- Bacterial aggregation is a strategy employed by many pathogens to establish infection. Mycobacterium avium subsp. hominissuis (MAH) undergoes a phenotypic change, microaggregation, when exposed to the respiratory epithelium. This aggregation is an important step in the pathogenesis of the infection, laying the foundation for biofilm formation. We therefore compared how non-aggregated, or planktonic bacteria, and microaggregated MAH can establish lung infections by evaluating mucosal epithelial cell and phagocytic cell responses. Human mucosal lung epithelial cells (BEAS-2B) recognition of MAH mostly occurs through toll-like receptors 1 and 2, which was confirmed through qRT-PCR, reverse-transcriptase PCR, and Western blotting. For both phenotypes, MAP Kinases 1 and 3 are phosphorylated at 30 minutes post infection, and active at the transcriptional level 2 hours post infection. Microaggregate infected BEAS-2B cells up-regulated CCL5, IL-1β, and TNF-α cDNA, while planktonic infected cells only up-regulated IL-1β cDNA at 2 hours post infection. Microaggregates are associated with increased uptake by macrophages (THP-1 cells) after 1 hour compared to planktonic bacteria (8.83% vs. 5.00%, P < 0.05). In addition, the microaggregate phenotype, when internalized by macrophages, had reduced intracellular growth compared to planktonic bacteria. The intracellular replication, however, increased 4-fold as determined at day 6 post infection, when the host cells were treated with microaggregate supernatant, obtained from the incubation of MAH with HEp-2 epithelial cells. Moreover, when microaggregate supernatant was used to form biofilm, planktonic and microaggregated bacteria had higher biofilm mass as compared to wild type MAH in HBSS. Microaggregate supernatant also induces the production of both pro- and anti-inflammatory cytokines, however, MAH infection decreased the level of pro-inflammatory cytokine secretion. The results suggest that epithelial recognition is occurring during MAH infection, and the microaggregate phenotype stimulates an inflammatory response. In addition, the initial bacterial interaction with the mucosal epithelium and development of the microaggregate phenotype has a potential role in pathogenesis, allowing for more robust biofilm formation and establishment of infection.