Macrolide-resistant Mycoplasma pneumoniae in South Korea: a strong association with M. pneumoniae type 1

Abstract

Mycoplasma pneumoniae is a main pathogen causing community-acquired pneumonia in children and young adults. Since the emergence of macrolide-resistant M. pneumoniae in the early 2000s in Japan, it has been increasingly reported worldwide as a growing problem in treatment for children. With increasing macrolide-resistant M. pneumoniae and limited data regarding its characterization and molecular analysis, we investigated the dominant M. pneumoniae strains during the recent outbreak in South Korea, and evaluated if there was an association between a specific type and macrolide resistance. Between October 2014 and December 2016 in South Korea, 249 respiratory specimens obtained from patients with confirmed M. pneumoniae pneumonia were genotyped the P1 adhesin gene, and the mutations associated with resistance (A2063G and A2064G) were tested by sequencing the targeted domain V regions of the 23S ribosomal RNA gene. Results revealed that M. pneumoniae type 1 were predominant, which was strongly associated with macrolide-resistance during the whole study period. This is the first study assessing whether M. pneumoniae subtype is related to macrolide resistance during the outbreak of M. pneumoniae.

Mycoplasma pneumoniae is a main pathogen causing community-acquired pneumonia (CAP) in children and young adults [1, 2–5]. Epidemic outbreaks occur every 3–7 years, while M. pneumoniae infection is endemic worldwide [1, 2]. Macrolides are the first choice of treatment in M. pneumoniae infection in children [6, 7]. Since the emergence of macrolide-resistant M. pneumoniae in the early 2000s in Japan [2, 8], macrolide-resistant M. pneumoniae infection has been increasingly reported in several countries, with prevalence now ranging from 0% to 10% in Europe and the USA, and ~69%–95% in Asia [2–10]. Due to potential risks of fluoroquinolones or doxycycline treatment in children, an increase in macrolide-resistant M. pneumoniae infection is a growing problem [3]. With increasing macrolide-resistant M. pneumoniae and limited data regarding its characterization and molecular analysis, we investigated the dominant M. pneumoniae strains during the recent outbreak in South Korea. Further, we examined whether there were differences between each strain in the presentation of clinical features. We also evaluated if there was an association between a specific type and macrolide resistance.

Between October 2014 and December 2016 in Asan Medical Center, Seoul, Korea, 8375 respiratory samples were obtained from CAP patients, who were diagnosed based on clinical symptoms or radiologic findings. Of these, 622 samples were positive for M. pneumoniae using the AmpliSens Mycoplasma pneumoniae/Chlamydophila pneumoniae-FRT PCR kit (InterLabService Ltd., Moscow, Russia). Of these 622 M. pneumoniae-positive samples, 249 samples were available for further testing (Table 1). Typing of M. pneumoniae isolates was performed by targeting the P1 adhesin gene with primer pairs as previously documented [6]. To identify major mutations associated with macrolide resistance (A2063G and A2064G), we amplified the domain V regions of the 23S ribosomal RNA gene by methods described previously [5]. Demographic and clinical data for all study populations were collected from electronic medical records. This work was approved by the Institutional Review Board. Informed consent was waived by the Institutional Review Board of Asan Medical Center because this study was performed retrospectively and did not require any extra clinical specimens. Descriptive statistics were performed in terms of quantitative and qualitative data and absolute frequency. Comparisons were conducted using the χ ² test or Fisher's exact test for categorical variables and Student's t test or Mann–Whitney test for continuous variables, as appropriate. A P value < 0.05 was defined as statistically significant. SPSS version 18.0 for Windows (SPSS Inc., Chicago, IL, USA) was used for data analysis.

Table 1. Patients' demographics and clinical features, according to sequencing type of P1 adhesin gene

WBC, while blood cell counts; ANC, absolute neutrophil counts; CRP, C-reactive protein.

Note: Data are presented as number (%) or mean ± standard deviation unless otherwise specified.

Overall, 180 (72.3%) of the 249 M. pneumoniae-positive specimens harboured mutations in the 23S ribosomal RNA gene. Genotyping revealed that M. pneumoniae subtype 1 was more prevalent during the entire outbreak, as follows: October 2014–June 2015, 34 (97.1%) of 35; July 2015–March 2016, 132 (80.0%) of 165; April 2016–December 2016, 35 (71.4%) of 49 (Fig. 1).

Fig. 1. Distribution of P1 adhesion gene subtypes of 249 M. pneumoniae between October 2014 and December 2016.

Patients' demographics and clinical features according to sequencing type of P1 adhesin gene were summarised in Table 1. Two hundred and one (80.7%) were classified as type 1 and 48 (19.3%) as type 2. Patients infected with type 1 were younger (8.9 years vs. 17.2 years, respectively, P < 0.001) and more likely to have longer fever (temperature ≥38 °C) duration (6.7 ± 3.6 days vs. 5.3 ± 4.7 days, respectively, P < 0.027), compared with patients infected with type 2. The most common clinical symptoms were cough, fever and sputum in both groups (98.4%, 98.0% and 79.5%, respectively). Chest radiographs of all patients were available, and lobar consolidation patterns were most common without statistically significant differences between these two types (60.7% in type 1 vs. 70.8% in type 2, P < 0.133). Frequencies of each clinical symptoms and laboratory findings, except for platelet counts, were not different between type 1 and type 2 of M. pneumoniae. Further, 169 (80.7%) of the type 1 were macrolide-resistant M. pneumoniae. Of these, the A2063G mutation was identified in 167 (98.8%), and the A2064G mutation was identified in two (1.2%) patients. Conversely, only 11 (19.3%) of the type 2 were macrolide-resistant M. pneumoniae, of which all had the A2063G mutation. The dominant macrolide-resistant genotype was type 1. Given that hospitalised patients have higher disease severity compared to outpatients, we evaluated the proportion of hospitalised patients in each type. The rate of each was 66.2% in type 1 and 52.1% in type 2 (P = 0.069).

In this study, a strong association between macrolide resistance and M. pneumoniae type 1 was observed. Previous studies attempted to clarify associations between M. pneumoniae type and macrolide resistance, but most of them did not determine the association between type and macrolide-resistant M. pneumoniae [1, 7]. Only one study in China with 53 clinical isolates documented an association between M. pneumoniae strain types and erythromycin resistance [3]. The present study represents the second study with a large group of clinical isolates that demonstrated relatedness between strain type and macrolide resistance. From the clinical data in this study, clinical presentation, laboratory findings and radiologic findings were similar between the groups infected with type 1 and type 2. In addition, our data indicated that type 1 was detected in over 80% of sequenced strains during the epidemic with the co-circulation of type 1 and type 2. Our group reported the substantially increased prevalence of macrolide-resistance of M. pneumoniae in children ranging from 2.9% in 2003 to 62.9% in 2011 [10]. Together with our data of a 76.3% macrolide-resistant M. pneumoniae rate during 2014–2016, the prevalence of macrolide-resistant M. pneumoniae increased continuously over 10 years. Even though most strains isolated in this study were type 1 of the P1 adhesin gene, it is still unclear whether the macrolide-resistant M. pneumoniae isolates originated from the same clone. Therefore, further studies regarding the isolate spread are necessary.

In summary, the dominant clinical strain of M. pneumoniae during a recent outbreak was type 1, which was more likely to infect younger patients. Given the current rapidly increasing trend of macrolide-resistant M. pneumoniae incidence, the continuing epidemiological monitoring of macrolide resistance is necessary to recognise macrolide resistance strains early and complement effective care against these infections.