Detection of Viable Streptococcus equi equi Using Propidium Monoazide Polymerase Chain Reaction.

The study investigates the effectiveness of propidium monoazide (PMA) enhanced quantitative real-time PCR (qPCR) in differentiating between viable and nonviable Streptococcus equi bacteria in clinical specimens. The findings suggest that this technique can be used to assess the viability of S. equi, although testing should ideally be performed on fresh specimens for optimal results.

Research Methodology

• The research was conducted on 57 clinical specimens, including both frozen and refrigerated, that were previously determined as positive (36 samples) or suspect positive (21 samples) for S. equi bacterial presence.
• The initial identification was conducted using SeeI qPCR, which was used as the gold standard method.
• These specimens were subjected to two versions of eqbE SEQ2190 triplex qPCR test, one without and one with pretreatment with propidium monoazide (PMA). PMA is a chemical compound employed to differentiate DNA obtained from viable and nonviable bacterial cells by making the DNA from nonviable cells unavailable for qPCR amplification.
• The research team compared the frequency of detection of S. equi in the specimens before and after PMA treatment and measured the cycle threshold values, which reflect the level of bacterial DNA in the samples.

Findings

• The results showed higher cycle thresholds when using PMA, which indicates a mix of heat-killed and viable cells. Higher cycle thresholds mean fewer copies of the bacterial DNA were detected in the sample, suggesting the presence of nonviable cells.
• A smaller fraction of specimens were identified as S. equi positive after PMA treatment, with 6 out of 57 positive for eqbE with PMA, compared to 13 out of 57 for eqbE without PMA. The same trend was observed for SEQ2190, with 10 positive specimens with PMA and 53 positive without PMA.
• Overall, both eqbE and SEQ2190 showed higher mean cycle thresholds after PMA treatment, suggesting that the PMA treatment effectively made a portion of the bacterial DNA unamplifiable due to it being sourced from nonviable cells.

Conclusions

• The results indicate that propidium monoazide-advanced quantitative real-time PCR (PMA qPCR) can successfully differentiate viable from nonviable S. equi bacteria in clinical samples.
• Although PMA facilitated qPCR can help determine the viability of S. equi, it’s recommended that testing should be conducted on fresh specimens to ensure accurate and reliable results.