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 filter 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, specific primers were used to identify Mycoplasma genus (16S rRNA gene) and M. pneumoniae strain (P1 gene) as shown in Table 1.
Table-1: primers sequences used in this study
Gene | Primer Sequence | | Product Size (bp) Reference |
16S rRNA P1 23S rRNA | GPO1 MGSO MPPIF MPPIR Mp-F1 Mp-R1 | F primer: 5' -ACTCCTACGGGAGGCAGCAGT-3' R primer : 5' TGCACCATCTGTCACTCTGTTAACCTC-3' F primer: 5'- AAAGGAAGCTGACTCCGACA-3' R primer: 5'-TGGCCTTGCGCTACTAAGTT-3' Mp-F1: 5‘- TAACTATAACGGTCCTAAGG − 3' Mp-R1: 5‘- CGCTACAACTGGAGCATAAGA − 3' | 713b ( 20) 450 bp (20) 793 bp (21) |
The PCR reaction was performed at final concentration of 25 µl including an initial denaturation at 94 °C for 5 min, 35 cycles comprised of denaturation at 94 °C for 35 seconds, primer binding to the target DNA at 56 °C for 40 seconds, elongation at 72 °C for 45 seconds, as well as a final elongation at 72° C for 5 minutes, according to the protocol, for M. pneumoniae; and also another PCR reaction at final concentration of 25 µl, including an initial denaturation at 94 °C for 4 min, 33 cycles comprised of denaturation at 94 °C for 45 seconds, primer binding to the target DNA at 55 °C for 45 seconds, elongation at 72 °C for 50 seconds, as well as a final elongation at 72° C for 5 minutes, according to the protocol for the 23S rRNA gene. The PCR products (ATCC: 29342) were finally electrophoresed. Finally, the PCR products positive for 23S rRNA gene were sequenced to detect mutations of domain V in 23S rRNA gene of MRMP, and sequences of 23S rRNA gene were analyzed. In order to prepare samples positive for P1 gene of M. pneumonia, the MIC was carried out by micro-dilution of Glucose PPLO broth containing 25% horse serum, 0.3% yeast extract, and 0.3% meat extract in 96 wells of microplates, after preparation of antibiotic macrolide suspension (erythromycin). Then, the microplates prepared for microscopic specimens were incubated for 5–6 days at 37 °C. Next, the MIC for erythromycin was determined based on color changes of Glucose PPLO broth enriched with horse serum, yeast extract, and meat extract. Glucose PPLO broth is a phenol red (a PH indicator) medium and M. pneumoniae growth causes the color of the medium to change from original purple to yellow.