Expression of Streptococcus pneumoniae Virulence-Related Genes in the Nasopharynx of Healthy Children

Abstract

Colonization and persistence in the human nasopharynx are prerequisites for Streptococcus pneumoniae disease and carriage acquisition, which normally occurs during early childhood. Animal models and in vitro studies (i.e. cell adhesion and cell cytotoxicity assays) have revealed a number of colonization and virulence factors, as well as regulators, implicated in nasopharyngeal colonization and pathogenesis. Expression of genes encoding these factors has never been studied in the human nasopharynx. Therefore, this study analyzed expression of S. pneumoniae virulence-related genes in human nasopharyngeal samples. Our experiments first demonstrate that a density of ≥104 CFU/ml of S. pneumoniae cells in the nasopharynx provides enough DNA and RNA to amplify the lytA gene by conventional PCR and to detect the lytA message, respectively. A panel of 21 primers that amplified S. pneumoniae sequences was designed, and their specificity for S. pneumoniae sequences was analyzed in silico and validated against 20 related strains inhabitants of the human upper respiratory tract. These primers were utilized in molecular reactions to find out that all samples contained the genes ply, pavA, lytC, lytA, comD, codY, and mgrA, whereas nanA, nanB, pspA, and rrgB were present in ∼91–98% of the samples. Gene expression studies of these 11 targets revealed that lytC, lytA, pavA and comD were the most highly expressed pneumococcal genes in the nasopharynx whereas the rest showed a moderate to low level of expression. This is the first study to evaluate expression of virulence- and, colonization-related genes in the nasopharynx of healthy children and establishes the foundation for future gene expression studies during human pneumococcal disease.

Increased Nasopharyngeal Density and Concurrent Carriage of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis Are Associated with Pneumonia in Febrile Children

“…Frozen NP samples were thawed at room temperature and then vortexed for 15 s. Two hundred microliters of the sample were mixed with 100 μl of TE buffer (10mM Tris-HCl, 1 mM EDTA, pH 8.0) containing 0.04 g/ml of lysozyme and 75 U/ml of mutanolysin, and then incubated for 1 h in a 37°C in water bath. The subsequent steps were carried out according to the Qiagen DNA mini kit protocol, as detailed elsewhere [20, 21]. DNAs were eluted in 100 μl of elution buffer and stored at -80°C.…”

Bacterial Density, Serotype Distribution and Antibiotic Resistance of Pneumococcal Strains from the Nasopharynx of Peruvian Children Before and After Pneumococcal Conjugate Vaccine 7

“…DNA was extracted using a QIAamp DNA mini kit (Qiagen, Valencia, CA) (19, 20). Bacterial density was quantified by qPCR reactions in a total of 25 μl volume.…”

Levofloxacin-Ceftriaxone Combination Attenuates Lung Inflammation in a Mouse Model of Bacteremic Pneumonia Caused by Multidrug-Resistant Streptococcus pneumoniae via Inhibition of Cytolytic Activities of Pneumolysin and Autolysin

“…The nucleotide sequence of the forward primer for the au-tolysin gene (LytA) was 5=-ACGCAATCTAGCAGATGAAGC-3=, and the sequence for the reverse primer was 5=-TGTTTGGTTGGTTATTCGTG C-3= (37). The reaction was started by an initial incubation at 42°C for 30 min and denaturation at 95°C for 5 min, followed by 35 cycles of 95°C for 15 s, 55°C for 30 s, and 68°C for 1 min and a final extension at 68°C for 10 min (38). The products were stored at Ϫ80°C until further use.…”