Comparative genomic sequencing to characterize Mycoplasma pneumoniae genome, typing, and drug resistance.

To analyze the characteristics of Mycoplasma pneumoniae as well as macrolide antibiotic resistance through whole-genome sequencing and comparative genomics. Thirteen clinical strains isolated from 2003 to 2019 were selected, 10 of which were resistant to erythromycin (MIC >64 µg/mL), including 8 P1-type I and 2 P1-type II. Three were sensitive (<1 µg/mL) and P1-type II. One resistant strain had an A→G point mutation at position 2064 in region V of the 23S rRNA, the others had it at position 2063, while the three sensitive strains had no mutation here. Genome assembly and comparative genome analysis revealed a high level of genome consistency within the P1 type, and the primary differences in genome sequences concentrated in the region encoding the P1 protein. In P1-type II strains, three specific gene mutations were identified: C162A and A430G in L4 gene and T1112G mutation in the CARDS gene. Clinical information showed seven cases were diagnosed with severe pneumonia, all of which were infected with drug-resistant strains. Notably, BS610A4 and CYM219A1 exhibited a gene multi-copy phenomenon and shared a conserved functional domain with the DUF31 protein family. Clinically, the patients had severe refractory pneumonia, with pleural effusion, necessitating treatment with glucocorticoids and bronchoalveolar lavage. The primary variations between strains occur among different P1-types, while there is a high level of genomic consistency within P1-types. Three mutation loci associated with specific types were identified, and no specific genetic alterations directly related to clinical presentation were observed.IMPORTANCEMycoplasma pneumoniae is an important pathogen of community-acquired pneumonia, and macrolide resistance brings difficulties to clinical treatment. We analyzed the characteristics of M. pneumoniae as well as macrolide antibiotic resistance through whole-genome sequencing and comparative genomics. The work addressed primary variations between strains that occur among different P1-types, while there is a high level of genomic consistency within P1-types. In P1-type II strains, three specific gene mutations were identified: C162A and A430G in L4 gene and T1112G mutation in the CARDS gene. All the strains isolated from severe pneumonia cases were drug-resistant, two of which exhibited a gene multi-copy phenomenon, sharing a conserved functional domain with the DUF31 protein family. Three mutation loci associated with specific types were identified, and no specific genetic alterations directly related to clinical presentation were observed.

Macrolide resistance of Mycoplasma pneumoniae in several regions of China from 2013 to 2019.

This paper retrospectively analysed the prevalence of macrolide-resistant Mycoplasma pneumoniae (MRMP) in some parts of China. Between January 2013 and December 2019, we collected 4,145 respiratory samples, including pharyngeal swabs and alveolar lavage fluid. The highest PCR-positive rate of M. pneumoniae was 74.5% in Beijing, the highest resistance rate was 100% in Shanghai, and Gansu was the lowest with 20%. The highest PCR-positive rate of M. pneumoniae was 74.5% in 2013, and the highest MRMP was 97.4% in 2019; the PCR-positive rate of M. pneumoniae for adults in Beijing was 17.9% and the MRMP was 10.48%. Among the children diagnosed with community-acquired pneumonia (CAP), the PCR-positive and macrolide-resistant rates of M. pneumoniae were both higher in the severe ones. A2063G in domain V of 23S rRNA was the major macrolide-resistant mutation, accounting for more than 90%. The MIC values of all MRMP to erythromycin and azithromycin were ≥ 64 µg/ml, and the MICs of tetracycline and levofloxacin were ≤ 0.5 µg/ml and ≤ 1 µg/ml, respectively. The macrolide resistance varied in different regions and years. Among inpatients, the macrolide-resistant rate was higher in severe pneumonia. A2063G was the common mutation, and we found no resistance to tetracycline and levofloxacin.

Proteomic and Phenotypic Studies of Mycoplasma pneumoniae Revealed Macrolide-Resistant Mutation (A2063G) Associated Changes in Protein Composition and Pathogenicity of Type I Strains.

Mycoplasma pneumoniae (MP) is an important respiratory pathogen, the prevalence of macrolide-resistant MP (mainly containing A2063G mutation in 23S rRNA) increased in recent years. Epidemiological studies suggest a higher prevalence of type I resistant (IR) strains than corresponding sensitive (IS/IIS) strains, but not type II resistant (IIR) strains. Here, we aimed to analyze the factors underlying the altered prevalence of IR strains. First, proteomic analyses exhibit the protein compositions were type specific, while more differential proteins were detected between IS and IR (227) than IIS and IIR strains (81). mRNA level detection suggested posttranscriptional regulation of these differential proteins. Differential protein-related phenotypic changes were also detected: (i) P1 abundance was different between genotypes (I < II, IR < IS), the adhesion of MPs showed accordance to P1 abundance within IS and IIS strains; (ii) type I, especially IR, strains had a higher proliferation rate, which is potentially associated with differential proteins participating in glycolysis and one carbon pool metabolisms; (iii) A549 cells infected with IR strains had lower activity of caspase-3 and higher levels IL-8, but the differences were not significant between groups (P > 0.05). Correlations of P1 abundance to caspase-3 activity and proliferation rate to the level of IL-8 were obtained. These results suggest changes in protein composition influenced the pathogenicity of MP, especially in IR strains, which may impact the prevalence of MP strains of different genotypes. IMPORTANCE The prevalence of macrolide-resistant MPs increased the difficulty in treatment of MP infections and posed potential threats to children's health. Epidemiological studies showed a high prevalence of IR-resistant strains (mainly A2063G in 23S rRNA) in these years. However, the trigger mechanisms for this phenomenon are not clear. In this paper, proteomic and phenotypic studies suggest that IR strains have reduced levels of multiple adhesion proteins and increased proliferation rate, which may lead to higher transmission rate of IR strains in the population. This suggests that we should pay attention to the prevalence of IR strains.

Efficacy and safety of Qinxiang Qingjie oral solution for the treatment of influenza in children: a randomized, double-blind, multicenter clinical trial.

Background: Qinxiang Qingjie (QXQJ), an oral solution containing various Chinese herbs, is indicated for pediatric upper respiratory tract infections. The treatment of influenza also shows potential advantages in shortening the duration of illness and improving symptoms. However, there is still a lack of high-quality clinical evidence to support this. The trial was to explore the efficacy and safety of QXQJ for treating pediatric influenza and provide an evidence-based basis for expanding its applicability. Methods: A randomized, double-blind, double-dummy, positive-controlled, multicenter clinical trial was conducted in 14 hospitals in China. Children aged 1-13 years with influenza and "exterior and interior heat syndromes" as defined by traditional Chinese medicine (TCM) were randomly assigned to two groups with 1:1 radio. Children in the test group received QXQJ oral solution and oseltamivir simulant, while the control group received oseltamivir phosphate granules and QXQJ simulant. The duration of treatment was five days, followed by a two-day follow-up period. The primary endpoint was the clinical recovery time. Secondary endpoints included the time to defervescence, incidences of complications and severe or critical influenza, negative conversion rate, improvement of TCM syndromes, and safety profiles of the therapeutics, which mainly contained the adverse clinical events and adverse drug reactions. Results: A total of 231 children were randomized to either the QXQJ (n=117) or oseltamivir (n=114) group. The FAS and PPS results showed that both groups experienced a median clinical recovery time of three days (P>0.05). The median time to defervescence of both groups were 36 hours in FAS and PPS (P>0.05), and two groups did not differ in terms of the other secondary endpoints (P>0.05). 14 patients (12.39%) in the QXQJ group and 14 patients (12.50%) in the oseltamivir group reported at least one adverse event, respectively. One serious adverse event occurred in the QXQJ group. There was no significant difference in the incidence of adverse events or adverse drug reactions between the groups. Conclusions: The efficacy of QXQJ oral solution was comparable to that of oseltamivir for treating influenza in children, with an acceptable safety profile. Trial Registration: Chinese Clinical Trial Registry ChiCTR1900021060.

Cell damage and neutrophils promote the infection of Mycoplasma pneumoniae and inflammatory response.

Mycoplasma pneumoniae (MP) is an important respiratory pathogen of human. The infection of MP can cause direct damage and immune damage in lung, resulting in Mycoplasma pneumoniae pneumonia (MPP). In this study, we aim to investigate the pathogenesis of MPP by detecting the proliferation of MP under conditions of cell damages and neutrophils in vitro. Firstly, we found the supplements of intracellular fluid, protein and RNA derived from intracellular fluid of A549 cells contribute to the survival of MP, thereby promoting the infection of MP. Cell damage can also significantly contribute to the survival of MP without supplements. At the same time, the additions of supplements contribute to apoptosis and the expression of IL-8 and IL-1ß. Further, we found live neutrophils show bactericidal activity to MP, and the phagocytosis of MP promotes apoptosis of neutrophils. When co-incubated with MP and A549 cells, the proliferation of MP in the high neutrophils proportion groups were accelerated with functional decline of neutrophils, and the level of extracellular IL-1ß showed a time and dose dependent manner to neutrophils. These results suggest that the release of intracellular nutrients by damaged cells and functional decline of neutrophils can promote the infection of MP and play roles in the activation of inflammatory response. Therefore, lung damage and infiltration of neutrophils would be important factors affecting the development of MPP.

Mechanism of Infantile Feire Kechuan Oral Solution against Mycoplasma pneumoniae infection of A549 cells.

BACKGROUND: Mycoplasma pneumoniae is a leading cause of community-acquired respiratory infections. Infantile Feire Kechuan Oral Solution (IFKOS) is effective for treatment of M. pneumoniae infection. The aim of this study was to explore the potential mechanism of IFKOS against M. pneumoniae infection in basal epithelial human lung adenocarcinoma A549 cells. METHODS: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine the effects of IFKOS on the viability of A549 cells infected with M. pneumoniae. Optical microscopy was used to observe cell morphology and a Muse cell analyzer was used to assess apoptosis and the cell cycle phase. Enzyme-linked immunosorbent assays were employed to assess the expression levels of interleukin (IL)-4, IL-6, IL-8, IL-17, tumor necrosis factor (TNF)-α, interferon (IFN)-α, and IFN-γ. RESULTS: Under certain conditions, M. pneumoniae infection reduced the viability and inhibited the proliferation of A549 cells, promoted early apoptosis, and arrested cells in the G0/G1 phase, thus shortening the S and G2/M phases (all p < 0.05). M. pneumoniae also upregulated expression of IL-8 and TNF-α and downregulated that of IL-6 (p < 0.05), which switched the immune balance of Th1/Th2 to Th1 cells. IFKOS (5.531 mg/mL) improved the viability and proliferation of M. pneumoniae-infected A549 cells, mitigated early apoptosis, and reversed cell cycle arrest in the G0/G1 phase, thereby extending the S and G2/M phases (all, p < 0.05). IFKOS downregulated expression of IL-8 and TNF-α and upregulated that of IL-6 (p < 0.01), thereby reversing the immune imbalance of Th1/Th2. Secretion of IL-4, IL-17, IFN-α, and IFN-γ was not observed. CONCLUSION: IFKOS played a protective role in the regulation of cell viability, apoptosis, the cell cycle, and Th1/Th2 immune imbalance induced by M. pneumoniae infection and conveyed an anti-inflammatory effect in A549 cells.

Serum cytokine profiling reveals different immune response patterns during general and severe Mycoplasma pneumoniae pneumonia.

Mycoplasma pneumoniae (MP) is an important human pathogen that mainly affects children causing general and severe Mycoplasma pneumoniae pneumonia (G/SMPP). In the present study, a comprehensive immune response data (33 cytokines) was obtained in school-age children (3-9 years old) during MPP, aiming to analyze the immune response patterns during MPP. At acute phase, changes of cytokines were both detected in GMPP (24/33) and SMPP (23/33) groups compared to the healthy group (p < 0.05), with 20 identical cytokines. Between MPP groups, the levels of 13 cytokines (IL-2, IL-10, IL-11, IL-12, IL-20, IL-28A, IL-32, IL-35, IFN-α2, IFN-γ, IFN-ß, BAFF, and TSLP) were higher and three cytokines (LIGHT, OPN and CHI3L1) were lower in the SMPP group than in the GMPP group (p < 0.05). Function analysis reveals that macrophage function (sCD163, CHI3L1) are not activated in both MPP groups; difference in regulatory patterns of T cells (IL26, IL27, OPN, LIGHT) and defective activation of B cells (BAFF) were detected in the SMPP group compared to the GMPP group. Besides, the level of osteocalcin; sIL-6Rß and MMP-2 are both decreased in MPP groups at acute and convalescent phases compared to the healthy group, among which the levels of sIL-6Rß and MMP-2 showed negative correlations (p < 0.1) to the application of bronchial lavage in SMPP group, indicating their roles in the development of MPP. At the convalescent phase, more cytokines recovered in GMPP (18) than SMPP (11), revealing better controlled immune response during GMPP. These results reveal different immune response patterns during GMPP and SMPP. In addition, the differentiated cytokines may serve as potential indicators of SMPP; early intervention on immune response regulations may be helpful in reducing the severity of SMPP.

Allele-specific real-time PCR testing for minor macrolide-resistant Mycoplasma Pneumoniae.

BACKGROUND: The point mutations in 23S rRNA gene of Mycoplasma pneumoniae (M. pneumoniae) can lead to high-level resistance to macrolides. This study aimed to evaluate allele-specific real-time PCR (ASPCR) to detect the resistance-related mutations located at positions A2063G and A2064G of 23S rRNA gene. METHODS: We detected 178 pharyngeal swab specimens and calculated the proportions of resistant and sensitive quasispecies using ASPCR assays. ASPCR assays can detect down to 10 copies of 23S rRNA gene and achieved sensitivities of < 0.1% for A2063G and A2064G. We also compared the findings of ASPCR with the results of nested PCR with sequencing. RESULTS: Of 178 samples, 164 were found to have M. pneumoniae including 90.85% (149/164) samples with macrolide-resistant M. pneumoniae (MRMP) quasispecies by ASPCR, while 153 were found to be M. pneumoniae-positive including 71.90% (110/153) samples with MRMP quasispecies by nested PCR with sequencing. Of the 164 M. pneumoniae-positive samples, 61.59% (101/164) had the mixed population of wild-type and mutant M. pneumoniae, and 56.44% (57/101) of the latter contained the mutations at low frequency (≤50%). CONCLUSION: ASPCR indicated that sensitive and resistant quasispecies coexisted in most of the M. pneumoniae positive samples. The ASPCR was a highly sensitive, accurate and rapid method for detecting the macrolide resistance-associated mutations and it could provide earlier and more drug-resistant information for M. pneumoniae research and the clinical therapy.

In vitro susceptibility test of Xiao'er Feire Kechuan Oral Solution to Mycoplasma pneumoniae.

The aim of this study was to evaluate the inhibitory effect of antibiotics and Xiao'er Feire Kechuan Oral Solution on Mycoplasma pneumoniae (MP) clinical isolates.Twenty clinical isolates containing A-to-G transition at position 2063 and 10 clinical isolates without mutations in 23S rRNA V regions were randomly selected. The international standard strain FH was chosen as control strain. The minimum inhibitory concentration (MIC) of macrolide, quinolones, tetracycline, and Xiao'er Feire Kechuan Oral Solution to MP clinical isolates were performed using broth microdilution method.In vitro antibiotic susceptibility test of MP clinical isolates showed that MP showed high resistance to macrolide antibiotics (erythromycin and azithromycin); MIC of both were more than 64 µg/mL. The MICs of erythromycin and azithromycin for clinical isolates without mutations in 23S rRNA V regions were ≤0.5 µg/mL. The MICs of tetracycline and levofloxacin for all clinical isolated strains were ≤2.0 µg/mL and ≤1.0 µg/mL, respectively. The MIC of Xiao'er Feire Kechuan Oral Solution was 13.828∼6.914 mg/mL.In vitro, the drug resistance of MP to macrolide antibiotics is higher, MP clinical isolates are sensitive to tetracycline and levofloxacin, and Xiao'er Feire Kechuan Oral Solution also has a certain inhibitory effect on the macrolide-resistant MP.

Epidemiology and mechanism of drug resistance of Mycoplasma pneumoniae in Beijing, China: A multicenter study.

Mycoplasma pneumoniae (M. pneumoniae) is one of the most common causes of community-acquired respiratory tract infections (RTIs). We aimed to investigate the prevalence of M. pneumoniae infection, antibiotic resistance and genetic diversity of M. pneumoniae isolates across multiple centers in Beijing, China. P1 protein was detected by Nested PCR to analyze the occurrence of M. pneumoniae in pediatric patients with RTI. M. pneumoniae isolates were cultured and analyzed by Nested-PCR to determine their genotypes. Broth microdilution method was used to determine the minimum inhibitory concentration (MIC) of antibiotics. Out of 822 children with RTI admitted to 11 hospitals in Beijing, 341 (41.48%) were positive for M. pneumoniae by Nested PCR and 236 (69.21%) samples had mutations in 23S rRNA domain V. The highest proportion of M. pneumoniae positive samples was observed in school-age children (118/190; 62.11%) and in pediatric patients with pneumonia (220/389; 56.56%). Out of 341 M. pneumoniae positive samples, 99 (12.04%) isolates were successfully cultured and the MIC values were determined for 65 M. pneumoniae strains. Out of these, 57 (87.69%) strains were resistant to macrolides, and all 65 strains were sensitive to tetracyclines or quinolones. M. pneumoniae P1 type I and P1 type II strains were found in 57/65 (87.69%) and 8/65 (12.31%) of cultured isolates, respectively. Overall, we demonstrated a high prevalence of M. pneumoniae infection and high macrolide resistance of M. pneumoniae strains in Beijing. School-age children were more susceptible to M. pneumoniae, particularly the children with pneumonia. Thus, establishment of a systematic surveillance program to fully understand the epidemiology of M. pneumoniae is critical for the standardized use of antibiotics in China.

Clinical Course and Factors Associated with Asthma Control in Children under Control-based Asthma Management: A Prospective Study.

In the current study, we sought to track the clinical course of children under control-based asthma management and focused on respiratory pathogens monitoring. We prospectively explored influencing factors for asthma control. 121 children with uncontrolled asthma between 3-14 years of age were recruited. Common respiratory pathogens were detected with pharyngeal swabs and serum aeroallergen-specific IgE was measured. Numeric asthma control scores, airway resistance and fractional concentrations of exhaled nitric oxide (FENO) were evaluated. A proper control-based asthma management plan was established by the study physician. Regular reviews were performed, with the above measurements retested at set time intervals. The proportion of patients achieving asthma control at 1 month and 3 months were 59% and 76% ; respectively (p=0.013). These patients exhibited significant improvement in numeric scores and lung function parameters. The prevalence of common respiratory pathogens did not significantly differ between reviews. The number of sensitized aeroallergens significantly increased with age (r=0.235, p=0.010). Children with a high visual analogue scale (VAS) score for asthma at baseline were less likely to achieve asthma control after 1 month, while those sensitized to more aeroallergens were more likely to achieve asthma control after 1 month (p=0.016 and 0.012). In summary, children with asthma showed significant improvements in control rates and lung function during control-based asthma management, independent of respiratory pathogens testing reults. Patients with high VAS scores and fewer sensitizations to aeroallergens had difficulty achieving short-term asthma control.

Clinical practices s of Traditional Chinese Medicine for acute upper respiratory tract infection in children.

OBJECTIVE: To develop Clinical practice s of Traditional Chinese Medicine (TCM) for acute upper respiratory tract infection (AURI) in children; TCM is used alone or administered together with antibiotics. METHODS: Under the guidance of evidence-based medicine concept, in strict accordance with the rules of international s development, as well as on the basis of evidence of clinical research of TCM, the s solicited opinions from clinical experts and methodologists in TCM and Western Medicine. GRADE standard was applied to form experts' consensus. RESULTS: The s standardized classification of TCM patterns and TCM treatments in children with AURI, including prescription, Chinese patent medicine, non-drug treatment and prevention. CONCLUSION: Follows the principle of ""evidence based, consensus supplemented, and experience referred"", these s were formulated, but the quality of evidence of included studies were relatively low. Further refinement of the s should be needed as deeper clinical studies as available in future.

Knowledge, attitude, and practices (KAP) and risk factors analysis related to cystic echinococcosis among residents in Tibetan communities, Xiahe County, Gansu Province, China.

Cystic echinococcosis (CE) is a global parasitic zoonosis caused by Echinococcus granulosus. The disease is highly endemic in western China, especially in Tibetan areas, because of poor economic development and hygiene conditions, limited community knowledge of CE, a large scale of dogs, and home slaughtering of livestock. Although many researchers have analyzed risk factors of CE transmission in Tibetan Plateau, there are rare reports of knowledge, attitude, and practice (KAP) of residents about CE in Tibetan communities. In our current study, community based cross-sectional study was conducted in three townships in Xiahe County, Gannan Tibetan Autonomous Prefectures of Gansu Province from May to September 2013. A total of 972 participants originating from Tibetan communities of 31 villages in the 3 townships were registered and data were collected using structured questionnaires. From the total of 972 study participants (457 males and 515 females), 65.9% heard of the disease CE. Most of them (96.1%) would like to accept CE inspection. About half of the peoples feed their dogs often and major of them do not play with the dogs. Risk factors included resident, knowing dog could be infected, knowing eating could be route of infection, oldest dog's age, usually feed your dog by self, feed dogs with internal organs. In general our findings showed that most of residents had positive attitude toward treatments of the disease, but their practice about disease prevention and control was low. Therefore, our study called for continued and strengthened education of changing the life style, especially the behaviors related to dogs.

Drug resistance mechanisms of Mycoplasma pneumoniae to macrolide antibiotics.

Throat swabs from children with suspected Mycoplasma pneumoniae (M. pneumoniae) infection were cultured for the presence of M. pneumoniae and its species specificity using the 16S rRNA gene. Seventy-six M. pneumoniae strains isolated from 580 swabs showed that 70 were erythromycin resistant with minimum inhibitory concentrations (MIC) around 32-512 mg/L. Fifty M. pneumoniae strains (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 of M. pneumoniae strains. 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.

P1 gene of Mycoplasma pneumoniae in clinical isolates collected in Beijing in 2010 and relationship between genotyping and macrolide resistance.

BACKGROUND: Mycoplasma pneumoniae is a common pathogen that caused community-acquired pneumonia (CAP). P1 protein served as major adhesion and immunodominant protein in Mycoplasma pneumoniae, but little about P1 gene was learned and the relationship between P1 genotype and macrolide resistance has yet to be explored. METHODS: The DNA sequence of the entire P1 gene from 35 strains isolated from clinical specimens collected in Beijing, China, in 2010 was determined. The resulting sequences were checked for known macrolide resistance mutations, such as A2063G, A2064G, C2617G in domain V of 23S rRNA. Antibiotic susceptibility test was done to further identify macrolide resistant strains. RESULTS: Thirty-four clinical strains were type 1, and were identical to type 1 reference strain MP129. Only one clinical strain, MpYYM22, was type 2, and proved to be variant 2c. One synonymous point mutation in the P1 type 1 gene from two isolates was identified relative to the MP129 P1 sequence at nucleotide position (nt) 552 (C>A), while another two isolates had missense mutations at nt 2504 (G>A). This point mutation caused an amino acid change from glycine to glutamic acid. An AGT tri-nucleotide variable-number tandem repeat (VNTR), coding for serine and repeating 6-11 times, up to 15-16 times, was found in the region between the RepMP4 and RepMP2/3 elements in the 35 isolates examined. All 35 clinical strains, including MpYYM22, demonstrated macrolide resistance with the range of minimum inhibitory concentration (MIC) of erythromycin from 64 to 256 µg/ml, having an A2063G transition in domain V of the 23S rRNA gene. CONCLUSIONS: P1 type 1 was the dominant type of Mycoplasma pneumoniae in Beijing in 2010, although variant 2c strains were present. More samples are needed to determine whether there is a relationship between the P1 genotype and macrolide resistance, as the 35 strains examined did not allow a conclusive result. However, the AGT tri-nucleotide VNTR may be a more informative locus for multi-locus VNTR analysis.

Molecular mechanisms of macrolide resistance in clinical isolates of Mycoplasma pneumoniae from China.

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.

[Application of nested PCR and sequencing technique to detect point mutations of the 23S rRNA gene of Mycoplasma pneumoniae].

OBJECTIVE: To establish a quick method to detect drug resistance of Mycoplasma pneumoniae (MP) and study the condition of drug resistance in MP infection. METHODS: MP 23S rRNA target gene in throat swab specimens from 200 patients with suspected MP infection was detected by using nested PCR and DNA sequencing. The result of 23S rRNA gene detection was confirmed by MP isolation and drug susceptibility test in vitro for reliability. RESULTS: Of the 200 clinical specimens, 64 were proved to be positive for MP through MP-IgM antibody, MP specific 16S rRNA nested PCR and MP isolation . The 23S rRNA gene was amplified and the gene sequence was compared with MP reference strain in Genbank, 26 were identical to the reference strain, 38 had a point mutation in 23S rRNA. Among them, 35 had A to G mutation at position 2063, 1 had A to C mutation at position 2063 and 2 had A to G mutation at position 2064, the percentage of drug resistance was 59.4%. The sensitivity of the gene detection method was 10(2) ccu/ml and it was confirmed to be reliable by MP isolation and drug susceptibility test. CONCLUSIONS: The gene detection method could detect MP drug resistant gene directly from clinical specimen, which has the advantages of high specificity, high sensitivity and quickness. It is of great significance for diagnosis of MP infection because MP isolation is difficult and time-consuming.