As key components of innate immune defense, macrophages are essential in controlling bacterial pathogens, including
group A Streptococcus (GAS). Despite this, only a limited number of studies have analyzed the recovery of GAS from within
human neutrophils and macrophages. Here, we determined the intracellular fate of GAS in human macrophages by using several
quantitative approaches. In both U937 and primary human macrophages, the appearance over time of long GAS chains revealed
that despite GAS-mediated cytotoxicity, replication occurred in viable, propidium iodide-negative macrophages. Whereas the
major virulence factor M1 did not contribute to bacterial growth, a GAS mutant strain deficient in streptolysin O (SLO) was impaired
for intracellular replication. SLO promoted bacterial escape from the GAS-containing vacuole (GCV) into the macrophage
cytosol. Up to half of the cytosolic GAS colocalized with ubiquitin and p62, suggesting that the bacteria were targeted by
the autophagy machinery. Despite this, live imaging of U937 macrophages revealed proficient replication of GAS after GCV rupture,
indicating that escape from the GCV is important for growth of GAS in macrophages. Our results reveal that GAS can replicate
within viable human macrophages, with SLO promoting GCV escape and cytosolic growth, despite the recruitment of autophagy
receptors to bacteria.