Nasal Colonization with Streptococcus pneumoniae Includes Subpopulations of Surface and Invasive Pneumococci

Abstract

We demonstrated that during colonization with Streptococcus pneumoniae the nasal mucosal tissues of mice support two populations of pneumococci. Transparent-phase pneumococci can be readily washed from the outer surface, while a second population composed of primarily opaque-phase pneumococci is released only by homogenization of the nasal tissue. The fact that the opaque phase has previously been associated with invasion and the fact that opaque-phase pneumococci were released by homogenization of previously washed nasal tissue suggest that the opaque-phase pneumococci may have invaded the nasal tissue. Consistent with this hypothesis was our observation that there was inflammation in portions of the nasal mucosa of the colonized mice but not in the mucosa of noncolonized mice, but this observation did not prove the hypothesis. If the opaque-phase pneumococci released from the nasal tissue were from within the tissue and/or if resistance of the opaque-phase subpopulation to antibody, complement, and phagocytes is essential for long-term carriage, it seems likely that the virulence factors of S. pneumoniae that are necessary for killing humans exist to facilitate carriage. Although this speculation is unproven, the observation that there are separate populations of pneumococci during colonization may help guide future attempts to understand the biology of nasal colonization by this pathogen.Humans acquire Streptococcus pneumoniae invariably by asymptomatic nasal colonization (15). Transmission to others is thought to be primarily from colonized individuals. Even so, pneumococci possess virulence factors, including capsule, PspA, PspC, and pneumolysin, that allow them to resist the opsonic effects of complement deposition and thereby to avoid phagocytosis (1,10,22,26). These virulence factors enable pneumococci to cause invasive disease, which results in more human deaths in developed countries than result from any other bacterial pathogen (12). However, since the reservoir of pneumococci is maintained primarily by human nasopharyngeal colonization, virulence factors that interfere with complement and phagocytosis make little sense at the mucosal surface, where there is little complement (28) and there are few phagocytes. One possible explanation for why pneumococci contain these disease-permitting virulence factors is that longterm nasal colonization involves more than simple surface adherence and actually requires invasion of mucosal tissue, where the pathogen has to defend itself against complement-dependent opsonization and phagocytosis.Pneumococci have a phase shift that allows them to exist in transparent and opaque phases (36). Transparent-phase pneumococci have been shown to be released from nasal cavities of mice and infant rats by gentle washing with physiological salt solutions (36). In contrast, the majority of pneumococci recovered from the blood or lung are opaque-phase organisms (6,19,34). Moreover, it has been shown that nasal colonization in rodents is established more readily by transparent-phas...

Streptococcus pneumoniae Isolates from Middle Ear Fluid and Nasopharynx of Children with Acute Otitis Media Exhibit Phase Variation

“…Previous studies in animal models have shown that transparent variants are released when nasal tissues are washed with physiological salt solutions, while the majority of S. pneumoniae isolates recovered from previously washed homogenized nasal tissue, from blood specimens, or from lung aspirates were in the opaque phase (1,12). In chinchillas pretreated with live influenza virus, inoculation with opaque pneumococci caused more severe middle ear infections than did inoculation with transparent pneumococci (10).…”

“…In contrast, opaque variants associated with greater amounts of capsular polysaccharide colonize poorly in animals and provide greater resistance to clearance by phagocytes but at the same time decrease the adherence of opaque variants to host cells (2, 4). Thus, the intrastrain phase shifts of pneumococci are hypothesized to be adaptations to different stages in the pathogenesis of infections (1,8,10,13,14). However, the phase shifts have never been clearly demonstrated with human samples.…”

“…Serial dilutions of each sample were plated on tryptic soy plates solidified with 1% agar containing 5,000 U/ml of catalase (Worthington Biochemical, Freehold, NJ) to allow for direct evaluation of individual colony morphology while limiting any growth of bacteria between extraction of the culture from the patient and the time it was plated on TSA (1,15). Transparent and opaque bacteria identified macroscopically on the TSA plates and classified by phase were subsequently confirmed to be pneumococci by streaking representative colonies on blood agar and examining them for colony morphology, alpha hemolysis, and sensitivity to optochin.…”

Effect of Concentrated Ambient Particles on Macrophage Phagocytosis and Killing of Streptococcus pneumoniae

“…One likely candidate is Streptococcus pneumoniae, the most frequent bacterial pathogen causing pneumonia in humans (21). Host defenses against pneumococcal pneumonia are complex and remarkably efficient, given the much greater rate of colonization of upper airways by pneumococci than pneumonic infection and the normal nocturnal aspiration of small amounts of bacteria-laden nasopharyngeal secretions (22,23). The lung macrophage plays a critical role in the initial host defense against bacterial infection (24,25), and is a likely target for air particle-mediated disruption of innate resistance.…”

A Bivalent Vaccine Based on a Replication-Incompetent Influenza Virus Protects against Streptococcus pneumoniae and Influenza Virus Infection

“…Forty-eight hours after infection, the supernatants were harvested and stored at Ϫ80°C until use. S. pneumoniae strain WU2 with serotype 3 and strain EF3030 with serotype 19F, which are virulent and relatively avirulent in mice, respectively (23,24), were grown in Todd-Hewitt Broth (Becton, Dickinson and Company) supplemented with 0.5% yeast extract (THY) to mid-log phase and washed twice with Dulbecco's phosphate-buffered saline (PBS) without CaCl 2 and MgCl 2 (Sigma-Aldrich). The bacteria were then suspended in THY containing 10% glycerol, aliquoted, and stored at Ϫ80°C until use.…”