Mutations in domain V of Mycoplasma pneumoniae 23S rRNA are not associated with clinical characteristics of M. pneumoniae pneumonia in children: a case control study

Mycoplasma pneumoniae (MP) is a common agent of community-acquired pneumonia in children and young adults that can lead to refractory or persistent Mycoplasma pneumoniae pneumonia (MPP). Macrolide-resistant MP harbors point mutations in domain V of 23S ribosomal Ribonucleic Acid (rRNA) with substitutions detected at positions 2063, 2064, 2067 and 2617. This study’s purpose is to investigate the prevalence and clinical characteristics of mutations in domain V of MP 23S rRNA. We sequenced the 23S rRNA domain V of MP strains collected from children with MPP. Clinical and laboratory data were also obtained, including gender, age, duration of fever, duration of fever after the start of macrolide therapy, MP-Deoxyribonucleic Acid (DNA) load at enrollment, leukocyte count, neutrophil count, and lymphocyte count, immunomodulators treatment and pulmonary complications. Of 276 strains, 255 harbored to no significant differences in This study provides the infecting MP strain in 92.39% of our patients harbor genetic mutations in the 23S rRNA domain V. The A2063G mutation has been found in all mutated strains, which those new mutations need further study. Moreover, all results indicate that MPP infected by these mutations in 23S rRNA domain V were not specificity in clinical features, laboratory results, pulmonary complications, development of refractory pneumonia. Instead, we found that MP DNA load and host immune response may drive the development of refractory pneumonia.


Background
The prevalence of infection with Mycoplasma pneumoniae (MP) is widely underestimated, as most patients infected with MP are seldom symptomatic and rarely seek medical attention. MP is considered one common agent of community-acquired pneumonia in children and young adults. MP epidemics tend to cycle every 3 to 7 years [1], and infection may lead to refractory or life-threatening pneumonia with pulmonary and extrapulmonary complications [2][3][4].
As MP has no cell wall, it is intrinsically resistant to beta-lactams, glycopeptides, and fosfomycin antibiotics that target the cell wall. Therefore, macrolides, tetracyclines, and fluoroquinolones are used instead. Macrolides are considered as first-line agents in children to avoid potential age-related side effects of other therapies (tetracyclines have possible adverse effects on enamel hypoplasia and bone, and fluoroquinolones may influence the growth of bone and articular cartilage) [5,6]. However, cases of refractory Mycoplasma pneumoniae pneumonia (MPP) have also steadily increased in recent years [2,3].  [6][7][8][9]. In particular, point mutations at nucleotide positions A2063, A2064, A2067 and C2617 in domain V, especially A to G transition at position 2063 (A2063G) and A to G transition at position 2064 (A2064G), confer strong resistance [10][11][12], whereas mutations at positions A2067 and C2617 confer lower levels of resistance [2,6,12].
Notably, since 2000, the prevalence of macrolide-resistant MPP has rapidly increased, especially in Asian countries [6]. Worryingly one study suggests that macrolide resistance was found in more than 90% of Chinese MP isolates, which all harbored gene mutations [13]. Our previous research found that the morbidity of macrolide-resistant MP is even up to 92.45% [14]. The excessive use and misuse of macrolides may contribute to these mutations [15]. Refractory pneumonia was also increased, which showing no clinical or radiological response to macrolides, and could be easier to become severe or fatal

3.Gene Amplification
23S rRNA domain V was amplified by nested PCR (Table 1). Amplification products, with expected size about 690 bp, were resolved on 1% agarose, visualized by ribonucleic acid staining, and imaged using a gel-imaging and analysis system. Amplification products were then shipped at 4 °C within 2 days for sequencing at Yingweijie, Shanghai, China.
Finally, DNA sequences were compared using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to strain M129 (ATCC 29342).  females) infected with non-mutated MP strains. There were no significant differences in age and sex distribution, as well as in pulmonary complications, duration of fever, duration of fever after the start of macrolide therapy, MP-DNA load at enrollment, hospitalization days and immunomodulators treatment. However, leukocyte and neutrophil count were significantly higher in patients infected with mutated strains, although lymphocyte count was comparable between groups (Table 3).

4.Clinical Characteristics of Refractory and Non-refractory MPP
On the basis of the clinical and radiologic findings, 64 patients (29males, 35 females) were deemed to have refractory MPP, while 212 (108 males, 104 females) were considered to have non-refractory MPP. There was no significant difference in gender between groups.
Notably, the mutation was found with comparable frequency between non-refractory (93.87%) and refractory cases (87.5%), P = 0.092. Moreover, MP-DNA load at enrollment and age were significantly higher in patients with refractory MPP (Table 4). However, a steadily increasing number of recent cases progress to refractory, severe, lifethreatening MP pneumonia [1,2]. Extensive use and misuse of macrolides may cause the rapid emergence of macrolide resistance [15]. Macrolides inhibit protein synthesis by binding to specific nucleotides of the 23S RNA in the 50S MP ribosomal subunit. Mutations at domain V of 23S RNA reduce the affinity of the macrolides for the ribosome, which develop macrolide resistance [6][7][8][9]. Since 2000, some studies have confirmed that this microbiological problem is increasing throughout the world, although the highest prevalence has been observed in East Asia. The published rate was reported to be 87.2% in Korea, 81.6% in Japan, and up to 90% in China [6,13]. Consistent with previous studies, we found that 92.39% clinical strains from children with MPP harbored A2063G mutation.
Notably, we didn't detect mutations in the other spots at positions 2064, 2067, or 2617.
However, several novel mutations were found, including a G to C transition at position 2601, a T insertion between positions 2589-2590 and 2612-2613, and a G insertion between positions 2586-2587. Further studies are required to test whether these new mutations contribute to macrolide resistance.
Previous studies focus on the minimum inhibitory concentrations of macrolides in vitro, and explore point mutations of MP clinical strains [10][11][12]. However, the clinical relevance of these mutations had not been clearly characterized and large sample studies were fewer. In this study, patients with MPP infected by mutant strains in the clinical, laboratory and radiologic characteristics were similar with patients without mutations.
These results hint the clinical symptoms, laboratory and radiologic characteristics of MPP are generally similar between mutations and without mutations. Previous studies on the comparisons of clinical manifestations between the groups also reported no significant differences [14,16,17] As we all know, refractory MPP is characterized by long duration of fever, severe pulmonary inflammatory response. In this study, the prevalence of mutations in non-refractory and refractory MPP were 93.87% and 87.5%, which suggested that the infection of mutant strains does not increase the refractoriness of MPP in children. In addition, some children infected mutant strain also were cured only treated by macrolide treatment. These reasons may be the anti-inflammatory of macrolides works synergistically, and use of immunomodulators improve prognosis regardless of macrolide resistance [18,19]. As previous research determined that the pathogenesis of MP are consist of direct damage mechanisms, immune damage and inflammatory damage [20] Thus, while there was some value in investigating the clinical significance of genetic mutations in MP, it is probably necessary to consider other risk factors that may trigger refractory, severe, or life-threatening pneumonia, such as a more robust host immune response with inflammatory cytokines, interleukins(IL), alexin, CD 4+ T cell and so on [21][22][23]. In refractory MPP, immunomodulators such as systemic corticosteroids or intravenous gamma immunoglobulin are considered to be an effective treatment option by reducing host inflammatory response [18,19]. Based on previous research, MP infection enhances mucin production and neutrophil recruitment, excretes inflammatory factors [20]. Mucus cell hypersecretion, especially goblet cell hyperplasia, has been shown in airways of MP infected mice [24]. Additionally, in children infected with MP, the expression levels of tumor necrosis factor-α (TNF-α), IL-1β, IL-6, IL-10, C1q, C3,C4 in serum increase to varying degrees [21,22]. Those   Declarations Ethics approval and consent to participate: The study protocol was approved by the ethics committee of Children's Hospital Affiliated to Nanjing Medical University.
Participant consent was written by the legal representatives of patients and informed consent forms were achieved.

Consent for publication:
The study has obtained consent to publish from children's parent or legal guardian.
Availablility of data and materials: All data generated or analyzed during this study are included in this published article and available form the corresponding author on reasonable request.