| Mycoplasma pneumoniae(MP)is one of the most common pathogens responsible for community-acquired pneumonia,accounting for approximately 10%～50% of cases.If left untreated,MP infection can recur and progress to refractory pneumonia,causing damage to multiple systems and affecting health.MP lacks a cell wall and is not sensitive to penicillin or cephalosporin antibiotics.Clinically,macrolides,tetracyclines,and quinolones are commonly used to treat MP.Macrolides are the first choice due to their high efficacy and low side effects.However,the widespread use and even abuse of macrolides have led to an increasing prevalence of resistance,with reported resistance rates ranging from 28% to 100% in various regions of China.There are currently no reports of quinolone resistance in clinical settings,and only one case of tetracycline resistance has been reported.Resistance can lead to prolonged fever and hospitalization time,increased incidence of complications,waste of medical resources,and economic losses.The main mechanism of MP resistance is a target site gene mutation,which reduces the affinity of drugs with the ribosome and causes resistance.Resistance can be evaluated by detecting the mutation status of resistant genes.Differences in MP genotypes can lead to different clinical symptoms.P1 type 1 and type 2 alternate every 10 years and occupy a dominant position alternately.The prevalence of genotypes may be related to MP outbreaks and resistance.However,there are no reports on the prevalence of MP resistance gene mutations,genotype prevalence,and their correlation with resistance in Yunnan province.In this study,the sequence differences between MP resistance sites and different genotypes were firstly determined by reviewing literature,designed specific primers,and established a nested PCR method for amplification,which was validated and evaluated for method specificity and sensitivity.Then,the established method was applied to detect resistance gene mutations and genotyping in 320 clinical samples collected from Kunming,Dali,and Yuxi in Yunnan in 2022.After gel electrophoresis detection of amplification products,Sanger sequencing was performed,and the results were analyzed by alignment.Finally,based on the detection results and clinical statistics,the first time the prevalence of resistance gene mutations and genotypes and their correlation with resistance in different regions were analyzed.The main results of this study are as follows:1.A nested PCR detection method was established to detect mutations in 21 gene loci associated with macrolide,quinolone,and tetracycline antibiotics that can lead to Mycoplasma pneumoniae(MP)resistance.Specificity evaluation showed that the primers did not cross-react with other pathogens except for MP.Sensitivity evaluation showed that the method could detect the target genes even after 10-fold serial dilutions of the samples.Sequencing results confirmed the accuracy of the mutation information.2.Through mutation detection of 320 clinical samples,it was found that the A2063 G mutation was the dominant type of resistance mutation,with a frequency of93.88%.Macrolide resistance was severe in all regions.No significant differences were found in the prevalence of resistance mutations among different genders,seasons,regions,populations,or age groups,according to the clinical data.Nine previously unreported mutations were discovered during the detection process.3.A nested PCR detection method was developed to genotype the P1 gene of MP.The sequencing results confirmed the accuracy of distinguishing between type 1 and type 2.Among 320 clinical samples,type 1 was the predominant type in Yunnan province,with an average frequency of 92.86%.The frequency increased with decreasing age.No significant differences were found in the prevalence of genotypes among different genders,seasons,regions,or age groups in the pediatric population.However,significant differences were found in the prevalence of genotypes among different genders,seasons,and regions in the adult population.Compared with type 2,type 1 was more prone to develop the A2063 G resistance mutation,with a significant difference.This study established a nested PCR assay for the detection of MP macrolide resistance gene mutations and P1 gene typing.The assay is simple to perform,highly specific,and sensitive,with an acceptable cost,making it suitable for testing many clinical samples.The results of the assay showed that the situation of MP macrolide resistance in Kunming,Dali,and Yuxi regions of Yunnan Province is more severe,with the main mechanism of resistance being the A2063 G mutation in the V region of 23 S r RNA.No strains resistant to tetracyclines or quinolones were found.Type 1 was the predominant genotype,and the MP genotypes in the three regions may be in the transition period from type 2 to type 1,with type 1 being more likely to develop resistance gene mutations.These data can provide clinical treatment strategies and priorities for the sample source areas,enabling patients to receive timely and effective treatment,and provide reference for local epidemiological investigations and MP outbreak prevention. |
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