Pretreatment of epithelial cells with live Streptococcus pneumoniae has no detectable effect on influenza A virus replication in vitro.

Influenza A virus (IAV) and Streptococcus pneumoniae (pneumococcus) are two major upper respiratory tract pathogens responsible for exacerbated disease in coinfected individuals. Despite several studies showing increased susceptibility to secondary bacterial infections following IAV infection, information on the direct effect of S. pneumoniae on IAV in vitro is unknown. This is an important area of investigation as S. pneumoniae is a common commensal of the human upper respiratory tract, present as an important coinfecting pathogen with IAV infection. A recent study showed that S. pneumoniae enhances human metapneumovirus infection in polarized bronchial epithelial cells in vitro. The aim of the current study was to determine whether treatment of epithelial cells with S. pneumoniae affects IAV replication using a standard immunofluorescence assay (IFA). For this study we used four IAV permissive epithelial cell lines including two human-derived cell lines, 12 pneumococcal strains including recent human clinical isolates which represent different genetic backgrounds and serotypes, and six IAV strains of varying genetic nature and pathogenic potential including the pandemic 2009 H1N1 virus. Our results suggested that pretreatment of MDCK cells with 7.5×10(6) colony-forming units (CFUs) of live S. pneumoniae resulted in gradual cell-death in a time-dependent manner (0.5 to 4 hr). But, pretreatment of cell lines with 7.5×10(5) and lower CFUs of S. pneumoniae had no detectable effect on either the morphology of cells or on the IAV replication. However, unlike in epithelial cell lines, due to influence of secreted host factors the effect of pneumococci on IAV replication may be different during coinfections in vivo in the human upper respiratory tract, and in vitro with primary human polarized bronchial epithelial cells

Effect of a range of <i>S. pneumoniae</i> inoculums on IAV replication.

<p>A representative study with MDCK cells pretreated with live <i>S. pneumoniae</i> (TIGR4) for 1 hr followed by infection with an IAV strain (SwH1N1/OH/2007/43266) for 20 hr. Infected cells were analyzed by both light microscopy and immunofluorescence microscopy: (A and C) cells pretreated with 7.5×10⁶ and 7.5×10⁵ CFUs of live TIGR4 strain, respectively, and analyzed by light microscopy; (B and D) similarly treated cells but immunostained and analyzed by immunofluorescence microscopy using a FITC filter. (E) MDCK cells were pretreated with five 10-fold dilutions (7.5×10⁶ to 7.5×10²) of live <i>S. pneumoniae</i> (TIGR4) for four time points (0.5–4 hr) and the effect on replication of IAV was assessed by IFA. Each bar represents the average FFU in triplicate wells ± SEM. An asterisk indicates a statistically significant (p<0.05) difference between FFU observed following pretreatment with THY medium and <i>S. pneumoniae</i> cultures for the stated time period.</p

Pneumococcal strains used in this study.

<p>Pneumococcal strains used in this study.</p

Immunofluorescence pictures of different IAV strains.

<p>MDCK cells were infected with indicated IAV strains at an approximate MOI of 0.01. The inoculum of each IAV strain was adjusted to obtain approximately 50–150 fluorescent focal units (FFU) per well following infection for 20 hr. Each FFU is from a single or multiple IAV infected cells in a single fluorescent area.</p

Calibration of a standard curves to quantify different strains of <i>S. pneumoniae.</i>

<p>Twelve different strains of <i>S. pneumoniae</i> were grown in THY medium and CFUs quantified based on OD value over the mid-exponential phase of growth. (A) A representative blood agar plate showing the number of CFUs at 16 hr of incubation in a series of 10-fold diluted <i>S. pneumoniae</i> broth culture in triplicate is shown. (B) Calibration curves were plotted for each strain to quantify 12 different <i>S. pneumoniae</i> strains in CFUs per mL over the OD₆₀₀ values from 0.2 and 0.6. Each data point on the graph represents the average value of two experiments.</p

Effect of pretreatment of epithelial cells with 12 live <i>S. pneumoniae</i> strains on replication of six IAV strains.

<p>The two indicated CFUs of 12 different <i>S. pneumoniae</i> strains were used to treat epithelial cell lines: (A) A549; (B) MDCK; (C) MK1; and (D) D562 for 1 hr, and following incubation cells were washed three times with PBS to remove bacteria and then infected with indicated six different IAV strains of both swine and human origin for 20 hr. The virus infected cells were analyzed by the IFA to determine the levels of viral replication. Each bar represents the average number of FFU from two or three independent experiments ± SEM.</p

Representative data showing the effect of pneumococcal (TIGR4) product (supernatant) on IAV (A/swine/Ohio/24366/07) replication in MDCK cells.

<p>Cells were treated (pre and/or post) with <i>S. pneumoniae</i> (TIGR4) culture supernatant (sup) at indicated dilutions and time, and infected with A/swine/Ohio/24366/07 at MOI 0.1. Cell culture supernatants harvested at 24 hr post-infection were analyzed to determine the viral titers using MDCK cells by the IFA method.</p