Emergence of Macrolide-Resistant Mycoplasma pneumoniae in Hong Kong Is Linked to Increasing Macrolide Resistance in Multilocus Variable-Number Tandem-Repeat Analysis Type 4-5-7-2.

Macrolide-resistant Mycoplasma pneumoniae (MRMP) is rapidly emerging in Asia, but information on the temporal relationship between the increase in macrolide resistance and changes in strain types is scarce. Between 2011 and 2014, M. pneumoniae infection was diagnosed by PCR as part of routine care in a health care region in Hong Kong. Testing was initiated by clinicians, mainly in patients with suspected M. pneumoniae pneumonia. Specimens positive for M. pneumoniae were retrospectively investigated by macrolide resistance genotyping and a four-locus (Mpn13 to -16) multilocus variable-number tandem-repeat analysis (MLVA) scheme. The overall percentage of M. pneumoniae-positive specimens was 17.9%, with annual rates ranging from 9.8% to 27.2%. The prevalence of MRMP had rapidly increased from 13.6% in 2011 to 30.7% in 2012, 36.6% in 2013, and 47.1% in 2014 (P = 0.038). Two major MLVA types, 4-5-7-2 and 3-5-6-2, accounted for 75% to 85% of the infections each year. MLVA types 4-5-7-2 and 3-5-6-2 predominated among macrolide-resistant and macrolide-sensitive groups, respectively. The increase in MRMP was mainly caused by increasing macrolide resistance in the prevalent MLVA type 4-5-7-2, changing from 25.0% in 2011 to 59.1% in 2012, to 89.7% in 2013, and to 100% in 2014 (P < 0.001). In conclusion, increasing MRMP in Hong Kong was linked to a single MLVA type, which was both prevalent and increasingly resistant to macrolides.

Comparison of pyrosequencing, Sanger sequencing, and melting curve analysis for detection of low-frequency macrolide-resistant mycoplasma pneumoniae quasispecies in respiratory specimens.

Macrolide-resistant Mycoplasma pneumoniae (MRMP) is emerging worldwide and has been associated with treatment failure. In this study, we used pyrosequencing to detect low-frequency MRMP quasispecies in respiratory specimens, and we compared the findings with those obtained by Sanger sequencing and SimpleProbe PCR coupled with a melting curve analysis (SimpleProbe PCR). Sanger sequencing, SimpleProbe PCR, and pyrosequencing were successfully performed for 96.7% (88/91), 96.7% (88/91), and 93.4% (85/91) of the M. pneumoniae-positive specimens, respectively. The A-to-G transition at position 2063 was the only mutation identified. Pyrosequencing identified A2063G MRMP quasispecies populations in 78.8% (67/88) of the specimens. Only 38.8% (26/67) of these specimens with the A2063G quasispecies detected by pyrosequencing were found to be A2063G quasispecies by Sanger sequencing or SimpleProbe PCR. The specimens that could be detected by SimpleProbe PCR and Sanger sequencing had higher frequencies of MRMP quasispecies (51% to 100%) than those that could not be detected by those two methods (1% to 44%). SimpleProbe PCR correctly categorized all specimens that were identified as wild type or mutant by Sanger sequencing. The clinical characteristics of the patients were not significantly different when they were grouped by the presence or absence of MRMP quasispecies, while patients with MRMP identified by Sanger sequencing more often required a switch from macrolides to an alternative M. pneumoniae-targeted therapy. The clinical significance of mutant quasispecies should be investigated further with larger patient populations and with specimens obtained before and after macrolide therapy.