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Streptococcus pneumoniae in a biofilm are hyper-adhesive, avirulent, and when used as a killed whole-cell vaccine confers protection against lethal challenge

ABSTRACT: For Streptococcus pneumoniae, biofilms have been suggested to promote long-term colonization of the nasopharynx and contribute to the pathology of recurrent middle ear infections. To date numerous studies have investigated the contribution of specific genetic determinants for the development of pneumococcal biofilms, however, studies examining the global changes that occur during biofilm development and how they contribute to disease are lacking. Using Scanning and Transmission electron microscopy we examined development of a mature pneumococcal biofilm in a continuous flow through reactor. We determined that a mature biofilm is formed in discrete stages, is marked by the formation of complex 3-dimensional structures, and is primarily composed of dead pneumococci. Using genomic microarrays we determined that pneumococci in mature biofilms down regulate genes involved in protein synthesis, energy production, metabolism, capsular polysaccharide production, and virulence. We confirmed these changes by testing bacterial resistance to antimicrobials, measuring capsule production by ELSIA, and immunoblotting for pneumolysin production. We determined that biofilm pneumococci are hyper-adhesive, binding to cell lines at levels 9 to 11-fold greater than planktonic counterparts. Using Western blot and ELISA, we determined that biofilm bacteria produce greater amounts of the adhesins PsrP, CbpA, and surface exposed phosphorylcholine. We subsequently determined that the hyper-adhesive phenotype was in part due to selection of the transparent phase variant during biofilm growth. Intranasal, intratracheal and intraperitoneal challenge of mice with biofilm and planktonic pneumococci determined that biofilm bacteria were highly attenuated for invasive disease but not nasopharyngeal colonization. Immunization of mice with ethanol-killed biofilm pneumococci of serotype 4 conferred protection against challenge with same isolate but not a serotype 3. ELISA for reactive IgG levels subsequently determined that biofilm pneumococci do not provide high levels of cross-reactive protein antigens. Together these studies suggest that biofilms do not directly contribute to disease but instead confer a protected mode of growth for the pneumococcus.

ORGANISM(S): Streptococcus pneumoniae

PROVIDER: GSE26976 | GEO | 2013/04/01

SECONDARY ACCESSION(S): PRJNA135919

REPOSITORIES: GEO

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Project description:For Streptococcus pneumoniae, biofilms have been suggested to promote long-term colonization of the nasopharynx and contribute to the pathology of recurrent middle ear infections. To date numerous studies have investigated the contribution of specific genetic determinants for the development of pneumococcal biofilms, however, studies examining the global changes that occur during biofilm development and how they contribute to disease are lacking. Using Scanning and Transmission electron microscopy we examined development of a mature pneumococcal biofilm in a continuous flow through reactor. We determined that a mature biofilm is formed in discrete stages, is marked by the formation of complex 3-dimensional structures, and is primarily composed of dead pneumococci. Using genomic microarrays we determined that pneumococci in mature biofilms down regulate genes involved in protein synthesis, energy production, metabolism, capsular polysaccharide production, and virulence. We confirmed these changes by testing bacterial resistance to antimicrobials, measuring capsule production by ELSIA, and immunoblotting for pneumolysin production. We determined that biofilm pneumococci are hyper-adhesive, binding to cell lines at levels 9 to 11-fold greater than planktonic counterparts. Using Western blot and ELISA, we determined that biofilm bacteria produce greater amounts of the adhesins PsrP, CbpA, and surface exposed phosphorylcholine. We subsequently determined that the hyper-adhesive phenotype was in part due to selection of the transparent phase variant during biofilm growth. Intranasal, intratracheal and intraperitoneal challenge of mice with biofilm and planktonic pneumococci determined that biofilm bacteria were highly attenuated for invasive disease but not nasopharyngeal colonization. Immunization of mice with ethanol-killed biofilm pneumococci of serotype 4 conferred protection against challenge with same isolate but not a serotype 3. ELISA for reactive IgG levels subsequently determined that biofilm pneumococci do not provide high levels of cross-reactive protein antigens. Together these studies suggest that biofilms do not directly contribute to disease but instead confer a protected mode of growth for the pneumococcus. Pneumococcal biofilms compared to planktonic control at 4, 12, 24, 48 hours. 3 biological replicates each of 4 and 12 hour time points, and 2 biological replicates each of 24 and 48 hour time points. Flip dye (technical replicates) performed for 4, 12, and 24 hour time points; no technical replicate performed for 48 hour time point due to limiting material. Ratios were determined by averaging across technical and biological replicates. The following hybridizations made up each biological replicate: 14090167.tav.annot and 14090190.tav.annot (4hr biol rep 1); 14090169.tav.annot and 14090176.tav.annot (4hr biol rep 2); 14090192.tav.annot and 14090188.tav.annot (4hr biol rep 3); 14087688.tav.annot and 14090180.tav.annot (12hr biol rep 1); 14090185.tav.annot and 14090168.tav.annot (12hr biol rep 2); 14090191.tav.annot and 14090174.tav.annot (12hr biol rep 3); 14090170.tav.annot and 14090175.tav.annot (24hr biol rep 2); 14090193.tav.annot and 14087687.tav.annot (24hr biol rep 3); 14090181.tav.annot (48hr biol rep 1); 14090187.tav.annot (48hr biol rep 2)

Project description:Background: Air-pollutants containing toxic particulate matters (PM) deposit in the respiratory tract and increases microbial infections. However, the mechanism underline is not well understood. In this study, we evaluated the effect of urban particles (UP) on S. pneumoniae in-vitro biofilm formation, colonization on Human middle ear epithelium cells (HMEECs) and in mouse nasal cavity and transition to middle ear and lungs. Methods: S. pneumoniae in vitro biofilms and planktonic growth was evaluated in metal ion free medium in presence of UP, and biofilms were quantified by CV-microplate assay, cfu counts and resazurin staining. Biofilm structures were analyzed using scanning electron microscope (SEM) and confocal microscopy (CM). Gene expressions of biofilms were evaluated using real time RT-PCR. Effects of UP exposure on S. pneumoniae colonization to HMEECs was evaluated using fluorescent in-situ hybridization (FISH), cell viability was detected by EZcyto kit, apoptosis in HMEECs were evaluated using Annexin-V/PI based cytometry analysis and reactive oxygen species (ROS) production were evaluated using Oxiselect kit. Alteration of HMEECs gene expressions on UP exposure or pneumococci colonization were evaluated using microarray. In vivo colonization of pneumococci in presence of UP and transition to middle ear and lungs were evaluated using intranasal mice colonization model. Results: UP exposure significantly (*p< 0.05) increased pneumococcal in vitro biofilms and planktonic growth. In presence of UP pneumococci formed organized biofilms with matrix, while in absence of UP bacteria was unable to form biofilms. The luxS, ply, lytA, comA, comB and ciaR genes involved in bacterial pathogenesis, biofilms formation and quorum sensing were up-regulated in pneumococci biofilms grown in presence of UP. The HMEECs viability was significantly (p<0.05) decreased and bacteria colonization was significantly (p<0.05) elevated in co-treatment (UP+S. pneumoniae) in compare to single treatment. Similarly, increased apoptosis and ROS produce were detected in HMEECs treated with UP+ pneumococci. The microarray analysis of HMEECs revealed that the genes involve in apoptosis and cell death, inflammation, immune response were up-regulated in co-treatment, those genes were unchanged or expressed in less fold in single treatments of UP or S. pneumoniae. In vivo study showed increased pneumococcal colonization to nasal cavity in presence of UP and higher transition of bacteria to middle ear and lungs in presence of UP.

Project description:Streptococcus pneumoniae is a Gram positive bacterium that causes severe invasive infection such as pneumonia, septicemia, meningitis and otitis media especially in children, the elderly and immune-compromised patients. Pneumococcal colonization and disease is often associated with biofilm formation. Bacteria in biofilms exhibit elevated resistance both to antibiotics and to host defense systems, which often results in persistent and difficult-to-treat infections. Therefore, the ongoing treat to human health posed by pneumococcal biofilms has prompted extensive research aimed to identify alternative targets and new antimicrobial agents that are effective against bacteria biofilms. The effective anti-biofilm strategies should include inhibition of microbial adhesion to the surface and of colonization, interference with the signal molecules modulating biofilm development and the disaggregation of the biofilm matrix. In this study, we examine the effect of DAM inhibitor small molecule pyrimidine-diones on streptococcus pneumoniae D-39 strain growth (planktonic and biofilm) and evaluate the changes in global gene expression using c-DNA microarray. The microarray analysis was performed on total RNA extracted from biofilms grown in 24-well microtiter plate with 7µm/ml pyrimidine-diones small molecule and control biofilms (biofilms grown without pyrimidine-diones small molecule). To validate the results of microarray, real-time RT-PCR was performed on 12 differentially expressed genes from six different functional groups. cDNA-microarray analysis detected a total of 259 genes that were significantly differentially expressed in biofilm growth with pyrimidine-diones small molecule. 204 genes were significantly down expressed and 55 genes were significantly up expressed in biofilms grown with 7µm/ml pyrimidine-diones small molecule. Among the 204 down expressed genes, 45 were hypothetical protein encoding gene and 159 were functional protein encoding genes. Of 55 up-regulated genes 21 were hypothetical genes and 34 were functional protein encoding genes. The functional annotation showed that gene involve in fatty acid metabolism, cell division, cell cycles, DNA metabolism, cell assembly were significantly down regulated and galactose metabolism related gene were up-expressed in biofilm grown with pyrimidine-diones small molecule.

Project description:Is there a universal genetically programmed defense providing tolerance to antibiotics when bacteria grow as biofilms? A comparison between biofilms of three different bacterial species by transcriptomic and metabolomic approaches uncovered no evidence of one. Single-species biofilms of three bacterial species (Pseudomonas aeruginosa, Staphylococcus aureus, and Acinetobacter baumannii) were grown in vitro for three days then challenged with respective antibiotics (ciprofloxacin, daptomycin, tigecycline) for an additional 24 h. All three microorganisms displayed reduced susceptibility in biofilms compared to planktonic cultures. Global transcriptomic profiling of gene expression comparing biofilm to planktonic and antibiotic-treated biofilm to untreated biofilm was performed. Extracellular metabolites including 18 amino acids, glucose, lactate, acetate, formate, and ethanol were measured to characterize the utilization of carbon sources between biofilms, treated biofilms, and planktonic cells. While all three bacteria exhibited a species-specific signature of stationary phase, no conserved gene, gene set, or common functional pathway could be identified that changed consistently across the three microorganisms. Across the three species, glucose consumption was increased in biofilms compared to planktonic cells and alanine and aspartic acid utilization were decreased in biofilms compared to planktonic cells. The reasons for these changes were not readily apparent in the transcriptomes. No common shift in the utilization pattern of carbon sources was discerned when comparing untreated to antibiotic-exposed biofilms. Overall, our measurements do not support the existence of a common genetic or biochemical basis for biofilm tolerance against antibiotics. Rather, there are likely myriad genes, proteins, and metabolic pathways that influence the physiological state of microorganisms in biofilms contributing to antibiotic tolerance. The Acinetobacter baumannii microarray data from the study described above is deposited here.

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Streptococcus pneumoniae in a biofilm are hyper-adhesive, avirulent, and when used as a killed whole-cell vaccine confers protection against lethal challenge

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