BacteriologyDetection of Mycoplasma pneumoniae P1 subtype variations by denaturing gradient gel electrophoresis
Introduction
Mycoplasma pneumoniae is the most common pathogen of community-acquired respiratory tract infections (Waites and Talkington, 2004). Epidemiological studies have shown that M. pneumoniae is responsible for 20–50% of community-acquired pneumoniae (Atkinson et al., 2008). Strains typing or subtyping by molecular methods is a powerful tool for surveillance and outbreak investigation. Several molecular typing methods have been developed. Historically, typing schemes of M. pneumoniae were based upon restriction fragment length polymorphism (RFLP) analysis (Cousin-Allery et al., 2000), multiple-locus variable-number tandem-repeat analysis assay (Dégrange et al., 2009, Dumke and Jacobs, 2011, Liu et al., 2012), pyrosequencing (Spuesens et al., 2010, Spuesens et al., 2012) and sequencing (Zhao et al., 2011). Among them, restriction fragment length polymorphism (RFLP) analysis of P1 gene (the gene encoding for the major adhesin of M. pneumoniae), which contains copies of repetitive elements (repMp4 and repMp2/3), is the most common genotyping method for M. pneumoniae molecular typing and identification of variants of each subtype (Cousin-Allery et al., 2000, Kenri et al., 1999). However, clinical isolates are poorly differentiated by PCR-based restriction fragment length polymorphism (PCR-RFLP) analysis as M. pneumoniae is a genetically homogeneous species (Dégrange et al., 2009).
Denaturing gradient gel electrophoresis (DGGE) was used to detect single base mutations in DNA products (Su et al., 2012). This method is based on the different level of migration of DNA fragments following strand separation caused by chemical denaturants. DGGE was now applied to investigate the fingerprint of samples (Matussek et al., 2011, McAuliffe et al., 2003, Muyzer, 1999, Oates et al., 2012) and adopted for the investigation of the microbial diversity of food-contact surfaces (Koo et al., 2013).
The aim of this study was to determine the P1 variants of M. pneumoniae in DNA samples from 109 isolates commonly associated with respiratory tract infections (RTI) collected during 2009–2011 in Shanghai, China, using a novel and rapid PCR-based denaturing gradient gel electrophoresis (DGGE) method.
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M. pneumoniae clinical isolates and DNA preparation
One hundred nine unique M. pneumoniae clinical isolates, which had never been described before, were obtained from bronchial aspirations with low respiratory infections (each specimen collected from one patient) from January 2009 to march 2011 in Shanghai. Culture and identification of M. pneumoniae was carried out as described previously (Liu et al., 2009). The Genomic DNAs of each isolate were extracted using TIANamp Bacteria DNA kit. Two reference strains of M. pneumoniae M129 (ATCC 29342,
Results of p1 gene subtyping by PCR-RFLP
The p1 gene PCR generated fragments of approximately 1686 bp (primers RepMp2/3 F and RepMp2/3R) and 1877 bp (primers RepMp4F and RepMp4R) from the 109 clinical isolates (data not shown). After digestion of these fragments with Rsa I/Taq I, 101 (92.7%) clinical isolates were classified into type I, and 8 clinical isolates (7.3%) were classified into type II (Fig. 1). Among these strains, 4 (4.0%) type I variants and 7 (87.5%) type II variants were identified.
Results of p1 gene variation by PCR-DGGE
Results of parallel gel electrophoresis
Discussion
PCR-based molecular techniques such as DGGE (Oates et al., 2012) and RFLP (Laguerre et al., 1994) have been widely used for study microbial communities. PCR-RFLP typing in P1 gene has played an important role in the subtyping of M. pneumoniae for a long time. However, now the concept has to be accepted that the power of discrimination for PCR-RFLP is limited. In this assay and previous assays, M. pneumoniae clinical isolates could only be divided into two types and the variations could not be
Acknowledgments
This work was supported by the National Natural Science Foundation of China (Grant No. 81000753 and 81072418).
References (23)
- et al.
Culture-independent multi-locus variable-number tandem-repeat analysis (MLVA) of Mycoplasma pneumoniae
J Microbiol Methods
(2011) - et al.
Characterization of macrolide resistance in Mycoplasma pneumoniae isolated from children in Shanghai, China
Diagn Microbiol Infect Dis
(2010) DGGE/TGGE a method for identifying genes from natural ecosystems
Curr Opin Microbiol
(1999)- et al.
Macrolide resistance determination and molecular typing of Mycoplasma pneumoniae by pyrosequencing
J Microbiol Methods
(2010) - et al.
Epidemiology, clinical manifestations, pathogenesis and laboratory detection of Mycoplasma pneumoniae infections
FEMS Microbiol Rev
(2008) - et al.
Molecular typing of Mycoplasma pneumoniae strains by PCR-based methods and pulsed-field gel electrophoresis. Application to French and Danish isolates
Epidemiol Infect
(2000) - et al.
Development of multiple-locus variable-number tandem-repeat analysis for molecular typing of Mycoplasma pneumoniae
J Clin Microbiol
(2009) - et al.
Mycoplasma pneumoniae P1 type 1- and type 2-specific sequences within the P1 cytadhesin gene of individual strains
Infect Immun
(2001) - et al.
Culture-independent molecular subtyping of Mycoplasma pneumoniae in clinical samples
J Clin Microbiol
(2006) - et al.
Identification of a new variable sequence in the P1 cytadhesin gene of Mycoplasma pneumoniae: evidence for the generation of antigenic variation by DNA recombination between repetitive sequences
Infect Immun
(1999)
Analysis of microbial diversity on deli slicers using polymerase chain reaction and denaturing gradient gel electrophoresis technologies
Lett Appl Microbiol
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Contributed equally to this work.