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A study investigating the role of tris buffer and organic cations in modifying the Gram-negative cell wall and enhancing the bactericidal effect of EDTA and HAPS. The study reveals that tris buffer plays an active role in the process by acting as an organic cation, and that EDTA and DDA appear to remove surface components of the Gram-negative cell wall. The document also discusses the effects of various organic cations on cell walls and the bactericidal activity of EDTA and HAPS against Escherichia coli.
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J. gen. Microbiol. (1967), 48, 391- Printed in Great Britain
Effects of Organic Cations on the Gram-negative Cell Wall and Their Bactericidal Activity with Ethylenediamine- tetra-acetate and Surface Active Agents
S U M M A R Y When Gram-negative cell walls were damaged by lysozyme and ethylene- diaminetetra-acetic acid (EDTA) in tris buffer, tris played an active role through its action as an organic cation. Other organic cations such as aliphatic amines and quaternary ammonium compounds were more effective than tris in modifying the cell wall and making it permeable to other solutes.
modified the cell wall of Escherichia coli so as to permit a more than 99.99 %
ineffective. Four other Gram-negative species were shown to be similarly susceptible to killing by this system. Together, EDTA and DDA appeared to remove surface components of the Gram-negative cell wall. Alone, DDA and other organic cations removed somatic antigens from the cell wall.
I N T R O D U C T I O N
cationic surface active agents, but are usually unaffected by those anionic and zwit- terionic agents which are active against Gram-positive organisms. It was suggested by Voss (1963) that the greater resistance of Gram-negative species may be due to the greater complexity of their cell walls, which exclude the agent from the interior of the cell. The Gram-negative cell wall may be so modified by treatment with EDTA in tris buffer at pH 8 as to perinit conversion of the cell by lysozyme to osmotically fragile
coli modified by treatment with EDTA and tris buffer, it was observed that such organisms may be killed by treatment with zwitterionic agents to which the organisms are normally resistant. As in the case of lysis by lysozyme, killing by the agent indicates changes induced in the cell wall by EDTA and tris buffer. Further study revealed that tris is not a physiologically inert buffer, but plays a specific role as an organic cation in modifying the permeability of the cell wall; other organic cations are much more effectivethan tris. Elucidation of the role of the organic cation forms the substance of this report.
METHODS
from the American Type Culture Collection with no discernible difference in results.
ammonia)-propane- I -sulphonate (HAPS), was prepared and purified in our labora- tories, and sterilized as a 0. r % solution in 0.85 % NaCl; it is much less soluble in water
coconut amine' (Armour). Some amines were derived from coconut or soybean oil, and thus contain a mixture of alkyl chains. Other compounds which were not available commercially were synthesized in our laboratories. Fatty amines were dissolved in
and plated on nutrient agar overlaid with plain agar to inhibit spreading. Exposure to
with EDTA, HAPS, dodecyltrimethylammoniumbromide (DTAB), or alkyldimethyl-
Na,CO, IOO pg./ml. Organisms were removed by centrifugation for 20 min. at 5000g. The supernatant fluids were tested for antigenic activity in double diffusion tests in
R E S U L T S
Tris was more effective than other buffers (e.g. phosphate buffer) in promoting the
compound, tris(hydroxymethy1) nitromethane, was inactive, indicating that it is the free amino group of tris buffer which affects the cell wall. Table I shows the results of a
molecular weight, more or less closely related to tris, were found to possess similar
mixture increased markedly. Concentrations of the amine buffers were decreased as activity increased ; therefore sodium triphosphate (Na,P,O,,) I 00 pg./ml. was added to maintain at pH 8.5-9-0. Maximum activity was reached with amines containing
methyl groups had little effect. Amines with two alkyl chains were less active. Such
Survival (%)
Test compound N,N-Dimethyldodecylamine (DDA) Decyl methyl sulphoxide Hexadecyl methyl sulphoxide Dodecyl methyl sulphone Dodecyl methyl sulphide Dodecyldimethylsulphoxonium methosulphate Dodecyldimethylsulphonium iodide 3-Tridecylp yridine Dodecyltrimethylammonium bromide Cetyltrimethylammonium bromide Cetylpyridinium chloride Alkyldimethylbenzylammonium chloride (Roccal) 2-Dodecyl- I , 3-bis-(trimethylammonio)- propane dibromide Dimethyldodecyl-3-(trimethylammonio)- propylammonium dibromide I -Dodecyl-2-imino-imidazolidine I ,2-Bis-(dimethylamino)-dodecane 2-Chlorodimethyldodecylamine
I Complete system 0- _> 6
6 6 6_ 0.008 I 0. > 6 0 ' 0 I 0. 0'0 I g 0.
0. 0- 0. 0.00 I 7 0'1 I
Test compound
38 68
32 88 78 0-
43 59 30 70 54
related compounds as coconut alkyl ethanolamine or the amides of coconut fatty acids were ineffective, while other compounds such as COCO- I ,3-propylene diamine and dodecyl piperidine were highly active. The increasing activity of the aliphatic amines with EDTA and HAPS was not due to the increasing bactericidal activity of the amhes alone as the length of the alkyl chain grew; the active amines effectively killed E. coli, in the presence of EDTA and HAPS, at concentrations at which the amines alone possessed only slight bactericidal activity. A further series of organic cations and related compounds, including DDA, is compared in Table 2. In this case, the compounds were tested in the presence of EDTA IOO ,ug./ml., HAPS, 20 ,ug./ml. and Na2C0, IOO pg./ml. to buffer the system at pH 10. Na,CO, was used instead of sodium triphosphate as a buffer to avoid combining the chelating effects of EDTA and triphosphate. Four non-ionic alkyl sulphur compounds were ineffective in increasing bactericidal activity, but two cationic sulphonium and sulphoxonium compounds were highly effective, as were a number of quaternary ammonium compounds and additional amines. It is evident that there was no specific
tericidal activity in the presence of EDTA, HAPS and an alkaline buffer.
pH 10. EDTA was used at IOO ,ug./ml., at which concentration it was present in excess; decrease to 10 pg./ml. caused no significant loss of activity. At 20 ,ug./ml. and above, HAPS alone was not appreciably bactericidal for E. coli; however, increasing the con- centration in the presence of the other components of the system increased killing, and decreasing the concentration decreased the killing. The concentration of Na,CO, was not critical. A number of alkaline buffers gave similar results in the range pH 9-10. EDTA could be replaced by a number of other chelating agents; however, only
effective as EDTA.
EDTA DDA Na,CO, HAPS (Pug.lml.) (Pug. 1mlJ (iuug.lm1.) (iug.lml.) Survival (%) I I 0 I I
2' 2' 2' 2'
0
I I I 0 I
20 20 20 20 0
0~ 42 I 8
34
0'
HAPS, which is regarded as the actual lethal agent in the bactericidal system through
could be replaced by other zwitterionic agents such as betaines, or by cationic quater- nary ammonium compounds. Many of the latter are themselves highly bactericidal, and perhaps fulfil the function of organic cation and surface active agent simul- taneously. Anionic and non-ionic surface active agents were ineffective as replace- ments for HAPS. The fact that each component of the bactericidal system containing EDTA, DDA, HAPS and Na,CO, was required for full activity is indicated by the data in Table 3. Omission of EDTA, DDA, or HAPS caused a sharp decrease in the killing of
The bactericidal action of the components of the EDTA +DDA +Na,CO, + HAPS
to one or two components under the conditions of the bactericidal test, removing the organisms by centrifugation, and then exposing them to the remainder of the system. The results of this study (Table 4) implied that EDTA and DDA acted jointly to exert a non-lethal effect on the cell wall, which thus became more permeable to HAPS. The surface active agent penetrates the wall and causes death of the organism, pre- sumably by damaging the cytoplasmic membrane. The role of Na,CO, was relatively non-specific, though it appeared to increase the effect of DDA more than that of
the usual concentration were exposed to the EDTA + DDA +Na,CO, +HAPS system for 10 min. at 37O. The N and P content of the supernatant fluid after centri- fugation was compared with that of a control suspension of organisms. It was found that 4-5 ,ug.N/ml. and 0.8 ,ug.P/ml. were released on killing the organisms. Ultraviolet ab- sorption curves showeda peak at 260 mp in the supernatant fluid of killed organisms,with an optical density of 0-2-0-3. Calculations based on adenine ribonucleotide as a model system indicated that this quantity of nucleic acid would yield about 0.4-0.6 ,ug.P/ml.
3
2 0 1 2 3 4 5 6 7 8 9 10 Time (min.) Fig. I. Survival of Escherichia coli on exposure at 37" to EDTA (100 pg./ml.)+DDA (2-5 ,ug./ml.) + Na,CO, ( IOO ,ug./ml.) + HAPS (20 pg./ml.).
Decrease in special density EDTA DDA tris Na,CO, lysozyme pH (660 mp) Viscosity? at (mg.1 fmg.) (mg.) (mg.) (mg.) value at 30min. 30min. 1'0 0 ' 2 I 0 ' 2 0. 1 7-8 0.21 + +
1'0 I 2 0 ' 1 8.1 0'20 + + + + 1'0 2.4 0'1 7'5 0.04^ - 1 ' 0 0'2 I 0'2 8.0 0'12 - 1.0 48 8.1 0 ' 1 I + +
1 ' 0 48 0-I 8.1 0.30 ++++
be expected from the increased viscosity which appeared on lysis of the suspensions. This viscosity was decreased rapidly by adding a trace of deoxyribonuclease. That the bactericidal activity of the EDTA + DDA +Na2C0, + HAPS system was
26 G. Microb. 48
negative species were also killed by this system. At the concentrations used, none of the
outermost layer of Vi antigen by some strains of Salmonella typhi and by the V dis- sociant of E. freundii appeared to confer some slight resistance to the killing effect.
Table 6. Bactericidal activity of a mixture of EDTA ( I 00 ,ug./ml.) + DDA ( 2 3 ,ug./ml) + Na,CO, ( I oo,ug./ml.) -k HAPS (20 ,ug./ml.) against organisms other than Escherichia coli, and the eflect of Vi antigen on survival Test organism Vi antigen % Survival Proteus mirabilis Pseudomanas aeruginosa Escherichia freundii (BALLERUP 107 w) E. freundii (BALLERUP I 07 v) Salmonella typhi 0 90 I S. typhi T Y ~ V S. typhi 9gg2.v S. typhi 12839
0. 0.
S. typhi. Removal of somatic antigens by organic cations
bromide (DTAB), alkyldimethylbenzylammonium chloride (Roccal), DDA, EDTA or
content of somatic antigens in these extracts was compared by the double diffusion method in agar, using rabbit antisera prepared against whole organisms. Comparison of the zones of precipitation showed that antigens were removed by DDA 50 ,ug./ml. and even more effectively by the other two cationic agents, DTAB and Roccal. Extraction by DDA 10 ,ug./d. was less effective, but still evident. Evidence of reaction consisted primarily of a broad, diffuse zone of precipitate; in some cases minor bands of precipitate also indicated the presence of two slower-moving antigenic components. Na,CO, had no perceptible effect on the extraction of antigens by the cations;
cell wall. D I S C U S S I O N
cell wall of Escherichia coli is lipoprotein, the somatic antigens which are responsible for specific agglutination of Gram-negative bacteria are known to be polysaccharide or
extension of somatic antigen for some distance beyond the cell wall of E. coli and Salmonella typhirnuriurn. It is probably an over-simplification to regard the outermost layer of the multilayered cell envelope as consisting solely of lipoprotein or lipopoly- saccharide. From biochemical and immunological evidence, both appear to be present.
more protection to the organism than does the simpler Gram-positive wall. Loss or decrease of somatic antigens at the cell surface decreases the resistance of Gram-
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