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Multiple-locus variable-number tandem-repeat analysis (MLVA) of macrolide-susceptible and -resistant Mycoplasma pneumoniae in children in Taiwan
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Journal of the Formosan Medical Association xxx (xxxx) xxx
Available online at www.sciencedirect.com
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Original Article
Multiple-locus variable-number tandemrepeat analysis (MLVA) of macrolidesusceptible and -resistant Mycoplasma pneumoniae in children in Taiwan Chun-Yi Lu a,1, Ting-Yu Yen a,1, Luan-Ying Chang a, Yi-Jen Liau a, Hong-Hsing Liu b,c, Li-Min Huang a,* a Department of Pediatrics, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan b Department of Pediatrics, En Chu Kong Hospital, Sanxia District, New Taipei City, Taiwan c Institute of Molecular and Genomic Medicine, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan
Received 28 September 2019; received in revised form 30 November 2019; accepted 16 December 2019
KEYWORDS Mycoplasma pneumoniae; MLVA; Molecular typing; Macrolide-resistant; Taiwan
Abstract Background/purpose: To date, molecular typing studies on Mycoplasma pneumoniae are limited. We evaluated the molecular types of Mycoplasma pneumoniae in pediatric patients in Taiwan in 2016. Methods: We used real-time quantitative PCR on respiratory specimens to identify M. pneumoniae in children with community-acquired pneumonia. The domain V of their 23S rRNA were sequenced for detection of macrolide-resistant point mutations. Molecular typing with multiple locus variable-number tandem repeat analysis (MLVA) was done for both macrolide-susceptible and -resistance M. pneumoniae samples. Results: M. pneumoniae was detected in 22% (180/826) respiratory samples during the study period. Among all M. pneumoniae-positive samples, 24% (43/180) had harbored macrolideresistant genotypes, and 86% (37/43) of them were A2063G mutation. Forty-two macrolideresistant strains and 20 randomly selected macrolide-susceptible strains underwent MLVA profiling. MLVA 4-5-7-2 was the most frequent type (32/62, 52%), followed by 4-5-7-3 (17/ 62, 27%) and 1-5-6-2 (9/62, 15%). There was a strong association between MLVA 4-5-7-2 and macrolide resistance (p < 0.001). In contrast, M 4-5-7-3 and 1-5-6-2 were related to macrolide susceptibility (p < 0.001, and p Z 0.025, respectively). Conclusion: Macrolide resistance was relatively low (24%) in this age group in 2016 in Taiwan, and A2063G was the dominant point mutation. MLVA 4-5-7-2 was associated with macrolide resistance.
* Corresponding author. No. 8, Chung-Shan South Road, Taipei, 10016, Taiwan. Fax. þ886 2 23147450. E-mail address: [email protected] (L.-M. Huang). 1 Chun-Yi Lu and Ting-Yu Yen contributed equally. https://doi.org/10.1016/j.jfma.2019.12.008 0929-6646/Copyright ª 2019, Formosan Medical Association. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article as: Lu C-Y et al., Multiple-locus variable-number tandem-repeat analysis (MLVA) of macrolide-susceptible and -resistant Mycoplasma pneumoniae in children in Taiwan, Journal of the Formosan Medical Association, https://doi.org/10.1016/ j.jfma.2019.12.008
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C.-Y. Lu et al. Copyright ª 2019, Formosan Medical Association. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).
Introduction Mycoplasma pneumoniae is one of the leading causes of respiratory tract infections (RTIs), especially community acquired pneumonia (CAP) in all age groups. Approximately 10e40% of CAP in pediatric patients is infected with M. pneumoniae.1,2 M. pneumoniae infection causes not only respiratory tract diseases but also extra-pulmonary illness, such as erythema multiforme, reactive arthritis, meningoencephalitis, myocarditis, and hemolytic anemia.3 It is well known that M. pneumoniae lacks a cell wall, and it is intrinsic resistant to b-lactam antibiotics. Therefore, macrolides are usually the first choice to treat children with CAP caused by M. pneumoniae. However, longterm excessive use of such antibiotics has provided selective pressure for macrolide-resistant M. pneumoniae (MRMP) strains worldwide. Since the first case of MRMP was reported in Japan in 2000, MRMP strains were increasing year by year in many countries.4 Macrolide resistance rate is relatively low in Europe and the United States at around 1e10%.5e8 The highest resistant rates occurred in Asia. It was reported to be as high as 90e100% in China and Japan.9e11 The mechanism of the development of MRMP is mainly caused by point mutations at certain sites of domain V on the 23S rRNA. Common mutation sites include A2063 and A2064, which usually induce high-level resistance; whereas mutation at C2617 was associated with low-level resistant.4 In Taiwan, the prevalence of MRMP strains was around 12e23% in 2010e2011,12,13 and all of these strains carried A2063G mutation. No further molecular genotyping study was available in Taiwan. Excessive use of macrolides and expansion of MRMP strains may account for the high rates observed in certain countries. M. pneumoniae epidemic occurred every 4e7 years.14 From recent studies, the most recent epidemic was reported across Japan, China, and England since the end of 2015,15e17 but there was no related study reported in Taiwan. Molecular typing for M. pneumoniae used to be mainly based on sequence differences in the P1 gene which codes for a cytadhesin protein that acts as an important immunogen of the bacteria. By P1 typing, M. pneumoniae could be divided into two distinct types (type 1 and 2) and further categorized into variants (variant 1, variant; 2a, 2b, 2c, 2d, and 2e).18 This approach has a significant disadvantage that its discriminative power is limited. In 2009, D’egrange et al. developed a new method, named multiple-locus variablenumber tandem repeat (VNTR) analysis (MLVA) that provided higher discriminatory power compared to previous methods and was increasingly used for M. pneumoniae typing.19 Afterwards, a modified culture-independent MLVA methods was proposed by Dumke at el in 2011.20 The original scheme was based on VNTR at five loci in the genome. One of the loci, Mpn1, carried an instability
problem, and was finally excluded in the recent proposal scheme.21e23 There has been limited studies focused on MLVA typing of M. pneumoniae worldwide, and none of them were from Taiwan. The aim of this cross-sectional study is to examine the MLVA typing of macrolide-susceptible and -resistant M. pneumoniae encountered in children in Taiwan, which might provide some insights about the emergence of MRMP in Taiwan and other countries.
Methods Sample collection Respiratory specimens were collected from patients 18 years old or younger with CAP for the purpose of routine clinical diagnosis on the clinician’s decision at a tertiary children’s hospital in Taipei, Taiwan. The specimens included sputum, throat swabs, bronchoalveolar lavage fluid, or pleural effusion. Positive samples obtained during January to December 2016 were used for MLVA analysis. Repeated samples from a given patient was excluded from the study. Institutional Review Boards in National Taiwan University Hospital approved this study (Approval number: 201605078RINA). Informed consents were obtained from the patients or their parents or guardians.
Nucleic acid extraction and detection of M. pneumoniae Genomic DNA from each specimen was extracted with Roche MagNA Pure LC total nucleic acid isolation kit
Figure 1 Monthly distribution of positive and negative M. pneumoniae case numbers and positive rates in Taiwan in 2016.
Please cite this article as: Lu C-Y et al., Multiple-locus variable-number tandem-repeat analysis (MLVA) of macrolide-susceptible and -resistant Mycoplasma pneumoniae in children in Taiwan, Journal of the Formosan Medical Association, https://doi.org/10.1016/ j.jfma.2019.12.008
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MLVA and macrolide-resistance of M. Pneumoniae in Taiwan according to the manufacturer’s instructions. M. pneumoniae was detected by an in-house real-time quantitative polymerase chain reaction (qPCR) assay, as previous described.12 Briefly, primers were targeted to the segment of 4596e4725 nucleotides, cytadhesin P1 gene (strain M129 subtype 1): MP-F (50 -CCA ACC AAA CAA CAA CGT TCA-30 ) and MP-R (50 -TAA CGG CAA CAC GTA ATC AGG TC-30 ). The probe (MP-pb) is 6FAM-ACC TTG ACT GGA GGC CGT TA-BHQ1using the LightCycler FastStart DNA Master HybProbe (Roche Molecular Biochemicals, Mannheim, Germany) and FRET probes in a Light Cycler (Roche Molecular Biochemicals). The detection limit of this in-house qPCR was 800 DNA copies/ml regarding the respiratory specimens.
Detection of the macrolide resistance For M. pneumoniae positive specimens, macrolide resistance was detected by amplification of the 1758e2769 nucleotides segment of 23S rRNA, followed by DNA sequencing for point mutation loci (A2063 G/C, A2064 G/C, C2617 G/A). The detail procedure was described previously.12
MLVA typing and minimum spanning tree analysis MLVA typing was done based on a previously described culture-independent method by Dumke et al. with slight modifications.20 Briefly, PCR was carried out using primers targeting the five selected loci containing tandem repeats (Mpn1, Mpn13, Mpn14, Mpn15, Mpn16). The PCR was
Table 1
completed by SapphireAmp Fast PCR Master Mix (Takara Bio Inc., Kyoto, Japan) in 35 cycles of denaturation for 10 s at 95 C, annealing of 10 s at 55 C, and elongation of 10 s at 72 C. PCR products were recovered by Gel/PCR DNA fragment extraction kit (Geneaid, Taiwan), and sequenced by automated DNA sequencing (ABI 3730). The number of repeat units from the sequence results was calculated to define the MLVA type.19 A minimum spanning tree (MST) analysis was performed using Python graph-tool (https:// graph-tool.skewed.de).
Statistical analysis SPSS Statistics version 25th was used for statistical analysis. Chi-square or Fisher’s exact tests was used for comparison of proportions across different groups. A p value of <0.05 was considered significant.
Results A total of 826 specimens from patients aged less than 18 years with CAP during study period were tested. We detected M. pneumoniae by real-time qPCR in 180 (22%) of all specimens. Fig. 1 shows the time distribution of positive and negative cases over different month of the year. The number of M. pneumoniae-positive specimens fluctuated from 9 to 34 in each month, whereas the detection rate increased from January with a peak of 35% in July. Of all M. pneumoniae-positive strains, 43 (24%) were macrolideresistant by detecting point-mutations in 23S rRNA. The
Association of MLVA profiles and macrolide resistance in 62 M. pneumoniae isolates from children with CAP.
MLVA profile 4-loci
3
All 5-loci
4-5-7-2 1-4-5-7-2 2-4-5-7-2 3-4-5-7-2 4-4-5-7-2 5-4-5-7-2 6-4-5-7-2 4-5-7-3 1-4-5-7-3 3-4-5-7-3 4-4-5-7-3 1-5-6-2 1-1-5-6-2 3-1-5-6-2 4-1-5-6-2 5-1-5-6-2 4-5-6-2 2-4-5-6-2 4-4-5-6-2 3-5-7-2 4-3-5-7-2
32 5 2 7 14 3 1 17 14 2 1 9 1 2 4 2 3 1 2 1 1
MSMP (N Z 20)
MRMP (N Z 42)
N (%)
N (%)
2 (10) 0 0 0 1 (5) 1 (5) 0 11 (55) 8 (40) 2 (10) 1 (5) 6 (30) 1 (5) 2 (10) 2 (10) 1 (5) 1 (5) 1 (5) 0 0 0
30 (72) 5 (12) 2 (5) 7 (17) 13 (31) 2 (5) 1 (2) 6 (14) 6 (14) 0 0 3 (7) 0 0 2 (5) 1 (2) 2 (5) 0 2 (5) 1 (2) 1 (2)
P valuea <0.001 0.165 >0.99 0.086 0.025 >0.99 >0.99 <0.001 0.048 0.101 0.323 0.025 0.323 0.101 0.588 0.545 >0.99 0.323 >0.99 >0.99 >0.99
MR mutation (N)
A2063G A2063G A2063G A2063G A2063G A2063G
(3), A2063T (2) (2) (7) (13) (2) (1)
A2063G (3), A2063T (2), A2064G (1)
A2063G (2) A2063G (1)
A2063G (1), A2063T (1) A2063G (1)
MLVA, Multiple Locus Variable-number Tandem Repeat Analysis; M type, MLVA type; MSMP, macrolide-sensitive M. pneumoniae; MRMP, macrolide-resistant M. pneumoniae, MR, macrolide-resistant, N, number. a p values were calculated by Chi-square or Fisher’s exact tests.
Please cite this article as: Lu C-Y et al., Multiple-locus variable-number tandem-repeat analysis (MLVA) of macrolide-susceptible and -resistant Mycoplasma pneumoniae in children in Taiwan, Journal of the Formosan Medical Association, https://doi.org/10.1016/ j.jfma.2019.12.008
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Figure 2 Distribution of MLVA types among macrolide-resistant M. pneumoniae (MRMP) and macrolide-susceptible M. pneumoniae (MSMP).
majority (37/43, 86%) of 23S rRNA mutation was A2063G, followed by A2063T (5/43, 12%) and A2064G (1/43, 2%). A subset of 63 specimens, including all 43 macrolideresistant M. pneumoniae (MRMP) and 20 randomly selected macrolide-susceptible M. pneumoniae (MSMP), were further analyzed by MLVA typing. We used a systematic random sampling method by picking up one specimen from every 7 consecutive macrolide-susceptible specimen. However, there was a MRMP sample unable to fulfill MLVA profile, and it was probably because of the low levels of M. pneumonia nucleic acid. Finally it was excluded in analysis. As a result, sixteen MLVA types were identified using the 5loci scheme (Table 1). When the Mpn1 loci were eliminated (the 4-loci scheme) according to the modified classification proposed by some studies,21e23 only 5 MLVA types were identified. The majority (30/42, 72%) of strains in the MRMP group could be matched to MLVA type 4-5-7-2 by the 4-loci scheme. Strains of this type could be further differentiated into 6 types by using the 5-loci scheme. The most prevalent 5-loci scheme MLVA type was 4-4-5-7-2 (13/42, 31%), followed by types 3-4-5-7-2 (7/42, 17%) and 1-4-5-7-3 (6/42,
14%). On the other hand, most strains in the MSMP group were either 4-5-7-3 (11/20, 55%) or 1-5-6-2 (6/20, 30%) by 4-loci scheme. There were only 2 strains belong to the 4-57-2 type, the most common type for MRMP. When 5-loci scheme was used, 1-4-5-7-3 is the most common MLVA type (8/20, 40%), but most of the MSMP strains could not be matched to any specific type. Difference in distributions of MLVA profiles between MSMP and MRMP is illustrated in Fig. 2. The relationships among MLVA types are demonstrated by a MST (Fig. 3). Red color indicates the number of the resistant samples out of the total in a particular MLVA type. It is shown that MLVA type 4-5-7-2 carried higher macrolide resistant rate, while isolates belonging to MLVA 4-5-7-3 and 1-5-6-2 were more macrolide sensitive. There was a statistically significant association between being MLVA type 4-4-5-7-2 and MRMP (p Z 0.025). Being type 1-4-5-7-3 showed a borderline significance of association with macrolide susceptibility (p Z 0.048). With the 4-loci scheme, MLVA type 4-5-7-2 was more dominant in MRMP group than in MSMP group (72% versus
Figure 3 Minimum spanning tree (MST) analysis of molecular features of 62 M. pneumoniae isolates. Each circle represents a particular MLVA type. The color of the circles indicates susceptible (green) and resistant (red) isolates.
Please cite this article as: Lu C-Y et al., Multiple-locus variable-number tandem-repeat analysis (MLVA) of macrolide-susceptible and -resistant Mycoplasma pneumoniae in children in Taiwan, Journal of the Formosan Medical Association, https://doi.org/10.1016/ j.jfma.2019.12.008
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MLVA and macrolide-resistance of M. Pneumoniae in Taiwan 10%, p < 0.001). In contrast, MLVA type 4-5-7-3 and 1-5-6-2 were more prevalent in the MSMP group than in MRMP group (55% versus 14%, p Z 0.001 and 30% versus 7%, p Z 0.025, respectively).
Discussion MLVA type 4-5-7-2 was a dominant stain of M. pneumoniae in many countries in Asia, Europe, America, and Australia.24e27 Our results showed a similar result in Taiwan. A significant difference in MLVA type distributions among macrolide-susceptible and -resistant M. pneumoniae strains was also shown by our study. MLVA type 4-5-7-2 is the predominant type among MRMP strains, while 4-5-7-3 and 15-6-2 are predominant MSMP types. Relationships between MLVA types and macrolide resistance have been reported before. In 2015, Ho et al. reported that increasing MRMP in Hong Kong was linked to the increase of a specific single MLVA type 4-5-7-2,28 and similar findings were also reported in China and Japan.24,25,29 However, MLVA type 4-5-7-2 is not always associated with macrolide-resistance. For example, MLVA type 4-5-7-2 was also the predominant type in the United States, European, Australia, and Thailand, where the prevalence of MRMP was low.26,27,30e32 The macrolide-resistance rate among MLVA type 4-5-7-2 may change with time. In Japan, the prevalence of A2063G mutation in MLVA type 4-5-7-2 strains was only 0.9% between 2004 and 2010, but increased dramatically to 83.8% between 2011 and 2014 in Japan.25 Macrolides usage serves as a major driving force for the increase of macrolide-resistant M. pneumoniae as it provides survival advantages for M. pneumoniae strains with macrolide resistance. The hypothesis is supported by a study showing M. pneumoniae may develop macrolide resistance during macrolide treatment.33 In some European countries where macrolides usage was more strictly restricted, macrolide resistant rates among M. pneumoniae remained low.34 The macrolide-resistant rates (24%) remained stable in Taiwan when compared with studies done earlier in 2011 from the same community.12,13 However, increase of MRMP is expected in Taiwan for several reasons. First, macrolide resistance rates are extremely high in some of the neighboring countries like China and Japan,9e11 import of MRMP is unavoidable. Second, in the era of universal childhood vaccination with 13-valent pneumococcal conjugate vaccine (PCV13), the incidence of pneumococcal diseases, including pneumonia, has been reduced substantially in Taiwan.35 M. pneumoniae might fill in the niche left by pneumococcus and becomes the most frequently encountered pathogen of CAP in children and adolescents. Frequent use of macrolides and increase of macrolide resistance is expected under such circumstances. Macrolides remain as the first line treatment for children with M. pneumoniae infection. Given the fact that the most prevalent mutation, A2063G, is associated with high-level resistance to macrolides,36 widespread use of macrolides for M. pneumoniae with this mutation is not only ineffective but also dangerous in terms of inducing more MRMP. Delayed usage of appropriate antibiotics against MRMP is related to poor clinical response and prolonged clinical
5 course.37 As a consequence, increase in severe CAP caused by MRMP is also expected. To break the vicious cycle, restraint yet decisive use of macrolides is important. Earlier use of antibiotics other than macrolide for treating M. pneumoniae with decreased macrolide susceptibility is important not only in treating the illness but also in preventing further increase of MRMP. Traveling to or residence in Asian countries like China or Japan might be a risk factor of macrolide resistance and justifies earlier use of second line antibiotics such as fluoroquinolones or tetracyclines when M. pneumoniae infection is highly suspected or diagnosed. There were some limitations in our study. First, all samples were from a single center in one year only are the major limitations of this study. The study results should be interpreted with caution due to possible selection bias. Second, MLVA typing was done in a fraction of all MSMP samples. More extensive surveillance studies are highly expected. In conclusion, this is the first study addressing MLVA profile and its relationships with macrolide resistance in pediatric patients in Taiwan. In 2016, M. pneumoniae was responsible for 22% pediatric patients with CAP. Macrolide resistance genes was detected in 24% strains in Taiwan in 2016. The majority of MRMP strains carried A2063G mutation (86%). The most common types were 4-5-7-2, 4-5-6-2, and 1-5-6-2. MRMP was associated with MLVA 4-5-7-2 stains.
Funding/Support statement The study is supported partly by grants from the National Taiwan University Hospital (107-A138, 108-A138, NTUH.106-S3366), and the Ministry of Science and Technology, Taiwan (107-2314-B-002-168, 108-3017-F-002-004). This work was also financially supported by the ‘Center of Precision Medicine’ from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (NTU108L901401).
Declarations of competing interest None.
Acknowledgments None.
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.jfma.2019.12.008.
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MLVA and macrolide-resistance of M. Pneumoniae in Taiwan replacement with a catch-up primary vaccination Program in Taiwan. Clin Infect Dis 2019 Oct 15;69(9):1581e7. 36. Morozumi M, Takahashi T, Ubukata K. Macrolide-resistant Mycoplasma pneumoniae: characteristics of isolates and clinical aspects of community-acquired pneumonia. J Infect Chemother 2010;16(2):78e86.
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Please cite this article as: Lu C-Y et al., Multiple-locus variable-number tandem-repeat analysis (MLVA) of macrolide-susceptible and -resistant Mycoplasma pneumoniae in children in Taiwan, Journal of the Formosan Medical Association, https://doi.org/10.1016/ j.jfma.2019.12.008
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Original Article
Multiple-locus variable-number tandemrepeat analysis (MLVA) of macrolidesusceptible and -resistant Mycoplasma pneumoniae in children in Taiwan Chun-Yi Lu a,1, Ting-Yu Yen a,1, Luan-Ying Chang a, Yi-Jen Liau a, Hong-Hsing Liu b,c, Li-Min Huang a,* a Department of Pediatrics, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan b Department of Pediatrics, En Chu Kong Hospital, Sanxia District, New Taipei City, Taiwan c Institute of Molecular and Genomic Medicine, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan
Received 28 September 2019; received in revised form 30 November 2019; accepted 16 December 2019
KEYWORDS Mycoplasma pneumoniae; MLVA; Molecular typing; Macrolide-resistant; Taiwan
Abstract Background/purpose: To date, molecular typing studies on Mycoplasma pneumoniae are limited. We evaluated the molecular types of Mycoplasma pneumoniae in pediatric patients in Taiwan in 2016. Methods: We used real-time quantitative PCR on respiratory specimens to identify M. pneumoniae in children with community-acquired pneumonia. The domain V of their 23S rRNA were sequenced for detection of macrolide-resistant point mutations. Molecular typing with multiple locus variable-number tandem repeat analysis (MLVA) was done for both macrolide-susceptible and -resistance M. pneumoniae samples. Results: M. pneumoniae was detected in 22% (180/826) respiratory samples during the study period. Among all M. pneumoniae-positive samples, 24% (43/180) had harbored macrolideresistant genotypes, and 86% (37/43) of them were A2063G mutation. Forty-two macrolideresistant strains and 20 randomly selected macrolide-susceptible strains underwent MLVA profiling. MLVA 4-5-7-2 was the most frequent type (32/62, 52%), followed by 4-5-7-3 (17/ 62, 27%) and 1-5-6-2 (9/62, 15%). There was a strong association between MLVA 4-5-7-2 and macrolide resistance (p < 0.001). In contrast, M 4-5-7-3 and 1-5-6-2 were related to macrolide susceptibility (p < 0.001, and p Z 0.025, respectively). Conclusion: Macrolide resistance was relatively low (24%) in this age group in 2016 in Taiwan, and A2063G was the dominant point mutation. MLVA 4-5-7-2 was associated with macrolide resistance.
* Corresponding author. No. 8, Chung-Shan South Road, Taipei, 10016, Taiwan. Fax. þ886 2 23147450. E-mail address: [email protected] (L.-M. Huang). 1 Chun-Yi Lu and Ting-Yu Yen contributed equally. https://doi.org/10.1016/j.jfma.2019.12.008 0929-6646/Copyright ª 2019, Formosan Medical Association. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article as: Lu C-Y et al., Multiple-locus variable-number tandem-repeat analysis (MLVA) of macrolide-susceptible and -resistant Mycoplasma pneumoniae in children in Taiwan, Journal of the Formosan Medical Association, https://doi.org/10.1016/ j.jfma.2019.12.008
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C.-Y. Lu et al. Copyright ª 2019, Formosan Medical Association. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).
Introduction Mycoplasma pneumoniae is one of the leading causes of respiratory tract infections (RTIs), especially community acquired pneumonia (CAP) in all age groups. Approximately 10e40% of CAP in pediatric patients is infected with M. pneumoniae.1,2 M. pneumoniae infection causes not only respiratory tract diseases but also extra-pulmonary illness, such as erythema multiforme, reactive arthritis, meningoencephalitis, myocarditis, and hemolytic anemia.3 It is well known that M. pneumoniae lacks a cell wall, and it is intrinsic resistant to b-lactam antibiotics. Therefore, macrolides are usually the first choice to treat children with CAP caused by M. pneumoniae. However, longterm excessive use of such antibiotics has provided selective pressure for macrolide-resistant M. pneumoniae (MRMP) strains worldwide. Since the first case of MRMP was reported in Japan in 2000, MRMP strains were increasing year by year in many countries.4 Macrolide resistance rate is relatively low in Europe and the United States at around 1e10%.5e8 The highest resistant rates occurred in Asia. It was reported to be as high as 90e100% in China and Japan.9e11 The mechanism of the development of MRMP is mainly caused by point mutations at certain sites of domain V on the 23S rRNA. Common mutation sites include A2063 and A2064, which usually induce high-level resistance; whereas mutation at C2617 was associated with low-level resistant.4 In Taiwan, the prevalence of MRMP strains was around 12e23% in 2010e2011,12,13 and all of these strains carried A2063G mutation. No further molecular genotyping study was available in Taiwan. Excessive use of macrolides and expansion of MRMP strains may account for the high rates observed in certain countries. M. pneumoniae epidemic occurred every 4e7 years.14 From recent studies, the most recent epidemic was reported across Japan, China, and England since the end of 2015,15e17 but there was no related study reported in Taiwan. Molecular typing for M. pneumoniae used to be mainly based on sequence differences in the P1 gene which codes for a cytadhesin protein that acts as an important immunogen of the bacteria. By P1 typing, M. pneumoniae could be divided into two distinct types (type 1 and 2) and further categorized into variants (variant 1, variant; 2a, 2b, 2c, 2d, and 2e).18 This approach has a significant disadvantage that its discriminative power is limited. In 2009, D’egrange et al. developed a new method, named multiple-locus variablenumber tandem repeat (VNTR) analysis (MLVA) that provided higher discriminatory power compared to previous methods and was increasingly used for M. pneumoniae typing.19 Afterwards, a modified culture-independent MLVA methods was proposed by Dumke at el in 2011.20 The original scheme was based on VNTR at five loci in the genome. One of the loci, Mpn1, carried an instability
problem, and was finally excluded in the recent proposal scheme.21e23 There has been limited studies focused on MLVA typing of M. pneumoniae worldwide, and none of them were from Taiwan. The aim of this cross-sectional study is to examine the MLVA typing of macrolide-susceptible and -resistant M. pneumoniae encountered in children in Taiwan, which might provide some insights about the emergence of MRMP in Taiwan and other countries.
Methods Sample collection Respiratory specimens were collected from patients 18 years old or younger with CAP for the purpose of routine clinical diagnosis on the clinician’s decision at a tertiary children’s hospital in Taipei, Taiwan. The specimens included sputum, throat swabs, bronchoalveolar lavage fluid, or pleural effusion. Positive samples obtained during January to December 2016 were used for MLVA analysis. Repeated samples from a given patient was excluded from the study. Institutional Review Boards in National Taiwan University Hospital approved this study (Approval number: 201605078RINA). Informed consents were obtained from the patients or their parents or guardians.
Nucleic acid extraction and detection of M. pneumoniae Genomic DNA from each specimen was extracted with Roche MagNA Pure LC total nucleic acid isolation kit
Figure 1 Monthly distribution of positive and negative M. pneumoniae case numbers and positive rates in Taiwan in 2016.
Please cite this article as: Lu C-Y et al., Multiple-locus variable-number tandem-repeat analysis (MLVA) of macrolide-susceptible and -resistant Mycoplasma pneumoniae in children in Taiwan, Journal of the Formosan Medical Association, https://doi.org/10.1016/ j.jfma.2019.12.008
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MLVA and macrolide-resistance of M. Pneumoniae in Taiwan according to the manufacturer’s instructions. M. pneumoniae was detected by an in-house real-time quantitative polymerase chain reaction (qPCR) assay, as previous described.12 Briefly, primers were targeted to the segment of 4596e4725 nucleotides, cytadhesin P1 gene (strain M129 subtype 1): MP-F (50 -CCA ACC AAA CAA CAA CGT TCA-30 ) and MP-R (50 -TAA CGG CAA CAC GTA ATC AGG TC-30 ). The probe (MP-pb) is 6FAM-ACC TTG ACT GGA GGC CGT TA-BHQ1using the LightCycler FastStart DNA Master HybProbe (Roche Molecular Biochemicals, Mannheim, Germany) and FRET probes in a Light Cycler (Roche Molecular Biochemicals). The detection limit of this in-house qPCR was 800 DNA copies/ml regarding the respiratory specimens.
Detection of the macrolide resistance For M. pneumoniae positive specimens, macrolide resistance was detected by amplification of the 1758e2769 nucleotides segment of 23S rRNA, followed by DNA sequencing for point mutation loci (A2063 G/C, A2064 G/C, C2617 G/A). The detail procedure was described previously.12
MLVA typing and minimum spanning tree analysis MLVA typing was done based on a previously described culture-independent method by Dumke et al. with slight modifications.20 Briefly, PCR was carried out using primers targeting the five selected loci containing tandem repeats (Mpn1, Mpn13, Mpn14, Mpn15, Mpn16). The PCR was
Table 1
completed by SapphireAmp Fast PCR Master Mix (Takara Bio Inc., Kyoto, Japan) in 35 cycles of denaturation for 10 s at 95 C, annealing of 10 s at 55 C, and elongation of 10 s at 72 C. PCR products were recovered by Gel/PCR DNA fragment extraction kit (Geneaid, Taiwan), and sequenced by automated DNA sequencing (ABI 3730). The number of repeat units from the sequence results was calculated to define the MLVA type.19 A minimum spanning tree (MST) analysis was performed using Python graph-tool (https:// graph-tool.skewed.de).
Statistical analysis SPSS Statistics version 25th was used for statistical analysis. Chi-square or Fisher’s exact tests was used for comparison of proportions across different groups. A p value of <0.05 was considered significant.
Results A total of 826 specimens from patients aged less than 18 years with CAP during study period were tested. We detected M. pneumoniae by real-time qPCR in 180 (22%) of all specimens. Fig. 1 shows the time distribution of positive and negative cases over different month of the year. The number of M. pneumoniae-positive specimens fluctuated from 9 to 34 in each month, whereas the detection rate increased from January with a peak of 35% in July. Of all M. pneumoniae-positive strains, 43 (24%) were macrolideresistant by detecting point-mutations in 23S rRNA. The
Association of MLVA profiles and macrolide resistance in 62 M. pneumoniae isolates from children with CAP.
MLVA profile 4-loci
3
All 5-loci
4-5-7-2 1-4-5-7-2 2-4-5-7-2 3-4-5-7-2 4-4-5-7-2 5-4-5-7-2 6-4-5-7-2 4-5-7-3 1-4-5-7-3 3-4-5-7-3 4-4-5-7-3 1-5-6-2 1-1-5-6-2 3-1-5-6-2 4-1-5-6-2 5-1-5-6-2 4-5-6-2 2-4-5-6-2 4-4-5-6-2 3-5-7-2 4-3-5-7-2
32 5 2 7 14 3 1 17 14 2 1 9 1 2 4 2 3 1 2 1 1
MSMP (N Z 20)
MRMP (N Z 42)
N (%)
N (%)
2 (10) 0 0 0 1 (5) 1 (5) 0 11 (55) 8 (40) 2 (10) 1 (5) 6 (30) 1 (5) 2 (10) 2 (10) 1 (5) 1 (5) 1 (5) 0 0 0
30 (72) 5 (12) 2 (5) 7 (17) 13 (31) 2 (5) 1 (2) 6 (14) 6 (14) 0 0 3 (7) 0 0 2 (5) 1 (2) 2 (5) 0 2 (5) 1 (2) 1 (2)
P valuea <0.001 0.165 >0.99 0.086 0.025 >0.99 >0.99 <0.001 0.048 0.101 0.323 0.025 0.323 0.101 0.588 0.545 >0.99 0.323 >0.99 >0.99 >0.99
MR mutation (N)
A2063G A2063G A2063G A2063G A2063G A2063G
(3), A2063T (2) (2) (7) (13) (2) (1)
A2063G (3), A2063T (2), A2064G (1)
A2063G (2) A2063G (1)
A2063G (1), A2063T (1) A2063G (1)
MLVA, Multiple Locus Variable-number Tandem Repeat Analysis; M type, MLVA type; MSMP, macrolide-sensitive M. pneumoniae; MRMP, macrolide-resistant M. pneumoniae, MR, macrolide-resistant, N, number. a p values were calculated by Chi-square or Fisher’s exact tests.
Please cite this article as: Lu C-Y et al., Multiple-locus variable-number tandem-repeat analysis (MLVA) of macrolide-susceptible and -resistant Mycoplasma pneumoniae in children in Taiwan, Journal of the Formosan Medical Association, https://doi.org/10.1016/ j.jfma.2019.12.008
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Figure 2 Distribution of MLVA types among macrolide-resistant M. pneumoniae (MRMP) and macrolide-susceptible M. pneumoniae (MSMP).
majority (37/43, 86%) of 23S rRNA mutation was A2063G, followed by A2063T (5/43, 12%) and A2064G (1/43, 2%). A subset of 63 specimens, including all 43 macrolideresistant M. pneumoniae (MRMP) and 20 randomly selected macrolide-susceptible M. pneumoniae (MSMP), were further analyzed by MLVA typing. We used a systematic random sampling method by picking up one specimen from every 7 consecutive macrolide-susceptible specimen. However, there was a MRMP sample unable to fulfill MLVA profile, and it was probably because of the low levels of M. pneumonia nucleic acid. Finally it was excluded in analysis. As a result, sixteen MLVA types were identified using the 5loci scheme (Table 1). When the Mpn1 loci were eliminated (the 4-loci scheme) according to the modified classification proposed by some studies,21e23 only 5 MLVA types were identified. The majority (30/42, 72%) of strains in the MRMP group could be matched to MLVA type 4-5-7-2 by the 4-loci scheme. Strains of this type could be further differentiated into 6 types by using the 5-loci scheme. The most prevalent 5-loci scheme MLVA type was 4-4-5-7-2 (13/42, 31%), followed by types 3-4-5-7-2 (7/42, 17%) and 1-4-5-7-3 (6/42,
14%). On the other hand, most strains in the MSMP group were either 4-5-7-3 (11/20, 55%) or 1-5-6-2 (6/20, 30%) by 4-loci scheme. There were only 2 strains belong to the 4-57-2 type, the most common type for MRMP. When 5-loci scheme was used, 1-4-5-7-3 is the most common MLVA type (8/20, 40%), but most of the MSMP strains could not be matched to any specific type. Difference in distributions of MLVA profiles between MSMP and MRMP is illustrated in Fig. 2. The relationships among MLVA types are demonstrated by a MST (Fig. 3). Red color indicates the number of the resistant samples out of the total in a particular MLVA type. It is shown that MLVA type 4-5-7-2 carried higher macrolide resistant rate, while isolates belonging to MLVA 4-5-7-3 and 1-5-6-2 were more macrolide sensitive. There was a statistically significant association between being MLVA type 4-4-5-7-2 and MRMP (p Z 0.025). Being type 1-4-5-7-3 showed a borderline significance of association with macrolide susceptibility (p Z 0.048). With the 4-loci scheme, MLVA type 4-5-7-2 was more dominant in MRMP group than in MSMP group (72% versus
Figure 3 Minimum spanning tree (MST) analysis of molecular features of 62 M. pneumoniae isolates. Each circle represents a particular MLVA type. The color of the circles indicates susceptible (green) and resistant (red) isolates.
Please cite this article as: Lu C-Y et al., Multiple-locus variable-number tandem-repeat analysis (MLVA) of macrolide-susceptible and -resistant Mycoplasma pneumoniae in children in Taiwan, Journal of the Formosan Medical Association, https://doi.org/10.1016/ j.jfma.2019.12.008
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MLVA and macrolide-resistance of M. Pneumoniae in Taiwan 10%, p < 0.001). In contrast, MLVA type 4-5-7-3 and 1-5-6-2 were more prevalent in the MSMP group than in MRMP group (55% versus 14%, p Z 0.001 and 30% versus 7%, p Z 0.025, respectively).
Discussion MLVA type 4-5-7-2 was a dominant stain of M. pneumoniae in many countries in Asia, Europe, America, and Australia.24e27 Our results showed a similar result in Taiwan. A significant difference in MLVA type distributions among macrolide-susceptible and -resistant M. pneumoniae strains was also shown by our study. MLVA type 4-5-7-2 is the predominant type among MRMP strains, while 4-5-7-3 and 15-6-2 are predominant MSMP types. Relationships between MLVA types and macrolide resistance have been reported before. In 2015, Ho et al. reported that increasing MRMP in Hong Kong was linked to the increase of a specific single MLVA type 4-5-7-2,28 and similar findings were also reported in China and Japan.24,25,29 However, MLVA type 4-5-7-2 is not always associated with macrolide-resistance. For example, MLVA type 4-5-7-2 was also the predominant type in the United States, European, Australia, and Thailand, where the prevalence of MRMP was low.26,27,30e32 The macrolide-resistance rate among MLVA type 4-5-7-2 may change with time. In Japan, the prevalence of A2063G mutation in MLVA type 4-5-7-2 strains was only 0.9% between 2004 and 2010, but increased dramatically to 83.8% between 2011 and 2014 in Japan.25 Macrolides usage serves as a major driving force for the increase of macrolide-resistant M. pneumoniae as it provides survival advantages for M. pneumoniae strains with macrolide resistance. The hypothesis is supported by a study showing M. pneumoniae may develop macrolide resistance during macrolide treatment.33 In some European countries where macrolides usage was more strictly restricted, macrolide resistant rates among M. pneumoniae remained low.34 The macrolide-resistant rates (24%) remained stable in Taiwan when compared with studies done earlier in 2011 from the same community.12,13 However, increase of MRMP is expected in Taiwan for several reasons. First, macrolide resistance rates are extremely high in some of the neighboring countries like China and Japan,9e11 import of MRMP is unavoidable. Second, in the era of universal childhood vaccination with 13-valent pneumococcal conjugate vaccine (PCV13), the incidence of pneumococcal diseases, including pneumonia, has been reduced substantially in Taiwan.35 M. pneumoniae might fill in the niche left by pneumococcus and becomes the most frequently encountered pathogen of CAP in children and adolescents. Frequent use of macrolides and increase of macrolide resistance is expected under such circumstances. Macrolides remain as the first line treatment for children with M. pneumoniae infection. Given the fact that the most prevalent mutation, A2063G, is associated with high-level resistance to macrolides,36 widespread use of macrolides for M. pneumoniae with this mutation is not only ineffective but also dangerous in terms of inducing more MRMP. Delayed usage of appropriate antibiotics against MRMP is related to poor clinical response and prolonged clinical
5 course.37 As a consequence, increase in severe CAP caused by MRMP is also expected. To break the vicious cycle, restraint yet decisive use of macrolides is important. Earlier use of antibiotics other than macrolide for treating M. pneumoniae with decreased macrolide susceptibility is important not only in treating the illness but also in preventing further increase of MRMP. Traveling to or residence in Asian countries like China or Japan might be a risk factor of macrolide resistance and justifies earlier use of second line antibiotics such as fluoroquinolones or tetracyclines when M. pneumoniae infection is highly suspected or diagnosed. There were some limitations in our study. First, all samples were from a single center in one year only are the major limitations of this study. The study results should be interpreted with caution due to possible selection bias. Second, MLVA typing was done in a fraction of all MSMP samples. More extensive surveillance studies are highly expected. In conclusion, this is the first study addressing MLVA profile and its relationships with macrolide resistance in pediatric patients in Taiwan. In 2016, M. pneumoniae was responsible for 22% pediatric patients with CAP. Macrolide resistance genes was detected in 24% strains in Taiwan in 2016. The majority of MRMP strains carried A2063G mutation (86%). The most common types were 4-5-7-2, 4-5-6-2, and 1-5-6-2. MRMP was associated with MLVA 4-5-7-2 stains.
Funding/Support statement The study is supported partly by grants from the National Taiwan University Hospital (107-A138, 108-A138, NTUH.106-S3366), and the Ministry of Science and Technology, Taiwan (107-2314-B-002-168, 108-3017-F-002-004). This work was also financially supported by the ‘Center of Precision Medicine’ from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (NTU108L901401).
Declarations of competing interest None.
Acknowledgments None.
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.jfma.2019.12.008.
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Please cite this article as: Lu C-Y et al., Multiple-locus variable-number tandem-repeat analysis (MLVA) of macrolide-susceptible and -resistant Mycoplasma pneumoniae in children in Taiwan, Journal of the Formosan Medical Association, https://doi.org/10.1016/ j.jfma.2019.12.008