Molecular detection and evaluation of resistance of Mycoplasma Pneumoniae to macrolide (Erythromycin) due to mutation in the 23S rRNA gene among Iranian Patients with respiratory infections

Objective: Mycoplasma pneumoniae is a common cause of community-acquired pneumonia. The global increased resistance of M. pneumoniae strains to macrolide (ML) has become a worrisome health problem. The widespread use of these drugs has led to increased rate of reported ML-resistant M. pneumoniae (MRMP) throughout the world. Therefore, this study was aimed to evaluate the resistance of M. pneumoniae against erythromycin due to mutations in the 23S rRNA gene of patients with respiratory infections in Iran. Results: According to the ndings of the present study, employing specic primers showed that 17 cases (17%) were positive for mycoplasma genus and 6 cases (6%) positive for M. pneumoniae species. Also, analysis of the sequence of 23S rRNA gene, revealed that one of the samples had mutations at positions A2431G and G2491A. Measuring the minimum inhibitory concentration (MIC), revealed that all samples positive for M. pneumoniae with 23S rRNA gene were sensitive to erythromycin, and no ML resistance was reported.


Introduction
Mycoplasma pneumoniae (M. pneumoniae) is one of the common causes of upper and lower respiratory tract infections, and is a major cause of community-acquired pneumonia (CAP) and atypical pneumonia which is responsible for mortality and morbidity among children and adults (1)(2)(3)(4). Since M. pneumoniae lacks cell wall, the infection is primarily treated with macrolide antibiotics (MLs), tetracycline, and uoroquinolones (5)(6)(7). Macrolides like erythromycin, azithromycin and clarithromycin are commonly considered as the rst-line treatment of M. pneumoniae infections, because alternative antibiotics, such as tetracycline and uoroquinolones have side effects (8,9). In addition to antimicrobial activity against M. pneumoniae, MLs have anti-in ammatory effects against M. pneumoniae infections, and are pharmacologically inhibitors of cytokines, particularly IL-8 (10,11). Because of limited therapeutic options, the global increase in resistance of M. pneumoniae to MLs is a major health concern worldwide. Moreover, the widespread application of these drugs has led to elevated rate of ML-resistant M. pneumoniae (MRMP) throughout the world (7,12,13). MRMP infection has been shown to cause prolonged treatment, persistent cough, and an elevated time of fever reduction compared with those infections sensitive to treatment (14,15). Currently, the MRMP has been reported more than 90% in Asia and 26% in the Europe (7,8,16). Undoubtedly, the emergence of MRMP is a serious health threat due to the widespread use of macrolide antibiotics in the treatment of M. pneumoniae infections, especially in Asia in recent years. Thus, it has attracted the attention of many scientists (13). The mechanism of resistance of M. pneumoniae to ML antibiotics has been as a point mutation (nucleotide displacement) of domain V in certain positions in 23S rRNA gene of MRMP (17,18). In recent years, a number of previous studies, exposed strains of M. pneumoniae strains to macrolides, and proposed that all strains with resistance mutations displayed high levels of resistance to ML antibiotics in broth dilution experiments. After Speci c Primer-Polymerase Chain Reaction (SSP-PCR) and analysis of the sequences of domain V of the 23Sr RNA gene in some positions, such as A2063G, A2064G, and A2064C, a series of A-to-G or A-to-G nucleotide displacements were identi ed. Therefore, resistance to MLs in M. pneumoniae can occur due to point mutations in the 23S rRNA gene (10,17,19). Therefore, this study was aimed to evaluate the resistance of M. pneumoniae against erythromycin due to mutations in the 23S rRNA gene of patients with respiratory infections in Iran.

Methods
In this study, 100 samples of throat swab were collected from patients with atypical pneumonia referring to Mostafa Khomeini and KhatamolAnbiah hospitals in Tehran province in 2018. For sampling, individuals diagnosed with clinical symptoms of respiratory infections by a lung specialist, such as weakness, lethargy, fatigue, persistent headache and dry cough, shortness of breath, sputum production, muscle pain, and no antibiotic use during the past month were targeted for recruitment. Samples were stored in transport pleuropneumonia-like organisms (PPLO) broth medium were obtained from Merck (Germany) to transfer them to the laboratory. In the laboratory, 1 ml of the transport medium was transferred following passing through the 0.45 µm lter in the main Glucose PPLO broth medium, containing 20% horse serum, 0.3% yeast extract, .3% meat extract, 0.33 mI penicillin, 0.5 ml polymyxin B, and 0.5 ml amphotericin B in 5-10% CO2, and were then incubated at 35 °C for 3 weeks. In this study, the standard strain was M. pneumoniae (ATCC: 29342) which was developed from the Molecular Biology Research Center of Baqiyatallah University of Medical Sciences. The kit was obtained from Roche Co. (Germany) was used to extract DNA from the Glucose PPLO broth medium in which clinical specimens were cultured and incubated at 37 °C for 3 weeks in 5-10% CO2. After extraction of DNA samples, speci c primers were used to identify Mycoplasma genus (16S rRNA gene) and M. pneumoniae strain (P1 gene) as shown in Table 1.

Results
The present study recruited 100 patients with respiratory infections, of whom 48 (48%) were males and 52 (52%) were females. The average age of the participants was 53.62 years, ranging from 17 years to 85 years. According to the ndings of our study, 14 subjects (14%) had M. pneumoniae colonies grown on PPLO agar. Figure 1 (Table 2). Therefore, mutations in domain V of 23S rRNA gene at A2431G and G2491A were not associated with resistance of M. pneumoniae against erythromycin.  (10,17). The prevalence of MRMP follows a diverse pattern in different countries, which is attributed to the excessive use of macrolides and age of the patients (12). ML resistance is associated with a point mutation in domain V of 23S rRNA gene and ribosomal proteins L22 and L14. Mutation affecting 23S rRNA gene at A2063G position is the most common mutation, followed by A2064G position, which is mostly responsible for signi cant ML resistance in M. pneumoniae (10,23). Results of the analysis of the sequence of 23S rRNA gene showed that point mutations at A2431G and G2491A positions were reported only in one specimen.
In addition, the broth microdilution MIC method revealed that all samples positive for M. pneumoniae, were sensitive to erythromycin, and no ML resistance was reported in specimens. The rate of MRMP among study participants was 0.6%, 0%. The prevalence of MRMP was reported as 2%, 50/1 in Switzerland, 1.5%, 87.1%, 202/176 in Japan, 97%, 33/32 in China, 1.2%, 167/2 in Germany, and 9.8%, 51/5 in France (7). Brown and colleagues conducted a study in England (2015), and reported mutations associated with ML resistance in samples positive for M. pneumoniae. They found that 43 samples of the total 60 specimens positive for M. pneumoniae had mutations in domain V of 23S rRNA gene, with ML resistance in four specimens (9.3%), which was not consistent with the results of our study (23).
Haruki and colleagues investigated hospitalized children in Japan (2014) and detected 33 cases of M. pneumoniae by PCR, of which 31 isolates were resistant to macrolide, and all isolates displayed point mutation at 2063 and 2064 in domain V of 23S rRNA, which was not in accordance with the present study. This difference in the results of these two studies may be due to the older age of the patients in our study (24). Liu and colleagues (2014) examined 580 samples of throat swab suspected for M. pneumoniae, and reported that 70 cases were resistant to erythromycin. They also revealed point mutation at 2063 and 2064 in domain V of 23SrRNA in all specimens positive for M. pneumoniae, which was not in accordance with our study (21). Zhou et al. (2015) also examined 650 samples of throat swab, and used PCR method. All sequences of the domain V of the 23SrRNA were investigated. They showed that 100 percent (71/71) of M. pneumoniae isolated from patients with (CAP) were resistant to erythromycin, clarithromycin, and azithromycin. In addition, all ML-resistant strains of M. pneumoniae had point mutation at A2063G in the domain V of 23S rRNA (25), which was not consistent with the results of our study, and it can be attributed to the larger sample size of this study. This was one of the rst studys conducted in Iran. Results of the analysis of the sequence of the 23S rRNA gene, showed that only one strain displayed point mutations at A2431G and G2491A. The broth microdilution MIC showed that all specimens positive for M. pneumoniae were resistant to erythromycin, and no ML resistance was observed. Besides, all point mutations affecting domain V of 23S rRNA at A2431G and G2491A were not associated with ML resistance to erythromycin. In conclusion, in order to control the emergence of ML resistance among M. pneumoniae strains, the use of these antibiotics should be restricted in Iran and reduce the widespread use. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.