Polysaccharide production in Pseudomonas cepacia

The effects of glucose, osmolarity, temperature and mode of growth on exopolysaccharide production in Pseudomonas cepacia was studied in batch culture using a chemically defined growth medium. Polymer production was maximal under conditions of a 2% (w v) glucose supplement, 0.4 ^m NaCl and an incubation temperature of 35 °C. In addition, polysaccharide composition and molecular weight varied with mode of growth. On agar culture there was a decrease in pyruvate and rhamnose content yet an increase in the amount of acetate compared to the polymer isolated from broth culture equivalents. The clincial implications of these results are discussed in relation to the potential pathogenicity of P. cepacia in cystic fibrosis patients.     

Fimbriation of Pseudomonas cepacia.

Fimbriae (pili) on the surface of bacteria have been suggested to facilitate adherence to mucosal epithelial surfaces. Three Pseudomonas cepacia cystic fibrosis isolates were screened for their ability to agglutinate erythrocytes (HA), a characteristic of some fimbrial types. One strain, designated PC103, was HA+, while another, PC109, was HA-. A fimbriated (f+) HA+ derivative of PC109 (PC2(13)) was selected by repeated erythrocyte adsorption. The two HA+ strains were shown by transmission electron microscopy to possess fimbriae which averaged 4.8 +/- 1.36 nm in width and 200 to greater than 2,100 nm in length (PCE2(13)) and 3.4 to 11.4 nm in diameter and 280 to 720 nm in length (PC103). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins prepared from PC103, PC109, and PCE2(13) indicated that the putative fimbrial subunit had a mass of 16 kDa. Western blot (immunoblot) analysis of sheared cell supernatants indicated that the 16-kDa subunit from PC103 and PCE2(13) reacted with antibody to the P. aeruginosa PAK pilin subunit. Southern blot analysis of a SalI digest of PC103 DNA showed DNA fragments which hybridized to P. aeruginosa PAK probes containing either the pilin structural gene (pilA) or the pilin accessory genes (pilB, -C, and -D) but not the conserved N-terminal region of pilA. A 15-kb band was common to both hybridizations, indicating that this fragment contains the PC103 fimbrial gene cluster. These results indicated the presence of homology between P. aeruginosa PAK and PC103 fimbriae but also suggested that the P. cepacia fimbriae are not type IV-like. The importance of fimbriae in adherence to A549 cells (type II pneumocytes) was assessed with PC109 (f-) and PCE2(13) (f+). PCE2(13) had an approximately 20-fold-higher level of adherence to A549 cells than PC109. This suggested that fimbriation of P. cepacia is associated with increased adherence in vitro.     

New serotypes of Pseudomonas cepacia

O and H serotyping of Pseudomonas cepacia has provided a suitable procedure for epidemiological studies. Our previous reports have described 7 O and 5 H antigens. The study of strains from another geographical origin led us to prepare antisera against those which could not be serotyped and thus to determine 2 new O and 2 new H specificities (O:8 and O:9, H:4 and H:8).     

Immunological nonidentity of Pseudomonas paucimobilis with Pseudomonas aeruginosa and Pseudomonas cepacia.

This investigation determined the serum agglutination activity and serum bactericidal response after rabbit immunization with Pseudomonas paucimobilis. Agglutination activity of antisera showed a twofold increase in titer from before immunization to 4 weeks post-immunization and peaked at 8 weeks post-immunization with a titer of 1:512. 2-Mercaptoethanol reduction of immunoglobulin M decreased agglutination titers. No major antigens were found to be common from crude antigen preparations of P. paucimobilis, Pseudomonas aeruginosa and Pseudomonas cepacia when tested with antisera to P. paucimobilis. Serum bactericidal activity was found in post-immunization antisera at 8 and 12 weeks against P. paucimobilis, with no activity present before immunization or at 4 weeks post-immunization. Antisera against P. paucimobilis showed no bactericidal activity against P. aeruginosa or P. cepacia.     

Pseudomonas cepacia septicemias: therapeutic difficulties

From Summer 1983 to Summer 1986, 34 cases of septicemia due to Pseudomonas cepacia could be detected in several intensive care units in the university hospital in Clermont-Ferrand (France). Intravascular catheters can be involved in the inoculation of this bacterial agent: a previous respiratory tract infection or a drained abscess can be the portal of entry of the bacteremia. Three patients died from the septicemia and the overall prognosis of the intensive care patients looks significatively worsened. The removing of the catheters and drains, the opening of an infected collection were useful but not sufficient to overcome. The choice of a good antibiotic was not easy; only ceftazidime, minocycline and cotrimoxazole have a fair activity in vitro. We only assessed the good results of ceftazidime. Pseudomonas cepacia is also resistant for many antiseptics. The large use of disinfecting procedures in intensive care units promotes the diffusion of this bacteria.     

O and H serotyping of Pseudomonas cepacia.

Procedures for the preparation, absorption, and titration of Pseudomonas cepacia O and H rabbit antisera are described. Seven O antigens (O1 to O7) for the slide agglutination test and five H antigens (H1, H3, H5, H6, and H7) for the agglutination and H immobilization tests were determined. Nearly 300 strains of P. cepacia isolated from hospitalized patients (a majority from Strasbourg hospitals) were serotyped. The use of P. cepacia serotyping as an epidemiological tool, especially in outbreak situations, was emphasized. Difficulties in obtaining monospecific H antisera are discussed.     

The importance of extracellular antigens in Pseudomonas cepacia infections

A clinical isolate of Pseudomonas cepacia from a cystic fibrosis patient was examined for its ability to produce extracellular toxic material. The organism was grown to stationary phase in a defined medium and toxic material was isolated by ultrafiltration, ion-exchange chromatography on DEAE-Sephacel and gel-filtration chromatography on Sepharose 4B. It consisted of a surface carbohydrate antigen, lipopolysaccharide and protein, and had an LD50 (when injected intraperitoneally into mice) of 395 +/- 20 micrograms. The toxicity appeared to be associated with the lipopolysaccharide portion of the complex, because boiling for 15 min and exposure to proteolytic enzymes had no effect on toxicity. However, saponification destroyed the toxicity of the compound. Studies employing radial immunodiffusion with the sera of mice infected with this organism demonstrated production of the complex in vivo at levels approaching those sufficient to produce death. When sublethal amounts of this complex were placed in the lungs of specific-pathogen-free rats, the lung pathology observed after 12, 24, 36 and 48 h was extensive. However, antibody generated in rabbits against this material could protect mice against the complex, as well as against challenge by the homologous organism. These data indicate that extracellular toxic material produced by P. cepacia may be responsible for the lethality and lung tissue destruction normally associated with an active pneumonia caused by this organism.     

Pyrimidine metabolism in Giardia lamblia trophozoites

The pyrimidine metabolism of Giardia lamblia trophozoites (Portland I strain) was studied using whole trophozoites and trophozoite homogenates. Pyrimidines and pyrimidine nucleosides were readily incorporated into nucleic acids. Orotic and aspartic acid incorporations were below the level of detection. Enzymes of the pyrimidine salvage pathway (i.e., thymidine and uridine phosphorylases and thymidine and uridine kinases) were detected in trophozoite homogenates, but the activities of de novo pyrimidine synthesis enzymes (i.e., carbamoyl-phosphate synthase, aspartate transcarbamoylase, dihydroorotase and dihydroorotate dehydrogenase) were below the level of detection in these same homogenates. The evidence presented supports the conclusion that G. lamblia trophozoites appear incapable of synthesizing pyrimidines de novo but are capable of salvaging preformed pyrimidines and pyrimidine nucleosides from the growth medium and the enzymes of this pyrimidine salvage pathway are not organelle associated.     

Serological classification of Pseudomonas cepacia by somatic antigen

Ten samples of antiserum against Pseudomonas cepacia were prepared by the intravenous immunization of rabbits with heat-killed organisms. Ten P. cepacia strains used for immunization were proven unique antigenic strains. Using these antisera, we serogrouped 127 strains of P. cepacia, and 114 strains (89.8%) fell under one of the ten serogroup. The most prevalent serogroup was C (26.8), the second most prevalent being D (18.1%). When we compared our serogroups with the serogroups of Monteil et al. (H. Monteil, C. Richard, and A. Heidt, Med. Mal. Infect. 11:544-547, 1981) and Heidt et al. (A. Heidt, H. Monteil, and C. Richard, J. Clin. Microbiol. 18:738-740, 1983), five out of seven of their serogroups were represented by our antisera.     

Effect of pyochelin on Pseudomonas cepacia respiratory infections

Exogenously supplied pyochelin influenced the virulence of Pseudomonas cepacia pyochelin-negative strains in a chronic pulmonary infection model in rats. Groups of rats were inoculated transtracheally with agar beads containing P. cepacia or P. aeruginosa strains, saturated with either pyochelin or PBS. Supplementation of the inocula with pyochelin had no effect on the number of bacteria recovered from the lungs. The availability of pyochelin, however, increased the degree of pathology observed in lungs infected with pyochelin-negative strains of P. cepacia. The area of pathological involvement in the lung was about 2-fold larger, when pyochelin was present. Inclusion of pyochelin in the inoculum had no effect on the degree of pathology observed in lungs infected with a pyochelin-positive P. aeruginosa strain. Pyochelin was shown to stimulate in vitro growth of P. cepacia, but it had no effect on production of lipase or protease, factors which may be involved in P. cepacia virulence. These studies support our hypothesis that pyochelin may be important for dissemination in P. cepacia infections.     

Typing of Pseudomonas cepacia by bacteriocin susceptibility and production.

The significance of Pseudomonas cepacia as an opportunistic pathogen in immunocompromised patients has become increasingly recognized. Particularly disturbing is its increased incidence, reported by several North American centers, in respiratory tract cultures from patients with cystic fibrosis. Epidemiological studies of P. cepacia have been hampered by a lack of typing methods. In this paper we report the development of a typing scheme based on bacteriocin production and susceptibility. For bacteriocin production, test isolates of P. cepacia were rapidly applied to the surfaces of agar plates with a multiple inoculator. After incubation of these test isolates for 5.5 h and their exposure to chloroform, indicator strains were applied in agar overlays without prior removal of the test strain growth. After 18 h of incubation, inhibition zones caused by bacteriocin activity were recognized. A similar procedure was used to examine the bacteriocin susceptibility of the test strain. The bacteriocin type of the test strain was defined based on its bacteriocin production as judged by zones of inhibition against a set of eight indicator strains and by susceptibility or resistance of the test strain to bacteriocin produced by six producer strains. Of 373 strains of P. cepacia, 95.2% were typed into a total of 44 type combinations. Bacteriocin typing provided a suitable procedure for epidemiological studies of colonization or infection by P. cepacia. The technique described in this paper was simple to perform, gave a result within 24 h, provided good strain discrimination, and was suitable for clinical, environmental, and phytopathogenic strains.     

Characterization of hemolysin in extracellular products of Pseudomonas cepacia.

Pseudomonas cepacia is recognized as an opportunistic pathogen in immunocompromised patients. We screened 120 strains of P. cepacia isolated from clinical specimens for production of extracellular products. About 70% of these strains produced lipase, protease, and lecithinase, but only 4% produced hemolysin. A hemolysin produced by P. cepacia JN106 was characterized. The hemolysin was most active against human erythrocytes. Horse, sheep, chicken, and rabbit erythrocytes were also susceptible. The hemolysin was heat labile and was inhibited by sterols but was not activated by 2-mercaptoethanol and dithiothreitol. Four hemolysin-negative mutants obtained by N-methyl-N'-nitro-N-nitrosoguanidine treatment produced the other extracellular products. A 58-kilobase-pair plasmid found in the parent strain was also found in the mutant strains, suggesting that the hemolysin gene resides on the chromosome.     

Pseudomonas cepacia adherence to respiratory epithelial cells is enhanced by Pseudomonas aeruginosa.

Pseudomonas aeruginosa and Pseudomonas cepacia are both opportunistic pathogens of patients with cystic fibrosis. The binding characteristics of these two species were compared to determine if they use similar mechanisms to adhere to respiratory epithelial cells. P. cepacia 249 was shown to be piliated, but there was no detectable homology between P. aeruginosa pilin gene probes and P. cepacia genomic DNA. P. cepacia and P. aeruginosa did not appear to compete for epithelial receptors. In the presence of purified P. aeruginosa pili, the adherence of 35S-labeled strain 249 to respiratory epithelial monolayers was unaffected, while that of P. aeruginosa PAO1 was decreased by 55%. The binding of P. cepacia 249 and 715j was increased by 2.4-fold and 1.5-fold, respectively, in the presence of an equal inoculum of PAO1. Interbacterial agglutination contributed to the increased adherence of P. cepacia, as the binding of 249 was increased twofold in the presence of irradiated PAO1. PAO1 exoproducts had a marked effect in enhancing the ability of the P. cepacia strains to adhere to the epithelial monolayers. A PAO1 supernatant increased the binding of 249 by eightfold and that of 715j by fourfold. Thus, there appears to be a synergistic relationship between P. aeruginosa and P. cepacia in which PAO1 exoproducts modify the epithelial cell surface, exposing receptors and facilitating increased P. cepacia attachment.     

A novel mucin-sulphatase activity found in Burkholderia cepacia and Pseudomonas aeruginosa.

Lung infections due to Burkholderia cepacia and Pseudomonas aeruginosa in patients with cystic fibrosis (CF) are common, are associated with respiratory morbidity and are a cause of mortality. Respiratory mucin in CF patients is highly sulphated, which increases its resistance to bacterial degradation. Desulphation increases the susceptibility of mucin to degradation by bacterial glycosidases and proteinases, and subsequent deglycosylation may facilitate bacterial colonisation by increasing available substrates and binding sites. This study determined whether clinical and environmental strains of B. cepacia and P. aeruginosa had the ability to desulphate mucin. Mucin-sulphatase activity was tested by incubating bacterial cell suspensions with 35S-sulphated mucins purified from LS174T and HT29-MTX human colon carcinoma cell lines. These mucins were also used to test for differences in substrate specificities. Mucin-sulphatase activity was detected in all nine B. cepacia strains and in four of six P. aeruginosa strains. There was strain variability in the level of mucin-sulphatase activity. Aryl-sulphatase activities of Pseudomonas isolates (determined with methylumbelliferyl sulphate) were c. 20-fold higher than those of B. cepacia strains, and were independent of mucin-sulphatase activity. This is the first report to demonstrate desulphation of mucin by B. cepacia and P. aeruginosa. It is concluded that B. cepacia and P. aeruginosa produce one or more cell-bound glycosulphatase(s), in addition to aryl-sulphatase activity. Mucin-sulphatase activity of B. cepacia and P. aeruginosa may contribute to their association with airway infections in patients with cystic fibrosis.     

Cervical osteomyelitis caused by Pseudomonas cepacia in an intravenous-drug abuser.

We report a case of vertebral osteomyelitis caused by Pseudomonas cepacia in a patient with a history of intravenous-drug abuse. P. cepacia infections are usually nosocomial, although community-acquired infections occur more commonly in intravenous-drug abusers than in the general population. Vertebral osteomyelitis caused by P. cepacia has not previously been reported.     

Association of Pseudomonas cepacia with chronic granulomatous disease.

Pseudomonas cepacia was recovered from a number of infected sites in three patients with chronic granulomatous disease of childhood. The organisms were identified on the basis of their oxidative utilization of a variety of carbohydrates and their positive beta-galactosidase and oxidase activities. They were resistant to most antimicrobial agents and moderately susceptible to chloramphenicol. Peripheral blood leukocytes isolated from two siblings with chronic granulomatous disease, including one of the patients in this series, failed to kill P. cepacia in vitro. Prolonged prophylactic and antimicrobial therapy may well have played a significant role in the colonization and infection of these patients with P. cepacia.     

Isolation of a novel siderophore from Pseudomonas cepacia.

A novel iron-binding compound was identified in ethyl acetate extracts of the supernates from Pseudomonas cepacia cultures. This compound, named azurechelin, was produced by 88% of P. cepacia strains isolated from the respiratory tract. Production of azurechelin was regulated by the iron concentration in the culture medium. Azurechelin enhanced the growth of P. cepacia in a medium containing transferrin 200 mg/L. Azurechelin released iron from transferrin in an equilibrium dialysis assay, suggesting that it could complete with transferrin for iron. Azurechelin could also stimulate iron uptake by P. cepacia. This siderophore appeared to have a novel structure with neither the typical characteristics of catechol nor of hydroxamate compounds.     

Purification and characterization of an extracellular protease from Pseudomonas cepacia.

An extracellular proteinase (PSCP) produced by Pseudomonas cepacia was purified from culture supernatants by ammonium sulfate precipitation, anion exchange chromatography on DEAE-Sephacel, and G200 gel filtration chromatography. The protease has an apparent Mr of 34,000 by electrophoresis. Substrates cleaved by the protease include gelatin, hide powder, and collagen but not human immunoglobulin G (IgG), IgM, secretory IgA, or IgA. The enzyme had the characteristics of a metalloprotease, a pH optimum of 6, and a temperature optimum of 45 degrees C. Intratracheal instillation of purified PSCP into rat lungs produced a bronchopneumonia characterized by polymorphonuclear cell infiltration and proteinaceous exudation into large airways. Rats responded immunologically to active immunization with PSCP, but this response was not protective against subsequent lung infection with P. cepacia. PSCP was shown to have antigenic similarity with Pseudomonas aeruginosa elastase by an immunoblotting technique. Sera from 10 cystic fibrosis patients, with and without a previous history of P. cepacia colonization, were shown to possess antibody reactive against PSCP.