Assessment of the in vitro growing dynamics and kinetics of the non-pathogenic J and pathogenic 11 and 232 Mycoplasma hyopneumoniae strains

Abstract

Information on the in vitro growth of pathogenic and non-pathogenic Mycoplasma hyopneumoniae (M. hyopneumoniae) strains is scarce and controversial. Despite its limitations, the colour changing units (CCU) assay is still considered the golden standard titration technique for M. hyopneumoniae culture. Thus, the aims of the present study were: (1) to describe the growth dynamics and kinetics of pathogenic and non-pathogenic M. hyopneumoniae strains, and (2) to monitor the strains' daily growth by ATP luminometry, CCU, colony forming units (CFU), and DNA quantification by real time quantitative PCR (qPCR) and by fluorescent double-stranded DNA (dsDNA) staining, to evaluate them as putative titration methodologies. The growth of the non-pathogenic J (ATCC^®25934™) type strain and the pathogenic 11 (ATCC^®25095™) reference strain and 232 strain was modelled by the Gompertz model. Globally, all three-strain cultures showed the same growing phases as well as similar maximal titres within a particular technique, but for CFU. However, the J strain displayed the fastest growing. During the logarithmic phase of growing, CCU, ATP and M. hyopneumoniae copy titres were strongly and linearly associated, and correlation between techniques could be reliably established. In conclusion, real-time culture titration by means of ATP or molecular assays was useful to describe the in vitro growth of the tested strains. Knowledge about the in vitro growth behaviour of a specific strain in a specific medium may provide several advantages, including information about the time required to reach maximal titres by the culture. Noteworthy, the obtained results refers to the three strains used, so extrapolation to other M. hyopneumoniae strains or culture conditions should be made cautiously.

Figure 1

ATP growth curves of M. hyopneumoniae strains J (filled triangle), 11 (filled square) and 232 (filled circle). Data points represent means of the 2 replicate ATP luminometry measurements (A and B) within each strain. The orange data points indicate when the original medium colour change was first visually detected in each strain culture.

Figure 2

CCU growth curves of M. hyopneumoniae strains J (filled triangle), 11 (filled square) and 232 (filled circle). Data points represent means of the 2 replicate CCU assay measurements (A and B) within each strain. The orange data points indicate when the original medium colour change was first visually detected in each strain culture.

Figure 3

Colonies of M. hyopneumoniae J strain on solid media. Colonies were photographed at 5 days post-inoculation (D5) of A undiluted culture, B 10⁻¹ and C 10⁻² dilution of the culture in ATCC medium. In B, the limit of the culture drop can be differentiated with the colonies observed within such limit. Once inoculated, solid plates were incubated for 7 days at 37 °C and 5% CO₂ and photographed using a microscope at ×40.

Figure 4

Counts of CFU and ATP concentration per millilitre of M. hyopneumoniae J strain culture. The continuous line represents the counts of CFU per millilitre whereas the discontinuous line depicts the ATP concentration per millilitre along the study period.

Figure 5

Mycoplasma hyopneumoniae strains J (filled triangle), 11 (filled square) and 232 (filled circle) copies per millilitre of dsDNA. Data points represent means of the 2 replicate fluorimetry measurements (A and B) within each strain. The orange data points indicate when the original medium colour change was first visually detected in each strain culture.

Figure 6

Mycoplasma hyopneumoniae strains J (filled triangle), 11 (filled square) and 232 (filled circle) copies per millilitre. Data points represent means of the 2 replicate qPCR measurements (A and B) within each strain. The orange data points indicate when the original medium colour change was first visually detected in each strain culture.

Figure 7

Distribution of average prediction error (APE) of M. hyopneumoniae titre data from distinct titration techniques. APE was defined by the Gompertz model for data obtained from the application of ATP luminometry, CCU, fluorimetry and qPCR techniques.