Anne Morris Hooke

Intranasal immunization with temperature-sensitive mutants protects granulocytopenic mice from lethal pulmonary challenge withPseudomonas aeruginosa

Intranasal (i.n.) immunization with two temperature-sensitive (ts) mutants ofPseudomonas aeruginosa protected, in a dose-related manner, granulocytopenic (GCP) mice challenged with a lethal dose of the wild-type (wt) organism. The number of ts mutants in oronasopharyngeal lavage fluids and stools decreased steadily in both normal and GCP mice after i.n. immunization. Intranasal immunization with 107 colony-forming units (CFU) of either mutant induced significant protection, whereas intraperitoneal (i.p.) immunization with similar doses induced lower protection. Protection induced by i.n. immunization was accompanied by increased levels of anti-P. aeruginosa IgA in lung lavage fluids. The results of this study demonstrate the usefulness of ts mutants ofP. aeruginosa for local immunization to protect GCP hosts from fatalP. aeruginosa pneumonia.

A nonhemolytic phospholipase C from Burkholderia cepacia

Abstract.Burkholderia cepacia is an opportunistic pathogen that causes serious pulmonary infections in cystic fibrosis patients. Although several potential virulence factors—a protease, lipase, and two phospholipases C (one hemolytic and one nonhemolytic)—have been identified, only two, the protease and the lipase, have been described in detail. The goal of this study was to purify and characterize a nonhemolytic phospholipase C secreted by B. cepacia strain Pc224c. The enzyme was concentrated from culture supernatants and purified by polyacrylamide gel electrophoresis. The 54-kDa protein was stable in the presence of sodium dodecyl sulfate (up to 10%) and at 4°, 22°, and 37°C; it was, however, inactivated at 100°C. The enzyme bound to glass, chromatography matrices, and polyvinylidene difluoride and cellulose membranes, suggesting that it is hydrophobic.  In a genetic approach, primers based on conserved sequences of a B. cepacia Pc69 hemolytic phospholipase C and both the Pseudomonas aeruginosa hemolytic and nonhemolytic proteins were designed to identify the Pc224c nonhemolytic phospholipase C gene. One polymerase chain reaction product was identified; it was sequenced and the sequence compared with sequences in the BLAST database. The best match was the Pseudomonas aeruginosa hemolytic phospholipase C. Ten additional B. cepacia strains were screened for the gene by Southern hybridization; five had the 4-kb band, suggesting that these strains have a similar form of the PLC gene. Nine of the ten strains reacted with the probe, suggesting that similar sequences were present, but in another form.

Regulation of Expression of the Nonhemolytic Phospholipase C of Burkholderia cepacia

Abstract.Burkholderia cepacia is an opportunistic pathogen that causes serious pulmonary infections in cystic fibrosis patients. We have purified and partially characterized one potential virulence factor for the organism—a nonhemolytic phospholipase C—and we studied the effect of iron restriction and choline and phosphate concentrations on the expression of phospholipase C. Iron limitation did not affect expression, the effect of choline was variable, and high phosphate concentrations repressed expression. Experiments with heat-treated spent culture supernatants suggested that autoinducers affected the expression of the phospholipase and two other potential virulence factors, a protease and a lipase. We screened 26 B. cepacia isolates for autoinducer activity: 11 induced violacein production in the autoinducer-deficient mutant Chromobacterium violaceum CV026. Spent supernatants from two strains, one that was positive in the C. violaceum assay and one that was negative, were tested for inducing early expression of phospholipase C, protease, and lipase in homologous and heterologous cultures. Expression of all three enzymes was increased or induced at an earlier stage in the growth curve in every case, suggesting not only that autoinducers were involved in the regulation of the expression of these enzymes, but also that the autoinducers were of two different classes.

The effect of liposomal cefoperazone against Pseudomonas aeruginosa in a granulocytopenic mouse model of acute lung infection

The therapeutic efficacy of liposomal cefoperazone againstPseudomonas aeruginosa was investigated in a granulocytopenic mouse model of acute lung infection. Granulocytopenia was induced in mice by intraperitoneal (i.p.) injection of 200 mg/kg cyclophosphamide. Mice were challenged by exposure to an aerosol containingP. aeruginosa and were treated i.p. with liposomal cefoperazone prepared by the dehydration-rehydration method. The half-life of free cefoperazone in the lungs following i.p. administration of the liposomal drug was significantly lengthened (13 min vs. 261 min), and the cefoperazone activity in the lungs remained above the MIC longer after administration of liposomal cefoperazone than after treatment with cefoperazone. Liposomal cefoperazone was more effective than cefoperazone alone in preventing death of granulocytopenic mice from lethal pulmonary challenge withP. aeruginosa (75% vs. 38% survival, p=0.031). Finally,P. aeruginosa was cleared faster from the lungs of mice treated with liposomal cefoperazone when compared with those treated with cefoperazone. This study shows that incorporation of cefoperazone into liposomes enhances the activity of the antibiotic againstP. aeruginosa in a granulocytopenic host. Die therapeutische Wirksamkeit von liposomalem Cefoperazon bei akuter Lungeninfektion durchPseudomonas aeruginosa wurde am Modell der granulozytopenischen Maus untersucht. Die Granulozytopenie wurde durch intraperitoneale (i.p.) Injektion von 200 mg/kg Cyclophosphamid hervorgerufen. Die Inokulation mitP. aeruginosa erfolgte durch Aerosol. Liposomales Cefoperazon, das mit der Dehydratations-Rehydratations-Methode hergestellt wurde, wurde intraperitoneal appliziert. Nach intraperitonealer Gabe von liposomalem Cefoperazon war die Halbwertszeit von freiem Cefoperazon in der Lunge signifikant länger als bei Applikation des Antibiotikums in freier Form (13 gegenüber 261 min.) und die Lungenkonzentration lag länger über den minimalen Hemmkonzentrationen. In der Prävention tödlicher Lungeninfektionen durchP. aeruginosa war die liposomale Präparation effektiver als freies Cefoperazon (Überlebensrate bei letaler Pseudomonas- Dosis 75% gegenüber 38%; p=0,031). Die Elimination vonP. aeruginosa aus der Lunge erfolgte bei Behandlung der Mäuse mit liposomalem Cefoperazon rascher als bei Gabe des freien Antibiotikums. Die Experimente zeigen, daß die Aktivität von Cefoperazon gegenP. aeruginosa-Infektionen bei Granulozytopenie durch Inkorporation in Liposomen gesteigert werden kann.SummaryThe therapeutic efficacy of liposomal cefoperazone againstPseudomonas aeruginosa was investigated in a granulocytopenic mouse model of acute lung infection. Granulocytopenia was induced in mice by intraperitoneal (i.p.) injection of 200 mg/kg cyclophosphamide. Mice were challenged by exposure to an aerosol containingP. aeruginosa and were treated i.p. with liposomal cefoperazone prepared by the dehydration-rehydration method. The half-life of free cefoperazone in the lungs following i.p. administration of the liposomal drug was significantly lengthened (13 min vs. 261 min), and the cefoperazone activity in the lungs remained above the MIC longer after administration of liposomal cefoperazone than after treatment with cefoperazone. Liposomal cefoperazone was more effective than cefoperazone alone in preventing death of granulocytopenic mice from lethal pulmonary challenge withP. aeruginosa (75% vs. 38% survival, p=0.031). Finally,P. aeruginosa was cleared faster from the lungs of mice treated with liposomal cefoperazone when compared with those treated with cefoperazone. This study shows that incorporation of cefoperazone into liposomes enhances the activity of the antibiotic againstP. aeruginosa in a granulocytopenic host.ZusammenfassungDie therapeutische Wirksamkeit von liposomalem Cefoperazon bei akuter Lungeninfektion durchPseudomonas aeruginosa wurde am Modell der granulozytopenischen Maus untersucht. Die Granulozytopenie wurde durch intraperitoneale (i.p.) Injektion von 200 mg/kg Cyclophosphamid hervorgerufen. Die Inokulation mitP. aeruginosa erfolgte durch Aerosol. Liposomales Cefoperazon, das mit der Dehydratations-Rehydratations-Methode hergestellt wurde, wurde intraperitoneal appliziert. Nach intraperitonealer Gabe von liposomalem Cefoperazon war die Halbwertszeit von freiem Cefoperazon in der Lunge signifikant länger als bei Applikation des Antibiotikums in freier Form (13 gegenüber 261 min.) und die Lungenkonzentration lag länger über den minimalen Hemmkonzentrationen. In der Prävention tödlicher Lungeninfektionen durchP. aeruginosa war die liposomale Präparation effektiver als freies Cefoperazon (Überlebensrate bei letaler Pseudomonas- Dosis 75% gegenüber 38%; p=0,031). Die Elimination vonP. aeruginosa aus der Lunge erfolgte bei Behandlung der Mäuse mit liposomalem Cefoperazon rascher als bei Gabe des freien Antibiotikums. Die Experimente zeigen, daß die Aktivität von Cefoperazon gegenP. aeruginosa-Infektionen bei Granulozytopenie durch Inkorporation in Liposomen gesteigert werden kann.

A nonhemolytic phospholipase C produced byPseudomonas cepacia

Pseudomonas cepacia Pc224c, a nonhemolytic strain originally isolated from the sputum of a cystic fibrosis patient, produced an extracellular, heat-labile phospholipase C activity, which was measured quantitatively on the synthetic substratep-nitrophenylphophorylcholine. Cell-free supernatants from cultures grown to late log phase in MOPS-minimal salts-Tryptose medium contained specific activity at least 38 times greater than that from cultures grown in Tryptose minimal medium, Tryptic Soy broth, or peptone medium. Production was inhibited by the presence of inorganic phosphate in the medium and enhanced by aeration of the culture. The PLC ofP. cepacia is nonhemolytic and does not exhibit lecithinase activity on egg-yolk agar.

Immunization with temperature-sensitive mutants of Actinobacillus pleuropneumoniae induces protective hemolysin-neutralizing antibodies in mice.

Abstract. The immunogenicity and protective potential of three temperature-sensitive mutants of Actinobacillus pleuropneumoniae were evaluated in mice with respect to antibodies against the capsular polysaccharide, lipopolysaccharide, outer membrane proteins, and hemolysin protein. Antibodies to the capsular polysaccharide and lipopolysaccharide could not be correlated with protection in the mice; there were no significant differences among the anti-capsular and anti-lipopolysaccharide antibody titers regardless of the severity of infection. Sera from mice immunized with the mutants and challenged with the wild type contained antibodies that reacted in immunoblots to four major outer membrane proteins (66, 39, 29, and 16 kDa) regardless of the severity of infection after challenge. Both the tight and coaster mutants synthesized and secreted the 105-kDa hemolysin protein exotoxin in vitro and in vivo; hemolysin protein neutralization titers and the blotting intensity of the sera, however, varied inversely with the severity of infection. Sera from mice surviving challenge with little to no lung involvement stained the hemolysin band more intensely and had significantly higher neutralization titers (P < 0.05) than sera from mice that either died or survived with severe pulmonary hemorrhage. These results confirm the importance of the hemolysin in pathogenesis and the need for including it in any vaccine preparation.

Temperature-sensitive mutants ofStaphylococcus aureus: Isolation and preliminary characterization

Temperature-sensitive (ts) mutants ofStaphylococcus aureus were isolated after mutagenesis with nitrosoguanidine and two cycles of enrichment with Penicillin G and D-Cycloserine. The mutants expressed tight, coasting, and leaky phenotypes on solid media. In broth, however, most exhibited coasting for a limited number of generations. The reversion frequency of selected ts mutants was less than 10−6. Intraperitoneal (i.p.) immunization with ts mutant G/1/2 conferred significant protection (0 dead/6 total vs. 7/7, immunized vs. control; p=0.0006) from lethal i.p. challenge with the parental wild-type (wt)S. aureus suspended in 5% porcine mucin, performed 28 days after i.p. administration of 108 colony-forming units. Protection induced by mutants of coasting phenotype was higher and lasted longer than that induced by mutants of the tight phenotype. The results of this study demonstrate that ts mutants ofS. aureus can be obtained and that ts mutants are able to induce protective immunity from subsequent challenge with the parental wt strain.

A microassay for the quantitative measurement of egg yolk-reactive enzyme activity produced byPseudomonas cepacia

A newly developed microassay offers a sensitive method for quantitating egg yolk reactivity in culture supernatants and samples prepared during enzyme purification. Equal volumes of supernatant, saline, egg yolk suspended in saline, and buffer were incubated in microtiter wells at 37°C, and the resulting turbidity was measured quantitatively with an ELISA reader at 410 nm. The microassay was used to screen culture supernatants from nine clinical isolates ofPseudomonas cepacia, and the results were compared with those obtained when the isolates were screened on egg yolk agar. The microassay was also used to detect egg yolk reactivity in ammonium sulfate-precipitated fractions of the culture supernatant of one strain, Pc224c, and to determine which fraction of egg yolk contained the substrate for the activity.

Diffuse pollution in Oxford (Ohio, USA) watershed and performance of 'street sweeping' as a 'best management practice' (BMP).

Experimental results are described to evaluate the diffuse pollution profile according to land use in the catchments and street sweeping as a best management practice (BMP). We studied the variation of pollutant concentrations in outfalls discharging runoff from residential, commercial and high-traffic areas and in street sweeping. Pollution profiles varied with the land use in the catchments and seasons along with other factors such as rainfall intensity, construction works and street maintenance. Microbial indicator organisms were relatively high in all three outfalls. Heavy metal concentrations were low with lead (Pb) as the predominant heavy metal. The organic and solid contents were low but non-degradable and persistent. Relatively high quantities of pollutants were found in street sweeps in all catchments suggesting street sweeping as an effective measure to control diffuse pollution. Regular and frequent sweeping is important as a BMP.

Spontaneous temperature-sensitive mutants ofPseudomonas cepacia: Isolation and utilization

Pseudomonas cepacia strain 224c was subjected to treatment with two alternating cycles of penicillin andd-cycloserine in order to isolate spontaneous temperature-sensitive (ts) mutants. Of 150 surviving clones tested, ten were ts mutants; these were partially characterized, and one, Pcp-5, was chosen for experiments to determine the in vivo growth rate of the wild-type (wt) and clearance from the peritoneal cavities of mice. This mutant ceases growth almost immediately after transfer from 29°C to the nonpermissive (36°C) temperature and has a reversion rate of 10−8. In vivo experiments were performed by inoculating mixtures of Pcp-5 and wt into the peritoneal cavities of ICR mice. The ts mutant was cleared quantitatively (90% by 4 h), and the mean generation time (MGT) of the wt in the murine peritoneal cavity was 94 min.