A Novel SimpleProbe PCR Assay for Detection of Mutations in the 23S rRNA Gene Associated with Macrolide Resistance in Mycoplasma genitalium in Clinical Samples

Macrolide-resistant strains ofMycoplasma genitaliumare an increasing problem throughout the world, and the implementation of a rapid and sensitive assay for mutation detection to guide treatment is needed. Macrolide-resistant strains have been shown to contain base substitutions in positions 2058 and 2059 (Escherichia colinumbering) in region V of the 23S rRNA gene. In this study, we present a SimpleProbe PCR followed by melting curve analysis to differentiate between macrolide-resistant mutants and wild types. The assay was performed on 159Mycoplasma genitalium-positive samples, and the results were compared with DNA sequencing. We also looked at the prevalence of macrolide-resistant strains in a Norwegian population. Of 139 samples characterized successfully by sequencing, 54 (39%) were wild types and 85 (61%) were mutants, consisting of 59 (42%) A2059G, 24 (17%) A2058G, 1 (1%) A2058T, and 1 (1%) A2059C mutation. The melting curve analysis correctly differentiated between wild-type and mutant strains in all cases, but it could not identify the different mutant types. The SimpleProbe PCR proved to be a simple, rapid, and reliable method for the detection of macrolide-resistant isolates ofMycoplasma genitaliumin a clinical setting.

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Molecular mechanisms of macrolide and fluoroquinolone resistance among Korean isolates of Mycoplasma genitalium over a period of five years 2014–2019

Journal of Medical Microbiology ◽

10.1099/jmm.0.001460 ◽

2021 ◽

Vol 70 (11) ◽

Author(s):

Yumi Seo ◽

Heeyoon Park ◽

Gilho Lee

Keyword(s):

Genetic Diversity ◽

Antimicrobial Resistance ◽

Type Species ◽

Mycoplasma Genitalium ◽

Quinolone Resistance ◽

23S Rrna ◽

Rrna Gene ◽

Content Type ◽

Link Type ◽

23S Rrna Gene

Antimicrobial resistance in Mycoplasma genitalium has become a global issue, and certain groups have a higher probability of acquiring resistant strains. Little is known about the genetic diversity and characteristics of the antimicrobial resistance-determining sites (ARDSs) of M. genitalium in the Korean population. Therefore, we examined the genetic diversity of the ARDSs of M. genitalium-positive urogenital samples obtained from Korean females (G1) and males (G2) visiting primary care clinics and DNA samples from referred males (G3) with persistent urethritis. From 2014 to 2019, 54 patients from G1, 86 patients from G2, and 68 patients from G3 were included in the study. Sanger sequencing was performed on the 2058/2059 sites in the 23S rRNA gene and quinolone resistance-determining regions (QRDRs) of M. genitalium . The rates of mutation in G1, G2, and G3 were 1.85, 5.81, and 48.53 %, respectively, for A2059G in the 23S rRNA gene (P<0.001); 1.85, 0, and 17.78 %, respectively, for M95R or I in gyrA (P<0.001); 0, 0, and 31.11 %, respectively, for D99N or G in gyrA (P<0.001); and 7.41, 16.28, and 30 %, respectively, for S83R or N or I in parC (P=0.015). A2059G significantly increased the risk of mutations at the gyrA95, gyrA99, and parC83 sites (all P<0.01). In conclusion, although the genetic diversity of the ARDSs of M. genitalium was variable among the groups, it was generally lower in isolates with macrolide resistance and higher in isolates with quinolone resistance in Korea compared with the isolates in other countries. The G3 group demonstrated increased genetic diversity at the A2059G, gyrA95, gyrA99, and parC83 sites.

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Macrolide-Resistant Mycoplasma pneumoniae in Adults in Zhejiang, China

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04308-14 ◽

2014 ◽

Vol 59 (2) ◽

pp. 1048-1051 ◽

Cited By ~ 31

Author(s):

Zibo Zhou ◽

Xiangzhi Li ◽

Xiaojian Chen ◽

Fangjun Luo ◽

Changwang Pan ◽

...

Keyword(s):

Mycoplasma Pneumoniae ◽

Rflp Analysis ◽

Zhejiang Province ◽

23S Rrna ◽

Rrna Gene ◽

Type I ◽

Content Type ◽

23S Rrna Gene ◽

Resistant Strains ◽

Domain V

ABSTRACTMycoplasma pneumoniaeis a major pathogen causing community-acquired pneumoniae (CAP), which is generally treated with macrolides. In recent years, however, although macrolide-resistantM. pneumoniaehas been reported frequently, particularly in China, very little is known about the prevalence of macrolide-resistantM. pneumoniaeinfection in adults. In this study, we survey the macrolide-resistantM. pneumoniaein adults in Zhejiang province and characterize the mechanisms of resistance to macrolide. Six hundred fifty throat swab samples were collected from adult patients with CAP from January 2012 to August 2014. These samples were assayed by nested PCR and then cultivated forM. pneumoniae. All isolates were sequenced to determine the mutation in domain V of the 23S rRNA gene. The activities of 10 antibiotics against macrolide-resistantM. pneumoniaeisolates were also investigatedin vitro. Moreover, restriction fragment length polymorphism (RFLP) analysis of the amplified P1 gene was used to type 50 resistant strains. One hundred percent (71/71) ofM. pneumoniaestrains isolated from adults with CAP were resistant to erythromycin (MIC = 128 to >256 μg/ml), clarithromycin (MIC = 128 to >256 μg/ml), and azithromycin (MIC = 32 to >64 μg/ml). Furthermore, all macrolide-resistantM. pneumoniaestrains identified had an A2063G mutation in domain V of the 23S rRNA gene. Forty-six resistant strains (92.0%) were classified into type I strain on the basis of P1 gene PCR-RFLP analysis. According to these findings, it is suggested that macrolide-resistantM. pneumoniaeinfection is very prevalence among adults in Zhejiang province. Thus, there is necessary to perform the epidemiological monitoring of macrolide-resistantM. pneumoniaein the future.

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Ribosomal Mutations as the Main Cause of Macrolide Resistance in Campylobacter jejuni and Campylobacter coli

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00314-11 ◽

2011 ◽

Vol 55 (12) ◽

pp. 5939-5941 ◽

Cited By ~ 29

Author(s):

Mirva Lehtopolku ◽

Pirkko Kotilainen ◽

Marjo Haanperä-Heikkinen ◽

Ulla-Maija Nakari ◽

Marja-Liisa Hänninen ◽

...

Keyword(s):

Macrolide Resistance ◽

23S Rrna ◽

Rrna Gene ◽

Campylobacter Coli ◽

Resistance Mutations ◽

Content Type ◽

Amino Acid Insertion ◽

23S Rrna Gene ◽

Ribosomal Protein L22 ◽

Resistant Strains

ABSTRACTThe aim of this study was to examine macrolide resistance mutations inCampylobacterspecies. In 76 strains studied, point mutation A to G at position 2059 of the 23S rRNA gene was detected in 30 of the 33 erythromycin-resistant strains. An amino acid insertion in the ribosomal protein L22 was found in one resistant strain without a 23S rRNA mutation. The A2059G mutation is the main cause of macrolide resistance inCampylobacterspecies.

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FEATURES OF THE OUTBREAK OF THE COMMUNITY-ACQUIRED PNEUMONIA CAUSED BY MYCOPLASMA PNEUMOINIAE AT CHILDREN IN KHABAROVSK REGION

Journal of microbiology epidemiology immunobiology ◽

10.36233/0372-9311-2018-1-39-45 ◽

2018 ◽

pp. 39-45

Author(s):

O. E. Guseva ◽

O. A. Lebedko ◽

E. B. Nagovitsyna ◽

M. N. Lazutkin ◽

E. A. Savitskaya ◽

...

Keyword(s):

Posterior Pharyngeal Wall ◽

Community Acquired Pneumonia ◽

23S Rrna ◽

Clinical Samples ◽

Winter Period ◽

Rrna Gene ◽

Pharyngeal Wall ◽

23S Rrna Gene ◽

Resistant Strains

Aim. To study the features of outbreak of community-acquired pneumonia caused by M. pneumoniae in children in the Khabarovsk region during the autumn-winter period 2016 - 2017. Materials and methods. A study was conducted of 30 clinical samples - smears from the posterior pharyngeal wall and sputum samples obtained from patients with community-acquired pneumonia due to M. pneumoniae. Samples were examined for the presence of mutations in the 23S rRNA gene of M. pneumoniae. Results. The age ofthe patients was from 2 to 16 years. In two cases, family foci of the incidence of community-acquired pneumonia occurred. In 19 samples, a mutation was detected in 23S rRNA of M. pneumoniae. Conclusion. The data obtained indicate that in the development of the outbreak of community-acquired pneumonia caused by M. pneumoniae in children in the Khabarovsk region in the autumn-winter period 2016 - 2017, macrolide-resistant strains of the pathogen were involved.

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961. Prevalence and Macrolide Resistance of Mycoplasma genitalium After Initiation of HIV Preexposure Prophylaxis

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz359.063 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S29-S29

Author(s):

Jens Van Praet ◽

Sanne Steyaert ◽

Stefaan Vandecasteele ◽

Barbara Van Den Bergh ◽

Hilde Mahieu ◽

...

Keyword(s):

Mycoplasma Genitalium ◽

Macrolide Resistance ◽

23S Rrna ◽

Rrna Gene ◽

Sexually Transmitted ◽

Preexposure Prophylaxis ◽

23S Rrna Gene ◽

Resistant Strains ◽

Taqman Array

Abstract Background Recent evidence shows that patients using HIV preexposure prophylaxis (PrEP) have an increased rate of bacterial sexually transmitted infections (STIs), including syphilis, chlamydia, and gonorrhea. The rate of Mycoplasma genitalium infections and the susceptibility of M. genitalium in patients on PrEP have been less well described. Methods We studied all patients who started on PrEP in the AZ Sint-Jan Hospital Bruges from January 6, 2017 to January 4, 2019. Patients were screened for M. genitalium and other bacterial STIs with rectal swabs, pharyngeal swabs, first-voided urine and blood collections at baseline and quarterly intervals after initiating PrEP. TaqMan array card technology was used to detect M. genitalium and determine macrolide-resistance mediating mutations in the region V of the 23S rRNA gene (A2058G, A2059G, A2058C, and others). Patients with an STI were treated based on a national guideline. Proportions were estimated using a Generalized Estimating Equations model with independent correlation structure. Results A total of 136 males and 1 female (median age, 40 years (interquartile range (IQR), 20–79)) were included in the study. All men were gay or bisexual. The median follow-up time was 11.3 months (IQR, 4.7–15.3). In total, 117 patients (85%) used PrEP daily at their last visit. The estimated proportion of patients with M. genitalium at baseline, 3 months, 6 months, 9 months, and 12 months was 7% (95% CI 4–13), 12% (95% CI 7–20), 7% (95% CI 4–15), 6% (3–15), and 6% (2–15). Thirty-two patients (23%) tested at least once positive for M. genitalium during the study period. The estimated percentage of macrolide resistance increased from 40% (95% CI 16–70) at baseline to 71% (95% CI 44–89), 67% (95% CI 27–92), 80% (95% CI 31–97), and 75% (95% CI 24–97) at 3 months, 6 months, 9 months, and 12 months, respectively. Conclusion After initiation of PrEP, the prevalence of M. genitalium in our cohort at quarterly screening was not increased compared with baseline. However, a nonsignificant trend of an increased percentage of macrolide-resistant strains was observed. Disclosures All Authors: No reported Disclosures.

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

Journal of Clinical Microbiology ◽

10.1128/jcm.00012-16 ◽

2016 ◽

Vol 54 (6) ◽

pp. 1593-1597 ◽

Cited By ~ 15

Author(s):

Gitte Qvist Kristiansen ◽

Jan Gorm Lisby ◽

Kristian Schønning

Keyword(s):

Mycoplasma Genitalium ◽

Antimicrobial Treatment ◽

Macrolide Resistance ◽

23S Rrna ◽

Rrna Gene ◽

Predictive Values ◽

Content Type ◽

Clinical Specimens ◽

Genotyping Assay ◽

23S Rrna Gene

Rapid and sensitive detection of macrolide resistance inMycoplasma genitaliumis required for the guidance of adequate antimicrobial treatment. Previous studies have confirmed that single-base mutations at position 2058 or 2059 in domain V of the 23S rRNA gene ofM. genitaliumresult in high-level macrolide resistance. Sequencing of PCR products remains the gold standard for the identification of mutations conferring resistance to macrolides but is laborious and time-consuming. The aim of the present study was to develop a 5′ nuclease genotyping assay to detect single nucleotide polymorphisms in the 23S rRNA gene ofMycoplasma genitaliumthat are associated with macrolide resistance by combining PCR with hydrolysis probes and subsequent endpoint genotyping analysis. The 5′ nuclease genotyping assay was used as a referral test to be used onM. genitalium-positive samples and was validated on 259 positive samples, of which 253 (97.7%) were successfully sequenced. With the newly developed assay, 237/259 (91.5%) investigatedM. genitalium-positive samples were genotyped. The positive and the negative predictive values were 100% when evaluated on successfully genotyped samples. The newly developed assay discriminated macrolide-resistantM. genitaliumin clinical specimens possessing A2058G, A2058C, A2058T, and A2059G mutations with a sensitivity of 94.4% (95% confidence interval [CI], 90.7% to 98.2%) and a specificity of 92.7% (95% CI, 87.8% to 97.6%) when evaluated on successfully sequenced samples. The assay can correctly guide antimicrobial treatment ofM. genitaliuminfections.

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Molecular Basis of Macrolide Resistance inCampylobacterStrains Isolated from Poultry in South Korea

BioMed Research International ◽

10.1155/2018/4526576 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9 ◽

Cited By ~ 3

Author(s):

Bai Wei ◽

Min Kang

Keyword(s):

Molecular Mechanisms ◽

Macrolide Resistance ◽

23S Rrna ◽

Rrna Gene ◽

Campylobacter Coli ◽

Erythromycin Resistance ◽

23S Rrna Gene ◽

Ribosomal Protein L22 ◽

Resistant Strains ◽

Azithromycin Resistance

We investigated the molecular mechanisms underlying macrolide resistance in 38 strains ofCampylobacterisolated from poultry. Twenty-seven strains were resistant to azithromycin and erythromycin, five showed intermediate azithromycin resistance and erythromycin susceptibility, and six showed azithromycin resistance and erythromycin susceptibility. FourCampylobacter jejuniand sixCampylobacter colistrains had azithromycin MICs which were 8–16 and 2–8-fold greater than those of erythromycin, respectively. The A2075G mutation in the 23S rRNA gene was detected in 11 resistant strains with MICs ranging from 64 to ≥ 512μg/mL. Mutations including V137A, V137S, and a six-amino acid insertion (114-VAKKAP-115) in ribosomal protein L22 were detected in theC. jejunistrains. Erythromycin ribosome methylase B-erm(B) was not detected in any strain. All strains except three showed increased susceptibility to erythromycin with twofold to 256-fold MIC change in the presence of phenylalanine arginine ß-naphthylamide (PAßN); the effects of PAßN on azithromycin MICs were limited in comparison to those on erythromycin MICs, and 13 strains showed no azithromycin MIC change in the presence of PAßN. Differences between azithromycin and erythromycin resistance and macrolide resistance phenotypes and genotypes were observed even in highly resistant strains. Further studies are required to better understand macrolide resistance inCampylobacter.

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Drug Resistance Mechanisms ofMycoplasma pneumoniaeto Macrolide Antibiotics

BioMed Research International ◽

10.1155/2014/320801 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7 ◽

Cited By ~ 3

Author(s):

Xijie Liu ◽

Yue Jiang ◽

Xiaogeng Chen ◽

Jing Li ◽

Dawei Shi ◽

...

Keyword(s):

Drug Resistance ◽

Resistance Mechanisms ◽

23S Rrna ◽

Macrolide Antibiotics ◽

Rrna Gene ◽

Site Mutation ◽

23S Rrna Gene ◽

Resistant Strains ◽

Domain V ◽

The 16S Rrna Gene

Throat swabs from children with suspectedMycoplasma pneumoniae(M. pneumoniae) infection were cultured for the presence ofM. pneumoniaeand its species specificity using the 16S rRNA gene. Seventy-sixM. pneumoniaestrains isolated from 580 swabs showed that 70 were erythromycin resistant with minimum inhibitory concentrations (MIC) around 32–512 mg/L. FiftyM. pneumoniaestrains (46 resistant, 4 sensitive) were tested for sensitivity to tetracycline, ciprofloxacin, and gentamicin. Tetracycline and ciprofloxacin had some effect, and gentamicin had an effect on the majority ofM. pneumoniaestrains. Domains II and V of the 23S rRNA gene and the ribosomal protein L4 and L22 genes, both of which are considered to be associated with macrolide resistance, were sequenced and the sequences were compared with the corresponding sequences in M129 registered with NCBI and the FH strain. The 70 resistant strains all showed a 2063 or 2064 site mutation in domain V of the 23S rRNA but no mutations in domain II. Site mutations of L4 or L22 can be observed in either resistant or sensitive strains, although it is not known whether this is associated with drug resistance.

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Neisseria oralis sp. nov., isolated from healthy gingival plaque and clinical samples

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.041731-0 ◽

2013 ◽

Vol 63 (Pt_4) ◽

pp. 1323-1328 ◽

Cited By ~ 23

Author(s):

William J. Wolfgang ◽

Teresa V. Passaretti ◽

Reashma Jose ◽

Jocelyn Cole ◽

An Coorevits ◽

...

Keyword(s):

Type Strain ◽

Type Species ◽

Novel Species ◽

Sequence Similarity ◽

23S Rrna ◽

Clinical Samples ◽

Rrna Gene ◽

The Novel ◽

Content Type ◽

Link Type

A polyphasic analysis was undertaken of seven independent isolates of Gram-negative cocci collected from pathological clinical samples from New York, Louisiana, Florida and Illinois and healthy subgingival plaque from a patient in Virginia, USA. The 16S rRNA gene sequence similarity among these isolates was 99.7–100 %, and the closest species with a validly published name was Neisseria lactamica (96.9 % similarity to the type strain). DNA–DNA hybridization confirmed that these isolates are of the same species and are distinct from their nearest phylogenetic neighbour, N. lactamica . Phylogenetic analysis of 16S and 23S rRNA gene sequences indicated that the novel species belongs in the genus Neisseria . The predominant cellular fatty acids were C16 : 0, summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH) and C18 : 1ω7c. The cellular fatty acid profile, together with other phenotypic characters, further supports the inclusion of the novel species in the genus Neisseria . The name Neisseria oralis sp. nov. (type strain 6332T = DSM 25276T = LMG 26725T) is proposed.

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