Respiratory Bacteria Stabilize and Promote Airborne Transmission of Influenza A Virus

Abstract

Influenza A virus (IAV) is a major pathogen of the human respiratory tract, where the virus coexists and interacts with bacterial populations comprising the respiratory tract microbiome. Synergies between IAV and respiratory bacterial pathogens promote enhanced inflammation and disease burden that exacerbate morbidity and mortality. We demonstrate that direct interactions between IAV and encapsulated bacteria commonly found in the respiratory tract promote environmental stability and infectivity of IAV. Antibiotic-mediated depletion of the respiratory bacterial flora abrogated IAV transmission in ferret models, indicating that these virus-bacterium interactions are operative for airborne transmission of IAV. Restoring IAV airborne transmission in antibiotic-treated ferrets by coinfection with Streptococcus pneumoniae confirmed a role for specific members of the bacterial respiratory community in promoting IAV transmission. These results implicate a role for the bacterial respiratory flora in promoting airborne transmission of IAV.IMPORTANCE Infection with influenza A virus (IAV), especially when complicated with a secondary bacterial infection, is a leading cause of global mortality and morbidity. Gaining a greater understanding of the transmission dynamics of IAV is important during seasonal IAV epidemics and in the event of a pandemic. Direct bacterium-virus interactions are a recently appreciated aspect of infectious disease biology. Direct interactions between IAV and specific bacterial species of the human upper respiratory tract were found to promote the stability and infectivity of IAV during desiccation stress. Viral environmental stability is an important aspect during transmission, suggesting a potential role for bacterial respiratory communities in IAV transmission. Airborne transmission of IAV was abrogated upon depletion of nasal bacterial flora with topical antibiotics. This defect could be functionally complemented by S. pneumoniae coinfection. These data suggest that bacterial coinfection may be an underappreciated aspect of IAV transmission dynamics.

Keywords: Streptococcus pneumoniae; microbiome; transmission.

FIG 1

Nasopharyngeal bacteria promote influenza virus desiccation stability. The indicated bacterial strain was preincubated with influenza virus PR8 (A to C) or A/California/04/2009 (H1N1) (D) or A/Wisconsin/67/2005 (H3N2) (E) followed by centrifugation and washes to remove nonassociated virus and desiccation in a SpeedVac (gray) or was not subjected to desiccation (black). Virus alone was prepared in a small volume (less than 10 μl) of either cell culture media or egg fluid and was directly desiccated in the SpeedVac. (A) Nasopharyngeal tract-colonizing bacteria provide differing degrees of IAV desiccation protection. (B) Pneumococcal viability does not affect desiccation promotion of IAV, as ethanol-killed or ΔspxB pneumococcal mutants with enhanced desiccation tolerance provided protection from IAV infectivity equivalent to that seen with live pneumococci. β-lactam-killed and lysed pneumococci did not promote viability retention. (C) Desiccation survival of IAV with encapsulated and noncapsulated strains of S. pneumoniae and H. influenzae. (D) Pneumococcal capsule and H. influenzae promote stability of A/California/04/2009 (H1N1). (E) H. influenzae serogroup B promotes stability of A/Wisconsin/67/2005 (H3N2). Bars represent means and error bars represent standard deviations of results from at least 6 biological replicates. P values were calculated by Mann-Whitney testing compared to virus desiccated in the absence of bacteria; the dotted line represents the limit of detection. NTHi, nontypeable H. influenzae.

FIG 2

Impact of mupirocin on ferret respiratory tract microbial community composition. (A) The bacterial content of the nasal passages was significantly lower after mupirocin treatment as measured by the number of bacterial 16S copies recovered on nasal swabs prior to and following treatment. (B) Microbiome content of Gram-positive and Gram-negative cocci (specifically, the relative frequencies of Moraxella, Neisseria, Lactococcus, Vagococcus, Enterococcus hirae, “Streptococcus fryi,” and Streptococcus suis) is significantly reduced following treatment. Each dot represents data from a swab collected from an individual ferret. Solid line indicates median for each group, dashed line represents limit of detection. Groups were compared by Mann-Whitney test with a P value of <0.05 being considered significant.

FIG 3

Nasopharyngeal bacteria promote airborne transmission of influenza virus. Donor ferrets were infected with influenza A virus A/California/04/2009 (H1N1) and paired 24 h postinfection with aerosol-contact ferrets in the same cage with perforated dividers separating the animals. (A, C, E, and G) Influenza virus burden in nasal lavage measured by 50% tissue culture infectious dose (TCID₅₀). (B, D, F, and H) Percentage of animals that seroconverted as measured by hemagglutination inhibition (HAI) assay titer greater than 1:80 dilution by day 21 postinfection. Black, donors; red, aerosol contacts. Dotted line represents limit of detection for TCID₅₀ assay. Day, day postinfection of donor animals. (A and B) Ferrets with no manipulation of the respiratory tract microbiota; n = 4 donors and 4 contacts. (C and D) Nostrils of both donor and contact ferrets were treated with mupirocin ointment; n = 5 donors and 5 contacts. (E and F) Donor ferret nostrils were treated with mupirocin ointment; n = 3 donors and 3 contacts. (G and H) Donor ferret nostrils were treated with mupirocin followed by colonization with 10⁶ CFU of S. pneumoniae strain BHN97Mup^RStrep^R. Each data point represents results determined for an individual ferret over time.