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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
By J. G. VOSS
Research Division, Miami Valley Laboratories,
The Proctor & Gamble Company, Cincinnati, Ohio $5239, U.S.A.
(Accepted for publication 20 March 1967)
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.
The most effective organic cations had one C,, to CI6 alkyl chain. At ~ H I o ,
EDTA roo pg./ml and N,N-dimethyldodecylamine (DDA) 2.5 ,ug./ml.
modified the cell wall of Escherichia coli so as to permit a more than 99.99 %
kill by 20,ug.fml. of a zwitterionic surface active agent, a-hydroxy-3-
(dimethylhexadecylammonio) propane- I -sulphonate (HAPS), in 10 min. at
37’. At these concentrations, the individual compounds were bactericidally
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
Gram-negative bacteria are generally susceptible to the bactericidal action of
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
rods (Voss, 1964) or ‘osmoplasts’ (Asbell & Eagon, 1966a). In studies on Escherichia
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.
392 J. G. VOSS
METHODS
Organisms. The test organisms were Escherichia coli (ATCC 10536), E. freundii,
Pseudomonas aeruginosa, Proteus mirabilis, and Salmonella typhi; S. typhi TY 2v and
0901 and E. freundii (BALLERUP 107) were obtained through the courtesy of the late
Dr P. R. Edwards (Communicable Disease Center, Public Health Service, Atlanta,
Ga.), S. typhi ggg2v and 12839 were obtained from the American Type Culture
Collection. Escherichia coli was obtained as ATCC 10536 and used in the earlier part of
this work; after the observation that this culture produced acid but no gas from
glucose or lactose, it was replaced with a fresh, gas-forming culture of ATCC 10536
from the American Type Culture Collection with no discernible difference in results.
Test compounds. EDTA was used as the disodium salt; solutions were adjusted to
pH 8.0 before sterilization. The zwitterionic agent 2-hydroxy-3-(dimethylhexadecyl-
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
alone. N,N-dimethyldodecylamine (DDA) was prepared by distillation of 'dimethyl
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
ethanol, and diluted with water before use; at values up to about pH 10, they are
present principally as cations.
Bactericidal tests. All organisms except Proteus ~ i r a b i l ~ s were grown in brain
heart infusion broth (Difco) for 24 hr at 37". Plate counts were made on brain heart
infusion agar incubated at 37" for 24 hr; P. mirabilis was grown in nutrient.broth,
and plated on nutrient agar overlaid with plain agar to inhibit spreading. Exposure to
bactericidal agents was done as described earlier (Voss, 1963) for 10 min. at 37" with
suspensions of about 60-1 20 million washed organisms/ml.
Lysis. Changes in optical density of suspensions of organisms at room temperature
were measured with a Coleman Junior spectrophotometer at 660 mp.
Precipitin tests. Escherichia coli was harvested from ten 150 mm. plates, washed,
and suspended in 400ml. water. These suspensions contained 3-4mg. dry wt.
organisms/ml. Portions were treated for 10 min. at 37" with DDA 10 and 50 pg./ml. or
with EDTA, HAPS, dodecyltrimethylammoniumbromide (DTAB), or alkyldimethyl-
benzylammonium chloride (Roccal), all 50 pg./ml. in the presence or absence of
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
agar, using as the source of antibody a 115 dilution of rabbit antiserum prepared
against whole E. coli organisms.
R E S U L T S
Activity of cations other than tris
Tris was more effective than other buffers (e.g. phosphate buffer) in promoting the
bactericidal activity of a mixture of EDTA and HAPS. The corresponding nitro
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
comparison of other amines and related compounds for activity in promoting the
bactericidal action of EDTA and HAPS against Escherichia coli at concentrations at
394 J. G. V O S S
which the latter two compounds together were ineffective. A number of amines of low
molecular weight, more or less closely related to tris, were found to possess similar
activity. As the length of the alkyl chain increased, the bactericidal activity of the
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
alkyl chains of 12-16 C atoms. The further substitution on the N atom of one or two
methyl groups had little effect. Amines with two alkyl chains were less active. Such
Table 2. Per cent survival of Escherichia coli in systems containing EDTA (ioo,ug./ml.) i-
Na2C03(100,ug./ml.) i- HAPS (20 ,ug.Iml.), and organic cations or related compounds
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
requirement for a positively charged N atom. A compound with an alkyl chain of C,,
Effects of organic cations on cell walls 395
or longer and a positively charged group at one end was sufficient to give high bac-
tericidal activity in the presence of EDTA, HAPS and an alkaline buffer.
Action of the bactericidal system
For further work, DDA was chosen as the organic cation; at a concentration of
2.5 ,ug./ml., it showed only very limited bactericidal activity against Escherichia coli at
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
N-hydroxyethylethylenediaminetriacetic acid (Versenol), diethylenetriaminepenta-
acetic acid (Versenex 80), and I ,2-diaminocyclohexane-N,N'-tetra-acetic acid were as
effective as EDTA.
Table 3. Eflect of omission of single components of the bactericidal system
on kill of Escherichia coli
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
its presumed effect on the cytoplasmic membrane (see Hotchkiss, 1946; Salton, 1g51),
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
Escherichia coli; lack of Na2C03caused a much smaller decrease.
The bactericidal action of the components of the EDTA +DDA +Na,CO, + HAPS
system against Escherichia coli was studied in greater detail by exposing the organisms
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
EDTA or HAPS. Although killing might be influenced by carry-over of test compounds
Eflects of organic cations on cell walls 397
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.
A major proportion of the P released may be accounted for as nucleic acid, as would
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.).
Table 5. Lysis of Escherichia coli by lysozyme, EDTA, and DDA or tris bufer"
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 0 ' 02 I 0'2 0-I 7-6 0.08 -
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 ++++
* 6 ml. washed bacterial suspension in 8 ml. total volume.
- ,no perceptible increase in viscosity; + + + +, highly viscous.
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
not restricted to Escherichia coli is shown by the data in Table 6. Four other Gram-
26 G. Microb. 48
398 J. G. VOSS
negative species were also killed by this system. At the concentrations used, none of the
individual components were actively bactericidal. It is of interest that possession of an
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.
* With DDA (I pg./ml.), at which concentration DDA alone was not actively bactericidal against
S. typhi. Removal of somatic antigens by organic cations
It seemed probable that EDTA and DDA removed some surface components of the
cell wall, thus rendering it more permeable to HAPS. To test this, washed suspensions
of Escherichia coli were treated for 10 min. at 37" with dodecyltrimethylammonium
bromide (DTAB), alkyldimethylbenzylammonium chloride (Roccal), DDA, EDTA or
HAPS (at 10 or 50,ug./ml.), in the presence or absence of Na,CO, ~oo,ug./ml.The
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;
Na,CO,, EDTA and HAPS were essentially ineffective in removing antigens from the
cell wall. D I S C U S S I O N
Although Weidel, Frank & Martin (1960) concluded that the outermost layer of the
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
lipopolysaccharide. Shands (I 965), by using ferritin-labelled antibody, demonstrated
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.
Martin (I 963) suggested that the complex wall of Gram-negative organisms affords
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-
400 J. G. VOSS
effective in removing somatic antigens under similar conditions. Perhaps antigens
suitable for use as vaccines could be prepared from a number of Gram-negative species
in this fashion.
The capable assistance of Rebecca Sosebee and R. Bush is gratefully acknowledged,
as are numerous constructive discussions with Dr H. H. Reller.
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