Protective effect of Lactobacillus casei on Pseudomonas aeruginosa infection in mice.

The protective effect of heat-killed Lactobacillus casei YIT9018 (LC 9018) against Pseudomonas aeruginosa infection in mice was compared with that of Corynebacterium parvum. Survival of mice after intraperitoneal (i.p.) infection with P. aeruginosa was augmented in mice that had been pretreated i.p. with LC 9018 5 days previously. Similar treatment of mice with C. parvum, however, was not effective at all. Moreover, mice became more susceptible to infection with P. aeruginosa after such treatment. Growth of P. aeruginosa in the peritoneal cavity and spleen was markedly inhibited in LC 9018-pretreated mice, whereas such inhibition of bacterial growth was not observed in C. parvum-treated mice. The protective effect of LC 9018 was observed in mice subjected to 800 rads of whole body irradiation but was abrogated when mice were treated with carrageenan. These results suggest that augmentation of the resistance of mice to P. aeruginosa was caused by the induction of activated macrophages. The number of macrophages detectable in the peritoneal cavity was almost the same in LC 9018- and C. parvum-treated mice. Growth of Listeria monocytogenes was inhibited by pretreatment with LC 9018. Inhibition of L. monocytogenes was also observed after the same pretreatment with C. parvum. It was suggested that macrophages activated with LC 9018 were involved in the protective immunity to P. aeruginosa.     

Protective effect of ren-shen-yang-rong-tang (Ninjin-youei-to) in mice with drug-induced leukopenia against Pseudomonas aeruginosa infection.

When 200 mg/kg of cyclophosphamide (CY) or 5-fluorouracil (5-FU) were given subcutaneously to mice, severe reduction of leukocyte numbers in the peripheral blood was observed on day 4 and from day 4 to day 8 after the treatment, respectively. Daily administration of ren-shen-yang-rong-tang (Japanese name: Ninjin-youei-to, NYT), (1 g/kg/day) and recombinant human granulocyte-colony stimulating factor (rhuG-CSF, 2 micrograms/mouse/day) either from day 0 to day 4 after treatment with CY or from day 0 to day 8 after treatment with 5-FU accelerated the recovery of peripheral leukocytes. Administration of NYT and rhuG-CSF inhibited decreases in the number of colony forming units in the spleen (CFU-S) in drug-treated mice. In mice infected intraperitoneally with a virulent strain of Pseudomonas aeruginosa 4 days and 8 days after treatment with CY and 5-FU, respectively, lethal doses were much lower (approximately 1/1000) than that in normal mice. Administration of NYT and rhuG-CSF to drug-treated mice inhibited the enhanced bacterial growth in the peritoneal cavity. Administration of NYT or rhuG-CSF to drug-treated mice enhanced the recovery of accumulation of leukocytes into the infected peritoneal cavity from their depressed state. Administration of NYT was more effective for improving the host resistance of 5-FU-treated mice than for improving that of CY-treated mice in contrast to rhuG-CSF which exhibited stronger effect in CY-treated mice than in 5-FU treated mice.     

Protective immunization against chronic Pseudomonas aeruginosa pulmonary infection in rats.

Rats were immunized systemically with various doses of the polyvalent Pseudomonas aeruginosa vaccine PEV-01. After a series of two or three doses (25 to 50 micrograms each) at 8- to 11-day intervals, animals were challenged intratracheally by the agarose bead technique with a serotype 5 P. aeruginosa strain at periods of 9 to 42 days. Immunized animals developed circulating antibodies (primarily immunoglobulin M) against vaccine components at levels significantly higher than challenged, nonimmunized controls (P less than 0.005). Eight to ten days postinfection, histological sections of lungs from immunized animals showed only minimal inflammation associated with infectious foci (agarose beads) as compared with the extensive pathological changes of airways and parenchyma seen in infected nonimmunized control animals. However, no significant reduction in bacterial numbers was observed. Such protection lasted at least 6 weeks after the final immunization. It is speculated that the vaccine may contain components of cell surface proteins and virulence exoproducts.     

Protective effect of recombinant murine granulocyte-macrophage colony-stimulating factor against Pseudomonas aeruginosa infection in leukocytopenic mice.

The effects of recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) against Pseudomonas aeruginosa infection in ICR mice were investigated. Mice were treated with cyclophosphamide (CPA) and were then injected intraperitoneally with rmGM-CSF three times daily, beginning on the day after CPA treatment, for 7 days. The number of peripheral blood leukocytes in both CPA- and rmGM-CSF-treated mice and control CPA-treated mice reached a nadir on day 4, when P. aeruginosa was injected intraperitoneally. The administration of rmGM-CSF significantly increased the proportion of survivors among mice infected with a lethal dose of P. aeruginosa. This effect was further analyzed by monitoring sequential changes in leukocyte count and bacterial growth in various organs. The number of bacteria in the peritoneal cavities, peripheral blood samples, and livers of GM-CSF-treated mice decreased to an undetectable level after a transient increase, and the number was significantly lower than that in control mice. In GM-CSF-treated mice, the neutrophil levels in peripheral blood started to increase 5 days after CPA administration and were consistently higher than those in controls. Furthermore, the neutrophils in GM-CSF-treated mice were more mature morphologically. Thus, the prophylactic effect of rmGM-CSF against P. aeruginosa infection may result from a rapid recovery of myelopoiesis and a partial enhancement of mature neutrophil function.     

Protective activity of immune sera against extracellular slime from Pseudomonas aeruginosa on experimental infection in mice

Slime-extracts from Pseudomonas aeruginosa strains induced in rabbit synthesis of antibodies which protected mice against lethal intraperitoneal challenge. The antislime sera produced the maximal protection when given simultaneously with challenge dose of bacteria or 3 to 24 h before inoculation of animals. Some antisera exhibited also marked activity in groups of mice passively immunized 48, 72 or even 96 h before challenge. No effect was observed when immune serum was administered after infection.     

Respiratory syncytial virus infection facilitates acute colonization of Pseudomonas aeruginosa in mice

Pseudomonas aeruginosa causes opportunistic infections in immunocompromised individuals and patients ventilated mechanically and is the major pathogen in patients with cystic fibrosis, in which it causes chronic infections. Epidemiological, in vitro and animal data suggest a role for respiratory virus infections in facilitating colonization and infection with P. aeruginosa. A study was undertaken to determine whether respiratory syncytial virus (RSV) infection could facilitate the initiation of an acute infection with P. aeruginosa in vivo. Balb/c mice were infected intranasally with P. aeruginosa, with and without simultaneous inoculation with RSV. Lung function measurements were undertaken using Whole Body Plethysmography and lungs were harvested 24 hr after inoculation. Mice exposed to RSV and P. aeruginosa showed 2,000 times higher colony‐forming units (CFU) counts of P. aeruginosa in the lung homogenates when compared to mice which were only infected with P. aeruginosa and lung function changes were more severe in co‐infected mice. Control mice receiving RSV alone showed no significant changes in lung function or cytokine production, and no inflammatory changes in the lung parenchyma. These results suggest that RSV can facilitate the initiation of acute P. aeruginosa infection without the RSV infection being clinically apparent. This could have implications for treatment strategies to prevent opportunistic P. aeruginosa lung infection. J. Med. Virol. 81:2096–2103, 2009. © 2009 Wiley‐Liss, Inc.     

Glycolipoprotein from Pseudomonas aeruginosa as a protective antigen against P. aeruginosa infection in mice.

After primary subcutaneous immunization of rabbits with glycolipoprotein from Pseudomonas aeruginosa BI, indirect hemagglutinating and bacterial agglutinating activities appeared in the antiserum 6 days after immunization and reached a peak between 15 and 20 days. Both these in vitro activities paralleled in vivo antipseudomonas-induced leukopenia and mouse passive-protection activities. Further experiments indicated that a functional association exists between the hemagglutinating and passive-protection activities, and that passive protection depends on activity levels in the plasma rather than in the peritoneum. After intraperitoneal injection in mice, in vitro and in vivo activities of antiglycolipoprotein serum declined in the peritoneal cavity as the plasma levels increased. After intravenous injection of the antiglycolipoprotein serum, initially high levels of in vitro and in vivo activity declined at approximately equal rates. Immunoglobulin G (IgG) and immunoglobulin M (IgM) fractions from 15-day antiglycolipoprotein serum were assayed for biological activity. Most of the hemagglutinating and bacterial agglutinating activity and all of the mouse passive-protection activity were found in the IgM fraction. Assay of antiglycolipoprotein serum after 2-mercaptoethanol inactivation of IgM showed that most of the in vitro and all of the passive-protection activities had been destroyed, again locating these activities principally in the IgM fraction of the original antiserum.     

Protective effect of saikosaponin A, saikosaponin D and saikogenin D against Pseudomonas aeruginosa infection in mice

Effects of saikosaponins and their genins on nonspecific resistance against Pseudomonas aeruginosa and Listeria monocytogenes infections were investigated. When mice were administered intraperitoneally (i.p.) saikosaponins one day before i.p. infection with P. aeruginosa, saikosaponins a and d induced a marked enhancement of nonspecific resistance at a dose of 10 micrograms/mouse. Also, saikogenin D, a secondary metabolite of saikosaponin d, showed an enhancing effect. The most effective condition for enhancing the nonspecific resistance was i.p. administration of saikosaponin d one day before i.p. or intravenous (i.v.) infection with P. aeruginosa, when mice were treated i.p., i.v., or subcutaneously with saikosaponin d 1, 4 or 7 days previously. Effect of saikosaponin d was weaker than that of formalin-killed bacilli of Propionibacterium acnes and lipopolysaccharide. On the other hand, effect of saikosaponin d on enhancement of nonspecific resistance against L. monocytogenes was not seen. Effector cells participating in the enhanced protection induced by saikosaponin d may be macrophages, since macrophages were a major component in peritoneal cells obtained from mice administered i.p. saikosaponin d 1 day earlier and intracellular bactericidal activity of peritoneal macrophages against P. aeruginosa increased.     

Experimental urinary tract infection with Pseudomonas aeruginosa in mice.

Urinary tract infection with Pseudomonas aeruginosa was induced in mice by transurethral inoculation of the organism into the bladder, followed by urethral obstruction for 6 h. The infection was mostly localized in the urinary organs. P. aeruginosa P9 was selected as the challenge organism from 10 laboratory strains of P. aeruginosa. After the inoculation of 10(7) colony-forming units of P. aeruginosa P9, transient bacteremia was observed in some of the mice from 6 h to 1 day after the inoculation. The number of organisms in the bladder tissue gradually decreased, whereas that in the kidneys increased to levels of 10(6) to 10(7) colony-forming units in 3 days, and these levels remained up to 2 weeks after the inoculation. The organisms gradually disappeared thereafter, and spontaneous recovery took place. The organisms could be recovered from the kidneys of 95% of the mice, and the gross lesions in the kidneys were observed in 77% of the mice 1 week after inoculation. The method developed here is simple and may be useful in the study of urinary tract infections due to P. aeruginosa and other species of bacteria.20     

Adaptive resistance to aminoglycoside antibiotics in Pseudomonas aeruginosa

Aminoglycoside-resistant variants of Pseudomonas aeruginosa strain PAO1 were readily selected by culturing the organism in medium containing increasing concentrations of gentamicin, tobramycin or amikacin until the strains were growing in a concentration of drug 128-fold greater than the minimal inhibitory concentration for the sensitive parent strain. These resistant strains exhibited characteristics previously associated with the impermeability type of resistance mechanism, i.e., they grew more slowly than the parent strain, the resistance was unstable in the absence of the antibiotic, and adaptation to one of the antibiotics conferred cross-resistance to other aminoglycosides. The adapted strains grew, with minimal morphological alterations, in concentrations of the various aminoglycosides that normally produced cell envelope damage, misshapen and filamentous cell formation, and cell lysis in the sensitive strain. Neither protein H1 nor phospholipid alterations appear to play a significant role in adaptive resistance to aminoglycoside antibiotics in this model system. The acquisition of adaptive resistance to the aminoglycoside antibiotics did not confer resistance to polymyxin B, another cationic antibiotic which is thought to share binding sites within the outer membrane with the aminoglycosides.     

Protection against experimental Pseudomonas aeruginosa infection in mice by active immunization with exotoxin A toxoids.

The immunoprophylactic effect of chemically inactivated Pseudomonas aeruginosa exotoxin A in experimental pseudomonas infections was studied. Exotoxin A toxoids were prepared by Formalin (f-TXD) or glutaraldehyde (g-TXD) treatment. Immunization of mice with three or four doses (10 micrograms each) of f-TXD and the synthetic adjuvant N-acetylmuramyl-L-alanyl-D-isoglutamine (50 micrograms) induced high levels of antiexotoxin A antibodies as measured by passive hemagglutination assay and enzyme-linked immunosorbent assay. Immunization with toxoid alone did not elicit antitoxin. A significant increase in survival time and survival rate (P less than 0.01) was seen in immunized (f-TXD) and in burned and infected mice (50 to 85%) as compared with control mice immunized with formalinized bovine serum albumin (6 to 20%). Virtually 100% survival was obtained when preinfection immunization weas combined with single-dose gentamicin treatment within 24 h of infection. Immunization with g-TXD increased survival time (P less than 0.01) but did not consistently increase survival rate, and the results were not as satisfactory as those with formalinized exotoxin. The data presented indicate that active immunization with formalinized exotoxin A toxoid and adjuvant induced protective immunity to various degrees against infections in mice and could be potentially useful in prophylaxis of P. aeruginosa infections.     

Therapeutic efficacy of granulocyte colony-stimulating factor alone and in combination with antibiotics against Pseudomonas aeruginosa infections in mice.

The therapeutic efficacy of granulocyte colony-stimulating factor (G-CSF) against an experimental intramuscular infection induced by Pseudomonas aeruginosa in mice was confirmed. Bacterial growth in the infected thigh muscle was suppressed by G-CSF treatment. The change in the number of peripheral blood polymorphonuclear leukocytes (PMN) after bacterial challenge was investigated. The results showed that G-CSF could stimulate stronger defense mechanisms after stimulation by bacterial challenge. In the G-CSF-treated group, more clusters of matured PMN were observed in the infected thigh muscle 6 h after bacterial challenge. Next, the correlation between the number of PMN in the blood at the time of infection and the therapeutic efficacy of antibiotics was investigated. The therapeutic efficacy of ceftazidime, a beta-lactam antibiotic, was affected by the number of blood PMN at the time of infection. In particular, a decrease of peripheral blood PMN at the time of infection resulted in a dramatic decrease in the efficacy of ceftazidime. The reduction in leukopenia by G-CSF remarkably strengthened the therapeutic effect of antibiotics in mice.     

Chronic colonization of rat airways with Pseudomonas aeruginosa.

Colonization of the airways of rats by Pseudomonas aeruginosa was established by treating the animals with hexamethylphosphoramide (HMPA) and inoculating with P. aeruginosa. Male Sprague-Dawley rats were given tap water (controls) or HMPA in the drinking water at 2 or 4 mg/ml. The ciliated cells of the airway epithelium were denuded, and microulcerative lesions in the epithelium were induced in the HMPA-treated rats. After 2 weeks of treatment, the rats were inoculated by transoral intratracheal instillation with 5 X 10(7) CFU of P. aeruginosa obtained from a cystic fibrosis patient. Two weeks after inoculation, P. aeruginosa was cultured from the airways, and scanning and transmission electron microscopy showed bacilli adhering to or invading the injured airway epithelium. P. aeruginosa was present in tracheal and intrapulmonary tissue homogenates of 9% of the P. aeruginosa-inoculated control rats (n = 22) as compared with 61% of the 2-mg/ml (n = 18) and 65% of the 4-mg/ml (n = 20) HMPA-treated rats (P less than 0.05). No dose-response relationship was found between 2 and 4 mg of HMPA per ml and colonization. Contamination of 47% of all of the rats with Mycoplasma pulmonis, as indicated by a positive enzyme-linked immunosorbent assay for immunoglobulin G, had no discernible significant effect on colonization by P. aeruginosa. These results indicate that colonization of the rat airway by P. aeruginosa can be achieved experimentally by treating the animals with HMPA. This research supports the hypothesis that colonization by P. aeruginosa may occur in airways where the ciliated epithelium has been injured and epithelial lesions exist.     

Effect of calcium chloride on experimental infection of mice with Pseudomonas aeruginosa.

Concomitant administration of Pseudomonas aeruginosa and calcium chloride to mice enhanced the virulence of some strains. The 50% lethal dose of P. aeruginosa 5 decreased by more than three orders of magnitude, regardless of the challenge route (intramuscular, subcutaneous, and intraperitoneal injection), while the 50% lethal dose of strain N10 did not decrease. When challenged intramuscularly with 10(4) organisms of strain 5 or strain N10 mixed with calcium chloride, both strains multiplied at the local site of injection. Strain 5 subsequently caused systemic infection, while strain N10 did not. The virulence-enhancing effect of calcium chloride can be successfully applied in protection assays for immunized mice challenged with strain 5.     

Protective effect of muramyl dipeptide analogs against infections of Pseudomonas aeruginosa or Candida albicans in mice.

Two analogs of N-acetylmuramyl-L-alanyl-D-isoglutamine (muramyl dipeptide) were found to give better protection than muramyl dipeptide against intraperitoneal Pseudomonas aeruginosa infection or intravenous Candida albicans infection in mice. The analogs tested were N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine and N-acetylmuramyl-L-alpha-amino-butyryl-D-isoglutamine. The optimum treatment was 80 mg/kg per day given once daily for 4 consecutive days before infection by the intraperitoneal, intravenous, or subcutaneous route. Dose response was limited. The compounds were not orally active. Synergism was seen between N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine and gentamicin. No postinfection protection was observed. A nonspecific stimulation of macrophage cells by muramyl dipeptide analogs may contribute to the protection because antiinfective activity against Listeria monocytogenes given intraperitoneally was achieved with CBA mice.     

Pseudomonas aeruginosa colonization in patients with spinal cord injuries.

The prevalence of Pseudomonas aeruginosa colonization of patients with spinal cord injury was studied annually from 1976 to 1980. The urethra, perineum, rectum, drainage bag, and urine of patients on the spinal cord injury service were cultured. A total of 224 men and 32 women were studied. Most patients were managed with an external urinary collection system or padding, with or without intermittent catheterization. P. aeruginosa was cultured from one or more body sites (urethra, perineum, or rectum) in 65% of men and 18% of women. Drainage bags on the beds were frequently colonized with P. aeruginosa (73%). Significant bacteriuria with P. aeruginosa was present in 19% of the men and 13% of the women. P. aeruginosa colonization of body sites in men was closely associated with the use of an external urinary collection system. Significantly greater urethral and perineal colonization was found in men using an external urinary collection system. P. aeruginosa serotype 11 was the predominant serotype for the first 3 years, and the number of patients colonized with serotype 11 increased with length of hospital stay. The prevalence of serotype 11 significantly decreased in the last 2 years. The antibiotic susceptibility of the strains of P. aeruginosa isolated from these patients did not change in the 5 years, except that there was increasing susceptibility to carbenicillin in later years. This increasing susceptibility to carbenicillin was a reflection of a decreased prevalence of serotype 11 in these patients, since serotype 11 was more resistant than other serotypes to carbenicillin.     

Antibody-independent protection against Pseudomonas aeruginosa infection in mice after treatment with a homologous strain vaccine

Formalin-killed cells of Pseudomonas aeruginosa strain M-24 elicited an antibody-independent protective effect against P. aeruginosa infection in mice. The effect was observed as early as 6 h after administration and 100% protection was obtained by 48 h. The protective effect could not be attributed to the production of specific antibody. In M-24-treated mice, the bacteria in the peritoneal cavity, blood and liver were eliminated 12 h after P. aeruginosa infection. This suggested that the protective effect was due to enhanced bacterial elimination. The percentage of macrophages in the peritoneal cavity was increased after M-24 administration. Furthermore, the enhanced bacterial elimination was abrogated by treatment of mice with 60Co-irradiation or carrageenan. These findings suggest the involvement of macrophages in the enhanced bacterial elimination observed. The chemiluminescence of peritoneal exudate cells from M-24-treated mice was markedly increased when compared with that of cells from untreated mice. The ability to kill P. aeruginosa in vitro was also greater in macrophages from mice treated with killed M-24 than in cells from proteose-peptone-treated mice. The M-24-treated mice showed enhanced nonspecific protection against infection with lethal doses of P. aeruginosa, Escherichia coli or Listeria monocytogenes. However, susceptibility to LPS in mice was not increased by M-24 treatment. These results suggest that macrophage activation without increasing LPS susceptibility was responsible for the antibody-independent protection induced by killed M-24.     

Risk factors for antimicrobial resistance and influence of resistance on mortality in patients with bloodstream infection caused by Pseudomonas aeruginosa

This study was conducted to evaluate risk factors for antimicrobial resistance and influence of resistance on mortality in Pseudomonas aeruginosa bacteremia. Data on 190 patients with P. aeruginosa bacteremia were analyzed retrospectively. Antimicrobial resistance to antipseudomonal antibiotics was evaluated. The main outcome measure was 30-day mortality. In P. aeruginosa bacteremia, resistance rates to piperacillin (PIP), ceftazidime (CAZ), ciprofloxacin (CIP), and imipenem (IPM) were 29 (56/190), 19 (36/190), 17 (32/190) and 15% (28/190), respectively. Prior uses of fluoroquinolones or carbapenems were independent risk factors for resistance to CIP and IPM, and prior use of extended-spectrum cephalosporins was a risk factor for PIP-R. An indwelling urinary catheter was a risk factor for PIP-R, CAZ-R, and CIP-R. An invasive procedure was a risk factor for CIP-R and IPM-R. The 30-day mortality rate was 44% (33/75) in patients infected by strains resistant to any of the antipseudomonal antibiotics, but 33.9% (39/115) in those by strains susceptible to all antipseudomonal antibiotics (p = 0.161). Among patients with bloodstream infection due to antimicrobial-resistant P. aeruginosa, those infected by IPM-R strains had the highest mortality (IPM-R, 53.6% vs. CAZ-R, 47.2% vs. CIP-R 46.9%, PIP-R, 39.3%). In this study regarding P. aeruginosa bacteremia, prior uses of fluoroquinolones, carbapenems, or extended-spectrum cephalosporins, a prior invasive procedure, and an indwelling urinary catheter were found to be associated with antimicrobial resistance. The patients with bloodstream infection caused by antimicrobial-resistant P. aeruginosa, especially to imipenem, had a tendency toward higher mortality than those infected by susceptible strains.     

Protection of immunosuppressed mice against infection with Pseudomonas aeruginosa by recombinant P. aeruginosa lipoprotein I and lipoprotein I-specific monoclonal antibodies.

Outer membrane protein I (OprI) is one of the major proteins of the outer membrane of Pseudomonas aeruginosa. The protective effect of OprI vaccination and that of three OprI-specific monoclonal antibodies (MAbs) against infection with P. aeruginosa were tested in immunosuppressed mice. The combination of Oprl and MAb 2A1 protected the mice against a challenge with a 96-fold 50% lethal dose. The binding site of MAb 2A1 was mapped, resulting in the identification of a protective epitope (amino acids 7 to 20).     

Protective immunity induced in mice by immunization with high-molecular-weight polysaccharide from Pseudomonas aeruginosa.

A high-molecular-weight alkali-labile polysaccharide (PS) isolated from the slime of immunotype 1 Pseudomonas aeruginosa was tested for its ability to protect mice from lethal challenge with the live, homologous organism. Intraperitoneal (i.p.) injection of 10 to 25 microgram of the PS protected 60 to 70% of the mice against challenge with up to 50 50% lethal dose units. Although single immunization of mice with up to 250 microgram of PS effected protective levels of only 70%, two successive immunizations provided 100% protection. Subcutaneous and intravenous immunization with PS also provided protection to i.p. challenges with immunotype 1 P. aeruginosa, but not to i.p. challenge with immunotype 4 P. aeruginosa. Although lipopolysaccharide (LPS) was found to be more immunogenic than PS in out studies, contamination of the alkali-labile PS with LPS did not account for the protection seen. Alkali treatment (0.1 N NaOH, 37 degrees C, 2 h) of the PS destroyed its protective effectiveness, while similarly treated LPS retained its capacity for inducing immunity in mice. Adsorption and passive protection studies with sera raised to either PS or a mixture of PS and LPS indicated that antibody directed to the alkali-labile PS antigen was capable of contributing to the protection of mice against challenge with P. aeruginosa.