Macrolide-resistant Mycoplasma pneumoniae in humans, Ontario, Canada, 2010-2011.

of M. pneumoniae has not been reported, although Macrolide- isolates development of such resistance after use of increased conof fluoroquinolones or doxycycline has been Resistant centrations demonstrated in in vitro settings (10,11). The Public Health Ontario Laboratory, which is the Mycoplasma reference microbiology laboratory for the province of Ontario, provides molecular testing for detection of M. pneupneumoniae in moniae for hospitalized and ambulatory patients. In August Humans, Ontario, 2011, the positivity rate for specimens with M. pneumoniae to 9.3% and peaked in December 2011 to 17.5%. Canada, 2010–2011 increased During the same time, increased numbers of cases of M. AliReza Eshaghi, Nader Memari, Patrick Tang, Romy Olsha, David J. Farrell, Donald E. Low, Jonathan B. Gubbay, and Samir N. Patel Antimicrobial drug resistance rates for Mycoplasma pneumoniae was determined in clinical specimens and isolates obtained during 2011–2012 in Ontario, Canada. Of 91 M. pneumoniae drug-resistant specimens, 11 (12.1%) carried nucleotide mutations associated with macrolide resistance in the 23S rRNA gene. None of the M. pneumoniae specimens were resistant to fluoroquinolones or tetracyclines. M ycoplasma pneumoniae is a major cause of community-acquired pneumonia among children and adults (1). Macrolides are recommended for treatment of M. pneumoniae pneumonia (1). High rates of macrolide-resistant M. pneumoniae have been reported in China (>90%) and Japan (87.1%) (2,3). In Europe, reports of macrolide resistance have ranged from 3% in Germany to 9.8% in France (4,5). In the United States, 8.2% of M. pneumonia–positive specimens identified during 2007–2010 were resistant to macrolides (6). M. pneumoniae confer macrolide resistance primarily as a result of nucleotide substitutions at specific positions in the V domain of the 23S rRNA gene. Mutations at nt 2063 (A2063T/G), 2064 (A2064G), and 2617 (C2617A/G) have been shown to be associated with increased MICs to macrolides, including erythromycin, azithromycin, and clarithromycin (2,3,7,8). Use of macrolides to treat macrolide-resistant M. pneumoniae result in lower effectiveness and increased clinical severity compared with macrolidesusceptible M. pneumoniae (9). In contrast to macrolides, resistance to quinolones or tetracyclines among clinical Author affiliations: Ontario Agency for Health Protection and Promotion, Toronto, Ontario, Canada (A. Eshaghi, N. Memari, P. Tang, R. Olsha, D.J. Farrell, D.E. Low, J.B. Gubbay, S.N. Patel); University of Toronto, Toronto (D.J. Farrell, D.E. Low, J.B. Gubbay, S.N. Patel); Mount Sinai Hospital, Toronto (D.E. Low, J.B. Gubbay); and The Hospital for Sick Children, Toronto (J.B. Gubbay) DOI: http://dx.doi.org/10.3201/eid1909.121466 pneumoniae were reported throughout Europe. In response to the increased positivity rate and lack of data for Canada on macrolide resistance in M. pneumoniae, we investigated antimicrobial drug susceptibility profiles of M. pneumoniae detected during February 2010–January 2012 by using molecular methods. In addition, available M. pneumoniae isolates were characterized by sequencing the P1 gene to determine the prevalence of circulating types in Ontario, Canada (12,13). The Study During February 1, 2010–January 31, 2012, a total of 2,898 respiratory specimens were tested for M. pneumoniae and Chlamydophila pneumoniae by using a multiplex testing real-time assay (ProPneumo-1 Assay; Gen-Probe Inc., San Diego, CA, USA). A total of 96 specimens were positive for M. pneumoniae, and 16 specimens were positive for C. pneumoniae. Among M. pneumoniae–positive specimens, 67 (70%) and 29 (30%) were from the upper and lower respiratory tract, respectively. Six (6.0%) specimens were collected from children < 4 years of age, 48 (50%) from persons 5–20 years of age, 19 (20%) from persons 21–40 years of age, 19 (20%) from persons 41–60 years of age, and 23 (24%) from persons >65 years of age. All M. pneumoniae– PCR positive specimens were cultured and 42 (44%) of the 96 primary specimens yielded positive isolates. Nested PCR amplification and DNA sequencing of the partial 23S rRNA gene were performed to detect mutations at nucleotide positions 2063, 2064, 2067, 2617 in the 23S rRNA gene, which are associated with macrolide resistance (2,8). In addition to macrolide resistance, molecular determinants of fluoroquinolones (gyrA and parC) and tetracycline (16S rRNA) resistance were also analyzed (10,11). For macrolide resistance, 91 (95%) of 96 specimens were amplified and analyzed for mutations. Mutations that have been associated with macrolide resistance were found in 11 (12.1%) of the 91 specimens (Table 1). Of the 11 isolates with a mutant genotype, 10 (90.9%) contained a mutation at nucleotide position 2063 (A2063G), and 2 (18.2%) specimens had a mutation at position 2064 (A2064G). In 4 isolates, a mixed population of wild type and mutant at position 2063 were identified on sequence chromatograms. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 9, September 2013 1525 DISPATCHES Table 1. Macrolide-resistant Mycoplasma pneumoniae identified in Ontario, Canada, 2010–2011* Substitution in 23s rRNA Specimen Specimen Patient ID no. Age, y/sex collection date source A2063 A2064 A2067 1 H72992–11 43/F 2011 Aug 8 SPT A/G A/G A 2 C706158–11 10/M 2011 Aug 25 NP A/G A A 3 K35611–11 38/F 2011 Sep 1 NP G A A 4 C751048–11 44/F 2011 Sep 8 BAL G A A 5 P54752–11 42/M 2011 Oct 6 NP G A A 6 P54912–11 12/M 2011 Oct 13 NP A/G A A 7 M29279–11 3/F 2011 Dec 9 NP G A A 8 N223472–11 5/M 2011 Dec 14 BAL G A A 9 N223473–11 5/M 2011 Dec 14 BAL A G A 10 C34899–12 10/F 2012 Jan 20 NP G A A 11 C63502–12 37/F 2012 Jan 23 BW A/G A A *ID, idenitifcation; SPT, sputum; NP, nasopharyngeal swab; BAL, bronchoavelor lavage; BW, bronchial washing. One specimen had wild type and co-mutations at positions 2063 and 2064. None of the specimens contained any mutations at positions A2067 or C2617. In addition to macrolide resistance, molecular determinants of fluoroquinolone and tetracycline resistance in M. pneumoniae were examined. A previous report showed that substitutions at position 99 (83 for Escherichia coli) of gyrA and positions 81, 83, and 87 (78, 80, and 84 for E. coli) of parC were associated with fluoroquinolone resistance (10). In our study, none of the isolates contained any mutations that have been associated with fluoroquinolones resistance. Similarly, amplification and sequencing of 16S rRNA gene regions encompassing the tetracycline binding site did not show any mutations at positions 968 (T968C) and 1193 (G1193A), which have been shown to be associated with tetracycline resistance among M. pneumoniae (11). Typing of M. pneumoniae isolates (42/96) by amplification and Sanger sequencing of almost the entire P1 adhesion gene was performed by using primer pairs ADH1/2, ADH3/4, and ADH2BF/R, which amplify 3 fragments of ≈2,280, 2,580 and 767 bp, respectively (13). Sequencing reac (...truncated)