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Objective To construct and identify Bifidobacterium bifidum-vectored outer membrane protein F-I[rBb(pGEX-OprF-I)] vaccine of Pseudomonas aeruginosa and observe its protection against Pseudomonas aeruginosa infection in mice. Methods OprF and OprI genes were amplified by PCR, then the OprF-I fusion gene obtained by gene SOEing was digested and ligated into the vector pGEX-1λT to construct the recombinant plasmid pGEX-OprF-I. The plasmid was transformed into Bifidobacterium bifidum (Bb) by electroporation, and the rBb(pGEX-OprF-I) vaccine was constructed and identified by double enzyme digestion and PCR. Expression products of the vaccine induced by IPTG were analyzed and identified by SDS-PAGE and Western blot analysis. Twenty-one BALB/c mice were randomly divided into rBb(pGEX-OprF-I) vaccine group, Bb-pGEX-1λT empty vector group and Bb control group. The 5×10 8 CFUs vaccine was intragastrically administered for 3 consecutive days per week for 3 weeks. All mice were challenged intranasally with 5×10 7 CFUs of PA01 strain at the 4th week after the first immunization. At the 2nd week after the challenge, all mice were sacrificed to count the lung bacteria loads. IgG levels in sera from the mice before immunization, 4th week after the first immunization and 2nd week after the challenge were detected by routine ELISA. Results A total of 1289 bp OprF-I fusion gene was amplified by PCR. Double enzyme digestion and PCR identification confirmed that the gene was ligated into pGEX-1λT and transformed into Bb, and the rBb(pGEX-OprF-I) vaccine was successfully constructed. SDS-PAGE showed that the fusion protein with a relative molecular mass (M r ) of about 68 000 could be expressed by IPTG-induced vaccine. Western blot analysis indicated that the protein could be specifically recognized by the sera of Pseudomonas aeruginosa-infected mice. The number of bacteria colonies in the lung of the mice immunized with rBb(pGEX-OprF-I) vaccine was significantly lower than that of the control group. The IgG levels in the sera of the immunized mice increased successively at 4th week after the first immunization and 2nd week after the challenge, and higher than that in the other control groups at the same time point. Conclusion The rBb(pGEX-OprF-I) vaccine has been successfully constructed, and it may take a certain protective effect on the mice against Pseudomonas aeruginosa infection.

Objective: To investigate CPC-nanoparticles of low concentrations in treatment of staphylococcus aureus and pseudomonas aeruginosa biofilms in vitro. Method: We established specific biofilms of staphylococcus aureus ATCC 25923 and pseudomonas aeruginosa ATCC 15692, and prepared CPC-nanoparticles and CPC micelle solutions of low concentrations(0.010%, 0.025% and 0.050%). AlamarBlue was used to test the viability of both planktonic staphylococcus aureus and pseudomonas aeruginosa and their biofilms after treatment for 5 minutes and 2 hours respectively in the bactericidal efficacy study.The interaction between CPC-nanoparticles and staphylococcus aureus and pseudomonas aeruginosa biofilms was observed by confocal laser scanning microscope(CLSM). Result: 0.010%, 0.025% and 0.050% CPC-nanoparticles and CPC-micelle solutions had significant bactericidal effect on planktonic staphylococcus aureus and pseudomonas aeruginosa after fiveminute exposure( P <0.05), and staphylococcus aureus and pseudomonas aeruginosa biofilms after both five-minute and two-hour treatments( P <0.05). In CLSM study, the size of staphylococcus aureus biofilms decreased, while dead bacteria of pseudomonas aeruginosa biofilms increased after two-hour treatment. Conclusion: CPC-nanoparticles had significant bactericidal effects on staphylococcus aureus and pseudomonas aeruginosa biofilms, which could be used in treatment of CRS., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Clinical Otorhinolaryngology Head and Neck Surgery.)

Objective: To construct the recombinant Bb-pGEX- OprI vaccine of Pseudomonas aeruginosa (Pa) outer membrane protein I (OprI) and study its protection effect in mice against Pa., Methods: The OprI gene was amplified by PCR,and cloned into pGEX-1λT to generate pGEX- OprI . The pGEX- OprI was transformed into Bifidobacterium bifidum (Bb) to construct recombinant Bb-pGEX- OprI vaccine by electroporation. After identification with double enzyme digestion,PCR and sequencing,the vaccine was then induced with IPTG,and its expression was analyzed and identified by SDS-PAGE and Western blot respectively. Twenty-one mice were randomly divided into 3 groups and vaccinated by intragastric administration with Bb-pGEX- OprI ,Bb-pGEX-1λT and Bb respectively. All mice were challenged with PA01 strain at 4 weeks after the first vaccination. At 2 weeks after the challenge,mice were sacrificed to separate their lungs,and the numbers of bacterial colonies in lungs were counted. Venous blood was collected before vaccination,at 4 weeks after the first vaccination and 2 weeks after the challenge of PA01 strain. The serum IgG,IgG subclasses and IgE were detected by routine ELISA., Results: The OprI gene of 194 bp was successfully amplified by PCR. Double enzyme digestion,PCR and sequencing confirmed that the OprI gene was successfully cloned into pGEX-1λT and pGEX- OprI was successfully transformed into Bb,constructing the Bb-pGEX- OprI vaccine. SDS-PAGE indicated that Bb-pGEX- OprI vaccine expressed an OprI-GST fusion protein with the relative molecular mass of approximately 32×10 3 . Western blot verified that the fusion protein could be specifically identified by the sera of mice infected with Pa. The number of bacterial colonies in lung of Bb-pGEX- OprI vaccine group was lower than that of Bb-pGEX-1λT or Bb control ( P <0.01). The levels of serum IgG,IgG2b,IgG3 and IgE in Bb-pGEX- OprI vaccine group rose at 4 weeks after the first vaccination and 2 weeks after the challenge successively. The levels of serum antibodies in Bb-pGEX- OprI vaccine group were higher than those in Bb-pGEX-1λT or Bb control at the same time point ( P <0.01 or P <0.05)., Conclusion: The recombinant Bb-pGEX- OprI vaccine was successfully constructed and produced an effective humoral immune response against the Pa infection., (CopyrightCopyright© by Editorial Board of Journal of Sichuan University (Medical Science Edition).)

Objective: To explore the effects of different beta-lactam antibiotics on the inducing of LPS release from gram-negative bacteria and on the protection of infected animals., Methods: Wistar rats were employed as the model and were inflicted by 30% TBSA III degree scalding and sepsis caused by PA103. The rats were randomly divided into 3 groups, i.e. simple antibiotic treatment group (A), treatment after sensitization with galactosamine (GalN) group (G) and treatment after blocking with carrageenan (CGN) group (C). The rats were injected intra-peritoneally with imipenem (IMP, 5 mg) and ceftazidime (CTZ, 10 mg) for single time, respectively. Same amount of aseptic normal saline was injected in the control group, and GalN (50 mg) was added in G and CGN (1 mg) in C groups. The blood bacterial concentration and plasma LPS levels were determined at different time points after the treatment by antibiotics. The mortality was observed in G and C groups at 10 days after treatment., Results: The blood bacterial amount could be decreased by both IMP and CTZ evidently. Large amounts of LPS released from PA103 could be induced by IMP and CTZ during their bactericidal process. But the plasma LPS level in rats treated by CTZ was markedly higher than that by IMP (P < 0.05 approximately 0.01). The mortality in G group treated by CTZ was much higher than that by IMP (P < 0.05). Nevertheless, the mortality in C groups was the same no matter CTZ or IMP was applied (P < 0.05)., Conclusion: There was no difference of the bactericidal power between IMP and CTZ. But CTZ was more powerful in inducing LPS release from bacteria than IMP. It was implied by the difference between these two antibiotics that IMP might be better choice in clinical application for burn infection due to its lower potential of inducing LPS release from the bacteria.

40 Wistar rats were scalded resulting in 15% TBSA full-thickness burn. Pseudomonas aeruginosa (10(9)/ml) was seeded on the wounds. The animals were divided into 4 groups. AgSD and ZnSD (with Azone) cream was applied to the wounds in the group I. The wounds were irradiated with negative ion current in the group II. The "moisture burn ointment" was applied to the wounds in the group III. Group IV consisted of controls without any treatment. Judging by changes in levels of LPS, TNF and ET, it was shown that the best result was obtained in the group I and II. The "moisture burn ointment" group yielded the poorest results.

In this study, we evaluated the prophylactic effect of Ceftazidime on bacterial translocation (BT) of Pseudomonas aeruginosa in scalded rat by qualitative and quantitative bacterial cultures of blood, organs and mesenteric lymph nodes (MLN). Ceftazidi-me was administered systemically at 3 h, 6 h and 12 h post injury respectively. The dynamic changes in levels of Ceftazidime in blood and organ tissues were also determined. The results showed that effective levels of Ceftazidime in blood, liver and mucosa of the small intestine were rapidly reached and maintained for more than 4 h, but no drug was found in MLN. The prophylactic effect on BT was significant when systemic administration started at 3 h post injury (P < 0.001) and 6 h post injury (P < 0.05). Although the incidence of BT was unchanged (P > 0.05) when systemic administration started at 12 h, the number of organisms present in livers and kidneys, except MLN, was dramatically reduced (P < 0.01).

45 male rabbits with 15% III degree burn were infected with Pseudomonas aeruginosa. They were divided into three groups: the prevention group, in which cpz (cefoperazone) was immediately administered after the scald at the dose of 25 mg/kg with an interval of 1/12 h for a total of 4 doses; the treatment group, in which cpz was administered 48 th after the scald with the dosage same as above; control group in which cpz was not administered. The findings were as follows: In the prevention group, gross observation showed dessication and crustation of the surface of the wound within 1 week after the scald. By employing immunofluorescent antibody it was found that P. A existed only in the superficial layer. However, ulceration and exudation were found in the control and treatment group within 3 or 4 days after the scald, and P. A was shown to have invaded the deep muscular tissue to cause intermuscular septal abscess. The positive rate of blood culture of bacteria in the prevention group (10/81) was significantly lower than that of the treatment (15/57) and the control groups (21/76, P < 0.05). The death rate of the prevention group (2/15) was significantly lower than that of the treatment (10/15) and the control groups (9/15, P < 0.05). The above finding showed that antibiotics specific to the infectious organism of the wound in extensive burn patients should be given in the early exudation stage in order to ward off invasive infection.

Thirty-six cases of pseudomonas aeruginosa septicemia were found in 5 years from 1986 to 1990. The main risk factors predisposing septicemia included severe underlying diseases and local primary infection or impaired anatomic barrier. The use of broad-spectrum antibiotics, cytotoxic or immunosuppressive therapy, and leukopenia were also important risk factors. The percentages of in vitro resistant strains to 8 antipseudomonal antibiotics varied from 18% to 47%. The overall effective rate was 42% in 26 patients receiving appropriate antibiotic therapy. The mortality attributable to pseudomonas aeruginosa septicemia was 61%. The prevention and antibiotic treatment of pseudomonas aeruginosa septicemia are discussed.

A controlled study was performed to evaluate the efficacy of human hyperimmune plasma (HIP) for protecting burned mice from Pseudomonas infection. The HIP was prepared from healthy volunteers immunized with endotoxin protein (Ep) of Pseudomonas aeruginosa. It contained antibody against Ep up to the titre higher than 1:128. Mice with full-thickness burn of 13% TBSA, infected by subeschar injection of 0.2 ml P. aeruginosa (CFU/ml), were treated with HIP at 6 hrs and 18 hrs postburn. The survival rate of the former on the 7th day was 45%, while in mice treated with normal plasma (NP) was 14% and in control mice 10% (P < 0.01). If the HIP was given 18h after burn, the survival rate was 43%, while in mice with NP was 39% and in control 42% (P > 0.05). The results indicate that HIP is able to protect burned mice from Pseudomonas infection giving at 6 hrs postburn (pre sepsis) but not at 18 hrs postburn (post sepsis).

From October 1987 to December 1989, bacteriological surveillance was done after open heart surgery in 100 patients with cardiovascular diseases (54 with congenital, 44 with rheumatic, and 2 with other types of heart disease). The time of bypass was 58 to 234 minutes (mean = 98.7 minutes). The positive rate of culture was 5% for blood, 40% for sputum, and 15% for pericardial drainage. Many types of bacteria were separated. Most of them were conditional pathogenic bacteria. Postoperative infection was found in 9 patients, (bacteremia in 1 and pulmonary infection in 8.) Tracheostomy was done in 5 cases. In discussion, the authors think that the low defensive ability of the patients with serious heart disease is usually further damaged by bacterial invasion after open heart surgery and that bacterial contamination should be reduced in order to prevent postoperative infection.