Antibiotic-induced microbiota disruption impairs neutrophil-mediated immunity to respiratory <i>Aspergillus fumigatus</i> infection in mice

ABSTRACT Aspergillus fumigatus is an ubiquitous environmental mold that forms airborne conidia and can cause life-threatening infections in immunocompromised individuals. Invasive aspergillosis occurs in patients with quantitative or qualitative neutrophil defects who often receive systemic antibiotics to prevent or manage bacterial infections. Antibiotic-induced bacterial dysbiosis has been linked to impaired neutrophil bactericidal activity and to intestinal commensal bacteria escape during systemic candidiasis, though it remains unclear whether receipt of antibacterial antibiotics impairs neutrophil-dependent defenses against inhaled mold pathogens in the lung. Herein, we measured the outcome of A. fumigatus challenge in C57BL/6J mice that were treated with different antibiotics in the drinking water for 3 weeks prior to experimental infection. We found that ampicillin, but not neomycin or vancomycin treatment, significantly increased murine mortality and lung fungal burden. The heightened susceptibility was associated with impaired fungal killing by lung-infiltrating neutrophils and monocytes, as well as reduced neutrophil production of NADPH oxidase 2 (NOX2)-dependent reactive oxygen species (ROS). These findings demonstrate that systemic antibiotic treatment can compromise pulmonary immunity against A. fumigatus and suggest that the host microbiota can enhance neutrophil fungicidal activity by promoting NOX2-mediated ROS production. IMPORTANCE Aspergillus fumigatus is an environmental mold that causes invasive pulmonary disease in immunocompromised individuals. Owing to limited diagnostic tools, a narrow arsenal of effective treatments, and rising antifungal resistance, the World Health Organization (WHO) has designated A. fumigatus as a critical priority fungal pathogen, highlighting the urgent need for further research. Patients with compromised immunity often receive broad-spectrum antibiotics to prevent or treat opportunistic infections, leading to significant disruption of the resident commensal microbiota. This antibiotic-induced dysbiosis has been linked to Clostridium difficile colitis and to intestinal overgrowth of vancomycin-resistant Enterococcus and Candida parapsilosis , preceding bloodstream infection. However, the impact of antibiotic treatment on susceptibility to invasive pulmonary aspergillosis remains undefined. In this study, we found that oral treatment with ampicillin, but not neomycin or vancomycin, significantly increased mortality in mice following A. fumigatus infection. Neutrophils from the lungs of ampicillin-treated mice also showed markedly impaired fungal killing. These findings raise the possibility that preserving microbiome integrity during antibiotic treatment could enhance immune protection against invasive aspergillosis in at-risk patient groups.