The Role of Tris in EDTA Toxicity and Lysozyme Lysis

Abstract

S U M M A R YTris and EDTA combined to form a homologue which has far greater chelating ability than EDTA alone. The weaker serine and NH,Cl homologues of EDTA replaced EDTA+tris in causing toxicity and permitting lysozyme lysis in Azotobacter vinelandii. Serine could effectively substitute for tris in these systems for Pseudomonas aeruginosa. These compounds had relatively little effect on Escherichia coli. The toxicity of EDTA+tris for these organisms could be alleviated by pre-incubation in physiological saline (0.15 M-NaCl). Subsequent lysozyme lysis was prevented by this treatment. The removal of metals from A . vinelandii cysts by EDTA+tris was also inhibited. The EDTA + tris + metallo chelate complexes were inactivated (dissociated) by saline. I N T R O D U C T I O NMany Gram-negative organisms which are resistant to lysozyme will lyse when ethylenediaminetetra-acetic acid (EDTA) and tris buffer (2-amino-2-hydroxymethylpropane-lY3-diol) are also present in addition to lysozyme (Repaske, 1956(Repaske, , 1958 McQuillen, 1960 a). Repaske first established a requirement for these compounds in this lytic system but did not determine the toxicity of the individual components. Socolofsky & Wyss (1961) were able to rupture cysts of Azotobacter vinelandii by using EDTA and tris. The released ' central bodies ' were not viable and were lysed by adding lysozyme. Goldschmidt & Wyss (1 966 : to be published), while studying this toxic effect in greater detail, observed that the EDTA+ tris system was toxic to vegetative organisms as well as to cysts even under conditions where cyst rupture did not occur (i.e. in 0.15 M-NaCl). EDTA suspended in water or 0.025 M-NaCl also ruptured the cyst exine, but the central bodies remained viable under these conditions. They also found that various other substances such as amines, amino acids and NH4C1 could replace tris in producing toxicity in the presence of EDTA; these compounds also substituted for tris in the tris+EDTA+lysozyme system. These results are presented in the present paper. During this work, other bacteria were tested for their sensitivity to the EDTA+ tris system (Goldschmidt, Goldschmidt & Wyss, 1967). Repaske (1958) noted that Escherichia coli was sensitive to his lytic system. Goldschmidt et al. (1967) found that male strains of E. coli (Hfr, male) were sensitive to the EDTA+ tris system (65-95 % kill within 4 min.) while female strains and E. coli B/r (which is ' F" ' and does not mate) were not killed. There are many reports in the literature discussing differ-* Present address :

Antimicrobial effect of combinations of EDTA-Tris and amikacin or neomycin on the microorganisms associated with otitis externa in dogs

“…Tris buffer enhances the effects of EDTA (Goldschmidt and Wyss, 1967). Gram-negative bacteria exposed to EDTA-Tris have increased permeability to extracellular solutes and leakage of intracellular solutes (Leive, 1968); are more sensitive to lysozyme, bactericides and antibiotics (Brown and Richards, 1965;Monkhouse and Graves, 1967;Russel, 1967;Wooley and Jones, 1983); and release periplasmic enzymes, cell membrane-associated proteins (Heppel, 1972) and lipopolysaccharide, protein, phospholipids and divalent cations from their cell walls .…”

Simple additives to increase the activity of chlorhexidine digluconate against urinary pathogens

“…This work was prompted by reports that EDT A could enhance the action of antiseptics (Brown & Richards, 1965;Caplin & Chapman, 1976;Goldschmidt et at., 1972) and that Tris increased the activity of EDTA (Voss, 1967;Goldschmidt and Wyss, 1967). It was hoped that the addition of these agents would enable the concentration of chlorhexidine to be reduced so that it was less erosive and also overcome any resistance that has been previously reported by Stickler (1974).…”

Studies on the Cell Walls of Pseudomonas Species Resistant to Ethylenediaminetetra-acetic Acid

“…Although EDTA has been found to extract lipopolysaccharide from Escherichia coli, the bacteria remained viable under the conditions used (Leive, 1965). Except when used in conjunction with organic cations (Wolin, 1966;Goldschmidt & Wyss, 1967;Voss, 1967), EDTA does not seem to be highly toxic for Gram-negative bacteria other than pseudomonads (Gray & Wilkinson, 1965 a;Wilkinson, 1967). This hypersensitivity to EDTA might prove to be a useful characteristic in the taxonomy of the pseudomonads (Shively & Hartsell, 1964;Wilkinson, 1967 (1956) without prior hydrolysis of samples, and was expressed as glucose.…”