Mycoplasma pneumoniae pneumonia in a mouse model

To investigate the pathogenesis of acute Mycoplasma pneumoniae infection, BALB/c mice were anesthetized with metofane, and M. pneumoniae was introduced intranasally on days 0, 1, and 2. Mice were sacrificed on days 0-15. A histopathologic scoring system defined inflammatory changes in the lungs on a scale of 0-26 (least to most severe). Broth cultures were positive for all nasal passage and bronchoalveolar lavage (BAL) specimens. Histopathologic scores ranged from 0 to 21. The mean log10 (cfu/mL) were 4.1-6.4 on days 1-10 and >/=1.7 on days 13-15 for nasal passage and BAL specimens. Serum polymerase chain reaction was negative. ELISA for serum IgM and immunoblots for M. pneumoniae antibody were positive in 21 (62%) of 34 and 33 (97%) of 34 infected animals, respectively, at days 8-15. ELISA for IgG antibody was negative. This mouse pneumonia model can be used to study the immunologic and therapeutic responses to acute M. pneumoniae infection.     

The efficacy of ciprofloxacin and doxycycline against experimental tularaemia

A group of patients with balance complaints (n = 16) was compared with a group of normal subjects (n = 17) by means of posturography, subjective assessments of balance, anxiety and unsteadiness when standing on a force platform with eyes closed. Postural instability was induced by vibratory stimulation of the calf muscles (20, 40, 60, 80 and 100 Hz). As a control condition, the arm (biceps) was stimulated at similar frequencies. In order to control for arousal, blood pressure and heart beat were assessed. Furthermore, questionnaire responses on psychological measures were collected. Results showed clear differences between the groups in terms of imbalance and self-reports. However, the 2 groups displayed similar increases of imbalance during calf stimulation and no increase during arm stimulation. Patients generally rated less increase of unsteadiness when the calf was stimulated than did the controls. No differences in arousal were found between the groups or within conditions. Results are discussed in terms of the proposed desynchrony between symptoms and complaints.     

Further studies on the efficacy of a live vaccine against mastitis caused by Streptococcus uberis

Three groups of dairy cows were immunized by subcutaneous (s.c.) administration of a preparation of live Streptococcus uberis (strain 0140J) and an intramammary infusion of a soluble surface extract derived from same the bacteria. Animals in Groups 1 and 2 received two s.c. vaccinations plus an intramammary inoculation. Animals in Group 3 received two s.c. vaccinations but did not receive the intramammary infusion. In addition to the vaccinated animals, each group also contained two non-vaccinated (control) animals. All animals were challenged experimentally by intramammary infusion (in two quarters per animal) of ca 100 c.f.u. of S. uberis (strain 0140J or C221) and monitored for clinical signs of disease, bacterial numbers in milk, somatic cell count in milk, and daily milk yield for the following 10 days. Animals in Group I were challenged with strain 0140J. Only one out of six challenged quarters of three vaccinated cows developed clinical disease compared to all (four out of four) quarters of non-vaccinated cows. Animals in Group 2 were challenged with strain C221. All challenged quarters of three vaccinated (six out of six) and two non-vaccinated (four out of four) cows developed clinical mastitis. Animals in Group 3 were challenged with strain 0140J. Five out of eight quarters on four vaccinated cows developed clinical mastitis but the onset was delayed in comparison with that in both non-vaccinated cows in which four out of four challenged quarters developed clinical mastitis. These results indicated that vaccination with live S. uberis protects against challenge with the homologous strain but was less effective against a heterologous strain. Reduced protection was also seen when the intramammary booster was omitted.     

Correlation between in vitro and in vivo activity of amoxicillin against Streptococcus pneumoniae in a murine pneumonia model

We studied the relationship between in vitro bacteriological parameters [minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) and killing rate, defined as the reduction in the inoculum within 1, 3 or 6 hr] and in vivo activity of amoxicillin against 12 strains of Streptococcus pneumoniae, with penicillin MICs of < 0.01 to 16 micrograms/ml, in a cyclophosphamide-induced neutropenic murine pneumonia model. Dose-response curves were determined for amoxicillin against each strain, and three quantitative parameters of in vivo amoxicillin activity were defined, i.e., maximal attainable antimicrobial effect attributable to the drug [i.e., reduction in log colony-forming units (CFU) per lung, compared with untreated controls], dose required to reach 50% of maximal effect and dose required to achieve a reduction of 1 log CFU/lung. We demonstrated a highly significant correlation between the dose required to reach 50% of maximal effect and MIC (Spearman r = 0.98, P < .0001) or MBC (Spearman r = 0.95, P < .0001) for amoxicillin against strains of S. pneumoniae with a wide range of amoxicillin MICs (0.01-8 micrograms/ml). Significant correlations between the dose required to achieve a reduction of 1 log CFU/lung and MIC (Spearman r = 0.98, P < .0001) or MBC (Spearman r = 0.95, P < .0001) were also observed. In contrast, there were no significant correlations between the maximal attainable antimicrobial effect attributable to the drug and MIC, MBC or killing rate or between killing rate and the dose required to reach 50% of maximal effect or the dose required to achieve a reduction of 1 log CFU/lung. We conclude that in vitro susceptibility test results (MICs and MBCs) correlated well with in vivo amoxicillin activity against pneumococcal strains, including highly penicillin-resistant strains, in this animal model. Furthermore, these data suggest that the estimated MIC breakpoints for amoxicillin against S. pneumoniae would be 2 micrograms/ml for intermediate-resistant and 4 micrograms/ml for resistant, although this remains to be confirmed in clinical studies.     

In vivo efficacies of amoxicillin and cefuroxime against penicillin-resistant Streptococcus pneumoniae in a gerbil model of acute otitis media

The comparative efficacies of amoxicillin and cefuroxime against acute otitis media caused by a penicillin-resistant (MIC, 2 micrograms/ml) Streptococcus pneumoniae strain were assessed in a gerbil model by challenging each ear with 10(7) bacteria through transbullar instillation. Each antibiotic was tested at two doses (5 and 20 mg/kg of body weight) administered at 2, 10, and 18 h postinoculation. Samples were obtained from the middle ear (ME) on days 3 and 7 postinoculation for determination of bacterial counts. Only amoxicillin, at both doses, was able to significantly halt the weight loss in animals, reducing both the number of culture-positive animals and the bacterial concentration in ME samples versus the values for untreated animals. Comparison of the efficacies between the antibiotics, determined by their ability to achieve culture-negative ME specimens, showed that amoxicillin at 5 mg/kg was significantly more active than cefuroxime at the same dose. The use of higher doses of either amoxicillin or cefuroxime did not produce significantly better results than those obtained with the lower dose but caused a greater inflammatory response. The more favorable results obtained with amoxicillin compared with those obtained with cefuroxime could be related to the antimicrobial susceptibility of the pneumococcal strain (MICs and minimum bactericidal concentrations of 1 and 1 microgram/ml and 4 and 4 micrograms/ml for amoxicillin and cefuroxime, respectively) as well as to the better pharmacokinetic parameters obtained with amoxicillin.     

Benzydamine protection in a mouse model of endotoxemia

OBJECTIVE: Previous studies have shown that benzydamine (40 mg/kg s.c.) is able to inhibit tumor necrosis factor (TNF) production and to reduce mouse lethality when administered before or concomitantly with LPS. The present study was designed to further investigate benzydamine activity against LPS-induced toxicity in terms of potency and therapeutic effects. METHODS: Female Balb/c mice were used. A dose-response curve of animal lethality versus endotoxin dose was performed (LD50 = 45 micrograms/mouse). Therapeutic effects were studied selecting the dose of LPS to achieve an LD100 (160 micrograms/mouse). Mortality was assessed daily and mice were followed for 8 days. The potential mode of action of therapeutically administered benzydamine was also investigated. TNF alpha and IL-1 beta levels were measured, at 5 h after LPS injection, both in sera and in lungs. Moreover, the drug was assayed in a TNF-dependent cytoxicity test. RESULTS: Benzydamine, administered at 20 mg/kg s.c. simultaneously with the endotoxin, significantly increased LPS LD50 up to 230 micrograms/mouse (p < 0.05). Moreover, the drug significantly protected mice against LPS-induced lethality when administered either 30 min or 4 h after endotoxin injection (p < 0.001). Benzydamine, therapeutically administered at 20 mg/kg s.c., significantly reduced TNF alpha and IL-1 beta production induced by LPS both in serum and lungs and it was shown to inhibit TNF-dependent cytoxicity on L929 cells. CONCLUSIONS: These results clearly demonstrate the therapeutic activity of benzydamine in a simple model of endotoxic shock. Available data confirm the potential role of benzydamine as an anti-cytokine agent and provide suggestions for novel therapeutic applications of this anti-inflammatory drug.     

Development of a model of low-inoculum Streptococcus pneumoniae intrapulmonary infection in infant rats

We have developed a model of low-inoculum Streptococcus pneumoniae infection in infant rats. We challenged 4-day-old Sprague-Dawley pups via intraperitoneal or intrapulmonary injection of S. pneumoniae serotypes 1, 3, 4, 5, 6b, 7f, 9v, 14, 19f, and 23f. To achieve bacteremia with low inocula, it was necessary to passage the isolates in rats. Inocula of the 10 S. pneumoniae serotypes producing bacteremia in 50% or more animals ranged from 1 to 400 CFU. Virulence was similar by intraperitoneal and intrapulmonary routes. Lung specimens from animals challenged by the intrapulmonary route grew S. pneumoniae and demonstrated histologic evidence of focal infection. Meningitis was detected in 20 to 50% of bacteremic animals, and mortality invariably followed bacteremia within 24 to 48 h. This model of intrapulmonary infection uses low inocula of S. pneumoniae and results in bacteremia, meningitis, and death in infant rats.     

Fc receptors are not required for antibody-mediated protection against lethal malaria challenge in a mouse model

The mechanisms by which Abs mediate protection during blood-stage malaria infections is controversial, with some evidence pointing to the direct effect of Abs on parasite invasion and growth, while other studies suggest that Abs act in cooperation with monocytes to achieve parasite inhibition. To determine whether the effector phase of protection in vivo to the rodent parasite Plasmodium yoelii yoelii requires Fc receptor bearing cells, we passively transferred immune sera into FcR gamma-chain knockout mice. Inflammatory macrophages from these knockout mice were unable to mediate phagocytosis or Ab-dependent cell-mediated cytotoxicity (ADCC) through Fc gamma RI, Fc gamma RII, or Fc gamma RIII. Passive transfer of either P. y. yoelii hyperimmune sera or anti-GST-PYC2 sera directed to the major merozoite surface protein (MSP-1) of this parasite enabled both BALB/cByJ mice and FcR gamma-chain-deficient mice to resist lethal P. y. yoelii 17XL (Py17XL) challenge. mAb302, a protective IgG3 Ab, also passively protected both strains of mice. Most of these samples contain Ab isotypes that would not be able to protect mice if their protective effects required Ab-dependent cell-mediated cytotoxicity. These results establish that, in this infection, protection is directly mediated by Abs and does not require the participation of Fc receptors.     

Standardization of an opsonophagocytic assay for the measurement of functional antibody activity against Streptococcus pneumoniae using differentiated HL-60 cells

Dopamine (DA) has been reported to depolarize neurons in the prefrontal cortex (PFC). To further characterize this effect of DA, we made whole cell recordings from PFC pyramidal cells in rat brain slices. As reported previously, DA depolarized most PFC cells tested. This effect of DA was concentration-dependent and persisted in the presence of synaptic blockade, indicating a direct effect of DA on the recorded cell. During DA-induced depolarization, PFC neurons consistently showed an increase in excitability, suggesting that the depolarization is not directly related to DA-induced inhibition of PFC neurons previously observed in vivo. Surprisingly, the effect of DA was not mimicked or blocked by several commonly used DA agonists and DA antagonists. The alpha and beta antagonists phentolamine and alprenolol and the atypical antipsychotic drug clozapine also showed no significant effect on DA-induced depolarization. These results suggest that DA-induced depolarization may be mediated by a nonspecific mechanism. However, it remains possible that there exists a new type of DA receptors in the PFC not sensitive to classical DA agonists and antagonists, particularly given the fact that DA applied in the same manner depolarized only PFC neurons but not those in the striatum or the substantia nigra.     

A novel resistance mechanism against beta-lactams in Streptococcus pneumoniae involves CpoA, a putative glycosyltransferase

Piperacillin resistance in Streptococcus pneumoniae was mediated by mutations in a novel gene, cpoA, that also confer transformation deficiency and a decrease in penicillin-binding protein la. cpoA is part of an operon located downstream of the primary sigma factor of S. pneumoniae. The deduced protein, CpoA, and the peptide encoded by the adjacent 3' open reading frame contained domains homologous to glycosyltransferases of procaryotes and eucaryotes that act on membrane-associated substrates, such as enzymes functioning in lipopolysaccharide core biosynthesis of gram-negative bacteria, RodD of Bacillus subtilis, which is involved in teichoic acid biosynthesis, and the human PIG-A protein, which is required for early steps of glycosylphosphatidylinositol anchor biosynthesis. This suggests that the cpo operon has a similar function related to cell surface components.     

Capsular transformation of a multidrug-resistant Streptococcus pneumoniae in vivo

OBJECTIVE: To evaluate the effects of a mandibular advancement device on apneas and sleep in mild, moderate, and severe obstructive sleep apnea. DESIGN: Prospective study. SUBJECTS: Forty-four of 47 patients included. INTERVENTION: Individually adjusted mandibular advancement devices. MEASUREMENTS: Polysomnographic sleep recordings for 1 night without the device and 1 night with it, with a median of 1 day and no changes in weight, medication, or sleep position between the recordings. RESULTS: The device reduced the median obstructive apnea-hypopnea index from 11 (range, 7 to 19) to 5 (range, 0 to 17) (p<0.001) in 21 patients with mild sleep apnea, from 27 (range, 20 to 38) to 7 (range, 1 to 19) (p<0.001) in 15 patients with moderate sleep apnea, and from 53 (range, 44 to 66) to 14 (range, 2 to 32) (p<0.05) in 8 patients with severe sleep apnea. The arousal index decreased and the sleep stage patterns improved in all severity groups. Twenty-eight of 44 patients were successfully treated with an obstructive apnea-hypopnea index of below 10 and a subjective reduction in snoring. Nine of 16 patients with treatment failure still reported a reduction in snoring. The success rate correlated inversely to the disease severity (r=-0.41; p<0.01). CONCLUSIONS: A mandibular advancement device reduces apneas and improves sleep quality in patients with obstructive sleep apnea, especially in those with mild and moderate disease. A follow-up sleep recording during treatment is necessary because of the risk of silent obstructive apneas without subjective snoring with the device.     

Evaluation of the antibody response in pigs vaccinated against Streptococcus suis capsular type 2 using a double-antibody sandwich enzyme-linked immunosorbent assay

OBJECTIVE: We assessed the value of dynamic sequential three-dimensional Fourier transformation (3DFT) MRI in differentiating various types of small liver tumors. MATERIALS AND METHODS: Forty-seven patients with 65 liver masses < 3 cm in size (42 hepatocellular carcinomas, 11 hemangiomas, 12 metastatic tumors) were studied by 3DFT fast imaging with steady-state precession (FISP) MRI [TR(ms)/TE(ms)/flip angle (degree): 20/8/30]. The slab thickness was 21-35 mm, and there were seven partitions. The 3DFT-FISP MR images were obtained immediately after 0.1 mmol/kg of gadopentetate dimeglumine was administered intravenously over 2-3 s (early phase), 60 s after (late phase I), and 120 s after (late phase II). RESULTS: Eighty-six percent of small hepatocellular carcinomas showed hyperintense enhancement relative to the surrounding liver parenchyma and iso- or hypointense enhancement with or without capsular enhancement in the late phase. Eighty-two percent of small hemangiomas showed peripheral globular enhancement in the early phase and total hyperintense or peripheral enhancement in the late phases. Ninety-two percent of the small metastatic liver tumors showed doughnut-like ring enhancement in the early phase. CONCLUSION: By dynamic 3DFT-FISP MRI, we were able to accurately evaluate the hemodynamics and morphological findings of each type of small liver tumor.     

Evaluation of antimicrobial and lipopolysaccharide-neutralizing effects of a synthetic CAP18 fragment against Pseudomonas aeruginosa in a mouse model

CAP18 (cationic antimicrobial protein; 18 kDa) is a neutrophil-derived protein that can bind to and inhibit various activities of lipopolysaccharide (LPS). The 37 C-terminal amino acids of CAP18 make up the LPS-binding domain. A truncated 32-amino-acid C-terminal fragment of CAP18 had potent activity against Pseudomonas aeruginosa in vitro. We studied the antimicrobial and LPS-neutralizing effects of this synthetic truncated CAP18 peptide (CAP18106-137) on lung injury in mice infected with cytotoxic P. aeruginosa. To determine its maximal effect, the CAP18106-137 peptide was mixed with bacteria just prior to tracheal instillation, and lung injury was evaluated by determining the amount of leakage of an alveolar protein tracer (125I-albumin) into the circulation and by the quantification of lung edema. The lung injury caused by the instillation of 5 x 10(5) CFU of P. aeruginosa was significantly reduced by the concomitant instillation of CAP18106-137. However, the administration of CAP18106-137 alone, without bacteria, induced lung edema, suggesting that it has some toxicity. Also, the peptide did not significantly reduce the number of bacteria that had been simultaneously instilled, nor did it significantly improve the survival of the infected mice. The addition of CAP18106-137 to aztreonam along with the bacteria did decrease the level of antibiotic-induced release of inflammatory mediators including tumor necrosis factor alpha, interleukin-6, and nitric oxide and also improved the survival of the mice. Therefore, more investigations are needed to confirm the toxicities and the therapeutic benefits of CAP18106-137 as an adjunctive therapy to antibiotics in the treatment of infections caused by gram-negative bacteria.     

Serotyping of Streptococcus pneumoniae by coagglutination with 12 pooled antisera

We report on the performance of a recently introduced commercial chessboard method using 12 antisera, in comparison with that of the 55-antiserum panel used in determining the serogroups and types (SGTs) of Streptococcus pneumoniae, both of which were carried out by a coagglutination technique. Of a total of 150 strains of S. pneumoniae studied, 135 (90%) belonged to the SGTs represented in the 23-valent pneumococcal vaccine; of these, 130 (96.3%) were identified as the same SGTs by both typing methods. The remaining five strains showed cross-reactivity with more than two pools by the chessboard method, but could be assigned to a single SGT by the Quellung test. The 96.3% concordance of the chessboard method suggests it can be adopted for determination of the SGTs of S. pneumoniae in laboratories.     

Strategies in the treatment of penicillin-resistant Streptococcus pneumoniae

The epidemiology, resistance mechanisms, susceptibility testing, treatment, prevention, and clinical importance of penicillin-resistant Streptococcus pneumoniae (PRSP) infection are discussed. PRSP is an established presence in the United States, with some geographic areas reporting decreased susceptibility in up to half of isolates. The mechanism of resistance to beta-lactam antibiotics in S. pneumoniae is genetic changes resulting in decreased binding of drug to the bacterial cell wall. Emerging PRSP strains have necessitated testing as a tool in selecting drugs for treating life-threatening infections. Opinions differ on how to treat these infections empirically. Non-life-threatening infections, such as otitis media, are still often treated successfully with amoxicillin, amoxicillin-clavulanate potassium, or a third-generation cephalosporin. Currently recommended initial treatment of pneumococcal pneumonia in otherwise healthy patients requiring hospitalization consists of cefuroxime, ceftriaxone, or cefotaxime; some authors continue to emphasize injectable penicillin. Once the mainstay of empirical treatment of pneumococcal meningitis, penicillin has largely been abandoned in favor of cefotaxime or ceftriaxone. Vaccination remains an underutilized strategy in atrisk populations. The clinical importance of penicillin resistance among pneumococci is still uncertain. Changing patterns in the susceptibility of S. pneumoniae to penicillin make selection of appropriate therapy increasingly difficult. Key considerations are the site of infection and the level of resistance.     

In vivo activities of amoxicillin and amoxicillin-clavulanate against Streptococcus pneumoniae: application to breakpoint determinations

The in vivo activities of amoxicillin and amoxicillin-clavulanate against 17 strains of Streptococcus pneumoniae with penicillin MICs of 0.12-8.0 mg/liter were assessed in a cyclophosphamide-induced neutropenic murine thigh infection model. Renal impairment was produced by administration of uranyl nitrate to prolong the amoxicillin half-life in the mice from 21 to 65 min, simulating human pharmacokinetics. Two hours after thigh infection with 10(5) to 10(6) CFU, groups of mice were treated with 7 mg of amoxicillin per kg of body weight alone or combined with clavulanate (ratio, 4:1) every 8 h for 1 and 4 days. There was an excellent correlation between the MIC of amoxicillin (0.03 to 5.6 mg/liter) and (i) the change in log10 CFU/thigh at 24 h and (ii) survival after 4 days of therapy. Organisms for which MICs were 2 mg/liter or less were killed at 1.4 to 4.2 and 1.6 to 4.1 log10 CFU/thigh at 24 h by amoxicillin and amoxicillin-clavulanate, respectively. The four strains for which MICs were >4 mg/liter grew 0.2 to 2.6 and 0.6 to 2. 3 logs at 24 h despite therapy with amoxicillin and amoxicillin-clavulanate, respectively. Infection was uniformly fatal by 72 h in untreated mice. Amoxicillin therapy resulted in no mortality with organisms for which MICs were 1 mg/liter or less, 20 to 40% mortality with organisms for which MICs were 2 mg/liter, and 80 to 100% mortality with organisms for which MICs were 4.0-5.6 mg/liter. Lower and higher doses (0.5, 2, and 20 mg/kg) of amoxicillin were studied against organisms for which MICs were near the breakpoint. These studies demonstrate that a reduction of 1 log10 or greater in CFU/thigh at 24 h is consistently observed when amoxicillin levels exceed the MIC for 25 to 30% of the dosing interval. These studies would support amoxicillin (and amoxicillin-clavulanate) MIC breakpoints of 1 mg/liter for susceptible, 2 mg/liter for intermediate, and 4 mg/liter for resistant strains of S. pneumoniae.     

Comparative efficacy evaluation of dicationic carbazole compounds, nitazoxanide, and paromomycin against Cryptosporidium parvum infections in a neonatal mouse model

The efficacies of dicationic carbazole compounds, nitazoxanide (NTZ), and paromomycin were evaluated against the AUCp1 isolate of Cryptosporidium parvum by using a neonatal mouse model. Compounds were solubilized or suspended in deionized water and administered orally by gavage to neonatal mice at a constant dose rate on days 0 to 5 (treatment started on day 0). Dose rates varied for individual carbazole compounds but ranged from 0.65 to 20 mg/kg of body weight. NTZ was tested at 100 and 150 mg/kg, and paromomycin was tested at 50 mg/kg. Efficacies were determined by comparing numbers of oocysts present in treated versus control mice at necropsy examination on day 6. Demonstrable efficacy was observed for several carbazole compounds, based on significant reductions in the numbers of oocysts recovered from treated mice versus control mice. Compounds 1, 7, and 10 (19.0 mg/kg) reduced oocyst passage in treated mice to less than 5% of that in control mice. Treatment with compounds 6, 8, and 9 (17.0 mg/kg) resulted in reductions of oocyst output to less than 10% of that in controls. Although they were not comparable in efficacy to compounds 1, 6, 7, 8, 9, and 10, treatment with other carbazole compounds resulted in statistically significant reductions in oocyst output in treated versus control mice. Compound 1 retained efficacy resulted in reduction of oocyst output to approximately 6% of that in controls when the dose was reduced to 5 mg/kg. Further reductions in the dose rate resulted in considerable reductions in anticryposporidial activity. Likewise, the efficacies of compounds 9 and 10 were reduced substantially when the doses were lowered to one-half the screening dose. Paromomycin yielded excellent activity (reduction of oocyst output to <2% of that in controls) at a dose of 50 mg/kg. NTZ yielded moderate efficacy as powder and injectable formulations administered at 100 mg/kg orally (reduction of oocyst output to 42 and 26% of that in controls, respectively). Oral administration of the injectable formulation of NTZ at a dose of 150 mg/kg resulted in improved efficacy (oocyst output, <5% of that in controls).