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

A maternal higher-complex carbohydrate diet increases bifidobacteria and alters early life acquisition of the infant microbiome in women with gestational diabetes mellitus

Gestational diabetes mellitus (GDM) is associated with considerable imbalances in intestinal microbiota that may underlie pathological conditions in both mothers and infants. To more definitively identify these alterations, we evaluated the maternal and infant gut microbiota through the shotgun metagenomic analysis of a subset of stool specimens collected from a randomized, controlled trial in diet-controlled women with GDM. The women were fed either a CHOICE diet (60% complex carbohydrate/25% fat/15% protein, n=18) or a conventional diet (CONV, 40% complex carbohydrate/45% fat/15% protein, n=16) from 30 weeks’ gestation through delivery. In contrast to other published studies, we designed the study to minimize the influence of other dietary sources by providing all meals, which were eucaloric and similar in fiber content. At 30 and 37 weeks’ gestation, we collected maternal stool samples; performed the fasting measurements of glucose, glycerol, insulin, free fatty acids, and triglycerides; and administered an oral glucose tolerance test (OGTT) to measure glucose clearance and insulin response. Infant stool samples were collected at 2 weeks, 2 months, and 4–5 months of age. Maternal glucose was controlled to conventional targets in both diets, with no differences in Homeostatic Model Assessment of Insulin Resistance (HOMA-IR). No differences in maternal alpha or beta diversity between the two diets from baseline to 37 weeks’ gestation were observed. However, women on CHOICE diet had higher levels of Bifidobacteriaceae, specifically Bifidobacterium adolescentis, compared with women on CONV. Species-level taxa varied significantly with fasting glycerol, fasting glucose, and glucose AUC after the OGTT challenge. Maternal diet significantly impacted the patterns of infant colonization over the first 4 months of life, with CHOICE infants showing increased microbiome alpha diversity (richness), greater Clostridiaceae, and decreased Enterococcaceae over time. Overall, these results suggest that an isocaloric GDM diet containing greater complex carbohydrates with reduced fat leads to an ostensibly beneficial effect on the maternal microbiome, improved infant gut microbiome diversity, and reduced opportunistic pathogens capable of playing a role in obesity and immune system development. These results highlight the critical role a maternal diet has in shaping the maternal and infant microbiome in women with GDM

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

Antibiotic eluting chitosan glycerophosphate implant in the setting of acute bacterial sinusitis: a rabbit model.

BACKGROUND: Topical therapy offers the potential for treatment of sinonasal disease with minimal systemic side effects. Chitosan glycerophosphate (CGP) is a mucoadhesive polymer that can be used as an antibiotic eluting sinonasal implant in the treatment of sinusitis. The purpose of this study was to assess the potential for CGP as an antibiotic impregnated implant in a rabbit model of acute bacterial sinusitis. METHODS: The Institutional Animal Care and Use Committee approved study of acute bacterial sinusitis in 12 New Zealand white rabbits using either Pseudomonas aeruginosa (n = 6) or Staphylococcus aureus (n = 6). CGP impregnated with 50 mg of either gentamicin or vancomycin was bilaterally implanted in two rabbits in each arm, respectively. The sinuses were irrigated with saline for 4 days and the lavage was collected for colony-forming unit (CFU) determination. Within each group, the CFU log reduction in the lavage was compared with that of rabbits receiving saline alone (n = 2) or a daily 80-microgram/mL gentamicin or vancomycin irrigation (n = 2) and analyzed using a Student\u27s t-test. RESULTS: Within the S. aureus group, the CFU log reduction using CGP + vancomycin (-2.57 +/- 0.21) was greater than vancomycin irrigation (-1.66 +/- 0.5; p = NS) and significantly greater than saline alone (2.46 +/- 0.97; p = 0.018). Within the P. aeruginosa group, the CFU log reduction using the CGP + gentamicin (-4.62 +/- 0.74) was greater than gentamicin irrigation (-4.09 +/- 0.70) and saline alone (-1.90 +/- 0.90); however, the results were not significant. In all rabbits receiving the CGP + antibiotic implant, no viable bacteria were present in the lavage by day 4. CONCLUSION: Placement of a single antibiotic impregnated CGP implant in the setting of an acute Gram-positive or Gram-negative bacterial sinusitis resulted in a greater log reduction of CFU than daily antibiotic irrigation and led to complete sterilization of the lavage within 4 days

Development of a novel and rapid antibody-based diagnostic for chronic staphylococcus aureus Infections based on biofilm antigens

Prosthetic joint infections are difficult to diagnose and treat due to biofilm formation by the causative pathogens. Pathogen identification relies on microbial culture that requires days to weeks, and in the case of chronic biofilm infections, lacks sensitivity. Diagnosis of infection is often delayed past the point of effective treatment such that only the removal of the implant is curative. Early diagnosis of an infection based on antibody detection might lead to less invasive, early interventions. Our study examined antibody-based assays against the Staphylococcus aureus biofilm-upregulated antigens SAOCOL0486 (a lipoprotein), glucosaminidase (a domain of SACOL1062), and SACOL0688 (the manganese transporter MntC) for detection of chronic S. aureus infection. We evaluated these antigens by enzyme-linked immunosorbent assay (ELISA) using sera from naive rabbits and rabbits with S. aureus-mediated osteomyelitis, and then we validated a proof of concept for the lateral flow assay (LFA). The SACOL0688 LFA demonstrated 100% specificity and 100% sensitivity. We demonstrated the clinical diagnostic utility of the SACOL0688 antigen using synovial fluid (SF) from humans with orthopedic implant infections. Elevated antibody levels to SACOL0688 in clinical SF specimens correlated with 91% sensitivity and 100% specificity for the diagnosis of S. aureus infection by ELISA. We found measuring antibodies levels to SACOL0688 in SF using ELISA or LFA provides a tool for the sensitive and specific diagnosis of S. aureus prosthetic joint infection. Development of the LFA diagnostic modality is a desirable, cost-effective option, potentially providing rapid readout in minutes for chronic biofilm infections

Effects of an LL-37-derived antimicrobial peptide in an animal model of biofilm Pseudomonas sinusitis.

BACKGROUND: LL-37, an innate immunity protein expressed within sinonasal mucosa, has in vitro antibacterial and antifungal properties as well as efficacy against preformed Pseudomonas aeruginosa biofilms. We hypothesize that a 24 amino acid peptide derivative of LL-37 will show efficacy against biofilm-forming P. aeruginosa in an established animal model of sinusitis. METHODS: Five groups of six New Zealand rabbits were each infected with P. aeruginosa (PAO-1) and fitted with irrigating catheters 7 days later. Each group was instilled with either one of three different concentrations of peptide, a positive control of topical tobramycin, or the carrier solution without the peptide once a day for 10 days. Nasal diluent was collected throughout the irrigation period to assess for persistence or resolution of infection by determining colony-forming units (CFU). At study end, sinus mucosa was harvested for histological assessment of inflammation and SEM evaluation for ciliary integrity and presence of biofilms. RESULTS: Topical tobramycin at 400x minimum inhibitory concentration and 2.5 mg/mL of peptide were effective in significantly lowering CFUs after 10 days of irrigation. Histological evaluation showed increased signs of inflammation in a dose-dependent manner within mucosa and bone of the groups receiving the peptide. SEM analysis showed ciliary loss in a dose-dependent manner. Biofilms were present in all groups except for the highest concentration of peptide and tobramycin. CONCLUSION: High concentrations of LL-37-derived peptide showed in vivo ability to eradicate Pseudomonas biofilms and decrease bacterial counts. However, increasing concentrations of peptide showed proinflammatory and ciliotoxic effects on sinus mucosa

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

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

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