Detection of influenza virus and Streptococcus pneumoniae in air sampled from co-infected ferrets and analysis of their influence on pathogen stability

Abstract

Secondary infection with Streptococcus pneumoniae has contributed significantly to morbidity and mortality during multiple influenza virus pandemics and remains a common threat today. During a concurrent infection, both pathogens can influence the transmission of each other, but the mechanisms behind this are unclear. In this study, condensation air sampling and cyclone bioaerosol sampling were performed using ferrets first infected with the 2009 H1N1 pandemic influenza virus (H1N1pdm09) and secondarily infected with S. pneumoniae strain D39 (Spn). We detected viable pathogens and microbial nucleic acid in expelled aerosols from co-infected ferrets, suggesting that these microbes could be present in the same respiratory expulsions. To assess whether microbial communities impact pathogen stability within an expelled droplet, we performed experiments measuring viral and bacterial persistence in 1 µL droplets. We observed that H1N1pdm09 stability was unchanged in the presence of Spn. Further, Spn stability was moderately increased in the presence of H1N1pdm09, although the degree of stabilization differed between airway surface liquid collected from individual patient cultures. These findings are the first to collect both pathogens from the air and in doing so, they provide insight into the interplay between these pathogens and their hosts.IMPORTANCEThe impact of microbial communities on transmission fitness and environmental persistence is under-studied. Environmental stability of microbes is crucial to identifying transmission risks and mitigation strategies, such as removal of contaminated aerosols and decontamination of surfaces. Co-infection with S. pneumoniae is very common during influenza virus infection, but little work has been done to understand whether S. pneumoniae alters stability of influenza virus, or vice versa, in a relevant system. Here, we demonstrate that influenza virus and S. pneumoniae are expelled by co-infected hosts. Our stability assays did not reveal any impact of S. pneumoniae on influenza virus stability, but did show a trend towards increased stability of S. pneumoniae in the presence of influenza viruses. Future work characterizing environmental persistence of viruses and bacteria should include microbially complex solutions to better mimic physiologically relevant conditions.

Keywords: Streptococcus pneumoniae; aerosols; influenza; persistence; stability; survival.

Fig 1

Co-infected ferrets shed H1N1pdm09 and Spn. Ferrets were infected with 10⁶ TCID₅₀ of H1N1pdm09 and subsequently infected 2 d later with 10⁷ CFU S. pneumoniae D39. (A) Nasal wash loads of H1N1pdm09 and Spn are shown for the days following initial H1N1pdm09 infection. (B) Condensation sampling with a Liquid Spot Sampler was used to collect infectious virus and bacteria shed by co-infected animals. Viral and bacterial loads were measured by TCID₅₀ and CFU assays, respectively. (C–E) Cyclone-based air samplers were used to fractionate and collect microbial genomic material shed from co-infected ferrets in (C) >4 µm droplets, (D) 1-4 µm droplets, and (E) <1 µm droplets. Quantitative PCR was used to measure genome copies for each microbe. For all graphs, orange symbols represent H1N1pdm09 (N = 3) and green symbols represent Spn (N = 3), with each animal indicated by a unique shape and the mean indicated by short, solid lines. Dotted lines denote the limit of detection (LOD) for H1N1pdm09 (orange) and Spn (green). Samples without infectious virus were placed at the LOD, and viable bacteria samples below the LOD were placed at 1/2 LOD. Samples without detectable genome copies were placed at 1/2 LOD (see supplemental materials and methods).

Fig 2

Stability of S. pneumoniae and influenza viruses in droplets. (A–D) Viral and bacterial loads of H1N1pdm09 and Spn were assessed after exposure of 10 × 1 µL droplets to 43% relative humidity (RH) at room temperature for 2 h. Microbes were suspended in ASL from four different HBE cell donors as indicated in A and B. Control loads were determined using 10 µL of bulk solutions in closed tubes at room temperature. (A) The stability of H1N1pdm09 in droplets containing H1N1pdm09 or H1N1pdm09/Spn measured by TCID₅₀ assay, and (C) log₁₀ decay for each individual ASL culture were determined. (B) The stability of Spn in droplets containing Spn or H1N1pdm09/Spn measured by CFU assay, and (D) log₁₀ decay for each individual ASL culture were determined. Differences were assessed using Welch’s unpaired t-test. (E) The RH and temperature were recorded every 15 min during stability experiments. Temperature (light green) and RH (light blue) for each ASL replicate are shown. The average temperature (dark green) and RH (dark blue) for all experiments are also included. Bacterial samples with no detection were placed at 1/2 the LOD.