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. 2021 Apr 16;24(5):102447. doi: 10.1016/j.isci.2021.102447

A multisite SNP genotyping and macrolide susceptibility gene method for Mycoplasmapneumoniae based on MALDI-TOF MS

Fei Zhao 1, Jianzhong Zhang 1, Xuemei Wang 2, Liyong Liu 1, Jie Gong 1, Zhixiang Zhai 2, Lihua He 1, Fanliang Meng 1, Di Xiao 1,3,∗
PMCID: PMC8105657  PMID: 33997713

Summary

In this study, a multisite SNP genotyping and macrolide (ML) susceptibility gene test method for Mycoplasma pneumoniae (M. pneumoniae) was developed based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The detection limit of this method for nucleic acids was 102 -103 copies/reaction. Six SNP site-based genotyping and 3 ML susceptibility sites could be detected simultaneously based on multiplex PCR and mass probe. Using the method constructed in this study, 141 Chinese clinical isolates were divided into 8 SNP types. All the SNP test results for the ML susceptibility gene were in line with those of the 23S rRNA sequencing results. With this method, the multisite SNP genotyping and ML susceptibility determination of M. pneumoniae can be completed simultaneously in one test, which greatly reduces the workload and cost, improves the genotyping ability of M. pneumoniae and deserves clinical application.

Subject areas: Microbiology, Microbial Genetics, Biotechnology

Graphical abstract

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Highlights

  • •

    An all-in-one genotyping and macrolide resistance testing method for M. pneumoniae

  • •

    Multisite SNP detection technology was used for genotyping and resistance testing

  • •

    The cost of M. pneumoniae genotyping and macrolide resistance detection was reduced

Microbiology;Microbial Genetics; Biotechnology

Introduction

Mycoplasma pneumoniae (M. pneumoniae), an important pathogenic bacterium of respiratory tract infection in children and adults, is responsible for approximately 10%–30% of community-acquired pneumonia (Jacobs et al., 2015; Loens et al., 2010). There has been epidemic spread of M. pneumoniae in many European and Asian countries since 2010 (Blystad et al., 2012; Chalker et al., 2011; Gadsby et al., 2012; Lenglet et al., 2012; Linde et al., 2012; Uldum et al., 2012). Generally, the prognosis of M. pneumoniae is good, with only a few pediatric cases progressing to refractory Mycoplasma pneumoniae pneumonia (RMPP) or severe Mycoplasma pneumoniae pneumonia (SMPP), resulting in severe complications and poor prognosis (Liu et al., 2018; Okumura et al., 2019). To date, macrolides (MLs) have been recommended as the first-line agent for treating clinical infection of M. pneumoniae infections, especially in children (Waites et al., 2009, 2017).

In the past two decades, the proportion of ML susceptibility in M. pneumoniae (MRMP) has shown an increasing trend, especially in Japan and the Republic of Korea (Hong et al., 2013; Kawai et al., 2013). In mainland China, the ML susceptibility ratio of M. pneumoniae is still high to some extent (Cao et al., 2010; Liu et al., 2009; Qu et al., 2013; Xin et al., 2009;Zhao et al., 2012).Studies have shown that the ML resistance mechanism of M. pneumoniae is very fixed and only closely related to 23S rRNA point mutations. The ML susceptibility of M. pneumoniae is closely related to the 23S rRNA mutation, among which the mutations at the 2063 and 2064 positions are correlated with high ML susceptibility (>64 μg/mL for erythromycin) and the mutation at the 2617 position is correlated with low ML susceptibility (Pereyre et al., 2016). Therefore, gene detection based on these mutation sites may serve as an alternative to antimicrobial susceptibility testing of M. pneumonia in vitro. To date, the most common methods of detecting ML resistant of M. pneumoniae strains include real-time PCR-HRM, PCR-RFLP, and pyrosequencing assays (Liu et al., 2014; Matsuoka et al., 2004; Nummi et al., 2015; Peuchant et al., 2009; Spuesens et al., 2010; Wolff et al., 2008).

Genotyping is an important method for studying the molecular biology and epidemiologic features of M. pneumoniae. There may be a close relationship between the transformation of M. pneumoniae genotypes in the population and the epidemics of M. pneumoniae, and genotyping is also an important technique for tracing the origin of M. pneumoniae local outbreaks. The M. pneumoniae genome is highly conserved, with a sequence similarity of up to 99% among strains. Based on single nucleotide polymorphisms (SNPs) and indels, two main subgroups of M. pneumoniae, genotype I and genotype II, can be clearly differentiated (Lluch-Senar et al., 2015; Xiao et al., 2015). Currently, the most common genotyping techniques for these two genotypes are based mainly on PCR-RFLP genotyping of the p1 gene (Dorigo-Zetsma et al., 2000), VNTR of the pl gene, and real-time PCR of the MPN459/MPNA5864 gene (Zhao et al., 2015). These techniques are easy to perform, but the discrimination ability is not adequate due to the presence of a single target site. Specifically, multiple-locus variable-number tandem-repeat analysis (MLVA) (Degrange et al., 2009) and multilocus sequence typing (MLST) (Brown et al., 2015) contribute to the genotyping of M. pneumoniae. However, these genotyping procedures are time-consuming and technically demanding, which hampers their applications. Both genotyping analysis and ML susceptibility nucleic acid testing for M. pneumoniae involve complicated procedures, and it is necessary to develop new methods that are rapid and inexpensive.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been utilized for detecting SNPs in recent decades. Multiplex PCR is used to amplify the genes containing the targets of SNPs. Subsequently, an extension mass probe has been utilized for the extension of SNP sites. Finally, MALDI-TOF MS is performed to identify the mass-to-charge ratio (m/z) of extended mass probes. Recently, MALDI-TOF MS has been commonly used in microbiological detection and analysis, and is considered a new gold standard for the identification of many kinds of microorganisms (Jang and Kim, 2018;Kostrzewa, 2018;Rahi et al., 2016). To date, SNP genotyping based on MALDI-TOF MS has been frequently conducted for the screening of hand-foot-and-mouth disease virus, hepatitis B virus (HBV) detection, and the human papillomavirus (HPV) genotyping and serves as an effective alternative to conventional techniques (Peng et al., 2013a, 2013b;Sjoholm et al., 2008). SNP analysis based on MALDI-TOF MS features high throughput, rapid detection, and simultaneous detection of multiple targets. Therefore, it is one of the most promising biomolecular techniques.

In this study, multiplex PCR coupled with MALDI-TOF MS (PCR-MALDI-TOF MS) was used to genotype and detect the antimicrobial susceptibility of M. pneumoniae.

Results

MALDI-TOF MS-based method establishment and optimization

Multiplex PCR coupled with MALDI-TOF MS assay was utilized to automate the detection of the nine SNPs and avoid sequencing the gene fragments. To avoid the formation of a dimer between the probes at the 23S rRNA2063 and 23S rRNA2064 sites, the quality probe at the 2064 site was designed at the reverse complementary strand. Therefore, the SNP of this site was the basis of the complementary strand (Table 1, Figure 1). MALDI-TOF MS-based amplification and identification were performed on 10 epidemiologically unrelated M. pneumoniae strains. Six SNP genotyping sites and three ML susceptibility gene sites were detected, and no MS peak was observed in the blank control. There was complete consistency between the MS SNP results of three ML susceptibility sites and the sequencing results of the strains. Among the 10 strains, there were 6 SNP types (0, 1, 3, 11, 15 and 32). Six odd number SNP types were classified into genotype I, and 4 even number SNP types were classified into genotype II (Table 2) . For example, there were 9 MS peaks for M129 in Figure 1A, representing 6 SNP typing mass probes and 3 ML susceptibility mass probes. The dotted line of the same color represents the mass spectrum peak for various mass probes extending the same base pairs in the SNP sites. The peak value represents the m/z of the MPE after extension. After multiplex PCR amplification and MPE probes extension for M129, the peak of the spectrum is shown in Figure 1B. The red arrow represents the quality value of the nine MPE probes after SNP site extension. The optimized concentration of each extension primer in the primer mixture was 6.9 μM (MPN1141461), 7.1μM (MPN3721112), 7.7μM (23S rRNA2063), 8.0μM (MPN21347), 8.6μM (MPN2801641), 9.2μM (23S rRNA2617), 9.5μM (MPN126470), 10.4μM (MPN262192), and 11.2μM (23S rRNA2064).

Table 1.

Primer sequences for M. pneumoniae SNP typing and susceptibility target site amplification and MPE probes for SNP detection

Target gene SNP Multiple-PCR
 Mass probe extension
Forward primer sequence Reverse primer sequence Mass probe sequence Mass probe mass (Da) Extension call Extended mass (Da) Extension call Extended mass (Da) Extension call Extended mass (Da) Extension call Extended mass (Da)
GENOTYPING MPN1141461 C/T acgttggatgCA
CCGAGTGTC
TTCGTCCTT		 acgttggatgG
GTTGACCCC
ACTACCCTTT		 CCCTTTTGC
GCTAACCG		 5097.4 C 5410.4 T 5394.4
MPN126470 C/T acgttggatgAA
ATTCCCCGT
AATCCGAGA		 acgttggatgGC
CTTCTGCTTGT
TAGGTAGATG		 AGTGATTT
AACGTTCC
ATTTTC		 6690.4 C 6963.4 T 7032.4
MPN21347 G/T acgttggatgA
TCAGTCGCT
GGTACTGTGG		 acgttggatgGC
TGTGCTTTTG
GCAACTTA		 TACTACCACC
GCAGTTGTT		 5738.8 G 6011.8 T 6035.8
MPN262192 C/T acgttggatgAC
CACTTAGAG
CCGGAACAA		 acgttggatgTT
GCTGAAACTG
ACCATCCA		 GCCCAATT
TAGTCTTAT
TTTGGAT		 7323.8 C 7636.8 T 7620.8
MPN2801641 G/T acgttggatgCTC
AATTAAACGCG
GTTTGG		 acgttggatgC
GGTTGAAATC
AACGGGTTA		 TTCCAAAT
GAAGTAAG
ACAC		 6118.0 G 6391.0 T 6415.0
MPN3721112 G/T acgttggatgAGTG
TTAGCGCGGTT
AATGC		 acgttggatgTC
GTGTTGTGAA
CCACCACT		 AATGACAC
CGCAAGACA		 5181.4 T 5523.4 G 5494.4
ML DETECTION 23S rRNA2063 A/T/C/G acgttggatgCCA
GGTACGGGTG
AAGACAC		 acgttggatgA
TTCCACCTTT
CGCATCAAC		 TTAGGCGCA
ACGGGACGG		 5589.6 G 5902.6 A 5886.6 C 5862.6 T 5931.6
a23S rRNA2064 A/T/C/G acgttggatgCC
AGGTACGGGTG
AAGACAC		 acgttggatgA
TTCCACCTTTC
GCATCAAC		 AGCTACAGT
AAAGCTTCA
CGGGGTCT		 7995.2 T 8337.2 C 8268.2 G 8308.2 A 8292.2
23S rRNA2617 A/T/C/G acgttggatgGC
TGTTCGCCGA
TTAAAGAG		 acgttggatgG
CGCTACAACT
GGAGCATAA		 CCGTCGT
GAGACAGG
TTGGTC		 6478.2 C 6751.2 G 6791.2 A 6775.2 T 6820.2
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a

The MPE extension probe for the SNP of 23S rRNA2064 was the reverse complementary strand, which detected the complementary bases for the SNP.

Figure 1.

Figure 1

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MS peak of the MPE probes

(A) MS peaks of 9 MPE probes without extension;

(B) SNP peak of the 9 MPE probes extended with M. pneumoniae M129.

Table 2.

Characteristics of the ten M. pneumoniae clinical isolates and strains selected for method establishment

Strain/Isolate Year of isolation Susceptibility to macrolides Sequence 23S rRNA mutation P1 gene type MLVA Geographical origin SNP typing ML SNP 23S rRNA mutation Reference
M129(ATCC29342) 1969 S None 1 4572 USA SNP1 None Himmelreich et al. (1996)
FH(ATCC15531) 1954 S None 2 3562 Boston, USA SNP0 None Xiao et al. (2015)
ICDC SY15-11 2012 S None 1 4572 Beijng, China SNP11 None Zhao et al., 2013b
ICDC P028 2008 S None 2 3562 Beijng, China SNP32 None Zhao et al., 2013b
ICDC 21075 2016 S None 2 3572 Beijng, China SNP0 None Zhao et al. (2019a)
ICDC AH-012 2017 R A2063G 1 4572 Fuyang, China SNP11 A2063G Zhao et al. (2019b)
ICDC SD14-08 2017 R A2063G 2 3562 Jinan, China SNP0 A2063G Zhao et al. (2019b)
ICDC SL066 2018 R A2064G 1 4572 Jilin, China SNP3 A2064G Zhao et al. (2019b)
ICDC JSSZ031 2018 R A2063G 1 4573 Soochow, China SNP15 A2063G Zhao et al. (2019b)
ICDC W081 2019 R A2063T 1 4573 Weihai, China SNP15 A2063T This study
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Specificity and detection limit of PCR-MALDI-TOF MS

There was no extension signal on the quality probe for the nucleic acids from the 20 non-M. pneumoniae pathogens, yielding a specificity of 100%. In addition, the nucleic acids of M. pneumoniae M129 strains subjected to 10-fold dilution (100-105copies/μl) were utilized for the validation of LDL, using 1 μL template. In the presence of an M. pneumoniae load of 103-105copies/reaction, all the nine SNP sites could be accurately identified after two tests. In the presence of a load of 102 copies/reaction, 23S rRNA2617 site could be accurately examined only once, while there was no MS peak in the other test. For MPN262, there was no MS peak after two tests, and no signals were noticed in the MS peak in the blank control (Figure 2). The detection limit of the nucleic acid derived from the M. pneumoniae strains was in the range of 102-103copies/reaction.

Figure 2.

Figure 2

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Detection of 9 SNP sites in the presence of different nucleic acid from M. pneumoniae based on the PCR-MALDI-TOF MS method

The test was performed twice for each concentration. a-g: of a concentration range of 105-100copies/μL and the blank control.

Nucleic acid detection of M. pneumoniae isolates

The genotyping and ML susceptibility of the nucleic acids from 141 M. pneumoniae isolates were evaluated using this method. The genotyping and ML susceptibility results are shown in Table 3. For the ML susceptibility sites, 106 were confirmed with the A2063G mutation, 3 with the A2064T mutation, 1 with the A2063T mutation, and 31 with no mutations. The MS findings were consistent with previous sequencing results and drug sensitivity tests. SNP genotyping indicated that there were eight genotypes for the 141 isolates, namely, SNP0, 1, 3, 11, 15, 26, 32, and 27. The 109 strains of genotype I showed an odd number SNP genotype (1, 3, 11, 15, 17), among which SNP 11, 15, and 27 were the major types. Thirty-two genotype II strains showed an even number SNP genotype (0, 26, 32), among which SNP32 was the major type. There were large variations in the number and proportion of the SNP genotypes in the M. pneumoniae isolates (Table 4).

Table 3.

Genotyping and macrolide susceptibility results of M. pneumoniae strains used in this study

Isolates name City year of isolation MLVA type Genotype Nucleotide of SNP
 SNP1-6 to digital string SNP type SNP (odd/even) 23S rRNA SNP
SNP1
 SNP2
 SNP3
 SNP4
 SNP5
 SNP6
 2063
 2064
 2617
aMPN1141461 MPN126470 MPN21347 MPN262192 MPN2801641 MPN3721112 23S rRNA2063 23S rRNA2064 23S rRNA2617
ICDC SD14-02 Jinan 2017 3562 II C C G C G G 000000 0 even A A C
ICDC SD14-07 Jinan 2017 3562 II C C G C G G 000000 0 even A A C
ICDC SD14-09 Jinan 2017 3562 II C C G C G G 000000 0 even A A C
ICDC SD14-26 Jinan 2017 3562 II C C G C G G 000000 0 even A A C
ICDC SD14-50 Jinan 2018 3662 II C C G C G G 000000 0 even G A C
ICDC SD14-58 Jinan 2018 3562 II C C G C G G 000000 0 even A A C
ICDC SD14-61 Jinan 2018 3562 II C C G C G G 000000 0 even G A C
ICDC SD14-69 Jinan 2018 3562 II C C G C G G 000000 0 even A A C
ICDC SD14-77 Jinan 2018 3562 II C C G C G G 000000 0 even A A C
ICDC SD14-13 Jinan 2017 3562 II C T T C T G 011010 26 even A A C
ICDC SD14-91 Jinan 2018 3562 II C T T C T G 011010 26 even A A C
ICDC SD14-03 Jinan 2017 4572 I C C G C T T 000011 3 odd A G C
ICDC SD14-01 Jinan 2017 4572 I C C T C T T 001011 11 odd G A C
ICDC SD14-06 Jinan 2017 4572 I C C T C T T 001011 11 odd G A C
ICDC SD14-22 Jinan 2017 4572 I C C T C T T 001011 11 odd G A C
ICDC SD14-23 Jinan 2017 4572 I C C T C T T 001011 11 odd G A C
ICDC SD14-37 Jinan 2018 4572 I C C T C T T 001011 11 odd G A C
ICDC SD14-88 Jinan 2018 4572 I C C T C T T 001011 11 odd A A C
ICDC SD14-45 Jinan 2018 4572 I C C T T T T 001111 15 odd G A C
ICDC SD14-14 Jinan 2017 4572 I C T T C T T 011011 27 odd G A C
ICDC SD14-25 Jinan 2017 4572 I C T T C T T 011011 27 odd G A C
ICDC SL002 Jilin 2017 4572 I C T T C T T 011011 27 odd G A C
ICDC SL008 Jilin 2017 4572 I C T T C T T 011011 27 odd G A C
ICDC SL011 Jilin 2017 4572 I C T T C T T 011011 27 odd G A C
ICDC SL019 Jilin 2017 4572 I C T T C T T 011011 27 odd G A C
ICDC SL033 Jilin 2018 4572 I C C G C T T 000011 3 odd G A C
ICDC SL037 Jilin 2018 4572 I C T T C T T 011011 27 odd G A C
ICDC SL044 Jilin 2018 4572 I C T T C T T 011011 27 odd G A C
ICDC SL061 Jilin 2018 4573 I C T T C T T 011011 27 odd G A C
ICDC SL066 Jilin 2018 4572 I C T T C T T 011011 27 odd G A C
ICDC SL069 Jilin 2018 4572 I C T T C T T 011011 27 odd G A C
ICDC SL075 Jilin 2018 4572 I C C T T T T 001111 15 odd G A C
ICDC SL083 Jilin 2018 4572 I C C T T T T 001111 15 odd G A C
ICDC SL087 Jilin 2018 4572 I C T T C T T 011011 27 odd G A C
ICDC SL091 Jilin 2018 4572 I C T T C T T 011011 27 odd G A C
ICDC SL093 Jilin 2018 4572 I C T T C T T 011011 27 odd G A C
ICDC SL121 Jilin 2018 4572 I C T T C T T 011011 27 odd G A C
ICDC SL134 Jilin 2018 4572 I C T T C T T 011011 27 odd G A C
ICDC SL144 Jilin 2018 4572 I C T T C T T 011011 27 odd G A C
ICDC SL145 Jilin 2018 4572 I C C T C T T 001011 11 odd G A C
ICDC SL181 Jilin 2018 4572 I C T T C T T 011011 27 odd G A C
ICDC SL182 Jilin 2018 4572 I C T T C T T 011011 27 odd G A C
ICDC SL199 Jilin 2018 4572 I C C T T T T 001111 15 odd G A C
ICDC SL200 Jilin 2018 4572 I C T T C T T 011011 27 odd G A C
ICDC SL251 Jilin 2018 4572 I C C T T T T 001111 15 odd G A C
ICDC SL266 Jilin 2018 4572 I C C T C T T 001011 11 odd G A C
ICDC SL277 Jilin 2018 4472 I C T T C T T 011011 27 odd G A C
ICDC SL281 Jilin 2018 4572 I C C T T T T 001111 15 odd G A C
ICDC SL288 Jilin 2018 4572 I C C T C T T 001011 11 odd G A C
ICDC SL309 Jilin 2018 4572 I C C T T T T 001111 15 odd G A C
ICDC SL331 Jilin 2018 4572 I C C T T T T 001111 15 odd G A C
ICDC BCH001 Beijing 2017 3562 II C C G C G G 000000 0 even A A C
ICDC BCH077 Beijing 2017 3562 II C C G C G G 000000 0 even A A C
ICDC BCH336 Beijing 2018 3562 II C C G C G G 000000 0 even G A C
ICDC BCH423 Beijing 2018 3562 II C C G C G G 000000 0 even A A C
ICDC BCH437 Beijing 2018 3562 II C C G C G G 000000 0 even A A C
ICDC BCH443 Beijing 2018 3562 II C C G C G G 000000 0 even A A C
ICDC BCH118 Beijing 2017 3562 II C T T C T G 011010 26 even A A C
ICDC BCH023 Beijing 2017 4572 I C C T C T T 001011 11 odd G A C
ICDC BCH031 Beijing 2017 4572 I C C T C T T 001011 11 odd G A C
ICDC BCH039 Beijing 2017 4572 I C C T C T T 001011 11 odd G A C
ICDC BCH145 Beijing 2018 4572 I C C T C T T 001011 11 odd G A C
ICDC BCH331 Beijing 2018 4572 I C C T T T T 001111 15 odd G A C
ICDC BCH012 Beijing 2017 4572 I C C T T T T 001111 15 odd G A C
ICDC BCH057 Beijing 2017 4572 I C C T T T T 001111 15 odd G A C
ICDC BCH112 Beijing 2017 4572 I C C T T T T 001111 15 odd G A C
ICDC BCH120 Beijing 2017 4573 I C C T T T T 001111 15 odd G A C
ICDC BCH133 Beijing 2018 4572 I C C T T T T 001111 15 odd G A C
ICDC BCH178 Beijing 2018 4572 I C C T T T T 001111 15 odd G A C
ICDC BCH215 Beijing 2018 4572 I C C T T T T 001111 15 odd G A C
ICDC BCH281 Beijing 2018 4472 I C C T T T T 001111 15 odd G A C
ICDC BCH399 Beijing 2018 4572 I C C T T T T 001111 15 odd G A C
ICDC BCH451 Beijing 2018 4572 I C C T T T T 001111 15 odd G A C
ICDC BCH008 Beijing 2017 4572 I C T T C T T 011011 27 odd G A C
ICDC BCH016 Beijing 2017 4572 I C T T C T T 011011 27 odd G A C
ICDC BCH019 Beijing 2017 4572 I C T T C T T 011011 27 odd G A C
ICDC BCH022 Beijing 2017 4572 I C T T C T T 011011 27 odd A A C
ICDC BCH090 Beijing 2017 4572 I C T T C T T 011011 27 odd G A C
ICDC BCH264 Beijing 2018 4572 I C T T C T T 011011 27 odd G A C
ICDC BCH271 Beijing 2018 4572 I C T T C T T 011011 27 odd G A C
ICDC BCH412 Beijing 2018 4572 I C T T C T T 011011 27 odd G A C
ICDC U016 Beijing 2008 3562 II C C G C G G 000000 0 even A A C
ICDC U098 Beijing 2008 3562 II C C G C G G 000000 0 even A A C
ICDC 12013 Beijing 2012 3562 II C C G C G G 000000 0 even A A C
ICDC 21055 Beijing 2012 3562 II C C G C G G 000000 0 even A A C
ICDC 21077 Beijing 2012 3562 II C C G C G G 000000 0 even A A C
ICDC 21109 Beijing 2013 3562 II C C G C G G 000000 0 even A G C
ICDC B117 Beijing 2013 3562 II C C G C G G 000000 0 even G A C
ICDC P0118 Beijing 2008 3562 II T C G C G G 100000 32 even A A C
ICDC P033 Beijing 2008 3562 II T C G C G G 100000 32 even A A C
ICDC P054 Beijing 2008 3562 II T C G C G G 100000 32 even A A C
ICDC 21010 Beijing 2012 4572 I C C G C G T 000001 1 odd G A C
ICDC SR15-22 Beijing 2015 4472 I C C G C G T 000001 1 odd G A C
ICDC U014 Beijing 2008 4572 I C C G C T T 000011 3 odd G A C
ICDC P396 Beijing 2009 4572 I C C G C T T 000011 3 odd G A C
ICDC 21001 Beijing 2012 4572 I C C T C T T 001011 11 odd G A C
ICDC 12066 Beijing 2013 4572 I C C T C T T 001011 11 odd T A C
ICDC B271 Beijing 2013 4572 I C C T C T T 001011 11 odd G A C
ICDC SY15-77 Beijing 2015 4572 I C C T T T T 001111 15 odd A G C
ICDC P005 Beijing 2008 4572 I C C T T T T 001111 15 odd A A C
ICDC 12075 Beijing 2013 4572 I C C T T T T 001111 15 odd A A C
ICDC B023 Beijing 2013 4572 I C C T T T T 001111 15 odd G A C
ICDC B370 Beijing 2013 4572 I C C T T T T 001111 15 odd G A C
ICDC B385 Beijing 2014 4572 I C C T T T T 001111 15 odd G A C
ICDC P135 Beijing 2008 4573 I C T T C T T 011011 27 odd G A C
ICDC 10577 Beijing 2012 4572 I C T T C T T 011011 27 odd G A C
ICDC 12038 Beijing 2012 4572 I C T T C T T 011011 27 odd G A C
ICDC 21000 Beijing 2012 4573 I C T T C T T 011011 27 odd G A C
ICDC 21114 Beijing 2013 4572 I C T T C T T 011011 27 odd G A C
ICDC B429 Beijing 2014 4572 I C T T C T T 011011 27 odd G A C
ICDC B434 Beijing 2014 4572 I C T T C T T 011011 27 odd G A C
ICDC JSSZ002 Soochow 2017 3562 II C C G C G G 000000 0 even A A C
ICDC JSSZ012 Soochow 2017 3562 II C C G C G G 000000 0 even A A C
ICDC JSSZ023 Soochow 2017 4572 I C C G C T T 000011 3 odd G A C
ICDC JSSZ038 Soochow 2017 4572 I C C G C T T 000011 3 odd G A C
ICDC JSSZ007 Soochow 2017 4572 I C C T C T T 001011 11 odd G A C
ICDC JSSZ010 Soochow 2017 4572 I C C T C T T 001011 11 odd G A C
ICDC JSSZ024 Soochow 2017 4572 I C C T C T T 001011 11 odd G A C
ICDC JSSZ028 Soochow 2017 4572 I C C T C T T 001011 11 odd G A C
ICDC JSSZ037 Soochow 2017 4572 I C C T C T T 001011 11 odd G A C
ICDC JSSZ041 Soochow 2017 4572 I C C T C T T 001011 11 odd G A C
ICDC JSSZ046 Soochow 2017 4572 I C C T C T T 001011 11 odd G A C
ICDC JSSZ047 Soochow 2017 4572 I C C T C T T 001011 11 odd G A C
ICDC JSSZ048 Soochow 2017 4572 I C C T C T T 001011 11 odd G A C
ICDC JSSZ051 Soochow 2017 4572 I C C T C T T 001011 11 odd G A C
ICDC JSSZ055 Soochow 2017 4573 I C C T C T T 001011 11 odd G A C
ICDC JSSZ064 Soochow 2017 4572 I C C T C T T 001011 11 odd G A C
ICDC JSSZ072 Soochow 2018 4572 I C C T C T T 001011 11 odd G A C
ICDC JSSZ078 Soochow 2018 4572 I C C T C T T 001011 11 odd G A C
ICDC JSSZ033 Soochow 2017 4572 I C C T T T T 001111 15 odd G A C
ICDC JSSZ067 Soochow 2018 4572 I C C T T T T 001111 15 odd G A C
ICDC JSSZ071 Soochow 2018 4572 I C C T T T T 001111 15 odd G A C
ICDC JSSZ073 Soochow 2018 4572 I C C T T T T 001111 15 odd G A C
ICDC JSSZ077 Soochow 2018 4572 I C C T T T T 001111 15 odd G A C
ICDC AH-001 Fuyang 2018 3562 II C C G C G G 000000 0 even A A C
ICDC AH-014 Fuyang 2018 3562 II C C G C G G 000000 0 even A A C
ICDC AH-007 Fuyang 2018 4572 I C C G C T T 000011 3 odd G A C
ICDC AH-004 Fuyang 2018 4572 I C C T C T T 001011 11 odd G A C
ICDC AH-006 Fuyang 2018 4572 I C C T C T T 001011 11 odd G A C
ICDC AH-008 Fuyang 2018 4472 I C C T C T T 001011 11 odd G A C
ICDC AH-010 Fuyang 2018 4572 I C C T C T T 001011 11 odd G A C
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The result information for columns 1–5 in the table comes from reference Zhao et al. (2013a), 2013b, Zhao et al. (2019b).

a

ATCC29342 (GenBank: U00089.2) was used as reference. The number after MPN is the gene number, and the superscript number indicates the position of SNP in the gene.

Table 4.

SNP genotyping of 141 Chinese M. pneumoniae isolates, macrolide susceptibility test results, and mimic genotyping of 79 international M. pneumoniae

Year No Genotype 1
 Genotype 2
 23S rRNA
SNP1 SNP3 SNP11 SNP15 SNP27 SNP0 SNP26 SNP32 A2063G A3064G A2063T None
Beijing, China 2008–2012 30 2 2 3 6 7 7 0 3 17 2 1 10
Beijing, China 2017–2018 30 0 0 4 11 8 6 1 0 23 0 0 7
Soochow, China 2017–2018 23 0 2 14 5 0 2 0 0 21 0 0 2
Jinan, China 2017–2018 21 0 1 6 1 2 9 2 0 10 1 0 10
Jilin, China 2017–2018 30 0 1 3 7 19 0 0 0 30 0 0 0
Fuyang, China 2017–2018 7 0 1 4 0 0 2 0 0 5 0 0 2
Total 2008–2018 141 2 7 34 30 36 26 3 3 106 3 1 31
International strains – 79 14 17 4 7 7 30 0 0 – – – –
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Detailed information on each M. pneumoniae strain is shown inTable 3.

Mimic SNP genotyping of international M. pneumoniae strains

There were 6 genotypes, namely, SNP 0,1,3,11,15 and 27, among the genomes of 79 international M. pneumoniae strains with sequences in the NCBI databases. There were 30 strains with SNP 0 genotypes, including AP012303.1, AP017318.1, AP017319.1, CP002077.1, CP010539.1, CP010540.1, CP010546.1, CP010547.1, CP010548.1, CP010549.1, CP010550.1, CP010551.1, CP017327.1, CP017329.1, CP017334.1, CP017335.1, CP017336.1, CP017337.1, CP017338.1, CP017339.1, CP017340.1, CP017341.1, CP017342.1, CP039761.1, CP039772.1, CP039775.1, CP039777.1, CP039781.1, CP039784.1, and LR214945.1. There were 14 SNP 1 genotype including CP003913.2, CP017330.1, CP017343.1, CP020689.1, CP020690.1, CP020691.1, CP020692.1, CP020693.1, CP020710.1, CP020711.1, CP020712.1, CP039787.1, CP039790.1, and U00089.2. There were 17 SNP 3 genotype including CP008895.1, CP010538.1, CP010541.1, CP010542.1, CP010543.1, CP010544.1, CP010545.1, CP017328.1, CP017331.1, CP017332.1, CP017333.1, CP039776.1, CP039779.1, CP039782.1, CP039783.1, CP039785.1, and CP039788.1. There were 4 SNP11 genotypes (i.e. CP013829.1, CP014267.1, CP039773.1, and CP039780.1). There were 7 SNP16 genotypes (CP039762.1, CP039763.1, CP039764.1, CP039765.1, CP039766.1, CP039767.1, and CP039769.1) and 7 SNP27 genotype (CP039768.1, CP039770.1, CP039771.1, CP039774.1, CP039778.1, CP039786.1, and CP039789.1). Among these genotypes, all the odd number SNP genotypes were genotype I, with SNP1 and SNP3 as the major types, and all the even number SNP genotypes were genotype II. Compared with the Chinese strains, the international strains lacked the genotypes SNP26 and SNP32 (Table 4).

Detection results for the nucleic acid positive clinical specimens of M. pneumoniae

Our assay was performed directly on clinical specimens using an increase in the template volume (5 μL) and PCR volume (20 μL). Among the 30 M. pneumoniae nucleic acid positive clinical specimens, 9 SNP sites were detected in 25 isolates (83.3%). The detection limit of the clinical specimens was 5.2×102 copies/reaction. In addition, the genotype and ML susceptibility results in the 25 isolates were consistent with the detection of M. pneumoniae. For the remaining 5 specimens, incomplete SNP profiles were obtained. On this basis, complete data interpretation could not be given.

Discussion

With the increased accessibility to markers through high-throughput whole-genome sequencing (WGS), SNP analysis is increasingly useful for evaluating drug susceptibility, evolution, and molecular epidemiology (Kuglik et al., 2014; Lipworth et al., 2019). To date, the single or small proportion of SNP site detection mainly relies on the first-generation sequencing technique or TaqMAN probe-based real-time PCR detection. For the analysis of multiple SNP sites that are extensively distributed, the WGS technique is the preferred option. However, these techniques are not adequate in the detection of more than tens or dozens of SNP sites. The MS technique facilitates these two SNP-based techniques. Multiplex PCR and the single base pair extension technique have proven to be superior in detecting more than ten SNP sites and are extensively utilized in the genotyping and identification of multiple microorganisms(Jang and Kim, 2018; Kostrzewa, 2018; Peng et al., 2013a, 2013b;Rahi et al., 2016;Sjoholm et al., 2008).

To date, there is still a lack of efficiency regarding genotyping techniques for M. pneumoniae compared with other pathogens. Currently, the most commonly utilized methods are based on the genotyping technique for the two genotypes. These methods are easy to perform, but their application in tracing pathogenic bacteria is hampered due to insufficient discriminatory capacity. In 2009, Degrange et al. established an MLVA genotyping technique using 5 VNTR sites, which divided M. pneumoniae into dozens of MLVA genotypes. However, the classification ability of this method is still limited in practical applications (Benitez et al., 2012;Degrange et al., 2009;Dumke and Jacobs, 2011;Kubota et al., 2015;Pereyre et al., 2013;Qu et al., 2013; Waller et al., 2014;Xue et al., 2014;Yan et al., 2014;Zhao et al., 2013a, 2013b). In 2015, Brown et al. (Brown et al., 2015) established a genotyping technique of MLST. MLST improves the typing ability to a certain extent, but such a technique is not extensively utilized, as it is technically demanding and expensive. In addition, there is a necessity for data uploading. In the same year, Touati et al. developed SNaPshot minisequencing technology based on the single base extension of a specially designed minisequencing primer, which promoted the discrimination of M. pneumoniae. However, the cost was still high, even though it was less than that presented in a previous study (4.2 Euro) (Touati et al., 2015). In this study, 6 SNP genotyping sites, which were derived from 1434 SNPs (Table S1) screened from 20 M. pneumoniae clinical isolates, were utilized as the targets. In addition, all three sites associated with ML susceptibility were included, and then MS-based amplification was established that could simultaneously accomplish M. pneumoniae genotyping of multiple sites and ML susceptibility analysis, which could test 96 samples simultaneously within 6 hr based on PCR amplification and extension (4.5 hr) and MS analysis (0.5 hr). The cost for MS was only 1.0 RMB (approximately 0.1 Euro), which was far less than that reported by Touati (Touati et al., 2015) Meanwhile, our method could provide more data on the ML susceptibility gene based on genotyping. To the best of our knowledge, this method provides the highest throughput for the M. pneumoniae genotyping and ML susceptibility gene testing and involves less time and expenditure than the current methods.

The selected SNP site, MPN3721112 (G1112T), was closely related to the two genotypes of M. pneumoniae, with T and G for genotype I and genotype II, respectively. According to the data interpretation standard, stains with an even number SNP genotype were classified into genotype II, while those with an odd number SNP genotype were classified into genotype I (Table S2). The SNP genotyping of the 141 Chinese isolates showed complete consistency, among which 109 genotype I strains showed an odd number SNP genotype and the 32 genotype II strains showed an even number SNP genotype. Moreover, for the 79 international strains, genome sequencing was consistent with the above rules (Table 3). This indicated that our method could improve the discriminatory capacity and present the genotyping traits of the two conventional genotypes of M. pneumoniae. For the detection of ML susceptibility sites of 23S rRNA2063, 23S rRNA2064 and 23S rRNA20617, 106 isolates showed A2063G, 3 showed A2064G, 1 showenA2063T, and 31 showed no mutations at the 2063/2064 sites. SNP ML susceptibility findings were consistent with the strain sequencing and ML sensitivity test findings (Table 3). The SNP genotyping results indicated that there were 4–7 SNP genotypes in the five selected cities (Table 4). There were differences in the SNP genotypes in different regions between 2017 and 2018. Specifically, SNP27 was the predominant type in Jilin City in northern China, while SNP14 was found in Suzhou in southern China, SNP15 in Beijing in central China, and SNP0 in Jinan in the mid-eastern part of China. The differences in SNP types for the same region in different years were relatively small. For instance, the genotype I M. pneumoniae strains between 2008 and 2012 as well as 2017 and 2018 in Beijing were predominantly of the SNP15 and SNP27 types, while for genotype II, SNP0 was the predominant type. For the 79 international strains, there were 6 SNP genotypes, among which 49 genotype I M. pneumoniae strains showed an odd number SNP type and 30 genotype II M. pneumoniae strains showed an even number SNP type. The international strains with an odd number SNP type were predominantly SNP1 and SNP3, while those of the Chinese strains were SNP11, SNP15, and SNP27, yielding a large variance. This further confirmed the discriminatory capacity of our method.

In addition to genotyping and the ML susceptibility test with pure culture M. pneumoniae nucleic acids, our method could be utilized for the analysis of nucleic acids derived from clinical specimens. The detection limit of the clinical specimens was 5.2×102 copies/reaction, which was relatively consistent with that of nucleic acids isolated from the purified M. pneumoniae strains (102-103copies/reaction). The detection limit was on the same order of magnitude (102 copies/reaction), which was similar to that of the multiplex PCR. The genotyping and ML susceptibility positive rate of the 30 clinical isolates with nucleic acid load after real-time PCR was 83.3%. This implied that our method contributed to the genotyping of the clinical isolates and the ML susceptibility test, which was appropriate for the rapid genotyping of M. pneumoniae with a slow growth and rapid detection of ML susceptibility.

In this study, we developed a multiplex PCR coupled with the MALDI-TOF MS method for the multisite genotyping and ML susceptibility gene testing of M. pneumoniae. The method was easy to perform with a high specificity and a low cost as well as a high throughput. Multisite genotyping and ML susceptibility gene testing could be accomplished simultaneously, with a strong discriminatory capacity for strains of genotype I and genotype II. The analysis of the ML susceptibility site was comprehensive and accurate and deserves clinical application. Meanwhile, the PCR-MALDI-TOF MS technique could meet the demands of various regimens, which may contribute to the microbial identification, genotyping, and drug susceptibility testing.

Limitations of the study

The sensitivity of the method constructed in this study is limited by the detection limit of multiplex PCR technology.

Resource availability

Lead contact

Further information and requests for resources should be directed to and will be fulfilled by the Lead Contact, Prof. Di Xiao, xiaodi@icdc.cn.

Material availability

This study did not generate nor use any new or unique reagents.

Data and code availability

The raw data of MALDI-TOF MS of all the samples used in this study are available on the project homepage at https://dx.doi.org/10.17632/b8pnzp2y6c.1.

Methods

All methods can be found in the accompanying Transparent methods supplemental file.

Acknowledgments

This study was funded by the National Natural Science Foundation of China (Grant No. 81772154), and The Major Infectious Diseases Such as AIDS and Viral Hepatitis Prevention and Control Technology Major Projects (Grant No. 2018ZX10712001&2018ZX10733402).

Author contributions

Z.F. and X.D. conceived and managed the project, analyzed the data, and wrote the manuscript. Z.J.Z searched the literature. W.X.M. acquired the MS data. L.L.Y. and G.J. operated the experiments. Z.Z.X. helped design the primers. H.L.H. and M.F.L. cultured the bacteria.

Declaration of interests

The authors declare no conflicts of interests.

Published: May 21, 2021

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2021.102447.

Supplemental information

Document S1. Transparent methods and Table S2
mmc1.pdf (622.6KB, pdf)
Table S1. Information of 1434 SNPs screened from 20 M. pneumoniae clinical isolates, related to figure 2
mmc2.xlsx (248.4KB, xlsx)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Transparent methods and Table S2
mmc1.pdf (622.6KB, pdf)
Table S1. Information of 1434 SNPs screened from 20 M. pneumoniae clinical isolates, related to figure 2
mmc2.xlsx (248.4KB, xlsx)

Data Availability Statement

The raw data of MALDI-TOF MS of all the samples used in this study are available on the project homepage at https://dx.doi.org/10.17632/b8pnzp2y6c.1.

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