Viable Bacteria In Lungs Research Articles

Antimicrobial effects of nitric oxide in murine models of Klebsiella pneumonia

RationaleInhalation of nitric oxide (NO) exerts selective pulmonary vasodilation. Nitric oxide also has an antimicrobial effect on a broad spectrum of pathogenic viruses, bacteria and fungi. ObjectivesThe aim of this study was to investigate the effect of inhaled NO on bacterial burden and disease outcome in a murine model of Klebsiella pneumonia. MethodsMice were infected with Klebsiella pneumoniae and inhaled either air alone, air mixed with constant levels of NO (at 80, 160, or 200 parts per million (ppm)) or air intermittently mixed with high dose NO (300 ppm). Forty-eight hours after airway inoculation, the number of viable bacteria in lung, spleen and blood was determined. The extent of infiltration of the lungs by inflammatory cells and the level of myeloperoxidase activity in the lungs were measured. Atomic force microscopy was used to investigate a possible mechanism by which nitric oxide exerts a bactericidal effect. Measurements and main resultsCompared to control animals infected with K. pneumoniae and breathed air alone, intermittent breathing of NO (300 ppm) reduced viable bacterial counts in lung and spleen tissue. Inhaled NO reduced infection-induced lung inflammation and improved overall survival of mice. NO destroyed the cell wall of K. pneumoniae and killed multiple-drug resistant K. pneumoniae in-vitro. ConclusionsIntermittent administration of high dose NO may be an effective approach to the treatment of pneumonia caused by K. pneumoniae.

Azithromycin Attenuates Lung Inflammation in a Mouse Model of Ventilator-Associated Pneumonia by Multidrug-Resistant Acinetobacter baumannii

Acinetobacter baumannii is one of the main pathogens that cause ventilator-associated pneumonia (VAP) and is associated with a high rate of mortality. Little is known about the efficacy of macrolides against A. baumannii. In order to confirm the efficacy of azithromycin (AZM) against VAP caused by multidrug-resistant A. baumannii (MDRAB), we used a mouse model that mimics VAP by placement of a plastic tube in the bronchus. AZM (10 and 100 mg/kg of body weight) was administered subcutaneously every 24 h beginning at 3 h after inoculation. Phosphate-buffered saline was administered as the control. Survival was evaluated over 7 days. At 48 h postinfection, mice were sacrificed and the numbers of viable bacteria in lungs and bronchoalveolar lavage fluid were compared. Histopathological analysis of lung specimens was also performed. The treatment groups displayed significantly longer survival than the control group (P < 0.05). AZM did not have an antimicrobial effect. Histopathological examination of lung specimens indicated that the progression of lung inflammation was prevented in the AZM-treated groups. Furthermore, total cell and neutrophil counts, as well as cytokine levels, in bronchoalveolar lavage fluid were significantly decreased (P < 0.05) in the AZM-treated groups. AZM may have a role for the treatment of VAP with MDRAB because of its anti-inflammatory effects.

In vivo efficacy and pharmacokinetics of tomopenem (CS-023), a novel carbapenem, against Pseudomonas aeruginosa in a murine chronic respiratory tract infection model

Tomopenem (CS-023) is a novel parenteral carbapenem with broad-spectrum activity against Gram-positive and -negative bacteria, as well as potent activity against drug-resistant pathogens, including penicillin-resistant Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. We compared the in vivo activity of tomopenem and that of meropenem in a chronic lower respiratory infection mouse model of P. aeruginosa. Mice with chronic airway infection by P. aeruginosa were treated with saline (as the control, twice daily), tomopenem (100 mg/kg, twice daily) or meropenem (100 mg/kg, twice daily) for 7 days. After treatment, the number of viable bacteria in lungs and histopathological findings were analysed. The pharmacokinetics of tomopenem and meropenem were also analysed after initial treatment. The number of viable bacteria in lungs treated with saline, tomopenem or meropenem was 4.21 +/- 1.28, 2.91 +/- 0.87 and 3.01 +/- 1.00 log(10) cfu/lung (mean +/- SEM), respectively (P < 0.05, control versus tomopenem- or meropenem-treated groups). In the histopathological examination of lung specimens, the control group had the features of chronic bronchial infection; however, tomopenem- and meropenem-treated groups had fewer inflammatory cells compared with the control group. The pharmacokinetic parameter of % time above MIC for tomopenem and meropenem was 16% and 17% in sera and 15% and 18% in lungs, respectively. Tomopenem significantly reduced the number of viable bacteria in a murine model of chronic airway infection by P. aeruginosa, compared with the control. Considering the longer half-life of tomopenem in humans compared with most other carbapenems, tomopenem treatment of chronic airway infection with P. aeruginosa is expected to be efficacious.

Inflammatory- and immune responses in relation to bacterial replication in mice following re-infections with Chlamydophila pneumoniae

Investigation of chronic infections with Chlamydophila pneumoniae. BALB/c mice were repeatedly infected with C. pneumoniae and tested during a 1-year period. Production of histamine, IFN-gamma, IL-6 and antibodies was monitored by ELISA. Live bacteria were cultured and DNA was detected by PCR. Cellular immunity was tested by ELISPOT. After re-infections, culture positivity and persistence of DNA in lungs and blood were shorter. Detection of DNA at late time points indicated persistent infection in a few mice. Histamine was produced after primary and re-infections, and the level correlated with the number of viable bacteria in lung. IFN-gamma, IL-6 levels, IgG2/IgG1 ratio, IgA titres, and level of chlamydial heat-shock protein antibodies were higher after re-infections. IgM antibodies were demonstrated even after re-infections. High number of IFN-gamma-producing splenocytes was observed after the third inoculation. These results promote an understanding of the patho- and immune mechanisms after C. pneumoniae re-infections.

Construction of a eukaryotic expression system for granulysin and its protective effect in mice infected with Mycobacterium tuberculosis

A full-length cDNA of granulysin was inserted into the pcDNA3.1(-) vector to construct a eukaryotic expression plasmid for granulysin. The recombinant plasmids were injected intramuscularly into mice infected with Mycobacterium tuberculosis to evaluate the protective effect of granulysin. Granulysin significantly decreased the weight index (WI) of the spleen, reduced the numbers of viable bacteria in lung and spleen, and reduced the lesions of lung tissue in granulysin-rDNA-immunized mice compared with those of control group mice. In vitro, the serum of the recombinant-plasmid-immunized mice inhibited the viability of M. tuberculosis by the physical disruption of cell membranes. Therefore, granulysin has a therapeutic effect against M. tuberculosis.

Immunization of mice with a Mycobacterium tuberculosis genomic expression library results in lower bacterial load in lungs after challenge with BCG

Tuberculosis is a serious infectious disease in many developing countries. The lack of an effective vaccine for preventing this disease has stimulated the search for new vaccine candidates against Mycobacterium tuberculosis. In the present work, the construction of a genomic expression library of M. tuberculosis in a eukaryotic expression vector was carried out. Immunization of Balb/c mice with a plasmid DNA pool from this library (containing 8360 clones) induced a significant IgG antibody response. Immunized mice were challenged by intratracheal route with 10(5) cfu of non-pathogenic Mycobacterium bovis BCG and were sacrificed 21 days post-challenge. Mice immunized with the genomic expression library showed a significant reduction of viable bacteria in lungs and less pulmonary tissue damage. Granulomas were not observed and the lungs had a more discrete perivascular inflammatory cell infiltrate compared to control mice. Results suggest that the genomic expression library contains genes encoding proteins that are protective against M. tuberculosis infection.

In vivo efficacies and pharmacokinetics of DX-619, a novel des-fluoro(6) quinolone, against Streptococcus pneumoniae in a mouse lung infection model.

DX-619 is a novel des-fluoro(6) quinolone with potent activity against gram-positive pathogens. The in vivo activity of DX-619 against Streptococcus pneumoniae was compared with those of fluoro(6) quinolones, sitafloxacin, and ciprofloxacin in a mouse model. Two strains of S. pneumoniae were used: a penicillin-sensitive S. pneumoniae (PSSP) strain and a penicillin-resistant S. pneumoniae (PRSP) strain. Furthermore, these strains showed intermediate susceptibilities to ciprofloxacin. In murine lung infections caused by PSSP, the 50% effective doses (ED50s) of DX-619, sitafloxacin, and ciprofloxacin were 9.15, 11.1, and 127.6 mg/kg of body weight, respectively. Against PRSP-mediated pneumonia in mice, the ED50s of DX-619, sitafloxacin, and ciprofloxacin were 0.69, 4.84, and 38.75 mg/kg, respectively. The mean +/- standard error of the mean viable bacterial counts in murine lungs infected with PSSP and treated with DX-619, sitafloxacin, ciprofloxacin (10 mg/kg twice daily), and saline (twice daily) were 1.75 +/- 0.06, 1.92 +/- 0.23, 6.48 +/- 0.28, and 7.57 +/- 0.13 log10 CFU/ml, respectively. Furthermore, the numbers of viable bacteria in lungs infected with PRSP and treated with the three agents and not treated (control) were 1.73 +/- 0.04, 2.28 +/- 0.17, 4.61 +/- 0.59, and 5.54 +/- 0.72 log10 CFU/ml, respectively. DX-619 and sitafloxacin significantly decreased the numbers of viable bacteria in the lungs compared to the numbers in the lungs of ciprofloxacin-treated and untreated mice. The pharmacokinetic parameter of the area under the concentration-time curve (AUC)/MIC ratio in the lungs for DX-619, sitafloxacin, and ciprofloxacin were 171.0, 21.92, and 1.22, respectively. The AUC/MIC ratio in the lungs was significantly higher for DX-619 than for sitafloxacin and ciprofloxacin. Our results suggest that DX-619 and sitafloxacin are potent against both PSSP and PRSP in our mouse pneumonia model.

온도 차이에 따른 수영훈련이 결핵균 감염정도에 미치는 영향

본 연구를 통하여 규칙적인 수영 트레이닝이 체중의 감소를 일으켰으며(온수 및 냉수), 특히 냉수 훈련이 큰 감소를 나타내었다. 온수 훈련군에서는 <TEX>$INF-\gamma$</TEX>의 감소를 유발하여 림프구의 증식을 Th0에서 Th2 쪽으로 유도한다는 사실을 알 수 있었으며, 이로 인해 결핵균을 감염시켰을 경우 오히려 폐와 비장에서 결핵균의 수가 현저히 증가하는 사실을 알 수 있어, 수영 훈련군에게서의 결핵균 감염 시 대조군에 비해 균수가 현저히 증가함을 알 수 있었다. 하지만 냉수 훈련군에서는 통제군에 비해 <TEX>$INF-\gamma$</TEX>의 현저한 증가를 보여, 결핵균 감염 시 폐에서 결핵균 수가 감소함을 확인 할 수 있었다. Tuberculosis is the leading infectious disease in the world. It is urgent to develop new vaccine and treating drugs. Besides vaccines, we want to know the effects of regular swim training on TB infection in the mouse model. This study was designed to examine the effects of regular swim training on lung and spleen TB counts and <TEX>$INF-\gamma$</TEX> activity in the trained mice at different temperature. The trained mice underwent a 10-wk endurance swim training (5 times/wk) in water at <TEX>$29\~33^{\circ}C$</TEX> (WWG) and <TEX>$21\~23^{\circ}C$</TEX>(CWG) for 60 min. And they were divided into 3 groups according to the regular swim training (CG; control, WWG; warm water group, and CWG; cold water group). Mice were challenged by aerosol infection with M. tuberculosis H37Rv using an inhalation device (Glas-Col, Terre Haute, Ind.) calibrated to deliver bacteria into lungs. Three weeks after immunization, the mice were challenged. Four weeks after challenge, the mice were sacrificed and the numbers of viable bacteria in lung and spleen were determined by plating serial dilution of whole organ homogenates on nutrient Middlebrook 7H11 agar (Difco, Detroit, MI). Colonies were counted after four weeks incubation at <TEX>$37^{\circ}C$</TEX>. All data were expressed as mean, standard deviation by using SPSS package program (win 10.0). The result through the statistical analysis of this data were summarized as follows; In the weight changes, there were significant differences among CG, WWG, and CWG following the swim training at different temperature, and CWG was the lowest. In the change of <TEX>$INF-\gamma$</TEX> following the swim training, there were significant differences (p<.05) among CG, WWG, and CWG after stimulated with media and CFP. In MTB counts, there were significant differences (p<.05) between CG and WWG in the lung. And also there were significant differences (p<.05) among CG, WWG, and CWG. These results suggest that regular swim training suppress Th1 immune response caused by decreased <TEX>$INF-\gamma$</TEX> level in the WWG, Also For the WWG, highly increased level of TB counts appear in the lung and spleen compare to CG.

Therapeutic effects of Ag85B and MPT64 DNA vaccines in a murine model of Mycobacterium tuberculosis infection

A new improved therapeutic strategy for tuberculosis is urgently needed. In our previous work DNA vaccines encoding secreted proteins Ag85B and MPT64 have been reported to protect mice from following H37Rv challenge by prompting the Th1 response and we consider whether these vaccines have the therapeutic effect through the same mechanism. In the present study these two DNA vaccines were tested in a mouse tuberculosis model to confirm their immunotherapeutic effect. C57BL/6 mice infected with Mycobacterium tuberculosis were treated with pCDNA3.1, pcD85B, pcDMPT64, pcD85B plus pcDMPT64, respectively. The numbers of viable bacteria in lung and spleen were counted as log 10 CFU/g. The level of IFN-γ, IL-4 and TNF-α released by spleen lymphocytes stimulated with PPD was detected with ELISA. Lungs and spleens were harvested for pathological analysis. The pcD85B group reduced the pulmonary and splenic bacterial loads of 1.2 and 0.7 logs, respectively compared with that of control mice, but the difference between pcDMPT64 group and control mice was not significant. Vaccination with pcD85B induced high level of IFN-γand TNF-α. No change of IL-4 level was found in all groups. The pathological change in lung in pcD85B group was slight, alveolar wall structure is clear and the lesions are constrained, while that in control group was extensive, alveoli and interalveolar septae are effaced. And there was no special change in spleen in all groups. In conclusion, Ag85B DNA vaccination has immunotherapeutic effects, and the effects may be associated with a switch to Th1 response and prompting production of cytokine TNF-α and INF-γ synchronously. Therefore, MPT64 DNA vaccination has no immuotheraputic effect on mice tuberculosis. Rather, the effects may be associated with its disability in switching improper immune status and with recalling a strong and early specific memory immune response against tuberculosis.

Combination therapy for chronic Pseudomonas aeruginosa respiratory infection associated with biofilm formation.

There had been no reports of investigations into biofilms in chronic respiratory infection in vivo. Recently, we established a new murine model of chronic respiratory infection with Pseudomonas aeruginosa. In the present study, we examined the bacteriological effect of combined clarithromycin and levofloxacin against chronic respiratory infection with P. aeruginosa. Scanning electron micrograph of the surface of the catheter intubated in mouse bronchus for 7 days demonstrated in vivo formation of a biofilm containing blood cells, complex fibrous structures and bacteria. Treatment with either clarithromycin alone or levofloxacin alone had no statistical effect on the number of viable bacteria in lung. The combined use of both drugs resulted in a significant decrease in the number of viable bacteria. The present experiment demonstrates that the newly established murine model was useful to investigate the treatment of biofilm-associated chronic respiratory infection with P. aeruginosa, and combination therapy with clarithromycin and levofloxacin was effective in biofilm-associated chronic respiratory infection.

Effects of Parenteral Application of Fish Oil versus Soy Oil Emulsions on Bacterial Clearance Functions

Background: The aim of this study was to test whether or not infusion of triglycerides containing ω-3 fatty acids (FAs) modifies immune function in terms of systemic clearance and organ distribution of injected Escherichia coli in a rabbit model. Materials and Methods: In groups of 6 rabbits each, a defined number of E. coli (1.3 × 10⁸ colony-forming units, CFU) was injected intravenously after 3 days of infusion with a lipid emulsion prepared from fish oil (Omegavenös^® 10%, 1.5 g/kg body weight/day), a soy oil preparation (Lipovenös^® 10%, 1.5 g/kg body weight/day), or after isotonic saline application (control). In order to compare dose-dependent effects, in a second experimental design a lower dose of each lipid emulsion (0.5 g/kg body weight) was infused over a 4-hour period before applicating E. coli . Parameters monitored were arterial pressure and rates of bacterial elimination from the blood. The animals were killed 3 h and 6 h after bacterial infection, respectively, and tissue samples of liver, spleen, lung, and kidney were collected for microbiological examinations. Results: Lipovenös infusion produced a significant delay in blood clearance compared with saline and Omegavenös treatment. The diminished systemic bacterial elimination after Lipovenös infusion was accompanied by increased numbers af viable bacteria in lung and spleen. In contrast to this, in the Omegavenös group bacterial counts in the lung did not increase and smaller amounts of viable bacteria were detected in organ homogenates 6 h after injection of E. coli . Conclusion: Compared with the soy oil preparation, ω-3 FAs induce improved bacterial clearance from the blood that is associated with significantly lower numbers of E. coli in the lung. Thus, appropriate nutrition providing ω-3 FA might serve as a pharmacological tool to improve host defence against bacterial infection in critically ill patients.

Therapeutic efficacy of liposomal clofazimine against Mycobacterium avium complex in mice depends on size of initial inoculum and duration of infection.

The therapeutic efficacy of liposomal clofazimine (L-CLF) against Mycobacterium avium complex (MAC) was evaluated in the acute and chronic infection models of the beige mouse (C57BL/6J bgj bgj). The maximum tolerated dose of L-CLF was inversely proportional to the infection level. L-CLF showed higher antibacterial activity than free clofazimine. Treatment with 25 mg of L-CLF per kg of body weight (intravenously) was started at days 1, 8, 15, and 22 postinfection and was studied at three levels of MAC infection (10(4), 10(5), and 10(6) bacilli/mouse). L-CLF treatment caused a significant (P < 0.05 to 0.001) reduction in the numbers of viable bacteria in lung, liver, and spleen at all infection levels, irrespective of time of treatment. However, the best results were obtained when an already established infection was treated (day 22). The organ-related differences in response to the treatment were also affected by the level of infection. A marked reduction in the numbers of CFU was observed in the lungs of mice with lower infection levels, whereas liver and spleen were treated more efficiently at higher infection levels. These studies might help in evaluations of host responses to therapy.