Dynamics of the contamination of mice during the treatment of burn sepsis due to Pseudomonas aeruginosa using tobramycin alone or combined with active and passive immunization

An experimental study was made with a view to finding out the possible bacteriological advantages of the combined use of tobramycin (Tb) and P. aeruginosa corpuscular polyvalent vaccine (PaCPV) or P. aeruginosa hyperimmune plasma (PaHIP) in burn sepsis caused by P. aeruginosa. The use of the median therapeutic dose of Tb (2.5 mg/kg body weight per day), alone or in combination with immunopreparations, ensured the survival rate of the animals equal to 100%. The contamination of the body with P. aeruginosa after treatment with Tb and PaHIP or PaCPV was lower than after the administration of Tb alone, this phenomenon becoming manifest starting from day 5 of observation in the first case and from day 10 in the second case. The combined use of Tb and immunopreparations (PaCPV or PaHIP) in acute P. aeruginosa infection proved to be more effective than treatment with Tb alone.     

Experimental basis for the combined use of tobramycin and immune preparations for treating acute Pseudomonas aeruginosa infections

The possibility of using tobramycin (Tb) in combination with P. aeruginosa polyvalent corpuscular vaccine (PaPCV) or pyocyanosis hyperimmune plasma (PHP) for the treatment of P. aeruginosa sepsis was experimentally studied. The combined use of Tb and PHP, administered in amounts corresponding to ED50 of each preparation used separately, ensured the survival of 90% of the infected mice, and the injected of PaPCV with ED50 of the antibiotic ensured the survival of 73% of the experimental animals. The combined use of Tb and the two immuno-preparations (PaPCV and PHP) for the treatment of P. aeruginosa infection proved to be more effective than their separate administrations.     

Protective effect of a corpuscular polyvalent Pseudomonas aeruginosa vaccine in generalized chronic Pseudomonas aeruginosa infection in mice with cyclophosphamide-induced leukopenia

The prophylactic effect of immunization with P. aeruginosa polyvalent corpuscular vaccine has been shown on the model of P. aeruginosa generalized chronic infection in mice with leukopenia induced by the intraperitoneal injection of cyclophosphamids. This effect is manifested by the increased resistance of the animals to sublethal doses of P. aeruginosa strain, as well as by more intense general and specific immunological responses in the infected animals (the increase of specific antibody titers, the number of leukocytes in the blood serum and the phagocytic activity of the cells of peritoneal exudate).     

Colonization resistance against Pseudomonas aeruginosa in gnotobiotic mice

Gnotobiotic (GB) mice were colonized with various groups of intestinal bacteria to determine which members of the indigenous flora would exert colonization resistance against Pseudomonas aeruginosa. P. aeruginosa was cultured from the faeces at levels of 10(3)-10(4) cells/g in GB mice inoculated with either the combination of bacteroides and clostridia obtained from conventional (CV) mice or the combination of bacteroides, lactobacilli and clostridia obtained from limited flora mice. The combination of lactobacilli and clostridia from CV mice also did not eliminate P. aeruginosa from GB mice. However, P. aeruginosa was not detected in the faeces of GB mice by 14 days after inoculation with the combination of bacteroides, lactobacilli and clostridia obtained from CV mice. Thus, a complex indigenous flora consisting of bacteroides, lactobacilli and certain clostridia obtained from CV mice but not clostridia obtained from limited flora mice is required to exert complete colonization resistance against P. aeruginosa in GB mice.     

Role of PAR2 in murine pulmonary pseudomonal infection

Proteinases can influence lung inflammation by various mechanisms, including via cleavage and activation of protease-activated receptors (PAR) such as PAR2. In addition, proteinases such as neutrophil and/or Pseudomonas-derived elastase can disarm PAR2 resulting in loss of PAR2 signaling. Currently, the role of PAR2 in host defense against bacterial infection is not known. Using a murine model of acute Pseudomonas aeruginosa pneumonia, we examined differences in the pulmonary inflammatory response between wild-type and PAR2(-/-) mice. Compared with wild-type mice, PAR2(-/-) mice displayed more severe lung inflammation and injury in response to P. aeruginosa infection as indicated by higher bronchoalveolar lavage fluid neutrophil numbers, protein concentration, and TNF-alpha levels. By contrast, IFN-gamma levels were markedly reduced in PAR2(-/-) compared with wild-type mice. Importantly, clearance of P. aeruginosa was diminished in PAR2(-/-) mice. In vitro testing revealed that PAR2(-/-) neutrophils killed significantly less bacteria than wild-type murine neutrophils. Further, both neutrophils and macrophages from PAR2(-/-) mice displayed significantly reduced phagocytic efficiency compared with wild-type phagocytes. Stimulation of PAR2 on macrophages using a PAR2-activating peptide resulted in enhanced phagocytosis directly implicating PAR2 signaling in the phagocytic process. We conclude that genetic deletion of PAR2 is associated with decreased clearance of P. aeruginosa. Our data suggest that a deficiency in IFN-gamma production and impaired bacterial phagocytosis are two potential mechanisms responsible for this defect.     

Enhanced killing of Pseudomonas aeruginosa by human polymorphonuclear leukocytes in presence of subinhibitory concentrations of carbenicillin and ticarcillin

The effect of carbenicillin and ticarcillin on the killing of Pseudomonas aeruginosa was studied with an in vitro system using peripheral blood polymorphonuclear (PMN) leukocytes collected from human donors. No corticosteroid was given to the donor prior to leukocytes collection by a continuous flow cell separator. The assay was carried out with or without serum. P. aeruginosa yield after a 4 hour-incubation was estimated by colony counting. In Hanks' balanced salt solution, P. aeruginosa strains 74 and 78 were resistant to human PMN leukocytes. The presence of subinhibitory concentrations of carbenicillin or ticarcillin (1/10th the minimal inhibitory concentration (MIC) for P. aeruginosa 74, 1/4th the MIC for P. aeruginosa 78) enhanced the bactericidal activity of human leukocytes. Difference between the numbers of bacteria recovered with PMN cells and without cells increased with concentration of carbenicillin or ticarcillin. The synergistic effect was not observed when serum (heated fetal calf serum or heated pooled human serum) was used. The mode of action of carbenicillin and ticarcillin on bactericidal activity of phagocytic cells was not elucidated, but we suggest the effect is due not to action on the phagocytic cells themselves but on the microorganisms.     

椒样薄荷、薄荷和苏格兰留兰香精油与抗生素的协同抑菌功能

以开花期的椒样薄荷（Mentha×piperita）、薄荷（M.haplocalyx）和苏格兰留兰香（M.×gentilis）叶片部位提取的精油为研究对象,通过GC-MS分析,并采用纸片扩散法研究了3种精油单独使用及与抗生素联合使用时对金黄色葡萄球菌、蜡状芽孢杆菌、大肠杆菌、绿脓杆菌和肺炎克雷伯氏菌的抑制情况。结果表明,（1）椒样薄荷与薄荷精油中含量最高的成分为薄荷醇、薄荷酮和异薄荷酮,苏格兰留兰香精油的主要成分为香芹酮和柠檬烯。薄荷和苏格兰留兰香精油符合欧洲药典与ISO标准,椒样薄荷需要继续改良以提高其精油品质与抑菌功能。（2）精油单独使用时,Pseudomonas aeruginosa ATCC15442对椒样薄荷精油和薄荷精油敏感;P.aeruginosa ATCC27853对薄荷精油和苏格兰留兰香精油敏感。精油与抗生素联合使用时抑菌范围和强度均有所改变：绿脓杆菌的2个菌株对精油与抗生素的组合最为敏感,其中,椒样薄荷精油与头孢他啶的组合对P.aeruginosa ATCC15442显示出最强的增效作用,薄荷精油与头孢他啶混合之后对P.aeruginosa ATCC27853出现拮抗作用。Staphylococcus aureus ATCC25923对所有精油以及精油与抗生素混合物均有抗性。（3）椒样薄荷、薄荷和苏格兰留兰香精油的不同成分及其含量差异不仅对精油品质有影响,而且影响精油对测试菌种的抑制作用,可考虑将其作为薄荷属植物品质育种的参考指标。     

Deficiency of mannose-binding lectin greatly increases susceptibility to postburn infection with Pseudomonas aeruginosa

Burn injury disrupts the mechanical and biological barrier that the skin presents against infection by symbionts like the Pseudomonas aeruginosa, a Gram-negative bacteria. A combination of local factors, antimicrobial peptides, and resident effector cells form the initial response to mechanical injury of the skin. This activity is followed by an inflammatory response that includes influx of phagocytes and serum factors, such as complement and mannose-binding lectin (MBL), which is a broad-spectrum pattern recognition molecule that plays a key role in innate immunity. A growing consensus from studies in humans and mice suggests that lack of MBL together with other comorbid factors predisposes the host to infection. In this study we examined whether MBL deficiency increases the risk of P. aeruginosa infection in a burned host. We found that both wild-type and MBL null mice were resistant to a 5% total body surface area burn alone or s.c. infection with P. aeruginosa alone. However, when mice were burned then inoculated s.c. with P. aeruginosa at the burn site, all MBL null mice died by 42 h from septicemia, whereas only one-third of wild-type mice succumbed (p = 0.0005). This result indicates that MBL plays a key role in containing and preventing a systemic spread of P. aeruginosa infection following burn injury and suggests that MBL deficiency in humans maybe a premorbid variable in the predisposition to infection in burn victims.     

椒样薄荷、薄荷和苏格兰留兰香精油与抗生素的协同抑菌功能

以开花期的椒样薄荷(Mentha × piperita)、薄荷(M. haplocalyx)和苏格兰留兰香(M. × gentilis)叶片部位提取的精油为研究对象, 通过GC-MS分析, 并采用纸片扩散法研究了3种精油单独使用及与抗生素联合使用时对金黄色葡萄球菌、蜡状芽孢杆菌、大肠杆菌、绿脓杆菌和肺炎克雷伯氏菌的抑制情况。结果表明, (1) 椒样薄荷与薄荷精油中含量最高的成分为薄荷醇、薄荷酮和异薄荷酮, 苏格兰留兰香精油的主要成分为香芹酮和柠檬烯。薄荷和苏格兰留兰香精油符合欧洲药典与ISO标准, 椒样薄荷需要继续改良以提高其精油品质与抑菌功能。(2) 精油单独使用时, Pseudomonas aeruginosa ATCC 15442对椒样薄荷精油和薄荷精油敏感; P. aeruginosa ATCC 27853对薄荷精油和苏格兰留兰香精油敏感。精油与抗生素联合使用时抑菌范围和强度均有所改变: 绿脓杆菌的2个菌株对精油与抗生素的组合最为敏感, 其中, 椒样薄荷精油与头孢他啶的组合对P. aeruginosa ATCC 15442显示出最强的增效作用, 薄荷精油与头孢他啶混合之后对P. aeruginosa ATCC 27853出现拮抗作用。Staphylococcus aureus ATCC 25923对所有精油以及精油与抗生素混合物均有抗性。(3) 椒样薄荷、薄荷和苏格兰留兰香精油的不同成分及其含量差异不仅对精油品质有影响, 而且影响精油对测试菌种的抑制作用, 可考虑将其作为薄荷属植物品质育种的参考指标。     

Human anti-Pseudomonas aeruginosa outer membrane proteins IgG cross-protective against infection with heterologous immunotype strains of P. aeruginosa.

In order to develop an effective means to treat and prevent Pseudomonas aeruginosa infections, we have purified P. aeruginosa outer membrane protein (Oprs)-specific human IgG antibody using a large-scale affinity column. In this study, we investigated the cross-protective activity of the purified anti-Oprs IgG against various immunotype strains of P. aeruginosa. The anti-Oprs IgG reacted with Oprs isolated from seven Fisher-Devlin immunotype strains of P. aeruginosa and was able to promote opsonophagocytic killing of all seven immunotype strains by human phagocytic cells. Administration of 500 microg anti-Oprs IgG to mice raised the LD50 of the P. aeruginosa strains by 8-250-fold, indicating the protective capacity against heterologous P. aeruginosa strains as well as homologous strains. In contrast, despite high titers against P. (aeruginosa Oprs, total serum IgG isolated from burn patient sera was no better than normal serum IgG in protecting mice from infection with P. aeruginosa. These data demonstrate that the affinity-purified human anti-Oprs IgG could afford protection against heterologous immunotype P. aeruginosa strains and provide a rationale to use anti-Oprs IgG as an adjunct for treatment of P. aeruginosa infections in humans.     

A类Ⅰ型清道夫受体调节小鼠腹腔巨噬细胞抗肺炎克雷伯菌和铜绿假单胞菌感染

本研究旨在探讨A类Ⅰ型清道夫受体(scavenger receptor class A type Ⅰ,SR-AI)在呼吸道感染常见病原体肺炎克雷伯菌及铜绿假单胞菌感染过程中的免疫调节功能。以肺炎克雷伯菌和铜绿假单胞菌临床分离株与野生型小鼠和SR-AI~(-/-)小鼠腹腔原代巨噬细胞互作,研究SR-AI在吞噬和炎症反应中的作用。荧光染料染色菌体及检测胞内荧光强度,数据显示SR-AI敲除后巨噬细胞对肺炎克雷伯菌的吞噬能力下降,但对铜绿假单胞菌的吞噬能力升高。采用实时定量荧光聚合酶链反应检测相关炎症因子mRNA水平,发现SRAI敲除后肺炎克雷伯菌和铜绿假单胞菌刺激巨噬细胞引发的炎症反应均增强。结果表明,SR-AI参与巨噬细胞对肺炎克雷伯菌的吞噬,但不参与对铜绿假单胞菌的吞噬,且可能抑制了肺炎克雷伯菌和铜绿假单胞菌引发的炎症反应。     

Benznidazole/Itraconazole Combination Treatment Enhances Anti-Trypanosoma cruzi Activity in Experimental Chagas Disease

The nitroheterocyclic drugs nifurtimox and benznidazole are first-line drugs available to treat Chagas disease; however, they have limitations, including long treatment courses and toxicity. Strategies to overcome these limitations include the identification of new drugs with specific target profiles, re-dosing regimens for the current drugs, drug repositioning and combination therapy. In this work, we evaluated combination therapy as an approach for optimization of the current therapeutic regimen for Chagas disease. The curative action of benznidazole/itraconazole combinations was explored in an established infection of the mice model with the T. cruzi Y strain. The activities of the benznidazole/itraconazole combinations were compared with the results from those receiving the same dosage of each individual drug. The administration of benznidazole/itraconazole in combination eliminated parasites from the blood more efficiently than each drug alone. Here, there was a significant reduction of the number of treatment days (number of doses) necessary to induce parasitemia suppression with the benznidazole/itraconazole combination, as compared to each compound administered alone. These results clearly indicate the enhanced effects of these drugs in combination, particularly at the dose of 75 mg/kg, as the effects observed with the drug combinations were four times more effective than those of each drug used alone. Moreover, benznidazole/itraconazole treatment was shown to prevent or decrease the typical lesions associated with chronic experimental Chagas disease, as illustrated by similar levels of inflammatory cells and fibrosis in the cardiac muscle tissue of healthy and treated mice. These results emphasize the importance of exploring the potential of combination treatments with currently available compounds to specifically treat Chagas disease.     

Experimentally determined safety and immunological activity of vaccine based on antigens isolated from Pseudomonas aeruginosa in medium K-4

The safety and immunological activity of P. aeruginosa vaccine were experimentally evaluated. The vaccine was prepared on the basis of the antigens of P. aeruginosa extracellular slime which was accumulated in medium K-4, obtained with the use of original technology. The immunization of animals with P. aeruginosa vaccine induced the synthesis of antibodies. The introduction of the vaccine in 2 or 3 injections resulted in a high level of antibody formation, differing with the use of various strains. Hyperimmune sera, obtained by the multiple immunization of rabbits with P. aeruginosa vaccine, ensured high protection of mice from P. aeruginosa infection. The vaccine proved to be safe when evaluated in experiments of acute and chronic toxicity, made on laboratory animals.     

IL-23 mediates inflammatory responses to mucoid Pseudomonas aeruginosa lung infection in mice

Patients with cystic fibrosis (CF) develop chronic Pseudomonas aeruginosa lung infection with mucoid strains of P. aeruginosa; these infections cause significant morbidity. The immunological response in these infections is characterized by an influx of neutrophils to the lung and subsequent lung damage over time; however, the underlying mediators to this response are not well understood. We recently reported that IL-23 and IL-17 were elevated in the sputum of patients with CF who were actively infected with P. aeruginosa; however, the importance of IL-23 and IL-17 in mediating this inflammation was unclear. To understand the role that IL-23 plays in initiating airway inflammation in response to P. aeruginosa, IL-23p19(-/-) (IL-23 deficient) and wild-type (WT) mice were challenged with agarose beads containing a clinical, mucoid isolate of P. aeruginosa. Levels of proinflammatory cytokines, chemokines, bacterial dissemination, and inflammatory infiltrates were measured. IL-23-deficient mice had significantly lower induction of IL-17, keratinocyte-derived chemokine (KC), and IL-6, decreased bronchoalveolar lavage (BAL) neutrophils, metalloproteinase-9 (MMP-9), and reduced airway inflammation than WT mice. Despite the reduced level of inflammation in IL-23p19(-/-) mice, there were no differences in the induction of TNF and interferon-gamma or in bacterial dissemination between the two groups. This study demonstrates that IL-23 plays a critical role in generating airway inflammation observed in mucoid P. aeruginosa infection and suggests that IL-23 could be a potential target for immunotherapy to treat airway inflammation in CF.     

Platelet-activating factor receptor contributes to host defense against Pseudomonas aeruginosa pneumonia but is not essential for the accompanying inflammatory and procoagulant response

Pseudomonas aeruginosa is a major cause of nosocomial pneumonia, which is associated with high morbidity and mortality. Because of its ubiquitous nature and its ability to develop resistance to antibiotics, it is a problematic pathogen from a treatment perspective. Platelet-activating factor receptor (PAFR) is involved in phagocytosis of several pathogens. To determine the role of PAFR in the innate immune response to P. aeruginosa pneumonia, pafr gene-deficient (PAFR-/-) mice and normal wild-type (Wt) mice were intranasally inoculated with P. aeruginosa. PAFR deficiency impaired host defense as reflected by increased bacterial outgrowth and dissemination in mice with a targeted deletion of the PAFR gene. PAFR-/- neutrophils showed a diminished phagocytosing capacity of P. aeruginosa in vitro. Relative to Wt mice, PAFR-/- mice demonstrated increased lung inflammation and injury as reflected by histopathology, relative lung weights and total protein concentrations in bronchoalveolar lavage fluid, which was accompanied by higher levels of proinflammatory cytokines in lung homogenates and plasma. In addition, PAFR deficiency was associated with exaggerated local and systemic activation of coagulation as determined by fibrin staining of lung tissue and pulmonary and plasma concentrations of thrombin-antithrombin complexes and D-dimer. These data suggest that PAFR is an essential component of an effective host response to P. aeruginosa pneumonia, at least partly via its contribution to the phagocytic properties of professional granulocytes. Additionally, our results indicate that PAFR signaling is not essential for the induction of a local and systemic inflammatory and procoagulant response to Pseudomonas pneumonia.     

Distinct synergistic action of piperacillin and methylglyoxal against Pseudomonas aeruginosa

The dicarbonyl compound methylglyoxal is a natural constituent of Manuka honey produced from Manuka flowers in New Zealand. It is known to possess both anticancer and antibacterial activity. Such observations prompted to investigate the ability of methylglyoxal as a potent drug against multidrug resistant Pseudomonas aeruginosa. A total of 12 test P. aeruginosa strains isolated from various hospitals were tested for their resistances against many antibiotics, most of which are applied in the treatment of P. aeruginosa infections. Results revealed that the strains were resistant to many drugs at high levels, only piperacillin, carbenicillin, amikacin and ciprofloxacin showed resistances at comparatively lower levels. Following multiple experimentations it was observed that methylglyoxal was also antimicrobic against all the strains at comparable levels. Distinct and statistically significant synergism was observed between methylglyoxal and piperacillin by disc diffusion tests when compared with their individual effects. The fractional inhibitory concentration index of this combination evaluated by checkerboard analysis, was 0.5, which confirmed synergism between the pair. Synergism was also noted when methylglyoxal was combined with carbenicillin and amikacin.     

Reducing bacterial resistance to antibiotics with ultrasound

The effect of erythromycin on planktonic cultures of Psedomonas aeruginosa, with and without application of 70 kHz ultrasound, was studied. Ultrasound was applied at levels that had no inhibitory effect on cultures of Ps. aeruginosa. Ultrasound in combination with erythromycin reduced the viability of Ps. aeruginosa by 1-2 orders of magnitude compared with antibiotic alone, even at concentrations below the minimum inhibitory concentration (MIC). Electron-spin resonance studies suggest that ultrasound induces uptake of antibiotic by perturbing or stressing the membrane. This application of ultrasound may be useful for expanding the number of drugs available for treating localized infections by rendering bacteria susceptible to normally ineffective antibiotics.     

Biological carrier improves passive protection of mice against Pseudomonas aeruginosa infection.

In a model experiment, the use of specific hyperimmune globulins (SHG) alone failed to protect mice against infection with homologous and heterologous P. aeruginosa serotypes. However, the therapeutic potential of SHG dramatically improved after its conjugation with A1(OH)3, used as a biological carrier. The binding of SHG to this carrier proved to be the most effective and stable combination in the treatment of acute pseudomonad infections in mice.     

In vitro interaction between mecillinam and piperacillin-tazobactam in the presence of azithromycin against members of the Enterobacteriaceae family and Pseudomonas aeruginosa

Mecillinam was tested in vitro alone or in combination with piperacillin-tazobactam and azithromycin against representative species of the Enterobacteriaceae family and Pseudomonas aeruginosa to extend its antibacterial spectrum, and to protect mecillinam from inactivating enzymes taking advantage of the presence of tazobactam. Drug interactions were studied by microdilution method, by selection of spontaneous resistant mutants on agar plates containing the drugs in combination and by time kill experiments. Against Enterobacteriaceae mecillinam and piperacillin-tazobactam showed synergistic interaction in 24/60 tests carried out by microdilution technology, in 4/16 by selecting resistant mutants and in 5/9 by time-kill experiments. P. aeruginosa reacted indifferently to the drug combinations, with few exceptions, when azithromycin was present a reduction of the MICs were recorded. Mecillinam reacted favourably in vitro in combination with piperacillin-tazobactam against not only strains included in its antibacterial spectrum but also against resistant Morganella morganii, Proteus spp and P. aeruginosa. The addition of azithromycin (8 mg/L) was beneficial for the drug combination increasing the bactericidal effect in the great majority of the cases. Only systematic in vivo studies may establish the clinical significance and benefits of the present observations.