Antimicrobial susceptibility and serotype distribution of Streptococcus pneumoniae isolated from patients with community-acquired pneumonia and molecular analysis of multidrug-resistant serotype 19F and 23F strains in Japan.

A nationwide study was undertaken to determine the susceptibility to penicillin and serotypes of Streptococcus pneumoniae in Japan. S. pneumoniae was isolated from 114 adult patients with community-acquired pneumonia over 22 months at 20 hospitals and medical centres in different regions in Japan. All but five isolates were from sputum. Forty-eight isolates (42.1%) were susceptible, 40 (35.1%) showed intermediate resistance (MIC, 0.12-1.0 microg/ml) and 26 (22.8%) were resistant (MIC, >or=2.0 microg/ml) to penicillin G. All isolates were susceptible to ceftriaxone (breakpoint 1 microg/ml), imipenem (4 microg/ml) and vancomycin (4 microg/ml). Most were resistant to erythromycin, clarithromycin and azithromycin; only two were resistant to levofloxacin. Differences were found in the distribution of serotypes among isolates showing susceptibility to penicillin (predominant types 3, 6B, and 19F), intermediate resistance (6B, 14, 19F, and 23F) and full resistance (19F and 23F). PFGE typing showed that 14 of the 25 strains of serotype 19F had a single DNA profile, pattern A, a pattern closely similar to that of the Taiwan multidrug-resistant 19F clone. Twelve pattern A strains were not susceptible to penicillin but carried the macrolide resistance gene mef(A). The DNA profiles of the 15 strains of 23F were also heterogeneous but six were highly similar (pattern b) yet distinct from the Spanish multidrug-resistant 23F clone although possibly related to the Taiwan multidrug-resistant 23F clone. The pattern b strains were not susceptible to penicillin and also harboured either mef(A) or erm(B). Our results indicate that multidrug-resistant pneumococci are spreading rapidly in Japan. Efforts to prevent the spread of the pandemic multidrug-resistant serotypes should be intensified.

Dexamethasone impairs pulmonary defence against Pseudomonas aeruginosa through suppressing iNOS gene expression and peroxynitrite production in mice.

To elucidate the in vivo mechanisms involved in the impairment in pulmonary defence as the result of treatment with glucocorticoids, we established fatal pneumonia with bacteraemia in dexamethasone (DEX)-treated mice by means of an intratracheal challenge of Pseudomonas aeruginosa. An increased neutrophil influx was observed in bronchoalveolar lavage (BAL) fluids from both untreated and DEX-treated mice. The complete suppression of an inducible isoform of nitric oxide synthase (iNOS) mRNA expression and tumour necrosis factor alpha (TNF-alpha) production during the early phase of pneumonia, but not CXC chemokine production, were found in the case of the DEX-treated mice. An immunohistochemical study with a specific antibody also revealed negative staining for nitrotyrosine in the lung tissue of DEX-treated mice, while the formation of nitrotyrosine, which indirectly indicates the generation of peroxynitrite with a potent bactericidal activity, was detected clearly in the bronchial epithelium as well as alveolar phagocytic cells of lung tissue from untreated mice. Furthermore, an intraperitoneal administration of S-methyl-isothiourea (SMT), a potent inhibitor of NOS, significantly decreased the survival and increased bacterial density in the case of untreated mice. In contrast, no significant effects on the survival and bacterial density in the lung and blood were found as the result of treatment with SMT in DEX-treated mice. Collectively, a complete repression of iNOS gene expression and a lack of the generation of peroxynitrite as well as an inhibition of TNF-alpha production in the lung appeared to be responsible for the progression of the fatal pneumonia due to P. aeruginosa in DEX-treated mice.

[Effect of stimulus size on binocular summation within the binocular visual field].

PURPOSE: To study the influence of stimulus sizes on binocular summation using the modified Octopus 201 combined with a space synoptophore. METHODS: Four normal subjects, aged 21 to 26 were tested. Using the SARGON program, we designed a new program to test 37 points in the central 6 degrees visual field. Sensitivity of the central 6 degrees visual field under monocular and binocular conditions was measured while the fusion patterns were displayed on the space synoptophore. The visual fields were measured at stimulus sizes 1, 3, and 5. RESULTS: The visual sensitivity under binocular conditions was higher than under monocular conditions for all the stimulus sizes. Binocular summation for stimulus size 1 was present in a flat form, for stimulus size 3 in a convex form, and for stimulus size 5 in a concave form in the central 6 degrees visual field. CONCLUSION: Binocular summation differed in stimulus size and retinal eccentricity. Binocular summation for stimulus size 3 increased in the fovea and it increased for stimulus size 5 in the peripheral area in the central 6 degrees visual field.

Asbestos exposure induces MCP-1 secretion by pleural mesothelial cells.

We showed previously that both crocidolite and chrysotile asbestos inhalation induced a persistent macrophage inflammatory response within the pleural space of the rat. We postulated that the stimulus for pleural macrophage recruitment after asbestos exposure was the induction of monocyte chemoattractant protein-1 (MCP-1) synthesis by pleural mesothelial cells. To test this hypothesis, rat pleural mesothelial cells (RPMC) were cultured with or without chrysotile or crocidolite asbestos fibers (8 micrograms/cm2) in the presence (50 ng/mL) or absence of either tumor necrosis factor-alpha (TNF-alpha) or interleukin-1 beta (IL-1 beta). MCP-1 mRNA expression was assessed by RT-PCR in RPMC cultured for 2 to 24 hours, and MCP-1 protein secretion was measured by ELISA in conditioned medium from 24-hour and 48-hour cultures. Crocidolite and chrysotile fibers induced MCP-1 mRNA expression in RPMC which was maximal after 12 hours in the absence of cytokines, but which peaked after 2 hours when RPMC were challenged with asbestos + TNF-alpha or IL-1 beta. Both types of asbestos also significantly increased MCP-1 protein secretion after 24 and 48 hours (P < .0001), an effect that was potentiated by cytokine stimulation. Rats exposed by inhalation to either chrysotile or crocidolite asbestos fibers also had greater amounts of MCP-1 protein in their pleural lavage fluid than did sham-exposed rats. These findings suggest that MCP-1 secretion by RPMC may have a role in the initiation and/or potentiation of asbestos-induced pleural injury.

Influence of synthetic antiendotoxin peptides on lipopolysaccharide (LPS) recognition and LPS-induced proinflammatory cytokine responses by cells expressing membrane-bound CD14.

Lipopolysaccharides (LPS) are proinflammatory bacterial products implicated in the pathogenesis of gram-negative sepsis and septic shock. Polymyxin B (PMB), a cyclic, cationic peptide antibiotic, inhibits biological activities of LPS through high-affinity binding to the lipid A moiety. Small synthetic peptides have been designed to mimic the primary and secondary structures of PMB to determine structural requirements for binding and detoxification of lipid A and to assess possible therapeutic potential. The purpose of this study was to compare and contrast the endotoxin-neutralizing activities of two synthetic antiendotoxin peptides (SAEP-2 and SAEP-4), PMB, and an LPS core-specific monoclonal antibody (MAb), WN1 222-5, based on their abilities to inhibit CD14-mediated target cell uptake of fluorescein isothiocyanate (FITC)-conjugated LPS, detected by flow cytometry and confocal microscopy, and LPS-induced production of the proinflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha), as measured by bioassays. PMB and SAEP-4 produced dose-dependent inhibition of FITC-LPS uptake by CD14-transfected Chinese hamster ovary fibroblasts (CHO-CD14 cells) and by human peripheral blood mononuclear cells. The anti-LPS MAb, WN1 222-5, also blocked LPS uptake by these cells and synergized with PMB and SAEP-4. LPS-induced IL-6 release was inhibited by PMB, SAEP-4, and MAb WN1 222-5, and these inhibitory activities were additive or synergistic. LPS-induced TNF-alpha release by PBMC was also inhibited by PMB and SAEP-4 alone and in combination with anti-LPS MAb. SAEP-2, in contrast, produced comparatively minor decrements in cellular uptake of LPS and LPS-induced cytokine responses, and did so only in the absence of serum, while a nonsense peptide exerted no discernible inhibitory effect on LPS uptake or LPS-induced cytokine expression in the presence or absence of serum. Thus, PMB and SAEP-4, like the LPS-reactive MAb, WN1 222-5, block proinflammatory activities of LPS in part by preventing LPS recognition by membrane-bound CD14-expressing target cells. Differences in peptide structure, however, like those exemplified by SAEP-2 and SAEP-4, may differentially affect the endotoxin-neutralizing potency of these peptides despite similar binding activity against lipid A, reflecting possible differences in peptide solubility or peptide regulation of intracellular signal transduction.

Asbestos exposure upregulates the adhesion of pleural leukocytes to pleural mesothelial cells via VCAM-1.

This study was designed to assess the effects of in vitro and in vivo asbestos exposure on the adhesion of rat pleural leukocytes (RPLs) labeled with the fluorochrome calcein AM to rat pleural mesothelial cells (RPMCs). Exposure of RPMCs for 24 h to either crocidolite or chrysotile fibers (1.25-10 microgram/cm(2)) increased the adhesion of RPLs to RPMCs in a dose-dependent fashion, an effect that was potentiated by interleukin-1beta. These findings were not observed with nonfibrogenic carbonyl iron particles. Crocidolite and chrysotile plus interleukin-1beta also upregulated vascular cell adhesion molecule-1 mRNA and protein expression in RPMCs, and the binding of RPL to asbestos-treated RPMCs was abrogated by anti-vascular cell adhesion molecule-1 antibody. PRLs exposed by intermittent inhalation to crocidolite for 2 wk manifested significantly greater binding to RPMCs than did RPLs from sham-exposed animals. The ability of asbestos fibers to upregulate RPL adhesion to RPMCs may play a role in the induction and/or potentiation of asbestos-induced pleural injury.

Endogenous tumor necrosis factor (TNF) alpha mediates neutrophil accumulation at the mid-phase of a murine model of Pseudomonas aeruginosa pneumonia.

To determine the role of endogenous tumor necrosis factor (TNF) alpha on neutrophil influx into the lungs in acute Pseudomonas aeruginosa pneumonia, we evaluated TNF alpha activity, inflammatory cell response and neutrophil chemotactic activity in the bronchoalveolar lavage fluids (BALFs) of P. aeruginosa-infected mice. In the case of fatal pneumonia, the TNF alpha activity in the BALFs appeared within 3 hr, peaked at 6-12 hr and attenuated within 24 hr after intratracheal challenging, while no TNF alpha activity was detected in the plasma. The elevation of TNF alpha activity in the BALFs was closely associated with neutrophil accumulation. Mirroring the TNF alpha activity response and the influx of neutrophils into the murine airway, the number of neutrophils in the BALFs increased within 3 hr, peaked at 6-12 hr and remained elevated up to 24 hr after challenging. Neutralization of the TNF alpha activity in the BALFs with anti-murine TNF antiserum decreased the level of neutrophil migration by BALF 45.0-49.7% at 6 hr and 49.3-54.2% at 12 hr, while the neutralizing antiserum had no effect on the level of neutrophil migration by BALFs at 3 and 24 hr. Furthermore, the intravenous administration of anti-murine TNF antiserum 2 hr before challenging significantly inhibited neutrophil migration into the lungs of mice with sublethal pneumonia (P < 0.05; compared with mice receiving pre-immune serum). These data suggest that intra-alveolar TNF alpha plays an important role in causing lung neutrophil accumulation at the mid-phase of murine P. aeruginosa pneumonia.

Coronary artery to pulmonary artery fistula with two giant saccular aneurysms in an elderly patient determined noninvasively.

An 87-year-old woman was admitted to our hospital on an emergency basis with atypical chest pain and dyspnea. She had a continuous precordial murmur. Electrocardiogram showed no evidence of myocardial ischemia, but chest X-ray showed marked enlargement of the cardiac silhouette and an abnormal calcified vascular structure. Computed tomography of the chest revealed large abnormal masses next to the heart. Two-dimensional echocardiography showed enlargement of the main trunk of the left coronary artery and 2 giant saccular aneurysms. Abnormal diastolic inflow to the main pulmonary trunk was also observed by color flow imaging. These findings were supported by data obtained using magnetic resonance imaging and transesophageal echocardiography. Based on the above findings, we diagnosed this case as a coronary artery fistula originating from the proximal left anterior descending artery associated with 2 giant saccular aneurysms draining into the pulmonary artery. To our knowledge, this is the oldest patient ever reported with such an anomaly. This case emphasizes that a good prognosis is possible even with a very pronounced visible structural abnormality.

Airway interleukin-8 in elderly patients with bacterial lower respiratory tract infections.

We investigated the interleukin-8 (IL-8) levels and neutrophil numbers in the sputum of 9 elderly patients with lower respiratory tract infections, including Pseudomonas aeruginosa infection, before and after treatment with various antimicrobial agents. The IL-8 levels in sputum supernatants and the neutrophil numbers in sputum smears from 9 patients decreased significantly after the elimination of the causative respiratory pathogens. We also demonstrated that human recombinant IL-8 at a range of 6.25-25 ng/ml significantly enhanced opsonophagocytic killing of P. aeruginosa immunotype-1 strain by human neutrophils in the presence of a serotype-specific anti-lipopolysaccharide monoclonal antibody and fresh normal human serum. These data suggest that the level of IL-8 production in the airways of patients with lower respiratory tract infections is dependent on bacterial densities, and indicate the important role of IL-8 not only in neutrophil migration but also in opsonophagocytic killing of bacteria in the lower respiratory tract.

Role of interleukin-8 (IL-8) and an inhibitory effect of erythromycin on IL-8 release in the airways of patients with chronic airway diseases.

To evaluate of the role of interleukin-8 (IL-8), a chemotactic cytokine, in the continuous neutrophil accumulation in the airways of patients with chronic airway disease (CAD) and persistent Pseudomonas aeruginosa infection, we investigated the cell population, IL-8 levels, IL-1 beta levels, tumor necrosis factor (TNF) activities, and neutrophil elastase (NE) activities of bronchoalveolar lavage (BAL) fluids in 17 CAD patients (with P. aeruginosa infections [CAD+PA], n = 9; without any bacterial infections [CAD-PA], n = 8) and 8 normal volunteers. We found significant elevations of neutrophil numbers, IL-8/albumin ratios, and NE/albumin ratios in BAL fluids from CAD patients, in the following rank order: CAD+PA > CAD-PA > normal volunteers. IL-1 beta/albumin ratios were elevated only in CAD+PA, while no TNF bioactivity was detected in BAL fluids. The neutrophil numbers correlated significantly with the IL-8/albumin ratios and NE/albumin ratios in the BAL fluids of CAD patients. When anti-human IL-8 immunoglobulin G was used for neutralizing neutrophil chemotactic factor (NCF) activities in BAL fluids, the mean reduction rate of NCF activities in CAD+PA patients was significantly higher than that in CAD-PA patients. We also evaluated the effects of low-dose, long-term erythromycin therapy in BAL fluids from three CAD+PA and two CAD-PA patients. Treatment with erythromycin caused significant reductions of neutrophil numbers, IL-8/albumin ratios, and NE/albumin ratios in BAL fluids from these patients. To elucidate the mechanism of erythromycin therapy, we also examined whether erythromycin suppressed IL-8 production by human alveolar macrophages and neutrophils in vitro. We demonstrated a moderate inhibitory effect of erythromycin on IL-8 production in Pseudomonas-stimulated neutrophils but not in alveolar macrophages. Our data support the view that persistent P. aeruginosa infection enhances IL-8 production and IL-8-derived NCF activity, causing neutrophil accumulation in the airways and the progressive lung injuries observed in patients with CAD. The clinical efficacy of erythromycin therapy for CAD patients might be partly mediated through a reduced IL-8 production, diminishing neutrophil accumulation and NE release in the airways.

[A case of Kartagener's syndrome with S. pneumoniae lung abscess].

A 38-year-old male with Kartagener's syndrome (KS) was admitted to our department for evaluation of recurrent pneumonia. Before admission the patient was diagnosed as having pneumonia in another hospital and received ofloxacin (300 mg/day). Fever and production of purulent sputum decreased initially but again increased in the middle of April. On admission the films of both X-ray and CT scan of the chest revealed several air-fluid levels and infiltrative shadows on the left lower lung field. The patient was diagnosed as lung abscess using bronchofiberscopy. Gram staining of the intrabronchial specimens revealed many Gram-positive cocci and neutrophils including phagocytosed bacteria. A new carbapenem (L-627, 600 mg/day), was started intravenously. After the therapy Streptococcus pneumoniae were eradicated soon from the sputum. At the same time the above symptoms including dyspnea on exertion subsided, and the findings of the chest X-ray and CT scan were also improved. Regarding KS the electron micrograph of the cilia showed the absence of the outer-dynein arms. While by both the saccharin test and the sputum cytology impaired mucociliary clearance was found. Lung abscess infrequently accompany KS. There are reports of respiratory infections in KS, but to our knowledge no report of lung abscess was found in KS. We present this case report describing lung abscess in KS.

Therapeutic effects of a human antiflagella monoclonal antibody in a neutropenic murine model of Pseudomonas aeruginosa pneumonia.

Human immunoglobulin G1 (IgG1) monoclonal antibodies (MAbs) reactive with type-specific Pseudomonas aeruginosa lipopolysaccharide (LPS) and flagella were compared for their protective activities against Fisher immunotype 2 P. aeruginosa pneumonia in neutropenic mice. The activity of the antiflagella MAb at a dose of 500 micrograms per mouse was comparable to that of the anti-LPS MAb at the same dose. In vivo protection was correlated with bacterial density in the lung tissue and blood of infected mice. In vitro data suggested that the protective activity of the antiflagella MAb was due more to inhibition of bacterial motility than to opsonophagocytosis of bacteria by alveolar macrophages. In contrast, the protective activity of the anti-LPS MAb was primarily related to alveolar macrophage-mediated opsonophagocytosis. Antiflagella MAb at a dose of 500 micrograms combined with oral sparfloxacin at a subtherapeutic dose of 62.5 micrograms produced a significant increase in survival (P < 0.05) compared with that produced by either agent alone or no treatment. The additive effects between the antiflagella MAb and sparfloxacin at sub-MICs on the inhibitory effects of bacterial motility supported the in vivo effect of the combination. These data suggest that human isotype-matched antiflagella and anti-LPS MAbs have similar protective activities against Pseudomonas pneumonia in neutropenic mice, despite discrete mechanisms of antibody-matched protection. In addition, in vivo synergy was demonstrated between antiflagella MAb and sparfloxacin in this model.

[Dynamic process of colonization by Haemophilus influenzae in children--is H. influenzae normal flora in the throat?].

Eighty-seven oropharyngeal isolates of Haemophilus influenzae were obtained by two time cultures six months apart from a total of 288 children who attend a kindergarten. We analyzed the strains by comparing their serotypes, biotypes, beta-lactamase activity and by performing electrophoresis of outer membrane proteins on polyacrylamide gels. Only nineteen strains were not identical, the rest of the 68 strains were classified into 23 types. During 6 months at least 15 types of strains lost from this group and 21 types of new strains were obtained. There were no children who had identical pairs of H. influenzae in their oropharynx during the 6 months. The classification of strains in oropharynx suggested that person-to-person transmission of nontypable H. influenzae can occur. We concluded that oropharyngeal colonization by nontypable H. influenzae is not a normal flora in children.

Effects of the combination of lipopolysaccharide-specific monoclonal antibodies and sparfloxacin against Pseudomonas aeruginosa pneumonia in neutropenic mice.

The effects of the combination of a murine monoclonal antibody (MAb) specific for the O side chain of Pseudomonas aeruginosa Fisher immunotype 1 lipopolysaccharide and sparfloxacin in a neutropenic mouse model of P. aeruginosa pneumonia were examined. Under the condition that neither MAb at a dose of 500 micrograms per mouse administered intravenously nor a suboptimal dose of oral sparfloxacin (5 mg/kg of body weight) protected mice from challenge with a fatal dose, the combination therapy with MAb and sparfloxacin caused a significant increase in the survival rate (P less than 0.001 compared with either treatment alone). The effect of the combination was closely correlated to bacterial killing in plasma and lung tissue of infected mice. In vitro, a significant MAb-dependent, complement-mediated killing of P. aeruginosa was documented in the presence of sparfloxacin at one-half the MIC, while the killing was not observed in the absence of sparfloxacin. These in vivo and in vitro data suggest the usefulness of combination therapy with a lipopolysaccharide-reactive immunoglobulin G MAb and sparfloxacin in neutropenic patients with P. aeruginosa pneumonia.

[Biochemical analysis of lipopolysaccharides from respiratory pathogenic Branhamella catarrhalis strains and the role of anti-LPS antibodies in Branhamella respiratory infections].

We characterized lipopolysaccharides (LPSs) from respiratory pathogenic Branhamella catarrhalis (BC) strains, and evaluated the protective property of anti-BC LPS antibody in BC respiratory infections. LPSs from four strains of BC were lipooligosaccharide having no O-side chain and a M(r) of 3 KDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). All of them produced different patterns, showing two to four bands on SDS-PAGE. We found high level of anti-BC IgG antibody in convalescent sera from a patient with BC respiratory tract infection by ELISA. This IgG antibody recognized BC LPSs on Western blots. Two respiratory pathogens of BC (strains; 87-122, 88-23) were tested in a bactericidal assay employing a convalescent sera. 87-122 strain was susceptible to antibody-dependent, complement-mediated killing, while 88-23 strain was resistant. The killing of 87-122 strain was inhibited by addition of the homologous BC LPS to the convalescent sera in a dose-dependent manner. These data support that anti-BC LPS antibody may mediate complement-lysis of some strains of BC, and play a protective role in BC respiratory infections.