Streptococcus pneumoniae: Microbiology and pathogenesis of infection

INTRODUCTION — Streptococcus pneumoniae occupies an important position in the history of microbiology [1]:

●The organism was first identified in 1881.

●Its role in causing lobar pneumonia was appreciated by the late 1880s.

●The central role of humoral immunity in host defense against extracellular organisms was first described for the pneumococcus.

●The first recognition that antibody directed to the capsular polysaccharide of a bacterium could be protective was shown for the pneumococcus; this observation forms the basis for many current bacterial vaccines.

●The discovery of DNA as the material for genetic exchange was made from the pneumococcus.

Despite the extensive study of this pathogen and the availability of a capsular polysaccharide vaccine for adults covering 23 different serotypes and protein-conjugated polysaccharide vaccines for children, S. pneumoniae remains a major cause of morbidity and mortality in children and older adults worldwide and is a principal bacterial cause of otitis media, pneumonia, meningitis, and bacteremia.

The microbiology and pathogenesis of S. pneumoniae infection will be reviewed here. The clinical syndromes of pneumococcal pneumonia and meningitis as well as issues in vaccination against S. pneumoniae are discussed separately. (See "Pneumococcal pneumonia in patients requiring hospitalization" and "Pneumococcal vaccination in adults" and "Clinical features and diagnosis of acute bacterial meningitis in adults".)

MICROBIOLOGY — S. pneumoniae is a fastidious gram-positive, alpha-hemolytic bacterium that grows best in 5 to 10 percent carbon dioxide, and requires a source of catalase (eg, blood) to grow on agar plates. The inability to make catalase has clinical significance since, unlike other pyogenic organisms, catalase-negative bacteria generate hydrogen peroxide and are killed by the phagocytic cells of patients with chronic granulomatous disease. (See "Primary disorders of phagocyte number and/or function: An overview", section on 'Chronic granulomatous disease'.)

In the laboratory, pneumococci are identified by partial lysis of sheep red blood cells (called alpha-hemolysis) on blood agar plates, killing by optochin, and lysis of colonies by bile salts (deoxycholate). The ability of deoxycholate and penicillin to dissolve the cell wall of the organism depends upon the normal presence of autolysin, an autolytic enzyme in pneumococci [2]. During growth in broth, this enzyme is triggered when the culture reaches high density, resulting in a characteristic autolysis and death of the bacteria. As an example, if a blood culture is allowed to incubate for 24 to 48 hours before sampling or subculturing, pneumococci may become undetectable. The natural ability to undergo DNA transformation and autolysis in the stationary phase is an attribute that pneumococci share with Haemophilus influenzae and Neisseria meningitidis, two other common invasive bacteria.

The complete genomic sequence of a clinical isolate of S. pneumoniae serotype 4 from a patient with meningitis was published in 1997; this was the first genome from a gram-positive bacterium to be sequenced in its entirety. A detailed annotation of 2236 potential coding regions, of which 64 percent have been assigned a potential biologic role, has been published [3]. The complete genome of an important laboratory strain R6 and a variety of clinical strains have also been described [4]. As new genomes are completed, updated sources for sequence analysis become available. Comparisons of these genomic sequences reveal a striking diversity within strains of S. pneumoniae. DNA composition of clinical isolates can vary by as much as 20 percent and still be classified as pneumococcus. This diversity is localized to regions of the chromosome that alter the virulence of the strain, referred to as pathogenicity islands [5,6].

PATHOGENESIS — Pneumococcus is a major cause of infection in children and older adults. Manifestations range from asymptomatic nasopharyngeal carriage to disease associated with the respiratory tract (otitis media, sinusitis, and pneumonia) or systemic disease (meningitis, endocarditis, and arthritis), often resulting in sepsis. Acute cardiac events have also been described in patients with pneumococcal disease [7] due to increased myocardial oxygen demand (type 2 myocardial infarction) or to increased inflammation in existing atherosclerotic plaques and subsequent rupture (type 1 myocardial infarction) [8,9]. Invasion of the myocardium with formation of microlesions that can lead to scarring, impaired cardiac contractility, and arrhythmias may also play a role [10,11].

A number of features of S. pneumoniae mediate its ability to produce infection, including surface components that enhance virulence and provoke a host inflammatory reaction. The following discussion will emphasize the pathogenetic mechanisms of the organism and host responses. The steps involved in the bacteriologic and pathologic progression of pneumococcal pneumonia are discussed separately. (See "Pneumococcal pneumonia in patients requiring hospitalization".)

Transmission — S. pneumoniae is a strictly human pathogen that is transmitted from host to host by intimate contact or aerosol. Recent evidence suggests that the bacteria adapt to survive in the aerosol outside the host [12]. In some cases, the bacteria can cotransport viruses on its surface, which facilitates the frequent coinfection of pneumococci and respiratory viruses [13]. Upon reaching the new host, pneumococci can deploy multiple strategies to achieve successful colonization of the nasopharynx, including releasing bacteriocins to kill other bacteria in the nasopharyngeal flora [14].

Capsule — With the (unexplained) exception of pneumococcal conjunctivitis, virtually all pneumococcal infection is caused by encapsulated strains. Surface capsular polysaccharides greatly inhibit uptake of S. pneumoniae by phagocytic cells. Injection of capsular polysaccharides into mammals stimulates the production of type-specific antibodies that provide protective immunity and also serves as the basis for serotyping of these organisms; more than 100 different pneumococcal serotypes have been identified. Prior to the routine use of pneumococcal conjugate vaccines, serotypes 6, 14, 18, 19, and 23 were the most prevalent, accounting for between 60 and 80 percent of infections depending upon the area of the world. In countries where conjugate pneumococcal vaccines are widely used, infection by vaccine strains have greatly decreased in frequency, but infections due to other serotypes (called 'replacement strains') have become more prevalent. Identification of pneumococci by serotyping is complicated by the ability of pneumococci to exchange capsules via genetic transformation.

The capsular polysaccharide, covalently linked to the glycan backbone of the cell wall, is the major antiphagocytic surface element of pneumococci and, consequently, the major virulence factor. The capsule locus has been sequenced [15], and the galU gene has been identified as essential for the biosynthesis of capsular polysaccharide [16]. Deletion mutants lacking the capsule locus are incapable of synthesizing a detectable capsule and are essentially avirulent for mice [17].

The capsule is shed from the bacterial surface as pneumococci enter the respiratory tract. Contact with antimicrobial peptides triggers the autolytic enzyme LytA to cleave the cell wall, releasing the attached capsule while not killing the bacteria. This outcome differs from bacterial lysis upon triggering of the autolysin by penicillin. Shedding of the capsule removes this key virulence determinant and reduces the ability of anticapsular antibody to opsonize the bacteria.

Adherence — Pneumococci, like other streptococcal species, avidly adhere to epithelial cells of the nasopharynx (picture 1); this adherence, called colonization, usually does not produce a symptomatic infection. Like other streptococci that use fibrillar structures to contact human cells, a minority of pneumococci display pili to facilitate adherence [18].

The pneumococcus also exports over a dozen choline-binding proteins, which are noncovalently linked to the bacterial cell wall scaffold and serve as ligands to traffic the bacteria from one body site to another and disarm host defenses. These proteins recognize and bridge to human cell surface carbohydrates and proteins creating a direct contact between bacteria and human cells [19,20]. Multifunctional members of this class are pneumococcal surface proteins A (PspA) and C (PspC; CbpA).

Different, multiple ligand-receptor pairings occur at different body sites [19]. Sialic acid is a prominent receptor in the conjunctiva, Eustachian tube, and nasopharynx, while the disaccharide N-acetylgalactosamine b1-4 galactose is an important ligand in the lower respiratory tract. Competitive inhibition of adherence by sugars leads to reduced pneumonia and bacteremia in animal models [21]. Once bound to host receptors, intracellular signaling pathways are activated to induce bacterial uptake, cytoskeletal rearrangements, and inflammation.

Preceding influenza infection strongly predisposes to secondary invasive pneumococcal infection [22]. The principal mechanism is damage to clearance of bacteria that have bypassed the glottis. In addition the influenza virus neuraminidase exposes pneumococcal receptors on lung cells [23], and influenza virus increases expression of bacterial genes that increase bacterial growth rate and metabolism [24].

Biofilm formation — Pneumococci form robust biofilms in the nasopharynx, sinus, and inner ear during colonization, sinusitis, and otitis media [25]. Biofilm formation is highly regulated depending on exogenous cues such as temperature. Biofilms have been tightly linked to the process of natural transformation whereby pneumococci become competent to exchange DNA in the environment.

Invasion — Pneumococci invade cells poorly, up to 10-fold less than other streptococci [26]. Clinical isolates exhibit a wide variability in invasive capacity [27]. Nonetheless, nasopharyngeal colonization is regularly followed by antibody production, indicating that bacteria have been taken up by host cells (presumably dendritic cells and/or macrophages), and antigen has been processed [28,29]. Invasion is promoted by the cell wall, adhesins, and the cytotoxin pneumolysin; in comparison, invasion is inhibited by capsular polysaccharide.

Phosphorylcholine is a key component of the pneumococcal cell wall teichoic acid. Intact pneumococci use phosphorylcholine to tether to the host cell platelet-activating factor (PAF) receptor, thereby inserting the bacteria into the PAF receptor uptake pathway in an endocytic vacuole [30]. Bacterial phosphorylcholine recognition of the PAF receptor is an example of mimicry since the natural ligand PAF itself contains phosphorylcholine. This invasion pathway is shared by virtually all respiratory pathogens. In parallel, pneumococci can invade cells using the macropinocytosis uptake pathway, a receptor-independent uptake mechanism that nonspecifically translocates bulk contents [31].

To initiate uptake of pneumococci, human cells must become activated and upregulate the PAF receptor on their surfaces [30]. PAF receptors are also upregulated on the vascular endothelium in sickle cell disease, which may play an important role in the predisposition of such patients to invasive pneumococcal infection [32]. Conversely, blockade or genetic deletion of PAF receptors impairs cellular uptake and protects against invasive infection [30,32]. Adhesion is further facilitated by a second adhesin, CbpA (PspC), which engages the host laminin receptor and activates endocytosis [33].

Once in the endocytic vacuole, pneumococci are transported to the basolateral cell surface, resulting in net transcytosis of the bacterium across the host cell and thus across epithelial and endothelial barriers (for instance, from the blood into the cerebrospinal fluid) [26]. Transcytosis without passing between cells or inducing cytotoxicity appears to be unique to pneumococci, as compared with other meningeal pathogens, and is initiated by binding to the PAF receptor [26]. Invasion is partially inhibited by PAF receptor antagonists [26]. Mice deficient in PAF receptor fail to develop pneumonia and meningitis in the face of bacteremia [34].

Regulatory mechanisms — Pneumococci use numerous regulatory mechanisms to change their surfaces in response to new host environments. Spontaneous phase variation changes gene expression when the bacteria penetrate the mucosal surface and enter the bloodstream, altering the surface coat to avoid host defenses. As an example, the amount of surface phosphorylcholine in pneumococci declines when the organism enters the bloodstream; phosphorylcholine is useful in attaching to the lung but detrimental in the blood, where C-reactive protein can bind and opsonize the bacterium. Other pulmonary pathogens, such as H. influenzae, Pseudomonas, and Mycoplasma, also can modulate the amount of surface phosphorylcholine [35].

Pneumococci change the proteins they express after exposure to heat shock, as occurs when they translocate from the nasal mucosa to the lungs, the brain, and the blood during infection. The S. pneumoniae heat shock protein, ClpP, appears to regulate the expression of pneumococcal virulence proteins, including pneumolysin and capsular polysaccharide. In a murine model, ClpP was required for colonization of the nasopharynx and for survival in host macrophages [36]. Immunization of mice with ClpP elicited a protective immune response against fatal systemic challenge with S. pneumoniae, making ClpP a potential vaccine candidate for human pneumococcal disease.

Pneumococci also can sense the density of other pneumococci in the environment and establish communication between the bacterial cells using small peptides similar to eukaryotic hormones [37]. These peptides signal all the bacteria, as a unified group, to undergo DNA transformation, adherence, or autolysis.

Virulence factors — As noted above, pneumococcal capsule serves as a principal virulence factor, inhibiting ingestion and killing by phagocytic cells. Pneumococci secrete a highly conserved, potent cytotoxin, pneumolysin, which binds to cholesterol and can indiscriminately form pores in membranes of mammalian cells, thereby killing them [38]. Pneumolysin also promotes intra-alveolar replication of pneumococci, penetration of pneumococci from the alveoli into the interstitium, and dissemination of the organisms into the bloodstream [39]. Intratracheal inoculation of pneumolysin into experimental animals produces all the histologic findings of pneumonia [40]. The capacity to induce pneumonia is greatly attenuated in pneumolysin-deficient (knock-out) strains [39,41]], and antibody to pneumolysin provides strong protection against experimentally induced pneumonia [42]. Pneumolysin is an important factor leading to neuronal loss during meningitis [43].

Pneumolysin and constituents of the cell wall produce symptoms of pneumococcal disease by stimulating a vigorous host inflammatory response. In fact, the disease is generally due to the inflammation caused by proliferation at a site where they do not belong. For example, accumulation of bacteria, plasma, and white blood cells produces the collection of fluid in alveoli that causes cough, sputum production, and fever and appears radiographically as consolidation during pneumonia.

Host inflammatory response — Intense inflammation is characteristic of sites of pneumococcal infection. Two major bacterial inflammatory components are the toxin Pneumolysin and the cell wall (teichoic acid-peptidoglycan complex). When pneumococcal cell wall, cytoplasm, and capsular polysaccharide are compared for inflammatory capacity, cell wall has the highest specific activity [20]. This activity can be diminished by the overlying capsule. The signs of infection induced by injection of cell wall mimic those of intact bacteria in animal models of meningitis, pneumonia, and otitis media, showing that the cell wall is an important cause of inflammation [20]. Cell wall fragments are prominently released during autolysis caused by antibiotics. Clinical strains that have defects in autolysis are termed tolerant and are associated with not only attenuated inflammation but also escape from bacterial killing with risk of clinical relapse [44,45].

The teichoic acid and lipoteichoic acid of the cell wall contribute strongly to host defense responses associated with acute inflammation. These components have the following effects:

●Activate the alternative pathway of the complement cascade

●Bind the acute-phase reactant C-reactive protein

●Activate procoagulant activity on the surface of endothelial cells

●Upon binding to epithelial and endothelial cells and macrophages, induce production of cytokines, nitric oxide, and chemokines

●Initiate the influx of neutrophils

Cell walls are recognized by the innate immune system in several ways. Cell wall-binding proteins in serum bind specific wall fragments and present them to human cell receptors [46]. The most prominent receptor on many cell types is Toll-like receptor 2 [47]. Downstream signaling leads to production of cytokines that initiate inflammation, with interleukin 1 being prominent. Evidence suggests that a cell wall-binding protein, Nod-2, also exists in the human cell cytoplasm [48-50]. Inflammatory signaling by various components of the cell wall leads to diverse manifestations of infection such as killing respiratory ciliated cells, promoting blood-brain barrier permeability, and induction of slow wave sleep. Viable pneumococci also induce apoptosis in several cell types in the brain and lung [51].

Factors mediating the influx of neutrophils are in part organ specific. As an example, P-selectin mediates rolling or slowing of circulating neutrophils, while intercellular adhesion molecule-1 (ICAM-1) contributes to the firm adhesion and emigration of neutrophils. Neutrophil emigration into the peritoneum during experimentally induced pneumococcal peritonitis in mice is markedly reduced with mutations in either P-selectin or ICAM-1 and is abolished in double mutants [52]. In contrast, neutrophil emigration into the alveoli during S. pneumoniae-induced pneumonia is not impaired in double mutants. This is consistent with the observation that, in the lungs, pneumococci induce neutrophil efflux in two ways: one dependent upon the CD18 family of leukocyte adhesion molecules and the other by an unknown mechanism independent of CD18 [53].

Attenuating the acute host response to pneumococci has direct clinical application in improving the outcome of disease. A major change in the therapy of meningitis arose from the observation that pneumococcal cell wall fragments are as bioactive as intact bacteria [54]. This observation provided an explanation for an increased host inflammatory response during lysis by antibiotic therapy and a rationale for reducing this unwanted side effect. Over the first few hours of antibiotic-induced bacterial death during meningitis, the leukocyte density in cerebrospinal fluid can increase one to two orders of magnitude [55]. This burst is sufficient to injure bystander host tissues, as evidenced by the significant attenuation of damage upon the inhibition of leukocyte recruitment [56]. The use of a short course of corticosteroids during the early phase of antibiotic therapy to inhibit this response has become accepted for pneumococcal meningitis in adults. (See "Dexamethasone to prevent neurologic complications of bacterial meningitis in adults".)

To truly affect the outcome of meningitis, the bacterial host interactions that cause neuronal death must be interrupted. In this context, the ability of pneumococci to induce human cell apoptosis is important. Evidence indicates that the inflammatory response to pneumococci leads to caspase-dependent human cell death, while direct toxicity of pneumolysin and hydrogen peroxide cause caspase-independent human cell death [43,57,58]. Both mechanisms may be inhibited in vitro and in vivo by treatment with a phosphatidylcholine analog, citicoline [59].

SUMMARY

●Microbiology – Streptococcus pneumoniae is a fastidious gram-positive, alpha-hemolytic bacterium that requires a source of catalase (eg, blood) to grow on agar plates. (See 'Microbiology' above.)

●Clinical manifestations – S. pneumoniae is a major invasive pathogen of children and older adults and is a principal cause of infection of the respiratory tract (otitis media, sinusitis, and pneumonia) and systemic disease (bacteremia, meningitis, endocarditis, and arthritis). (See 'Pathogenesis' above.)

●Capsule – A surface polysaccharide capsule inhibits ingestion and killing by host cells and, therefore, serves as the principal virulence factor of pneumococci. These polysaccharides are antigenically unique and serve as a basis for serologic typing; more than 100 serotypes of S. pneumoniae have been identified. Combinations of capsular types form the basis of highly effective polysaccharide and protein-conjugated polysaccharide vaccines. (See 'Capsule' above.)

●Adherence and invasion – Pneumococci avidly adhere to epithelial cells of the nasopharynx; this colonization is usually asymptomatic and induces protective antibodies. Spread to lungs, blood, and brain involves surface proteins that enable adherence to host receptors to progress to transmigration through epithelial and endothelial barriers. (See 'Adherence' above and 'Invasion' above.)

●Regulatory mechanisms – Pneumococci use many regulatory mechanisms to change their surfaces in response to new host environments, including shedding of capsule and upregulation of adhesins to bind to host cells. (See 'Regulatory mechanisms' above.)

Pneumococci secrete a potent cytotoxin, pneumolysin, which binds to cholesterol and can indiscriminately form pores in membranes of eukaryotic cells. The toxin is a major source of host cell damage in disease. (See 'Regulatory mechanisms' above.)

●Virulence factors – Pneumolysin and cell wall (peptidoglycan, teichoic acid, and lipoteichoic acid complex) activate strong innate immune responses, thereby stimulating inflammation that is prominent in causing symptomatic disease. (See 'Virulence factors' above.)

●Host inflammatory response – The success of antibiotic therapy depends on bacterial killing by autolysis, which by necessity releases highly inflammatory bacterial debris. Clinical strains that fail to lyse are termed tolerant and may be a source of clinical failure. (See 'Host inflammatory response' above.)