Antimicrobial chemotherapy of Mycoplasma genitaliumpositive. urethritis. Review

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1 For reprint orders, please contact Antimicrobial chemotherapy of Mycoplasma genitaliumpositive non-gonococcal urethritis Expert Rev. Anti Infect. Ther. 10(7), (2012) Takashi Deguchi*, Shin Ito, Noriyasu Hagiwara, Mitsuru Yasuda and Shin-ichi Maeda Department of Urology, Graduate School of Medicine, Gifu University, Gifu, Japan *Author for correspondence: Tel.: Mycoplasma genitalium is an important pathogen of acute non-gonococcal urethritis (NGU) in men and plays a significant role in persistent or recurrent NGU. In the management of patients with M. genitalium-positive NGU, eradication of the mycoplasma from the urethra is necessary to prevent persistent or recurrent NGU. Therefore, M. genitalium should be considered for antimicrobial chemotherapy of NGU. This article reviews the in vitro antimicrobial activities of antibiotics against M. genitalium and the efficacies of various antibiotic regimens against M. genitalium-positive NGU, including the doxycycline and azithromycin regimens recommended as first-line treatments for NGU in the guidelines. Selection of macrolide-resistant M. genitalium by treatment with the single-dose regimen of 1-g azithromycin and mechanisms of macrolide resistance in M. genitalium are discussed. The effectiveness of the moxifloxacin regimen against persistent or recurrent NGU, unsuccessfully treated with azithromycin and/or doxycycline regimens, is emphasized. KEYWORDS: Mycoplasma genitalium Urethritis, one of the most common sexually transmitted infectious diseases among heterosexual men, is classified as gonococcal or nongonococcal depending on the presence or absence of Neisseria gonorrhoeae. Chlamydia trachomatis is a major pathogen of non-gonococcal urethritis (NGU), and Trichomonas vaginalis could be a pathogen causing symptomatic or asymptomatic NGU in men [1]. In addition, adeno virus [2], Haemophilus species [3] and Bacteroides ureolyticus [4] have been suggested to be associated with NGU. Some studies have shown a significant association between U. urealyticum (biovar 2) and NGU, and that the presence of U. parvum (biovar 1) in the male urethra might be due to colonization [5]. Mycoplasma genitalium, which was first isolated in urethral cultures from two men with acute NGU in 1981 [6], has been recognized as an important pathogen of acute NGU independent from C. trachoamtis [7 11]. In addition, the mycoplasma has been suggested to be associated with cervicitis, endometritis, salpingitis and pelvic inflammatory diseases in women [9 11]. In the current guidelines for treatment of sexually transmitted diseases, NGU is not classified into subgroups on the basis of detected pathogens [12,13,101,102]. Although most patients with NGU associated with C. trachomatis respond to antibiotic therapy, NGU in which C. trachomatis is not detected, sometimes persists or recurs after treatment [14]. T. vaginalis could be one of the pathogens responsible for persistent or recurrent NGU [1]. In most cases, however, the cause of such persistent and recurrent NGU is usually unknown. Therefore, management of men with persistent or recurrent NGU has been one of the most difficult problems in venereology. Some studies, however, have suggested that failure to eradicate M. genitalium from the urethra might be responsible for persistent or recurrent NGU [15 18]. Thus, M. genitalium should be taken into account at the initial stages of antimicrobial chemotherapy for treating NGU. In this review, the authors selected literature from peer-reviewed journals listed in /ERI Expert Reviews Ltd ISSN

2 Deguchi, Ito, Hagiwara, Yasuda & Maeda MEDLINE and from resources cited in those articles from 1981 to February The selection criteria for this review placed specific emphasis on the following: antimicrobial activities of various antibiotics against M. genitalium; and observations made based on a discussion of antimicrobial chemotherapies of M. genitalium-positive NGU. M. genitalium in acute NGU Tully et al. recently discovered a mycoplasma in the human urogenital tracts of men with acute NGU [6]. The new species of mycoplasma was named M. genitalium in 1983 [19]. M. genitalium shared several morphologic, biologic and antigenetic properties with Mycoplasma pneumoniae. The mycoplasma had the ability to attach to glass, plastic surfaces and epithelial cells. Furthermore, it was shown to penetrate cells. In animal models of primates, most male chimpanzees inoculated intraurethrally with M. genitalium developed an obvious genital tract infection accompanied by a urethral polymorphonuclear leukocyte response and a four-fold or greater increase in antibody production. Taylor-Robinson et al. reported, using a culture technique, presumptive data of the existence of M. genitalium in seven out of 22 (31.8%) men with acute NGU and in two out of 20 (10.0%) men without urethritis [20]. M. genitalium had been proposed as a cause of human NGU, but its role in the etiology of NGU was not established because of the immense difficulty in isolating it from clinical specimens. PCRbased assays have facilitated detection of M. genitalium in clinical specimens [21,22]. Both the prevalence of the mycoplasma in men with acute NGU and in asymptomatic men has been determined by PCR assays. In most studies, M. gentialium has been significantly detected more often in men with acute NGU than in men without urethritis. Most recently, the median prevalence of mycoplasma among men with acute NGU, which was calculated from 34 studies published between 1993 and 2011, was reported to be 15% (range: 5 42%) [11]. The median prevalence among men with acute nonchlamydial NGU was reported to be 25% (range: 10 38%). Many studies have supported the proposition that M. genitalium can cause acute NGU [14,102]. M. genitalium in persistent or recurrent NGU In regard to the prevalence of M. genitalium in patients with persistent or recurrent NGU, Hooton et al. used a DNA probe to examine urethral specimens and detected the mycoplasma in seven out of 32 (21.9%) patients [15]. The prevalence was significantly higher than in men with acute NGU (8.7%) or without urethritis (9.2%). When Taylor-Robinson et al. used a PCR-based assay and detected M. genitalium in first-passed urine samples from 15 out of 78 (19.2%) patients with persistent or recurrent NGU, they reported that the occurrence of M. genitalium coincided with the objective presence of urethritis [16]. Horner et al. treated patients with M. genitalium-positive NGU with doxycycline, and at follow-up after treatment, four out of 14 men were positive for M. genitalium [17]. Of these four men, three had objective evidence of persistent inflammation, and the fourth, who had no evidence of inflammation, relapsed 2 weeks later. The treatment of 12 men with M. genitalium-positive NGU who were given levofloxacin at a dosage of 100 mg thrice daily for 14 days was reported [18]. Eight were persistently positive for M. genitalium after treatment. Among these eight, NGU persisted in one, and signs and symptoms of acute NGU disappeared and urethral smears became negative for leukocytes in the other seven. However, five of these seven men returned with recurrent NGU. They were subsequently treated with minocycline or clarithromycin, and after the second treatment, one remained positive for M. genitalium despite alleviation of symptoms and no evidence of urethral inflammation. This patient had a later recurrence of NGU. In these five men with recurrent M. genitalium-positive NGU, the authors monitored longitudinal changes in M. genitalium load from the beginning of the treatment to the period of recurrence of NGU with a TaqMan assay to quantify copies of the M. genitalium 16S rrna gene in their urine [23]. M. genitalium loads in men with M. genitaliumpositive NGU were suppressed to below the detection level of the TaqMan assay during levofloxacin therapy, and signs and symptoms of urethritis disappeared. At the end of the treatment, the mycoplasma loads became detectable by the TaqMan assay, but there were still no signs and symptoms. Later, regrowth of M. genitalium persisting in hosts and a possible association of an increase in the M. genitalium load with emergence of signs and symptoms of NGU were reported. Various other findings have also suggested that M. genitalium may play a significant role in persistent or recurrent NGU [7,9 11]. In the management of patients with M. genitalium-positive NGU, eradication of the mycoplasma from the urethra would be essential to prevent persistent or recurrent NGU [7,9 11]. In vitro antimicrobial susceptibilities Mycoplasmas including M. genitalium are normally susceptible to antibiotics that inhibit protein synthesis but are resistant to those that act on bacterial cell wall components because of the absence of cell walls. Because of the difficulty in isolating M. genitalium from clinical samples, there have been some reports on the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of various antibiotics determined for American Type Culture Collection (ATCC) reference strains and for a small number of clinical strains of M. genitalium [24 34]. MICs were tested either by the broth microdilution or quantitative TagMan PCR methods [26]. The respective MICs determined by the two methods were similar so that they could be compared with each other [26]. MIC The MICs of antibiotics for strains of M. genitalium, including seven ATCC reference strains (G37, M30, R32, TW10-5G, TW10-6G, TW48-5G and UTMB-10G) and some clinical isolates, have been determined (TABLE 1). Erythromycin and some newer macrolides, such as clarithromycin and azithromycin, are highly active against M. genitalium. Telithromycin and solithromycin have also been reported to be highly active [29,30]. Tetracyclines, including doxycycline and minocycline, are highly active but are slightly less active than the macrolides. 792 Expert Rev. Anti Infect. Ther. 10(7), (2012)

3 Antimicrobial chemotherapy of Mycoplasma genitalium-positive non-gonococcal urethritis Review Table 1. Minimal inhibitory concentrations of antimicrobial agents for American Type Culture Collection reference strains and clinical strains of Mycoplasma genitalium. Study (year) Number of strains ATCC reference strains MIC (g/ml) Ref. EM CAM AZM TC DOXY MINO CPFX LVFX GFLX MFLX STFX Renaudin et al. (1992) [24] Bébéar et al. (2008) ( ) (0.12 1) 1 (1 4) (0.25 4) ( ) [31] Hamasuna et al. (2009) [26] Clinical isolates Bébéar et al. (2008) ( ) (0.12 2) (1 4) (0.25 1) ( ) [31] Jensen et al. (2008) # [28] Hamasuna et al. (2009) [26] g/ml). # 793

4 Deguchi, Ito, Hagiwara, Yasuda & Maeda Fluoroquinolones have been shown to have a wide range of antimicrobial activities against the mycoplasma strains. Ciprofloxacin has low activity against M. genitalium, levofloxacin has moderate activity, and gatifloxacin, moxifloxacin and sitafloxacin have high activity [26,28,31]. The activities of other newer quinolones, such as sparfloxacin, grepafloxacin, gemifloxacin, trovafloxacin and garenoxacin, have been reported to be as high as that of gatifloxacin [32 34]. Recent studies on antimicrobial susceptibilities of current clinical strains of M. genitalium have been reported (TABLE 1). The MICs of tetracycline and doxycycline for these strains ranged from to 2 or 1 g/ml, respectively. Some isolates decreased their susceptibility to tetracyclines compared with the reference strains. The MICs of azithromycin and clarithromycin ranged from to 250 g/ml and from to 128 g/ml, respectively, indicating that some macrolide-resistant isolates emerged in clinical practice [26]. By contrast, moxifloxacin and sitafloxacin were still highly active against the clinical strains, including the macrolide-resistant strains. In one study, clinical isolates were analyzed for the macrolide resistance-associated mutations in 23S rrna and the ribosomal proteins L4 and L22 [28]. An A2058G or A2059G mutation in the 23S rrna conferred higher MICs of macrolides to clinical strains of M. genitalium. However, moxifloxacin remained highly active against the strains harboring an A2058G or A2059G mutation in the 23S rrna. The mechanisms of macrolide resistance in M. genitalium are reviewed in detail in the Macrolides section below. MBC Bébéar et al. reported MICs and MBCs of antibiotics for seven ATCC reference strains and seven current clinical isolates of M. genitalium (TABLE 1) [31]. Bactericidal activity was identified as the MBC:MIC ratio being no greater than four. The MICs of doxycycline ranged from 0.06 to 0.12 g/ml for the ATCC reference strains and from 0.03 to 0.12 g/ml for the clinical strains. However, its MBCs ranged from 0.12 to 1 g/ml for the ATCC reference strains and from 0.12 to 2 g/ml for the clinical strains. In one of the seven ATCC reference strains, the MBC:MIC ratio of doxycycline was 16, whereas in three of the seven clinical strains, the MBC:MIC ratios were eight. The prevalence of clinical strains of M. genitalium, against which doxycycline is not bactericidal, might be relatively high. Such low bactericidal activity of doxycycline could be associated with treatment failures with doxycycline regimens that have frequently been reported in clinical studies of M. genitalium infections. The MBCs of erythromycin for the strains of M. gentialium studied ranged from to 0.03 g/ml, and its MBC:MIC ratios were four or less. Among the fluoroquinolones, including moxifloxacin, ofloxacin, levofloxacin, gatifloxacin, gemi floxacin and garenoxacin, moxifloxacin was reported to show the lowest MBCs for both the ATCC ( g/ml) and clinical ( g/ml) strains. For these fluoroquinolones, the respective MBC:MIC ratios for all the strains studied were four or less. Erythromycin and the fluoroquinolones were bactericidal against the M. genitalium strains, including clinical isolates. Tetracyclines The current guidelines recommend a 7-day regimen of a twicedaily dose of 100 mg of doxycycline for the treatment of NGU [12,13,101,102]. For the treatment of M. genitalium-positive infections, several reports have addressed the bacteriological efficacy of various tetracycline regimens (TABLE 2) [35 41]. Tetracycline regimens Johannisson et al. reported that treatment with 500 mg of oral tetracycline twice daily for 10 days successfully eradicated M. genitalium in only five out of 13 (38.5%) men with NGU [37]. A regimen of one 200-mg dose of doxycycline given on the first day and one 100-mg dose given once daily for the next 8 or 13 days resulted in microbiological cure rates of 17.1 and 71.4%, respectively. Gambini et al. reported that 100 mg of doxycycline given orally twice daily for 7 days eradicated M. genitalium in 33 out of 35 (94.3%) men with NGU [36]. This regimen had been expected to be an appropriate treatment for M. genitaliumpositive NGU [7]. In recent studies, however, eradication rates of M. genitalium with the same regimen of doxycycline were reported to range from 30.8 to 45.2% in men with NGU [40,41]. A regimen of one 100-mg dose of minocycline given for 7 days resulted in a microbiological cure rate of 50% [42]. Treatment of M. genitalium infections with tetracycline regimens, including that of doxycycline recommended for the treatment of NGU in the guidelines, have not been so effective against NGU caused by this microorganism. Tetracycline resistance In vitro tests of the susceptibility of current clinical isolates of M. genitalium to tetracyclines showed the emergence of some isolates with decreased susceptibility (TABLE 1). Tetracycline and doxycycline MICs for these were reported to be 4 and 1 g/ml, respectively [26]. In clinical strains of Mycoplasma hominis and U. urealyticum isolated from genital infections, resistance to tetracyclines associated with the tetm gene has been reported [43,44]. However, tetracycline resistance of M. genitalium has not been assessed in detail. Macrolides The current guidelines recommend a single-dose regimen of 1 g of azithromycin for treatment of NGU. They also recommend an erythromycin regimen as an alternative treatments [12,13,101,102]. The in vitro tests have shown that macrolides, including erythromycin, clarithromycin and azithromyicn, are highly active against mycoplasmas. However, only azithromycin regimens have been used for treatment of M. gentialium infections, and their efficacies against the mycoplamsa have been assessed. The eradication rates of M. genitalium in cases of genitourinary tract infections treated with azithromycin regimens are shown in TABLE 3 [27,36,39,40,45 48]. Azithromycin regimens A single 1-g dose of azithromycin has been considered as one of several relevant treatments for treatment of M. genitalium 794 Expert Rev. Anti Infect. Ther. 10(7), (2012)

5 Antimicrobial chemotherapy of Mycoplasma genitalium-positive non-gonococcal urethritis Review infections. Microbiological eradication rates of M. genitalium with a single 1-g dose of azithromycin when given as the first-line treatment range from 66.7 to 86.9% (TABLE 3) [27,36,39 41,45 48]. Falk et al. reported that the extended 5-day regimen of azithromycin, 500 mg on day 1 and 250 mg on the following 4 days, was very effective as a first-line treatment against M. genitalium infections in men with NGU or women with cervicitis [38]. The eradication rate of M. genitalium was 100%. However, Jernberg et al. reported that the eradication rate of this same extended 5-day regimen when given as the first-line treatment against M. genitalium infections in men with NGU or women with cervicitis was 77.6% [48]. They also reported an eradication rate of 73.7% for the regimen of azithromycin comprising a single 1-g dose on day 1 and an additional 1-g dose after 5 6 days. The microbiological effectiveness of the extended regimens, including the extended 5-day regimen, was not superior to that of the regimen of a single 1-g dose of azithromycin. No randomized trials comparing the efficacies of the extended regimens of azithromycin with that of the single 1-g dose regimen have been performed for the first-line treatment of M. genitalium infections. The superiority of the extended regimens of azithromycin would be inconclusive. For M. genitalium infections in male or female patients that had been unsuccessfully treated with doxycycline regimens, the extended 5-day regimen of azithromycin was effective as the second-line treatment. The eradication rates of M. genitalium with this extended 5-day regimen given as second-line therapy were reported to be % [38,39,49]. However, both Jernberg et al. and Jensen et al. reported that the eradication rates of the extended regimens of azithromycin given as the second- or thirdline treatment for M. genitalium infections, for which treatment with a single 1-g dose of azithromycin as the first-line treatment had failed, were % [28,48]. Azithromycin resistance Macrolides, including azithromycin, inhibit bacterial protein biosynthesis by reverse binding to the 50S subunit of bacterial ribosomes. In some bacterial species, the genes that encode efflux pumps confer resistance to macrolides [50]. The presence of the 23S rrna methylase genes, which modify the ribosomal targets, is associated with macrolide resistance [50]. However, modification of the macrolide binding site in the 50S subunit of bacterial ribosomes is the most widespread macrolide resistance mechanism [51]. Azithromycin treatment failure was reported in cases of M. genitalium-positive NGU, and macrolide-resistant clinical strains of M. genitalium were isolated from some treatment failure cases initially in Australia, Sweden and Norway [28]. In these strains, mutations in the 23S rrna gene associated with macrolide resistance and mutations in ribosomal protein genes of L4 and L22 were found. The mutations in the residues of the 23S rrna gene of M. genitalium corresponding to A2058 and A2059 in region V of the 23S rrna gene of Escherichia coli are critical for the binding of macrolides and can confer high-level resistance to macrolides, including azithromycin, because this species possesses only one copy of the rrna Table 2. Microbiological outcomes of tetracycline regimens in treatment of Mycoplasma genitalium-positive infections. Study (year) Horner et al. (1993) Gambini et al. (2000) Johannisson et al. (2000) et al. (2003) Björnelius et al. (2008) et al. (2009) et al. (2011) Regimen for 1 day mg once daily for 13 days daily for 7 days Tetracycline 500 mg twice daily for 10 days for 1 day mg once daily for 8 days, or lymecycline 300 mg twice daily for 7 days for 1 day mg once daily for 8 days daily for 7 days daily for 7 days with/without tinidazole 2 g once daily for 1 day Eradication rate, n (%) Ref. 10/14 (71.4) [35] 33/35 (94.3) [36] 5/13 (38.5) [37] 10/30 (33.3) [38] 13/76 (17.1) [39] 14/31 (45.2) [40] 12/39 (30.8) [41] gene operon [52]. Jensen et al. reported that the seven clinical isolates with azithromycin MICs of 8 or 32 g/ml harbored an A2058G or A2059G substitution in the 23S rrna gene [28]. The authors also found this identical mutation of A2058G or A2059G in the 23S rrna gene in M. genitalium DNAs in the urine specimens from men with NGU in Japan [53,54]. Yew et al. also detected the A2059G mutation in M. genitalium DNA in the genital specimen from a man with recurrent NGU in New Zealand [55]. For ribosomal proteins, macrolide resistance-associated missense mutations in L4 and L22 tend to be localized to Gln62 Gly66 in L4 and Arg88 Ala93 in L22 of E. coli, which are closest to the macrolide binding site [56]. In some bacterial species, however, macrolide-resistant mutations outside of these clusters have also been reported [57 59]. The amino acid substitutions reported in recent studies are H69R, Pro81Ser, Val84Gly, Glu128Gly, Tyr135Pro and N172S in L4, and Ser81Thr, Met82Lys, Gly93Glu, Asp109Glu, Asn112Asp, Arg114Kys and E123K in L22. Some of these amino acid alterations in L4 and L22 ribosomal proteins could be responsible for the development of macrolide resistance. Selection of azithromycin resistance Jensen et al. reported that a single-dose regimen of 1-g azithromycin selected M. genitalium strains harboring A2058G or A2059G in the 23S rrna gene from respective pre-treatment strains with the wild-type 23S rrna gene in three patients with NGU [28]. The authors also reported that a single-dose regimen 795

6 Deguchi, Ito, Hagiwara, Yasuda & Maeda Table 3. Microbiological outcomes of azithromycin regimens in treatment of Mycoplasma genitalium-positive infections. Study (year) Regimen Eradication rate, n (%) Ref. First-line treatment Second-, third- or fourth-line treatment Gambini et al. (2000) Azithromycin 1 g once daily for 1 day 14/17 (82.4) [36] et al. (2003) et al. (2006) Azithromycin 500 mg once daily for 1 day mg once daily for 4 days Azithromycin 500 mg once daily for 1 day mg once daily for 4 days 8/8 (100) 8/8 (100), [38] 19/19 (100) [49] Bradshaw et al. (2006) Azithromycin 1 g once daily for 1 day 23/32 (71.9) [45] Björnelius et al. (2008) Azithromycin 1 g once daily for 1 day 33/39 (84.6) [39] Azithromycin 500 mg once daily for 1 day mg once daily for 4 days 45/47 (95.7), Stamm et al. (2007) Azithromycin 1 g once daily for 1 day 6/7 (85.7) [46] Bradshaw et al. (2008) Azithromycin 1 g once daily for 1 day 101/120 (84.2) [47] Jernberg et al. (2008) Azithromycin 1 g once daily for 1 day 144/183 (78.7) [48] Azithromycin 500 mg once daily for 1 day mg once daily for 4 days Azithromycin 1 g once daily for 1 day + 1 g once daily for 1 day after 5 7 days 76/98 (77.6) 8/23 (34.8), 28/38 (73.7) Jensen et al. (2008) 0/4 (0) [28] Azithromycin 500 mg once daily for 1 day mg once daily for 4 6 days 0/3 (0) et al. (2009) Azithromycin 1 g once daily for 1 day 20/23 (86.9) [40] et al. (2011) Azithromycin 1 g once daily for 1 day with/without tinidazole 2 g once daily for 1 day 30/45 (66.7) [41] Hagiwara et al. (2011) Azithromycin 1 g once daily for 1 day 25/30 (83.3) [27] of 1-g azithromycin selected the mutants harboring the identical mutations in four out of 11 treatment failure cases of NGU by examining the associated urine specimens for the mutations in the 23S rrna gene [54]. Yew et al. also found an A2059G mutation in M. genitalium DNA in five out of nine genital samples from nine men with persistent NGU [55]. They demonstrated the selection of the strain with an A2059G mutation by a singledose azithromycin treatment in one man and suggested that the single-dose treatment might select similar mutants in the other four men. The single-dose regimen of 1-g azithromycin has been recommended as a presumptive treatment for non-gonococcal genitourinary tract infections [12,13,101,102]. However, such high prevalence of the emergence of macrolide-resistant mutants after the single-dose treatment with 1-g azithromycin would be a matter of serious concern. However, there are no reports that the extended 5-day regimen of azithromycin selected azithromycinresistant strains. The extended regimen might decrease the risk of macrolide resistance selection during treatment of M. genitalium infections. For the prevention of the emergence and spread of macrolide-resistant M. genitalium, however, there have not been any randomized trials to conclude the superiority of the extended regimen of azithromycin. Clinical impact of azithromycin resistance The authors reported a man with NGU infected with the strain of M. genitalium harboring both the A2059G (E. coli numbering) mutation in the 23S rrna gene and the Gly93Glu change in L22 [53]. He was treated with a single dose of 1-g azithromycin and was clinically and microbiologically cured of NGU. However, Jensen et al. reported that a single-dose regimen of 1-g azithromycin failed to eradicate M. genitalium harboring an A2058G mutation in men with NGU [28]. Yew et al. reported that in two men with NGU caused by M. genitalium harboring an A2059G mutation, NGU was cured with an extended course of azithromycin [55]. However, Jensen et al. reported that the mutants of M. genitalium harboring A2058G or A2059G 796 Expert Rev. Anti Infect. Ther. 10(7), (2012)

7 Antimicrobial chemotherapy of Mycoplasma genitalium-positive non-gonococcal urethritis Review could not be eradicated by the extended azithromycin regimens of either 500 mg on day 1 and 250 mg on the following 4 days or 1 g on day 1 and 500 mg on the following 6 days [28]. Another extended regimen of azithromycin 1 g weekly for 3 weeks was not effective against persistent NGU after a single-dose regimen of 1-g azithromycin or the extended 5-day azithromycin regimen, and selected the mutants harboring an A2058G or A2059G mutation in the 23S rrna gene [28]. The emergence and spread of such clinical mutants could be a threat in the treatment of M. genitalium infections with macrolides. Fluoroquinolones Fluoroquinolones are expected to exert a bactericidal effect on M. genitalium. The in vitro tests have shown them to have a wide range of antimicrobial activities against this mycoplasma. In relation to this aforementioned antimycoplasmal activity of fluoroquinolones, gatifloxacin, moxifloxacin and sitafloxacin are highly active against M. genitalium, but ofloxacin and levofloxacin, the L-isomer of ofloxacin, which are recommended for treatment of NGU in the guidelines [12,13,101,102], are less active. The eradication rates of M. genitalium in cases of genitourinary tract infections treated with several different regimens of fluoroquinolones are shown in TABLE 4 [25,28,47,48,60,61]. For other fluoroquinolones, including sparfloxacin, greparfloxacin, gemifloxacin, trovafloxacin and garenozacin, their efficacies against M. gentialium infections have not been evaluated, although they have been reported to be as active as gatifloxacin against M. genitalium in vitro. Ofloxacin & levofloxacin regimens In Japan, multiple and low doses of levofloxacin were frequently used to treat patients with NGU. The authors reported the low eradication rates (31.3 and 50.0%) of M. genitalium in men with NGU treated with the 7- and 14-day regimens of 100-mg levofloxacin three-times daily, respectively [25], and the high prevalence of recurrent NGU after treatment with the 14-day regimen [18]. It could be surmised that the regimen of 100 mg levofloxacin three-times daily for 14 days is effective for suppressing mycoplasmal growth and achieving clinical improvement but that this regimen is insufficient for eradicating the mycoplasma, resulting in frequent recurrence of NGU after treatment. In a recent report, the ofloxacin regimen of 200 mg twice daily for 10 days eradicated the mycoplasma in four out of nine patients, including men with NGU and women with cervicitis [48]. Takahashi et al. also reported that a regimen of levofloxacin 500 mg once daily for 7 days eradicated M. genitalium in only three out of five men with M. genitalium-positive NGU [60]. From the moderate antimycoplasmal activities of levofloxacin and ofloxacin seen in vitro and microbiological outcomes of their regimens in the treatment of M. genitalium infections, these fluoroquinolones would not appear to be appropriate for treatment of M. genitalium infections. Gatifloxacin regimens According to the previously reported results, gatifloxacin at 200 mg twice daily for 7 or 14 days had higher eradication rates for M. genitalium in men with NGU [25]. Hamasuna et al. also reported an eradication rate of 83% against M. genitalium in Table 4. Microbiological outcomes of fluoroquinolone regimens in treatment of Mycoplasma genitalium-positive infections. Study (year) Regimen Eradication rate, n (%) Ref. First-line treatment Second-, third-, fourth-, fifth- or sixth-line treatment et al. (2005) 5/16 (31.3) [25] 9/18 (50.0) Tosufloxacin 150 mg three-times daily for 14 days 5/7 (71.4) Gatifloxacin 200 mg twice daily for 7 days 22/24 (91.7) Gatifloxacin 200 mg twice daily for 14 days 6/6 (100) Jernberg et al. (2008) 4/9 (44.4) 21/36 (58.3), [48] 3/3 (100) 24/24 (100), Bradshaw et al. (2008) 9/9 (100), [47] Jensen et al. (2008) 11/11 (100) [28] et al. (2011) 3/5 (60.0) [60] Hamasuna et al. (2011) Gatifloxacin 200 mg twice daily for 7 days 15/18 (83.3) [61] Ito et al. (2012) Sitafloxacin 100 mg twice daily for 7 days 6/6 (100) 5/5 (100) # [62] with Mycoplasma genitalium harboring an A2058G or A2059G mutation in the 23S rrna. # 797

8 Deguchi, Ito, Hagiwara, Yasuda & Maeda men with NGU treated with a regimen of gatifloxacin 200 mg twice daily for 7 days [61]. However, this fluoroquinolone is no longer available because of its side effects. Moxifloxacin regimens Jensen et al. [28] and Bradshaw et al. [47] both reported that persistent M. genitalium-positive NGU following failure of treatment with azithromycin regimens was cured with a regimen of moxifloxacin 400 mg once daily for 10 days. Jernberg et al. reported that the 7-day regimen of moxifloxacin 400 mg once daily was highly effective as the second-, third- or fourth-line treatment for persistent M. genitalium-positive genito urinary tract infections in men and women unsuccessfully treated with azithro mycin and/or ofloxacin regimens [48]. These good microbio logical outcomes with moxifloxacin regimens could be derived from the high activity of this fluoroquinolone against M. genitalium strains, including those with macrolide resistance conferred by mutations in the 23S rrna gene. Sitafloxacin regimens In in vitro susceptibility tests, sitafloxacin was as active as moxifloxacin against M. genitalium strains, including reference strains and currently isolated strains [26]. In the recent study, the authors reported 89 patients with NGU with a 100-mg twicedaily dose (200-mg total daily dose) regimen of sitafloxacin to assess its efficacy against NGU [62]. In all 11 patients positive for M. genitalium, including five with persistent or recurrent NGU, the mycoplasma was eradicated by this sitafloxacin regimen. Sitafloxacin was supposed to be a promising agent for the treatment of M. genitalium infections. However, clinical studies for treating M. genitalium-positive NGU with sitafloxacin regimens are limited by the small number of patients. Further studies are required to assess sitafloxacin treatment of M. genitalium infections. Fluoroquinolone resistance Fluoroquinolones act by binding to their target enzymes, DNA gyrase and topoisomerase IV, and interfere with DNA replication. DNA gyrase and topoisomerase IV are composed of two GyrA and two GyrB subunits, and two ParC and two ParE subunits, respectively. The central mechanism of fluoroquinolone resistance involves alterations of the GyrA subunit of DNA gyrase and/or the ParC subunit of topoisomerase IV in bacterial species, including mycoplasmas and ureaplasmas [63 65]. Alterations of the GyrB and ParE subunits play a complementary role in the development of fluoroquinolone resistance. In recent studies, the authors detected amino acid changes S83N, D87Y and D87V in ParC, corresponding to changes at amino acid positions 80 and 84 in E. coli in M. genitalium DNA in three out of 28 urine specimens from men with NGU who had not received antibiotic treatment during the 3 months preceding their admission to the clinic [66,67]. These alterations in ParC located within the region analogous to the quinolone resistance-determining region of GyrA are related to resistance in various bacterial species, including other mycoplasmas and ureaplasmas. The authors did not isolate those strains and were, therefore, unable to determine their susceptibilities to fluoroquinolones or to confirm the association of the alterations in GyrA or ParC with fluoroquinolone resistance. In Japan, several fluoroquinolones have been developed, and some of them have antimicrobial activity against C. trachomatis. Therefore, low and multiple doses of fluoroquinolones with moderate activity against M. genitalium, such as the regimen of levofloxacin 100 mg three-times daily, are used so frequently to treat NGU that the mutants of M. genitalium with decreased susceptibility to fluoroquinolones could have been selected in clinical practice. Selection of fluoroquinolone resistance In previous studies, the eradication rates of M. genitalium with levofloxacin regimens were low, which could be mainly due to moderate activity of levofloxacin against M. genitalium and its low dose regimen [18,25]. Although clinical fluoroquinoloneresistant strains were not isolated from clinical samples, the authors assumed the emergence of fluoroquinolone-resistant M. genitalium in the clinical setting and then analyzed the association of failure of treatment with levofloxacin regimens with fluoroquinolone resistance [68]. The authors examined M. genitalium DNA in pre- and post-treatment urine specimens from men with NGU treated with the regimen of levofloxacin 100 mg three-times daily for 14 days for the presence of amino acid changes in the quinolone resistance-determining region of GyrA and the analogous region of ParC. In one man who remained positive for M. genitalium after treatment despite alleviation of symptoms and no evidence of urethral inflammation, the pretreatment M. genitalium had no alterations in either GyrA or ParC. The post-treatment mycoplasma, however, had an amino acid change of D87N in ParC. This patient did not receive antimicrobial agents after completing 14 days of levofloxacin treatment. However, he returned to the clinic 4 days later with recurrent symptoms. At the time of NGU recurrence, his firstpass urine was positive for M. genitalium, and the mycoplasma that was detected in the urine also harbored a D87N change in ParC. Treatments with fluoroquinolones with moderate activity against M. genitalium or with suboptimal doses of fluoroquinolones could increase the risk of fluoroquinolone resistance selection after treatment. Clinical impact of fluroroquinolone resistance In the previous study, one man with acute NGU was treated with the regimen of levofloxacin 100 mg three-times daily for 14 days but had persistent NGU after treatment [68]. The pretreatment M. genitalium showed no alterations in GyrA but did have an amino acid change of D87Y in ParC, corresponding to a change at amino acid position 84 in E. coli. In the post-treatment of M. genitalium, the amino acid sequences of GyrA and ParC were the same as the pre-treatment sequences. Although the alteration in ParC could lead to treatment failure of M. genitalium-positive NGU with the levofloxacin regimen, it is unknown how seriously the emergence of clinical strains of M. genitalium harboring such a single amino acid change 798 Expert Rev. 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9 Antimicrobial chemotherapy of Mycoplasma genitalium-positive non-gonococcal urethritis Review in ParC could threaten the efficacy of other fluoroquinolones with high activity against M. genitalium, such as moxifloxacin and sitafloxacin, in the treatment of M. genitalium infections. However, the acquisition of a single amino acid change in GyrA or ParC might be the first step in the development of clinically significant resistance to fluoroquinolones. Subsequently, the accumulation of amino acid changes in GyrA and ParC could be induced by serial exposure of strains with a single amino acid change in GyrA or ParC to fluoroquinolones and could bring about stepwise increases in the level of fluoroquinolone resistance [63 65]. To maintain the efficacy of fluoroquinolone regimens, care must be taken to select suitable fluoroquinolones with high activity against M. genitalium and to use them in optimal doses for the treatment of genitourinary tract infections. Expert commentary M. genitalium is considered an important pathogen of genitourinary tract infections in men and women. In urethritis in men, the mycoplasma that is significantly associated with persistent or recurrent NGU should be taken into account when treating NGU. The current guidelines recommend a single-dose regimen of 1-g azithromycin or a 7-day regimen of a twice-daily dose of 100-mg doxycycline for treatment of NGU. They also recommend an erythromycin, levofloxacin or ofloxacin regimen as alternative treatments. These regimens have been effective in the treatment of chlamydia-positive NGU. For M. genitalium infections, however, treatment with the doxycycline regimen has not been so effective. Microbiological eradication of M. genitalium with the 1-g dose regimen of azithromycin has been reported to be %. In addition, the emergence of macrolide-resistant clinical strains of M. genitalium selected by the azithromycin regimen has been reported in clinical practice. The single-dose treatment of 1-g azithromycin, which could be suboptimal for eradication of M. genitalium, might increase the risk of macrolide resistance selection after treatment. The initial use of azithromycin regimens with higher doses or longer dose durations could be considerable to assure the efficacy of eradication of M. genitalium and reduce the risk of macrolide resistance selection in M. genitalium infections. For fluoroquinolones, ofloxacin and levofloxacin, which are recommended in the guidelines, are less active than gatifloxacin, moxifloxacin and sitafloxacin. Moxifloxacin, in particular, has been shown to be a highly effective agent against M. genitalium-positive NGU. Therefore, the initial use of moxifloxacin could be a sensible alternative option. Pointof-care testing for C. trachomatis at the time of treatment for NGU is difficult, and no sensitive and rapid tests for detection of genital mycoplasmas and ureaplasmas are available in clinical settings. In addition, many patients with urogenital infections do not return for a test of cure. Thus, patients with NGU should be treated presumptively with the regimens that are effective against infections caused not only by C. trachomatis but also by genital mycoplasmas and ureaplasmas. However, it should be considered whether azithromycin regimens with higher doses or longer dose durations or a moxifloxacin regimen would be appropriate for the initial treatment of all NGU. There are also concerns that such treatments may lead to the development of further resistance to azithromycin and to fluoroquinolones in M. genitalium [69]. Thus, further studies are required to establish a new treatment algorithm for NGU, including diseases positive for genital mycoplasmas and ureaplasmas. The development of new diagnostic methods that could be available as pointof-care testing for pathogens in the clinical setting is also required to carry out optimal treatment specific to the pathogen. Nevertheless, at the present time, the single-dose treatment of 1-g azithromycin would appear to be an appropriate firstline treatment for NGU, and if trichomonas infections can be ruled out, the regimen of moxifloxacin 400 mg once daily for 7 days would appear to be a reasonable second-line treatment of persistent or recurrent NGU. Five-year view The selection of macrolide-resistant M. genitalium by the singledose regimen of 1-g azithromycin and the subsequent spread of macrolide-resistant M. genitalium would be a matter of serious concern. As the prevalence of macrolide-resistant M. genitalium increases, fluoroquinolones, including moxifloxacin, would be commonly used in place of macrolides. Subsequently, fluoroquinolone-resistant strains would be selected. As a matter of fact, some strains that are resistant to both azithromycin and moxifloxacin have been detected [10]. Multidrug-resistant M. genitalium would emerge and prevail in clinical practice, resulting in difficulty in the treatment of M. genitalium infections. In vitro tests of antimicrobial activities of antibiotics have shown some new agents that are highly active against M. genitalium. For example, solithromycin, which is the newest macrolide derivative called a fluoroketolide, was reported to be highly active against azithromycin-susceptible strains of M. genitalium. This agent maintained in vitro activity against macrolide-resistant M. pneumoniae isolates that altered binding sites in the 23S rrna, but it has not been examined for its activity against macrolide-resistant M. genitalium isolates. It is expected that regimens with such new antibiotics would be examined for clinical and microbiological efficacy in the treatment of M. genitalium infections. However, until promising new alternative antibiotics for the treatment of M. genitalium infections are developed, the drug resistance of M. genitalium must be controlled in an efficient and timely manner to prolong the efficacy of existing antibiotics. Surveillance for antimicrobial resistance in clinical strains of M. genitalium is crucial for determining subsequent treatment guidelines. However, isolation of M. genitalium in culture remains a labor-intensive and time-consuming task. In previous studies, the authors attempted to apply a molecular approach that did not involve culturing M. genitalium and testing of in vitro antimicrobial susceptibility to assess fluoroquinolone or macrolide resistance of clinical strains of M. genitalium. To monitor the antimicrobial susceptibilities of clinical strains of M. genitalium, such a non-culture approach to assess antimicrobial resistance of 799

10 Deguchi, Ito, Hagiwara, Yasuda & Maeda M. genitalium, clinical strains should be used until culturing of the mycoplasma from clinical specimens and testing of its in vitro antimicrobial susceptibility can be performed easily in laboratories [53,54,66,67]. Ideally, point-of-care testing for the pathogens, including C. trachomatis, M. genitalium, U. urealyticum and T. vaginalis, at the time of treatment for non-gonococcal urogenital infections in men and women should be developed and made available in clinical settings. According to a new treatment algorithm for genitourinary tract infections based on detected pathogens, appropriate treatments could be administered against respective infections, including that of M. genitalium. Financial and competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript. Key issues Mycoplasma genitalium is an important pathogen of acute non-gonococcal urethritis (NGU). M. genitalium may play a significant role in persistent or recurrent NGU. In the management of patients with M. genitalium-positive NGU, eradication of the mycoplasma from the urethra would be essential to prevent persistent or recurrent NGU. The treatment of NGU with 100 mg of doxycycline given orally twice daily for 7 days as recommended in the guidelines has not been so effective against NGU caused by M. genitalium. M. genitalium with the single 1-g dose of azithromycin, recommended as the first-line treatment for NGU in the guidelines, have been reported to be %. M. genitalium with the extended 5-day regimen of azithromycin, 500 mg on day 1 and 250 mg on the following 4 days, when given as the first-line treatment, have been reported to be %. As a second-line treatment, the extended regimen of azithromycin was effective against M. genitalium infections unsuccessfully treated with doxycycline regimens but was ineffective against those unsuccessfully treated with the single-dose regimen of 1-g azithromycin. In clinical strains of M. genitalium, mutations in the 23S rrna gene were responsible for macrolide resistance. M. genitalium with mutations in the 23S rrna gene associated with macrolide resistance were isolated from azithromycin treatment failure cases. M. genitalium was selected by the single-dose regimen of 1-g azithromycin. The 7-day regimen of moxifloxacin 400 mg once-daily was highly effective against persistent M. genitalium-positive NGU unsuccessfully treated with azithromycin regimens as the second-, third- or fourth-line treatment. At present, the single-dose treatment of 1-g azithromycin should be a first-line treatment for NGU, and if trichomonas infections can be ruled out, the regimen of moxifloxacin 400 mg once daily for 7 days should be a second-line treatment of persistent or recurrent NGU. To monitor the antimicrobial susceptibilities of clinical strains of M. genitalium, a non-culture approach to assess antimicrobial resistance of M. genitalium clinical strains would be useful until culturing of the mycoplasma from clinical specimens and testing of their in vitro antimicrobial susceptibility can be performed easily in laboratories. References Papers of special note have been highlighted as: 1 Krieger JN. Trichomoniasis in men: old issues and new data. Sex. Transm. Dis. 22(2), (1995). 2 Swenson PD, Lowens MS, Celum CL, Hierholzer JC. Adenovirus types 2, 8, and 37 associated with genital infections in patients attending a sexually transmitted disease clinic. J. Clin. Microbiol. 33(10), (1995). 3. 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