Lung antibacterial defense mechanisms in infant and adult rats: implications for the pathogenesis of group B streptococcal infections in the neonatal lung

We investigated factors that may contribute to lung infections in infants by studying the intrapulmonary responses to aerosols of three different types of organisms--group B streptococcus with and without type-specific capsule, Pseudomonas aeruginosa, and Staphylococcus aureus--in infant (12-h-old or 24-36-h-old) and adult (150 g, 6-w-old) rats. After aerosol exposure, the lung clearance rate of each organism varied inversely with the age of the animals, and the magnitude of the clearance defect was related more strongly to animal age than to the bacterial species. Fewer alveolar macrophages from infant animals phagocytosed each type of organism in vivo, and the rate of neutrophil accumulation in the lungs of infant animals was delayed. The neonatal lung functioned effectively, however, as an antibacterial barrier, as newborn animals survived an aerosolized inoculum that exceeded the LD50 by the subcutaneous route.     

Lipid Peroxidation of Lung Surfactant in Experimental Neonatal Group B Streptococcal Pneumonia

Group B streptococcal (GBS) pneumonia, with neutrophilic granulocytes immigrating into the lungs, may occur in neonates. The incidence is particularly high among preterm infants, who often are treated with exogenous surfactant. We have previously demonstrated in vitro that neutrophils stimulated by GBS cause lipid peroxidation (LPO) and functional impairment of lung surfactant. The present study aimed at evaluating LPO of exogenous lung surfactant (Curosurf) and the protective effect of the natural antioxidant, vitamin E in immature ventilated newborn rabbits with experimental neonatal GBS pneumonia. There was a prominent proliferation of GBS in the lungs of animals treated with surfactant and ventilated for 5 h. GBS-infected rabbits had a higher LPO of lung lavage fluid than non-infected ones. The LPO could be diminished using vitamin E, which, however, did not affect bacterial proliferation. During the 5-h incubation period, mean lung-thorax compliance values were significantly lower in GBS-infected than in noninfected animals. We speculate that addition of vitamin E to exogenous surfactant preparations may improve their resistance to LPO and make them more suitable for treatment of neonates with pneumonia.     

A mutation in the mip gene results in an attenuation of Legionella pneumophila virulence

Infection by Legionella pneumophila is believed to depend upon its ability to multiply within host alveolar macrophages. To investigate this, a site-specific mutation was introduced into a gene (mip) that encodes a 24,000-Da surface protein; an 80-fold loss of infectivity for both U937 cells and explanted human alveolar macrophages was observed. Further phenotypic analysis of the mutant strain has failed to show alterations in bacterial factors (e.g., proteinase, lipopolysaccharide) that have suspected roles in virulence. To substantiate that this mutation also results in reduced virulence in animals, the lethality and clinical illnesses produced by the parent and mutant L. pneumophila strains were compared in guinea pigs after intratracheal inoculation. The mutant strain produced fewer illnesses, slower-progressing disease, and fewer lethal infections than either the parent strain or a derivative of the mutant strain with the wild-type mip gene reintroduced. When sublethal inocula of the three strains were used, the mutant bacteria were recovered in slightly lower numbers from lung homogenates and in significantly lower numbers from the spleen, at 48 h, than were the other two test strains. Thus mip seems to be necessary for full virulence of L. pneumophila and may represent the first genetically defined virulence factor in this species.     

Pathophysiology and histopathology of group B streptococcal sepsis in Macaca nemestrina primates induced after intraamniotic inoculation: evidence for bacterial cellular invasion

Four pregnant Macaca nemestrina dams at 140-145 days of gestation received an intraamniotic inoculation of group B streptococci (GBS). All four premature infants were born by cesarean delivery, were bacteremic at birth, and showed symptoms of GBS sepsis similar to infected human infants with early-onset disease. Three infants did not receive antibiotics and died of GBS sepsis by 10 h of age despite mechanical ventilation and fluids for blood pressure support. Penicillin treatment of the fourth infant prolonged survival and decreased the requirement for supportive therapy. Quantitative cultures and histopathology were done on all four infants. Transmission electron microscopy of lung tissue demonstrated GBS within membrane-bound vacuoles of type I and II alveolar epithelium and interstitial fibroblasts. This model should be useful for studying the early steps in the pathogenesis of early-onset GBS infections. GBS may enter alveolar epithelial cells to transit this barrier and ultimately disseminate via the blood-stream.     

Combined treatment with surfactant and specific immunoglobulin reduces bacterial proliferation in experimental neonatal group B streptococcal pneumonia.

Neonates suffering from group B streptococcal (GBS) pneumonia often lack type-specific opsonizing antibodies. We studied the influence of combined intratracheal treatment with surfactant and a specific antibacterial polyclonal antibody (IgG fraction) on bacterial proliferation and lung function in an animal model of GBS pneumonia. Near-term newborn rabbits received an intratracheal injection of either the specific IgG antibody, nonspecific IgG, surfactant, a mixture of surfactant and the antibody, or 0.9% saline. At 30 min the rabbits were infected with a standard dose (10(8)) of the encapsulated GBS strain 090 Ia. After 5 h of mechanical ventilation the mean estimated increase in bacterial number in lung homogenate (log10 colonies/g) was 0.76 in the antibody group, 0.92 in the nonspecific IgG group, 0.55 in the surfactant group, and 1.29 in the saline group. A mean decrease in bacterial number (-0.05) was observed in the group that received combined treatment with surfactant and antibody (p < 0.05 versus all other groups). Lung-thorax compliance was significantly higher in both groups of surfactant-treated animals compared with saline or IgG treatment. We conclude that in experimental neonatal GBS pneumonia combined treatment with surfactant and a specific immunoglobulin against GBS reduced bacterial proliferation more effectively than either treatment alone.     

Lung Function and Bacterial Proliferation in Experimental Neonatal Pneumonia in Ventilated Rabbits Exposed to Monoclonal Antibody to Surfactant Protein A

Surfactant protein A (SP-A) increases the resistance of surfactant to inhibition by plasma and other proteins. In a previous study we found that a monoclonal anti-SP-A antibody (R 5) increased the sensitivity of surfactant to inhibition by fibrinogen in vivo and in vitro. SP-A has been shown to stimulate microbial phagocytosis and killing by alveolar macrophages. We hypothesized that using R 5 to inactivate SP-A in an animal model mimicking congenital group B streptococcal (GBS) pneumonia might result in increased bacterial proliferation and a deterioration in lung function. Newborn near term rabbits were delivered by Cesarean section, anesthetized, tracheotomized, and ventilated for 5 h in a plethysmograph system allowing measurement of dynamic lung-thorax compliance. Postnatally the animals received one intratracheal injection (5 ml/kg) of R 5, nonspecific IgG, or normal saline. At 30 min all animals received a standard dose of an encapsulated GBS strain by intratracheal injection. The number of bacteria (mean log₁₀ CFU/g lung ± S.D.; CFU = colony forming unit) was evaluated in lung homogenates. Histologic lung sections were judged by light microscopy. Bacterial proliferation was similar in rabbits treated with the monoclonal antibody (9.33 ± 0.39; n= 14) and in control animals receiving saline (9.16 ± 0.35; n= 14) or nonspecific IgG (9.26 ± 0.31; n= 11). No significant differences were noted on the histologic analysis or in measurements of lung function. We conclude that intratracheal instillation of a monoclonal anti-SP-A antibody did not increase bacterial proliferation in GBS-infected newborn rabbits. These findings suggest that SP-A does not play an important role in protection against encapsulated GBS strains in the neonatal period. Accepted for publication: 20 June 1997     

Phagocytosis and killing of common bacterial pathogens of the lung by human alveolar macrophages

To investigate factors that determine susceptibility of the lungs to infection with common respiratory pathogens, we studied phagocytosis and killing of nontypable Haemophilus influenzae, H. influenzae type b, Streptococcus pneumoniae types III, VI, and XIV, an unencapsulated variant of S. pneumoniae type III, and Staphylococcus aureus Cowan I, by using human alveolar macrophages obtained by bronchoalveolar lavage of healthy nonsmokers. After opsonization with 10% pooled human serum, mean uptake (+/- standard deviation) of nontypable H. influenzae (67.5% +/- 15.0%), unencapsulated S. pneumoniae type III (71.2% +/- 4.8%) and S. aureus (79.1% +/- 10.2%) was significantly greater (P less than .01) than that of H. influenzae type b (40.1% +/- 15.0%), and S. pneumoniae types III (4.4% +/- 3.1%), VI (11.8% +/- 9.6%), or XIV (8.7% +/- 7.0%). Nontypable H. influenzae was ingested after opsonization with much less pooled human serum than was H. influenzae type b, and uptake of encapsulated S. pneumoniae was not enhanced by as much as 80% pooled human serum. Intracellular killing of unencapsulated S. pneumoniae type III and nontypable H. influenzae was rapid and complete and corresponded to the degree of phagocytosis, but despite a high uptake, S. aureus were killed slowly and incompletely. The virulence of S. pneumoniae and H. influenzae as lung pathogens is thus determined jointly by encapsulation and the inadequate opsonizing effect of normal human serum, whereas that of S. aureus may be related to the organism's relative resistance to intracellular killing by alveolar macrophages.     

Virulence studies, in mice, of transposon-induced mutants of Staphylococcus aureus differing in capsule size

We used three related strains of Staphylococcus aureus to determine whether capsule size influenced bacterial virulence. Strain SA1 mucoid elaborated a large capsule demonstrable by transmission electron microscopy (TEM). Nonmucoid isolates were derived from strain SA1 mucoid by Tn551 insertional mutagenesis. By TEM, strain JL24 produced a "microcapsule," whereas strain JL25 was unencapsulated. Strain SA1 mucoid had a 50% lethal dose for mice greater than 3,000-fold lower than that of strains JL24 and JL25. Quantitative cultures of blood and kidney from animals challenged intravenously revealed that strain SA1 mucoid was cleared less readily from the bloodstream and kidneys than the nonmucoid mutants. In an in vitro assay, only strain SA1 mucoid demonstrated antibody-dependent, complement-mediated opsonophagocytosis by human leukocytes. Strains JL24 and JL25 were opsonized for phagocytosis by complement alone. Thus a highly encapsulated strain of S. aureus was more virulent in mice than two related nonmucoid strains. The microencapsulated mutant was not more virulent than the unencapsulated mutant.     

Neonatal mouse model of group B streptococcal infection.

Neonatal mice were infected with type III group B streptococcal (GBS) strain M781 by the intraperitoneal route. Age-related susceptibility to challenge was seen within the first 5 days of life. Quantitative blood cultures demonstrated a rapid increase in bacterial numbers during the first 30 h after challenge. Infected pups showed clinical signs of septicemia, and most succumbed within 48 h of challenge. Histopathologic evaluation of the neonates showed bacterial infection within 1 day after challenge. Pregnant adult mice were given a single inoculation of serum raised in rabbits against a tetanus toxoid-conjugated type III GBS polysaccharide vaccine. This serum passively protected 100% of the offspring. This neonatal mouse model of GBS infection and protection may be suitable for study of various forms of intervention.     

The contribution of pneumococcal cell wall to the pathogenesis of experimental otitis media

We studied the contribution of pneumococcal cell wall to the pathogenesis of otitis media in chinchillas after middle ear inoculation of killed, encapsulated type 7F Streptococcus pneumoniae; killed, unencapsulated R6 S. pneumoniae; and isolated R6 pneumococcal cell wall. Ears inoculated with encapsulated and unencapsulated pneumococci had significantly higher concentrations of polymorphonuclear and mononuclear leukocytes and lysozyme in middle ear fluid and developed more epithelial metaplasia and granulation tissue than did saline-inoculated ears. The mean concentration of lysozyme in middle ear fluid was higher in ears inoculated with killed, unencapsulated than encapsulated pneumococci. The middle ear mucoperiosteum of ears inoculated with pneumococcal cell wall showed significantly more polymorphonuclear leukocytes, epithelial metaplasia, subepithelial congestion, and granulation tissue than did control ears. Because nonviable, unencapsulated pneumococci and pneumococcal cell wall caused middle ear inflammation in the chinchilla model of otitis media, it is possible that cell envelope and cell wall components released during bacterial lysis may contribute to chronic otitis media with effusion in humans.     

Bacterial evasion of the antibody response: human IgG antibodies neutralize soluble but not bacteria-associated group B streptococcal C5a-ase.

Most strains of group B streptococci (GBS) possess an enzyme that inactivates the human anaphylatoxin C5a by cleaving a heptapeptide from the carboxyl terminus of C5a. This enzyme, called GBS C5a-ase, has been purified to homogeneity and cleaves and inactivates C5a in physiologic buffer. The enzymatic activity of soluble C5a-ase is completely inhibited, however, in the presence of plasma or serum from normal human adults. The neutralization of soluble C5a-ase by plasma and serum results largely from naturally occurring IgG antibodies directed against C5a-ase. IgG does not neutralize C5a-ase present on intact encapsulated type III GBS but does neutralize the C5a-ase activity associated with a transposon-induced mutant strain of type III GBS that lacks capsule. The location of GBS C5a-ase on the surface of encapsulated type III GBS permits the C5a-ase to inactivate C5a while evading neutralization by IgG antibodies.     

The relative role of bacterial cell wall and capsule in the induction of inflammation in pneumococcal meningitis

The relative contribution of bacterial components to the induction of inflammation during Streptococcus pneumoniae meningitis is unknown. Several strains of pneumococci with differences in cell surface characteristics (capsule or cell wall) were compared for the effect on the inflammatory response evoked during infection of the cerebrospinal fluid (CSF) in vivo. The presence of bacterial capsular polysaccharide was not necessary for bacterial growth in CSF in vivo but correlated with greater CSF bacterial density. CSF inflammatory changes began to appear when the bacterial concentration exceeded 10(5) cfu/ml, regardless of the pneumococcal strain. CSF inflammatory changes could be invoked by cisternal instillation of 10(5)-10(6) cell equivalents of whole, heat-killed unencapsulated strains or their isolated cell walls but not by similar concentrations of heat-killed encapsulated strains or isolated capsular polysaccharide. Hypoglycorrhachia was observed only during inflammation caused by live bacteria. The inflammatory response characteristic of naturally acquired pneumococcal meningitis can be reproduced by challenge with both encapsulated and uncapsulated bacteria. The bacterial cell wall appears to be the most potent pneumococcal surface component in inducing CSF inflammation.     

Inhibition of neutrophil elastase reduces lung injury and bacterial count in hamsters

Neutrophil elastase (NE) has been recognized as a double-edge sword as it can be both host-defensive and pro-inflammatory depending on its tissue level and microenvironment. The present study was designed to examine whether inhibition of NE activity by sivelestat, a specific NE inhibitor with low cellular permeability, is beneficial for acute lung injury induced by Streptococcus pneumoniae in hamsters. Intratracheal inoculation of live S. pneumoniae (1.5 x 10(7) CFU/Lung) into hamsters caused acute lung injury characterized by an increase in lung alveolar permeability and leukocytes accumulation in the lungs. These pathophysiological changes, which were associated with elevation of NE activity in the bronchoalveolar lavage fluid (BALF), were transient but remained high 4-22 h post-bacterial inoculation. Intravenous infusion of sivelestat at 3mg/kg/h 0-22 h after bacterial inoculation reduced the increase in NE activity and lung alveolar permeability, as indicated by leakage of pre-injected human serum albumin into BALF, without affecting the number of leukocytes in BALF and lung interstitial tissue. Interestingly, the number of bacteria in BALF and lung interstitial tissue was also reduced by sivelestat to less than 10% of the control values. Furthermore, S. pneumoniae-induced decrease in the level of surfactant protein D (SP-D), a protein that protects against bacterial infection, was preserved by sivelestat treatment with a significant negative correlation between NE activity and SP-D content in BALF. These results suggest that sivelestat is beneficial in acute lung injury associated with lung bacterial infection and that this inhibitor rather decreases infection by preserving host defense system.     

Enhanced immediate inflammatory response to Streptococcus pneumoniae in the lungs of mice with pulmonary emphysema

Background and objective:   Pulmonary emphysema is associated with frequent respiratory infections but little is known about the reasons for this susceptibility to bacterial infection. We previously demonstrated an impaired inflammatory response to Streptococcus pneumoniae in an experimental emphysema mouse model at 24 h, or longer following bacterial inoculation. Toll-like receptors (TLR) have been recognized as regulators in the inflammatory response. We examined the expression of TLR on alveolar macrophages in experimental emphysema mice and evaluated the immediate inflammatory response of the emphysematous lung to streptococcal infection.
Methods:   Elastase was administered once into mice trachea to induce pulmonary emphysema. Three weeks later, expression of TLR-2 and TLR-4 in the BAL cells was examined by immunostaining. Following the intratracheal inoculation of Streptococcus pneumoniae , pro-inflammatory cytokine concentrations were measured in the BAL fluids of the control and emphysema mice.
Results:   The expression of TLR-2 and TLR-4 was significantly elevated in the alveolar macrophages of emphysema mice. Six hours after infection, neutrophils in the BAL fluid of emphysema mice were significantly increased, and the levels of tumour necrosis factor-α, IL-1β and IL-6 were significantly elevated, compared with the control mice. At 3 h post inoculation, macrophage inflammatory protein-2 levels were significantly elevated.
Conclusions:   The immediate inflammatory response in the emphysematous lung is significantly enhanced in response to streptococcal infection. This may be partly attributed to the increased expression of TLR in the alveolar macrophages of emphysema mice.     

Role of pulmonary phagocytes in host defense against group B streptococci in preterm versus term rabbit lung.

Intrapulmonary clearance of group B streptococci (GBS) occurred in term rabbits 4 and 8 h after infection; GBS growth was evident in preterm rabbits at 8 h. Bronchoalveolar lavage revealed 17-fold higher numbers of pulmonary alveolar macrophages (PAM) in term versus preterm animals immediately after infection, whereas polymorphonuclear leukocyte (PMNL) recruitment was 13-fold greater in preterm than term rabbits at 8 h. Anti-CD18 monoclonal antibody R15.7 did not reduce PMNL influx or GBS killing in term animals. R15.7 failed to inhibit PMNL influx but augmented GBS growth in preterm animals. R15.7 significantly impaired GBS phagocytosis by preterm and term PMNL in vitro but had no effect on ingestion of GBS by preterm and term PAM. Thus, GBS infection initiates PMNL recruitment into lungs of preterm rabbits by CD18-independent mechanisms, but phagocytosis of GBS by PMNL is largely CD18-dependent. The poorer outcome of GBS pneumonia in preterm versus term newborns may result from low levels of PAM, thereby mandating recruitment of PMNL as a second phagocytic defense.     

Flamingo cadherin: a putative host receptor for Streptococcus pneumoniae

Streptococcus pneumoniae fructose bisphosphate aldolase (FBA) is a cell wall-localized lectin. We demonstrate that recombinant (r) FBA and anti-rFBA antibodies inhibit encapsulated and unencapsulated S. pneumoniae serotype 3 adherence to A549 type II lung carcinoma epithelial cells. A random combinatorial peptide library expressed by filamentous phage was screened with rFBA. Eleven of 30 rFBA-binding phages inhibited 90% of S. pneumoniae adhesion to A549 cells. The insert peptide sequence of 9 of these phages matched the Flamingo cadherin receptor (FCR) when aligned against the human genome. A peptide comprising a putative FBA-binding region of FCR (FCRP) inhibited 2 genetically and capsularly unrelated pairs of encapsulated and unencapsulated S. pneumoniae strains from binding to A549 cells. Moreover, FCRP inhibited S. pneumoniae nasopharyngeal and lung colonization and, possibly, pneumonia development in the mouse intranasal inoculation model system. These data indicate that FBA is an S. pneumoniae adhesin and that FCR is its host receptor.     

Interaction between lung surfactant and nitric oxide production by alveolar macrophages stimulated by group B streptococci

The major etiologic agent in neonatal pneumonia and meningitis is group B streptococci (GBS). Nitric oxide (NO) production by alveolar macrophages (AM) in response to Gram-positive bacteria such as GBS and the effect of surfactant on this production have received little attention. We studied production of NO by GBS-stimulated AM using the Griess reaction, the effect of lung surfactant on this NO production, and the possible lipid peroxidation (LPO) of surfactant caused by NO. The LPO test was used to measure surfactant peroxidation. Heat-killed and live GBS were found to stimulate NO production by rat alveolar macrophages, and the presence of interferon gamma (IFN-gamma) increased this stimulation in a synergistic manner. Curosurf(R), the natural surfactant used in our study, significantly reduced NO production in various sets of experiments. Lipid peroxidation of surfactant was noted when NO was produced by stimulated AM, a phenomenon that could be suppressed by NG-monomethyl L-arginine (L-NMMA), the inhibitor of NO synthase. In the lung of GBS-infected neonates, nitric oxide produced by AM might contribute to the destruction of surfactant caused by inflammatory cells. Pediatr Pulmonol. 2000; 30:106- 113.     

Serotypes VI and VIII predominate among group B streptococci isolated from pregnant Japanese women

Infection by group B streptococcus (GBS) is an important cause of bacterial disease in neonates, pregnant women, and nonpregnant adults. Whereas serotypes Ia, Ib, II, III, and V are most commonly associated with colonization and disease in the United States, strains of other serotypes have been isolated from patients in Japan. By use of an inhibition ELISA, the serotypes of 73 vaginal colonizing GBS strains isolated from healthy pregnant Japanese women were investigated. Twenty-six (35.6%) were type VIII, 18 (24.7%) were type VI, and the remaining 29 were distributed among more traditional serotypes. Strains were also tested by immunoblot for the presence of GBS surface proteins. Fifty-three (72.6%) of the 73 strains expressed one or more laddering GBS proteins. These data show that type VI and VIII GBS strains are common vaginal isolates in pregnant Japanese women and that one or more laddering proteins are present in most GBS strains.     

Use of a dynamic in vitro attachment and invasion system (DIVAS) to determine influence of growth rate on invasion of respiratory epithelial cells by group B Streptococcus

Expression of capsular polysaccharide (CPS) and some surface proteins by group B Streptococcus (GBS) is regulated by growth rate. We hypothesized that precise control of GBS growth, and thus surface-expressed components, could modulate the ability of GBS to invade eukaryotic cells. To test this hypothesis, a dynamic in vitro attachment and invasion system (DIVAS) was developed that combines the advantages of bacterial growth in continuous culture with tissue culture. Tissue culture flasks were modified with inlet and outlet ports to permit perfusion of GBS. Encapsulated type III GBS strains M781 and COH1 and strains COH1-11 and COH1-13 (transposon mutants of COH1 that express an asialo CPS or are acapsular, respectively) were grown in continuous culture in a chemically defined medium at fast mass doubling time (t(d) = 1.8 h) and slow (t(d) = 11 h) growth rates, conditions previously shown to induce and repress, respectively, type III CPS expression. Encapsulated GBS strains invaded A549 respiratory epithelial cells 20- to 700-fold better at the fast than at the slow growth rate, suggesting a role for CPS. However, unencapsulated GBS were also invasive but only when cultured at the fast growth rate, which indicates that GBS invasion is independent of CPS expression and can be regulated by growth rate. Growth rate-dependent invasion occurred when GBS was grown in continuous culture under glucose-defined, thiamine-defined, and undefined nutrient limitations. These results suggest a growth rate-dependent regulation of GBS pathogenesis and demonstrate the usefulness of DIVAS as a tool in studies of host-microbe interactions.     

Perinatal age determines the severity of retarded lung development induced by starvation

Susceptibility of the lung to caloric restriction is age-dependent, with more permanent damage occurring during the phases of growth and differentiation. Because the guinea pig is born with more well-developed alveoli than are other rodents, the postnatal lung of this species may better resist alveolar hypoplasia than the prenatal lung. Control animals were raised from sows provided food ad libitum during and after normal gestations (66 to 68 days). Starvation groups received 50% rations of control food intakes during 1 of three 21-day periods: prenatal starvation, with sows rationed during their last trimester (Day 45 to term); neonatal starvation, with nursing sows rationed during the 21 days postpartum before weaning; weanling starvation, with animals starved from 21 to 42 days postpartum. Lungs were fixed in situ with glutaraldehyde and analyzed for pulmonary morphometrics. At the end of starvation and before refeeding, lungs of prenatal and weanling starvation groups were significantly reduced for tissue volumes, alveolar and capillary surface areas, and pulmonary diffusing capacity. Recovery with feeding was complete for most parameters in the starved weanlings by maturity, but animals starved prenatally showed residual starvation effects as adults. The neonatally starved animals showed minimal effects of starvation on lung dimensions, both acutely and as adults. Morphologically, the lungs of some prenatally starved neonates were apparently retarded, at least to the saccular phase, and correlated with significant increases in the number of stillborn litters and in neonatal mortality within hours of parturition.