Survival and safety evaluation of Bifidobacterium longum subsp. longum ZS-8 in healthy adults, determined using PMAxx-qPCR and amplicon sequencing.

Species-level quantitative PCR (qPCR) provides in-depth knowledge of oral probiotics in the human gastrointestinal tract (GIT). However, it lacks the capability to differentiate exogenous strains from native microbiota, nor can it distinguish between live and dead bacteria. In this study, we employed improved propidium monoazide (PMAxx)-qPCR to evaluate the survival and colonization of Bifidobacterium longum subsp. longum ZS-8 (designated ZS-8) on the strain level in the GIT and its impact on human gut microbiota. By spiking in live and dead ZS-8, we demonstrated that strain-level PMAxx-qPCR could identify and quantify the viable ZS-8 in fecal samples accurately. Using this method, we found that, in healthy humans, oral administration of ZS-8 can transiently survive in the GIT, and multi-layer seamless capsules (MLSC) significantly improve the gastrointestinal tolerance and survivability of ZS-8 compared to its powder form. Furthermore, through selective cultivation and PMAxx-microbiome sequencing, we investigated the response of gut viable microbiome to ZS-8. Results showed that, while the microbiota diversity and total viable counts of Bifidobacterium and Lactobacillus remained stable, certain indigenous species of Bifidobacterium and Lactobacillus increased in abundance, confirming ZS-8's probiotic potential in healthy individuals. Overall, our study demonstrates the effectiveness of combining strain-specific comparative genomics with PMAxx-qPCR for evaluating probiotic survival and colonization in the human gut and highlights the safety of ZS-8 oral administration in healthy individuals.

Importance: The survival and colonization of probiotics in the gut are critical for their functional efficacy, yet conventional species-level quantitative PCR (qPCR) fails to distinguish exogenous strains from native microbiota or differentiate live from dead bacteria. By integrating strain-specific comparative genomics with propidium monoazide (PMAxx)-qPCR, we precisely quantified the viability of Bifidobacterium longum ZS-8 at the strain level in the human gut after its oral administration. Our study demonstrated that 1.53-6.90% of cells surviving transit and multi-layer seamless capsules (MLSC) significantly enhanced the gastrointestinal tolerance of ZS-8. While ZS-8 administration did not alter gut microbiota diversity or total viable counts of Bifidobacterium and Lactobacillus, it selectively increased the abundance of specific indigenous beneficial species. This method overcomes the dual limitations of traditional techniques (strain-level specificity and viability discrimination), providing a robust tool for probiotic research. Furthermore, our findings confirm the safety of ZS-8 in healthy individuals and its potential to modulate gut ecology, offering a scientific foundation for personalized probiotic development and clinical translation.