Faculty Bibliography

During mucosal colonization, epithelial cells are concurrently exposed to numerous microbial species. Epithelial cytokine production is an early component of innate immunity and contributes to mucosal defence. We have previously demonstrated a synergistic response of respiratory epithelial cells to costimulation by two human pathogens, Streptococcus pneumoniae and Haemophilus influenzae. Here we define a molecular mechanism for the synergistic activation of epithelial signalling during polymicrobial colonization. H. influenzae peptidoglycan synergizes with the pore-forming toxin pneumolysin from S. pneumoniae. Radiolabelled peptidoglycan enters epithelial cells more efficiently in the presence of pneumolysin, consistent with peptidoglycan gaining access to the cytoplasm via toxin pores. Other pore-forming toxins (including anthrolysin O from Bacillus anthracis and Staphylococcus aureus alpha-toxin) can substitute for pneumolysin in the generation of synergistic responses. Consistent with a requirement for pore formation, S. pneumoniae expressing pneumolysin but not an isogenic mutant expressing a non-pore-forming toxoid prime epithelial responses. Nod1, a host cytoplasmic peptidoglycan-recognition molecule, is crucial to the epithelial response. Taken together, these findings demonstrate a role for cytosolic recognition of peptidoglycan in the setting of polymicrobial epithelial stimulation. We conclude that combinations of extracellular organisms can activate innate immune pathways previously considered to be reserved for the detection of intracellular microorganisms.

Expression of a polysaccharide capsule is required for the full pathogenicity of many mucosal pathogens such as Streptococcus pneumoniae. Although capsule allows for evasion of opsonization and subsequent phagocytosis during invasive infection, its role during mucosal colonization, the organism's commensal state, remains unknown. Using a mouse model, we demonstrate that unencapsulated mutants remain capable of nasal colonization but at a reduced density and duration compared to those of their encapsulated parent strains. This deficit in colonization was not due to increased susceptibility to opsonophagocytic clearance involving complement, antibody, or the influx of Ly-6G-positive cells, including neutrophils seen during carriage. Rather, unencapsulated mutants remain agglutinated within lumenal mucus and, thus, are less likely to transit to the epithelial surface where stable colonization occurs. Studies of in vitro binding to immobilized human airway mucus confirmed the inhibitory effect of encapsulation. Likewise, pneumococcal variants expressing larger amounts of negatively charged capsule per cell were less likely to adhere to surfaces coated with human mucus and more likely to evade initial clearance in vivo. Removal of negatively charged sialic acid residues by pretreatment of mucus with neuraminidase diminished the antiadhesive effect of encapsulation. This suggests that the inhibitory effect of encapsulation on mucus binding may be mediated by electrostatic repulsion and offers an explanation for the predominance of anionic polysaccharides among the diverse array of unique capsule types. In conclusion, our findings demonstrate that capsule confers an advantage to mucosal pathogens distinct from its role in inhibition of opsonophagocytosis--escape from entrapment in lumenal mucus.

BACKGROUND: The heptavalent pneumococcal conjugate vaccine (PCV7) has decreased the incidence of invasive pneumococcal disease among children in the United States. In the postlicensure period, the impact of non-PCV7 serotypes against pediatric pneumococcal bacteremia is unknown. METHODS: Episodes of bacteremia due to Streptococcus pneumoniae and other respiratory pathogens (ORP), namely Neisseria meningitidis, Haemophilus influenzae, and Moraxella catarrhalis, were identified in children <18 years old at the Children's Hospital of Philadelphia from January 1999 to May 2005. For pneumococci, serotype distribution and antibiotic resistance were compared. RESULTS: A total of 188 episodes of pneumococcal bacteremia and 55 episodes of ORP bacteremia were identified. By comparing data from 1999-2000 with data from 2001 to May 2005, we found that the incidence of pneumococcal bacteremia decreased by 57%. The incidence of bacteremia caused by ORPs was unchanged; 1.43 episodes (95% confidence interval [CI], 0.84-2.29 episodes) to 1.25 (95% CI, 0.88-1.71) per 10,000 emergency department visits. Vaccine serotypes caused 85% of episodes of bacteremia in 1999-2000, compared with 34% of episodes of bacteremia in 2001 to May 2005 (P<.01). The percentage of isolates nonsusceptible to penicillin increased from 25% to 39% (P<.05). The percentage of episodes of pneumococcal bacteremia caused by vaccine-related serotypes--those of the same serogroup but not of the same serotype as PCV7--increased from 6% of episodes in the prelicensure period to 35% of episodes in the postlicensure period (P<.01). Rates of serotype pneumococcal bacteremia caused by nonvaccine serotypes were not statistically different between the 2 periods. CONCLUSIONS: The overall incidence of pneumococcal bacteremia decreased by 57% after the introduction of PCV7. During the postlicensure period, there were significant decreases in the incidence of pneumococcal bacteremia caused by vaccine serotypes; however, rates of penicillin resistance and bacteremia due to vaccine-related serotypes increased.

BACKGROUND: The heptavalent pneumococcal conjugate vaccine was licensed in the United States in 2000 for use in infants and children. Postlicensure surveillance revealed substantial regional and national decreases in invasive pneumococcal disease. It is not known whether widespread vaccine use has led to a concomitant decrease in invasive pneumococcal disease-associated hospitalization rates. OBJECTIVE: We examined national trends in rates of hospitalization among both children and adults with invasive pneumococcal disease. METHODS: Data from the 1998-2003 National Hospital Discharge Survey and population estimates from the National Center for Health Statistics were used to calculate rates of hospital discharge for patients admitted with invasive pneumococcal disease, defined as meningitis or bacteremia caused by Streptococcus pneumoniae. RESULTS: Rates of hospital discharge for patients admitted with invasive pneumococcal disease decreased during the study period from a peak of 12.03 discharges per 100,000 population in 1999 to 5.60 discharges per 100,000 population in 2003 (P < .001). Rates of hospital discharge for persons admitted with pneumococcal meningitis decreased from 1.60 discharges per 100,000 person-years in the prelicensure period to 0.53 discharges per 100,000 person-years in the postlicensure period. For persons > or = 65 years of age but not for those < 4 years of age, the rates of hospital discharge for those admitted with pneumococcal bacteremia were significantly lower during the postlicensure period, compared with the prelicensure period. CONCLUSIONS: Hospital discharge rates for persons admitted with invasive pneumococcal disease, including meningitis, have decreased since introduction of the heptavalent pneumococcal conjugate vaccine. The decrease was driven in part by the reduction of invasive pneumococcal disease-associated hospitalizations in the subgroup aged > or = 65 years.

Epithelial cells act as an interface between human mucosal surfaces and the surrounding environment. As a result, they are responsible for the initiation of local immune responses, which may be crucial for prevention of invasive infection. Here we show that epithelial cells detect the presence of bacterial pore-forming toxins (including pneumolysin from Streptococcus pneumoniae, alpha-hemolysin from Staphylococcus aureus, streptolysin O from Streptococcus pyogenes, and anthrolysin O from Bacillus anthracis) at nanomolar concentrations, far below those required to cause cytolysis. Phosphorylation of p38 MAPK appears to be a conserved response of epithelial cells to subcytolytic concentrations of bacterial poreforming toxins, and this activity is inhibited by the addition of high molecular weight osmolytes to the extracellular medium. By sensing osmotic stress caused by the insertion of a sublethal number of pores into their membranes, epithelial cells may act as an early warning system to commence an immune response, while the local density of toxin-producing bacteria remains low. Osmosensing may thus represent a novel innate immune response to a common bacterial virulence strategy.

Since mucosal surfaces may be simultaneously colonized by multiple species, the success of an organism may be determined by its ability to compete with co-inhabitants of its niche. To explore the contribution of host factors to polymicrobial competition, a murine model was used to study the initiation of colonization by Haemophilus influenzae and Streptococcus pneumoniae. Both bacterial species, which occupy a similar microenvironment within the nasopharynx, persisted during colonization when given individually. Co-colonization, however, resulted in rapid clearance of S. pneumoniae from the upper respiratory tract, associated with increased recruitment of neutrophils into paranasal spaces. Systemic depletion of either neutrophil-like cells or complement was sufficient to eliminate this competitive effect, indicating that clearance was likely due to enhanced opsonophagocytic killing. The hypothesis that modulation of opsonophagocytic activity was responsible for host-mediated competition was tested using in vitro killing assays with elicited neutrophil-like cells. Components of H. influenzae (but not S. pneumoniae) stimulated complement-dependent phagocytic killing of S. pneumoniae. Thus, the recruitment and activation of neutrophils through selective microbial pattern recognition may underlie the H. influenzae-induced clearance of S. pneumoniae. This study demonstrates how innate immune responses may mediate competitive interactions between species and dictate the composition of the colonizing flora.

The epithelial surfaces of the upper respiratory tract are continuously exposed to a wide variety of commensal microorganisms. In addition to acting as a physical barrier, epithelial cells respond to specific microbial products with the generation of signals, such as cytokines, that trigger inflammation. Because they are common components of the nasopharyngeal flora that share the potential to cause disease, we investigated the effects of Haemophilus influenzae and Streptococcus pneumoniae, alone and in combination, on human respiratory epithelial cells in culture and in a murine model of nasopharyngeal colonization. Exposure of A549 or Detroit 562 epithelial cells to both S. pneumoniae and H. influenzae led to a synergistic increase in production of IL-8, the major neutrophil chemokine in the airway, through an NF-kappaB-dependent mechanism. Likewise, nasal cocolonization of mice caused a synergistic rise in local production of macrophage inflammatory protein 2 in nasal lavage fluid and subsequent recruitment of neutrophils. This synergistic effect depended on production of the pore-forming cytolytic toxin, pneumolysin, by S. pneumoniae and activation of host p38 mitogen-activated protein kinase. Although both H. influenzae and S. pneumoniae have ligands for Toll-like receptors (TLRs) TLR2 and TLR4, synergistic activation was TLR2- and TLR4-independent. Thus, epithelial surfaces are capable of amplifying proinflammatory responses during concurrent stimulation by multiple microbial species. These synergistic responses, demonstrated both in vitro and in vivo, may contribute to inflammation of heavily colonized mucosal barriers.

Ocular manifestations of cat scratch disease are uncommon. The diagnosis is usually made on the basis of increasing Bartonella henselae serum antibody titers. We report a child presenting with orbital abscess and osteomyelitis who was diagnosed with hepatosplenic cat scratch disease by detection of B. henselae DNA in the orbital abscess fluid.

IgA, the major class of Ig in secretions, classically functions by interfering with microbial attachment to host tissues. Many mucosal pathogens, including Streptococcus pneumoniae, express an IgA1 protease that may circumvent the protective effects of this Ig subclass. Because these proteases are specific for human IgA1, we generated human mAbs to the major surface antigen of the pneumococcus, its capsular polysaccharide, and tested their effect in a colonization model of bacterial adherence to respiratory epithelial cells in culture. Rather than inhibiting adherence, type-specific IgA1 markedly enhanced bacterial attachment to host cells, but only when cleaved by IgA1 protease. Neither antibodies of protease-insensitive subclasses (IgA2 and IgG) nor those directed against heterologous capsules had such activity. The adherence-promoting properties of cleaved antibodies correlated with the cationic characteristics of their variable segments, suggesting that bound Fab fragments may neutralize the inhibitory effect of negatively charged capsules on adhesive interaction with host cells. Coating of pneumococci with anticapsular polysaccharide antibody unmasked the bacterial phosphorylcholine ligand, allowing for increased adherence mediated by binding to the platelet activating factor receptor on epithelial cells. In addition, our findings provide evidence for a novel function of bacterial IgA1 proteases. These enzymes may enable pathogens to subvert the antigen specificity of the humoral immune response to facilitate adhesive interactions and persistence on the mucosal surface.