A Single-Tube Multiple-Locus Variable-Number Tandem-Repeat Analysis of Mycoplasma pneumoniae Clinical Specimens by Use of Multiplex PCR-Capillary Electrophoresis

Abstract

In this study, we developed a single-tube multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) assay to type Mycoplasma pneumoniae directly from respiratory samples collected from children with respiratory infections. The multiplex PCR included four fluorescently primed VNTRs (Mpn13, Mpn14, Mpn15, and Mpn16) and was carried out in a single tube. A total of 137 M. pneumoniae-positive specimens, collected in 2013 from Beijing, China, were divided among four types (M4-5-7-2, M4-5-6-2, M3-5-6-2, and M5-5-7-2) using the amended MLVA system. The most prevalent genotype was M4-5-7-2. No correlation was found between macrolide resistance in the M. pneumoniae samples and the MLVA types. To our knowledge, this is the first study to type and analyze M. pneumoniae clinical specimens using multiplex PCR-capillary electrophoresis in a single tube. This novel low-cost method can be used to rapidly type M. pneumoniae clinical specimens directly and shows great potential for monitoring outbreaks of M. pneumoniae. Mycoplasma pneumoniae is one of the most common etiological agents of community-acquired pneumonia and other respiratory tract infections (1). Clinical symptoms include mild to severe pneumonia and even death. M. pneumoniae infections cause worldwide epidemics every 3 to 7 years, lasting up to 2 years at a time (2, 3). Rapid molecular typing of strains is critical to identify the source of an outbreak and monitor the progress of the epidemic. Multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) is a common method for genotyping infectious agents as it has greater discriminatory power than other methods, such as restriction fragment length polymorphism (RFLP), pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) (4-8). Degrange et al. (9) successfully used MLVA to investigate M. pneumoniae isolates and grouped 265 strains into 26 MLVA types. Many studies use a multiplex approach with five loci (Mpn1, Mpn13, Mpn14, Mpn15, and Mpn16) and fluorescently labeled primers in two solutions to type M. pneumoniae isolates (9-15). Because cultivation of M. pneumoniae is difficult, other studies have used nested PCR in the MLVA approach to directly amplify the regions from the clinical specimens (16, 17). However, because of the instability of the VNTR locus Mpn1, we proposed an amended MLVA nomenclature system based on the remaining four VNTR loci in our preliminary study (17).In this study, we typed 137 M. pneumoniae-positive clinical specimens collected from pediatric patients in Beijing, China, in 2013 using the amended MLVA system, which connects multiplex PCR (mPCR) with capillary electrophoresis (CE) in a single tube. We also analyzed macrolide resistance in all of the typed strains. MATERIALS AND METHODSSamples and reference strains. A total of 477 clinical specimens were collected from pediatric patients at the Affiliated Children's Hospital of the Capital Institute of Pediatrics in Beijing, China, in 2013. Each specimen was collected from a separate patient. The speci...

Emergence of Macrolide-Resistant Mycoplasma pneumoniae in Hong Kong Is Linked to Increasing Macrolide Resistance in Multilocus Variable-Number Tandem-Repeat Analysis Type 4-5-7-2

“…In the United States, 13 (68.4%) of 19 MRMP isolates identified during CDCassisted investigations across the country between 2006 and 2013 belonged to MLVA type 4-5-7-2 (15). In China, this MLVA type accounted for Ͼ90% of the MRMP isolates identified in Beijing from 2010 to 2013 (12,22,24). Of the 21 distinct types that can be distinguished by the amended four-locus MLVA scheme (5, 6, 9-15, 22, 24-26) (see Table S1 in the supplemental material), MRMP has been detected in 12 types.…”

“…The MLVA type was designated by the numeric combination of the number of tandem repeats at four loci (Mpn13 to -16), as suggested previously (13,15). The number of repeats was rounded up to an integer value (9,12). The number of repeats in each locus (1 to 2 specimens for each product size) was confirmed by Sanger sequencing.…”

“…In 2009, a multilocus variable-number tandem-repeat analysis (MLVA) scheme based upon five loci (Mpn1 and Mpn13 to -16) was developed for the molecular typing of M. pneumoniae (9). It was initially used for an investigation of isolates but was later modified for directly typing M. pneumoniae in respiratory specimens (10)(11)(12)). An amended 4-locus MLVA scheme was later proposed after studies raised concerns on the instability of the Mpn1 locus (13,14).…”

Inter- and Intra-Strain Variability of Tandem Repeats in Mycoplasma Pneumoniae Based on Next-Generation Sequencing Data

“…Following the first report of its use in M. pneumoniae in 2009 [15], many studies over the world have consistently shown that MLVA typing is clearly more powerful than the standard P1 typing system in differentiating clinical strains [25,[32][33][46][47][48], and meanwhile there have been rising concerns about its stability, discriminability and correlation to clinical and epidemiological parameters [25,[31][32][33]. These concerns motivated us to seek deeper understanding of We have demonstrated that intrastrain variation occurred in different degrees in all 13 VNTR loci surveyed except for 3 loci (Mpn15, Mpn16 and Mpn103) (Table 2).…”

Comparison of Molecular Characteristics of Mycoplasma pneumoniae Specimens Collected from the United States and China

“…Currently, P1 restriction fragment length polymorphisms (RFLP) and multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) are the most widely used methods for strain typing for this pathogen (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). We improved the MLVA method by amplifying four VNTRs (without Mpn1) in one tube for epidemic and outbreak analyses (11).…”