For more than a century, blood agar plates have been the only test for beta-hemolysis. Although blood agar cultures are highly predictive for bacterial pathogens, they are too slow to yield actionable information. Here, we show that beta-hemolytic pathogens are able to lyse and release fluorophores encapsulated in sterically stabilized liposomes whereas alpha and gamma-hemolytic bacteria have no effect. By analyzing fluorescence kinetics, beta-hemolytic colonies cultured on agar could be distinguished in real time with 100% accuracy within 6 h. Additionally, end point analysis based on fluorescence intensity and machine-extracted textural features could discriminate between beta-hemolytic and cocultured control colonies with 99% accuracy. In broth cultures, beta-hemolytic bacteria were detectable in under an hour while control bacteria remained negative even the next day. This strategy, called beta-hemolysis triggered-release assay (BETA) has the potential to enable the same-day detection of beta-hemolysis with single-cell sensitivity and high accuracy.
Although Clostridium novyi-NT is an anti-cancer bacterial therapeutic which germinates within hypoxic tumors to kill cancer cells, the actual germination triggers for C. novyi-NT are still unknown. In this study, we screen candidate germinants using combinatorial experimental designs and discover by serendipity that D-valine is a potent germinant, inducing 50% spore germination at 4.2 mM concentration. Further investigation revealed that five D-valine analogs are also germinants and four of these analogs are enantiomeric pairs. This stereoflexible effect of L- and D-amino acids shows that spore germination is a complex process where enantiomeric interactions can be confounders. This study also identifies L-cysteine as a germinant, and hypoxanthine and inosine as co-germinants. Several other amino acids promote (L-valine, L-histidine, L-threonine and L-alanine) or inhibit (L-arginine, L-glycine, L-lysine, L-tryptophan) germination in an interaction-dependent manner. D-alanine inhibits all germination, even in complex growth media. This work lays the foundation for improving the germination efficacy of C. novyi-NT spores in tumors.
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