Regulation of virulence gene expression resulting from Streptococcus pneumoniae and nontypeable Haemophilus influenzae interactions in chronic disease

Chronic rhinosinusitis (CRS) is a common inflammatory disease of the sinonasal cavity mediated, in part, by polymicrobial communities of bacteria. Recent molecular studies have confirmed the importance of Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi) in CRS. Here, we hypothesize that interaction between S. pneumoniae and NTHi mixed-species communities cause a change in bacterial virulence gene expression. We examined CRS as a model human disease to validate these polymicrobial interactions. Clinical strains of S. pneumoniae and NTHi were grown in mono- and coculture in a standard biofilm assay. Reverse transcriptase real-time PCR (RTqPCR) was used to measure gene expression of key virulence factors. To validate these results, we investigated the presence of the bacterial RNA transcripts in excised human tissue from patients with CRS. Consequences of physical or chemical interactions between microbes were also investigated. Transcription of NTHi type IV pili was only expressed in co-culture in vitro, and expression could be detected ex vivo in diseased tissue. S. pneumoniae pyruvate oxidase was up-regulated in co-culture, while pneumolysin and pneumococcal adherence factor A were down-regulated. These results were confirmed in excised human CRS tissue. Gene expression was differentially regulated by physical contact and secreted factors. Overall, these data suggest that interactions between H. influenzae and S. pneumoniae involve physical and chemical mechanisms that influence virulence gene expression of mixed-species biofilm communities present in chronically diseased human tissue. These results extend previous studies of population-level virulence and provide novel insight into the importance of S. pneumoniae and NTHi in CRS

Associations between respiratory illnesses and secondhand smoke exposure in flight attendants: A cross-sectional analysis of the Flight Attendant Medical Research Institute Survey

Abstract Background Secondhand tobacco smoke (SHS) is associated with increased risk of respiratory illness, cancer, and cardiovascular disease. Prior to smoking bans on airlines in the late 1980s, flight attendants were exposed to a significant amount of SHS. In the present study, we examine associations between flight attendant SHS exposure and development of respiratory illnesses and cardiovascular disease. Methods Between December 2006 and October 2010, three hundred sixty-two flight attendants completed an online questionnaire with information regarding experience as a flight attendant, medical history, smoking history, and SHS exposure. Rates of illnesses in flight attendants were compared with an age and smoking history matched population sample from NHANES 2005-2006. Logistic regression analysis was used to examine the association of reported medical conditions and pre-ban years of exposure. Results Compared with the sample from NHANES 2005-2006, flight attendants had increased prevalence of chronic bronchitis (11.7% vs. 7.2%, p < 0.05), emphysema/COPD (3.2% vs. 0.9%, p < 0.03), and sinus problems (31.5% vs. 20.9%, p < 0.002), despite a lower prevalence of medical illnesses including high blood pressure, diabetes, high cholesterol, heart failure, cancer, and thyroid disease. Amongst flight attendants who reported never smoking over their lifetimes, there was not a significant association between years of service as a flight attendant in the pre-smoking ban era and illnesses. However, in this same group, there was a significantly increased risk of daily symptoms (vs. no symptoms) of nasal congestion, throat, or eye irritation per 10-year increase of years of service as a flight attendant prior to the smoking ban (OR 2.14, 95% CI 1.41 - 3.24). Conclusions Flight attendants experience increased rates of respiratory illnesses compared to a population sample. The frequency of symptoms of nasal congestion, throat or eye irritation is associated with occupational SHS exposure in the pre-smoking ban era

Tobacco Smoke Mediated Induction of Sinonasal Microbial Biofilms

Cigarette smokers and those exposed to second hand smoke are more susceptible to life threatening infection than non-smokers. While much is known about the devastating effect tobacco exposure has on the human body, less is known about the effect of tobacco smoke on the commensal and commonly found pathogenic bacteria of the human respiratory tract, or human respiratory tract microbiome. Chronic rhinosinusitis (CRS) is a common medical complaint, affecting 16% of the US population with an estimated aggregated cost of $6 billion annually. Epidemiologic studies demonstrate a correlation between tobacco smoke exposure and rhinosinusitis. Although a common cause of CRS has not been defined, bacterial presence within the nasal and paranasal sinuses is assumed to be contributory. Here we demonstrate that repetitive tobacco smoke exposure induces biofilm formation in a diverse set of bacteria isolated from the sinonasal cavities of patients with CRS. Additionally, bacteria isolated from patients with tobacco smoke exposure demonstrate robust in vitro biofilm formation when challenged with tobacco smoke compared to those isolated from smoke naïve patients. Lastly, bacteria from smoke exposed patients can revert to a non-biofilm phenotype when grown in the absence of tobacco smoke. These observations support the hypothesis that tobacco exposure induces sinonasal biofilm formation, thereby contributing to the conversion of a transient and medically treatable infection to a persistent and therapeutically recalcitrant condition

Real time PCR for expression of <i>S. pneumoniae</i> housekeeping and virulence genes show differential gene regulation in co-culture with NTHi.

<p><i>S. pneumoniae</i> were grown in mono- and co-culture biofilms and expression of <i>spxB, ply,</i> and <i>pavA</i> are recorded and normalized to expression of the 16s rRNA gene using the ΔΔCt method. Up and down-regulation are displayed as ΔΔCt in panel A. Fold change from the reference gene are displayed in panel B. Changes are considered significant when p≤0.05. Significance is indicated by *. Here, we show significant up-regulation of <i>spxB</i> in co-culture with NTHi, and down-regulation of <i>ply,</i> and <i>pavA.</i></p

List of primer sequences and target gene function.

<p>Genes for NTHi and <i>S. pneumoniae</i> are listed along with primers sequences (5′-3′), gene target, function, and GenBank accession number, if applicable.</p

Chemical factors secreted in co-culture conditions regulate the expression of key virulence genes.

<p>Conditioned media from <i>S. pneumoniae/</i>NTHi 24h co-cultures was used to treat 24h single species biofilms (Conditioned Media column). Cells were grown in 100% media, and treated with 100%, or 10% media to distinguish the effects of nutrient depletion. The ΔΔCt is presented in Panel A. Fold change from 16s gene expression is displayed in Panel B. Conditioned media did not induce <i>pilA</i> expression. <i>S. pneumoniae spxB</i> and the 16s rRNA genes were down-regulated in the presence of CM compared to the media controls. Student's t-test determined significant down-regulation of <i>spxB</i> only when conditioned media was used.</p

Real time PCR analysis of expression of NTHi type IV pili.

<p>NTHi were grown in mono- and co-culture biofilms and expression of 16s rRNA reference gene and <i>pilA</i> are recorded and the ΔΔCt calculated. Fold change, or 2^ΔΔCt, from the reference gene are displayed in panel B. Standard deviation is presented, and changes are considered significant when p≤0.05, determined by Students t-test. Significance is indicated by *.</p

Bacterial isolates used in this work.

<p>Culture result represents the bacterial isolate recovered from the initial Clinical culture or resultant from the Biofilm culture. Tobacco exposure is expressed a packs of cigarettes smoked per day X years smoking (PY). One patient, #1690, lived with a smoker and was thus designated as second hand smoker (SHS). CNS: Coagulase negative <i>Staph</i>.</p

Tobacco biofilm index.

<p>Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015700#pone-0015700-g001" target="_blank">figure 1A</a> was normalized by creating a ratio of smoke to sham exposed biofilm formation. Value of <1 demonstrates biofilm inhibition while value >1 reflects biofilm induction.</p

Biofilm formation after repetitive smoke exposure.

<p>Bacteria isolated from smoke naïve patients were subjected to daily sham or smoke exposure (5 cigarettes/3 h) and then subjected to the biofilm detection assay. Data is represented as the ratio of smoke exposure to sham exposure. *, #, and $ indicate significant differences (p<0.05) between day 1 and 2, day 1 and 3, and day 1 and 4, respectively. Indicate the number of replicates.</p