Detection of Mycoplasma pneumoniae Nucleic Acid and Drug Resistance Gene

Summary

This protocol provides a quick and reliable way to detect Mycoplasma pneumoniae infections and identify antibiotic resistance. It helps practitioners choose the right treatments faster, improving care for patients with pneumonia and supporting better use of antibiotics

Abstract

Mycoplasma pneumoniae (Mp) represents one of the most prevalent pathogens responsible for community-acquired pneumonia (CAP) in pediatric populations. The detection of Mycoplasma pneumoniae resistance genes provides robust technical support and a theoretical foundation for the precise clinical diagnosis and treatment of MP infections. Accurate diagnosis and evaluation of drug resistance in Mp infection are essential for clinical treatment. Common detection methods for Mycoplasma pneumoniae include culture, antibody detection, and molecular biology-based assays. Mp culture is the gold standard for diagnosis but requires a specific medium, is time-consuming, and has low sensitivity. Antibody-based detection of Mp is widely used; however, false negatives may occur in the early stage of infection. Molecular biology-based detection provides rapid turnaround, low risk of contamination, high sensitivity, high specificity, and is not limited by the timing of sample collection or immune status. It has therefore been recognized as a new gold standard for diagnosing Mycoplasma pneumoniae infection. Macrolide agents are the first-choice treatment for Mp infection in children. However, with the extensive use of macrolides in respiratory tract infections in children, the incidence of drug-resistant Mycoplasma pneumoniae infection has been increasing. Patients infected with resistant strains experience significantly longer fever duration, extended hospitalization, and prolonged fever after administration compared with those infected with sensitive strains. Mutation sites identified to date include 2063, 2064, 2067, and 2617. In China, point mutations have been documented only at positions 2063 and 2064, with an A-to-G substitution at position 2063 being the most common. For these sites, polymerase chain reaction (PCR) combined with fluorescent probe technology has been employed to detect the nucleic acids and drug resistance mutations of Mycoplasma pneumoniae in human sputum samples. Detection of Mycoplasma pneumoniae resistance genes offers crucial technical support and a theoretical basis for accurate diagnosis and effective treatment of Mp infection.

Introduction

Mycoplasma pneumoniae (Mp) is the smallest pathogenic microorganism capable of independent survival, situated at an intermediary position between bacteria and viruses. It is chiefly disseminated by respiratory droplets or aerosols and constitutes the second most common pathogen associated with community-acquired pneumonia (CAP) in children¹, responsible for 20% to 40% of CAP instances². Mp shows inherent resistance to β-lactam antibiotics, such as penicillin and cephalosporins, due to its absence of a cell wall, which these antibiotics target by obstructing cell wall synthesis. The principal therapeutic modalities for Mp infections comprise agents that obstruct protein synthesis, including macrolides and tetracyclines, alongside pharmaceuticals that impede DNA replication, such as tetracyclines and fluoroquinolones^3,4,5. Therefore, the prompt laboratory identification of Mp is essential for informing the judicious application of antibiotics. Contemporary laboratory detection methods for Mp encompass isolation and culture, antigen detection, antibody detection, and genetic diagnostic techniques.

As a traditional detection method, isolation and culture continue to be the definitive standard for diagnosing Mycoplasma pneumoniae infection. Kashyap et al. observed that culturing Mp from throat swabs or sputum specimens is a dependable diagnostic technique⁶. This approach encounters considerable obstacles, such as Mp's rigorous cultural prerequisites, extended culture periods, and typically low positive detection rates, which restrict its routine clinical use. Mp antigen detection depends on Mp-specific antibodies. Miyashita et al. used monoclonal antibodies from various Mp strains as coating agents in a double-antibody sandwich ELISA and evaluated their efficacy using PCR⁷. The findings revealed no substantial difference between the two methodologies. However, the necessity for highly specific and high-titer monoclonal antibodies constrains the extensive implementation of this method.

Following Mp infection, the immune system generates specific IgM and IgG antibodies. IgM antibodies generally become detectable within 1 week following infection and reach their peak at 3 to 4 weeks, rendering them valuable diagnostic indicators for early infection. However, false-negative results may arise in the initial stages owing to diminished antibody levels⁸. Conversely, IgG antibodies manifest later and typically signify a previous infection^9,10. Serological detection of Mp is classified into non-specific and specific antibody assays. While non-specific antibody tests are simple to administer, their specificity is limited, as infections from other pathogens, including rickettsia and adenovirus, can also increase cold agglutinin levels, resulting in false-positive outcomes. Specific antibody assays encompass the complement fixation test, particle agglutination test, indirect hemagglutination test, indirect immunofluorescence assay, and enzyme-linked immunosorbent assay (ELISA). ELISA is extensively utilized in China owing to its established technology and commercial accessibility, providing high sensitivity and operational simplicity, thereby serving as a practical and dependable method for diagnosing Mp infections in routine clinical practice¹¹.

Real-time fluorescent quantitative PCR is distinguished by its swift processing time, elevated specificity, robust sensitivity, and absence of cross-reactivity. This method identifies Mp DNA in samples of sputum, throat swab, or bronchial lavage fluid, and requires 1-100 ng of DNA per reaction. Detection was performed using a real-time PCR instrument. The DNA template experiences denaturation at higher temperatures, followed by primer annealing and fluorescent probe hybridization. Gene amplification technology enables the replication of nucleic acid fragments at extremely low concentrations to millions of copies in a brief timeframe, attaining a detection sensitivity of ≥10 CFU/mL¹². In comparison to conventional PCR, real-time fluorescent quantitative PCR reduces contamination risks while enabling high-throughput and automated analysis. Therefore, it has emerged as the favored technique for direct Mp detection in clinical practice¹³. Due to its heightened sensitivity, stringent measures are necessary to avert contamination, which could result in false-positive or false-negative outcomes.

In clinical treatment, macrolides constitute the primary therapeutic intervention for Mycoplasma pneumoniae pneumonia (MPP). The excessive use and improper application of antibiotics have imposed selective pressure, leading to the emergence of macrolide-resistant Mp. This resistance is a significant contributor to refractory MPP. Macrolide resistance in Mycoplasma pneumoniae is notably elevated in East Asia, particularly in China and Japan, whereas Europe and the United States exhibit relatively lower resistance rates. Pediatric patients generally demonstrate elevated resistance rates compared to adults, necessitating heightened scrutiny¹⁴. Since 2000, the incidence of Mycoplasma pneumoniae infections (MRMPI) has escalated significantly on a global scale, especially in East Asia. In China, MRMPI strains constitute 69% to 95% of cases¹⁵, substantially contributing to severe disease, treatment failure, complications, and long-term sequelae. Research indicates that mutations in the 23S rRNA gene inhibit macrolide binding to target sites, consequently diminishing drug efficacy. Frequent mutation loci encompass positions 2063, 2064, 2067, and 2617. In China, mutations have been identified exclusively at positions 2063 and 2064^16,17. A 2016 study indicated that macrolide resistance rates in Beijing, Harbin, Shanghai, and Xinjiang varied between 66.7% and 86.7%, while Nanjing exhibited a lower rate of 20%¹⁸. Macrolide-resistant strains have emerged in various regions, with resistance rates reported as follows:8.3%-9.8% in France, 3.0%-3.6% in Germany, 8.2% in the United States, 0.9%-2.9% in Denmark, 12.1% in Canada, 20% in Italy, 22% in Israel, 8% in Spain, and 9.3% in the United Kingdom^{19,20,21,22,23,24,25,26,27}. This trend highlights the worldwide proliferation of macrolide resistance. In 2008, Hindley et al. reported 46 instances of macrolide-resistant Mycoplasma pneumoniae, with 40 instances demonstrating the A2063G mutation, one instance presenting the A2063C mutation, and five instances exhibiting the A2064G mutation²⁸. Comparable resistance mutations have been identified in Japan and the United States, with the primary mutations occurring at positions 2063 and 2064²⁹. Research suggests a relationship between mutation sites and drug resistance phenotypes: A2063G mutation imparts resistance to 14-membered macrolides (ML), A2064G mutation imparts resistance to both 14- and 16-membered ML, C2617G imparts resistance to 14- and 15-membered ML, and A2067G mutation imparts resistance to 16-membered ML³⁰. Monitoring Mp resistance is crucial for the early identification of drug-resistant infections and for informing clinical treatment choices. Studies have shown that in vitro antimicrobial susceptibility testing can forecast clinical drug effectiveness³¹. In 2011, the American Clinical and Laboratory Standards Institute promulgated guidelines advocating the microdilution method for ascertaining the minimum inhibitory concentration (MIC) to evaluate macrolide resistance³². However, the determination of MIC may require several weeks, thereby constraining its clinical utility in directing real-time antibiotic therapy.

Sanger sequencing is a direct technique for identifying mutations in the 23S rRNA gene of Mp. This method uses fluorescently labeled nucleotides and electrophoretic analysis to ascertain DNA sequences, facilitating the identification of Mp genetic variations. Sanger sequencing, while capable of simultaneously identifying multiple mutations and quantifying the ratios of resistant and susceptible strains, is labor-intensive and time-consuming, thereby constraining its clinical utility^33,34. In contrast, pyrosequencing provides a more accurate assessment of DNA sequence variations by identifying pyrophosphate release during nucleotide incorporation. This method can identify MRMPI strains and quantify the ratios of resistant and susceptible strains in clinical samples with greater accuracy than Sanger sequencing. In addition, pyrosequencing enables longitudinal monitoring of individual patients, allowing for real-time evaluation of developing resistance and informing treatment modifications³³. However, its clinical application is constrained by the necessity for specialized apparatus, proficient personnel, and prolonged processing durations.

Based on restriction fragment length polymorphism (RFLP) analysis of the P1 gene, Mp is categorized into two types: type I (further divided into five subtypes: 1a-1e) and type II (further divided into three subtypes: 2a-2c). This test can detect 8 gene subtypes of Mycoplasma pneumoniae³⁵. This method facilitates the qualitative identification of Mycoplasma pneumoniae and macrolide resistance mutations (23S rRNA 2063 (A:G) and 2064 (A:G) mutation sites) in pediatric sputum specimens, providing a swift and straightforward supplementary diagnostic instrument for Mycoplasma infections and antibiotic resistance surveillance.

Protocol

All study protocols were reviewed and approved by the Medical Ethics Committee of Heyuan People's Hospital (Approval No. YXYJLL-LW2025001). All patients provided written informed consent prior to participation.

1. Specimen collection

NOTE: Exercise caution in maintaining aseptic technique during sample collection.

1. Collect specimens of sputum of the patient or suspected patient. Obtain samples within 3 days of the patient's disease onset.
2. Instruct the patient to rinse his mouth with water, breathe deeply, retain his breath for 5 s, and exhale gradually.
3. Ask the patient to exhale deeply once more, attempt to expel the sputum, deposit it into a designated plastic cup, and secure the lid. The sputum should be promptly delivered to the clinical laboratory at room temperature (RT). Store the samples at 2-8 °C in a refrigerator.

2. Preparation of amplification reagents

1. In the PCR laboratory's reagent preparation area, thaw the buffer, primer probe, and water from the kit at 4 °C after retrieving them from the -20 °C freezer. Remove the enzyme, gently mix it by shaking, and then centrifuge at 1,006 x g for 10 s.
2. Retrieve a sterile nuclease-free centrifuge tube, label it, and prepare the reaction system for each individual as outlined in Table 1. The total number of PCR reactions must equal the sum of the samples, one weak positive quality control, one quality control product W, and one negative extraction control.
3. Determine the quantity of each reagent, incorporate it into a suitably sized centrifuge tube, mix thoroughly, and centrifuge briefly at low speed. Dispense 20 µL of reagent into each PCR reaction tube and relocate it to the sample processing area.

3. Sample treatment

1. Treat 1 mL of sputum samples with 2 mL of sputum digestion buffer, vortex-mixed thoroughly, and incubate for 30 min until complete liquefaction is achieved.
2. Based on the sample quantity, add 5 µL of the internal standard sequentially to the 1^st and 7^th columns of the nucleic acid extraction plate, followed by the addition of 200 µL of liquefied samples, normal saline, and MM weak positive quality control in succession.
3. Retrieve a nucleic acid extraction plate using an automated nucleic acid extraction instrument. Add the extracted nucleic acids after carrying out downstream assays in columns 6 and 12 of the extraction plate.
4. Sample Addition: In the PCR laboratory's sample preparation area, add extracted nucleic acid into the 6^th and 12^th columns of the extraction plate. Subsequently, transfer 5.0 µL of the prepared sample nucleic acid to the PCR reaction tube, which contains the PCR reaction mixture (including sample, strong and weak positive quality controls, quality control W, and extraction negative control).
5. Secure the PCR reaction tube liquid and perform immediate centrifugation to confirm that the liquid is properly sealed and devoid of bubbles.

4. Amplification

1. Insert the reaction tube into the fluorescence PCR detector and configure the cycle parameters as follows in Table 2.
2. Acquire fluorescence signals in FAM (2063 or 2064 A:G mutation), VIC (Mycoplasma pneumoniae), and CY5 (Internal standard), with data collection set at 64 °C.

5. Data analysis

NOTE: This study employed quantitative real-time PCR (qPCR), with representative results indicating A:G resistance-associated mutations at positions 2063 or 2064 of Mycoplasma pneumoniae. Patients testing positive should avoid macrolide antibiotics in clinical treatment.

1. After the reaction, the instrument autonomously analyzes and archives the results. Check that the target genes for negative control exhibit FAM fluorescence Ct ≥ 35.33 and VIC fluorescence Ct ≥ 35.01, while the target genes for quality control W demonstrate FAM fluorescence Ct ≥ 35.33 and VIC fluorescence Ct ≥ 35.01. The Ct values for FAM and VIC fluorescence in weakly positive quality control products ranged from 29.00 to 34.00.
2. Generally, the Ct value of the internal standard is ≤ 35.00. If a characteristic S amplification curve is observed but the internal standard is not amplified due to the elevated concentration of the target gene, report it directly as a positive sample, or re-evaluate the sample through a 10-fold gradient dilution.
3. Classify the sample exhibiting UNDECT or an absence of a typical S-type amplification curve as negative.
4. Deem samples negative when FAM fluorescence (2063 or 2064 A:G mutation) Ct ≥ 35.33, VIC fluorescence (Mycoplasma pneumoniae) Ct ≥ 35.01, or when there is no typical S-type amplification curve. Deem samples are positive when their Ct value meets Table 3 and exhibit a characteristic S-type amplification curve.
   NOTE: The outcomes of sample detection are contingent upon the quality of sample collection, processing, transportation, and preservation; any errors may result in false negative results. Failure to control cross-contamination during sample processing may result in false positive outcomes.

Results

Following liquefaction, nucleic acid extraction, and fluorescence PCR amplification of sputum samples, the Start value, End value, and Threshold value of the baseline were modified after the analysis of the amplification curve. The Start value was 3, and the End value was 20.

The representative results of this experiment were positive for Mp nucleic acid, and an A : G resistance mutation occurred at 2063 or 2064 sites, suggestin...

Discussion

PCR and its derivative techniques demonstrate superior sensitivity and specificity compared to serological methods in the identification, typing, and detection of drug resistance in Mycoplasma pneumoniae (MP). Moreover, PCR-based techniques provide ease of operation and reduced expenses, rendering them a viable alternative for routine clinical diagnostics³⁶. Mp is a notable pathogen associated with CAP and demonstrates a considerable prevalence³⁷. Choosing suitable...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Automated nucleic acid extraction instrument	Guangzhou Da 'an Gene Co., Ltd.	Smart 32
Filter Tips	Coring Life Science Co., Ltd.	29822001	0-10µL
Filter Tips	Coring Life Science Co., Ltd.	03023001	1-50µL
Filter Tips	Coring Life Science Co., Ltd.	05623001	5-300µL
Microtubes	Coring Life Science Co., Ltd.	14124934	1.5mL
Mycoplasma pneumoniae nucleic acid and drug resistance mutation site detection kit ( fluorescence PCR method )	Jiangsu Mole Biotechnology Co., Ltd.	20241111
Nuclear acids extracting reagent(magnetic beads method)	Shengxiang Biotechnology Co., Ltd.	G24004
PCR tubes	DN Biotech(Hong Kong)  Co., Ltd.	6121003	0.2mL
Real-time fluorescent PCR instrument	BIO-RAD	CFX96