De novo mutations drive the spread of macrolide resistant Mycoplasma genitalium : mathematical modelling study

The rapid spread of antimicrobial resistance in sexually transmitted infections caused by Mycoplasma genitalium is a growing concern. It is not yet clear to what degree macrolide resistance in M. genitalium results from the emergence of de novo mutations or the transmission of resistant strains. We analysed epidemiological data and developed a compartmental model to investigate the contribution of de novo macrolide resistance mutations to the spread of antimicrobial resistant M. genitalium. We fitted the model to data from France, Sweden and Denmark and estimated treatment rates and the time point of azithromycin introduction. In a meta-analysis of six studies, we estimated that de novo resistance develops in 12% (95% CI 7-17%, I2 44%) of azithromycin treated M. genitalium infections. Our model shows that the high probability of de novo resistance accelerates the spread of antimicrobial resistant M. genitalium in comparison with lower probabilities. The estimated per capita treatment rate in France was lower than in Denmark and Sweden but confidence intervals for the three estimates overlap. The estimated dates of introduction of azithromycin in each country are consistent with published reports. We conclude that clinical management strategies for M. genitalium should seek to limit the unnecessary use of macrolides

Phenotypic and genotypic antimicrobial susceptibility patterns of the emerging human respiratory pathogen Mycoplasma amphoriforme isolated from the UK and Denmark

Methods Seven isolates of M. amphoriforme were examined for antimicrobial susceptibility to seven antibiotics using the microbroth dilution assay in line with the CLSI guidelines for mycoplasmas. Each isolate was additionally subjected to WGS to identify resistance-associated mutations. Based on the consensus sequences from the genomic data, PCR primers were designed, and tested, for the amplification of the QRDR within the parC gene. Results Of the seven isolates investigated, four (57%) were resistant to moxifloxacin (0.5–1 mg/L) and levofloxacin (1–2 mg/L), compared with those that were susceptible (0.03–0.06 and 0.006 mg/L, respectively). Isolate H29 was resistant to five of the seven antibiotics tested: moxifloxacin, 0.5 mg/L; levofloxacin, 2 mg/L; azithromycin, 64 mg/L; erythromycin, 128 mg/L; and clindamycin, 64 mg/L. All isolates were susceptible to tetracycline (0.06 mg/L) and lefamulin (0.001–0.004 mg/L). Mutations from genomic data confirmed the presence of an S89F mutation within the ParC protein among all fluoroquinolone-resistant isolates and an A2059G mutation in the 23S rRNA gene in the macrolide- and lincosamide-resistant isolate H29. Conclusions To the best of our knowledge, this is the first time where phenotypic and genotypic resistance data have been paired for M. amphoriforme confirming a correlation between the two. These data suggest the need for focused testing and resistance determination of isolates from high-risk patients given the backdrop of a high prevalence of antimicrobial resistance

Mycoplasma genitalium PCR: Does Freezing of Specimens Affect Sensitivity?▿

Mycoplasma genitalium is an established cause of sexually transmitted infections. Studies of disease associations are often performed on archived specimens, but little is known about the effect of storage of specimens on the detection of M. genitalium. Genital swab and first-void urine specimens submitted for detection of M. genitalium were tested on the day of receipt. Remnants of positive original specimens as well as DNA preparations were stored at −20°C for up to 18 months. A total of 361 M. genitalium-positive specimens were available. PCR after repeat DNA preparation was performed for 262 specimens. The sensitivity after repeat DNA preparation was 90%, and the median decrease in DNA load was 155 genome equivalents (geq) (P < 0.0001). For 327 specimens, PCR could be repeated on the primary DNA preparation. The sensitivity of PCR after storage was 95%, and the median decrease in DNA load was 13.5 geq (P < 0.0001). The specimens yielding negative results at repeat testing had a significantly lower median DNA load in the primary analysis than those with a repeat positive test (P < 0.0001). For 228 specimens, PCR could be performed both on the primary DNA preparation and after repeat DNA preparation. The median DNA load was lower after repeat DNA extraction than after repeat testing of the stored DNA extract (P < 0.0001). In conclusion, the M. genitalium DNA load as well as the detection rate decreased after storage. This was more pronounced in clinical specimens stored frozen than in stored DNA extracts, particularly in those with an initial low DNA load

Mutations in ParC and GyrA of moxifloxacin-resistant and susceptible Mycoplasma genitalium strains.

Macrolide or fluoroquinolone-resistant Mycoplasma genitalium is spreading worldwide. We aimed to determine the influence of single nucleotide polymorphisms (SNPs) in the quinolone resistance determining regions (QRDR) of parC and gyrA in cultured M. genitalium strains. In addition, we examined the prevalence of macrolide- and fluoroquinolone resistance mediating mutations in specimens collected from Japanese male patients with urethritis in two time-periods between 2005-2009 and 2010-2017, respectively, by sequencing the QRDR of parC and gyrA and domain V of the 23S rRNA gene. The minimum inhibitory concentrations (MIC) of moxifloxacin, sitafloxacin, ciprofloxacin, levofloxacin, doxycycline, minocycline, azithromycin and clarithromycin were determined in 23 M. genitalium strains. Three cultured strains had elevated MICs for moxifloxacin at 16, 4 and 2 mg/L and had SNPs with the amino-acid change Ser83→Ile in ParC (p<0.001) and 3 kinds of SNPs with amino-acid changes Asp99→Asn, Gly93→Cys and Met95→Ile in GyrA, respectively. Among a total of 148 M. genitalium positive urine specimens, the prevalence of A2058G and A2059G SNPs in the 23S rRNA gene and any SNPs in ParC increased from 4.8% and 22.6% in 2005-2009 to 42.2% and 53.1% in 2010-2017, respectively. If M. genitalium is considered multi-drug resistant in clinical specimens carrying SNPs in the 23S rRNA gene and Ser83→Ile in ParC, the prevalence of multi-drug resistance is 12.5% in 2010-2017 in Japan. In conclusion, the SNP resulting in Ser83→Ile in ParC is closely related to moxifloxacin resistance even though other factors may also affect treatment outcomes by moxifloxacin. The prevalence of circulating multi-drug resistant M. genitalium strains with macrolide- and fluoroquinolone-resistance is dramatically increasing in Japan

Changes in the vaginal microbiota following antibiotic treatment forMycoplasma genitalium,Chlamydia trachomatisand bacterial vaginosis

The human vagina harbor a rich microbiota. The optimal state is dominated by lactobacilli that help to maintain health and prevent various diseases. However, the microbiota may rapidly change to a polymicrobial state that has been linked to a number of diseases. In the present study, the temporal changes of the vaginal microbiota in patients treated for sexually transmitted diseases or bacterial vaginosis (BV) and in untreated controls were studied for 26 days. The patients included 52 women treated with azithromycin, tetracyclines or moxifloxacin for present or suspected infection withChlamydia trachomatisorMycoplasma genitalium. Women with concurrent BV were also treated with metronidazole. The controls were 10 healthy women of matching age. The microbiota was analyzed by 16S rRNA gene deep sequencing, specific qPCRs and microscopy. There was generally good correlation between Nugent score and community state type (CST) and qPCR confirmed the sequencing results. By sequencing, more than 600 different taxa were found, but only 33 constituted more than 1 parts per thousand of the sequences. In both patients and controls the microbiota could be divided into three different community state types, CST-I, CST-III and CST-IV. Without metronidazole, the microbiota remained relatively stable regarding CST although changes were seen during menstrual periods. Administration of metronidazole changed the microbiota from CST-IV to CST-III in approximately 50% of the treated patients. In contrast, the CST was generally unaffected by azithromycin or tetracyclines. In 30% of the BV patients,Gardnerella vaginaliswas not eradicated by metronidazole. The majority of women colonized withUreaplasma parvumremained positive after azithromycin whileU.urealyticumwas eradicated

Correlation between MICs for selected antimicrobials and SNPs in domain V of the 23S rRNA gene and the QRDRs of GyrA (NA: 127–402, AA: 57–134) and ParC (NA: 164–483, AA: 55–161) among 23 <i>M</i>. <i>genitalium</i> isolates.

<p>(Numbering of amino acids in <i>M</i>. <i>genitalium</i> numbering; <i>E</i>. <i>coli</i> numbering in brackets).</p