Multilocus Variable-Number Tandem-Repeat Analysis of Mycoplasma pneumoniae Clinical Isolates from 1962 to the Present: a Retrospective Study

Abstract

In this study, we evaluated a recently developed multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) method for the molecular typing of Mycoplasma pneumoniae. The method is based on GeneScan analysis of five VNTR loci throughout the genome which define a specific genotype based on the number of tandem repeats within each locus. A retrospective analysis of 154 M. pneumoniae clinical isolates collected over the last 50 years and a limited (n ‫؍‬ 4) number of M. pneumoniae-positive primary specimens acquired by the CDC was performed using MLVA. Eighteen distinct VNTR types were identified, including two previously unidentified VNTR types. Isolates from several M. pneumoniae community outbreaks within the United States were also analyzed to examine clonality of a specific MLVA type. Observed in vitro variability of the Mpn1 VNTR locus prompted further analysis, which showed multiple insertions or deletions of tandem repeats within this locus for a number of specimens and isolates. To our knowledge, this is the first report showing variation within the Mpn1 locus, thus affecting precise and reliable classification using the current MLVA typing system. The superior discriminatory capability of MLVA provides a powerful tool for greater resolution of M. pneumoniae strains and could be useful during outbreaks and epidemiological investigations. Mycoplasma pneumoniae is one of the most common bacterial etiologies of community-acquired pneumonia (CAP), representing 15 to 20% of cases in some studies (28). It is also a significant cause of community-wide outbreaks which are reported to occur in 3-to 7-year intervals with various incidence rates (10,12,28). Upper and lower respiratory tract symptoms are often mild and self-limiting; however, occasional extrapulmonary complications may develop, sometimes resulting in death (5,7,23).Strain subtyping is an important epidemiological tool for surveillance and outbreak investigations. Until recently, molecular typing of M. pneumoniae had been hindered by its minimal and highly uniform genome. Molecular typing was often restricted to a single gene encoding the P1 protein. Methods such as PCR-restriction fragment length polymorphism (RFLP) analysis of the P1 gene (4, 18) and real-time PCR followed by high-resolution melt analysis (HRM) targeting the region of M. pneumoniae repetitive elements 2 and 3 (RepMp2/3) of the P1 gene (19,20) were used to genotype M. pneumoniae into two subtypes (PCR-RFLP) and variants of subtypes 1 and 2 (PCR-HRM). Both sequencing (9) and pyrosequencing (6, 21, 22) techniques have also been developed to differentiate subtypes of M. pneumoniae. Recently, Degrange et al. (6) developed a multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) method for M. pneumoniae based on whole-genome analysis that was able to differentiate 26 distinct VNTR types. This procedure takes advantage of the variation in the copy numbers of tandemly repeated sequences within five different loci within the genome and has proven to be highly discriminatory (...

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

“…The detection of extensive intrastrain copy number variation in Mpn1 (Supplementary Table 3) supports the instability of Mpn1 as reported previously [25,33], whereas the absence or presence of low-level intrastrain variation in other four loci in the current MVLA scheme suggests a relatively high stability of these loci. Of note, despite the absence of report of instability of Mpn13 and Mpn14, we detected two or more repeat copy numbers in both loci in a few isolates (Table 2).…”

“…The Mpn16 marker in the current MLVA scheme showed no or little discriminatory ability in this study (Tables 1 & 2) and previous reports [15,25,32,46], and can be replaced by any of the seven new VNTR markers. Of the nine new potential MLVA schemes, MVLA-8 had the highest stability though with a relatively low discriminatory power and MLVA-9 showed the highest discriminatory power though with a low stability.…”

“…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).…”

“…These loci have been frequently used as the targets for molecular typing to help understand the epidemiology and pathogenesis of both pathogens [13][14][15][16][17][18]. Of note, numerous studies of VNTRs in M. genitalium have demonstrated extensive intrastrain repeat number variations despite its genome size being the smallest among all sequenced mycoplasma species [14,[18][19][20][21][22][23][24], whereas there has been a scarcity of reports of such variations in M. pneumoniae [25] and other mycoplasma species. It is unknown whether this difference reflects intrinsic genomic divergence between different species or technical limitations of the detection methods used.…”

“…However, there have been rising concerns of instability at one of these five loci (Mpn1) [25,33] though the extent of instability in this locus and other loci is not well understood. In addition, both P1 and MLVA typing have been shown to be insufficient for meaningful differentiation and lack of epidemiological and clinical relevance [25,32]. There is a clear need to characterize in more detail the variability of VNTRs and identify new VNTR markers with higher stability and discriminatory power [31,34].…”