Escherichia coli BarA-UvrY regulates the pks island and kills Staphylococci via the genotoxin colibactin during interspecies competition.

Wound infections are often polymicrobial in nature, biofilm associated and therefore tolerant to antibiotic therapy, and associated with delayed healing. Escherichia coli and Staphylococcus aureus are among the most frequently cultured pathogens from wound infections. However, little is known about the frequency or consequence of E. coli and S. aureus polymicrobial interactions during wound infections. Here we show that E. coli kills Staphylococci, including S. aureus, both in vitro and in a mouse excisional wound model via the genotoxin, colibactin. Colibactin biosynthesis is encoded by the pks locus, which we identified in nearly 30% of human E. coli wound infection isolates. While it is not clear how colibactin is released from E. coli or how it penetrates target cells, we found that the colibactin intermediate N-myristoyl-D-Asn (NMDA) disrupts the S. aureus membrane. We also show that the BarA-UvrY two component system (TCS) senses the environment created during E. coli and S. aureus mixed species interaction, leading to upregulation of pks island genes. Further, we show that BarA-UvrY acts via the carbon storage global regulatory (Csr) system to control pks expression. Together, our data demonstrate the role of colibactin in interspecies competition and show that it is regulated by BarA-UvrY TCS during interspecies competition.

Enterococcus faecalis alters endo-lysosomal trafficking to replicate and persist within mammalian cells.

Enterococcus faecalis is a frequent opportunistic pathogen of wounds, whose infections are associated with biofilm formation, persistence, and recalcitrance toward treatment. We have previously shown that E. faecalis wound infection persists for at least 7 days. Here we report that viable E. faecalis are present within both immune and non-immune cells at the wound site up to 5 days after infection, raising the prospect that intracellular persistence contributes to chronic E. faecalis infection. Using in vitro keratinocyte and macrophage infection models, we show that E. faecalis becomes internalized and a subpopulation of bacteria can survive and replicate intracellularly. E. faecalis are internalized into keratinocytes primarily via macropinocytosis into single membrane-bound compartments and can persist in late endosomes up to 24 h after infection in the absence of colocalization with the lysosomal protease Cathepsin D or apparent fusion with the lysosome, suggesting that E. faecalis blocks endosomal maturation. Indeed, intracellular E. faecalis infection results in heterotypic intracellular trafficking with partial or absent labelling of E. faecalis-containing compartments with Rab5 and Rab7, small GTPases required for the endosome-lysosome trafficking. In addition, E. faecalis infection results in marked reduction of Rab5 and Rab7 protein levels which may also contribute to attenuated Rab incorporation into E. faecalis-containing compartments. Finally, we demonstrate that intracellular E. faecalis derived from infected keratinocytes are significantly more efficient in reinfecting new keratinocytes. Together, these data suggest that intracellular proliferation of E. faecalis may contribute to its persistence in the face of a robust immune response, providing a primed reservoir of bacteria for subsequent reinfection.

Cellular chaining influences biofilm formation and structure in group A Streptococcus.

Group A Streptococcal (GAS) biofilm formation is an important pathological feature contributing to the antibiotic tolerance and progression of various GAS infections. Although a number of bacterial factors have been described to promote in vitro GAS biofilm formation, the relevance of in vitro biofilms to host-associated biofilms requires further understanding. In this study, we demonstrate how constituents of the host environment, such as lysozyme and NaCl, can modulate GAS bacterial chain length and, in turn, shape GAS biofilm morphology and structure. Disruption of GAS chains with lysozyme results in biofilms that are more stable. Based on confocal microscopy, we attribute the increase in biofilm stability to a dense and compact three-dimensional structure produced by de-chained cells. To show that changes in biofilm stability and structure are due to the shortening of bacterial chains and not specific to the activity of lysozyme, we demonstrate that augmented chaining induced by NaCl or deletion of the autolysin gene mur1.2 produced defects in biofilm formation characterized by a loose biofilm architecture. We conclude that GAS biofilm formation can be directly influenced by host and environmental factors through the modulation of bacterial chain length, potentially contributing to persistence and colonization within the host. Further studies of in vitro biofilm models incorporating physiological constituents such as lysozyme may uncover new insights into the physiology of in vivo GAS biofilms.