Antimicrobial resistance (AMR) of Mycoplasma genitalium (M. genitalium) to antibacterial regiments is a growing problem with global implications for clinical guidelines and treatment [1–6]. As Jensen and Bradshaw (2015) argue, clinical monitoring and effective reporting on antimicrobial resistance-mediating 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 circumstances of AMR-related mutations in China. Here, we contribute to global efforts to address this gap in AMR surveillance by investigating the presence of mutations to macrolide and fluoroquinolone, two commonly uses medical regiments for bacterial intervention treatments in Guangzhou, China.
What diseases are caused by or associated with M. genitalium?
M. genitalium is commonly associated with sexually transmitted infections (STIs) and often presents as a co-infection other STIs [5, 8–10]. It was first isolated from urethral specimens in male patients with Nongonococcal urethritis (NGU) in 1981 by Tully et al [11]. M. genitalium belongs to the order Mycoplasma in the class Mollicute, which is the smallest known prokaryotic microorganism capable of self-replication [12]. Substantial evidence supports research of M. genitalium being a major cause of STI, including urethritis [13], Mucopurulent cervicitis (MPC) [14], endometritis [15], and pelvic inflammatory disease (PID) [16, 17]. M. genitalium is also a suspected cause of reactive arthritis and proctitis [18]. The 2009 European guidelines on NGU management identified M. genitalium and Chlamydia trachomatis (C. trachomatis) as leading causes of NGU [19]. In reviewing recent prevalence rates collected from studies across diverse medical facilities in England and reported in journals from 2006–2016, Horner and Martin [20], argue that NGU is the most common clinical manifestation of M. genitalium [20]. Based on available data, they project that if 125,000 male patients were to become infected with M. genitalium in England, 5.2% would likely develop NGU during a one-year infection period [20]. In Australia, a report published in 2006 provides support for this pattern from earlier periods– male patients with NGU tested significantly more frequently for M. genitalium compared to male patients without NGU at a rate of 9–1% [21]. In the US, a study from 2009 also supports this trend– males with urethritis tested positive for M. genitalium at 22.4% vs males without urethritis having positive results at a rate of 7.3% [22].
What Are Known Or Suspected Factors For M. Genitalium?
According to a recent meta-analysis of 63 epidemiological studies published from 1991 to 2016 [23], the incidence of M. genitalium infection appears related to the income-level of reporting localities. In high-income countries (HIC) in US and Western Europe such as Denmark, Great Britain, Norway, and Russian Federation, the calculated summary incidence rate was extremely low, at 1.3% [23], ranging between 1% to 3% for both men and women in general populations [24–26]. However, in comparison, general populations living in low-and-middle-income countries (LMIC), such as Honduras, Vietnam, Kenya, Madagascar, and Tanzania experienced much higher disease burden. The calculated summary incident rate was almost three times higher, at 3.9%, for the same period [23].
Many studies from earlier waves of research, like the meta-analysis described above [23], report not finding significant differences in M. genitalium prevalence and risk factors between men and women. However, mounting evidence suggest differences by sex for acquiring an M. genitalium infection [17, 27]. According to Wang et al., among 2753 outpatients (2161 males, 592 females) seeking treatment at an STI clinic in Nanjing, the M. genitalium infection rate was 7.95% [28]. Focusing solely on M. genitalium infection alone, male patients had a significantly higher infection rate at 62.30% compared to female patients at 36.84% [28]. Similarly, Qin et al [29] from our STI facility found an infection rate of 7.94% (209/2,633) from swabs of patients’ genital tract, with males experiencing significantly higher positive tests at 8.94% (n = 1958) compared to female patients at 5.04% (n = 675) [29].
Moreover, there is a growing consensus from research in China and internationally, of other characteristics contributing to increased risk of M. genitalium infections. Characteristics linked to increased infection include experiencing problems with fertility for both men [9, 30] and women [31], abnormal pregnancy status [32], and from being members of vulnerable populations, including men-who-have-sex-with men (MSM) [33], female sex workers (FSW) [34], and people living with HIV [35]. As reported in this journal, a study from Shenyang, a city in north China, reported that M. genitalium infection is a significantly correlated to HIV infection among MSM living in China [33].
Overall, the high rates of infection in LMIC, and patterns of co-infecting STIs coupled with high disease burden among vulnerable populations, even for people living in HIC, are causes for concern. These considerations have spurred clinicians and public health agencies around the globe to call for concerted management of M. genitalium guidelines and treatment as a means of mitigating AMR-related problems [1–6].
AMR Problems Related To M. Genitalium Treatment Interventions
Due to the lack of a cell wall, M. genitalium is hardy, readily surviving the onslaught of antibiotics, such as penicillin, typically used in first-wave and multi-wave medical treatment interventions prescribed in clinical setting [36]. Taking advantage of in vitro tests that demonstrate M. genitalium sensitive to tetracyclines, macrolides, and new fluoroquinolones [37], current treatment interventions and guidelines often recommend these medications. According to the 2016 European NGU guidelines, patients who test positive for urethritis should be tested for C. trachomatis and M. genitalium via nucleic acid amplification testing [38]. Since a single-dose treatment of azithromycin may result in the development of antimicrobial resistance in M. genitalium, the 2015 UK NGU guidelines and the 2016 European M. genitalium guidelines were no longer recommended azithromycin 1 g as first line therapy [39, 40]. Hence, it is troubling that mounting evidence illustrating M. genitalium drug-resistance increases with even just a single-dose treatment of azithromycin [2, 41–43]. The macrolide sensitive M. genitalium eradication rate was 85% (82–88%, in the 12 studies prior to 2009) compared to 67% (57–77%, in the 9 studies since 2009) [44]. In locations with high incidence of NGU, the practice of widespread use of a single-dose antimicrobial treatment can be problematic. This is the case for Greenland, where 55,000 inhabitants have tested positive for NGU, with common treatment intervention consisting of single dose azithromycin therapy, from 1998 to 2005. Unfortunately, macrolide resistance rate of M. genitalium has been reported to be 100% in the region [45]. Fluoroquinolone moxifloxacin, another medication extensively used as a second-line bactericidal, has a cure rate approaching 100% in infections with susceptible strains [46]. In recent years, an increasing moxifloxacin treatment failure rate has also been noted, first in the Asia-Pacific region [7] and later in Australia [47]. Indications point to an emergence of fluoroquinolone resistant strains [47–49]. The elimination rate of moxifloxacin for M. genitalium infection has decreased from 100–89% since 2010 [47].
Genetically, mutations resulting in macrolide resistance are primarily attributed to single-nucleotide polymorphism (SNP) at positions A2058 or A2059 in region V of the 23S rRNA gene [2, 50]. Fluoroquinolone resistance is attributed to alternations 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) [51]. 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 [41, 52–54]. 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 [55].
Multidrug resistance is present in both macrolide and fluoroquinolone resistance-associated mutations in M. genitalium being reported in Japan, Australia and New Zealand since 2008 [41, 52, 56, 57]. This disturbing trend suggesting that the AMR dilemma attributable to M. genitalium is spreading and becoming even more virulent [41, 52, 56, 57].
Clinical Research On M. Genitalium Antimicrobial Resistance In China
Although many published reports focus on M. genitalium prevalence and risk-factors across diverse sub-populations, at the time of this study, there are only three locations actively conducting AMR-related research in clinical settings in China. There are two published reports, one study of macrolide and quinolone resistant among men attending an STI center in the Nanjing vicinity, a city on the coastline central coastline, near the China Centers for Disease Control and Prevention [58, 59], published in Chinese and English. The second study is on macrolide and tetracycline mutations among men seeking care at an infertility clinic in Changsha, in the interior of China [60], published in English.
Our location, an STI center based in a hospital in Guangzhou, constitutes the third AMR study site. Our facility is situated in Guangdong, the biggest province in China, with more than 100 million people living within this region of south China. Our hospital is located in Guangzhou, a city at the geographic center of Guangdong. As a major city in the Pearl River Delta, Guangzhou is well-integrated with nearby metropolises of Hong Kong, Macau, and Shenzhen, each within a one-hour commuting radius. Guangzhou is the earliest foreign trade port, so there are many foreign exchanges and high population movement. This region is an international hub for travel, trade, and commerce and a major destination for migrants and their concomitant illnesses. As a provincial level STI center, our doctors are referred patients from all over the region when care providers from feeder hospitals throughout the region are unable to resolve medical ailments locally.
Servicing the medical needs of such a diverse population, our study focuses on macrolide and fluoroquinolone resistance-associated mutations in M. genitalium. While the two published studies are the first to address AMR mutations in China, we build on current knowledge in two keyways. 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 contributing data on a major urban migration destination in south China. Second, we expand on AMR surveillance by being the first to report macrolide and fluoroquinolone-associated mutations in men and women population in China.
Looming AMR dilemma: A near clinical failure for treating M. genitalium
Along with conducting this study, our team recently shared a case-report describing the dire conditions of AMR confronted by patients [Unpublished]. A particularly virulent case was treated at our facility in Guangzhou in 2017– a patient experiencing a 2-month-long history of persistent urethral irritation and urethral discharge was referred to our clinic. From the referral clinic, we collected the medical history for a 48-year-old male who presented at our clinic for C. trachomatis. Doctors at prior facility had prescribed one combination treatment of a 7-day course of azithromycin and a subsequent 7-day course of minocycline. After these initial treatments, the patient reported mild improvement of urethral irritation, but the symptoms never fully resolved, and urethral discharge persisted despite the C. trachomatis tests coming back with negative results in post-treatment assessments.
Unfortunately, after 8 weeks of treatment, the patient was referred to our provincial-level STI center. We conducted a test for C. trachomatis from the first void urine sample via Cobas 4800 chlamydia/NG Amplification/Detection Assay (Roche Molecular Systems Inc, New Jersey, USA). Physical examination of the genital area revealed redness of the penile meatus, and his underwear was contaminated with a small amount of discharge, symptomatic indicators of M. genitalium infection. Collection of first void urine sample was then analyzed and was negative for C. trachomatis and N. gonorrhoeae.
During the following 10 months, the patient was treated with a series of antibiotics to resolve M. genitalium symptoms There were a total of six treatment regiments. When the diagnostic lab work indicated M. genitalium positive results, the patient was (1) started on antofloxacin (a fluoroquinolone antibiotic produced in China) 200 mg once a day for 7 days, followed by azithromycin 500 mg once a day for 7 days; (2) when M. genitalium results came back positive, the second regiment consisted of moxifloxacin 400 mg once daily for 7 days, followed by minocycline 100 mg twice daily for 14 days; (3) persistent M. genitalium infection after the second round of treatments resulted in a prescription for antofloxacin at 200 mg once daily combined with rifampin 450 mg once a day for 21 days; (4) when this regiment also failed to clear the M. genitalium infection, the patient was put on azithromycin 500 mg once a day for 21 days. (5) After exhausting orally administered medications, when the results came back positive again, our team started the patient on intramuscular spectinomycin 2 g once a day for 7 days. Although there were promising results initially, with M. genitalium test screening coming back negative on day 11 and 22, the infection results came back positive again on day 52. (6) The last regiment, doxycycline 100 mg twice a day for 14 days, yielded M. genitalium negative results during screenings on day 19, 81 and 116 after doxycycline treatment. His symptoms almost resolved except for occasional urethral irritation. During the more than 10 months of treatment, he occasionally had protected vaginal sex with his wife. Cervical swabs from his wife were negative for C. trachomatis, N. gonorrhoeae, and M. genitalium.
During his intensive course of care, our team collected and analyzed several laboratory specimens. A urethral swab sample was taken prior to starting the 21-day treatment of azithromycin. M. genitalium had amino acid changes of S83I in parC, and G93C in gryA. M. genitalium had A2059G mutation in positions 2059 (E. coli numbering) of the 23S rRNA gene. No mutations were found in the gyrB. Despite interventions using a combination of azithromycin, minocycline, moxifloxacin and spectinomycin, this case was a near clinical failure for effective treatment of persistent M. genitalium infection. From his extended schedule of treatments, it is evident that this patient suffered from AMR complications, and is a harbinger of looming AMR problems in China.
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 efficacious regiments for combating M. genitalium infections and managing AMR across global settings [7]. Unfortunately, there is sparse data and low awareness of the trends of antimicrobial resistance of M. genitalium in China. The aim of this study is to support and contribute to AMR research in south China by analyzing the prevalence and diversity of mutations associated with macrolide and fluoroquinolone resistance among M. genitalium in positive clinical specimens in Guangzhou, China.