Genotyping Mycoplasma Pneumoniae In Respiratory Sample From Children

Background:Mycoplasma pneumoniae is a type of prokaryotic cell biology with no cell wall, which is between cells and viruses. Mycoplasma pneumoniae is not only one of an important pathogen which results in tracheitis, bronchitis and primary atypical pneumonia in children, but also the common pathogen resulting in community-acquired pneumonia in adults. Epidemic outbreaks were revealed in time-intervals of 3-7 years followed by endemic phases in the epidemiology of M. pneumoniae infections. Naturally people could acquire immunity after MP infection with a range of 2 to 10 years.Mycoplasma pneumoniae infected host mainly by the way of adhesion and colonization on respiratory tract epithelial cells. The process of adhesion is completed by the top structure of the particular MP adhesion organelles. The P1 protein is considered to be the major adhesin of Mycoplasma pneumoniae. In early studies, only two types of M. pneumoniae P1 genes were assumed to exist, that is P1 type 1 and P1 type 2. Later, Dorigo-Zetsmaet et al reported that they found 5 subtypes among clinical specimens of P1 type 1 and 3 subtypes among clinical specimens of P1 type 2. Dorigo-Zetsma et al sequenced the P1 gene of MP clinical isolates and found there may be recombination between P1 type 1 and P1 type 2. In addition, the reports about the variations of MP P1 gene appeared frequently. In recent years, the incidence of M. pneumoniae is rising. Refractory M. pneumoniae pneumonia was also reported frequently. Whether MP subtypes change or antigen gene mutation play a role remains unclear until now.At present, although there have been a few reports on M. pneumoniae subtypes and the variation of P1 gene in other countries, China have no related reports. Therefore, we genotyped Mycoplasma pneumoniae in nasopharyngeal aspirate from children with pneumonia, aiming to understand epidemiology of MP subtype and the variation of P1 gene.Objectives:Our study aimed to genotype Mycoplasma pneumoniae (M. pneumoniae) based on P1 cytadhesin genes directly from clinical specimens from children with pneumonia in Zhejiang Province and to understand epidemiology of MP subtype and the variation of P1 gene.Methods:300 nasopharyngeal aspirate (NPA) were collected from children with MPP hospitalized in Children's Hospital of Zhejiang University school of Medicine during the period from February to December in 2009.We genotyped and sequenced the P1 gene.1. Collected the NPA clinical specimens from children with MPP; and isolated the DNA of MP for fluorescence quantitative PCR. We selected the specimens whose concentration was 1.0*10^4or above (including 1.0* 10^4), and then drawn the supernatant after 4000rpm/15min centrifugation to 1.5ml eppendorf tube, frozen the supernatant in-80℃refrigerator; 2. Designed the primers of ADH1/ADH2 and ADH3/ADH4, then to PCR reaction; Toke 15μl PCR products in 2% agarose gel (containing 1μg/ml ofethidium bromide) in the electrophoresis and photographed with a gel imaging analysis system;3. PCR-RFLP:the PCR products were digested with HaeⅢ(or HhaⅠ,HpaⅡ,Sau3A) endonuclease; the products of PCR-RFLP were analyzed on a 2% agarose gel.4. Randomly selected the P1 type 1, P1 type 2 specimens, the reference strain MP-129 and the reference strain MP-FH to sequence.5. Collected and compared the clinical data of MPP children with different subtypes:sex, age, clinical manifestations, pulmonary complications, laboratory test results and treatment conditions, et al.Result:1. The P1 gene PCR generated fragments of approximately 2,280bp and 2,580bp from MP-129, MP-FH reference strain and 300 clinical specimens;2. After digestion of these PCR products with HaeⅢ, we found that reference strain of MP129 and 297 clinical specimens showed the banding pattern characteristic for P1 type 1,and only 3 clinical specimens and MP-FH showed the banding pattern characteristic for P1 type 2;3. After PCR-RFLP, we found all the P1 type 1 specimens as subtype lb and the 3 P1 type 2 specimens as subtype 2a;4. The sequences of the P1 type 1 specimen showed very high similarity to MP129 strain except 1 P1 type 1 specimen. One nonsynonymous point mutations was identified in this specimen. The sequences of the P1 type 2 specimen were completely identical to MP-FH strain5. The cases with P1 type 2 M. pneumoniae pneumonia showed severe clinical course and improved after macrolide therapy. Conclusion:1. We successfully genotyped MP by PCR-RFLP based on PI adhension protein gene directly from 300 clinical specimens and revealed predominance of P1 type 1 subtype lb in Zhejiang Province last year;2. P1 gene remained relatively stable with the emergence of new mutations;3. The genotypes of MP may have some relationship with the clinical severity.