A strain of Pseudomonas cepacia that survived for 14 years (1963 to 1977) as a contaminant in an inorganic salt solution which contained commercial 0.05% benzalkonium chloride (CBC) as an antimicrobial preservative, was compared to a recent clinical isolate of P. cepacia. Ammonium acetate was present in the concentrated stock CBC solution, and served as a carbon and nitrogen source for growth when carried over into the salts solution with the CBC. The isolate's resistance to pure benzalkonium chloride was increased step-wise to a concentration of 16%. Plate counts showed 4 x 10(3) colony-forming units per ml in the salts solution. Comparison of growth rates, mouse virulence, antibiotics resistance spectra, and substrate requirements disclosed no differences between the contaminant and a recently isolated clinical strain of P. cepacia. The results indicate that it is critical that pharmaceutical solutions containing benzalkonium chloride as an antimicrobial preservative be formulated without extraneous carbon and nitrogen sources or be preserved with additional antimicrobial agents.
Resistant cells of
Pseudomonas aeruginosa
and a waterborne
Pseudomonas
sp. (strain Z-R) were able to multiply in nitrogen-free minimal salts solution containing various concentrations of commercially prepared, ammonium acetate-buffered benzalkonium chloride (CBC), a potent antimicrobial agent. As the CBC concentration increased, growth increased until a point was reached at which the extent of growth leveled off or was completely depressed. Minimal salts solutions of pure benzalkonium chloride (PBC) containing no ammonium acetate did not support bacterial growth. When ammonium acetate was added to PBC solutions in the same concentrations found in CBC solutions, growth patterns developed that were comparable to those found with CBC. Likewise, (NH
4
)
2
SO
4
added to PBC solutions supported growth of both organisms.
P. aeruginosa
was initially resistant to CBC levels of 0.02% and it was adapted to tolerate levels as high as 0.36%. Strain Z-R was naturally resistant to 0.4% CBC. Since ammonium acetate, carried over by the CBC used in drug formulations and disinfectant solutions, has the potential to support the growth of resistant bacteria and thus make possible the risk of serious infection, it is suggested that regulations allowing the presence of ammonium acetate in CBC solution be reconsidered.
Cells of Pseudomonas aeruginosa resistant to benzalkonium chloride (BC) underwent unique ultrastructural reorganizations when they were grown in the presence of 1 mg of BC/ml. The resistant cells usually contained a single, centrally positioned pseudovacuole. The pseudovacuole was surrounded by a diffuse substance that spread irregularly throughout the cytoplasm. The presence of the pseudovacuole seemed to cause a physical compartmentalization of the cytoplasm into random pockets of ribosomes and nuclear material. Contained within the pseudovacuole was a horseshoe-shaped, electron-dense body which was bounded by a trilaminar membrane 5.2 nm in width. These bodies averaged 77 nm when measured through the long axis. The surfaces of resistant cells were covered by an additional layer not found in sensitive cells. Thin sections of sensitive cells which had been treated with 1 mg of BC/ml showed little or no lysis. The cytoplasm appeared to be deeply stained and coagulated. Ribosomes were no longer distinctly visible. Although the cell wall remained intact, the cell membrane was dissolved and fragmented. BC-grown resistant cells could not be successfully stained by standard techniques; however, details were demonstrated with the aid of a combination of 1.5% glutaraldehyde, 1% osmium tetroxide, and 1% phosphotungstic acid prepared in 0.1 M sodium dimethylarsonate buffer (pH 6.8).
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