RESUMO
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.
Assuntos
Antibacterianos , Farmacorresistência Bacteriana , Macrolídeos , Mycoplasma pneumoniae , Pneumonia por Mycoplasma , Mycoplasma pneumoniae/efeitos dos fármacos , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/isolamento & purificação , Humanos , China/epidemiologia , Macrolídeos/farmacologia , Estudos Retrospectivos , Criança , Antibacterianos/farmacologia , Pré-Escolar , Adolescente , Adulto , Feminino , Masculino , Pneumonia por Mycoplasma/epidemiologia , Pneumonia por Mycoplasma/microbiologia , Pneumonia por Mycoplasma/tratamento farmacológico , Pessoa de Meia-Idade , Adulto Jovem , Testes de Sensibilidade Microbiana , Idoso , Lactente , Prevalência , RNA Ribossômico 23S/genética , Idoso de 80 Anos ou maisRESUMO
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.
Assuntos
Mycoplasma pneumoniae , Pneumonia por Mycoplasma , Células A549 , Humanos , Pulmão/patologia , Mycoplasma pneumoniae/genética , Neutrófilos/metabolismo , Pneumonia por Mycoplasma/patologiaRESUMO
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 AâG 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.
Assuntos
Antibacterianos , Farmacorresistência Bacteriana , Genoma Bacteriano , Testes de Sensibilidade Microbiana , Mycoplasma pneumoniae , Pneumonia por Mycoplasma , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/efeitos dos fármacos , Mycoplasma pneumoniae/classificação , Mycoplasma pneumoniae/isolamento & purificação , Humanos , Antibacterianos/farmacologia , Genoma Bacteriano/genética , Pneumonia por Mycoplasma/microbiologia , Pneumonia por Mycoplasma/tratamento farmacológico , Farmacorresistência Bacteriana/genética , Masculino , Feminino , Sequenciamento Completo do Genoma , Pessoa de Meia-Idade , Macrolídeos/farmacologia , Adulto , Mutação , RNA Ribossômico 23S/genética , Genômica , Idoso , Eritromicina/farmacologiaRESUMO
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.
Assuntos
Macrolídeos , Mycoplasma pneumoniae , Criança , Humanos , Mycoplasma pneumoniae/genética , Macrolídeos/farmacologia , Caspase 3/genética , RNA Ribossômico 23S/genética , Virulência , Interleucina-8 , Proteômica , Farmacorresistência Bacteriana/genética , Testes de Sensibilidade Microbiana , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , MutaçãoRESUMO
Background: Mycoplasma pneumoniae is an important infectious pathogen of lower respiratory tract infection in children and adolescents. Macrolide resistant M. pneumoniae (MRMP) has become increasingly prevalent, and identifying pathogen resistance genes is crucial for treatment. Case Presentation: We report a patient with severe refractory M. pneumoniae pneumonia (MPP). The failure of initial clinical treatment prompted the re-analysis of metagenomic next-generation sequencing (mNGS) data for macrolide-resistant gene. Macrolide-resistance 23S ribosomal RNA gene was confirmed with read depth of 64 X for the A2063G mutation, which can decrease the affinity of macrolide with M. pneumoniae ribosome resulting in macrolide resistance. Furthermore, antimicrobial susceptibility testing demonstrated that M. pneumoniae was resistant to macrolide. PCR confirmatory test about M. pneumoniae resistance A2063G mutation, clinical treatment course and prognosis with altered treatment strategy, and M. pneumoniae antimicrobial susceptibility confirmed that the severe refractory MPP was due to macrolide resistant M. pneumoniae. Conclusion: As a new molecular level detection, mNGS is an effective method for detecting M. pneumoniae resistance genes. Early recognition of macrolide resistance and suitable antibiotics strategy is of vital importance for the prognosis of severe refractory MPP.
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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.
Assuntos
Antibacterianos/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Mycoplasma pneumoniae/efeitos dos fármacos , Pneumonia por Mycoplasma/tratamento farmacológico , Células A549 , Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Pneumonia por Mycoplasma/imunologia , Pneumonia por Mycoplasma/microbiologia , Células Th1/imunologia , Células Th2/imunologiaRESUMO
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.
Assuntos
Mycoplasma pneumoniae , Pneumonia por Mycoplasma , Criança , Humanos , Pré-Escolar , Metaloproteinase 2 da Matriz , Citocinas , ImunidadeRESUMO
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 64âµg/mL. The MICs of erythromycin and azithromycin for clinical isolates without mutations in 23S rRNA V regions were ≤0.5âµg/mL. The MICs of tetracycline and levofloxacin for all clinical isolated strains were ≤2.0âµg/mL and ≤1.0âµg/mL, respectively. The MIC of Xiao'er Feire Kechuan Oral Solution was 13.828â¼6.914âmg/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.