Molecular mechanisms of macrolide resistance in clinical isolates of Mycoplasma pneumoniae from China.
ABSTRACT: Fifty clinical Mycoplasma pneumoniae strains were isolated from 370 children with respiratory tract infections. Four strains were susceptible to macrolides, while the other 46 (92%) were macrolide resistant. The molecular mechanism of resistance was shown to be associated with point mutations in 23S rRNA at positions 2063 and 2064.
Project description:We have found that early corticosteroid therapy was effective for reducing morbidity during five Korea-wide epidemics. We evaluated the clinical and laboratory parameters of 56 children who received early corticosteroid treatment for pneumonia that was caused by macrolide-resistant Mycoplasma pneumoniae (M. pneumoniae) or macrolide-sensitive M. pneumoniae between July 2019 and February 2020. All subjects had dual positive results from a PCR assay and serological test, and received corticosteroids within 24–36 h after admission. Point mutation of residues 2063, 2064, and 2067 was identified in domain V of 23S rRNA. The mean age was 6.8 years and the male:female ratio was 1.2:1 (31:25 patients). Most of the subjects had macrolide-resistant M. pneumoniae (73%), and all mutated strains had the A2063G transition. No significant differences in clinical and laboratory parameters were observed between macrolide-resistant and macrolide-sensitive M. pneumoniae groups that were treated with early dose-adjusted corticosteroids. Higher-dose steroid treatment may be needed for patients who have fever that persists for >48 h or increased biomarkers such as lactate dehydrogenase concentration at follow-up despite a usual dose of steroid therapy.
Project description:Mycoplasma pneumoniae pneumonia (MPP) is one of the most common forms of community-acquired pneumonia in children and adolescents. Outbreaks of MPP occur in 3- to 7-year cycles worldwide; recent epidemics in Korea occurred in 2006-2007, 2011, and 2015-2016. Although MPP is known to be a mild, self-limiting disease with a good response to macrolides, it can also progress into a severe and fulminant disease. Notably, since 2000, the prevalence of macrolide-resistant MPP has rapidly increased, especially in Asian countries, recently reaching up to 80%-90%. Macrolide-resistant Mycoplasma pneumoniae (MRMP) harbors a point mutation in domain V of 23S rRNA with substitutions mainly detected at positions 2063 and 2064 of the sequence. The excessive use of macrolides may contribute to these mutations. MRMP can lead to clinically refractory pneumonia, showing no clinical or radiological response to macrolides, and can progress to severe and complicated pneumonia. Refractory MPP is characterized by an excessive immune response against the pathogen as well as direct injury caused by an increasing bacterial load. A change of antibiotics is recommended to reduce the bacterial load. Tetracyclines or quinolones can be alternatives for treating MRMP. Otherwise, corticosteroid or intravenous immunoglobulin can be added to the treatment regimen as immunomodulators to down-regulate an excessive host immune reaction and alleviate immune-mediated pulmonary injury. However, the exact starting time point, dose, or duration of immunomodulators has not been established. This review focuses on the mechanism of resistance acquisition and treatment options for MRMP pneumonia.
Project description:In recent years, Mycoplasma pneumoniae strains that are clinically resistant to macrolide antibiotics have occasionally been encountered in Japan. Of 76 strains of M. pneumoniae isolated in three different areas in Japan during 2000 to 2003, 13 strains were erythromycin (ERY) resistant. Of these 13 strains, 12 were highly ERY resistant (MIC, > or =256 microg/ml) and 1 was weakly resistant (MIC, 8 microg/ml). Nucleotide sequencing of domains II and V of 23S rRNA and ribosomal proteins L4 and L22, which are associated with ERY resistance, showed that 10 strains had an A-to-G transition at position 2063 (corresponding to 2058 in Escherichia coli numbering), 1 strain showed A-to-C transversion at position 2063, 1 strain showed an A-to-G transition at position 2064, and the weakly ERY-resistant strain showed C-to-G transversion at position 2617 (corresponding to 2611 in E. coli numbering) of domain V. Domain II and ribosomal proteins L4 and L22 were not involved in the ERY resistance of these clinical M. pneumoniae strains. In addition, by using our established restriction fragment length polymorphism technique to detect point mutations of PCR products for domain V of the 23S rRNA gene of M. pneumoniae, we found that 23 (24%) of 94 PCR-positive oral samples taken from children with respiratory infections showed A2063G mutation. These results suggest that ERY-resistant M. pneumoniae infection is not unusual in Japan.
Project description:Mycoplasma pneumoniae is a major pathogen causing community-acquired pneumoniae (CAP), which is generally treated with macrolides. In recent years, however, although macrolide-resistant M. pneumoniae has been reported frequently, particularly in China, very little is known about the prevalence of macrolide-resistant M. pneumoniae infection in adults. In this study, we survey the macrolide-resistant M. pneumoniae in 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 for M. pneumoniae. All isolates were sequenced to determine the mutation in domain V of the 23S rRNA gene. The activities of 10 antibiotics against macrolide-resistant M. pneumoniae isolates were also investigated in 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) of M. pneumoniae strains isolated from adults with CAP were resistant to erythromycin (MIC=128 to >256 ?g/ml), clarithromycin (MIC=128 to >256 ?g/ml), and azithromycin (MIC=32 to >64 ?g/ml). Furthermore, all macrolide-resistant M. pneumoniae strains 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-resistant M. pneumoniae infection is very prevalence among adults in Zhejiang province. Thus, there is necessary to perform the epidemiological monitoring of macrolide-resistant M. pneumoniae in the future.
Project description:Mycoplasma pneumoniae is usually susceptible to macrolides, but macrolide-resistant strains have been found frequently in recent years. Mutations in domain V of the 23S rRNA gene of M. pneumoniae interfere with the binding of macrolides to rRNA and mediate macrolide resistance. In this study, we developed a rapid and inexpensive method that combines nested PCR (nPCR), single-strand conformation polymorphisms (SSCPs), and capillary electrophoresis (CE) to detect macrolide-resistant mutants directly from throat swabs. nPCR was used to specifically amplify M. pneumoniae 23S rRNA gene fragments containing mutations, and amplicons were analyzed by CE-SSCP for macrolide resistance mutations, with results confirmed by sequencing. From January to December 2009, 665 throat swabs were collected in Beijing, China, yielding 110 samples that tested positive for M. pneumoniae by nPCR and serological testing. We randomly selected 64 positive throat swabs for CE-SSCP analysis. The A2063G mutation was found in 57 samples, and a coexisting T2611C mutation was identified in 1 sample. An A2063T mutation was identified in 1 sample. The total mutation rate was 91%. All mutant samples identified by nPCR-CE-SSCP were sequenced. The nPCR-CE-SSCP method could identify macrolide-resistant mutants directly from clinical samples. This is the first report of an nPCR-CE-SSCP assay for the detection of dominant mutations that confer macrolide resistance on M. pneumoniae. This approach would allow clinicians to choose appropriate therapy rapidly and could be used as a screening method for genetic mutations related to antibiotic resistance.
Project description:Introduction:Community-acquired pneumonia (CAP) is a global disease responsible for a large number of deaths, with significant economic impact. As diagnostic tools have increased in sensitivity, understanding of the etiology of CAP has begun to change. Mycoplasma pneumoniae is one of the major pathogens causing CAP. Macrolides and related antibiotics are first-line treatments for M. pneumoniae. Macrolide resistance has been spreading for 15 years and now occurs in worldwide. We undertook the first study on macrolide resistance of M. pneumoniae in Yantai. This may be helpful to determine the appropriate therapy for CAP in this population. Objective:To investigate the rate and mechanism of macrolide resistance in Yantai. Methods:Pharyngeal swab samples were collected from adult CAP patients. Samples were assayed by polymerase chain reaction (PCR) and cultivated to test for M. pneumoniae. Nested PCR was used to specifically amplify M. pneumoniae 23S rRNA gene fragments containing mutations, and amplicons were analyzed by CE-SSCP for macrolide resistance mutations. Results were confirmed by sequencing. Twenty-seven strains of M. pneumoniae were isolated and the activities of nine antibiotics against M. pneumoniae were tested in vitro. Results:Out of 128 samples tested, 27 were positive for M. pneumoniae. Mycoplasma 100% macrolides resistance to Mycoplasma pneumoniae. The mechanism of macrolides resistance was A2063G point mutation in the sequence directly binding to macrolides in the 23S rRNA V domain in vitro. The mean pyretolytic time for the fluoroquinolone group was 4.7 ±2.9 d, which was significantly shorter than 8.2 ±4.1 d for the azithromycin group. Conclusions:Macrolides are not the first-line treatment for M. pneumoniae respiratory tract infections in Yantai.
Project description:Mycoplasma pneumoniae is a significant cause of community-acquired pneumonia, which is often empirically treated with macrolides or azalides such as erythromycin or azithromycin. Recent studies have discovered the existence of macrolide-resistant strains within the population that have been mapped to mutations within the domain V region of the 23S rRNA gene. Currently, identification of these resistant strains relies on time-consuming and labor-intensive procedures such as restriction fragment length polymorphism, MIC studies, and sequence analysis. The current study reports two distinct real-time PCR assays that can detect the A2063G or A2064G base mutation (A2058G or A2059G by Escherichia coli numbering) conferring macrolide resistance. By subjecting the amplicon of the targeted domain V region of the 23S rRNA gene to a high-resolution melt curve analysis, macrolide-resistant strains can quickly be separated from susceptible strains. Utilizing this method, we screened 100 clinical isolates and found 5 strains to possess mutations conferring resistance. These findings were concordant with both sequencing and MIC data. This procedure was also used successfully to identify both susceptible and resistant genotypes in 23 patient specimens. These patient specimens tested positive for the presence of M. pneumoniae by a separate real-time PCR assay, although the bacteria could not be isolated by culture. This is the first report of a real-time PCR assay capable of detecting the dominant mutations that confer macrolide resistance on M. pneumoniae, and these assays may have utility in detecting resistant strains of other infectious agents. These assays may also allow for clinicians to select appropriate treatment options more rapidly and may provide a convenient method to conduct surveillance for genetic mutations conferring antibiotic resistance.
Project description:In areas with high prevalence of macrolide-resistant <i>Mycoplasma pneumoniae</i> (MRMP) pneumonia, treatment in children has become challenging. This study aimed to analyze the efficacy of macrolides and doxycycline with regard to the presence of macrolide resistance. We analyzed children with MP pneumonia during the two recent epidemics of 2014-2015 and 2019-2020 from four hospitals in Korea. Nasopharyngeal samples were obtained from children with pneumonia for MP cultures and polymerase chain reaction (PCR). Macrolide resistance was determined by the analysis of 23S rRNA gene transition. Time to defervescence and to chest X-ray improvement were analyzed. Of 145 cases, the median age was 5.0 years and MRMP accounted for 59 (40.7%). Among macrolide-susceptible MP (MSMP), 78 (90.7%) were treated with macrolides and 21 (35.6%) in the MRMP group with doxycycline. In MRMP pneumonia, shorter days to defervescence (2 vs. 5 days, <i>p</i> < 0.001) and to chest X-ray improvement (3 vs. 6 days, <i>p</i> < 0.001) in the doxycycline group than in the macrolide group was observed, whereas no differences were observed among children with MSMP pneumonia. Compared to macrolides, treatment with doxycycline resulted in better outcomes with a shorter time to defervescence and to chest X-ray improvement among children with MRMP pneumonia.
Project description:Rapid and sensitive detection of macrolide resistance in Mycoplasma genitalium is 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 of M. genitalium result 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 of Mycoplasma genitalium that 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 on M. 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%) investigated M. 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-resistant M. genitalium in 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 of M. genitalium infections.
Project description:Eighteen out of 45 children were reported to have a respiratory illness during an outbreak at a temporary dormitory in a nursery school in China in 2011. To study the outbreak and to determine the risk factors for infection, an epidemiological investigation was performed. A standardized questionnaire was completed for a total of 45 children with the help of their guardians and parents. In addition, acute- and convalescent-phase serum samples and throat swabs from the children were taken for laboratory diagnosis. The diagnosis of a Mycoplasma-like illness was based on the following clinical criteria. The criteria were onset of illness after 31 May 2011, characterized by a cough, fever(>37.5 °C), or at least 3 of the following symptoms: fever, sore throat, cough or expectoration, and runny or stuffy nose. PCR-restriction fragment length polymorphism (PCR-RFLP), determination of MICs, and sequencing were performed to determine the genotype, antibiotic resistance, and sequence polymorphisms of the isolated strains, respectively. The paired sera revealed that 15 patients were infected with Mycoplasma pneumoniae. Epidemiology confirmed that this was a point source outbreak, characterized by a short incubation period, a high secondary attack rate, and a long period of hospitalization. PCR-RFLP analysis revealed that the 12 isolated strains of M. pneumoniae shared the same subtype P1 gene, and 23S rRNA sequence analysis showed that these strains harbored two macrolide-resistant gene-related point mutations at position 2063 and 2617. In this outbreak, the major risk factor was the distance between the bed of the first patient and the beds of close contacts (beds less than three meters apart). The strains isolated in this study were found to harbor two point mutations conferring macrolide resistance, indicating the importance of pathogen and drug resistance surveillance systems.