Macrolide-Resistant Mycoplasma pneumoniae in Humans, Ontario, Canada, 2010–2011

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 isolates of M. pneumoniae has not been reported, although development of such resistance after use of increased concentrations of fluoroquinolones or doxycycline has been demonstrated in in vitro settings (10,11).
The Public Health Ontario Laboratory, which is the reference microbiology laboratory for the province of Ontario, provides molecular testing for detection of M. pneumoniae for hospitalized and ambulatory patients. In August 2011, the positivity rate for specimens with M. pneumoniae increased to 9.3% and peaked in December 2011 to 17.5%. During the same time, increased numbers of cases of M. 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).
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. 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 reactions were performed in both directions by using the Big Dye Terminator Cycle Sequencing Ready Reaction DNA Sequencing Kit in an ABI 3730 or 3750 automated sequencer (Applied Biosystems, Foster City, CA, USA). P1 gene sequencing identified 6-11 variable number tandem repeat AGT sequences. Using sequence typing and comparing homology of nucleotide and amino acid sequences to reference sequences, we found that 16 (38%) isolates belonged to type 1 and shared high homology at nucleotide (99.7%-99.9%) and amino acid (99.5%-99.9%) levels. Seven of the P1 type 1 isolates had a point mutation in E179K, and 3 of these isolates also had a second mutation Q1232E compared with the type 1 reference strain (M129). Twenty-six (62%) isolates were characterized as type 2 and were differentiated within 3 variants (Table 2). Among 5 resistant isolates that were typed, 3 belonged to type 1 and 2 belonged to type 2.

Conclusions
In this study, 12.1% of M. pneumoniae-positive specimens contained mutations that are associated with macrolide resistance, and most (90.9%) specimens had a mutation at nt 2063. This finding is not surprising because this mutation has been shown to be predominant among macrolide-resistant M. pneumoniae and has been associated with high-level resistance (erythromycin MIC >64 mg/L) (2,3,14). None of the specimens contained any mutations at positions 2067 or 2617 because mutations at these positions are rare. Previous studies have shown reduced efficacy rate of macrolides for treating infections with M. pneumoniae isolates containing 3 mutations (7,9).
Typing of the P1 gene showed no clear association between macrolide-resistant isolates and specific subtype  because 3 of the 5 resistant isolates belonged to type 1 and the remaining 2 belonged to type 2; there was no evidence of clonality. This finding is consistent with reports in which macrolide-resistant M. pneumoniae isolates of both types were found (2,5,8). However, according to a recent study in China, where macrolide-resistant M. pneumoniae is highly prevalent (>90%), most isolates were type 1 (15). None of the M. pneumoniae isolates contained any gyrA gene mutations associated with fluoroquinolone resistance or any 16S rRNA gene mutations associated with tetracycline resistance. These findings are consistent with those of a study in which none of the isolates were resistant to fluoroquinolones or minocycline (2).
Dr Eshaghi is a research technologist at the Public Health Ontario Laboratory with extensive experience in molecular microbiology. His research interests are characterization of antimicrobial drug resistance and respiratory viral infections.