A pilot study for monitoring changes in the microbiological component of metalworking fluids as a function of time and use in the system.

This article describes the results of a pilot study to examine changes in the biological component of metalworking fluids (MWF) as a function of use. Fluid samples were taken from two newly charged systems, designated BT-7415 and BT-7707, at 1-week intervals for 8 weeks and characterized with respect to the kinds and numbers of bacteria present and presence of soluble protein in cell-free supernatants. In addition, lipid extracts of pelleted cells from fluids in BT-7415 were examined by gas chromatography/mass spectroscopy for the kinds and relative amounts of phospholipid fatty acids (PLFA) present. A total of 19 different bacterial species was cultured and identified, more than half (12/19) of which were gram-negative. Total colony-forming units (CFU) reached levels of 2.2 x 10(3)/mL in BT-7415 and 2.4 x 10(5)/mL in BT-7707. The most common genus isolated was Pseudomonas. Estimations of cell numbers based on total biomass from PLFA in samples from BT-7415 indicated 1.1 x 10(7)/mL after 8 weeks of use. Both the numbers of PLFA identified and the amounts of each detected in BT-7415 increased as the fluids were used. The chromatograms were dominated by two fatty acids, the amounts of which increased with time. These fatty acids, 18:2 omega 6 and 18:1 omega 9c, are not commonly associated with pseudomonads. This suggests that there is an important component of the biological consortium in MWF is not being detected by currently used culture techniques. There was no soluble protein detected in any of the samples from either system.

Inhibition of rat alveolar macrophage phagocytic function by a Pseudomonas cepacia lipase.

The effects of purified Pseudomonas cepacia lipase on rat pulmonary alveolar function and morphology were examined. Lipase (2.5-20 micrograms/ml) adversely effected the phagocytic function of rat pulmonary alveolar macrophages in a dose-dependent manner. The lipase itself was not directly cytotoxic to these cells. Alveolar macrophages, in the absence of lipase, phagocytosed c. 35% of a given population of opsonised P. cepacia in 30 min when the ratio of bacteria:phagocyte was 10:1. Phagocytosis of P. cepacia by rat pulmonary alveolar macrophages was significantly reduced when the cells were either pre-incubated with the lipase or when phagocytosis occurred in the presence of the lipase. This was confirmed by transmission electronmicroscopy. These functional changes were associated with marked alterations of the macrophage morphology. Scanning electronmicroscopy showed that macrophages exposed to the P. cepacia lipase had fewer specialised surface structures and did not spread on plastic surfaces as well as untreated macrophages. The effects of the lipase were lost after heat inactivation, which indicates that the effects of the P. cepacia lipase were due to its enzymic activity. These results suggest that, if sufficient quantities of the enzyme are produced in vivo, lipase may be an important virulence factor for P. cepacia, allowing the organism to evade phagocytic cells.

The effects of purified 25-kDa lipase from a clinical isolate of Pseudomonas cepacia in the lungs of rats.

Nanogram quantities of a 25-kDa lipase purified from culture supernatants of Pseudomonas cepacia 90ee, a sputum isolate from a cystic fibrosis (CF) patient, were placed in the lungs of healthy rats. The resulting pathological changes included large amounts of proteinaceous exudate, the accumulation of polymorphonuclear leukocytes and red blood cells, and disorganization of alveolar structure. Pseudomonas cepacia 90ee immobilized in agar beads was also placed in the lungs of rats in a model of chronic infection. This resulted in bronchopneumonia and a milder inflammatory response than that elicited by the purified enzyme.

3-deoxy-D-manno-2-octulosonic acid in the lipopolysaccharide of various strains of Pseudomonas cepacia.

Six clinical isolates of Pseudomonas cepacia (representing the five serotypes of the organism) were examined for the presence of 3-deoxy-D-manno-2-octulosonic acid (KDO) in their lipopolysaccharide (LPS). Purified LPS was examined for the presence of KDO by the thiobarbituric acid (TBA) assay and by gas chromatography. All strains possessed KDO. One strain possessed KDO that was detectable by the TBA assay after mild acid hydrolysis with 0.04 M H2SO4 at 100 degrees C for 20 min. The other strains also possessed KDO but it was only demonstrable by the TBA assay after strong acid hydrolysis (4 M HCl for 60 min at 100 degrees C). All six purified LPS preparations were shown to possess KDO by two separate gas chromatography procedures. LPS isolated from the six strains of P. cepacia was toxic for mice.

Production of an extracellular toxic complex by various strains of Pseudomonas cepacia.

Six isolates of Pseudomonas cepacia, representing various serotypes of the organism and possessing similar degrees of virulence in mice, were examined for their production of an extracellular toxic complex (ETC) in vitro. This compound is lethal for mice and produces extensive lung pathology in rats; it is composed of a surface carbohydrate antigen, lipopolysaccharide and protein. All six isolates produced the ETC. The LD50 values for the six ETC preparations ranged from 395 micrograms for strain 61g to 1750 micrograms for strain 90ee. Only two of the six ETC preparations contained ketodeoxyoctonate detectable by the methods used, and these two were the most toxic. Rabbit antiserum to the ETC of a serotype D strain could significantly protect mice only against serotype D strains. Examination of the various phases of growth of P. cepacia showed that there was extracellular release of the ETC beginning in the early logarithmic phase and continuing through the late stationary phase. The presence of the ETC in the supernatant fluids was due to release of this material rather than to cell lysis. In addition, at least one strain of P. cepacia was shown to produce an alginic acid-like compound.

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.

Production of lipase by clinical isolates of Pseudomonas cepacia.

Ten clinical isolates of Pseudomonas cepacia from the sputum of cystic fibrosis patients were examined for the ability to produce lipase. Lipase substrates used included egg yolk agar, four different polyoxyethylene sorbitans (Tweens), and p-nitrophenylphosphorylcholine, a chromogenic substrate used to assay for phospholipase C. Lipase activity was detected in the filtrates of organisms grown to the exponential phase in either tryptose minimal medium or chemically defined medium. Lipase activity increased in the filtrates if the cultures were allowed to proceed into the stationary phase. None of the isolates produced phospholipase C. Lipase activity on Tween 20 ranged from 41.6 X 10(-3) to 640.0 X 10(-3) U/micrograms of protein. The activity was similar or slightly lower when Tween 40, 60, or 80 was used as the substrate. There was no correlation between lipase activity on Tween and that demonstrated on egg yolk agar. Lipase activity increased as pH increased from 7.0 to 9.0. Boiling for 5 min resulted in 66% loss of enzyme activity. The remaining activity continued to decrease with increasing boiling time. The enzyme was purified by gel filtration on Sephadex G-200, and the resultant preparation, when subjected to polyacrylamide gel electrophoresis, resulted in a single protein band (molecular weight, approximately 25,000) from which lipase activity could be eluted. The purified lipase was not cytotoxic to HeLa cells, nor was it toxic when injected intravenously into mice.