Using 16S rDNA Sequencing Technology to Preliminarily Analyze Intestinal Flora in Children with Mycoplasma pneumoniae Pneumonia.

Objective: We investigated changes in the intestinal flora of children with Mycoplasma pneumoniae pneumonia (MPP). Methods: Between September 2019 and November 2019, stool samples from 14 children with MPP from The Fourth Hospital of Baotou city, Inner Mongolia Autonomous Region, were collected and divided into general treatment (AF) and probiotic (AFY) groups, according to the treatment of "combined Bifidobacterium, Lactobacillus, Enterococcus, and Bacillus cereus tablets live". High-throughput 16S rDNA sequencing was used to identify intestinal flora. Results: Intestinal flora abundance and diversity in children with MPP were decreased. Both Shannon and Simpson indices were lower in the AF group when compared with healthy controls ( P < 0.05). When compared with healthy controls, the proportion of Enterorhabdus was lower in the AF group, while the proportion of Lachnoclostridium was higher ( P < 0.05). The proportion of Bifidobacteria and Akkermansia was lower in the AFY group but Enterococcus, Lachnoclostridium, Roseburia, and Erysipelatoclostridium proportions were higher. The proportion of Escherichia coli- Shigella in the AFY group after treatment was decreased ( P < 0.05). Conclusions: The intestinal flora of children with MPP is disturbed, manifested as decreased abundance and diversity, and decreased Bifidobacteria. Our probiotic mixture partly improved intestinal flora disorders.

Data mining and systematic pharmacology to reveal the mechanisms of traditional Chinese medicine in Mycoplasma pneumoniae pneumonia treatment.

Traditional Chinese Medicine (TCM) is widely used in the treatment of Mycoplasma pneumoniae Pneumonia (MPP) in East Asia. However, our current understanding of the underlying molecular mechanism remains dispersive and promiscuous. In this study, a systematic pharmacological approach combined with literature data mining was applied for drug similarity evaluation, drug half-life evaluation, oral bioavailability prediction, drug target exploration, Gene Ontology (GO) analysis, KEGG pathway enrichment and network construction, thus providing the rationale for its clinical performance. Five mostly studied herbs, including Ephedra Herba, Amygdalus communis Vas, Platycodon grandiforus, Licorice and Scutellariae Radix, were selected from the literature. Total ninety-three active ingredients, which are expected to be the effective components for MPP treatment, were screened out. Interrelationship between active compounds, drug targets and signaling pathways were analyzed to reveal the therapeutic effect of TCM in detail. Of importance, we found that TNF, ß2AR and PTGS2 play pivotal role in TCM mediated MPP inhibition. And mechanistically, epithelial apoptosis (defensive barrier function), GPCR signaling (symptom amelioration) and immune pathways (innate signaling and adaptive Th17 response) are critically involved. Our work, achieved through systematic pharmacology and data mining, enlarges the knowledge of TCM in MPP therapy, and could provide valuable insights for further drug discovery studies.

Mycoplasma pneumoniae Macrolide Resistance and MLVA Typing in Children in Beijing, China, in 2016: Is It Relevant?

OBJECTIVE: The aim of this study is to investigate the macrolide resistance rate and molecular type with multiple-locus variable-number tandem-repeat analysis (MLVA) of Mycoplasma pneumoniae of Beijing in 2016 in pediatric patients. METHODS: Real-time quantitative polymerase chain reaction (PCR) was used to identify M. pneumoniae, and MLVA was performed. The domain V of the 23S rRNA was sequenced to detect macrolide-resistant point mutations. We also investigated the activities of antibiotics against M. pneumoniae isolates in vitro. RESULTS: The PCR detection rate of M. pneumoniae in children in Beijing was 40%, and the macrolide resistance rate was 66%. The A2063G mutation in the 23S rRNA V region is the dominant mutation (137/146, 93.84%), whereas the A2064G mutation is rare (9/146, 6.16%). Seventy-three samples were typed successfully by MLVA typing, including 86.3% (63/73) were MLVA type 4-5-7-2, and 13.7% (10/73) were MLVA type 3-5-6-2. No other types were found. No strains were resistant to levofloxacin or tetracycline. CONCLUSION: In 2016, a specific decrease in the macrolide resistance rate occurred in Beijing. The detection rate and macrolide resistance rate of outpatients are lower than those of inpatients. The A2063G mutants M. pneumoniae have high levels of resistance to erythromycin and azithromycin. The primary MLVA type is 4-5-7-2, followed by 3-5-6-2. No other MLVA types were detected. No strains resistant to tetracycline or levofloxacin were found in vitro.

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.

Establishment of a Predictive Diagnostic Model for Acute Mycoplasma Pneumoniae Infection in Elderly Patients with Community-acquired Pneumonia.

We established a diagnostic model to predict acute Mycoplasma pneumoniae (M. pneumonia) infection in elderly Community-acquired pneumonia (CAP) patients. We divided 456 patients into acute and non-acute M. pneumoniae infection groups. Binary logistic regression and receiver operating characteristic (ROC) curves were used to establish a predictive model. The following independent factors were identified: age ⋝ 70 years; serum cTNT level ⋝ 0.05 ng/mL; lobar consolidation; mediastinal lymphadenopathy; and antibody titer in the acute phase ⋝ 1:40. The area under the ROC curve of the model was 0.923 and a score of ⋝ 7 score predicted acute M. pneumoniae infection in elderly patients with CAP. The predictive model developed in this study has high diagnostic accuracy for the identification of elderly acute M. pneumoniae infection.

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.

[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.

[Effect of zhidan huayu oral liquid on mycoplasmal pneumonia in mice].

OBJECTIVE: To study the effect of Chinese herbal medicine for activating blood circulation to remove stasis in treating mycoplasmal pneumonia (MP) in mice. METHODS: One hundered and thirty-five BALB/C mice were randomly divided into the control group, the MP model groups IF1 and IF2, the Rexithromycin treated groups LH1 and LH2, and the Rexithromycin plus Zhidan Huayu oral liquid treated groups LZ1 and LZ2. The changes of pathologic scoring, graphic analysis and thrombus counting of lung were observed. RESULTS: In the 3rd day of treatment, the pathologic scores in LH1 and LZ1 were significantly lower and their values of graphic analysis were significantly higher than those in IF1 (P < 0.01 and P < 0.05 respectively), but with inflammation of lung significantly milder than that in IF1. The difference of therapeutic effect between LH1 and LZ1 was insignificant. In the 4th day of treatment, pathologic scores in LZ2 was significantly lower and value of graphic analysis higher than those in IF2 respectively (P < 0.01), with the improvement better than that of LH2 (P < 0.05). In 3rd and 4th day of treatment, the difference of thrombus counting between the Rexithromycin treated groups and the model groups was insignificant (P > 0.05), but it was significantly lower in the combined treated groups than that in the model groups (P < 0.05). CONCLUSION: Zhidan Huayu oral liquid could assist Rexithromycin to alleviate the condition of mice with MP, its mechanism may be related with the effect of reducing thrombosis and improving microcirculation.