Direct Detection of Macrolide Resistance in Mycoplasma genitalium Isolates from Clinical Specimens from France by Use of Real-Time PCR and Melting Curve Analysis

Touati, Arabella; Peuchant, Olivia; Jensen, Jørgen Skov; Bebear, Cécile; Pereyre, Sabine

doi:10.1128/jcm.03318-13

Cited by 87 publications

(92 citation statements)

References 32 publications

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“…subset a genotype for 99/105 (94.3%) samples. However, the need for a preselection of samples with a relatively high bacterial load may suggest that sensitivity may limit the utility of this assay in clinical routine (11,19).…”

Section: Discussionmentioning

confidence: 99%

“…Previously published primers were used for amplification of the PCR product and sequencing of the domain V of the 23S rRNA gene encompassing the 2058, 2059, and 2062 positions (numbers referring to Escherichia coli sequence) (11). PCR was performed on an Eppendorf Mastercycler (Eppendorf, Hamburg, Germany) using the Qiagen OneStep reverse transcriptase PCR (RT-PCR) kit (Qiagen, Hilden, Germany) with the following cycling settings: 50°C for 30 min and 95°C for 15 min followed by 40 cycles of 94°C for 15 s, 55°C for 30 s, 72°C for 60 s, and finally 72°C for 10 min with cooling at 4°C.…”

Section: Methodsmentioning

confidence: 99%

“…This method is best suited to process samples in bulk and may result in unacceptable reporting times. Highresolution melting (HRM) analysis is another powerful tool to detect mutations in a target sequence, and PCR assays using HRM analysis for detecting mutations associated with macrolide resistance have recently been reported (10,11). An alternative method for identifying known mutations is genotyping assays using differentially labeled hydrolysis probes targeting wild-type and mutated alleles (12,13).…”

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confidence: 99%

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A 5′ Nuclease Genotyping Assay for Identification of Macrolide-Resistant Mycoplasma genitalium in Clinical Specimens

2016

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M ycoplasma genitalium is a sexually transmitted bacterium that causes nongonococcal urethritis in men and has been associated with cervicitis and pelvic inflammatory disease in women (1). The current recommended treatment of M. genitalium infection in Scandinavia is an extended course of oral macrolide. However, if empirical treatment of nongonococcal urethritis is initiated, usually only a single dose of azithromycin is given as treatment for suspected Chlamydia infection. Single-dose azithromycin treatment of M. genitalium infection is associated with a suboptimal treatment response and, in the case of treatment failure, with the development of M. genitalium macrolide resistance (2, 3). Macrolide-resistant M. genitalium strains are consequently prevalent, and resistance rates of 41% in a cohort of men with urethritis in London (4) and 38% in a recent population-based Danish survey (5) have been reported. In proven M. genitalium infection, macrolide susceptibility reporting is therefore necessary to guide therapy (6).Macrolide resistance in M. genitalium is strongly associated with the mutations A2058G and A2059G in the gene encoding 23S rRNA (3,7,8). In a recent Dutch study, a high proportion of the A2058T mutation causing resistance was observed (9). Resistance-associated mutations can be identified by PCR amplification and sequencing. This method is best suited to process samples in bulk and may result in unacceptable reporting times. Highresolution melting (HRM) analysis is another powerful tool to detect mutations in a target sequence, and PCR assays using HRM analysis for detecting mutations associated with macrolide resistance have recently been reported (10, 11). An alternative method for identifying known mutations is genotyping assays using differentially labeled hydrolysis probes targeting wild-type and mutated alleles (12,13). In the present study, we report such a 5= nuclease genotyping assay for the detection of the macrolide resistance-associated mutations in M. genitalium. The assay was used to determine macrolide resistance in 259 samples that had previously tested positive for the presence of M. genitalium, and the results obtained using this assay were compared to sequencing of the region of interest in the 23S rRNA gene. The 5= nuclease genotyping assay was highly accurate compared to sequencing. MATERIALS AND METHODSM. genitalium detection. Between 8 October 2013 and 12 September 2014, the Department of Clinical Microbiology received 3,147 samples to test for the presence of M. genitalium. Testing was done using a laboratory-developed quantitative PCR (qPCR) using hydrolysis probes targeting the pdhD and mgpB genes of M. genitalium and an additional sampleprocessing control. The primers and probes of this laboratory-developed test have previously been published (14-16). In the multiplex qPCR, the final concentrations of M. genitalium-specific primers and of probes were 500 nM and 100 nM, respectively. Reactions were done in a total volume of 20 l with LightCycler 480 probes master (Roche A...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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A 5′ Nuclease Genotyping Assay for Identification of Macrolide-Resistant Mycoplasma genitalium in Clinical Specimens

2016

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“…A more recent characterization of S. aureus strains isolated from Czech CF patients showed high rates (29%) of strains with ribosomal mutations conferring resistance to MLS B antibiotics with the majority in 23S rRNA (23%) (Tkadlec et al 2015). Jensen et al 2008;Ito et al 2011;Chrisment et al 2012;Gesink et al 2012;Twin et al 2012;Touati et al 2014;Bissessor et al 2015 Lucier et al 1995;Matsuoka et al 2004;Liu et al 2009b;Peuchant et al 2009;Xin et al 2009;Cao et al 2010;Kawai et al 2013;Ye et al 2013; (Rantala et al 2005) characterized by polymerase chain reaction (PCR) did not harbor an efflux mechanism, mef(E) or mef(A), or a target modification mechanism, erm(B) or erm(A) subclass erm(TR), but did have previously identified A2059G mutations in one or more 23S rRNA genes. As had been shown previously, erythromycin minimum inhibitory concentrations (MICs) increased with an increasing number of rrl alleles containing A2059G (Tait-Kamradt et al 2000a), whereas a C2611T mutation was present in all four alleles (Rantala et al 2005).…”

Section: S Rrna Mutationsmentioning

confidence: 99%

Resistance to Macrolide Antibiotics in Public Health Pathogens

Fyfe

Grossman

Kerstein

et al. 2016

Cold Spring Harb Perspect Med

172

140

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Macrolide resistance mechanisms can be target-based with a change in a 23S ribosomal RNA (rRNA) residue or a mutation in ribosomal protein L4 or L22 affecting the ribosome's interaction with the antibiotic. Alternatively, mono-or dimethylation of A2058 in domain V of the 23S rRNA by an acquired rRNA methyltransferase, the product of an erm (erythromycin ribosome methylation) gene, can interfere with antibiotic binding. Acquired genes encoding efflux pumps, most predominantly mef(A) þ msr(D) in pneumococci/streptococci and msr(A/B) in staphylococci, also mediate resistance. Drug-inactivating mechanisms include phosphorylation of the 2 0 -hydroxyl of the amino sugar found at position C5 by phosphotransferases and hydrolysis of the macrocyclic lactone by esterases. These acquired genes are regulated by either translation or transcription attenuation, largely because cells are less fit when these genes, especially the rRNA methyltransferases, are highly induced or constitutively expressed. The induction of gene expression is cleverly tied to the mechanism of action of macrolides, relying on antibiotic-bound ribosomes stalled at specific sequences of nascent polypeptides to promote transcription or translation of downstream sequences. Macrolide antibiotics are polyketides composed of a 14-, 15-, or 16-membered macrocyclic lactone ring (14-, 15-, and 16-membered) to which several sugars and/or side chains have been attached by the producing organism or as modifications during semisynthesis in the laboratory (Figs. 1 and 2). Newer semisynthetic derivations, like ketolides telithromycin, and solithromycin, have a C3-keto group in place of the C3 cladinose (akin to naturally occurring pikromycin) (Brockmann and Henkel 1950) and an 11,12-cyclic carbamate with an extended alkyl -aryl side chain that increases the affinity of the antibiotic for the ribosome by 10-to 100-fold (Hansen et al. 1999;Dunkle et al. 2010); in the case of solithromycin, a fluorine substituent at C2 provides an additional ribosomal interaction (Llano-Sotelo et al. 2010). Macrolides continue to be important in the therapeutic treatment of community-acquired pneumonia (Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and atypicals Legionella pneumophila, Mycoplasma pneumoniae, Chlamydia pneumoniae), sexually transmitted diseases (Neiserria gonorhoeae, Chlamydia trachomatis, Mycoplasma genitalium), shigellosis, and salmonellosis. With solithromycin heading for a new drug application (NDA) filing in 2016 and having the in vitro potency to treat erythromycin-resistant

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“…Although there is no U.S food and drug administration (FDA) approved commercial detection system, the availability of molecular methods for research and commercial purposes has altered our ability to derive valid information about the pathogenicity of this bacterium. There have been more studies in recent years researching into its pathogenicity and treatment [2][3][4][5][6]. The most recent Centers for Disease Control and Prevention (CDC) Sexually Transmitted Diseases treatment guidelines discussed it under emerging issues [1].…”

Section: Introductionmentioning

confidence: 99%

Prevalence and Clinical Significance of Mycoplasma genitalium in Gynecologic Patients

Goje¹,

Munoz²,

Nolte³

et al. 2017

J AIDS Clin Res

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Objective: Mycoplasma genitalium has been recognized as a cause of male urethritis, and there is now evidence suggesting it causes cervicitis and pelvic inflammatory disease (PID) in women.Methods: Prevalence, risk factors and co-infections with other sexually transmitted pathogens were collected in a cross-sectional study looking at 400 women at the gynecologic clinics of a university medical center in the United States. Bacterial vaginosis and trichomoniasis were diagnosed using Amsel's criteria, gram stain and trichomonas culture respectively. Cervicitis and PID were clinically diagnosed. After testing for Chlamydia trachomatis and Neisseria gonorrhoeae, the residual cervical swab transport medium (Gen-Probe/Hologic ® ) was stored at -70°C. Stored samples were later analyzed for M. genitalium by a research use only transcription-mediated amplification assay using procedures similar to those established for APTIMA Combo2 assay for C. trachomatis and N. gonorrhoeae (Gen-Probe/Hologic ® ). Results:The overall prevalence of infection with C. trachomatis, N. gonorrhoeae, T. vaginalis and M. genitalium was found to be 7.8%, 1.8%, 10.43% and 8.9%, respectively. Prevalence of M. genitalium was comparable to that of C. trachomatis and greater than the prevalence of N. gonorrhoeae. Univariate analysis of M. genitalium status showed that participants with lower condom use had an increased probability of M. genitalium (p=0.037). Conclusion:Prevalence of M. genitalium was comparable to C. trachomatis in our study, but more research is needed to clarify pathogenicity.

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Direct Detection of Macrolide Resistance in Mycoplasma genitalium Isolates from Clinical Specimens from France by Use of Real-Time PCR and Melting Curve Analysis

Cited by 87 publications

References 32 publications

A 5′ Nuclease Genotyping Assay for Identification of Macrolide-Resistant Mycoplasma genitalium in Clinical Specimens

A 5′ Nuclease Genotyping Assay for Identification of Macrolide-Resistant Mycoplasma genitalium in Clinical Specimens

Resistance to Macrolide Antibiotics in Public Health Pathogens

Prevalence and Clinical Significance of Mycoplasma genitalium in Gynecologic Patients

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