Miyuki Morozumi

Macrolide-resistant Mycoplasma pneumoniae: characteristics of isolates and clinical aspects of community-acquired pneumonia

Mycoplasma pneumoniae is one of the main pathogens causing community-acquired respiratory tract infections in children and adults. Macrolide (ML) antibiotics are recognized generally as first-choice agents for M. pneumoniae infections, and these antibiotics were thought to have excellent effectiveness against M. pneumoniae for many years. In 2000, however, M. pneumoniae showing resistance to macrolides was isolated from clinical samples obtained from Japanese pediatric patients with community-acquired pneumonia (CAP). Since then, prevalence of ML-resistant M. pneumoniae isolates in pediatric patients has increased rapidly. In 2007, ML-resistant M. pneumoniae isolates were obtained from Japanese adults with CAP; numbers of such isolates also have gradually increased in Japan. Recently, similar antimicrobial resistance in M. pneumoniae has begun to emerge worldwide. In this review, we focus on changes of ML-resistant M. pneumoniae from year to year and consider resistance mechanisms as well as clinical features of patients with resistant M. pneumoniae infection.

Increased macrolide resistance of Mycoplasma pneumoniae in pediatric patients with community-acquired pneumonia.

ABSTRACT Among 380 Mycoplasma pneumoniae isolates from 3,678 pediatric patients with community-acquired pneumonia, 50 macrolide-resistant strains had an A2063G transition in domain V of the 23S rRNA, whereas 5 had an A2064G transition. These resistant strains increased rapidly from April 2002 to December 2006.

Increased macrolide resistance of Mycoplasma pneumoniae in France directly detected in clinical specimens by real-time PCR and melting curve analysis

OBJECTIVES Mycoplasma pneumoniae is a common aetiological agent of community-acquired respiratory tract infections for which macrolides are the treatment of choice. In France, only two macrolide-resistant isolates were reported in 1999. In contrast, several recent data reported that macrolide-resistant M. pneumoniae isolates have been spreading since 2000 in Japan. Mutations A2058G (Escherichia coli numbering), A2058C, A2059G, A2062G, C2611A and C2611G in domain V of the 23S rRNA gene were associated in vivo or in vitro with this resistance. The aim of this study was to determine whether macrolide resistance of M. pneumoniae is emerging in France. PATIENTS AND METHODS We developed a duplex real-time PCR for the detection of the six 23S rRNA mutations associated with macrolide resistance in M. pneumoniae and a simplex real-time PCR for the identification of the A2058G mutation, the most common one. Both methods rely on fluorescence resonance energy transfer coupled to melting curve analysis and are directly applicable to clinical samples. The duplex real-time PCR assay, first validated on 40 genetically characterized M. pneumoniae strains, was then applied directly on 248 French respiratory tract clinical samples. RESULTS Among M. pneumoniae-positive specimens collected before 2005, no macrolide-resistant M. pneumoniae isolate was detected. In contrast, among 51 samples collected between 2005 and 2007, five (9.8%) yielded a resistant genotype, suggesting a recent increase in macrolide-resistant M. pneumoniae isolates in France. CONCLUSIONS The epidemiological monitoring of macrolide resistance in this species has become necessary in France and Europe, and will be made easier by using these PCR assays.

Simultaneous Detection of Pathogens in Clinical Samples from Patients with Community-Acquired Pneumonia by Real-Time PCR with Pathogen-Specific Molecular Beacon Probes

ABSTRACT In this study, real-time PCR with pathogen-specific molecular beacons (MB) and primers was evaluated for prediction of community-acquired pneumonia (CAP) causative agents, detecting six main CAP agents, Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, and Streptococcus pyogenes, simultaneously. The PCR assay was evaluated for fresh clinical specimens from infants and children (n = 389) and from adults (n = 40). The MB probes and primers are both pathogen specific, namely, the lytA gene for S. pneumoniae, the mip gene for L. pneumophila, and 16S rRNA genes for the remaining four organisms. DNA extraction of clinical specimens was performed with a commercially available EXTRAGEN II kit, and amplification was performed with Stratagene Mx3000P. The limit of detection for these pathogens ranged from 2 copies to 18 copies. The whole process from DNA extraction to the analysis was finished in less than 2 h. The obtained sensitivity and specificity of this real-time PCR study relative to those of conventional cultures were as follows: 96.2% and 93.2% for S. pneumoniae, 95.8% and 95.4% for H. influenzae, 100% and 100% for S. pyogenes, and 100% and 95.4% for M. pneumoniae, respectively. The sensitivity and specificity for M. pneumoniae relative to those of a serologic assay were 90.2% and 97.9%, respectively. In six clinical samples of C. pneumoniae, the real-time PCR gave positive predictable values, and in those cases, elevation of the titer value was also observed. In conclusion, we demonstrated that a real-time PCR assay with pathogen-specific MB is useful in identifying CAP causative agents rapidly and in examining the clinical course of empirical chemotherapy in a timely manner, supporting conventional culture methods.

Rapid Effectiveness of Minocycline or Doxycycline Against Macrolide-Resistant Mycoplasma pneumoniae Infection in a 2011 Outbreak Among Japanese Children

BACKGROUND Mycoplasma pneumoniae is a major pathogen causing community-acquired pneumonia in children and young adults. Outbreaks typically occur at intervals of several years. In 2011, a widespread outbreak was associated with macrolide-resistant M. pneumoniae (MRMP) in Japanese children, often those of school age. METHODS Two hundred fifty-eight children were diagnosed with M. pneumoniae-associated pneumonia based on chest radiography, real-time polymerase chain reaction (PCR), and antibody titers between January and December 2011. Mycoplasma pneumoniae cultures obtained from nasopharyngeal samples using appropriate broth were subjected to real-time PCR, by which decreases in M. pneumoniae in patients treated with minocycline (MIN), doxycycline (DOX), or tosufloxacin (TFX) were calculated. Mutations of the 23S ribosomal RNA gene that confer high resistance to macrolides in M. pneumoniae were identified by DNA sequencing. RESULTS Among 202 M. pneumoniae isolates from M. pneumoniae-associated pneumonia patients, 176 (87.1%) were MRMP. Macrolide-resistant M. pneumoniae infection was significantly related to school age (P < .01) and initial administration of macrolides (P < .01). Minocycline or DOX (n = 125) or TFX or levofloxacin (n = 15) was used for definitive treatment of MRMP patients. Minocycline or DOX was significantly more effective than TFX (P ≤ .05) in achieving defervescence within 24 hours and in decreasing numbers of M. pneumoniae DNA copies 3 days after initiation. CONCLUSIONS Macrolides are inappropriate as first-choice agents against MRMP in terms of shortening the clinical course and decreasing M. pneumoniae. Control and prevention of MRMP outbreaks in children require early decreases in M. pneumoniae as well as improvement of clinical findings.

Comprehensive detection of causative pathogens using real-time PCR to diagnose pediatric community-acquired pneumonia

We have developed a real-time reverse transcription-PCR (RT-PCR) method to detect 13 respiratory viruses: influenza virus A and B; respiratory syncytial virus (RSV) subgroup A and B; parainfluenza virus (PIV) 1, 2, and 3; adenovirus; rhinovirus (RV); enterovirus; coronavirus (OC43); human metapneumovirus (hMPV); and human bocavirus (HBoV). The new method for detection of these viruses was applied simultaneously with real-time PCR for the detection of six bacterial pathogens in clinical samples from 1700 pediatric patients with community-acquired pneumonia (CAP). Of all the patients, 32.5% were suspected to have single bacterial infections; 1.9%, multiple bacterial infections; 15.2%, coinfections of bacteria and viruses; 25.8%, single viral infections; and 2.1%, multiple viral infections. In the remaining 22.6%, the etiology was unknown. The breakdown of suspected causative pathogens was as follows: 24.4% were Streptococcus pneumoniae, 14.8% were Mycoplasma pneumoniae, 11.3% were Haemophilus influenzae, and 1.4% were Chlamydophila pneumoniae. The breakdown of viruses was as follows: 14.5% were RV, 9.4% were RSV, 7.4% were hMPV, 7.2% were PIV, and 2.9% were HBoV. The new method will contribute to advances in the accuracy of diagnosis and should also result in the appropriate use of antimicrobials.

Emergence of Macrolide-Resistant Mycoplasma pneumoniae with a 23S rRNA Gene Mutation

ABSTRACT A total of 195 Mycoplasma pneumoniae strains were isolated from 2,462 clinical specimens collected between April 2002 and March 2004 from pediatric outpatients with respiratory tract infections. Susceptibilities to six macrolide antibiotics (ML), telithromycin, minocycline, levofloxacin, and sitafloxacin were determined by the microdilution method using PPLO broth. A total of 183 M. pneumoniae isolates were susceptible to all agents and had excellent MIC90s in the following order: 0.00195 μg/ml for azithromycin and telithromycin, 0.0078 μg/ml for clarithromycin, 0.0156 μg/ml for erythromycin, 0.0625 μg/ml for sitafloxacin, 0.5 μg/ml for minocycline, and 1 μg/ml for levofloxacin. Notably, 12 ML-resistant M. pneumoniae strains were isolated from patients with pneumonia (10 strains) or acute bronchitis (2 strains). These strains showed resistance to ML with MICs of ≥1 μg/ml, except to rokitamycin. Transition mutations of A2063G or A2064G, which correspond to A2058 and A2059 in Escherichia coli, in domain V on the 23S rRNA gene in 11 ML-resistant strains were identified. By pulsed-field gel electrophoresis typing, these strains were classified into groups I and Vb, as described previously (A. Cousin-Allery, A. Charron, B. D. Barbeyrac, G. Fremy, J. S. Jensen, H. Renaudin, and C. Bebear, Epidemiol. Infect. 124:103-111, 2000). These findings suggest that excessive usage of MLs acts as a trigger to select mutations on the corresponding 23S rRNA gene with the resultant occurrence of ML-resistant M. pneumoniae. Monitoring ML susceptibilities for M. pneumoniae is necessary in the future.

Serotype and antibiotic resistance of isolates from patients with invasive pneumococcal disease in Japan

Invasive pneumococcal disease (IPD) is of concern in Japan, where the heptavalent pneumococcal conjugate vaccine (PCV7) is unavailable. We determined serotypes, genotypes indicating beta-lactam resistance, and antibiotic susceptibilities of 496 isolates from normally sterile sites in patients (193 children, 303 adults) from 186 institutions between August 2006 and July 2007. Disease presentations included sepsis (46.2%), pneumonia (31.5%), and meningitis (17.5%). Mortality was 1.4% in children and 22.1% in adults, many of whom had underlying diseases. In children, serotype 6B (22.5%) was followed by 19F (14.1%), and 14 (13.1%); potential coverages of PCV7 and PCV13 were 75.4% and 93.7%, respectively. In adults, serotype 12F (14.3%) was followed by 3 (11.3%), and 6B (10.3%); 23-valent polysaccharide vaccine (PPV23) coverage was 85.4%. Most serotype 12F strains were gPISP, with pbp2b gene alteration; carbapenem had an excellent MIC90. PCV7 is recommended for children and PPV23 for adults to increase prevention against IPD.

A comparative clinical study of macrolide-sensitive and macrolide-resistant Mycoplasma pneumoniae infections in pediatric patients.

In recent years, the increased prevalence of macrolide-resistant Mycoplasma pneumoniae (MR-M. pneumoniae) has become a significant issue in Japan. We isolated 94 strains of M. pneumoniae, and determined the minimum inhibitory concentrations (MICs) of macrolides and other antimicrobial agents for these strains. We also performed a comparative clinical evaluation of macrolide efficacy for cases of MR-M. pneumoniae infections and cases of macrolide-sensitive Mycoplasma pneumoniae infections (MS-M. pneumoniae). Of the 94 isolates of M. pneumoniae, 64 (68.1%) were classified as MS-M. pneumoniae and 30 (31.9%) as MR-M. pneumoniae strains. The clinical study included an assessment of 47 pediatric cases of MS-M. pneumoniae and 22 pediatric cases of MR-M. pneumoniae. The patient demographics, such as sex, age, the period from the onset of the infection to the first examination, laboratory findings, diagnosis, and the severity of symptoms, showed no significant difference between the two study groups. However, the efficacy of macrolide treatment was 91.5% for MS-M. pneumoniae and 22.7% for MR-M. pneumoniae, a statistically significant difference (P < 0.01). Although M. pneumoniae infection is generally considered a treatable condition, the increasing prevalence of macrolide-resistant strains of M. pneumoniae has become a significant clinical issue in pediatric patients, and it is therefore necessary to give careful consideration to the appropriate antimicrobial therapy for MR-M. pneumoniae infection.

Rapid detection of eight causative pathogens for the diagnosis of bacterial meningitis by real-time PCR

We aimed to detect causative pathogens in cerebrospinal fluid (CSF) collected from patients diagnosed with bacterial meningitis by real-time polymerase chain reaction (PCR). In addition to Streptococcus pneumoniae, Haemophilus influenzae, and Mycoplasma pneumoniae described previously, five other pathogens, Neisseria meningitidis, Escherichia coli, Streptococcus agalactiae, Staphylococcus aureus, and Listeria monocytogenes, were targeted, based on a large-scale surveillance in Japan. Results in CSF from neonates and children (n = 150), and from adults (n = 18) analyzed by real-time PCR with molecular beacon probes were compared with those of conventional culturing. The total time from DNA extraction from CSF to PCR analysis was 1.5 h. The limit of detection for these pathogens ranged from 5 copies to 28 copies per tube. Nonspecific positive reactions were not recognized for 37 microorganisms in clinical isolates as a negative control. The pathogens were detected in 72.0% of the samples by real-time PCR, but in only 48.2% by culture, although the microorganisms were completely concordant. With the real-time PCR, the detection rate of H. influenzae from CSF was high, at 45.2%, followed by S. pneumoniae (21.4%), S. agalactiae (2.4%), E. coli (1.8%), L. monocytogenes (0.6%), and M. pneumoniae (0.6%). The detection rate with PCR was significantly better than that with cultures in patients with antibiotic administration (χ2 = 18.3182; P = 0.0000). In conclusion, detection with real-time PCR is useful for rapidly identifying the causative pathogens of meningitis and for examining the clinical course of chemotherapy.