Nested PCR-linked capillary electrophoresis and single-strand conformation polymorphisms for detection of macrolide-resistant Mycoplasma pneumoniae in Beijing, China

Abstract

Mycoplasma pneumoniae is usually susceptible to macrolides, but macrolide-resistant strains have been found frequently in recent years. Mutations in domain V of the 23S rRNA gene of M. pneumoniae interfere with the binding of macrolides to rRNA and mediate macrolide resistance. In this study, we developed a rapid and inexpensive method that combines nested PCR (nPCR), single-strand conformation polymorphisms (SSCPs), and capillary electrophoresis (CE) to detect macrolide-resistant mutants directly from throat swabs. nPCR was used to specifically amplify M. pneumoniae 23S rRNA gene fragments containing mutations, and amplicons were analyzed by CE-SSCP for macrolide resistance mutations, with results confirmed by sequencing. From January to December 2009, 665 throat swabs were collected in Beijing, China, yielding 110 samples that tested positive for M. pneumoniae by nPCR and serological testing. We randomly selected 64 positive throat swabs for CE-SSCP analysis. The A2063G mutation was found in 57 samples, and a coexisting T2611C mutation was identified in 1 sample. An A2063T mutation was identified in 1 sample. The total mutation rate was 91%. All mutant samples identified by nPCR-CE-SSCP were sequenced. The nPCR-CE-SSCP method could identify macrolide-resistant mutants directly from clinical samples. This is the first report of an nPCR-CE-SSCP assay for the detection of dominant mutations that confer macrolide resistance on M. pneumoniae. This approach would allow clinicians to choose appropriate therapy rapidly and could be used as a screening method for genetic mutations related to antibiotic resistance.

FIG. 1.

Superimposed CE electropherograms of nPCR products. Line A, mixture of dsDNA₃₄₂ (primers 342F and 342R) with the pBR322-HaeIII DNA marker; line B, mixture of dsDNA₃₀₃ (primers 303F and 303R) with the pBR322-HaeIII DNA marker; peaks 1 to 5, 184-bp, 192-bp, 213-bp, 234-bp, and 267-bp fragments of the pBR322-HaeIII DNA marker.

FIG. 2.

Superimposed CE electropherograms of ssDNA from nPCR products. Line A, ssDNA of 342-bp products. Three peaks were observed, corresponding to one dsDNA and two ssDNA peaks. Line B, ssDNA of 303-bp products. Line C, mixture of ssDNAs of 342-bp and 303-bp products. Four peaks, corresponding to ssDNA_303-1, ssDNA_303-2, ssDNA_342-1, and ssDNA_342-2, were observed.

FIG. 3.

Superimposed CE electropherograms of ssDNA₃₀₃ (A) and ssDNA₃₄₂ (B) from mutated and wild-type samples. Shown are electropherograms of ssDNA₃₀₃ from a mutated clinical specimen (line a), the M. pneumoniae reference strain (line b), and a wild-type isolate (line c) and of ssDNA₃₄₂ from a mutated clinical specimen (line 1), the M. pneumoniae reference strain (line 2), and a wild-type isolate (line 3).

FIG. 4.

CE reproducibility test. Shown are superimposed CE electropherograms of a mixture of dsDNA₃₄₂ and dsDNA₃₀₃.

FIG. 5.

Multiple alignment of the 23S rRNA genes from some clinical samples and M. pneumoniae M129 and FH. Partial sequences of the peptidyltransferase (domain V) from positions 2041 to 2091 and 2562 to 2612 are presented. M. pneumoniae numbering is used. The nucleotide sequence of M129 is given according to GenBank accession no. X68422. The reference strain FH was sequenced, and the results are shown. Dots indicate identical nucleotides. The nucleotides at positions 2063 and 2611 are underlined.