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

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          Abstract

          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.

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          Most cited references 34

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          Mycoplasma genitalium: from Chrysalis to multicolored butterfly.

          The history, replication, genetics, characteristics (both biological and physical), and factors involved in the pathogenesis of Mycoplasma genitalium are presented. The latter factors include adhesion, the influence of hormones, motility, possible toxin production, and immunological responses. The preferred site of colonization, together with current detection procedures, mainly by PCR technology, is discussed. The relationships between M. genitalium and various diseases are highlighted. These diseases include acute and chronic nongonococcal urethritis, balanoposthitis, chronic prostatitis, and acute epididymitis in men and urethritis, bacterial vaginosis, vaginitis, cervicitis, pelvic inflammatory disease, and reproductive disease in women. A causative relationship, or otherwise strong association, between several of these diseases and M. genitalium is apparent, and the extent of this, on a subjective basis, is presented; also provided is a comparison between M. genitalium and two other genital tract-orientated mollicutes, namely, Mycoplasma hominis, the first mycoplasma of human origin to be discovered, and Ureaplasma species. Also discussed is the relationship between M. genitalium and infertility and also arthritis in both men and women, as is infection in homosexual and immunodeficient patients. Decreased immunity, as in HIV infections, may enhance mycoplasmal detection and increase disease severity. Finally, aspects of the antimicrobial susceptibility and resistance of M. genitalium, together with the treatment and possible prevention of mycoplasmal disease, are discussed.
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            Use of TaqMan 5' nuclease real-time PCR for quantitative detection of Mycoplasma genitalium DNA in males with and without urethritis who were attendees at a sexually transmitted disease clinic.

            Mycoplasma genitalium is a cause of nongonococcal urethritis, particularly in patients not infected with Chlamydia trachomatis. A quantitative 5' nuclease assay (TaqMan PCR) was developed and validated. The assay detected a fragment of the MgPa adhesin gene by use of a TaqMan MGB (minor groove binder) probe and included an internal processing control to detect PCR inhibition. Urethral swab specimens and first-void urine samples from M. genitalium-positive men were examined, and the M. genitalium DNA load was correlated to symptoms and signs. The assay consistently detected <5 genome copies without cross-reactions with other mycoplasmas. Urine and urethral swab specimens from men with urethritis had higher M. genitalium DNA loads than specimens from men without urethritis. However, a very broad overlap of DNA loads between patients with and without urethritis was observed. Urethral swab specimens from patients with urethral discharge had a significantly higher DNA load than specimens from patients without discharge. This correlation was not found in first-void urine specimens.
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              Azithromycin treatment failure in Mycoplasma genitalium-positive patients with nongonococcal urethritis is associated with induced macrolide resistance.

              Mycoplasma genitalium is a common cause of nongonococcal urethritis. Treatment trials have shown that doxycycline is inefficient, whereas a 5-day course of azithromycin eradicates the bacterium from 95% of infected men. The aim of the study was to establish the reason for the occasional treatment failures. Seven M. genitalium strains isolated from men who experienced azithromycin treatment failure were tested for in vitro susceptibility to macrolides with use of a cell culture-based method. The genetic basis for the drug resistance was established by sequencing parts of the 23S ribosomal RNA gene and the genes encoding the L4 and L22 proteins. Nine sets of specimens obtained before and after treatment from patients who experienced azithromycin treatment failure were examined with use of sequencing of polymerase chain reaction products. The 7 strains that were isolated from patients who experienced treatment failure with azithromycin had minimum inhibitory concentrations >8 microg/mL for azithromycin and erythromycin. Three different mutations at positions 2058 and 2059 (Escherichia coli numbering) in region V of the 23S rRNA gene were found. Of the 9 patients with specimens obtained before and after treatment, only 2 had an initial specimen in which the mutation was present, indicating that drug resistance was induced as the result of an inappropriate dosage of azithromycin. Development of macrolide resistance was shown to correlate with subsequent azithromycin treatment failure. The genetic basis for the drug resistance was shown to be mutations in region V of the 23S rRNA gene, which is well described in other Mollicutes. These findings raise concern about the use of single-dose azithromycin treatment of nongonococcal urethritis of unknown etiology.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Formal analysisRole: InvestigationRole: MethodologyRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: InvestigationRole: VisualizationRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: InvestigationRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: Supervision
                Role: Funding acquisitionRole: Writing – review & editing
                Role: Supervision
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                8 June 2018
                2018
                : 13
                : 6
                Affiliations
                [1 ] Department of Urology, University of Occupational and Environmental Health, Kitakyushu, Japan
                [2 ] Disease Control and Prevention Center, International Health Care Center, National Center for Global Health and Medicine, Tokyo, Japan
                [3 ] Sonezaki Furubayashi Clinic, Osaka, Japan
                [4 ] Research Unit for Reproductive Tract Microbiology, Statens Serum Institut, Copenhagen, Denmark
                Miami University, UNITED STATES
                Author notes

                Competing Interests: The authors have read the journal’s policy and have declared that no competing interests exist.

                ‡ These authors are shared first authors on this work.

                PONE-D-17-39625
                10.1371/journal.pone.0198355
                5993279
                29883482
                © 2018 Hamasuna et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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                Figures: 0, Tables: 3, Pages: 10
                Product
                Funding
                Funded by: the Japan Society for the Promotion of Science (JSPS) KAKENHI
                Award ID: 26462456
                Award Recipient :
                This work was funded by grants from the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 26462456 and 17K11193. Dr. Hamasuma R obtained fund and designed the study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                Non-coding RNA
                Ribosomal RNA
                Biology and life sciences
                Biochemistry
                Ribosomes
                Ribosomal RNA
                Biology and life sciences
                Cell biology
                Cellular structures and organelles
                Ribosomes
                Ribosomal RNA
                People and Places
                Geographical Locations
                Asia
                Japan
                Biology and Life Sciences
                Anatomy
                Body Fluids
                Urine
                Medicine and Health Sciences
                Anatomy
                Body Fluids
                Urine
                Biology and Life Sciences
                Physiology
                Body Fluids
                Urine
                Medicine and Health Sciences
                Physiology
                Body Fluids
                Urine
                Research and Analysis Methods
                Specimen Preparation and Treatment
                Biology and Life Sciences
                Organisms
                Bacteria
                Mollicutes
                Mycoplasma
                Biology and Life Sciences
                Genetics
                Mutation
                Biology and Life Sciences
                Microbiology
                Microbial Control
                Antimicrobial Resistance
                Medicine and Health Sciences
                Pharmacology
                Antimicrobial Resistance
                Medicine and Health Sciences
                Pharmacology
                Drugs
                Antimicrobials
                Biology and Life Sciences
                Microbiology
                Microbial Control
                Antimicrobials
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