Isolation site influences virulence phenotype of serotype 14 Streptococcus pneumoniae strains belonging to multilocus sequence type 15

Abstract

Streptococcus pneumoniae is a diverse species causing invasive as well as localized infections that result in massive global morbidity and mortality. Strains vary markedly in pathogenic potential, but the molecular basis is obscured by the diversity and plasticity of the pneumococcal genome. We have previously reported that S. pneumoniae serotype 3 isolates belonging to the same multilocus sequence type (MLST) differed markedly in in vitro and in vivo phenotypes, in accordance with the clinical site of isolation, suggesting stable niche adaptation within a clonal lineage. In the present study, we have extended our analysis to serotype 14 clinical isolates from cases of sepsis or otitis media that belong to the same MLST (ST15). In a murine intranasal challenge model, five ST15 isolates (three from blood and two from ears) colonized the nasopharynx to similar extents. However, blood and ear isolates exhibited significant differences in bacterial loads in other host niches (lungs, ear, and brain) at both 24 and 72 h postchallenge. In spite of these differences, blood and ear isolates were present in the lungs at similar levels at 6 h postchallenge, suggesting that early immune responses may underpin the distinct virulence phenotypes. Transcriptional analysis of lung tissue from mice infected for 6 h with blood isolates versus ear isolates revealed 8 differentially expressed genes. Two of these were exclusively expressed in response to infection with the ear isolate. These results suggest a link between the differential capacities to elicit early innate immune responses and the distinct virulence phenotypes of clonally related S. pneumoniae strains.

FIG 1

Comparative virulence of ST15 blood and ear isolates. Groups of mice were challenged intranasally with the three blood isolates (ST15/4495, ST15/4534, and ST15/4559) and the two ear isolates (ST15/9-47 and ST15/51742), and numbers of pneumococci in the nasopharynx, lungs, brain, and ear at either 24 h or 72 h postchallenge were determined. For ST15/4495 and ST15/9-47, 16 challenged mice were examined at each time point compared with 5 mice at each time point for the remaining strains. Blood isolates are represented by open gray symbols; ear isolates are represented by filled black symbols. The horizontal bars denote the median CFU for each group; the dotted horizontal lines indicate the lower limit of detection, and symbols on the lines denote that no bacteria were detected. Differences in infection rates (number of infected versus number of uninfected mice) were analyzed using the Fisher exact test. **, P < 0.01; ****, P < 0.0001.

FIG 2

In vivo competition between ST15/9-47 (ear isolate) and ST15/4495 (blood isolate). Data shown are pooled from two competition experiments performed with 5 mice per group for each time point (24 h and 72 h postchallenge). In one experiment, mice were challenged with equal numbers (5 × 10⁷ CFU each) of ST15/4495 and ST15/9-47:pAL3 (open symbols); in the other experiment, mice were challenged with equal numbers of ST15/4495:pAL3 and ST15/9-47 (solid symbols). The competitive index (CI) is shown as log₁₀ CI for each tissue of each mouse. The horizontal dotted lines denote the geometric mean CI for each tissue. Statistical differences between the log-transformed geometric mean CI and a hypothetical value of 0 (ratio of 1:1) in each niche were analyzed using the two-tailed Student's t test. ns, not significant; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.

FIG 3

Peripheral blood leukocyte counts. (A and B) Leukocytes isolated from peripheral blood collected at 6 h from control (sham-infected) mice or from those infected with the blood isolate (ST15/4495) or the ear isolate (ST15/9-47) were examined by FACS analysis after staining with anti-Ly-6G (A) or anti-F4/80 (B), as described in Materials and Methods. Data are presented as the product of geometric mean fluorescence intensity and the total number of positive cells (GMFIn) for the respective marker (± SE). (C) Alternatively, blood films were subjected to differential cell counts, and neutrophil numbers are expressed as a percentage of total leukocyte levels (mean ± SE). Statistical significance is indicated as follows: *, P < 0.05; **, P < 0.01 (Student's paired, two-tailed t test).

FIG 4

Lung histopathology. HE-stained lung sections from control (sham-infected) mice or from those infected with the blood isolate (ST15/4495) or the ear isolate (ST15/9-47) at 6 h were examined by light microscopy. (A) Representative sections from each group are shown. Bar, 0.1 mm. Slides were also scored (blind) according the following 8-point scheme: congested capillaries (fine arrows) were scored 0 to 2; thickened alveolar wall (arrowheads) were scored 0 to 2; swollen cuboidal epithelial cells of the bronchioles (thick arrows) were scored 0 to 2; and secretions in the alveolar and bronchiole space (open arrows) were scored 0 to 2. (B) Data were examined with Student's unpaired two-tailed t test and are presented as mean ± SE for each group.

FIG 5

Neutrophil infiltration. Lung tissue from infected or control mice was fixed, sectioned, and labeled with rat anti-mouse Ly-6G or F4/80, followed by Alexa-488-conjugated anti-rat IgG and examined by fluorescence microscopy. Bar, 50 μm.