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Antimicrobial Agents and Chemotherapy, September 2000, p. 2514-2517, Vol. 44, No. 9
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Antimicrobial Evaluation of N-Alkyl
Betaines and
N-Alkyl-N,N-Dimethylamine
Oxides with Variations in Chain Length
Christine R.
Birnie,1
Daniel
Malamud,2,3 and
Roger L.
Schnaare1,*
Department of Pharmaceutical Sciences,
Philadelphia College of Pharmacy, University of the Sciences in
Philadelphia,1 and Department of
Biochemistry, School of Dental Medicine, University of
Pennsylvania,2 and Biosyn,
Inc.,3 Philadelphia, Pennsylvania 19104
Received 13 January 2000/Returned for modification 13 March
2000/Accepted 5 June 2000
 |
ABSTRACT |
Alkyl betaines and alkyl dimethylamine oxides have been shown to
have pronounced antimicrobial activity when used individually or in
combination. Although several studies have been conducted with these
compounds in combinations, only equimolar concentrations of the
C12/C12 and C16/C14
chain lengths for the betaine and the amine oxide, respectively, have
been investigated. This study investigates the antimicrobial activity
of a wide range of chain lengths (C8 to C18)
for both the betaine and amine oxide and attempts to correlate their
micelle-forming capabilities with their biological activity. A broth
microdilution method was used to determine the MICs of these compounds
singly and in various molar ratio combinations. Activity against both
Staphylococcus aureus and Escherichia coli was
investigated. Antimicrobial activity was found to increase with
increasing chain length for both homologous series up to a point,
exhibiting a cutoff effect at chain lengths of approximately 16 for
betaine and 14 for amine oxide. Additionally, the C18 oleyl derivative of both compounds exhibited activity in the same range as
the peak alkyl compounds. Critical micelle concentrations were correlated with MICs, inferring that micellar activity may contribute to the cutoff effect in biological activity.
| |
INTRODUCTION |
As more resistant organisms continue
to emerge in society, the identification of additional antimicrobial
agents becomes increasingly more important. Compounds such as
surfactants are an area to be investigated. Betaines and amine oxides,
two types of amphoteric surfactants, have been shown to exhibit
antimicrobial activity against a variety of microorganisms (7, 16,
18, 25). Although each of these compounds has shown pronounced
activity alone, they have also been used in combination to exhibit a
synergistic effect (6).
An equimolar mixture of N-alkyl betaine and
N-alkyl-N,N-dimethylamine oxide was
patented in 1978 in a compound called C31G (17). With chain
lengths ranging from C8 to C18 and buffered in
a citrate buffer, C31G was first shown to have pronounced wound healing
and deodorizing effects, as well as antimicrobial sensitivity. Further
studies showed C31G has exhibited pronounced activity not only against
bacteria, but also against yeasts, fungi, sperm, and enveloped viruses
(4, 6, 14, 23). Although several studies have been published
about this compound in reference to the extent of antimicrobial
activity, little work has been conducted with any other chain lengths
besides the following two chain-length combinations: (i)
C12 betaine-C12 amine oxide and (ii)
C16 betaine-C14 amine oxide. Additionally, only
an equimolar ratio of the two components has been investigated.
The structures of these two components are shown in Fig.
1. The variation in length of the long
hydrocarbon tail is thought to influence the extent of antimicrobial
activity. Like most other surfactants, they are believed to be membrane
perturbants, disrupting the cell membrane of the microorganism
(26). It is believed that interaction with the surface of
the microorganism is a function of the polar head groups of the
betaine, amine oxide, or mixture of these molecules and that the
hydrocarbon tail subsequently becomes integrated with the lipid bilayer
of the cell membrane. This integration causes a disruption in the
membrane and inevitably causes leakage of the cell contents. The length
of the alkyl chain of the surfactants is thought to contribute to the
extent of this membrane disruption, because the higher chain lengths
may be incorporated into the lipid bilayers of the plasma membrane. The
increased hydrophobic effect of these longer chain tails may aid in
this disruption (16).
In an effort to find an optimal combination of betaine and amine oxide,
our study evaluated the extent of antimicrobial activity of a
homologous series of betaines, amine oxides, and combinations of these
compounds. Being surfactants, their micelle-forming capability is also
correlated with their biological activity.
| |
MATERIALS AND METHODS |
Betaine and amine oxides.
Compounds ranging in chain length
from 8 to 18 carbons for N-alkyl betaine and
N-alkyl-N,N-dimethylamine oxide were
obtained from two manufacturers, McIntyre Group, Ltd. (University Park, Ill.), and Stepan Co. (Northfield, Ill.). Although not all chain lengths were available, a representative group of samples was acquired.
In addition to the alkyl straight chains, oleyl derivatives [C18(o)] of both betaine and amine oxide were also
obtained for analysis. Table 1 shows the
compounds tested and their manufacturers.
Microorganisms.
Escherichia coli (ATCC 25922) and
Staphylococcus aureus (ATCC 25923) were obtained from the
American Type Culture Collection (ATCC) and used as representative
gram-negative and gram-positive organisms. Both microorganisms were
maintained in Mueller-Hinton agar and broth, buffered to pH 4.8 for
E. coli, and maintained at pH 7.3 for S. aureus.
Citric acid buffer (2.5 mM) at pH 5.5 and 7.3 was used as a diluting
solution as needed.
Microorganisms were stored in freezer vials at
10°C under
conditions recommended by the ATCC. As needed, samples were thawed,
warmed to 37°C, and streaked by dilution on agar petri plates
for
isolation. Inoculated plates were placed in a humidified incubator
at
37°C for 24 h, during which time colonies formed. With a sterile
loop, colonies were picked and dispersed in broth. After 24 h
of
incubation, the concentration of microorganisms was adjusted
to a
turbidity equal to that of a 0.5 McFarland standard, adjusted
by
diluting the overnight culture to a concentration equivalent
to 80%
transmittance (625 nm). This standardized suspension has
been shown to
contain approximately 10
8 CFU/ml (
19).
Antimicrobial evaluation.
A microdilution plate method was
used according to National Committee for Clinical Laboratory Standards
methods with Mueller-Hinton broth (19). In 96-well dishes,
concentrations of N-alkyl betaine and
N-alkyl-N,N-dimethylamine oxide ranged
from 104 to 1 µM, with the final well column containing
no active sample solution. The standardized microorganism suspension
was added to each well, and plates were incubated in a 37°C
humidified incubator for 24 h before evaluation. The MICs were
determined based on visual observation of turbid and nonturbid wells.
Samples were run in duplicate.
CMCs.
Selected critical micelle concentrations (CMCs) were
determined by using measured surface tension values as a function of concentration. Surface tension measurements at 25°C were determined by the Wilhelmy plate method on a Rosano surface tensiometer. The CMCs
were determined by plotting the surface tension against the log of the
concentration. The CMC is noted as the sharp change in decreasing
surface tension as the concentration of surface active agent is increased.
| |
RESULTS |
Betaine.
The antimicrobial activity of a homologous series of
N-alkyl betaines was evaluated against S. aureus
and E. coli. Table 2 shows the
MICs of these compounds. Antimicrobial activity was very poor at lower
chain lengths, with MICs of C8 betaine of 2.3 × 104 µM for S. aureus and 1.2 × 104 µM for E. coli. The MICs of the betaine
series decreased with increasing chain length, plateauing at the higher
chain lengths
around C16 for both microorganisms. The
C16 compound exhibited some of the best activity, with MICs
of 61 and 120 µM for S. aureus and E. coli,
respectively.
Amine oxide.
Table 3 shows the
MIC results for the series of homologous
N-alkyl-N,N-dimethylamine oxides. Like
the betaine series, the amine oxide series followed a similar trend of
increased activity with increased chain length. Again exhibiting very
poor activity at the low chain lengths, the MICs of the C8
amine oxide were 2.9 × 104 and 3.6 × 104 µM for S. aureus and E. coli,
respectively. The activity also increased with chain length, up to
approximately C14 to C16, and then tailed off
at the higher chain lengths. For the amine oxide series, activity
peaked at a chain length of C14 against E. coli, at a MIC of 31 µM, and plateaued at C14 to
C16 against S. aureus, at a MIC of 62 µM. In
addition, the C18 oleyl compound showed excellent activity
against both microorganisms, matching the alkyl chain's peak activity.
Being unsaturated in chemical structure, the oleyl compounds do not
exhibit the poor solubility problems usually associated with the
C18 stearyl compounds.
CMCs.
The CMCs for N-hexadecyl betaine and
N-tetradecyl-N,N-dimethylamine
oxide were determined by using surface tension measurements as a
function of concentration. The remaining CMCs have been collected from
the literature and are shown in Table 4.
It is well known in the literature that a linear relationship exists
between chain length and CMC (22). For a homologous series,
the following equation has been used: Log CMC = k1 N + k2, where N
is equal to the number of carbon atoms in the alkyl chain and
k1 and k2 are constants.
A linear relationship was determined for the betaine series in this
study, yielding the equation Log CMC = 0.447 N + 4.10 (R2 = 0.995). The amine oxide series
also followed a linear trend, yielding the equation Log CMC = 0.438 N + 3.89 (R2 = 0.998). Even with compiled CMCs, determined under different conditions, the linear correlation is still very good.
| |
DISCUSSION |
Cutoff effect.
Unlike the log-linear relationship between
increasing alkyl chain length and CMC for an entire homologous series,
both the betaine and amine oxide series showed this linear relationship with the antimicrobial activity only at the lower chain lengths. Figures 2 and
3 show the relationship of concentration
versus alkyl chain length, comparing both the MIC and the CMC. The MICs of both the betaine and the amine oxide series for both microorganisms exhibited a plateauing or parabolic effect with increasing alkyl chain
lengths, occurring at chain lengths of approximately 14 to 16. This
phenomenon is consistent with current literature regarding biological
activity, with numerous studies documenting this type of response with
homologous series of long-chain amphiphilic molecules (13, 22,
24).
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FIG. 2.
Correlation of MIC with CMC for a homologous series of
N-alkyl betaines.  , CMC;  , MIC for S. aureus;  , MIC for E. coli.
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FIG. 3.
Correlation of MIC with CMC for a homologous series of
N-alkyl-N,N-dimethylamine oxides. ,
CMC; , MIC for S. aureus; , MIC for E. coli.
|
|
Ferguson was one of the first to document this type of effect in 1939 (
8) when compiling a combination of studies relating
to
homologous series of compounds. He referred to this change
in
biological activity as exhibiting a "cutoff effect" at higher
chain
lengths.
Several theories have been postulated as to why this cutoff effect
occurs. Janoff and Pringle (
12,
20) have associated
this
cutoff with a limit in solubility. They proposed that as
the alkyl
chain increases, lipid solubility increases at a rate
faster than the
change in partition coefficient (lipid/aqueous).
At these higher chain
lengths, partitioning is limited, making
the concentration at the site
of action insufficient to have a
significant effect on the membrane of
the cell wall (
12,
20).
Other accounts attribute this to a
decrease in perturbation of
the membrane at higher chain lengths,
proposing that the longer
alkyl chain molecules better mimic molecules
in the lipid bilayer,
causing less of a disruption in the membrane
(
7).
Although numerous theories exist, it is likely that in the case of the
betaine and amine oxide series, the cutoff in biological
activity is
due to the micellar action of these compounds. Ross
and coworkers first
suggested this theory, investigating alkylbenzyldimethylammonium
chlorides of various chain lengths (
22). As surfactants
increase
in chain length, their tendency toward micelle formation is
greater,
noted by the lower CMCs at higher chain lengths. This tendency
to form micelles becomes greater than the tendency to move toward
the
interface (the membrane), and thus the concentration at the
site of
action becomes decreased. Also, as the size of the diffusing
species
increases from the size of a monomer to that of micelles,
their
diffusibility and permeation abilities will decrease, affecting
their
action on the microbial cell
wall.
Comparing the CMC and the MIC in Fig.
2 and
3, the linear CMC line
intersects the MIC line at the cutoff point for both the
betaine and
amine oxide series with both microorganisms investigated.
At chain
lengths below the cutoff point, the MICs of both compounds
are below
the CMC line, implying that the monomeric species of
these compounds
are responsible for an antimicrobial response.
Although some micelles
may be present at concentrations below
the CMC, it is not likely they
are significant enough to produce
an effect. In the case of the amine
oxide series, the MICs for
E. coli are up to 2 orders of
magnitude lower than the CMC. At
these concentrations, it is not likely
that the presence of micelles
is
significant.
At chain lengths above the cutoff point, the MIC is not reached until
well above the CMC. Possessing a much lower CMC than
the short-chain
homologues, fewer monomers will be present at
these concentrations,
apparently less than are needed to produce
a significant biologic
effect. Increased overall concentrations
are needed to obtain the
desired bactericidal
effects.
Effects of differences in bacterial strains.
It is also to be
noted that both the betaine and amine oxide series show similar trends
in activity for both S. aureus and E. coli, with
both organisms exhibiting a cutoff effect in approximately the same
location. These results are advantageous in a compound selection
process, in which only one chain length that exhibits the best
broad-spectrum activity can be selected for further development.
Although the similar effect on both the gram-positive and gram-negative
organisms is preferred, it is not always the case
with different
strains of microorganisms. Ferguson noted that
the most resistant
organisms will often exhibit a cutoff effect
much lower in alkyl chain
length, while Lien and Hansch more specifically
concluded that
gram-positive organisms preferred a more lipophilic
molecule than the
gram-negative one (
15). This has been attributed
to the cell
wall difference between bacterial types and strains.
E. coli, a gram-negative rod, exhibits a more complex cell wall
than
gram-positive organisms such as
S. aureus (
5).
Although
both gram-positive and -negative organisms have a similar
cytoplasmic
membrane inside the outer wall, containing both
phospholipids
and membrane proteins, the outer walls are very
different. Gram-positive
bacteria have a very simple cell wall,
consisting mainly of a
mesh-like structure, while the gram-negative
bacterial cell walls
contain a layer of peptidoglycan between an outer
membrane and
the cytoplasmic membrane. This outer membrane contains
lipopolysaccharides
which are cross-bridged by divalent cations,
believed to aid in
the stabilization of the outer membrane and also
make the membrane
more impermeable to lipophilic molecules
(
3).
Oleyl compounds.
In addition to the cutoff peak of the
homologous series, the oleyl compounds showed very good antimicrobial
activity. The compounds are unsaturated and exhibit a much greater
aqueous solubility than that of the other high chain lengths,
particularly the C18 stearyl. They likely possess the right
lipophilic/hydrophilic balance to allow the molecule to adequately
disrupt the cell wall of the microorganism. The CMC of the
9-octadecyl-N,N-dimethylamine oxide has been
shown to be 128 µM (11), which is comparable to the CMC of
the most antimicrobially active alkyl compounds. Additionally, the CMC
of this derivative is greater than the MIC, further supporting the
contention that the antimicrobial aspects of these compounds are
primarily due to that of the monomer.
Conclusions.
Overall, by comparing the MIC and CMC of the
betaine and amine oxide series, this study has provided a better
understanding of the relationship between the biological activities of
these compounds correlated with their micelle-forming capabilities. It
was shown that the majority of compounds provide excellent antimicrobial activity in their monomeric forms, but at chain lengths
above the cutoff point, compounds must be in both a micellar form and
monomeric form to exhibit a similar antimicrobial effect. Additionally,
a range of chain lengths that exhibited some of the best antimicrobial
activity was identified for both compounds. For both the betaine and
amine oxide, compounds in the range of C14 to
C16 were shown to be among the most effective of the alkyl compounds in addition to the unsaturated C18 oleyls.
| |
ACKNOWLEDGMENT |
This work was supported in part by the Biosyn Graduate Research Fellowship.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Pharmaceutical Sciences, Philadelphia College of Pharmacy, University of the Sciences in Philadelphia, Philadelphia, PA 19104. Phone: (215)
596-8942. Fax: (215) 895-1100. E-mail:
r.schnaa{at}usip.edu.
| |
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Antimicrobial Agents and Chemotherapy, September 2000, p. 2514-2517, Vol. 44, No. 9
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
