Enterococcus faecalis and Staphylococcus aureus mixed species infection attenuates pathogen-specific neutrophil responses and impairs bacterial clearance

Enterococcus faecalis is an opportunistic pathogen that is frequently co-isolated with other microbes in catheterized urinary tract infections and chronically infected wounds. While E. faecalis can subvert the host immune response and promote the survival of other microbes via interbacterial synergy, little is known about the impact of immune suppression mediated by E. faecalis and how E. faecalis impacts the survival of co-infecting microbes. We hypothesized that E. faecalis can attenuate neutrophil-mediated responses in mixed-species infection to promote survival of the co-infecting species. Here, we show that E. faecalis and Staphylococcus aureus mono-species infection activates intracellular ROS production and NET formation, respectively, enabling effective neutrophil-mediated control of the microbial infection. Growth of both bacterial species was enhanced during co-infection in neutrophils in vitro and in wounds in vivo. E. faecalis reduced S. aureus -induced NET formation and S. aureus suppressed E. faecalis- induced intracellular ROS production. When the species ratios were skewed, the neutrophil reaction profile resembled that elicited by the more abundant species, favoring enhanced survival of the less abundant species. These findings highlight the complexity of the immune response to polymicrobial infections and show that attenuated pathogen-specific immune responses contribute to microbial survival in the mammalian host. ### Competing Interest Statement The authors have declared no competing interest. * Abbreviations: Abbreviations : Definition ATP : Adenosine triphosphate BHI : Brain-heart infusion CAUTI : Catheter-associated urinary tract infection CFU : Colony-forming unit DCFDA : Dichlorofluorescin diacetate dpi : Days post infection DPI : Diphenyleneiodonium DMSO : Dimethyl sulfoxide E.f. : Enterococcus faecalis H3Cit : Citrullinated histone H3 HBSS : Hanks’ balanced salt solution hpi : Hours post infection MACS : Magnetic-activated cell sorting MOI : Multiplicity of infection MPO : Myeloperoxidase NE : Neutrophil elastase NET : Neutrophil extracellular trap NMS : Normal mouse serum NS : Not significant PAD4 : Peptidylarginine deiminase 4 PAMP : Pathogen-associated molecular pattern PBS : Phosphate buffered saline ROS : Reactive oxygen species S.a. : Staphylococcus aureus TBHP : tert-butyl hydrogen peroxide TSB : Tryptic soy broth