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Human pleural fluid is a potent growth medium for Streptococcus pneumoniae.

ABSTRACT: Empyema is defined by the presence of bacteria and/or pus in pleural effusions. However, the biology of bacteria within human pleural fluid has not been studied. Streptococcus pneumoniae is the most common cause of pediatric and frequent cause of adult empyema. We investigated whether S. pneumoniae can proliferate within human pleural fluid and if growth is affected by the cellular content of the fluid and/or characteristics of pneumococcal surface proteins. Invasive S. pneumoniae isolates (n = 24) and reference strain recovered from human blood or empyema were inoculated (1.5×106CFU/mL) into sterile human malignant pleural fluid samples (n = 11). All S. pneumoniae (n = 25) strains proliferated rapidly, increasing by a median of 3009 (IQR 1063-9846) from baseline at 24hrs in all pleural effusions tested. Proliferation was greater than in commercial pneumococcal culture media and concentrations were maintained for 48hrs without autolysis. A similar magnitude of proliferation was observed in pleural fluid before and after removal of its cellular content, p = 0.728. S. pneumoniae (D39 strain) wild-type, and derivatives (n = 12), each with mutation(s) in a different gene required for full virulence were inoculated into human pleural fluid (n = 8). S. pneumoniae with pneumococcal surface antigen A (?psaA) mutation failed to grow (2207-fold lower than wild-type), p<0.001, however growth was restored with manganese supplementation. Growth of other common respiratory pathogens (n = 14) across pleural fluid samples (n = 7) was variable and inconsistent, with some strains failing to grow. We establish for the first time that pleural fluid is a potent growth medium for S. pneumoniae and proliferation is dependent on the PsaA surface protein and manganese.

SUBMITTER: Popowicz ND

PROVIDER: S-EPMC5708656 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Human pleural fluid is a potent growth medium for Streptococcus pneumoniae.

Popowicz Natalia D ND Lansley Sally M SM Cheah Hui M HM Kay Ian D ID Carson Christine F CF Waterer Grant W GW Paton James C JC Brown Jeremy S JS Lee Y C Gary YCG

PloS one 20171130 11

Empyema is defined by the presence of bacteria and/or pus in pleural effusions. However, the biology of bacteria within human pleural fluid has not been studied. Streptococcus pneumoniae is the most common cause of pediatric and frequent cause of adult empyema. We investigated whether S. pneumoniae can proliferate within human pleural fluid and if growth is affected by the cellular content of the fluid and/or characteristics of pneumococcal surface proteins. Invasive S. pneumoniae isolates (n = ...[more]

PMID: 29190798

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Project description:BACKGROUND:Streptococcus agalactiae (group B Streptococcus) is a bacterial pathogen that causes severe intrauterine infections leading to fetal morbidity and mortality. The pathogenesis of GBS infection in this environment is poorly understood, in part because we lack a detailed understanding of the adaptation of this pathogen to growth in amniotic fluid. To address this knowledge deficit, we characterized the transcriptome of GBS grown in human amniotic fluid (AF) and compared it with the transcriptome in rich laboratory medium. METHODS:GBS was grown in Todd Hewitt-yeast extract medium and human AF. Bacteria were collected at mid-logarithmic, late-logarithmic and stationary growth phase. We performed global expression microarray analysis using a custom-made Affymetrix GeneChip. The normalized hybridization values derived from three biological replicates at each growth point were obtained. AF/THY transcript ratios representing greater than a 2-fold change and P-value exceeding 0.05 were considered to be statistically significant. PRINCIPAL FINDINGS:We have discovered that GBS significantly remodels its transcriptome in response to exposure to human amniotic fluid. GBS grew rapidly in human AF and did not exhibit a global stress response. The majority of changes in GBS transcripts in AF compared to THY medium were related to genes mediating metabolism of amino acids, carbohydrates, and nucleotides. The majority of the observed changes in transcripts affects genes involved in basic bacterial metabolism and is connected to AF composition and nutritional requirements of the bacterium. Importantly, the response to growth in human AF included significant changes in transcripts of multiple virulence genes such as adhesins, capsule, and hemolysin and IL-8 proteinase what might have consequences for the outcome of host-pathogen interactions. CONCLUSIONS/SIGNIFICANCE:Our work provides extensive new information about how the transcriptome of GBS responds to growth in AF, and thus new leads for pathogenesis research.

Project description:The polysaccharide capsule of Streptococcus pneumoniae defines over ninety serotypes, which differ in their carriage prevalence and invasiveness for poorly understood reasons. Recently, an inverse correlation between carriage prevalence and oligosaccharide structure of a given capsule has been described. Our previous work suggested a link between serotype and growth in vitro. Here we investigate whether capsule production interferes with growth in vitro and whether this predicts carriage prevalence in vivo. Eighty-one capsule switch mutants were constructed representing nine different serotypes, five of low (4, 7F, 14, 15, 18C) and four of high carriage prevalence (6B, 9V, 19F, 23F). Growth (length of lag phase, maximum optical density) of wildtype strains, nontypeable mutants and capsule switch mutants was studied in nutrient-restricted Lacks medium (MLM) and in rich undefined brain heart infusion broth supplemented with 5% foetal calf serum (BHI+FCS). In MLM growth phenotype depended on, and was transferred with, capsule operon type. Colonization efficiency of mouse nasopharynx also depended on, and was transferred with, capsule operon type. Capsule production interfered with growth, which correlated inversely with serotype-specific carriage prevalence. Serotypes with better growth and higher carriage prevalence produced thicker capsules (by electron microscopy, FITC-dextran exclusion assays and HPLC) than serotypes with delayed growth and low carriage prevalence. However, expression of cpsA, the first capsule gene, (by quantitative RT-PCR) correlated inversely with capsule thickness. Energy spent for capsule production (incorporation of H3-glucose) relative to amount of capsule produced was higher for serotypes with low carriage prevalence. Experiments in BHI+FCS showed overall better bacterial growth and more capsule production than growth in MLM and differences between serotypes were no longer apparent. Production of polysaccharide capsule in S. pneumoniae interferes with growth in nutrient-limiting conditions probably by competition for energy against the central metabolism. Serotype-specific nasopharyngeal carriage prevalence in vivo is predicted by the growth phenotype.

Project description:Streptococcus pneumoniae is the dominant cause of community-acquired pneumonia worldwide. Invasion of the pleural space is common and results in increased mortality. We set out to determine the bacterial and host factors that influence invasion of the pleural space. In a murine model of pneumococcal infection, we isolated neutrophil-dominated samples of bronchoalveolar and pleural fluid containing bacteria 48?hours after infection. Using dual RNA sequencing (RNA-seq), we characterized bacterial and host transcripts that were differentially regulated between these compartments and bacteria in broth and resting neutrophils, respectively. Pleural and lung samples showed upregulation of genes involved in the positive regulation of neutrophil extravasation but downregulation of genes mediating bacterial killing. Compared to the lung samples, cells within the pleural space showed marked upregulation of many genes induced by type I interferons, which are cytokines implicated in preventing bacterial transmigration across epithelial barriers. Differences in the bacterial transcripts between the infected samples and bacteria grown in broth showed the upregulation of genes in the bacteriocin locus, the pneumococcal surface adhesin PsaA, and the glycopeptide resistance gene vanZ; the gene encoding the ClpP protease was downregulated in infection. One hundred sixty-nine intergenic putative small bacterial RNAs were also identified, of which 43 (25.4%) small RNAs had been previously described. Forty-two of the small RNAs were upregulated in pleura compared to broth, including many previously identified as being important in virulence. Our results have identified key host and bacterial responses to invasion of the pleural space that can be potentially exploited to develop alternative antimicrobial strategies for the prevention and treatment of pneumococcal pleural disease.IMPORTANCE The factors that regulate the passage of bacteria between different anatomical compartments are unclear. We have used an experimental model of infection with Streptococcus pneumoniae to examine the host and bacterial factors involved in the passage of bacteria from the lung to the pleural space. The transcriptional profile of host and bacterial cells within the pleural space and lung was analyzed using deep sequencing of the entire transcriptome using the technique of dual RNA-seq. We found significant differences in the host and bacterial RNA profiles in infection, which shed light on the key factors that allow passage of this bacterium into the pleural space.

Dataset Information

Human pleural fluid is a potent growth medium for Streptococcus pneumoniae.

Publications

Human pleural fluid is a potent growth medium for Streptococcus pneumoniae.

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