Abstract
A total of 201 Mycoplasma pneumoniae clinical isolates from Beijing, China, isolated from 2008 to 2011, were clustered into 16 multiple-locus variable-number tandem-repeat analysis (MLVA) types, of which 6 new MLVA types have never been reported previously. Type 1 isolates based on p1 gene genotyping were mainly MLVA types E, J, P, U, and X. There was no correlation between macrolide-resistant Mycoplasma pneumoniae and their MLVA type.
TEXT
Mycoplasma pneumoniae is an important pathogen for human respiratory tract infection, especially for community-acquired pneumonia (CAP), of which M. pneumoniae infection accounts for a ratio of 10% to 30% (1, 2). The genotyping of clinical isolates is an important means for understanding the epidemiology of M. pneumoniae and the analysis of its breakout. Except for fewer adhesion-related protein-coding genes, the genomic sequences of M. pneumoniae are highly homogeneous, so the previously reported genotyping target site of M. pneumoniae is mainly a p1 adhesion protein gene. Commonly used PCR-restriction fragment length polymorphism (PCR-RFLP) technology primarily classified M. pneumoniae into type 1 and type 2 (3, 4). With the in-depth study of the RepMp2/3 and RepMp4 repeat sequence within the p1 gene, more variants of type 1 and 2 isolates were found by molecular subtyping and sequencing (5–8). However, the methods mentioned above have limited ability for understanding the M. pneumoniae epidemiological investigations. In 2009, Dégrange et al. established the multiple-locus variable-number tandem-repeat analysis (MLVA) technique using 265 M. pneumoniae clinical isolates and classified M. pneumoniae into 26 MLVA types based on 5 stable variable-number tandem-repeats (VNTRs) of the M. pneumoniae genome (9); this method greatly improved the ability to identify M. pneumoniae types. Recently, studies involving MLVA genotyping and method improvement have identified new MLVA types (10–13). In the present study, we use this method to make an MLVA genotyping analysis against 201 M. pneumoniae clinical isolates collected from Beijing, China, from 2008 to 2011. This study may help to understand the variations in M. pneumoniae genotyping in Beijing and investigate the relevance between p1 gene-based typing and MLVA typing methods and the relationship of macrolide-resistant M. pneumoniae isolates to their MLVA types.
M. pneumoniae isolates and DNA extraction.
A total of 201 M. pneumoniae clinical isolates were collected from throat swab specimens of respiratory tract infection patients at the Beijing Chao-Yang Hospital, Beijing Children's Hospital, and Beijing Centers for Disease Control and Prevention from 2008 to 2011. M. pneumoniae was isolated in Mycoplasma selective broth medium (Oxoid) at 37°C. All pure-cultivated isolates were identified by real-time PCR (14). Bacterial DNA from cultivated M. pneumoniae isolates was extracted using the QIAamp DNA minikit (Qiagen), and the aliquots were placed in a refrigerator at −20°C.
p1 gene-based genotyping of M. pneumoniae.
Sixty of 201 M. pneumoniae clinical isolates were genotyped by full-length sequencing of the p1 gene in our previous report (8); the other 141 isolates were genotyped by a previously reported method (15, 16). Results indicated that 184 (91.5%) isolates were type 1, 2 (1.0%) isolates were variant 2a, 15 (7.5%) isolates were variant 2c, and no type 2 isolates were detected.
Sequencing analysis of the 23S rRNA gene.
The domain V of 23S rRNA, related to M. pneumoniae macrolide resistance, was detected using a previously reported method (17). Results indicated that 24 (11.9%) isolates were macrolide sensitive without mutation and 177 (88.1%) isolates were macrolide resistant with mutation within the domain V of 23S rRNA, of which the numbers of A2063G, A2064G, and A2064T mutant isolates were 171, 5, and 1, respectively.
M. pneumoniae MLVA genotyping.
A total of 201 M. pneumoniae clinical isolates were analyzed using the MLVA assay (9), with M129 as a reference strain. Using nonfluorescent primers, PCR amplification against 5 VNTRs from 10 randomly selected isolates was performed to validate the creditability of MLVA genotyping. All data were analyzed using BioNumerics (version 6.5) software. Results indicated that 201 M. pneumoniae clinical isolates were divided into 16 MLVA types, of which 10 types were reported previously, and the other 6 types were new MLVA types, as shown in Table 1.
Table 1.
Results of MLVA, macrolide susceptibility and p1 gene typing of 201 M. pneumoniae clinical isolates from 2008 to 2011 in Beijing, China
| MLVA typea | No. of isolates by year of distribution |
No. of isolates by macrolide susceptibility |
No. of isolates by p1 gene-based typing |
Total | |||||
|---|---|---|---|---|---|---|---|---|---|
| 2008 | 2009 | 2010 | 2011 | Resistant | Sensitive | Type 1 | Variant 2 | ||
| E | 0 | 3 | 7 | 3 | 13 | 0 | 12 | 1 | 13 |
| G | 2 | 0 | 0 | 0 | 0 | 2 | 0 | 2 | 2 |
| J | 5 | 0 | 9 | 7 | 20 | 1 | 21 | 0 | 21 |
| M | 7 | 0 | 1 | 2 | 4 | 6 | 1 | 9 | 10 |
| P | 7 | 3 | 13 | 14 | 35 | 2 | 36 | 1 | 37 |
| U | 25 | 5 | 18 | 19 | 65 | 2 | 66 | 1 | 67 |
| V | 1 | 0 | 0 | 2 | 1 | 2 | 0 | 3 | 3 |
| X | 4 | 8 | 10 | 6 | 25 | 3 | 28 | 0 | 28 |
| Z | 1 | 0 | 3 | 1 | 5 | 0 | 5 | 0 | 5 |
| (4/4/5/7/3)b | 1 | 0 | 0 | 0 | 1 | 0 | 1 | 0 | 1 |
| NEW1(2/4/5/7/3) | 1 | 0 | 1 | 0 | 2 | 0 | 2 | 0 | 2 |
| NEW2(3/4/5/7/3) | 4 | 0 | 0 | 0 | 1 | 3 | 4 | 0 | 4 |
| NEW3(4/4/4/7/2) | 0 | 0 | 0 | 1 | 1 | 0 | 1 | 0 | 1 |
| NEW4(5/4/5/7/3) | 1 | 1 | 1 | 0 | 2 | 1 | 3 | 0 | 3 |
| NEW5(5/4/4/7/2) | 1 | 0 | 0 | 0 | 0 | 1 | 1 | 0 | 1 |
| NEW6(8/4/5/7/2) | 1 | 0 | 1 | 1 | 2 | 1 | 3 | 0 | 3 |
| Total | 61 | 20 | 64 | 56 | 177 | 24 | 184 | 17 | 201 |
The MLVA type of reference strain M129 was not included in this table. Reference strain M129 belonged to MLVA type P (4/4/5/7/2).
This MLVA type reported by Chalker et al. in 2012 (11).
Detection of VNTRs in the p1 gene from 25 MLVA type U M. pneumoniae isolates.
Using the method we reported previously (18), PCR amplification was performed against the VNTR region in the p1 gene from 25 MLVA type U M. pneumoniae isolates identified during the period of August to December 2008 from Beijing Chao-Yang Hospital. The PCR products were sequenced by the Beijing Genomics Institute (BGI). Results from the sequence alignment indicated the numbers of VNTRs in the p1 gene from the same 25 MLVA type U M. pneumoniae isolates were different, ranging from 6 to 14, as shown in Table 2.
Table 2.
The AGT VNTRs in the p1 gene from 25 MLVA type U M. pneumoniae isolates collected during the period of August to December 2008
| Isolate | Sample collection date | Patienta | MLVA type | p1 gene-based type | Macrolide susceptibilityb | No. of VNTRs in p1 gene |
|---|---|---|---|---|---|---|
| U017 | 8/2/2008 | URI | U | 1 | R | 7 |
| P005 | 8/8/2008 | CAP | U | 1 | R | 7 |
| P015 | 8/13/2008 | CAP | U | 1 | R | 6 |
| P023 | 8/15/2008 | CAP | U | 1 | R | 10 |
| P030 | 8/21/2008 | CAP | U | 1 | R | 7 |
| P078 | 9/8/2008 | CAP | U | 1 | R | 6 |
| P084 | 9/10/2008 | CAP | U | 1 | R | 7 |
| P083 | 9/10/2008 | CAP | U | 1 | R | 10 |
| P088 | 9/21/2008 | CAP | U | 1 | R | 9 |
| P087 | 9/22/2008 | CAP | U | 1 | R | 10 |
| P092 | 9/26/2008 | CAP | U | 1 | R | 7 |
| P093 | 10/7/2008 | CAP | U | 1 | R | 8 |
| P103 | 10/20/2008 | CAP | U | 1 | R | 8 |
| P106 | 10/21/2008 | CAP | U | 1 | R | 6 |
| P108 | 10/22/2008 | CAP | U | 1 | R | 14 |
| P105 | 10/29/2008 | CAP | U | 1 | R | 7 |
| P112 | 11/5/2008 | CAP | U | 1 | R | 11 |
| P116 | 11/17/2008 | CAP | U | 1 | R | 7 |
| P119 | 11/26/2008 | CAP | U | 1 | R | 7 |
| P120 | 11/27/2008 | CAP | U | 1 | R | 8 |
| P121 | 12/3/2008 | CAP | U | 1 | R | 7 |
| P123 | 12/5/2008 | CAP | U | 1 | R | 10 |
| P124 | 12/8/2008 | CAP | U | 1 | R | 6 |
| F022 | 12/18/2008 | URI | U | 1 | R | 10 |
| P132 | 12/26/2008 | CAP | U | 1 | R | 8 |
URI, upper respiratory tract infection.
R, macrolide resistant.
In the present study, 201 M. pneumoniae isolates were genotyped using the traditional p1 gene-based typing method. Data indicated that 91.5% were type 1 and 88.6% (163/184) were MLVA types E, J, P, U, and X (Fig. 1A). There were no type 2 isolates but only 17 variant 2 isolates, which mainly focus on V, M, and G MLVA types in Beijing. The minimum-spanning tree (MST) results from our study were consistent with previous reports (9, 10), which prompted some correlations between MLVA and p1 gene-based genotyping at the gene level. Since the study by Liu et al. (19) has a lack of unified MLVA typing criteria, we could not compare our MLVA results with their study with regional M. pneumoniae isolates in China. A total of 177 macrolide-resistant M. pneumoniae isolates covered 15 of 16 MLVA types (Fig. 1B). Four of 5 isolates with an A2064G mutation, isolated in 2008, belong to MLVA types M, P, U, and X; another one, isolated in 2011, was MLVA type P. Such results further indicated that there was no correlation between macrolide-resistant M. pneumoniae isolates and their MLVA type. In addition, MLVA typing results also indicated that macrolide-resistant M. pneumoniae isolates were not from the same clones, and similar results were also reported in Shanghai, China (19).
Fig 1.
MST of the MLVA profiles of 201 M. pneumoniae isolates. Each circle represents a unique MLVA type, as indicated by a letter and with the number of isolates corresponding to this type given in parentheses. The circle size is proportional to the number of isolates belonging to the indicated MLVA type. The distance between neighboring MLVA types is expressed as the number of allelic changes. The MLVA type labeled with an asterisk was reported by Chalker et al. in 2012 (11). The colors of the circles in panel A represent the p1 gene-based types: white for type 1, gray for type variant 2a, and black for type variant 2c. In panel B, the color white represents macrolide-resistant M. pneumoniae isolates and the color black represents macrolide-sensitive M. pneumoniae isolates.
The current MLVA method has largely overcome the limitation of the p1 gene-based typing methods to distinguish M. pneumoniae at the molecular level and may be able to provide more information to M. pneumoniae molecular epidemiological studies. However, the idea of p1 gene-based typing methods still has an important role for the study of the mechanism of immunity and the epidemiological investigations of M. pneumoniae. Because of the limited number of VNTRs identified using the Microorganisms Tandem Repeats Database (http://minisatellites.u-psud.fr) in the M. pneumoniae M129 genome and the unstable Mpn1 locus during culture or within the host (10), the currently established MLVA method is still not ideal. In this study, the number of VNTRs in the p1 gene from the same 25 MLVA type U M. pneumoniae isolates, isolated in 2008, was further analyzed. The results indicated that the 25 type U M. pneumoniae isolates were from at least 7 clones, but not the same clone (Table 2). Therefore, it is very important to more fully understand the molecular characteristics of M. pneumoniae by constantly improving the variety of typing methods and mutual validation.
Footnotes
Published ahead of print 5 December 2012
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