OBJECTIVE: To study the drug resistance of METHODS: BALF specimens were collected from 245 children with RMPP who were admitted to the Children's Hospital Affiliated to Zhengzhou University from March 2016 to December 2020. A rapid cultured drug sensitivity assay was used to detect the resistance of MP isolates to nine commonly used antimicrobial drugs. The real-time PCR was used to measure MP DNA. The direct sequencing was used to detect gene mutations in MP 23SrRNA V region central ring. RESULTS: Among the 245 BALF specimens, 207 tested positive for MP DNA, with a positive rate of 84.5%. The results of drug susceptibility test showed that the children with RMPP had a resistance rate of > 70% to macrolide antimicrobial drugs, with the highest resistance rate to clarithromycin, followed by roxithromycin, clindamycin, acetylspiramycin, erythromycin, and azithromycin, and these children had a resistance rate of < 5% to quinolone antimicrobial drugs. Among the 207 MP DNA-positive specimens, 41 (19.8%) had no drug-resistance gene mutations and 166 (80.2%) had drug-resistance gene mutations, among which 154 (74.4%) had an A→G mutation at 2063 locus of 23SrRNA V region central ring, 7 (3.4%) had an A→G mutation at 2064 locus, and 5 (2.4%) had mutations in both 2063 and 2064 loci. Among the 166 specimens with point mutations of the MP 23SrRNA gene, 159 (95.8%) had point mutations at 2063 locus. The A→G point mutation at 2063 locus of 23SrRNA V region central ring had a great impact on resistance to macrolide antimicrobial drugs. There was a significant difference in the distribution of alleles at 2063 locus between the children with resistance to clarithromycin, roxithromycin, clindamycin, acetylspiramycin, erythromycin, and azithromycin ( CONCLUSIONS MP in the BALF of children with RMPP has a relatively high resistance rate to macrolide antimicrobial drugs. Resistance to macrolide antimicrobial drugs is closely associated with the A→G point mutation in the 23SrRNA gene, and the point mutation at 2063 locus of 23SrRNA V region central ring may affect the drug-resistance mechanism of MP.
Anti-Bacterial Agents/pharmacology* ; Bronchoalveolar Lavage Fluid ; Child ; Drug Resistance, Bacterial/genetics* ; Humans ; Mycoplasma pneumoniae/genetics* ; Pneumonia, Mycoplasma/drug therapy*

OBJECTIVEMutations in 23S rRNA gene are known to be associated with macrolide resistance in Mycoplasma pneumoniae (M. pneumoniae). However, these mutations alone do not fully explain the high resistance rates in Asia. The aim of this study was to investigate other possible mutations involved in macrolide resistance in M. pneumoniae.

METHODSThe whole genomes of 10 clinical isolates of M. pneumoniae with macrolide resistance were sequenced by Illumina HiSeq2000 platform. The role of the macrolide-specific efflux transporter was assessed by efflux-pump inhibition assays with reserpine and carbonyl cyanide m-chlorophenyl-hydrazone (CCCP).

RESULTSA total of 56 single nucleotide polymorphisms (SNPs) were identified in 10 clinical isolates in comparison to the reference strains M129 and FH. Strikingly, 4 of 30 SNPs causing non-synonymous mutations were clustered in macrolide-specific efflux system gene macB encoding macrolide-specific efflux pump protein of the ATP-binding cassette transporter family. In assays of the minimal inhibitory concentrations (MIC) of macrolide antibiotics in the presence of the efflux pump inhibitors caused a significant decrease of MICs, even under detectable levels in some strains.

CONCLUSIONOur study suggests that macrolide efflux pump may contribute to macrolide resistance in M. pneumoniae in addition to the common point mutations in 23S rRNA gene.

Anti-Bacterial Agents ; pharmacology ; Drug Resistance, Bacterial ; genetics ; Genome-Wide Association Study ; Macrolides ; pharmacology ; Microbial Sensitivity Tests ; Mutation ; Mycoplasma pneumoniae ; drug effects ; genetics

OBJECTIVETo investigate the association of drug resistance of Mycoplasma pneumoniae (MP) with DNA load and genotypes in children with MP pneumonia.

METHODSA total of 230 children who were hospitalized and diagnosed with MP pneumonia between January 2012 and December 2016 were enrolled. Throat swabs were collected from the 230 children, and a rapid drug sensitivity assay was used to determine the sensitivity of clinical isolates of MP to nine commonly used antibacterial agents. Quantitative real-time PCR was used to measure MP-DNA load in throat swabs. PCR sequencing was used to determine the genotype of 2063 locus of the MP 23S rRNA V domain.

RESULTSOf the 230 children, 86 (37.4%) had genotype A in 2063 locus, 134 (58.3%) had genotype G, 8 (3.5%) had genotype C, and 2 (0.9%) had genotype T. Mutant strains (genotype G+C+T) had a significantly higher MP-DNA load than wild-type strains (genotype A) (P<0.05). The strains resistant to erythromycin, azithromycin, clarithromycin, and clindamycin had a significantly higher MP-DNA load than non-resistant strains (P<0.05). MP had a high drug resistance rate to macrolide antibiotics. More than 60% of the cases with resistance to macrolides were found to have A2063G mutations. MP was rarely resistant to quinolones (less than 2%).

CONCLUSIONSMutations in 2063 locus of the MP 23S rRNA V domain may result in the resistance of MP to macrolides and the change in DNA load and can be used as a basis for selecting drugs for MP.

Child ; Child, Preschool ; DNA, Bacterial ; analysis ; Drug Resistance, Bacterial ; Female ; Genotype ; Humans ; Infant ; Male ; Mycoplasma pneumoniae ; drug effects ; genetics ; Pneumonia, Mycoplasma ; drug therapy ; microbiology

The role of atypical bacteria and the effect of antibiotic treatments in acute bronchitis are still not clear. This study was conducted at 22 hospitals (17 primary care clinics and 5 university hospitals) in Korea. Outpatients (aged > or = 18 yr) who had an acute illness with a new cough and sputum (< or = 30 days) were enrolled in 2013. Multiplex real-time polymerase chain reaction (RT-PCR) was used to detect five atypical bacteria. A total of 435 patients were diagnosed as having acute bronchitis (vs. probable pneumonia, n = 75), and 1.8% (n = 8) were positive for atypical pathogens (Bordetella pertussis, n = 3; B. parapertussis, n = 0; Mycoplasma pneumoniae, n = 1; Chlamydophila pneumoniae, n = 3; Legionella pneumophila, n = 1). Among clinical symptoms and signs, only post-tussive vomiting was more frequent in patients with atypical pathogens than those without (P = 0.024). In all, 72.2% of the enrolled patients received antibiotic treatment at their first visits, and beta-lactams (29.4%) and quinolones (20.5%) were the most commonly prescribed agents. In conclusion, our study demonstrates that the incidence of atypical pathogens is low in patients with acute bronchitis, and the rate of antibiotic prescriptions is high.
Anti-Bacterial Agents/therapeutic use ; Bordetella parapertussis/genetics/*isolation & purification ; Bordetella pertussis/genetics/*isolation & purification ; Bronchitis/drug therapy/*microbiology ; Chlamydophila pneumoniae/genetics/*isolation & purification ; Community-Acquired Infections/microbiology ; Female ; Humans ; Hypertension/complications ; Legionella pneumophila/genetics/*isolation & purification ; Male ; Middle Aged ; Mycoplasma pneumoniae/genetics/*isolation & purification ; Real-Time Polymerase Chain Reaction ; Republic of Korea ; Sputum/microbiology

OBJECTIVEMycoplasma pneumoniae (MP) is an important pathogen for community-acquired pneumonia in children. MP infection was considered to be self-limited, but many severe refractory MP pneumonia cases have been reported in recent years. The reason for variation in severity of MP pneumonia remains unclear. MP virulence including drug-resistance and host immunologic function are important influencing factors. The present study aimed to clarify relationship between local MP load and severity of MP pneumonia.

METHODMP DNA was quantitatively detected by fluorescent real-time PCR in bronchoalveolar lavage fluid (BALF) from 77 children with MP pneumonia. They were classified into groups of low MP load ( < 10(3)/ml, n = 14) , moderate MP load (10(3)-10(6)/ml, n = 22) and high MP load ( > 10(6)/ml, n = 41) . Clinical symptoms, main laboratory and imaging results of children among the three groups were compared.

RESULTWhen compared with low load group and moderate load group, high load group had longer fever duration (7 d, 10 d vs. 12 d) , longer time to normalization of temperature with macrolide administration (4 d, 8 d vs. 10 d) , more patients with high fever (50.0%, 68.2% vs. 87.8%) and longer duration of fever than 10 d (35.7%, 50.0% vs. 73.2%).Statistically significant difference existed in CRP among the three groups (1.0 mg/L, 11.5 mg/L, 34 mg/L). Large field of consolidation or atelectasis were found in 58.5% of high load patients, much higher than 22.7% in moderate load and 14.3% in low load patients. Bilateral or massive pleural effusion was not found in low load group, while in moderate load and high load group, they were 13.6% and 24.4%. However, no significant difference was found in symptoms and main laboratory and imaging results among different age groups in high load patients.

CONCLUSIONThere is a close relationship between MP load in BALF and clinical characteristics in children with MP pneumonia. Those with high MP load have a more severe process.

Adolescent ; Bacterial Load ; Bronchoalveolar Lavage Fluid ; microbiology ; Child ; Child, Preschool ; Colony Count, Microbial ; DNA, Bacterial ; genetics ; Female ; Humans ; Infant ; Male ; Mycoplasma pneumoniae ; genetics ; isolation & purification ; Pneumonia, Mycoplasma ; microbiology ; pathology ; Real-Time Polymerase Chain Reaction ; Severity of Illness Index

Amino Acid Sequence ; Animals ; Bacterial Proteins ; genetics ; metabolism ; Bacterial Toxins ; genetics ; metabolism ; Base Sequence ; Community-Acquired Infections ; microbiology ; Gene Expression Regulation, Bacterial ; Humans ; Lung ; microbiology ; pathology ; Mycoplasma pneumoniae ; Pneumonia, Mycoplasma ; microbiology ; Respiratory Distress Syndrome, Adult ; microbiology

BACKGROUND: This study aimed to evaluate the prevalence of Mycoplasma pneumoniae in primary and tertiary care hospitals and its macrolide resistance rate. METHODS: Nasopharyngeal swabs were collected from 195 pediatric patients in primary and tertiary care hospitals from October to November 2010. The AccuPower MP real-time PCR kit (Bioneer, Korea) was used for the detection of M. pneumoniae. Direct amplicon sequencing was performed to detect point mutations conferring resistance to macrolides in the 23S rRNA gene. RESULTS: Among the 195 specimens, 17 (8.7%) were M. pneumoniae positive, and 3 of the strains (17.6%) obtained from these 17 specimens displayed the A2063G mutation in 23S rRNA. Three macrolide-resistant M. pneumoniae isolates were isolated from patients hospitalized at the primary care hospital. The positive rates of M. pneumoniae for the primary and tertiary care hospitals were 12.1% (15/124) and 2.8% (2/71), respectively (P=0.033). CONCLUSIONS: The positive rate of M. pneumoniae in the primary care hospital was higher than that in the tertiary care hospital. Simultaneous detection of M. pneumoniae and macrolide-resistant mutation genes in the 23S rRNA by real-time PCR is needed for rapid diagnosis and therapy of M. pneumoniae infections.
Anti-Bacterial Agents/*pharmacology ; Child, Preschool ; Drug Resistance, Bacterial/*drug effects ; Female ; Humans ; Infant ; Infant, Newborn ; Macrolides/*pharmacology ; Male ; Mycoplasma pneumoniae/genetics/*isolation & purification ; Nasopharynx/microbiology ; Pneumonia, Mycoplasma/epidemiology/microbiology ; Primary Health Care ; RNA, Ribosomal, 23S/analysis ; Reagent Kits, Diagnostic ; Real-Time Polymerase Chain Reaction ; Tertiary Healthcare

BACKGROUNDMycoplasma 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.

METHODSThe 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.

RESULTSThirty-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.

CONCLUSIONSP1 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.

Anti-Bacterial Agents ; pharmacology ; DNA, Bacterial ; Drug Resistance, Bacterial ; Genotype ; Humans ; Macrolides ; pharmacology ; Microbial Sensitivity Tests ; Mycoplasma pneumoniae ; drug effects ; genetics ; metabolism

OBJECTIVETo investigate the infection rate and genotypes of Mycoplasma pneumoniae (MP) by examining bronchoalveolar lavage fluid from children with community acquired pneumonia (CAP).

METHODSPolymerase chain reaction (PCR) was used for detecting MP in bronchoalveolar lavage fluid from 220 children hospitalized with CAP, and the accuracy was confirmed by quantitative real-time PCR. Positive samples were digested with HaeⅡ and Hae Ⅲ and compared with standard strain to analyze the genotypes of MP from positive samples. The accuracy of genotyping was confirmed by sequencing the amplified products of some randomly selected positive samples.

RESULTSThe positive rate of MP in 220 samples was 55.0% (121/220). MP infection occurred mostly in preschool and school-age children (63.5%, 101/159), and the lowest positive rate was seen in children aged under 6 months (20%, 1/5). The positive rate showed no significant differences between sexes and between seasons. Sixty randomly selected MP-positive samples showed a genotype of P1 type 1 after restriction digestion, which was further confirmed by sequencing of 4 samples.

CONCLUSIONSMP is one of the main pathogens of pneumonia in children, and the MP infection rate is significantly correlated with age. The dominant genotype of MP in children is P1 type 1.

Adolescent ; Child ; Child, Preschool ; Female ; Genotype ; Hospitalization ; Humans ; Infant ; Male ; Mycoplasma pneumoniae ; classification ; genetics ; Phylogeny ; Pneumonia, Mycoplasma ; microbiology ; Real-Time Polymerase Chain Reaction ; Seasons

We describe 2 cases of pneumonia caused by the same macrolide-resistant Mycoplasma pneumoniae in siblings. M. pneumoniae was identified using real-time PCR. Direct sequence analysis of the 23S rRNA gene revealed a point mutation in V domain (A2063G) of the 23S rRNA gene.
Anti-Bacterial Agents/pharmacology ; Child ; Child, Preschool ; Drug Resistance, Bacterial/drug effects ; Humans ; Macrolides/pharmacology ; Male ; Mutation ; Mycoplasma pneumoniae/*genetics/isolation & purification ; Pneumonia, Mycoplasma/*diagnosis/microbiology/radiography ; RNA, Ribosomal, 23S/*analysis ; Real-Time Polymerase Chain Reaction ; Sequence Analysis, RNA ; Siblings