Mitsuru Yasuda

Selection of Mycoplasma genitalium strains harbouring macrolide resistance-associated 23S rRNA mutations by treatment with a single 1 g dose of azithromycin

Objective A single 1 g dose regimen of azithromycin has been recommended for the treatment of Mycoplasma genitalium infections. The authors evaluated whether this regimen could select M genitalium strains with macrolide resistance after treatment for M genitalium-positive non-gonococcal urethritis. Methods In seven men with non-gonococcal urethritis, who were infected with M genitalium without macrolide resistance-associated mutations but experienced microbiological azithromycin treatment failure, M genitalium DNAs in their post-treatment urine specimens were examined for mutations in the 23S rRNA gene and the ribosomal protein genes of L4 and L22. To assess the relatedness of M genitalium strains before and after treatment, their DNAs in pretreatment and post-treatment urine were genotyped by analysing short tandem repeats of an AGT/AAT unit in the MG309 gene and single nucleotide polymorphisms in the MG191 gene. Results In four of seven patients, M genitalium in post-treatment urine had an A-to-G transition at nucleotide position 2071 or 2072, corresponding to 2058 or 2059 in the 23S rRNA gene of Escherichia coli. In one of the four strains, Pro81Ser in the ribosomal protein L4 accompanied the mutation in the 23S rRNA gene. The genotyping of M genitalium DNAs suggested that these four post-treatment strains were selected from the respective closely related or identical pretreatment strains without macrolide resistance-associated mutations by the treatment. Conclusions The single 1 g dose treatment of azithromycin could select M genitalium strains harbouring macrolide resistance-associated mutations. For M genitalium, this regimen might increase the risk of macrolide resistance selection after treatment.

Macrolide Resistance–associated 23S rRNA Mutation in Mycoplasma genitalium, Japan

To the Editor: Mycoplasma genitalium is now recognized as a serious pathogen in sexually transmitted infections (1,2). Azithromycin regimens have been commonly used for treatment of M. genitalium infections (3). However, failure of azithromycin treatment has been reported in cases of M. genitalium–positive nongonococcal urethritis (NGU) (4,5), and macrolide-resistant strains of M. genitalium have been isolated from case-patients in Australia, Sweden, and Norway for whom azithromycin treatment has failed (4,5). In these strains, mutations in the 23S rRNA gene were associated with macrolide resistance, and mutations in ribosomal protein genes L4 and L22 were also found (5). Surveillance for antimicrobial resistance of M. genitalium is essential to identify antimicrobial resistant strains and to then determine appropriate treatment. Coculture of patient specimens with Vero cells has improved the primary isolation rate of M. genitalium from clinical specimens and offered some current clinical strains for antimicrobial drug susceptibility testing (6). To determine their antimicrobial susceptibilities, a molecular real-time PCR method has been developed (7,8). However, isolating M. genitalium from clinical specimens and antimicrobial drug susceptibility testing of clinical isolates remain labor-intensive, time-consuming tasks. In addition, no methods are available to directly determine antimicrobial drug susceptibilities of M. genitalium in clinical specimens. To monitor macrolide susceptibilities in clinical strains of M. genitalium in Japan, therefore, we examined M. genitalium DNA found in the urine of men with NGU for the presence of macrolide resistance–associated mutations in the 23S rRNA gene and the ribosomal protein genes L4 and L22. n nThis retrospective study was approved by the Institutional Review Board of the Graduate School of Medicine, Gifu University, Gifu, Japan. We collected pretreatment urine specimens from 308 men with NGU who had visited a urologic clinic (iClinic) in Sendai, Japan, during 2006 through 2008 and stored the specimens at –70°C. Each man gave informed consent. Twenty-five of 58 urine specimens confirmed to be positive for M. genitalium by PCR-based assay were randomly chosen for this study and subjected to DNA purification. The 23S rRNA gene and the ribosomal proteins genes L4 and L22 of M. genitalium were amplified from the purified DNA by PCR as reported previously and then sequenced (5). n nIn 1 specimen, we found an A-to-G transition at nucleotide position 2072 in the 23S rRNA gene of M. genitalium, corresponding to position 2059 in Escherichia coli (Table). An A2059 (E. coli numbering) residue in region V of the 23S rRNA gene is critical for the binding of macrolides (9). Mutations of A2058, A2059, and other 23S rRNA residues within the macrolide-binding site can confer a high-level resistance to macrolides in several bacterial species, including M. genitalium (5,9). Therefore, M. genitalium strains that harbor the A2059G (E. coli numbering) mutation in the 23S rRNA gene could be highly macrolide resistant. We also found a T-to-G transition at nucleotide position 2199 in the 23S rRNA gene of M. genitalium, corresponding to position 2185 in E. coli, in 3 specimens, but this mutation has not been associated with macrolide resistance in other bacterial species (9). n n n nTable n nMutations in the 23S rRNA gene and amino acid changes in L4 and L22 ribosomal proteins of 25 Mycoplasma genitalium strains in the pretreatment urine specimens of men with nongonococcal urethritis, Japan n n n nWe found amino acid changes in L4 and L22 ribosomal proteins in M. genitalium in 9 specimens. L4 and L22 ribosomal proteins each have extended loops, which converge to form a narrowing in the exit tunnel adjacent to the macrolide-binding site (10). Therefore, 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 (10). All of the amino acid changes in L4 of M. genitalium found in this study corresponded to those at the downstream regions from Gln62-Gly66 in L4 of E. coli. Of the amino acid changes in L22 of M. genitalium, the only Gly93Glu change found in M. genitalium harboring the A2059G (E. coli numbering) mutation in the 23S rRNA gene was located within the region corresponding to Arg88-Ala93 in L22 of E. coli. In this strain, therefore, the Gly93Glu change in L22 might contribute to the increase of macrolide resistance. The patient with NGU, whose specimen exhibited this strain of M. genitalium that harbored both the A2059G (E. coli numbering) mutation in the 23S rRNA gene and in which the Gly93Glu change in L22 was detected, was given a single dose of 1 g azithromycin and was clinically cured of NGU. However, the present study suggests that M. genitalium strains with high-level macrolide resistance might have already emerged in clinical settings in Japan. The emergence and spread of such a clinical mutant could threaten the ability of macrolides to treat M. genitalium infections. We should continue monitoring macrolide resistance of M. genitalium clinical strains. The nonculture approach used in our study will be useful until culturing of mycoplasmas from clinical specimens and antimicrobial drug susceptibility testing can be performed easily in laboratories.

Emergence and spread of drug resistant Neisseria gonorrhoeae.

PURPOSEnThe emergence and spread of Neisseria gonorrhoeae with resistance to oral antibiotics have led to difficulty in treating gonorrhea. We review drug resistance in N. gonorrhoeae with a particular emphasis on resistance to fluoroquinolones, cefixime and azithromycin.nnnMATERIALS AND METHODSnLiterature selected from peer reviewed journals listed in MEDLINE(R)/PubMed(R) from 1943 to 2009 and from resources cited in those articles was reviewed comprehensively.nnnRESULTSnDue to the spread of fluoroquinolone resistant N. gonorrhoeae fluoroquinolones are no longer recommended for the treatment of gonorrhea. The emergence of N. gonorrhoeae with a mosaic penicillin-binding protein 2 associated with oral cephalosporin resistance has threatened cefixime treatment for gonorrhea. Emergence of N. gonorrhoeae with high level resistance to azithromycin has also been documented. However, injectable antibiotics (sepctinomycin and ceftriaxone) retain their activity against N. gonorrhoeae. To monitor drug resistance in N. gonorrhoeae several national and international programs have become functional.nnnCONCLUSIONSnOral regimens for the treatment of gonorrhea are limited. At present to our knowledge ceftriaxone is the most reliable and available agent for the treatment of gonorrhea. To prevent the further emergence and international spread of drug resistance, and allow for the selection of appropriate treatments, a comprehensive global program is needed including surveillance for drug resistance in N. gonorrhoeae and collection of patient epidemiological data. Clinicians should effectively treat patients with gonorrhea, always being conscious of local trends of drug resistance in N. gonorrhoeae, and should perform culture and antimicrobial susceptibility testing in those with persistent gonorrhea after treatment.

New Clinical Strain of Neisseria gonorrhoeae with Decreased Susceptibility to Ceftriaxone, Japan.

To the Editor: In 2009, 2010, and 2013, Neisseria gonorrhoeae strains H041 (ceftriaxone MIC of 2 mg/L), F89 (ceftriaxone MIC of 1 mg/L), and A8806 (ceftriaxone MIC of 0.5 mg/L) were isolated from samples from patients in Japan (1), France (2) and Australia (3), respectively. In Japan, no other clinical N. gonorrhoeae strains with decreased susceptibility to ceftriaxone were reported until 2014, when clinical strain {type:entrez-nucleotide,attrs:{text:GU140106,term_id:268370439,term_text:GU140106}}GU140106 (ceftriaxone MIC of 0.5 mg/L) was isolated from a man in in Nagoya, Japan. We report details of this case and sequencing results of the penA gene for the strain. The study was approved by the Institutional Review Board of the Graduate School of Medicine, Gifu University, Japan. n nN. gonorrhoeae strain {type:entrez-nucleotide,attrs:{text:GU140106,term_id:268370439,term_text:GU140106}}GU140106 was isolated from a urethral swab sample from a man with acute urethritis. The man had received fellatio, without condom use, from a female sex worker in Nagoya in December 2013. He visited our clinic in January 2014 for urethral discharge. Culture of a urethral swab sample was positive for N. gonorrhoeae. We used the Cobas 4800 CT/NG Test (Roche Molecular Systems Inc., Pleasanton, CA, USA) to test a first-voided urine sample; results were positive for N. gonorrhoeae but negative for Chlamydia trachomatis. The infection was treated with a single-dose regimen of ceftriaxone (1 g) administered by intravenous drip infusion. Two weeks later, the man reported no symptoms, and his first-voided urine sample was negative for leukocytes. The test-of-cure for N. gonorrhoeae was not performed. The female sex worker could not be examined for the presence of N. gonorrhoeae strain {type:entrez-nucleotide,attrs:{text:GU140106,term_id:268370439,term_text:GU140106}}GU140106 in her pharynx. n nThe strain was confirmed to be a gonococcal species by testing with Gonochek-II (TCS Biosciences Ltd, Buckingham, UK), the HN-20 Rapid system identification test (Nissui, Tokyo, Japan), and the Aptima Combo 2 assay for CT/NG (Hologic, Inc., Bedford, MA, USA) and by 16S rRNA gene sequencing and porA pseudogene PCR (4). MICs of antimicrobial drugs for {type:entrez-nucleotide,attrs:{text:GU140106,term_id:268370439,term_text:GU140106}}GU140106 were as follows, as determined by using the agar dilution method: 2.0 mg/L for penicillin G, 1.0 mg/L for tetracycline, 2.0 mg/L for cefixime, 0.5 mg/L for ceftriaxone, 8.0 mg/L for levofloxacin, 0.5 mg/L for azithromycin, and 32.0 mg/L spectinomycin. The strain was determined to be resistant to penicillin G, tetracycline, cefixime, ceftriaxone, and levofloxacin, according to criteria of the European Committee on Antimicrobial Susceptibility Testing (5) n nThe penA gene of strain {type:entrez-nucleotide,attrs:{text:GU140106,term_id:268370439,term_text:GU140106}}GU140106 was sequenced as previously described (6); results showed the presence of a novel mosaic penicillin-binding protein 2 (PBP2; GenBank accession no. {type:entrez-nucleotide,attrs:{text:LC056026,term_id:965690016,term_text:LC056026}}LC056026) (Figure). Multilocus sequence typing (MLST) and N. gonorrhoeae multiantigen sequence typing (NG-MAST) of {type:entrez-nucleotide,attrs:{text:GU140106,term_id:268370439,term_text:GU140106}}GU140106 were performed as previously reported (8,9). MLST assigned strain {type:entrez-nucleotide,attrs:{text:GU140106,term_id:268370439,term_text:GU140106}}GU140106 to sequence type 7363, the same as strains H041 and A8806 (1,2). NG-MAST assigned strain {type:entrez-nucleotide,attrs:{text:GU140106,term_id:268370439,term_text:GU140106}}GU140106 to sequence type 6543. MLST and NG-MAST results for {type:entrez-nucleotide,attrs:{text:GU140106,term_id:268370439,term_text:GU140106}}GU140106 differed from those for F89 (3). n n n nFigure n nSequences of altered amino acids in penicillin-binding protein 2 (PBP2) of Neisseria gonorrhoeae strains with decreased susceptibility to oral cephalosporins and strains with resistance to ceftriaxone. Strain {type:entrez-nucleotide,attrs:{text:GU140106,term_id:268370439,term_text:GU140106}} ... n n n nSince the naming of the mosaic PBP2 associated with decreased susceptibilities to oral cephalosporins as pattern X (6), various PBP2 mosaic structures have been discovered. Mosaic PBP2 structures are basically composed of fragments analogous to PBP2s in Neisseria species. Before strain H041 emerged, strains harboring mosaic PBP2s had been resistant to oral cephalosporins but susceptible to ceftriaxone. H041 (ceftriaxone MIC of 2 mg/L) had additional novel amino acid changes, including A311V, V316P, and T483S, in its mosaic PBP2. The presence of substitutions A311V, V316P, and T483S was reported to be responsible for resistance to ceftriaxone (7). Like strain H041, strains {type:entrez-nucleotide,attrs:{text:GU140106,term_id:268370439,term_text:GU140106}}GU140106 and A8806 (ceftriaxone MICs of 0.5 mg/L) had substitutions A311V and T483S, but instead of substitution V316P, they had substitution V316T. In addition, {type:entrez-nucleotide,attrs:{text:GU140106,term_id:268370439,term_text:GU140106}}GU140106 had several changes in positions 227–281 that were not present in other strains. These alterations might also contribute to the decreased susceptibility to ceftriaxone. n nOn the basis of pharmacodynamic analyses (10), a 1-g dose of ceftriaxone (the recommended first-line treatment for gonorrhea in Japan) would be effective against genital gonorrhea caused by strains exhibiting decreased susceptibility to ceftriaxone (e.g., strains {type:entrez-nucleotide,attrs:{text:GU140106,term_id:268370439,term_text:GU140106}}GU140106 and A8806). However, such strains could be resistant to lower-dose regimens, including 250-mg and 500-mg doses of ceftriaxone. n nThis N. gonorrhoeae strain, {type:entrez-nucleotide,attrs:{text:GU140106,term_id:268370439,term_text:GU140106}}GU140106, was isolated from the urethra of a man who received fellatio from a female sex worker; thus, the bacteria could have derived from her pharynx. N. gonorrhoeae strain H041 was previously isolated from the pharynx of a female sex worker (1). To prevent the emergence and spread of ceftriaxone-resistant N. gonorrhoeae, pharyngeal gonorrhea must be treated. It is uncertain whether a 1-g dose of ceftriaxone would be effective against pharyngeal gonorrhea caused by strains with decreased susceptibility to ceftriaxone, and this regimen might facilitate the selection of such strains from oral cephalosporin-resistant strains in the pharynx. The emergence of N. gonorrhoeae {type:entrez-nucleotide,attrs:{text:GU140106,term_id:268370439,term_text:GU140106}}GU140106 in Japan suggests that new strategies (not just increased ceftriaxone doses), including combination treatment with ceftriaxone and another class of antimicrobial drugs and multiple dose regimens of ceftriaxone, might be required to treat pharyngeal gonorrhea.

Management of Pharyngeal Gonorrhea Is Crucial To Prevent the Emergence and Spread of Antibiotic-Resistant Neisseria gonorrhoeae

We read with great interest the paper recently published by Unemo et al. that reported a second strain (F89) of Neisseria gonorrhoeae with a high level of ceftriaxone resistance, for which the ceftriaxone MIC was 1 μg/ml, that was isolated in France in 2010 ([10][1]). The first high-level

Contribution of Topoisomerase IV Mutation to Quinolone Resistance in Mycoplasma genitalium

ABSTRACT The mechanism of quinolone resistance in Mycoplasma genitalium remains poorly understood due to difficulties with in vitro culture, especially of clinical isolates. In this study, to confirm the association between mutations in topoisomerases and antimicrobial susceptibilities to quinolones, ciprofloxacin-resistant mutant strains were selected using the cultivable type strain ATCC 33530. Sequence analysis revealed that the mutant strains harbored mutations in topoisomerase IV: Gly81Cys in ParC, Pro261Thr in ParC, or Asn466Lys in ParE. The MICs of all quinolones tested against the mutant strains were 2- to 16-fold higher than those against the wild-type strain. No cross-resistance was observed with macrolides or tetracyclines. We determined the inhibitory activities of quinolones against DNA gyrase and topoisomerase IV in order to investigate the correlation between antimicrobial susceptibility and inhibitory activity against the target enzymes, considered the primary targets of quinolones. Furthermore, using enzymatic analysis, we confirmed that Gly81Cys in the ParC quinolone resistance-determining region (QRDR) contributed to quinolone resistance. This is the first study to isolate quinolone-resistant mutant strains of M. genitalium harboring substitutions in the parC or parE gene in vitro and to measure the inhibitory activities against the purified topoisomerases of M. genitalium.

Antimicrobial chemotherapy of Mycoplasma genitalium-positive non-gonococcal urethritis

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.

Male non-gonococcal urethritis: From microbiological etiologies to demographic and clinical features.

To detect microorganisms responsible for male acute urethritis and to define the microbiology of non‐gonococcal urethritis.

A single 2 g oral dose of extended-release azithromycin for treatment of gonococcal urethritis

OBJECTIVESnWe treated gonococcal urethritis in men with a single 2 g dose of azithromycin extended-release formulation (azithromycin-SR) to determine its microbiological outcomes and tolerability.nnnPATIENTS AND METHODSnWe enrolled 189 Japanese men with gonococcal urethritis between April 2009 and December 2013. The patients were given a single 2 g dose of azithromycin-SR. Microbiological efficacy was evaluated by the results of the post-treatment molecular testing of Neisseria gonorrhoeae. MIC testing was performed only for pretreatment isolates of N. gonorrhoeae collected from the patients.nnnRESULTSnWe evaluated 130 patients for microbiological outcomes. Of these patients, 122 (93.8%) were judged to be microbiologically cured on the basis of negative test results. All isolates for which the azithromycin MICs were ≤0.25 mg/L were eradicated, whereas 5 of 12 isolates for which the MICs were 1 mg/L persisted after the treatment. Forty-six adverse events occurred in 41 patients. However, all adverse events were classified as mild.nnnCONCLUSIONSnThe eradication rate of N. gonorrhoeae was 93.8% in men with gonococcal urethritis treated with a single 2 g dose of azithromycin-SR. The breakpoint MIC of a 2 g dose of azithromycin-SR for gonococcal urethritis associated with clinical treatment failures appeared to be 1 mg/L. With regard to side effects of higher doses of azithromycin, the 2 g dose of azithromycin-SR appeared to improve tolerability. However, the widespread use of a high-dose regimen of azithromycin might lead to the development of further resistance to azithromycin.

Drug Resistance-Associated Mutations in Mycoplasma genitalium in Female Sex Workers, Japan

Mycoplasma genitalium was detected in 21 (14.1%) of 149 vaginal swab samples and in 1 (0.7%) of 149 throat washing samples from female sex workers during 2013–2014 in Japan. Prevalences of M. genitalium with macrolide resistance–associated 23S rRNA mutations and fluoroquinolone resistance–associated parC alterations were 47.1% and 36.8%, respectively.