Assessment of Streptococcus pneumoniae capsule in conjunctivitis and keratitis in vivo neuraminidase activity increases in nonencapsulated pneumococci following conjunctival infection

Abstract

Purpose: The pneumococcal capsule is required for pathogenesis in systemic infections, yet reports show most conjunctivitis outbreaks are caused by nonencapsulated pneumococci, while keratitis infections are caused by encapsulated strains. This study aims to determine the effect of capsule in pneumococcal keratitis and conjunctivitis in rabbit models of infection.

Methods: A capsule-deficient isogenic mutant was created using homologous transformation. Parent and mutant strains were injected within the upper bulbar conjunctiva (conjunctivitis) or into the corneal stroma (keratitis) of New Zealand white rabbits. Clinical examinations were performed 24 and 48 hr post-infection at which time corneas or conjunctivae were removed, homogenized, and plated to determine the recovered bacterial load. Whole eyes were removed for histological examination. The neuraminidase activity was determined following in vitro and in vivo growth.

Results: There were no significant differences in clinical scores between the eyes infected with the parent or mutant for either infection, nor was there a difference in the amount of bacteria recovered from the cornea. In the conjunctivae, however, the mutant strain was cleared by the host faster than the parent strain. Histological examination showed slightly more infiltrating polymorphonuclear leukocytes (PMN) and macrophages in the conjunctivae infected with the parent strain. The neuraminidase activity of both strains was not significantly different when the strains were grown in vitro. However, the neuraminidase activity of the parent was significantly less than that of the mutant at 3 and 12 hr post conjunctival infection.

Conclusions: Although more outbreaks of pneumococcal conjunctivitis are tied to nonencapsulated S. pneumoniae strains, this study showed that an encapsulated strain was capable of establishing conjunctivitis in a rabbit injection model and survive attack by the host immune system longer than its nonencapsulated isogenic mutant. Nonetheless, the nonencapsulated pneumococci had an increased neuraminidase activity level in vivo when compared to the parent strain.

FIGURE 1

(A) Average clinical scores of keratitis infection at 24 and 48 hr post-infection caused by S. pneumoniae parent strain K1544 and mutant strain K1544ΔCAP. p > 0.05 at both time points. SLE scores are reported as the mean scores ± standard errors of the means. (B) Pictures show representative eyes with keratitis caused by S. pneumoniae parent strain K1544 and its isogenic mutant strain K1544ΔCAP. Eyes were scored and photographed 24 and 48 hr post-infection. Representative histology pictures of eyes infected intrastromally with the (C) parent strain K1544 or (D) mutant strain K1544ΔCAP at 48 hr post-infection.

FIGURE 2

Log CFU recovered from (A) corneal homogenates and (B) conjunctival homogenates throughout the course of infection with S. pneumoniae parent strain K1544 and S. pneumoniae mutant strain K1544ΔCAP. (***) indicates significant difference in recovered bacterial load between the two strains, p < 0.05.

FIGURE 3

(A) Average clinical scores of conjunctival infection at 24 and 48 hr post-infection caused by S. pneumoniae parent strain K1544 and mutant strain K1544ΔCAP. There were no significant differences in scores between the groups, p ≥ 0.05. Scores are reported as the mean scores ± standard errors of the means. (B) Representative eyes with conjunctivitis caused by S. pneumoniae parent strain K1544 and its isogenic mutant K1544ΔCAP. Eyes were scored and photographed 24 and 48 hr post-infection.

FIGURE 4

Representative histology pictures of (A) bulbar conjunctivae or (B) palpebral conjunctivae that were infected with parent strain K1544 and mutant strain K1544ΔCAP at 24 and 48 hr post-infection. A non-infected negative control (C) is also shown. The first column at each time point is stained with hematoxylin and eosin while the second column is stained with a mouse anti-rabbit monoclonal antibody specific for macrophages and granulocytes. Black arrow indicates PMN. White arrow indicates macrophage. S: sclera; R: retina.

FIGURE 5

Neuraminidase activity of parent strain K1544 and mutant strain K1544ΔCAP following growth in THY or recovered from infected conjunctivae 3 and 12 hr post-infection. After growth in THY and at 3 hr post-infection, there were 1 × 10⁷ CFU/mL per strain. At 12 hr post-infection, there were 1 × 10⁴ CFU/mL per strain. A student t-test was used to analyze the data. (***) indicates significant differences in the absorbance between groups, p < 0.05. Results are reported as the mean absorbances ± standard errors of the means.