Imagine grappling with a respiratory infection that's surging back after years of quiet, and your standard treatment is losing its edge—welcome to the unfolding story of Mycoplasma pneumoniae outbreaks in Canada, where antibiotic resistance is turning heads and sparking urgent questions about public health strategies.
As an early release article, keep in mind that this isn't the final version; any updates will appear in the official online publication once it's released.
The authors hail from esteemed institutions: McMaster University in Hamilton, Ontario, Canada (Z. Fatima, P. Jayaratne, D. Leto, M. Smieja); the Research Institute of St. Joe’s Hamilton, also in Hamilton (Z. Fatima, A. Arrabi, M. Smieja, M.R. Hasan); and the Hamilton Regional Laboratory Medicine Program in Hamilton (P. Jayaratne, C. Rutherford, D. Leto, M. Smieja, M.R. Hasan).
Mycoplasma pneumoniae, often called walking pneumonia because it doesn't always knock you down with obvious symptoms, is a sneaky bacterium that triggers infections in the upper and lower respiratory tract, hitting children especially hard. It cycles through quiet, everyday periods and explosive outbreaks. While it can cause milder issues like tracheobronchitis, its real punch comes in pneumonia, which makes up about 4% to 8% of bacterial pneumonias caught in the community during those calmer endemic times (1). Macrolides, a class of antibiotics including azithromycin and erythromycin, have long been the frontline defense against this bug. But globally, rising resistance to these drugs is complicating treatment, creating a tough puzzle for doctors (2). Since COVID-19 restrictions eased in 2023, M. pneumoniae cases and outbreaks have spiked dramatically around the world (3–5). In Ontario, Canada, there's been a delayed but record-breaking uptick in detections, with positivity rates soaring to as high as 30% since May 2024 (6). In this study, we dove into the macrolide resistance levels and the P1 cytadhesin types—think of P1 as a key protein on the bacterium's surface that helps it stick to human cells—of M. pneumoniae strains during the 2024–2025 outbreak in Hamilton, Ontario, Canada. We compared these to samples collected before the COVID-19 pandemic to spot any changes.
From January 2024 through April 2025, the Microbiology Laboratory at the Hamilton Regional Laboratory Medicine Program received 4,297 nasopharyngeal swab samples from 3,717 patients for M. pneumoniae testing via a custom PCR test developed in-house. After eliminating duplicates, we had 417 positive samples. We checked all of these for macrolide resistance using PCR genotyping, a method that detects genetic markers of resistance. For a deeper look, we performed P1 cytadhesin typing on roughly 25% of the positive samples each month (totaling 110), by amplifying and sequencing the RepMP4 region of the P1 gene using advanced nanopore technology from Oxford Nanopore (available at https://nanoporetech.com). Full details are in the Appendix. To broaden our view, we also tested additional positive samples from 2013–2020 for resistance (45 samples) and P1 types (23 samples). We used statistical tests like the χ² test with Yates correction to check for meaningful differences in detection rates and resistance across groups, adjusting for smaller sample sizes—again, see the Appendix for the nitty-gritty.
Diving into the data, Figure 1 shows that in 2024, an average of 14.2% of tested patients (381 out of 2,680) came back positive for M. pneumoniae, a stark contrast to 0.34% in 2022 (2 out of 576) and 0.36% in 2023 (2 out of 555). Starting in May 2024, positivity climbed steadily, peaking at 22.5% in September. From there, it dropped gradually to under 5% by January 2025, even as testing volume kept rising through December 2024. Macrolide resistance rates fluctuated monthly, hitting an overall 11.8% of positives from January 2024 to April 2025, with a high of 50% in July 2024 (check Figure 1, panel A, and Appendix Table 2). Our PCR genotyping pinpointed just one mutation linked to resistance: A2063G, which causes high-level resistance to macrolides (meaning erythromycin MIC over 64 mg/L). This echoes findings from a 2011–2012 Ontario study where over 90% of isolates had the same mutation (8).
As you might expect, detection rates were notably higher—around 20%—in kids aged 5 to under 18 compared to other age groups. Resistance in this age bracket was about 11% of positives, similar to younger children (under 5) and adults aged 18 to under 65 (see Figure 1, panel B). But here's where it gets intriguing: in patients over 65, 50% of positive strains showed macrolide resistance, significantly more than in the 5–<18 group (p = 0.02). Even though we only had a small number of samples from this older group (n=6), the elevated resistance might tie to more frequent macrolide prescriptions in elderly patients, who often deal with chronic respiratory issues.
Comparing resistance rates between pre-pandemic (2013–2020) and post-pandemic (2024–2025) periods, we found 17.8% resistance before the pandemic and 11.8% after—not a statistically significant difference (p = 0.24) (Figure 1, panel C). And this is the part most people miss: the molecular fingerprints tell a different story. Through P1 typing, 89 out of 110 strains (81%) were P1-1 type, while 21 (19.1%) were P1-2. Resistance was way higher in P1-1 strains at 29.9%, compared to just 7.7% in P1-2 (p = 0.04) (Figure 1, panel D). From 2013–2020, 78.3% were P1-1, rising slightly to 81% in 2024–2025, with no significant shift overall (p = 0.85). Within P1-2 strains, we saw variants like 2k, 2b, and 2g/2j in the earlier period. The 2g/2j variant dominated (50%) in both eras, but 2c/2k variants jumped to 36.4% post-pandemic (Figure 1, panel E).
Figure 2 illustrates our phylogenetic analysis of the RepMP4 region, revealing that nearly all P1-1 strains (95.1%), including 16 from 2017–2020, grouped on a unique branch, distinct from Ontario strains reported over a decade ago (8). That earlier work, from Public Health Ontario Lab, covered samples across the province, including Hamilton (S.N. Patel, pers. comm., 2025 Jul 28). Only two current outbreak strains (MPON05 and MPON71) matched older ones, suggesting P1-1 has evolved locally over time. Among P1-2, 4 strains (15.4%) clustered with known P1-2b variants, and one linked to earlier P1-2c. Plus, we uncovered that P1-2 variants 2g/2j were present in Ontario as far back as 2013, a first for the region.
Of course, no study is perfect. One limitation is that we only typed P1 on a subset of samples and relied on RepMP4 sequencing, which might lump some P1-2 variants into broader groups like 2g/2j or 2c/2k. Also, pre-pandemic samples for genotyping were limited. But despite steady resistance rates, our results highlight a significant evolution in M. pneumoniae's molecular makeup since 2011–2012. Back then, P1-1 accounted for just 38.1% of strains, with P1-2 at 61.9% (8), while now about 80% of our 2024–2025 typed strains are P1-1. Unlike the earlier study, which found no resistance link to P1 types, we see a clear association with higher resistance in P1-1. Post-pandemic, P1-2 variants also diversified more, with 2c/2k expanding. That said, our data focuses solely on Hamilton; other Ontario areas might differ.
Interestingly, the P1-1 vs. P1-2 ratios from 2013–2020 (Figure 1, panel E) don't match the 2011–2012 report (8), possibly because most of our pre-pandemic samples (22 out of 23) came from 2017–2020, with just one from 2013. This hints that the shift toward P1-1 dominance might have started before the pandemic.
In summary, our research offers a fresh look at macrolide resistance and P1 genotypes in M. pneumoniae from Hamilton, Ontario, Canada, about a decade after the last provincial survey. Resistance levels seem unchanged, but the P1 types circulating here have shifted dramatically. Healthcare providers and public health experts need to stay alert to these changes, as they could influence how we diagnose, treat, and prevent M. pneumoniae infections across Ontario.
But here's where it gets controversial: Is this surge in P1-1 strains and linked resistance a sign of natural evolution, or could treatment practices be driving it? And what if global travel and immunity shifts from the pandemic are amplifying these variants—could we be seeing the start of a new dominant strain?
Dr. Fatima, a postdoctoral fellow at the Research Institute of St. Joe’s and McMaster University, specializes in genomic tracking of infectious diseases. Dr. Jayaratne, an associate professor at McMaster University, focuses on creating molecular tests for infections.
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Suggested citation for this article: Fatima Z, Jayaratne P, Arrabi A, Rutherford C, Leto D, Smieja M, et al. Macrolide resistance and P1 cytadhesin genotyping of Mycoplasma pneumoniae during outbreak, Canada, 2024–2025. Emerg Infect Dis. 2025 Dec [date cited]. https://doi.org/10.3201/eid3112.250872
1These first authors contributed equally to this article.
The conclusions, findings, and opinions expressed by authors contributing to this journal do not necessarily reflect the official position of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors' affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.
What do you think? Is the rise in macrolide resistance a red flag for changing antibiotic guidelines, or are we overreacting? Could the P1-1 dominance signal a need for new vaccines or surveillance strategies? Share your thoughts in the comments—do you agree with our findings, or see a different angle?
