dMycoplasma genitalium is a significant sexually transmitted pathogen, causing up to 25% of cases of nongonococcal urethritis in men, and it is strongly associated with cervicitis and pelvic inflammatory disease in women. Currently, the usual first-line treatment is the macrolide antibiotic azithromycin, but an increasing incidence of treatment failure over the last 5 years suggests the emergence of antibiotic resistance. The mutations responsible for macrolide resistance have been found in the 23S rRNA gene in numerous M. genitalium populations. A second-line antibiotic, the fluoroquinolone moxifloxacin, was thought to be a reliable alternative when azithromycin began to fail, but recent studies have identified mutations that may confer fluoroquinolone resistance in the genes parC and gyrA. The aim of this study was to determine the prevalence of antibiotic resistance in M. genitalium in Sydney, Australia, by detecting relevant mutations in the 23S rRNA gene, parC, and gyrA. M. genitalium-positive DNA extracts of specimens, collected from patients attending sexual health clinics in Sydney, were tested by PCR amplification and DNA sequence alignment. The 186 specimens tested included 143 initial patient specimens and 43 second, or subsequent, specimens from 24 patients. We identified known macrolide resistance-associated mutations in the 23S rRNA gene in 43% of the initial patient samples and mutations potentially associated with fluoroquinolone resistance in parC or gyrA sequences in 15% of the initial patient samples. These findings support anecdotal clinical reports of azithromycin and moxifloxacin treatment failures in Sydney. Our results indicate that further surveillance is needed, and testing and treatment protocols for M. genitalium infections may need to be reviewed. Mycoplasma genitalium, one of the smallest known self-replicating organisms (1), is an obligate parasite, preferring the human genital tract as its host environment (2-4). In men, M. genitalium is a significant cause of both acute and chronic nongonococcal urethritis (NGU) (5-7) and is estimated to account for 15% to 25% of cases of NGU in some populations (2). In women, M. genitalium has been significantly associated with both cervicitis (2, 8-10) and pelvic inflammatory disease (2,(11)(12)(13)(14), and it has been identified in up to 7.3% of women in high-risk populations (15). In Australia, M. genitalium has been reported to account for between 4.5% and 9% of cases of NGU (16, 17), while it was identified in 4% of women attending a Sydney sexual health clinic and was the second most common cause of cervicitis (9).The usual treatment regime for clinically diagnosed NGU and cervicitis is a single dose of 1 g azithromycin (AZM) (18). An extended regime (1.5 g over 5 days) is often prescribed if singledose AZM is unsuccessful, but there is contention regarding the effectiveness of this extended treatment (19,20). Alarmingly, studies investigating the effectiveness of either 1 g or 1.5 g AZM treatment have reported microbiological treatment f...

Increasing azithromycin treatment failure in sexually transmitted Mycoplasma genitalium infection, is linked to macrolide resistance and second-line treatment relies on the fluoroquinolone, moxifloxacin. We recently detected fluoroquinolone and macrolide resistance-associated mutations in 15% and 43%, respectively, of 143 initial M. genitalium PCR-positive specimens. For a subset of 33 Western Sydney Sexual Health Centre patients, clinical information and results of sequence analysis of M. genitalium macrolide and fluoroquinolone target genes - the 23S rRNA gene, and parC and gyrA, respectively - were used to examine whether mutations were associated with treatment failure. Macrolide resistance-associated mutations correlated with microbiological (p = 0.013) and clinical (p = 0.024) treatment failure, and fluoroquinolone resistance-associated mutations with microbiological moxifloxacin treatment failure (p = 0.005). We describe the first reported cases of clinical and microbiological moxifloxacin treatment failure. Failure of first- and second-line antibiotic treatment of M. genitalium infection is occurring and likely to increase with current treatment strategies.