Comparative evaluation and validation of rapid quantification methods for Mycoplasma hyopneumoniae: development of a PMA-based viability qPCR assay

Mycoplasma hyopneumoniae (M. hyopneumoniae), the primary etiological agent of swine enzootic pneumonia, causes significant economic losses in the pork industry. This fastidious pathogen exhibits extremely slow growth in vitro, complicating its quantification. Several quantification methods, including color-changing units (CCU), colony-forming units (CFU), flow cytometry, and ATP luminometry, are documented in the literature, with CCU being the gold standard. However, the correlation among these techniques has not been thoroughly evaluated. Additionally, viability quantitative polymerase chain reaction (v-qPCR) using propidium monoazide (PMA) or ethidium monoazide (EMA) offers a rapid and sensitive alternative for detecting viable bacteria. This study aims to evaluate and compare different methods for quantifying M. hyopneumoniae, validate an accurate real-time quantitative approach, and develop a tailored v-qPCR assay for this challenging pathogen. The in vitro growth kinetics of three M. hyopneumoniae strains (232, J, and 2010) were evaluated using CCU, CFU, flow cytometry, and ATP luminometry. A confocal laser scanning microscopy (CLSM) protocol was established for direct quantification of M. hyopneumoniae in culture media and used to validate flow cytometry-based quantification under controlled conditions. Finally, a rapid v-qPCR assay was developed and optimized for viable M. hyopneumoniae quantification. The comparison of M. hyopneumoniae growth kinetics across CFU, CCU, flow cytometry, and ATP luminometry demonstrated similar growth dynamics and high assay correlation. Flow cytometry and CLSM quantification showed a strong correlation for strain 232 (r = 0.9973) and a moderate correlation for strain J (r = 0.8933). The detection of live M. hyopneumoniae by v-qPCR correlated strongly with viable cell numbers detected by flow cytometry (R² values: 232: 0.9726; J: 0.8628; 2010: 0.9933, p < 0.05). The limit of detection of the v-qPCR assay for the reference strain 232 was 5 × 10⁴ viable M. hyopneumoniae cells/mL. This study presents the first validated PMA-based v-qPCR assay for mycoplasma species, along with established flow cytometry and CLSM protocols for rapid and accurate differentiation of viable and non-viable M. hyopneumoniae cells. These methods significantly reduce quantification time from the four weeks required by CCU or CFU to just a few hours. Furthermore, upon validation in clinical specimens, v-qPCR can potentially serve as a valuable tool in M. hyopneumoniae eradication programs.