Multiple-Locus Variable-Number Tandem-Repeat Analysis of Mycoplasma pneumoniae Clinical Specimens and Proposal for Amendment of MLVA Nomenclature

Sun, Hongmei; Xue, Guanhua; Yan, Chao; Li, Shaoli; Cao, Ling; Yuan, Yang; Zhao, Huiru; Feng, Yinchang; Wang, Liqiong; Fan, Zhaoyang

doi:10.1371/journal.pone.0064607

Cited by 53 publications

(103 citation statements)

References 25 publications

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“…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).…”

Section: Discussionsupporting

confidence: 88%

“…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).…”

Section: Discussionmentioning

confidence: 99%

“…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].…”

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confidence: 99%

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

Zhang

Song

Marella³

et al. 2016

Future Microbiol.

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Aim:To characterize inter-and intra-strain variability of variable-number tandem repeats (VNTRs) in Mycoplasma pneumoniae to determine the optimal multilocus VNTR analysis scheme for improved strain typing. Methods: Whole genome assemblies and next-generation sequencing data from diverse M. pneumoniae isolates were used to characterize VNTRs and their variability, and to compare the strain discriminability of new VNTR and existing markers. Results: We identified 13 VNTRs including five reported previously. These VNTRs displayed different levels of inter-and intra-strain copy number variations. All new markers showed similar or higher discriminability compared with existing VNTR markers and the P1 typing system. Conclusion: Our study provides novel insights into VNTR variations and potential new multilocus VNTR analysis schemes for improved genotyping of M. pneumoniae.

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Section: Discussionsupporting

confidence: 88%

Section: Discussionmentioning

confidence: 99%

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

Zhang

Song

Marella³

et al. 2016

Future Microbiol.

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“…MLVA types were reported using four loci (Mpn13, Mpn14, Mpn15, and Mpn16) as previously suggested (20)(21)(22). High-resolution melt (HRM) analysis for identification of the macrolide susceptibility genetic profile was performed with a slight modification of the original procedure (9); the modification consisted of using a nested PCR approach to allow standardization of input material into the HRM reactions.…”

Section: Methodsmentioning

confidence: 99%

Investigations of Mycoplasma pneumoniae Infections in the United States: Trends in Molecular Typing and Macrolide Resistance from 2006 to 2013

2015

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Mycoplasma pneumoniae is a leading cause of respiratory infections, including community-acquired pneumonia (CAP). Currently, pathogen-specific testing is not routinely performed in the primary care setting, and the United States lacks a systematic surveillance program for M. pneumoniae. Documentation of individual cases and clusters typically occurs only when severe illness and/or failure to improve with empirical antibiotic therapy is observed. Outbreaks, some lasting for extended periods and involving a large number of cases, occur regularly. However, many more likely go unrecognized due to the lack of diagnostic testing and structured reporting. We reviewed data from 17 investigations of cases, small clusters, and outbreaks of M. pneumoniae M ycoplasma pneumoniae is a common respiratory pathogen in adults and children worldwide and is a leading cause of community-acquired pneumonia (CAP). The majority of infections are self-limiting, and empirical treatment without pathogen-specific diagnostic testing is common in the primary care setting. In fact, the current treatment guidelines do not recommend diagnostic testing for suspected CAP of atypical bacterial etiology in adults in the outpatient setting unless a change in the patient treatment regimen is anticipated (1). The recommendation to perform diagnostic testing for M. pneumoniae infection in children is classified as a weak recommendation and likely not routinely performed, in part due to a lack of available diagnostic tests in the primary care setting and at state and local public health laboratories (2). The estimates of the actual number of cases occurring annually are inexact due to the lack of systematic surveillance and reporting. Although historically noted to occur in 3-to 7-year cycles, outbreaks of M. pneumoniae are common and may last for several months as a result of the long incubation period and prolonged carriage after resolution of symptoms (3-5).Excessive or inappropriate antibiotic use provides selective pressure for the development of antimicrobial resistance. In M. pneumoniae infections, macrolide resistance is an emerging threat worldwide, and in some parts of the world, Ͼ90% of M. pneumoniae infections are caused by resistant strains (6, 7). In the United States, the incidence of infection with macrolide-resistant M. pneumoniae strains is not well defined, although the trait has been consistently reported over the past decade (8-10). Macrolide-resistant M. pneumoniae infection has been associated with increased febrile period, increased duration of persistent cough, and extended antibiotic therapy compared to macrolide-sensitive strains (11-16).Methods for molecular typing of M. pneumoniae strains have been developed, including discrimination of two major types and variants based on the sequence of the P1 adhesion molecule gene and identification of multiple types using multilocus variablenumber tandem-repeat analysis (MLVA) to examine four or five polymorphic loci. Despite development of these sophisticated typing methods, character...

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“…However, the rapid evolution of VNTRs and the recently reported lack of stability of the marker Mpn1 (16), the most discriminant VNTR of the five VNTRs used in this method, are limitations of this technique. Recently, an amendment of the MLVA-5 nomenclature system, which eliminates the unstable Mpn1 marker and bases the typing method on the remaining 4 VNTRs (named the MLVA-4 typing method here), has been proposed (17). With MLVA-4, M. pneumoniae strains were separated into 25 types.…”

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confidence: 99%

Molecular Epidemiology of Mycoplasma pneumoniae: Genotyping Using Single Nucleotide Polymorphisms and SNaPshot Technology

et al. 2015

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i Molecular typing of Mycoplasma pneumoniae is an important tool for identifying grouped cases and investigating outbreaks. In the present study, we developed a new genotyping method based on single nucleotide polymorphisms (SNPs) selected from the whole-genome sequencing of eight M. pneumoniae strains, using the SNaPshot minisequencing assay. Eight SNPs, localized in housekeeping genes, predicted lipoproteins, and adhesin P1 genes were selected for genotyping. These SNPs were evaluated on 140 M. pneumoniae clinical isolates previously genotyped by multilocus variable-number tandem-repeat analysis (MLVA-5) and adhesin P1 typing. This method was also adapted for direct use with clinical samples and evaluated on 51 clinical specimens. The analysis of the clinical isolates using the SNP typing method showed nine distinct SNP types with a Hunter and Gaston diversity index (HGDI) of 0.836, which is higher than the HGDI of 0.583 retrieved for the MLVA-4 typing method, where the nonstable Mpn1 marker was removed. A strong correlation with the P1 adhesin gene typing results was observed. The congruence was poor between MLVA-5 and SNP typing, indicating distinct genotyping schemes. Combining the results increased the discriminatory power. This new typing method based on SNPs and the SNaPshot technology is a method for rapid M. pneumoniae typing directly from clinical specimens, which does not require any sequencing step. This method is based on stable markers and provides information distinct from but complementary to MLVA typing. The combined use of SNPs and MLVA typing provides powerful discrimination of strains. Mycoplasma pneumoniae is the second leading cause of community-acquired pneumonia behind Streptococcus pneumoniae. This bacterium affects both the upper and lower respiratory tracts of individuals of all age groups, and infections occur endemically and epidemically worldwide (1, 2). Many outbreaks of M. pneumoniae respiratory infections have been reported in the community and in closed or semiclosed settings, such as military bases, hospitals, religious communities, schools, and institutions for the mentally disabled and may be associated with considerable morbidity (1, 3-6). Since 2010, a substantial increased incidence of M. pneumoniae infections has been reported in several countries (7-9). Molecular typing methods for M. pneumoniae have been developed for the identification of grouped cases and investigation of outbreaks (10). Until recently, the most common typing methods for M. pneumoniae were based on the analysis of single nucleotide polymorphisms (SNPs) within the gene encoding the major immunogenic protein P1, which is involved in adhesion to host cells. Several methodologies were used, including PCR-restriction fragment length polymorphism (11), amplification and gene sequencing (12), real-time PCR with high-resolution melt analysis (13), and pyrosequencing (14). However, due to the homogeneity of the M. pneumoniae species, few polymorphisms have been identified, and P1-based typing methods allow th...

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Multiple-Locus Variable-Number Tandem-Repeat Analysis of Mycoplasma pneumoniae Clinical Specimens and Proposal for Amendment of MLVA Nomenclature

Cited by 53 publications

References 25 publications

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

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

Investigations of Mycoplasma pneumoniae Infections in the United States: Trends in Molecular Typing and Macrolide Resistance from 2006 to 2013

Molecular Epidemiology of Mycoplasma pneumoniae: Genotyping Using Single Nucleotide Polymorphisms and SNaPshot Technology

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