Macrolide and fluoroquinolone associated mutations in Mycoplasma genitalium in a retrospective study of male and female patients seeking care at a STI Clinic in Guangzhou, China, 2016-2018

Background Antimicrobial resistance in M. genitalium is a growing clinical problem. We investigated the mutations associated with macrolide and fluoroquinolone resistance, two commonly used medical regimens for treatment in China. Our aim is to analyze the prevalence and diversity of mutations among M. genitalium-positive clinical specimens in Guangzhou, south China. Methods A total of 154 stored M. genitalium positive specimens from men and women attending a STI clinic were tested for macrolide and fluoroquinolone mutations. M. genitalium was detected via TaqMan MGB real-time PCR. Mutations associated with macrolide resistance were detected using primers targeting region V of the 23S rRNA gene. Fluoroquinolone resistant mutations were screened via primers targeting topoisomerase IV (parC) and DNA gyrase (gyrA). Results 98.7% (152/154), 95.5% (147/154) and 90.3% (139/154) of M. genitalium positive samples produced sufficient amplicon for detecting resistance mutations in 23S rRNA, gyrA and parC genes, respectively. 66.4% (101/152), 0.7% (1/147) and 77.7% (108/139) samples manifested mutations in 23S rRNA, gyrA and parC genes, respectively. A2072G (59/101, 58.4%) and S83I (79/108, 73.1%) were highly predominating in 23S rRNA and parC genes, respectively. Two samples had amino acid substitutions in gyrA (M95I and A96T, respectively). Two samples had two amino acid substitutions in parC (S83I + D87Y). 48.6% (67/138) of samples harbored both macrolide and fluoroquinolone resistance-associated mutations. The most common combination of mutations was A2072G (23S rRNA) and S83I (parC) (40/67, 59.7%). One sample had three amino acid changes in 23S rRNA, gyrA and parC genes (A2072G + A96T + S83I). Conclusions The high antimicrobial resistance rate of M. genitalium in Guangzhou is a very worrying problem and suggests that antimicrobial resistance testing and the development of new antibiotic regimens are crucially needed.


Background
Antimicrobial resistance (AMR) of Mycoplasma genitalium (M. genitalium) is a growing problem with global implications for clinical guidelines and treatment [1][2][3][4][5][6]. As Jensen and Bradshaw (2015) argue, clinical monitoring and effective reporting on antimicrobial resistancemediating mutations in M. genitalium across geographic regions and populations are crucial for developing effective treatments in managing M. genitalium infections and AMR-mediation across global settings [7]. Yet, despite being one of the most populous countries in the world, there is sparse data on the prevalence of AMR-related mutations in China. Here, we contribute to global efforts to address this gap in AMR surveillance. We investigate the rates of AMR mutations associated with macrolide and fluoroquinolone treatment failure in M. genitalium, expanding the discussion on the use of these antibiotics in Guangzhou, China. Given these alarmingly high rates, it is very important to understand the background of antibiotic use in this region.
Due to the lack of a cell wall, the treatment choice of M. genitalium was limited to tetracyclines, macrolides, or later-generation fluoroquinolones [7,27]. According to the 2016 European NGU guidelines, patients with urethritis should be tested for C. trachomatis and M. genitalium via nucleic acid amplification testing [28]. Since a single-dose treatment of azithromycin may result in the development of antimicrobial resistance in M. genitalium [29], the 2015 UK NGU guidelines, the 2016 European M. genitalium guidelines and the Australian STI management guidelines (2018) no longer recommend azithromycin 1 g as first line therapy [30][31][32]. Hence, it is troubling that mounting evidence indicates M. genitalium drug-resistance increases with even just a singledose treatment of azithromycin [2,[33][34][35]. Although a single dose of AZM can result in the development of resistance, AZM can be used in conjunction with doxycycline for susceptible infections (no 23S mutation, where screening is available). The fluoroquinolone moxifloxacin, another medication extensively used as a secondline bactericidal, has a cure rate approaching 100% in infections with susceptible strains [36]. In recent years, a decline in the efficacy of moxifloxacin has also been noted, first in Japan [7] then in Australia [37][38][39]. The elimination rate of moxifloxacin for M. genitalium infection has decreased from 100 to 89% since 2010 [37].
Genetically, mutations resulting in macrolide resistance are primarily attributed to single-nucleotide polymorphism (SNP) at positions A2071 or A2072 in region V of the 23S rRNA gene [2,40]. Fluoroquinolone resistance is attributed to alternations of the gyrA subunit in DNA gyrase (which is composed of two gyrA and two gyrB subunits), or the parC subunit of topoisomerase IV (which is composed of two parC and two parE subunits) [41]. Compared with parC mutations, gyrA mutations likely have a less-severe effect on reducing the susceptibility of the bacterium for fluoroquinolone [42,43]. Moxifloxacin resistant M. genitalium isolates, primarily causing amino acid changes at positions S83 and D87 (M. genitalium numbering) of parC, are similar to those found in other fluoroquinolone resistant bacteria [33,[44][45][46]. AMR studies of fluoroquinolone resistance in M. genitalium DNA tend to amplify the quinolone-resistance determining region (QRDR) of the gyrA gene and the corresponding region of the parC gene from M. genitalium DNA [47]. Antibiotic resistance of M. genitalium to both macrolides and quinolones have been found in Japan, Australia, and New Zealand since 2008 [33,38,44,48,49]. This disturbing trend suggests that the AMR dilemma attributable to M. genitalium is spreading and becoming even more virulent [33,44,48,49].
Clinical monitoring and effective reporting of findings and research of antimicrobial resistance-mediating mutations in M. genitalium across geographic regions and populations are crucial for the development of efficacious treatment for combating M. genitalium infections and managing AMR across global settings [7]. Unfortunately, there is sparse data and low awareness of the patterns of antimicrobial resistance of M. genitalium in China. The aim of this study is to support and contribute to AMR research by analyzing the prevalence and diversity of mutations associated with macrolide and fluoroquinolone resistance among M. genitalium in positive clinical specimens in Guangzhou, China.

Study population and specimens
A total of 154 M. genitalium positive clinical specimens were collected from patients attending a STI clinic at Dermatology Hospital, Southern Medical University, Guangzhou, China. The collecting period was from December 2016 to December 2018. The samples included urethral swabs and/or rectal swabs from male patients and cervical swabs from female patients.
M. genitalium was detected via TaqMan MGB realtime polymerase chain reaction (PCR) as described by Jensen et al. [50]. DNA extracted by using DNA extraction kit (Suzhou Bacme Biotech Co.) from M. genitalium positive samples were tested for the mutations associated with macrolide and quinolone resistance in the same day or stored at − 20°C prior to testing. Samples in our study were collected with the permission of STI patients as part of standard protocol for diagnostics and treatment. Specimens were then processed and stored with no identifiable patient data. As part of hospital protocol, de-identified patient samples are stored in the STI clinic biobank for surveillance, diagnosis, and research purposes. This study utilizes specimens from this biobank.
Detection of macrolide and fluoroquinolone resistanceassociated mutations in 23S rRNA, gyrA, and parC From extracted DNA, mutations associated with macrolide resistance were detected using primers targeting region V of the 23S rRNA gene (nucleotides 1992-2138) [40]. Fluoroquinolone resistance mutations in the gyrA (nucleotides 172-402) and parC (nucleotides 164-483) genes were screened using primers as reported previously [51,52]. Details of the primer sequencing and thermo-cycling parameters for amplification are provided in Table 1.

Data analysis
The mutation sequencing data in 23S rRNA, gyrA and parC genes from M. genitalium positive DNA specimens were analyzed with the software program BioEdit (http://www.mbio.ncsu.edu/bioedit/bioedit.html).
The genome sequence of M. genitalium strain G37 (GenBank accession no. NC_000908.2) was used as a reference strain. Amino acid changes in the QRDRs of the gyrA and parC genes in this study were compared with previous studies [6,24,26,33,38,41].

Ethics approval
The study was approved by the Ethics Committee of Dermatology Hospital of Southern Medical University (approval no. GDDHLS -20,171,203, 13/12/2017). Informed consent was waived as the committee believes that this research presents no potential risk because the study does not contain identifiable data that would cause harm or result in a breach of confidentiality.
Although, none of the gyrA mutations have been associated with increased fluoroquinolone MICs, amino acid alterations in gyrA (M95I, A96T) were only detected in two samples. To our knowledge, this specific amino acid change (A96T) has not been reported elsewhere in the AMR literature. However, mutation at the next position (99 in gyrA) have been described in previous reports, and the positions are within the QRDRs, indicating its association with fluoroquinolone resistance [45]. Mutations of parC at positions S83 (except S83N) and D87 have been associated with increased MICs of moxifloxacin and parC mutations typically associated with fluoroquinolone resistance were detected in 77.7% (108/139) of samples. Of these, the most frequent mutation was S83I (n = 79), accounting for 73.1% of 108 samples. In two samples, two amino acid substitutions in parC (S83I + D87Y) were present. As shown in Table 2, substitutions in parC are more common than in gyrA.   Of the 138 samples undergoing complete analysis for both the 23 s RNA and parC genes, 48.6% (67/138) harbored both macrolide and fluoroquinolone resistanceassociated mutations. Combining the 23S rRNA and parC mutations, 15 genotypes were identified (Table 3).

Discussion
The mutations in 23S rRNA and parC have been identified as the cause of failure of macrolide and quinolone in the treatment of M. genitalium [53,54]. In our study, 66.4% (101/152) and 77.7% (108/139) of samples manifested mutations in 23S rRNA and parC genes, and A2072G (59/101, 58.4%) and S83I (79/108, 73.1%) were highly predominating in 23S rRNA and parC genes, respectively. More worryingly, the proportion of mutations in both 23S rRNA and parC genes was as high as 48.6%. This suggests that nearly half of these samples are resistant to both macrolide and quinolones. We also reported other mutations in parC and gyrA genes. However, the significance of these mutations requires further study.
Within the last 10 years, M. genitalium eradication rate has declined gradually [55,56]. The resistance rate of M. genitalium has been described as a rising phenomenon in many countries [42,57]. At the time of this study, there are only three locations actively conducting AMRrelated research in clinical settings in China. The earliest published macrolide-associated mutations in M.genitalium in China collected samples from 18 symptomatic NGU patients [58]. In this research, the 23S rRNA mutation rate was 94.4%, with A2072G being the most common (55.6%), A2071G the second most (27.8%), and A2071T as the third most common mutation (11.1%), with no double-mutations detectable [58]. Later, in the same hospital, 358 M. genitalium positive samples were collected. The 23S rRNA mutation rate was 88.9%, with A2072G being the most common (61.9%), A2071G the second most (17.6%), and no double-mutations were detected [59]. The parC mutation rate was 90.4%, S83 → I was the most common mutation (83.7%) [59]. The double mutation in parC (G248A + G259T) was detected [59]. The gyrA mutation rate was 13.0%, with M95 → I being the most common (5.3%), three double-mutations in G244A + G285A, G285A + A309G, and G285A + A317G were detectable [59]. Another earlier study collected samples among men seeking care at an infertility clinic in Changsha, a city in the interior of China [60]. The macrolide mutations rate was similarly extremely high at 96.7% [60]. The two most common mutations in the Nanjing study are also the most frequent mutations in Changsha, that is, A2072G (60.0%) and A2071G (20.0%) [60]. Unlike in Nanjing, the analysis conducted on specimens from Changsha detected double-mutations, and these mutations are frequent enough to be the third most common set of mutations (A2071T + A2072G at 11.7%). Our location, a STI center based in a hospital in Guangzhou, constitutes the third AMR site. Our facility is a provincial STI center situated in Guangzhou, the capital city of Guangdong Province. Guangzhou is an international hub for travel, trade, and commerce and a major destination for migrants and their concomitant illnesses. Servicing the medical needs of such a diverse population, we focus on macrolide and fluoroquinolone resistance-associated mutations in M. genitalium. We extend current knowledge in two key ways. First, we continue monitoring and reporting efforts on macrolide and fluoroquinolone resistance, expanding on reports from the two prior studies based in central and interior China, by adding a major urban migration destination in south China. Second, we expand on AMR surveillance by being the first to report on macrolide and fluoroquinolone-associated mutations in men and women in China.
The 23S rRNA mutation is associated with macrolide resistance [61]. We found that SNPs in region V of the 23S rRNA gene were observed in 101 (66.4%) samples from male and female patients with M. genitalium-positive infection in 2016-2018. Mutations mainly occurred at positions A2071 and A2072 mainly to G (C or T is relatively less). With the exception of a study from Greenland, the mutation frequency (66.4%) [62] observed was higher than frequencies reported by Russia and Estonia (0.7~10%) [63], South Africa (10%) [64], southern Sweden (13%) [65], France (17%) [66], Japan (42.2%) [42], southern USA (48%) [67], Norway (56%) [68], and Denmark (57%) [68].  [26], and Australia (79.4%) [70]. It is widely reported that M. genitalium expressed a diversity of mutations linked to fluoroquinolone resistance-associated in gyrA and parC gene [49,53,68]. Similar to extant studies, mutations in the QRDR of the gyrA gene of our samples were rarely detected [53,71]. The amino acid changes (M95 → I and A96 → T) in gyrA were found in our specimens. The M to I transition at position 95 of gyrA (G to C at nucleotide position 285) was first reported in 2013 by Tagg et al. [49], most commonly observed from 2013 to 2017 in Japan, and have been reported in moxifloxacin-resistant strains of M. pneumoniae, M. hominis, and Ureaplasma spp [49,57,72,73]. To our knowledge, a gyrA A96 → T mutation in the core of the QRDR has not previously been described in M. genitalium and its association with resistance to fluoroquinolone remains unknown. The amino acid changes at G81, S83 and D87, have been previously reported as being associated with fluoroquinolone resistance in M. genitalium and other closely related organisms [44,46,49]. Although the majority of published reports have shown the parC S83N and S83I substitution as the two most prevalent base changes at position 248, we find that the S83I substitution accounted for 71.8% (79/110), significantly higher than reports from Japan (13.0-23.2%) [42], New Zealand (16.7%) [48], and southwestern France (9.1%) [66]. Among the 139 samples successfully amplified DNA sequences of parC gene, we observed an exorbitantly high mutation rate of 77.7%.
Additionally, 48.6% (67/138) of samples were multidrug resistant and contained both macrolide and fluoroquinolone resistance related SNPs. If SNP on parC is strictly limited to S83I, the multidrug resistance rate was 36.1% (50/138). In Japan, the prevalence of multidrug resistance with A2071G or A2072G in the 23S rRNA and amino-acid change in S83 or D87 of parC has been reported in up to 21.8% from 2010 to 2017 [42]. Our data showed very high prevalence (47.8%) of the same mutation. This trend of multidrug resistance presents challenges for clinicians because of a lack of suitable alternative therapy after azithromycin and moxifloxacin failure. Pristinamycin as the only third-line treatment has been reported to be only about 75% effective and is not readily available in China [27].
The high prevalence of mutations in macrolide and quinolone resistance-associated genes observed in our study might be related to the study population and to antibiotic overuse in China. The clinical samples were collected from the STI clinic of Dermatology Hospital, Southern Medical University. As a provincial level STI center, our doctors are referred patients from all over the region when doctors from feeder hospitals are unable to resolve medical ailments locally. These patients likely experienced several prior courses of antibiotic treatment. In addition, in China, it is incredibly easy for the public to obtain antibiotic prescriptions and purchase antibiotics in pharmacies. Data show that antibiotic use in children and hospitalized patients in China is very high [74]. These factors further exacerbate the problems of antibiotics resistance confronting health facilities today [75].

Limitations
An important limitation of the study is the lack of epidemiological and clinical information, as well as information about treatment received and clinical evolution of the patients. The significance of several novel mutations in the parC and gyrA genes remains unknown. Nonetheless, the prevalence of mutations associated to macrolide and fluoroquinolone resistance in our study related to phenotypic testing has been previously reported in several studies. Our prevalence rate is a calculation based on a sample of patients seen by clinicians at our STI clinic. During patient intake and consultation, we did not collect patients' history of previous antibiotic use. We hypothesized that patients at our STI clinic were more likely to have previously used antibiotics than the general population, so there might be a possibility of overestimating the prevalence rate when extended to the general population. Secondarily, we lack data for a large sample epidemiological survey of M. genitalium, since samples studied were collected mainly from a single clinic. Hence, our findings might not be representative or readily generalizable to the larger population living in Guangzhou.

Conclusions
In conclusion, the high mutation rate of M. genitalium reported in this study is a very worrying problem. For patients with M. genitalium infection, antimicrobial resistance testing is crucial. The occurrence of drugresistant strains is of great public health concern. The development of new antibiotic regimens for M. genitalium infections are urgently needed. curated the data reviewed and edited the manuscript. YYL1, HRC, YHL performed the laboratory testing, collected and validated the data. CML, YYL2 collected and curated the data, drafted the manuscript, and secured funding. LST, HPZ, LGY provided overall leadership to the study, participated in the analysis, and secured funding. YYL1 corresponding to Yinyuan Lan and YYL2 corresponding to Yuying Liao. All authors have read and approved the final manuscript.

Authors' information
Authors are cited in the same order that they are cited in the title page. . The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Availability of data and materials
The datasets generated and/or analysed during the current study are available in the GenBank repository, accession number to datasets are BankIt2402413, BankIt2402439 and BankIt2402448.

Ethics approval and consent to participate
The study was approved by the Ethics Committee of Dermatology Hospital of Southern Medical University (approval no. GDDHLS -20171203, 13/12/ 2017). Informed consent was waived, because the committee believes that the research presents no potential risk for harm resulting from a breach of confidentiality.

Consent for publication
Not applicable.