Pneumococcal meningitis in children: relationship of antibiotic resistance to clinical characteristics and outcomes.

BACKGROUND: The relationship of antibiotic susceptibility to clinical outcome in children with pneumococcal meningitis is uncertain. Previous studies have been limited by inclusion of relatively few patients infected with nonsusceptible pneumococci and inconsistent use of empiric vancomycin. METHODS: Medical records of 86 children with culture-confirmed pneumococcal meningitis at a single institution from October, 1991, to October, 1999, were retrospectively reviewed, and differences in presentation and outcome based on antibiotic susceptibility of pneumococcal isolates were assessed. RESULTS: Of 86 isolates 34 were nonsusceptible to penicillin (12 resistant). Of 60 isolates for which cefotaxime susceptibility data were available, 17 were nonsusceptible (12 resistant). Antibiotic susceptibility was not significantly associated with death, intensive care unit admission, mechanical ventilation, focal neurologic deficits, seizures, secondary fever, abnormal neuroimaging studies or hospital days. Children with penicillin-resistant isolates had significantly higher median blood leukocyte counts (24,100/microliter vs. 15,700/microliter, P = 0.03) and lower median CSF protein concentrations (85 mg/dl vs. 219 mg/dl, P = 0.04), were more likely to have a CSF glucose concentration of > or = 50 mg/dl (7 of 11 vs. 15 of 68, P = 0.009) and had lower rates of sensorineural hearing loss (1 of 8 vs. 25 of 40, P = 0.02) than children with isolates that were not resistant to penicillin. Children with cefotaxime-nonsusceptible isolates had an increased median duration of primary fever compared with those with nonsusceptible strains (6 days vs. 3.5 days, P = 0.02). CONCLUSIONS: In children with pneumococcal meningitis, penicillin resistance was associated with a reduced risk of hearing loss, while cefotaxime resistance was associated with a longer duration of fever. Other outcome measures were not significantly influenced by the antibiotic susceptibility of pneumococcal isolates.

Immunomodulation by exogenous surfactant: effect on TNF-alpha secretion and luminol-enhanced chemiluminescence activity by murine macrophages stimulated with group B streptococci.

Group B streptococci (GBS) are important pathogens in neonatal sepsis and pneumonia. GBS stimulate alveolar macrophages to produce inflammatory cytokines and free oxygen radicals, which can damage the lungs. In several studies, use of exogenous surfactant in term babies has improved outcome related to sepsis and respiratory failure. The role(s) of exogenous surfactant in modulating the inflammatory response produced by this microbe was examined. Tumor necrosis factor alpha (TNF-alpha) production and luminol-enhanced chemiluminescence (LCL), a measure of respiratory burst, were investigated. For measuring TNF-alpha release, RAW 264.7 murine macrophages were pre-incubated with bovine surfactant and stimulated with either lipopolysaccharide, live or heat-killed GBS type Ia. LCL was measured after macrophages were pre-incubated with or without surfactant overnight, then stimulated with GBS or phorbol myristate acetate. Lipopolysaccharide and GBS stimulated TNF-alpha secretion from macrophages that was suppressed by exogenous surfactant in a dose-dependent fashion. GBS and phorbol myristate acetate also increased LCL from macrophages, which was significantly suppressed by pre-incubation of macrophages with exogenous surfactant. We conclude that GBS type Ia stimulates TNF-alpha release and LCL from RAW 264.7 cells and that these responses are suppressed by surfactant. Suppression of inflammatory mediators by exogenous surfactant might improve respiratory disease associated with GBS.

Effects of antibiotic class on the macrophage inflammatory response to Streptococcus pneumoniae.

Antibiotic choice can alter host inflammation during invasive bacterial infections. Previous studies of gram-negative organisms concluded that antibiotic-mediated release of bacterial cell wall components amplifies inflammation. Less has been reported about antibiotic effect on gram-positive organisms. This study explored the hypothesis that Streptococcus pneumoniae would induce greater macrophage inflammatory mediator production when killed with cell wall active antibiotics rather than protein synthesis inhibitors. Stimulation of RAW 264.7 murine macrophages with pneumococci and oxacillin led to significantly higher inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF) accumulation than did the same concentrations of pneumococci and clindamycin. Neither antibiotic alone or in combination with lipopolysaccharide acted directly on macrophages to modify the immune response. Endotoxin contamination did not confound the results, as preincubation with polymyxin B did not change iNOS or TNF protein levels. Thus, the antimicrobial mechanism of action affects macrophage inflammatory mediator production after stimulation with pneumococci.

Ceramide-mediated stimulation of inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF) accumulation in murine macrophages requires tyrosine kinase activity.

In macrophages, bacterial lipopolysaccharide (LPS) has been noted to mimic certain effects of the sphingolipid ceramide, suggesting that ceramide may be involved in macrophage activation by LPS and/or that LPS utilizes ceramide-related signaling pathways. Putative downstream targets of ceramide include a ceramide-activated (serine/threonine) protein kinase (CAPK) and phosphatase (CAPP). However, the potential role of tyrosine phosphorylation pathways in macrophage response to ceramide has not been examined. Herein we report that cell-permeable analogs of ceramide up-regulate both inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF) production in RAW 264.7 murine macrophages. Herbimycin A and genistein, potent natural inhibitors of protein tyrosine (but not serine/threonine) phosphorylation, block ceramide-induced iNOS and TNF production. Furthermore, the highly src-family selective pyrazolopyrimidine inhibitor PP1 also blocks ceramide-induced iNOS and TNF production in RAW 264.7 cells. We found that PP1 also inhibits ceramide-mediated tyrosine phosphorylation of the src-family kinase hck. These data indicate that src-related tyrosine kinases play a critical role in macrophage activation by ceramide.

Isolation and characterization of vancomycin-tolerant Streptococcus pneumoniae from the cerebrospinal fluid of a patient who developed recrudescent meningitis.

The emergence of tolerance to vancomycin has recently been reported in Streptococcus pneumoniae, the most common cause of bacterial meningitis. A vancomycin- and cephalosporin-tolerant strain of S. pneumoniae, the Tupelo strain, was isolated from the cerebrospinal fluid of a patient who then developed recrudescence of meningitis despite treatment with vancomycin and a third-generation cephalosporin. The Tupelo strain evidenced no lysis in the exponential or stationary phase of growth when exposed to vancomycin and only minimal loss of viability. Further characterization revealed normal autolysin expression, localization, and triggering by detergents, indicating that the defect leading to tolerance in the Tupelo strain is in the control pathway for triggering of autolysis. Because tolerance is a precursor phenotype to resistance and may lead to clinical failure of antibiotic therapy, these observations may have important implications for vancomycin use in infections caused by S. pneumoniae.

The src family-selective tyrosine kinase inhibitor PP1 blocks LPS and IFN-gamma-mediated TNF and iNOS production in murine macrophages.

Tyrosine phosphorylation pathways are essential components of the process of macrophage activation and the resultant production of inflammatory mediators such as tumor necrosis factor (TNF) and nitric oxide (NO). Several lines of evidence suggest that members of the src family of protein tyrosine kinases play important roles in macrophage activation by gram-negative bacterial lipopolysaccharide (LPS) or the cytokine interferon-gamma (IFN-gamma), but targeted disruption of three members of the src family (hck, fgr, and lyn) in mice failed to demonstrate a requirement for these particular kinases in macrophage activation. We report that the pyrazolopyrimidine PP1, a src family-selective tyrosine kinase inhibitor, potently inhibits the production of TNF and inducible nitric oxide synthase (iNOS) in RAW 264.7 murine macrophages stimulated with LPS, rlFN-gamma, or LPS + rIFN-gamma. Furthermore, the tested concentrations of PP1 inhibit LPS- and rlFN-gamma-mediated tyrosine phosphorylation of the hck tyrosine kinase and its putative substrate, vav, but fail to block rlFN-gamma-mediated JAK2 tyrosine phosphorylation. These findings provide additional support for a model of macrophage activation involving one or more src-related kinases. Selective inhibitors of this signaling pathway should be studied in animal models of sepsis.

Specific inhibitors of p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways block inducible nitric oxide synthase and tumor necrosis factor accumulation in murine macrophages stimulated with lipopolysaccharide and interferon-gamma.

Whether p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase cascades are required for inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF) accumulation in RAW 264.7 murine macrophages exposed to lipopolysaccharide (LPS) plus recombinant interferon-gamma (rIFN-gamma) was investigated. By use of Western blotting for iNOS detection and ELISA for quantitation of TNF secretion, three selective inhibitors of these pathways were tested (the p38 inhibitors SB202190 and SB203580 and the MEK 1,2/ERK inhibitor PD98059). Dose-related inhibition of iNOS production was demonstrated when inhibitors were added 1 h before, simultaneously with, or 1 h after LPS plus rIFN-gamma stimulation. In contrast, inhibition of TNF secretion was observed only when cells were preincubated with these agents. Thus, both the p38 and ERK pathways are involved in the up-regulation of iNOS and TNF production by murine macrophages, and specific inhibitors of these pathways block macrophage iNOS production even when added 1 h after activation of these cells.

Successful medical therapy for deeply invasive facial infection due to Pythium insidiosum in a child.

Pythiosis occurs in animals and humans who encounter aquatic habitats that harbor Pythium insidiosum. Drug therapy for deeply invasive infections with this organism has been ineffective in humans and animals; patients have been cured only by radical surgical debridement. A 2-year-old boy developed periorbital cellulitis unresponsive to antibiotic and antifungal therapy. The cellulitis extended to the nasopharynx, compromising the airway and necessitating a gastrostomy for feeding. P. insidiosum was isolated from surgical biopsy specimens of the affected tissue. On the basis of in vitro susceptibility studies of the isolate, the patient was treated with a combination of terbinafine and itraconazole. The infection resolved over a period of a few months. The patient remained well 1.5 years after completing a 1-year course of therapy. Cure of deep P. insidiosum infection is feasible with drug therapy.

Pneumococci stimulate the production of the inducible nitric oxide synthase and nitric oxide by murine macrophages.

The role of nitric oxide (NO) in the pathophysiology of gram-positive sepsis is uncertain. In inflammatory conditions, high-output NO production is catalyzed by the enzyme inducible nitric oxide synthase (iNOS). The ability of 2 strains of pneumococci, pneumococcal cell wall preparations, and purified pneumococcal capsule (Pnu-Imune 23) to trigger the production of iNOS protein and NO in RAW 264.7 murine macrophages was tested. Live pneumococci, oxacillin-killed pneumococci, and pneumococcal cell wall preparations stimulated the production of iNOS and NO by RAW 264.7 cells in the presence, but not the absence, of low concentrations of recombinant murine interferon-gamma. In contrast, purified pneumococcal capsule induced little or no iNOS or NO production by these cells. Thus, pneumococci stimulate high-output NO production by murine macrophages. The potential role of NO in the pathogenesis of pneumococcal sepsis deserves further study.

Genital mycoplasmas stimulate tumor necrosis factor-alpha and inducible nitric oxide synthase production from a murine macrophage cell line.

Ureaplasma urealyticum and Mycoplasma hominis, two genital mycoplasmas, are the most common organisms isolated in the perinatal period and both either cause or are associated with poor perinatal outcomes. We speculate that these microbes could increase inflammation by stimulating macrophages to produce tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase because of their propensity to interact with the host's immune system. To test this hypothesis, RAW 264.7 cells, a murine macrophage cell line, were coincubated for 16 h with either U. urealyticum or M. hominis, and LPS and sterile broth were used as controls. Lipopolysaccharide (LPS) and both mycoplasmas induced TNF-alpha production, which was concentration-dependent, whereas sterile broth had little effect. TNF-alpha production was not inhibited by the addition of polymyxin B, excluding the possibility of contaminating endotoxin in this effect. Inducible nitric oxide synthase was produced only in the presence of recombinant inteferon-gamma. We conclude that both viable and nonviable U. urealyticum and M. hominis are capable of TNF-alpha induction from murine macrophages and that LPS is not involved in this event. Also, the genital mycoplasmas are capable of stimulating inducible nitric oxide synthase production from murine macrophages. We speculate that the genital mycoplasmas produce perinatal disease by producing proinflammatory mediators by their interaction with inflammatory cells and either induce or act as a catalyst and augment inflammation which in turn leads to a poor pregnancy outcome.

Exogenous bovine surfactant suppresses tumor necrosis factor-alpha release by murine macrophages stimulated by genital mycoplasmas.

Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine that appears to play a significant role in the development of neonatal chronic lung disease (CLD). Inflammation and CLD are also associated with respiratory tract colonization with genital mycoplasmas. The possible protective roles of surfactant in mitigating the inflammatory response to these microbes were investigated. Murine RAW 264.7 macrophages were preincubated with an exogenous surfactant and exposed overnight to sterile media, lipopolysaccharide (LPS), Mycoplasma hominis, or Ureaplasma urealyticum. Macrophages released TNF-alpha in response to challenge with LPS, U. urealyticum, and M. hominis in a concentration-dependent fashion. Surfactant suppressed LPS and M. hominis induced TNF-alpha production in a dose-dependent manner but suppressed U. urealyticum-mediated TNF-alpha production only at the higher dose tested. Similar effects were seen in hyperoxia (95% O2). Thus, exogenous bovine surfactant significantly inhibits the production of TNF-alpha by murine macrophages stimulated with genital mycoplasmas and bacterial LPS.

Bacterial LPS and IFN-gamma trigger the tyrosine phosphorylation of vav in macrophages: evidence for involvement of the hck tyrosine kinase.

We and others have previously reported that tyrosine kinases play key roles in the activation of macrophages by both bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). However, little is known regarding the substrates of tyrosine phosphorylation that mediate macrophage activation and the resultant production of inflammatory mediators. In lymphocytes and other hematopoietic lineages, tyrosine phosphorylation of the proto-oncogene vav appears to be an essential component of cell activation. In this study, we demonstrate that both LPS and rIFN-gamma trigger the prompt, dose-dependent tyrosine phosphorylation of vav in murine RAW 264.7 macrophages. In addition, vav is physically associated with the src-related kinase hck in murine macrophages, and antisense oligonucleotides specific for murine hck block both LPS and rIFN-gamma-mediated vav phosphorylation. These findings suggest that hck probably mediates vav tyrosine phosphorylation during macrophage activation and that LPS and rIFN-gamma-mediated signaling pathways partially overlap.

Viridans streptococcal isolates from patients with septic shock induce tumor necrosis factor-alpha production by murine macrophages.

Viridans streptococci are an important cause of bacteremia and septic shock in neutropenic patients, especially patients receiving chemotherapeutic agents that induce severe mucositis. The mechanisms by which viridans streptococci cause septic shock are unclear. We hypothesized that septic shock due to viridans streptococci is attributable to host cytokine production. Three clinical isolates of viridans streptococci were evaluated for their ability to induce production of tumor necrosis factor-alpha (TNF-alpha) by RAW 264.7 murine macrophages. These three strains of viridans streptococci induced TNF-alpha in a dose-dependent fashion, and the kinetics of TNF-alpha induction were similar to those observed with a clinical isolate of Escherichia coli.

Differential effects of pentoxifylline and interleukin-10 on production of tumor necrosis factor and inducible nitric oxide synthase by murine macrophages.

The abilities of pentoxifylline and recombinant interleukin-10 (rIL-10) to inhibit tumor necrosis factor (TNF) and inducible nitric oxide synthase (iNOS) production in RAW 264.7 murine macrophages were compared. Pentoxifylline consistently inhibited the accumulation of both TNF and iNOS in a dose-dependent manner whether the stimulus was bacterial lipopolysaccharide (LPS), recombinant interferon-gamma (rIFN-gamma), or LPS plus rIFN-gamma. Similarly, rIL-10 consistently reduced TNF production by cells stimulated with LPS, rIFN-gamma, or LPS plus rIFN-gamma. However, rIL-10 weakly inhibited LPS-induced iNOS production but failed to block (and often augmented) rIFN-gamma-induced iNOS production. Combinations of pentoxifylline and rIL-10 led to additive or synergistic inhibition of TNF but not iNOS production; in fact, rIL-10 appeared to interfere with the ability of pentoxifylline to block iNOS accumulation. These data suggest that combinations of antiinflammatory agents may have unanticipated effects on inflammatory mediator production.

Repeated invasive pneumococcal infections in young children without apparent underlying immunodeficiency.

During a 30-month interval at LeBonheur Children's Medical Center, 394 patients had a blood or cerebrospinal fluid culture positive for Streptococcus pneumoniae. Sixteen of these episodes (4%) were repeated infections; 6 of these 16 patients had sickle cell disease. Six of the remaining 10 patients had immunologic evaluations of varying completeness; no immunodeficiency was identified by these tests or on follow-up. Nine of the ten previously healthy patients with repeated pneumococcal disease were less than 2 years of age. In our experience, repeated invasive pneumococcal infections in otherwise healthy young children were relatively common (10/394, or 2.5% of patients with invasive pneumococcal infections) and did not indicate the presence of an unsuspected immunodeficiency.

Streptococcal pyrogenic exotoxins A (SpeA) and C (SpeC) stimulate the production of inducible nitric oxide synthase (iNOS) protein in RAW 264.7 macrophages.

Streptococcal pyrogenic exotoxins A (SpeA) and C (SpeC) are members of a family of superantigens produced by group A streptococci that appear to play a key role in the pathogenesis of streptococcal toxic shock syndrome. Since it is known that nitric oxide (NO) and tumor necrosis factor (TNF) are largely responsible for the shock and multiple organ dysfunction of Gram-negative sepsis, we hypothesized that SpeA and/or SpeC could trigger the production of inducible nitric oxide synthase (iNOS) and/or TNF by murine macrophages. We exposed RAW 264.7 macrophages to increasing concentrations of SpeA or SpeC alone and in combination with recombinant murine interferon-gamma (rIFN gamma) for 16-24 h. We found that both SpeA and SpeC triggered iNOS production in the presence of low concentrations of rIFN gamma, while neither provoked iNOS accumulation in the absence of rIFN gamma. Neither SpeA nor SpeC (with or without rIFN gamma) reproducibly induced TNF production by these murine macrophages. These data indicate that two streptococcal exotoxins up-regulate iNOS production by murine macrophages and suggest that nitric oxide production may play an important role in the pathogenesis of streptococcal toxic shock syndrome.

Lipoteichoic acid from viridans streptococci induces the production of tumor necrosis factor and nitric oxide by murine macrophages.

Bacterial endotoxin or lipopolysaccharide is the major proinflammatory component of gram-negative bacteria, but the components of gram-positive bacteria that trigger the inflammatory cascade are poorly understood. Lipoteichoic acid (LTA) purified from 2 strains of viridans streptococci induced the accumulation of tumor necrosis factor (TNF) mRNA and protein by the murine macrophage cell line RAW 264.7 in a dose- and time-dependent manner. Furthermore, in the presence of recombinant interferon-gamma, LTA from both strains of viridans streptococci provoked the accumulation of inducible nitric oxide (NO) synthase mRNA and the production of NO. Together these observations indicate that LTA can trigger macrophage activation and the production of TNF and NO and suggest that LTA may be an important determinant of the host inflammatory response to gram-positive infection.

Expression of the activated (Y501-F501) hck tyrosine kinase in 32Dcl3 myeloid cells prolongs survival in the absence of IL-3 and blocks granulocytic differentiation in response to G-CSF.

The hematopoietic cell kinase (hck), a member of the src family of intracellular, membrane-associated protein tyrosine kinases, is primarily expressed in mature granulocytes and monocyte/macrophages. Hck kinase activity plays an essential role in macrophage activation and may be an important signaling molecule in granulocytes. To examine the potential role of hck in hematopoietic differentiation pathways, retroviral vectors were used to express wild-type and mutant forms of p59hck in the hck-negative, interleukin-3 (IL-3) -dependent murine myeloid cell line, 32Dcl3. Constitutive expression of an activated form of hck markedly prolonged the viability of 32Dcl3 cells in the absence of IL-3 but failed to abrogate the requirement for IL-3 for proliferation. Moreover, enforced expression of the activated hck kinase (and less so, the wild-type kinase) blocked granulocytic differentiation of 32Dcl3 cells in response to granulocyte colony-stimulating factor. These findings indicate that up-regulation of hck expression is not required for (and may interfere with) granulocytic differentiation.

Serious infections due to penicillin-resistant Streptococcus peneumoniae in two children with nephrotic syndrome.

Children with nephrotic syndrome are susceptible to the development of invasive bacterial infections, particularly those caused by Streptococcus peneumoniae. Recently, penicillin-resistant pneumococcal infections in children have occurred in increased frequency in some regions. We have seen two children with nephrotic syndrome who developed penicillin-resistant pneumococcal bacteremia and/or peritonitis. A change in the initial therapy from penicillin in the usual dose in these patients must be considered.