In vitro assessment of chemotaxis by peripheral blood neutrophils from adult and senescent C57BL/6 mice: correlation with in vivo responses to pulmonary infection with type 3 Streptococcus pneumoniae

To assess the effects of advanced age on the ability of circulating neutrophils to respond to biologically relevant chemoattractants, cells were isolated from the peripheral blood of pathogen and disease-free C57BL/6 mice and evaluated in a microchemotaxis chamber. The responses of granulocytes obtained from senescent mice (26-28 months) to the chemotactic peptide, FMLP, and to leukotriene B4 were similar to those found with cells from the younger animals (8-10 months). In contrast, the migration of neutrophils in response to sonicated type 3 Streptococcus pneumoniae was significantly greater with cells from the older animals. Similarly, the chemotactic response of neutrophils to zymosan-activated serum was greater with cells and serum from the senescent animals; however, the enhanced chemotaxis exhibited by granulocytes from the aged mice was a consequence of serum factors. Following the deposition of viable type 3 S. pneumoniae into the lower respiratory tract, the neutrophil influx at 24 h after challenge was significantly greater in the senescent mice; however, age-related differences in survival rates and LD50 were not detected. Thus, in the C57BL/6 mouse, senescence is not associated with deficiencies in the response of neutrophils in vitro to chemoattractants that contribute to lung host defense against the pneumococcus; further, in this murine strain, advanced age does not result in an attenuation of the pulmonary inflammatory reaction to infection with type 3 S. pneumoniae.     

Oral immunization with bacterial lysate against infection with Streptococcus pneumoniae in mice

The protective effect of oral immunization against infection with Streptococcus pneumoniae was investigated in mice. Two bacterial lysates, one with an additional lysate of Candida albicans, were investigated. Intranasal inoculation of adult Balb-C mice with a S. pneumoniae type I strain resulted in a lethal infection, with deaths occurring from the 2nd until the 6th day after infection. Oral immunization resulted in a significant decrease in mortality rate (18-48% reduction). No significant difference in mortality rates was observed between the groups immunized with different lysates in the same concentrations.     

In vitro and in vivo activity of first generation cephalosporins against Leptospira

Third generation cephalosporins are commonly used in the treatment of leptospirosis. The efficacy of first generation cephalosporins has been less well-studied. Susceptibility testing of 13 Leptospira strains (11 serovars) to cefazolin and cephalexin was conducted using broth microdilution. Median minimal inhibitory concentration (MIC) for cefazolin and cephalexin ranged from < 0.016 to 2 μg/mL (MIC(90) = 0.5 μg/mL) and from 1 to 8 μg/mL (MIC(90) = 8 μg/mL), respectively. Efficacy of cefazolin and cephalexin in an acute lethal hamster model of leptospirosis was studied. Survival rates for cefazolin were 80%, 100%, and 100%, and survival rates for cephalexin were 50%, 80%, and 100% (treated with 5, 25, and 50 mg/kg per day for 5 days, respectively). Each treatment group showed improved survival compared with no treatment (P < 0.01), and none of the therapies, regardless of dose, was statistically significantly different than doxycycline. These results support a potential role for first generation cephalosporins as alternative therapies for leptospirosis.     

Effect of high dose oral zinc in mice with severe infection with Streptococcus pneumoniae

Zinc is important for normal function of the immune system and inflammation increases the demand for zinc. We hypothesized that high doses of zinc given during acute pneumococcal illness would alter the severity of infection. 24 six-week-old BALB/c mice were anaesthetized and infected intranasally with Streptococcus pneumoniae. Zinc intake was controlled by administering zinc through an intragastric tube. One group was given normal doses (5 microg/d) and the other group high doses of zinc (225 microg/d). We counted the number of bacteria from venous blood at 24 and 48 h, and from heart puncture and nasal wash at 72 h after intranasal challenge. Mice given excess zinc had 2.65 micromol/l, i.e. 25% higher (p= 0.05) mean plasma zinc concentration compared to those given normal amounts. 75% of mice in both groups developed pneumococcal bacteraemia. There were no differences in the numbers of S. pneumoniae colony forming units (CFUs) in blood or nasal wash between the groups. Thus, high doses of zinc did not alter the severity of systemic pneumococcal infection in mice.     

Antibacterial activity of four cephalosporins in an experimental infection in relation to in vitro effect and pharmacokinetics

The in vitro activity of four cephalosporins was compared with their effects in an experimental thigh infection (cefuroxime and cefamandole against Escherichia coli and cefamandole, ceftriaxone, and ceftazidime against Klebsiella pneumoniae) in granulocytopenic mice. The effect in vitro (ER) was defined as the difference between the growth rate without antibiotic and the growth rate at the steepest part of a 3-h growth curve in the presence of an antibiotic. The relation between concentration and ER was described with the Hill equation. Using pharmacokinetic parameters of the plasma concentrations in vivo and those of the Hill equation the corresponding time course of ER was calculated and by integration with respect to time (0tERdt), an estimate was obtained of the effect on bacteria. For all four antibiotics this estimate was significantly correlated with the actual values of the effect in vivo (EN), defined as the difference in numbers of bacteria between controls and antibiotic-treated animals at 4 h.     

The effect of dietary fish oil on survival after infection with Klebsiella pneumoniae or Streptococcus pneumoniae

Dietary fish oil is believed to have a beneficial effect in various infections and in autoimmune disorders. This effect may correspond to an altered immune response. In order to discover whether the effect of dietary fish oil is different in various infections, we studied the survival of mice fed fish oil or corn oil supplemented diets and infected in the lungs with either Klebsiella pneumoniae or Streptococcus pneumoniae. 120 NMRI mice were divided into 4 groups, of which 2 groups were fed a fish oil supplemented diet and 2 a corn oil supplemented diet. After 6 weeks the mice were infected in the lungs with Klebsiella pneumoniae (fish oil groups and corn oil groups) or with Streptococcus pneumoniae serotype 3 (both groups). The survival rate was monitored. The experiment was performed twice. The survival of the mice fed fish oil enriched diet and infected with Klebsiella pneumoniae was significantly better compared with the mice fed corn oil enriched diet (p = 0.0001 and p = 0.0013). No difference was found between the mice fed corn oil enriched diet or fish oil enriched diet and infected with Streptococcus pneumoniae serotype 3 (p = 0.74 and p = 0.15). Our results indicate that dietary fish oil has a beneficial effect on survival of mice after experimental pneumoniae when infected with Klebsiella pneumoniae, but not after infection with Streptococcus pneumoniae serotype 3.     

Haemophilus influenzae and Streptococcus pneumoniae in children with acute respiratory infection

The incidence of Haemophilus influenzae and Streptococcus pneumoniae in children with acute respiratory infection (ARI) under 5 years was carried out by throat swab culture, blood culture, body fluid or tissue culture in 688 patients from a community, 744 patients from a teaching hospital in Bangkok, 766 normal children from the community and 303 children from a hospital well baby clinic. H. influenzae was found in the throats of 15-20% of patients and in the throats of 4-6% of normal children (p less than 0.001 for both hospital and community patients). Only 12/332 strains (3.6%) of H. influenzae were type b. The rest of H. influenzae were non type b. The most common biotype of H. influenzae non type b was biotype II. S. pneumoniae was found in hospital patients in highly significant numbers compared to the controls (12% vs 4%). No significant difference was observed in strains from the community patients.     

In vitro and in vivo correlation of clotting protease activity: Effect of heparin

The thrombogenicity of three highly purified proteases (thrombin, activated Factor X, and activated Factor IX) was determined quantitatively in an animal model. The minimal amounts required to produce a standard score 4 thrombus were 1.1 nmol for thrombin, 0.12 nmol for activated Factor X, and 0.018 nmol for activated Factor IX. After the administration of heparin at 5, 10, and 20 units/kg in rabbits, the thrombogenicity of each of these proteases decreased progressively. The heparin-induced inhibition of thrombosis decreased in the order, activated Factor IX > activated Factor X > thrombin at each heparin concentration. These differences were statistically significant.These in vivo data provide support for the following hypotheses originally developed from in vitro experiments: (i) activation of the blood coagulation system, which proceeds through a cascade mechanism, incorporates biochemical amplification; (ii) the inhibition of activated Factor IX by antithrombin III in the presence of heparin is an important reaction in the prevention of thrombosis; (iii) less heparin is required to inhibit thrombosis prior to thrombin generation than afterward; (iv) an increase in the reactivity of antithrombin III reflects a decreased tendency to thrombosis while a decrease in this reactivity reflects an increased tendency to thrombosis.     

In vitro activity of fosfomycin against Haemophilus influenzae,Streptococcus pneumoniae and Neisseria species

Summary The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of fosfomycin were determined by an agar dilution technique for 100 strains each ofHaemophilus influenzae andStreptococcus pneumoniae, and for 40 strains ofNeisseria species andBranhamella catarrhalis. The median MIC ofH. influenzae was 0.13 mg/l (range 0.008–64 mg/l), ofS. pneumoniae 13.3 mg/l (range 8–128 mg/l), and ofNeisseria includingB. catarrhalis 27.0 mg/l (range 16–128 mg/l). The MBCs were very close to the MICs.

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In-vitro-Aktivität von Fosfomycin gegen Haemophilus influenzae, Streptococcus pneumoniae und Neisseriaspecies

Zusammenfassung Die minimalen Hemmkonzentrationen (MHK) und minimalen bakteriziden Konzentrationen (MBK) von Fosfomycin wurden mittels einer Agardilutionstechnik für je 100 Stämme vonHaemophilus influenzae undStreptococcus pneumoniae und 40 Stämme vonNeisseria species undBranhamella catarrhalis bestimmt. Die mittlere MHK vonH. influenzae war 0,13 mg/l (Bereich 0,008–64 mg/l), vonS. pneumoniae 13,3 mg/l (Bereich 8–128 mg/l) und vonNeisseria einschließlichB. catarrhalis 27,0 mg/l (Bereich 16–128 mg/l). Die MBKs lagen sehr nahe an den MHKs.

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This paper is dedicated to Professor Walter Marget in honour of his 60th birthday.     

Streptococcus pneumoniae and Streptococcus pyogenes isolated from a paediatric population in Great Britain and Ireland: the in vitro activity of telithromcycin versus comparators

OBJECTIVES: To investigate the antimicrobial susceptibility profiles of clinical isolates of Streptococcus pneumoniae and Streptococcus pyogenes, isolated from children within Great Britain and Ireland (Northern Ireland and Eire), with particular reference to the new oral ketolide telithromycin. To determine the distribution of macrolide resistance genes within the erythromycin resistant population. METHODS: MICs were determined using NCCLS microbroth dilution methodology and macrolide resistance mechanisms were investigated using PCR. RESULTS: Penicillin susceptibility was found to be 92.6% in S. pneumoniae isolates ( n=831; 3.7% intermediate, MIC 0.12-1 mg/l, 3.7% resistant, MIC >2.0 mg/l) and 100% in S. pyogenes isolates (n=1333) 8.8% of S. pneumoniae and 2.5% of S. pyogenes isolates demonstrated erythromycin-A resistance (EryA(R)). One hundred percent of S. pneumoniae and 99.8% of S. pyogenes isolates were susceptible to telithromycin (MIC相似文献   

In vitro activity of the new ketolide telithromycin and other antibiotics against Streptococcus pneumoniae in Belgium

In Belgium more than 17% of the invasive pneumococci are not susceptible to penicillin, and more than 38% not to macrolides. The most prevalent mechanism of macrolide resistance in Europe is modification of the drug target site leading to cross-resistance to lincosamides and group B streptogramines (MLSB resistance). Telithromycin is the first antibiotic of the family of ketolides, which differ from erythromycin by having a 3-keto group instead of the neutral sugar L-cladinose. We tested the susceptibility of 637 pneumococci, recently isolated from patients in Belgium, to telithromycin and five other antibiotics. Data generated by this study show that telithromycin inhibits 98.4% of pneumococci at a breakpoint concentration of 1 mg/L in spite of a high percentage (> 30%) of strains with the MLSB constitutive type of resistance. Susceptibilities to the five comparator drugs were: penicillin (81.8%), tetracycline (67.0%), levofloxacin (98.9%), erythromycin (61.5%) and clindamycin (66.6%). Consequently telithromycin looks to have considerable potential for the empiric treatment of community-acquired respiratory tract infections.     

The classical pathway is the dominant complement pathway required for innate immunity to Streptococcus pneumoniae infection in mice

The complement system is an important component of the innate immune response to bacterial pathogens, including Streptococcus pneumoniae. The classical complement pathway is activated by antibody-antigen complexes on the bacterial surface and has been considered predominately to be an effector of the adaptive immune response, whereas the alternative and mannose-binding lectin pathways are activated directly by bacterial cell surface components and are considered effectors of the innate immune response. Recently, a role has been suggested for the classical pathway during innate immunity that is activated by natural IgM or components of the acute-phase response bound to bacterial pathogens. However, the functional importance of the classical pathway for innate immunity to S. pneumoniae and other bacterial pathogens, and its relative contribution compared with the alternative and mannose-binding lectin pathways has not been defined. By using strains of mice with genetic deficiencies of complement components and secretory IgM we have investigated the role of each complement pathway and natural IgM for innate immunity to S. pneumoniae. Our results show that the proportion of a population of S. pneumoniae bound by C3 depends mainly on the classical pathway, whereas the intensity of C3 binding depends on the alternative pathway. Furthermore, the classical pathway, partially targeted by the binding of natural IgM to bacteria, is the dominant pathway for activation of the complement system during innate immunity to S. pneumoniae, loss of which results in rapidly progressing septicemia and impaired macrophage activation. These data demonstrate the vital role of the classical pathway for innate immunity to a bacterial pathogen.     

The interaction of Streptococcus pneumoniae with intact human respiratory mucosa in vitro.

The interaction of Streptococcus pneumoniae with human ciliated upper respiratory mucosa was studied in an agar-embedded organ culture of nasal turbinate tissue, which only exposed the intact epithelial surface and its secretion. The ciliary beat frequency, measured along the edge of the organ culture, was slowed by 13% in the presence of S. pneumoniae after 16 h (p less than 0.05) compared with the control, and by 24% after 24 h (p less than 0.01). Light microscopy showed bacteria in a thickened gelatinous layer, which obscured the surface of the organ culture. Transmission and scanning electron microscopy confirmed the association of bacteria with the gelatinous layer above an epithelial surface which showed only minor changes compared to uninfected control organ cultures. Contact between bacteria and normal or damaged epithelial cells was not seen. S. pneumoniae in organ culture developed projections from their surface, which were not present after broth culture. S. pneumoniae interactions with epithelial-derived secretions, the formation of a thickened gelatinous layer, and the effects of bacterial toxins on ciliary motility, may be important during colonization of the respiratory tract.     

Corticotrophin-releasing activity of desmopressin in Cushing's disease: lack of correlation between in vivo and in vitro responsiveness

Desmopressin (DDAVP), an arginine vasopressin analogue, markedly stimulates ACTH secretion in patients with Cushing's disease, in contrast to its minimal effect in normal subjects. However, little is known about the mechanisms underlying this action and it appeared to be of interest to evaluate the effect of DDAVP on ACTH-secreting pituitary adenomas in vitro, in comparison with its effect in the same patients in vivo. Pituitary adenomas from 14 patients with Cushing's disease were incubated with DDAVP, corticotrophin-releasing hormone (CRH) and DDAVP together with vasopressin receptor antagonists or CRH. Incubation with DDAVP induced a modest dose-dependent increase in ACTH concentrations which appeared maximal at 10 nM. CRH stimulated ACTH to a greater extent compared with DDAVP and potentiated the effect of DDAVP alone. The DDAVP-induced ACTH increase appeared blunted by vasopressin V(2) and V(3) receptor antagonists. V(3) receptor gene expression was detected by RT-PCR in all adenoma samples except for two which were not responsive to DDAVP in vitro but responsive to the peptide in vivo. Surprisingly, no difference in the in vitro ACTH secretory response was observed between in vivo DDAVP-responsive (ACTH peak>150% baseline) and -unresponsive (ACTH peak<120% baseline) patients, suggesting that the pituitary adenoma is not the sole mediator of the ACTH-releasing effect of DDAVP. In conclusion, the marked stimulatory effect of DDAVP observed in patients with Cushing's disease appears to be mainly dependent on an extrapituitary action, possibly the inhibition of a corticotrophin release-inhibitory factor.     

Serotype distribution and penicillin-binding protein genes of Streptococcus pneumoniae in children with invasive pneumococcal infection

Between August 1998 and July 2005, we studied the serotypes and mutation of penicillin-binding protein (PBP) genes of 46 strains of Streptococcus pneumoniae isolated from children with invasive pneumococcal infection in Hokkaido. The clinical diagnosis was pneumonia in 16 cases, occult bacteremia in 15, meningitis in 8, upper respiratory infection in 6, and arthritis in 1. Patients ranged in age from 2 months to 9 years old. Prevalent serotypes were 6B (39.1%), 23F (17.4%) 6A (8.7%), and 19F (8.7%). The serotype coverage rate by the 7-valent pneumococcal vaccine was greater than 70% in children. Mutation of 3 genes (Penicillin-binding S. pneumoniae, PRSP) was detected in 18 isolates, mutation of 10 genes (pbp1a and pbp2x, or pbp2x and pbp2b) in 2, and of pbp2x alone in 15. PRSP was found in serotypes 6A, 6B, and 23F, and the rate of PRSP in 6A, 6B, and 23F was 75.0%, 62.5%, and 55.6%, respectively.