified as MSM and 29 (46.8%) as MSW (Table 1). The median age of all patients was 27 years (interquartile range, 23–32.5), with MSM being significantly older compared with MSW (P<0.001). The majority reported their region of origin as either the Netherlands (37.1%) or Central and South America (35.5%). MSM had a higher proportion of patients originating from Asia, whereas MSW exhibited a significantly greater representation from Central and South America (P<0.05). Human immunodeficiency virus (HIV) status was available for 54/62 (87.1%) patients, with 9 (14.5%) testing positive, all of whom were MSM. Co-infections with C. trachomatis and N. gonorrhoeae were observed in 14.5% and 9.7% of all patients, respectively. MSW demonstrated a significantly higher prevalence of C. trachomatis co-infection (27.6%) compared with MSM (P<0.01). Additionally, a history of previous urethritis was reported by 48.5% of MSM, which was significantly higher than that in MSW (P<0.05). MRAMs were identified in 47 of 62 patients (75.8%), being 84.8% and 65.5% for MSM and MSW, respectively. The overall prevalence of QRAMs was observed in 24.2% of patients, with a higher prevalence of 30.3% in MSM. QRAMs were exclusively attributed to mutations in the parC gene, as no mutations in the gyrA gene were identified.

Table 1. Patient characteristics.

	All patients (n=62)	MSM (n=33)	MSW (n=29)	P-value
Age, median (IQR)	27 (23–32.5)	30 (26–36.5)	24 (22.5–27.5)	<0.001*
Region of origin, n (%)
The Netherlands	23 (37.1)	13 (39.4)	10(34.5)	<0.05†
Europe, outside the Netherlands	6 (9.7)	5 (15.2)	1 (3.4)
Africa	4 (6.5)	2 (6.1)	2 (6.9)
Asia	6 (9.7)	6 (18.2)	0 (0)
Central and South America	22 (35.5)	7 (21.2)	15 (51.7)
North America	0 (0)	0 (0)	0 (0)
Oceania	0 (0)	0 (0)	0 (0)
Unknown	1 (1.6)	0 (0)	1 (3.4)
HIV status, n (%)
Negative	45 (72.6)	23 (69.7)	22 (75.9	<0.001†
Positive	9 (14.5)	9 (27.3)	0 (0)
Unknown	8 (12.9)	1 (3.0)	7 (24.1)
Education level, n (%)
Low	11 (17.7)	3 (9.1)	8 (27.6)	0.238†
Middle	11 (17.7)	6 (18.2)	5 (17.2)
High	31 (50.0)	18 (54.5)	13 (44.8)
Unknown	9 (14.6)	6 (18.2)	3 (10.3)
No. of sexual partners last 3 months, median (IQR)	4.5 (2–10)	5 (3–14.5)	4 (2-7)	0.150*
C. trachomatis co-infection, n (%)
Negative	53 (85.5)	32 (97.0)	21 (72.4)	<0.01†
Positive	9 (14.5)	1 (3.0)	8 (27.6)
N. gonorrhoeae co-infection, n (%)
Negative	56 (90.3)	29 (87.9)	27 (93.1)	0.679†
Positive	6 (9.7)	4 (12.1)	2 (6.9)
Previous antibiotic use last3 months, n (%)
No	46 (74.2)	21 (63.6)	25 (86.2)	0.079‡
Yes	16 (25.8)	12 (36.6)	4 (13.8)
Previous urethritis in last2 years, n (%)
No	40 (64.5)	17 (51.5)	23 (79.3)	<0.05‡
Yes	22 (35.5)	16 (48.5)	6 (20.7)
Macrolide resistance (MRAM), n (%)
Mutation	47 (75.8)	28 (84.8)	19 (65.5)	0.070*
Wildtype	15 (24.2)	5 (15.2)	10 (34.5)
Fluoroquinolone resistance (QRAM), n (%)
Mutation	15 (24.2)	10 (30.3)	5 (17.2)	0.254*
Wildtype	47 (75.8)	23 (69.7)	24 (82.8)

*Mann–Whitney U test

†Fischer’s exact test

‡Chi-square test

HIV, human immunodeficiency virus; IQR, interquartile range; MRAM, macrolide resistance-associated mutation; MSM, men who have sex with men; MSW, men who have sex with women; QRAM, fluoroquinolone resistance-associated mutation.

Molecular typing of M. genitalium isolates and antibiotic resistance

For the mgpB locus, 24 distinct STs were observed among the 62 patients included in this study (DI=0.9101), including 13 new STs (ST262–ST275) (Table S1, available in the online Supplementary Material). Of the 13 newly identified STs, 10 (76.9%) were isolated from patients in the MSW group. None of the observed STs matched the reference G37 strain (ST1). ST4 showed the highest prevalence among MSM, accounting for 45.5% of the cases, whereas ST2 was the most prevalent type among MSW, with a prevalence of 20.7% (Fig. 1a, b). Isolates with mgpB ST4 harboured MRAMs in 14 of 16 cases (87.5%), whereas none contained QRAMs (Fig. 2). In contrast, isolates with mgpB ST2 carried MRAMs in only 2/7 cases (28.6%) and QRAMs in 1/7 cases (14.3%). ST7 was among the most abundant ST for both MSM and MSW, with a prevalence of 6.1% and 10.3%, respectively. For mgpB ST7 isolates, MRAMs were present in 4/5 cases (80.0%) and QRAMs in all 5 cases (100%).

Fig. 1. Distribution of most prevalent sequence types (STs) for mgpB in men who have sex with men (MSM) (a) and men who have sex with women (MSW) (b) and number of short tandem repeats (STRs) for MG309 in MSM (c) and MSW (d). Only STs/STRs with a frequency count of ≥2 isolates are indicated.

Fig. 2. Phylogenetic tree of mgpB sequences from 62 M. genitalium isolates (black). Three previously documented isolates from PubMLST were included as references (red), including the reference strain (G37), M6282 (male with urethritis from Japan) and M6320 (male with urethritis from Australia). The tree is annotated with sample name, sex preference, macrolide resistance (MRAM), fluoroquinolone resistance (QRAM), mgpB sequence type (ST) and MG309 short tandem repeat (STR) number. The maximum-likelihood tree was constructed using the TPM2u+F+I substitution method with 1,000 bootstraps. Clusters A and B could be identified, containing 21 and 40 M. genitalium isolates, respectively. Isolate MGU057 was designated as an outlier due to its low bootstrap value (Fig. S1) and was, therefore, excluded from the two phylogenetic clusters.

Analysis of the MG309 locus identified 12 distinct numbers of STRs across the 62 patients included in this study (DI=0.8149), varying from 8 to 22 repeats (Table S2). Five isolates, all derived from MSW patients, exhibited an STR12, consistent with the reference G37 strain. For isolates from both MSM and MSW, STR10 was most prevalent, accounting for 45.5% and 31.0%, respectively (Fig. 1c–d). Isolates with STR10 contained MRAMs in 17 of 24 cases (70.8%), whereas isolates from 8 of 24 cases (33.3%) contained QRAMs (Fig. 2). The lowest STR number of 8 was observed in two MSW patients, corresponding to isolates MGU057 and MGU138. In contrast, the highest STRs of 21 and 22 were identified in isolates MGU030 and MGU023, both originating from MSM patients. Notably, these isolates shared the identical mgpB ST275.

The mgpB/MG309 typing method identified 42 different genotypes in 62 M. genitalium isolates, resulting in a DI of 0.9736 (Table S3). Genotype 4.10, comprising ST4 and STR10, was highly prevalent among MSM, accounting for 27.3% of MSM cases. Isolates with genotype 4.10 contained MRAMs in 7 of 9 cases (77.8%), whereas none harboured QRAMs (Fig. 2). Otherwise, genotypes within both MSM and MSW did not exhibit any significant outlier; instead, the genotypes were relatively homogeneously distributed, with frequencies ranging from 0 to 3. The MSM and MSW groups shared only genotypes 4.13, 5.10 and 7.10, while all other genotypes were exclusive to either the MSM or MSW group.

Phylogenetic clustering and epidemiological associations

The phylogenetic tree based on mgpB sequences (Fig. 2) revealed two distinct clusters among M. genitalium isolates. MGU057 was classified as an outlier due to low bootstrap values (Fig. S1), resulting in a cluster A comprising 21 isolates and a cluster B containing 40 isolates. The analysis of the association between these phylogenetic clusters and several epidemiological determinants (Table 2) showed that cluster A included a significantly higher proportion of MSW isolates, whereas cluster B was predominantly composed of isolates from MSM (P<0.001). The prevalence of MRAMs (P<0.01) and QRAMs (P<0.05) was significantly higher in cluster B (87.5% and 35.0%, respectively) compared with cluster A (57.1% and 4.8 %). Moreover, all 13 cases of dual-resistant isolates were observed in cluster B, significantly exceeding those in A, where none were observed (P<0.01). Finally, frequencies of the mgpB STs 2, 4, 5, 8 and 263 were significantly different between the two clusters (P<0.001). No statistically significant differences were noted between clusters in the frequency of specific point mutations for either MRAM or QRAM, type of sexual partner, region of origin, HIV status, N. gonorrhoeae co-infection, C. trachomatis co-infection, previous antibiotic use in the last 3 months, previous urethritis diagnosis in the last 2 years, or MG309 STR.

Table 2. Differences in epidemiological determinants per phylogenetic cluster.

	Cluster A (n=21)	Cluster B (n=40)	P-value
Sex preference, n (%)			<0.001*
Men who have sex with men	4 (19.0)	29 (72.5)
Men who have sex with women	17 (81.0)	11 (27.5)
Type of sexual partner, n (%)			0.552*
Casual partner	12 (57.1)	21 (52.5)
Steady partner	2 (9.5)	8 (20.0)
Both	7 (33.3)	11 (27.5)
Region of origin, n (%)			0.052*
The Netherlands	8 (38.1)	14 (35.0)
Europe, outside the Netherlands	0 (0)	7 (15.0)
Africa	3 (14.3)	0 (0)
Asia	0 (0)	6 (15.0)
Central and South America	9 (42.9)	13 (32.5)
North America	0 (0)	0 (0)
Oceania	0 (0)	0 (0)
Unknown	1 (4.8)	0 (0)
HIV status, n (%)			0.649*
Negative	15 (71.4)	29 (72.5)
Positive	2 (9.5)	7 (17.5)
Unknown	4 (19.0)	4 (10.0)
N. gonorrhoeae co-infection, n (%)			0.688*
Negative	20 (95.2)	35 (87.5)
Positive	1 (4.8)	5 (12.5)
C. trachomatis co-infection, n (%)			0.080*
Negative	15 (71.4)	37 (92.5)
Positive	6 (28.6)	3 (7.5)
Previous antibiotic use last3 months, n (%)			0.358*
No	18 (85.7)	27 (67.5)
Yes	3 (14.3)	13 (32.5)
Previous urethritis in last2 years, n (%)			0.067*
No	17 (81.0)	22 (55.0)
Yes	4 (19.0)	18 (45.0)
Macrolide resistance (MRAM), n (%)			<0.01*
Wildtype	9 (42.9)	5 (12.5)
Mutation	12 (57.1)	35 (87.5)
A2058G	3 (14.3)	10 (25.0)
A2059G	6 (28.6)	24 (60.0)
A2058C	0 (0)	0 (0)
A2059C	0 (0)	0 (0)
A2058T	3 (14.3)	1 (2.5)
Fluoroquinolone resistance (QRAM), n (%)			<0.05*
Wildtype	20 (95.2)	26 (65.0)
Mutation	1 (4.8)	14 (35.0)
S83I	0 (0)	6 (15.0)
S83R	0 (0)	0 (0)
D87N	1 (4.8)	6 (15.0)
D87Y	0 (0)	2 (5.0)
Dual MRAMandQRAM, n (%)			<0.01*
No	21 (100)	27 (67.5)
Yes	0 (0)	13 (32.5)
mgpB ST			<0.001*
2	7 (33.3)	0 (0)
4	0 (0)	16 (40.4)
5	3 (14.3)	0 (0)
7	0 (0)	5 (12.5)
145	0 (0)	4 (10.0)
261	0 (0)	3 (7.5)
263	3 (14.3)	0 (0)
MG309 STR			0.849*
9	2 (9.5)	4 (10.0)
10	8 (38.1)	16 (40.0)
11	3 (14.3)	5 (12.5)
12	3 (14.3)	2 (5.0)
13	2 (9.5)	3 (7.5)

*Fisher’s exact test. MGU057 was excluded from the two phylogenetic clusters. QRAM solely entails mutations in the parC gene. MgpB STs with frequency counts of ≥3 are shown. MG309 STRs numbers with frequency counts of ≥5 are shown.

HIV, human immunodeficiency virus; MRAM, macrolide resistance-associated mutation; QRAM, fluoroquinolone resistance-associated mutation; ST, sequence type; STR, short tandem repeat.

Discussion

This study aimed to explore the molecular epidemiology based on mgpB/MG309 typing among M. genitalium isolates collected from men diagnosed with NGU, comparing two groups of MSM and MSW. In addition, antibiotic resistance was evaluated to investigate a potential link between genotypes, antimicrobial resistance profiles, and epidemiological determinants.

Based on mgpB typing only, our findings demonstrated two distinct phylogenetic clusters of M. genitalium isolates, with significant differences between clusters based on sexual preference and antibiotic resistance. Cluster A was predominantly composed of isolates from MSW (81.0%), whereas cluster B primarily consisted of isolates from MSM (72.5%), highlighting the significant difference in the distribution of sexual preference between the two clusters. This is in accordance with Guiraud et al. [11], who identified distinct genetic clustering in a large group of M. genitalium isolates from MSM and women. Three distinct clusters were observed based on mgpB STs, with one cluster showing a significantly higher proportion of MSM relative to the other clusters, which consisted predominantly of isolates from women. Moreover, Fernández-Huerta et al. [21] performed mgpB typing on 54 asymptomatic individuals and demonstrated two clusters, where one cluster significantly correlated with infections occurring in women and MSW, and a second cluster mostly entailed infections in bisexual men and MSM. Collectively, these findings suggest that mgpB typing can differentiate between distinct sexual networks of MSM and MSW. This association is not apparent for the MG309 locus, considering the majority of samples in both clusters contained STR10. Including MG309 could provide additional data on direct transmission routes, although, in this study, the added value is limited, possibly due to the sample size and lower genetic diversity.

In the mgpB phylogenetic tree, branch lengths for MSM isolates were notably longer compared with MSW, reflecting a higher genetic distance among MSM isolates. Several factors may have contributed to this, including the high levels of genetic recombination identified in M. genitalium. In populations with enhanced transmission rates, such as MSM, recombination events may occur more frequently, contributing to the increased genetic diversity [2,22]. Furthermore, the increased prevalence of antibiotic resistance in MSM populations may have created a stronger selective pressure on M. genitalium isolates, leading to more frequent mutations [23].

The overall prevalence of macrolide and fluoroquinolone resistance in the study population was 75.8% and 24.2%, respectively. The resistance of both macrolides and fluoroquinolones was significantly elevated in cluster B (87.5% and 35.0%, respectively), which predominantly comprised samples from MSM, compared with cluster A (57.1% and 4.8%, respectively). This is consistent with previous studies evaluating the prevalence of macrolide-resistant M. genitalium isolates across different populations [11,21, 23]. The high levels of resistance observed in this group could be attributed to the high transmission rate of resistant M. genitalium isolates within MSM communities but may also reflect the acquisition of resistance after increased antibiotic use [24,25]. This is particularly problematic in populations with increased risk of STIs, especially when individuals are regularly screened and treated with azithromycin-targeting pathogens other than M. genitalium [18,26]. To prevent further undesirable spread of macrolide resistance, some guidelines have recommended discontinuing azithromycin as part of the presumptive management of STIs [27,28].

This study identified 13/24 novel mgpB STs among the collected M. genitalium isolates, based on PubMLST. Ten new STs originated from MSW isolates, likely reflecting a sampling bias in existing M. genitalium ST databases, which predominantly include MSM isolates due to prior study focus. ST2 was the predominant mgpB ST within the MSW group, accounting for 20.7% of the cases. This ST exhibited notably lower levels of MRAMs and QRAMs, 28.6% and 14.3%, respectively, relative to ST4, ST7, and ST145. Similar findings have been reported by Guiraud et al. [11] who observed MRAMs in 7.4% and QRAMs in 24.1% of ST2 isolates. The mgpB ST7 was detected in both MSM and MSW groups. This ST exhibited high levels of multidrug resistance, with MRAMs and FRAMs recorded at 80% and 100 %, respectively. In contrast, previous studies have reported significantly lower resistance rates for ST7; however, the overall prevalence of this ST remains low across various studies [11,18].

Within the MSM group, mgpB ST4 was most common (45.5%), consistent with other studies reporting a dominance of ST4 within MSM populations [11,12, 18], suggesting the spread of a specific clone [18,21, 29]. The reason for this increased prevalence remains unknown; however, a hypothesis could be that the increased prevalence is attributed to the widespread occurrence of this type in a self-contained sexual network [21]. ST4 showed a high level of MRAM prevalence of 87.5%, while no FRAM mutations were detected. Other studies have equally demonstrated that ST4 often harbours MRAMs, while in contrast, FRAMs are rare [11,18].

While certain mgpB STs exhibit distinct trends in antibiotic resistance, these patterns may be more strongly associated with sexual preference than with inherent genotypic characteristics of M. genitalium isolates themselves. For instance, mgpB ST4 demonstrated elevated levels of macrolide resistance but was exclusively identified in MSM, a population with a documented higher burden of antibiotic resistance [11,21, 23]. Conversely, ST2, which displayed relatively low levels of antibiotic resistance, was predominantly observed in MSW, a group generally associated with lower antibiotic resistance profiles. This suggests that resistance is likely to arise through independent mutations, supporting previous findings that the development of antibiotic resistance in M. genitalium is multiclonal, with no strong correlation between genotypes and antibiotic resistance [7,22, 24, 30].

In contrast with previous studies, which primarily included M. genitalium isolates from asymptomatic individuals [11,12, 18, 21], this study focused exclusively on isolates from individuals diagnosed with NGU. Notably, the most frequently identified mgpB STs in this study were also reported in earlier studies that were not limited to symptomatic patients, suggesting that there is no apparent correlation to pathogenicity.

A strength of this study includes the comprehensive collection of epidemiological data, co-infections and genotypic analyses, which substantially strengthens the robustness of our findings. There are some limitations to our study that should be addressed. First, this study lacks detailed information on sexual interactions between patients, which constrained our ability to fully evaluate direct transmission between closely related isolates. Second, a group of women infected with M. genitalium was missing in order to elaborate on whether the isolates would closely cluster to the isolates originating from MSW.

In conclusion, we described the molecular typing and antibiotic resistance of M. genitalium isolates from MSM and MSW NGU patients in the Netherlands. Molecular typing revealed clusters differentiated by sexual preference and antibiotic resistance profiles. These findings provide evidence regarding the molecular epidemiology and transmission dynamics for M. genitalium infections within sexual networks, underlining the need to expand the monitoring of antibiotic resistance in these populations. Moreover, our data suggest that resistance is likely to arise through independent mutations, supporting the multiclonal spread of antibiotic resistance in M. genitalium. Future studies using next-generation sequencing are needed to further explore the relationship between sexual transmission and genetic diversity in M. genitalium.

Abbreviations

DI

discriminatory index

MRAMs

macrolide resistance-associated mutations

MSM

men who have sex with men

MSW

men who have sex with women

NGU

non-gonococcal urethritis

PubMLST

public databases for molecular typing and microbial genome diversity

qPCR

quantitative polymerase chain reaction

QRAMs

quinolone resistance-associated mutations

SNPs

single nucleotide polymorphisms

ST

sequence type

STI

sexually transmitted infection

STRs

short tandem repeats