JCM Accepts, published online ahead of print on 30 May 2012 J. Clin. Microbiol. doi:10.1128/jcm.00678-12 Copyright 2012, American Society for Microbiology. All Rights Reserved. 1 2 Multi-clonal origin of macrolide-resistant Mycoplasma pneumoniae isolates determined by multiple-locus variable-number tandem-repeat analysis 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Running title: Multi-clonal origin of macrolide-resistant M. pneumoniae Yang Liu, 1 Xinye Ye, 1 Hong Zhang, 2 Xiaogang Xu, 1 Minggui Wang 1* Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai 200040, P. R. China 1 ; Shanghai Children s Hospital, Shanghai Jiaotong University, Shanghai 200040, P. R. China 2 *Corresponding author. Mailing address: Institute of Antibiotics, Huashan Hospital, Fudan University, 12 M. Wulumuqi Rd., Shanghai 200040, People s Republic of China. Phone: 8621-52888195. Fax: 8621-62482859. E-mail: mgwang@fudan.edu.cn. Key Words: Subtyping; Macrolide resistance; Antimicrobial resistance epidemiology 1
20 Abstract 21 There was a high percentage of macrolide resistance in Mycoplasma pneumoniae clinical 22 isolates in China. The genetic relatedness of macrolide-resistant M. pneunomiae strains was 23 24 25 26 investigated using the multi-locus variable-number tandem-repeat assay (MLVA). Among 152 M. pneunomiae isolates, the 137 macrolide-resistant strains were clustered into 15 MLVA types indicating that the high macrolide-resistance rate in M. pneumoniae is a result of the dissemination of the multiple resistant clones. Downloaded from http://jcm.asm.org/ on November 17, 2018 by guest 2
27 Mycoplasma pneumoniae is one of the most common etiological agents of 28 community-acquired respiratory tract infections, and macrolides are the first-choice 29 antibiotics for treatment of M. pneumoniae infections (14). Prior to the year 2000, macrolide 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 resistance in M. pneunomiae is rare around the world. However, several studies from China described high percentage of macrolide-resistant isolates of M. pneumoniae, ranging from 69% to 92%, obtained from both children and adults between 2003 and 2010 (1, 7, 8, 15). While it is apparent that macrolide-resistant M. pneumoniae isolates are spreading rapidly in certain parts of Asia, the epidemiological mechanism is still unknown. There was no clear association between the macrolide-resistant M. pneumoniae isolates and the P1-RFLP subtypes or PFGE subtypes in previous studies (1). Different typing methods such as conventional PCR (6), restriction fragment length polymorphism (RFLP) (3), pulsed-field gel electrophoresis (3) were developed to differentiate subtypes of M. pneumoniae. However, M. pneumoniae is a highly homogeneous organism and all these methods have a limited power of discrimination. Multi-locus variable-number tandem-repeat analysis (MLVA), which determines the number of tandem repeat sequences (TR) at different loci in a bacterial genome, is highly discriminative and has been successfully applied in typing M. pneumoniae clinical isolates, including the macrolides resistant strains, in Europe (13). In this study, MLVA was used to determine the genetic relationships of the 137 macrolide-resistant M. pneumoniae strains from Shanghai, China in a 5 year period. 46 M. pneumoniae strains. One hundred and fifty-two unique M. pneumoniae clinical 47 isolates were obtained from bronchial aspirates of children with lower respiratory infections 48 in Shanghai, China from December 2005 to July 2009. The distribution of the isolates over 3
49 the time of these 5 years was as follows: a total of 3 to 10 strains were collected in March, 50 June, July and August of these 5 years; 11 to 16 strains in February, October, December and 51 September; 20 to 21 strains in January, April and May. M. pneumoniae culture, PCR 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 amplification of the P1 gene for species identification and antimicrobial susceptibility testing were carried out as described previously (7). The CLSI breakpoint (erythromycin resistance, 1µg/ml) was used to define an isolate as resistant (2). A total of 137 macrolide resistant isolates (90.1%) were identified from our collection. PCR-RFLP analysis. P1 gene PCR-RFLP typing on all 152 clinical strains of M. pneumoniae was performed as previously described (7). M. pneumoniae clinical isolates could only be classified in two types by PCR-RFLP typing method. Of 152 clinical strains, 138 strains were belonged to type I, 12 were type II and 2 could not be identified. Variable-number tandem-repeat (VNTR) locus selection and analysis. A total of 5 VNTR loci with core sequences >9 bp were selected according to the website (http://minisatellites.u-psud.fr/aspsamp/base_ms/interrogation.php, see Table S1 in the supplemental material) and the corresponding PCR primers that were the same as previously described were used for MLVA typing (4). PCR reactions were carried out in a 50 μl volume containing 25 μl of 2 GC PCR buffer (TaKaRa Biotechnology, Dalian, China), 200 μm of each of the four dntps, 10 μm of each primer set, 0.5U TaKaRa La Taq polymerase (TaKaRa Biotechnology, Dalian, China), 5 μl of template DNA and 9.5 μl of water. PCR conditions 68 were as follows: initial denaturation at 95 C for 10 min, and then 40 cycles of 95 C for 1 min, 69 53 C for 1 min and 72 C for 1 min, followed by a final polymerase extension step at 72 C for 70 10 min. All PCR products were sequenced and copy numbers were calculated according to the 4
71 number of repeats for each locus. Two reference strains of M. pneumoniae M129 (ATCC 72 29342) and FH (ATCC15531) were also included as the positive control for the MLVA 73 analysis. 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 All data were input into BioNumerics version 6.5 software (Applied Maths, Belgium), then cluster analysis and minimal spanning tree (MST) analysis were performed. The 152 M. pneumoniae clinical isolates were divided into 17 MLVA types (Table 1). Of these 152 isolates, 137 macrolide resistant strains formed 15 MLVA types. There were 8 most common MLVA types in the resistant isolates, each containing more than 10 resistant strains and accounting for 84% of the total 137 isolates (Table 1). There was no evidence of resistant clonal outbreaks according to diversified MLVA patterns during the 5-years study period. The diversified pattern was also observed in isolates collected in the same time period, e.g. 11 resistant isolates from September, 2008, 15 resistant strains from April, 2009 and 21 resistant strains from May, 2009 were clustered into 7, 8 and 9 MLVA types, respectively. MST population modeling also indicated the diversity among the tested isolates, and no dominant MLVA type could not be determined (Figure 1). Diversity of M. pneumoniae strains. Hunter-Gaston index (HGI) of MLVA typing method was calculated. The formula was as follows: HGI = 1- [nj (nj-1)]/[n (N-1)], where N refers to the total number of experimental strains and nj is the number of strains belonging. Diversity indices of the 5 loci were between 0.000 and 0.812 (see Table S2 in the 90 supplemental material). Locus VNTR1 had higher HGI than others. The numbers of alleles of 91 the 5 loci were between 1 and 7. Loci VNTR1 had the largest number of alleles (n=7). MST 92 modeling and cluster analysis showed the diversity among the tested isolates and no dominant 5
93 MLVA type could not be determined (Figure 1). 94 Our earlier studies indicated that more than 90% of M. pneumoniae strains isolated in 95 Shanghai, China were resistant to macrolides (7, 8). It is known that most of the macrolide 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 resistant M. pneunomiae strains are caused by point mutations in 23S rrna (1, 7, 8, 15). This study used MLVA as an individual identification method to type a large number of macrolide resistant M. pneunomiae isolates for determining the origin of the resistant isolates, whether from a single colony or from multiple clones. In recent years, typing schemes based on MLVA have been designed and implemented for a number of micro-organisms of public health importance (9, 10, 11, 12). Degrange et al. first applied this MLVA method in typing M. pneumoniae isolates and were able to group 265 strains (12 strains were macrolide-resistant) into 26 MLVA types (4). The discrimination potential of MLVA was shown to be much higher than the typing approach based on the differences in the P1 encoding gene. However, MLVA analysis on the limited isolates from France and Japan did not reveal any link between a particular MLVA type and macrolide resistance (4). The present study was the largest MLVA typing worldwide on the macrolide resistant isolates so far. The 137 macrolide-resistant isolates were clustered into 15 MLVA types and none of them could be determined as dominant, indicating the absence of a particular emerging macrolide-resistant clone, and the high rate of macrolide resistance in China is resulted from the dissemination of multiple resistant clones, instead of the spreading of a single colony. 112 113 ACKNOWLEDGEMENTS 114 This work was supported by the National Natural Science Foundation of China (Grant No. 6
115 116 81000753). We thank very much Ken B. Waites and Li Xiao for critical review of the manuscript. 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 References 1. Cao B., et al. 2010. High prevalence of macrolide resistance in Mycoplasma pneumoniae isolates from adult and adolescent patients with respiratory tract infection in China. Clin. Infect. Dis. 51: 189-194. 2. Clinical and laboratory Standards Institute. 2011. Methods for antimicrobial susceptibility testing for human mycoplasmas; approved guideline M43-A. Clinical and Laboratory Standards Institute, Wayne, PA, USA. 3. Cousin-Allery A., et al. 2000. Molecular typing of Mycoplasma pneumoniae strains by PCR-based methods and pulsed-field gel electrophoresis. Application to French and Danish isolates. Epidemiol. Infect. 124: 103-111. 4. Degrange S., et al. 2009. Development of multiple-locus variable-number tandem-repeat analysis for molecular typing of Mycoplasma pneumoniae. J. Clin. Microbiol. 47: 914 923. 5. Dumke R., and E. Jacobs. 2011. Culture-independent multi-locus variable-number tandem-repeat analysis (MLVA) of Mycoplasma pneumoniae. J. Microbiol. Methods. 86: 393-396. 134 6. Kong F., S. Gordon, and G.L. Gilbert. 2000. Rapid-cycle PCR for detection and typing 135 of Mycoplasma pneumoniae in clinical specimens. J. Clin. Microbiol. 38: 4256 4259. 136 7. Liu Y., et al. 2009. Antimicrobial susceptibility of Mycoplasma pneumoniae isolates and 7
137 molecular analysis of macrolide-resistant strains from Shanghai, China. Antimicrob. 138 Agents Chemother. 53: 2160-2. 139 8. Liu Y., et al. 2010. Characterization of macrolide resistance in Mycoplasma pneumoniae 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 isolated from children in Shanghai, China. Diagn. Microbiol. Infect. Dis. 67: 355-358. 9. Schouls L.M., et al. 2005. Increase in genetic diversity of Haemophilus influenzae serotype b (Hib) strains after introduction of Hib vaccination in The Netherlands. J. Clin. Microbiol. 43: 2741 2749. 10. Schouls L.M., et al. 2009. Multiple-locus variable number tandem repeat analysis of Staphylococcus aureus: comparison with pulsed-field gel electrophoresis and spa-typing. PLoS One 4: e5082. 11. Schwartz S., et al. 2009. Identification of P1 variants of Mycoplasma pneumoniae by use of high-resolution melt analysis. J. Clin. Microbiol. 47: 4117 4120. 12. Spuesens E.B., et al. 2010. Macrolide resistance determination and molecular typing of Mycoplasma pneumoniae by pyrosequencing. J. Microbiol. Methods. 82: 214-222. 13. Vergnaud G., and C. Pourcel. 2006. Multiple locus VNTR (variable number of tandem repeat) analysis (MLVA). In E. Stackebrandt (ed.), Molecular identification, systematics and population structure of prokaryotes. Springer-Verlag, Berlin, Germany, p.83-104. 14. Waites K.B., and D.F. Talkington. 2004. Mycoplasma pneumoniae and its role as a human pathogen. Clin. Microbiol. Rev. 17: 697 728. 156 15. Xin D.L., et al. 2009. Molecular mechanisms of macrolide resistance in clinical isolates 157 of Mycoplasma pneumoniae from China. Antimicrob. Agents Chemother. 53: 2158-2159. 158 159 8
160 Table 1. MLVA type distribution of 152 M. pneumoniae clinical isolates MLVA Macrolide Macrolide Year distribution(number of resistant strains) type a susceptible b (n) resistant c (n) 2005 2006 2007 2008 2009 161 162 163 1 2 0 0 0 0 0 0 2 0 14 0 0 0 3 11 3 3 17 0 2 6 0 9 4 1 3 3 0 0 0 0 5 0 13 0 1 1 8 3 6 0 4 0 0 1 1 2 7 4 1 0 0 0 0 1 8 1 17 0 3 3 2 9 9 1 13 0 2 3 5 3 10 1 0 0 0 0 0 0 11 1 20 0 3 5 8 4 12 1 11 0 1 2 5 3 13 0 10 0 0 2 4 4 14 0 1 0 0 0 1 0 15 0 5 0 1 1 2 1 16 0 2 0 0 0 1 1 17 0 6 0 0 0 3 3 total 15 137 3 13 24 43 54 a The MLVA types of two reference strains were not included in this table. Reference strain Mp M129 belonged to MLVA type of 13 and reference strain Mp FH alone showed a separate MLVA type. 164 b Susceptible, erythromycin MIC 0.5µg/ml. 165 c Resistant, erythromycin MIC 1µg/ml. All erythromycin-resistant strains had MIC 64µg/ml in 166 present study. 9
167 10
168 Figure legend: 169 Figure 1. Minimum spanning tree of the MLVA profiles of 152 M. pneumoniae isolates. Each 170 circle denotes a particular MLVA type (MT) indicated by a number corresponding to this genotype 171 172 173 in parentheses. The size of the circle is proportional to the number of isolates belonging to the indicated MLVA genotype. Neighboring genotypes have the same distance. Downloaded from http://jcm.asm.org/ on November 17, 2018 by guest 11
174 175 Figure 1 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 MT4 (4) MT17 (6) MT9 (14) MT14 (13) MT6 (4) MT12 (12) MT8 (18) MT11 (21) MT1 (2) MT2 (14) MT10 (1) MT13 (10) MT15 (5) MT7 (5) MT14 (1) MT3 (20) MT16 (2) 12