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The antimicrobial activity of C2-C18 fatty acids was determined in vitro in cultures of two strains of Escherichia coli grown on glucose. Antimicrobial activity was expressed as IC50 (a concentration at which only 50% of the initial glucose in the cultures was utilized). Utilization of glucose was inhibited by caprylic acid (IC50 0.30-0.85 g/L) and capric acid (IC50 1.25-2.03 g/L). Neither short-chain fatty acids (C2-C6) nor fatty acids with longer chain (C12-C18) influenced substrate utilization. Caproic acid, however, decreased cell yield in cultures of E. coli in a dose-dependent manner. No inhibition of glucose utilization was produced with unsaturated fatty acids, oleic and linoleic. Calcium ions added in excess reversed the antimicrobial effect of capric acid, but not that of caprylic acid. Antimicrobial activity of caprylic and capric acid decreased when the bacteria were grown in the presence of straw particles, or repeatedly subcultured in a medium containing these compounds at low concentrations. Counts of viable bacteria determined by plating decreased after incubation with caprylic and capric acid (30 min; 1 g/L) at pH 5.2 from > 10(9) to approximately 10(2)/mL. A reduction of a mere 0.94-1.96 log10 CFU was observed at pH 6.5-6.6. It can be concluded that caprylic acid, and to a lesser extent also capric acid, has a significant antimicrobial activity toward E. coli. Effects of other fatty acids were not significant or absent.
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Folia Microbiol. 48 (6), 731–735 (2003) http://www.biomed.cas.cz/mbu/folia/
Susceptibility of Escherichia coli to C
2
C
18
Fatty Acids
M. MAROUNEK
a,b
*, E. SKŘIVANOVÁ
b
, V. RADA
c
a
Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, 104 00 Prague-Uhříněves, Czechia
e-mail marounek@iapg.cas.cz
b
Research Institute of Animal Production, 104 01 Prague-Uhříněves, Czechia
c
Department of Microbiology and Biotechnology, Czech University of Agriculture in Prague, 165 21 Prague, Czechia
Received 7 May 2003
Revised version 6 October 2003
ABSTRACT. The antimicrobial activity of C
2
–C
18
fatty acids was determined in vitro in cultures of two
strains of Escherichia coli grown on glucose. Antimicrobial activity was expressed as IC
50
(a concentration
at which only 50 % of the initial glucose in the cultures was utilized). Utilization of glucose was inhibited
by caprylic acid (IC
50
0.30–0.85 g/L) and capric acid (IC
50
1.25–2.03 g/L). Neither short-chain fatty acids
(C
2
–C
6
) nor fatty acids with longer chain (C
12
–C
18
) influenced substrate utilization. Caproic acid, however,
decreased cell yield in cultures of E. coli in a dose-dependent manner. No inhibition of glucose utilization
was produced with unsaturated fatty acids, oleic and linoleic. Calcium ions added in excess reversed the
antimicrobial effect of capric acid, but not that of caprylic acid. Antimicrobial activity of caprylic and capric
acid decreased when the bacteria were grown in the presence of straw particles, or repeatedly subcultured in
a medium containing these compounds at low concentrations. Counts of viable bacteria determined by
plating decreased after incubation with caprylic and capric acid (30 min; 1 g/L) at pH 5.2 from >10
9
to
10
2
/mL. A reduction of a mere 0.94–1.96 log
10
CFU was observed at pH 6.5–6.6. It can be concluded that
caprylic acid, and to a lesser extent also capric acid, has a significant antimicrobial activity toward E. coli.
Effects of other fatty acids were not significant or absent.
Young animals are susceptible to the development of digestive disorders caused by Escherichia
coli, salmonellas and other feed-borne pathogens. Antibiotics are used to control enteritis infections but their
use has been viewed critically. This had led to a renaissance of the research into the use of nonantibiotic
compounds with bactericidal activity (Lauková and Mareková 2001; Morovský et al. 2001; Pantev et al.
2002). Fatty acids and their derivatives are among the candidate replacements for antibiotics (Jalč et al. 2002;
Ricke 2003). Some of them have a long history of being utilized as additives and preservatives, preventing
food and feed deterioration (acetic, propionic, lactic and sorbic acid). Antimicrobial activity of fatty acids
has been known for a long time. Fatty acids facilitate the transport of protons across the bacterial membrane
and thus collapse the proton gradient. This results in depletion of cellular energy followed by denaturation
of acid-sensitive proteins and nucleic acids. Nieman (1954) concluded that (i) fatty acids with a chain length
of around C
12
were the most active, (ii) inhibitory properties of unsaturated fatty acids were more pro-
nounced than those of saturated acids, and (iii) G
+
-bacteria were more susceptible to the action of fatty acids
than G
-bacteria. Antimicrobial activity of saturated C
4
–C
18
fatty acids and oleic acid in aerobically grown
cultures of E. coli was examined by Hassinen et al. (1951). Bacterial growth was inhibited by capric (C
10
)
and lauric (C
12
) acid. Caprylic acid (C
8
) inhibited the growth of E. coli only slightly. Other saturated fatty
acids and oleic acid did not inhibit the growth of the strain tested. In experiments of Sprong et al. (2001)
capric and lauric acid appeared to be effective bactericidal bovine milk fat components, active against
E. coli and other enteropathogens. As for short-chain fatty acids (SCFA; <C
8
), the results of Hassinen et al.
(1951) are not consistent with the results of some later studies. Prohászka (1980) suggested that undissocia-
ted SCFA were responsible for the inhibitory action of the rabbit cecal contents against E. coli strains, and
on the same basis Wallace et al. (1989) explained the failure of E. coli to colonize the ovine rumen. Cherring-
ton et al. (1990) observed a bacteriostatic effect of 5 mmol/L propionic acid in cultures of E. coli at pH 5.0.
Similarly, Mortensen et al. (1999) reported that SCFA in concentrations normally found in the human colon
had an inhibitory effect on the growth of E. coli.
*Present address: Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, 142 20 Prague, Czechia.
732 M. MAROUNEK et al. Vol. 48
The aim of our study was to determine the susceptibility of two strains of E. coli to C
2
–C
18
fatty
acids. Furthermore, the effect of calcium ions, the presence of feed particles in the testing medium, adapta-
tion effect, and effect of pH were examined.
MATERIALS AND METHODS
E. coli strains CCM 3954 (ATCC 25922) and CCM 4225 (ATCC 35218) were obtained from the
Czech Collection of Microorganisms (Brno, Czechia). The former strain is a clinical isolate, both strains
being reference strains for testing of antimicrobials. The bacteria were grown on a medium containing
(in g/L): K
2
HPO
4
·3H
2
O 5.9, KH
2
PO
4
4.5, NaHCO
3
3.0, (NH
4
)
2
SO
4
2.9, NaCl 0.9, MgSO
4
·7H
2
O 0.09,
CaCl
2
0.09, bactopeptone 6, glucose 5, yeast extract 3. A trace metal solution (1 mL) containing nitrilotri-
acetic acid (Clark and Holms 1976) was also added. Experiments were carried out as quintuplicates.
1st experiment. The medium was dispensed to gas-tight glass flasks containing acetic, propionic,
butyric, isobutyric, valeric, isovaleric, caproic, caprylic, capric, lauric, myristic, palmic, stearic, oleic and
linoleic acid (0, 0.1, 0.2, 0.3, 0.5, 1, 2, 3, 5 g/L), together with an equivalent amount of 5 mol/L NaOH. The
flasks were filled with CO
2
, closed by rubber stoppers and autoclaved at 110 °C for 45 min. Inoculated cul-
tures were incubated in triplicates at 37 °C for 1 d. Then the pH was measured using the pH03 pH-meter
from Labio Praha (Prague) and residual glucose was determined enzymically by the oxidase–peroxidase
method using a commercial kit Oxochrom Glucose from Lachema Brno (Czechia). The amount of residual
glucose was expressed relative to the initial glucose concentration and plotted against the concentration of
the fatty acid. The IC
50
was the concentration at which only 50 % of the initial glucose was utilized. This
method avoids problems caused by opalescence or turbidity of media containing
C
8
-fatty acids. In control
cultures and those containing C
2
–C
6
-fatty acids the growth was assessed by measuring absorbance A
640
at
the end of incubation.
2nd experiment. The bacteria were cultivated in media containing caprylic and capric acid (for con-
centrations see above), supplemented with CaCl
2
at 0.09 (control) or 1.8 g/L. After a 1-d incubation, the pH
was measured and residual glucose determined (see above).
3rd experiment. The bacteria were grown in media containing caprylic and capric acid and supple-
mented with particles of wheat straw at 0 or 10 g/L. The average size of straw particles (determined by dry
sieving) was 0.2 mm. the IC
50
was determined in 1-d cultures.
4th experiment. The bacteria were transferred 10
×
in media containing low concentrations of
caprylic and capric acid (3
×
lower than IC
50
found in the 1st experiment). IC
50
was then determined in the
cultures of both strains using adapted and nonadapted inocula.
5th experiment. Caprylic and capric acid were added to overnight-grown cultures of both strains at
1 g/L as 20 % (W/W) Me
2
SO solutions. Controls received an equivalent amount of the solvent. The cultures
were incubated at 37 °C in a water bath for 30 min; treated cultures were incubated at pH 5.2 and 6.5–6.6.
In the latter cultures, the pH was increased by addition of NaOH. After incubation, samples were removed
and serially diluted with the Wilkins–Chalgren broth (Oxoid). The number of viable bacteria was determi-
ned by streaking 0.1 mL of an appropriate dilution on Wilkins–Chalgren agar plates using sterile glass rods.
Inoculated plates were incubated at 37 °C for 1 d. Colonies were counted and means and SD calculated. The
significance of differences between control and treated cultures was evaluated by the t-test.
RESULTS
Table I top presents fatty acid concentration causing a 50 % decrease in the glucose utilization by
given E. coli strain. Both strains examined were susceptible to caprylic and capric acid. Control cultures and
those containing shorter (C
2
–C
6
) or longer (C
12
–C
18
) fatty acids utilized glucose completely. This was
accompanied by a pH decrease from 6.7 at the beginning of the incubation to 5.6 ± 0.1 at the end. The inhi-
bitory concentration of caprylic acid was not influenced by CaCl
2
addition; on the contrary, capric acid lost
its antimicrobial activity in cultures supplemented with CaCl
2
at 1.8 g/L (data not shown).
The antimicrobial activity of C
8
- and C
10
-acids decreased when the bacteria were grown in the pre-
sence of straw particles (Table I middle), or repeatedly grown in the presence of low fatty acid concentrat-
ions (Table I bottom). Caproic acid (C
6
) decreased the absorbance of E. coli cultures in a dose-dependent
manner while the effect of valeric acid (C
5
) was less pronounced (Fig. 1). Other SCFA had no influence on
growth of the two strains.
2003 EFFECT OF FATTY ACIDS ON E. coli 733
Incubation of bacteria with C
8
- and C
10
-acids at pH 5.2 led to a considerable reduction in the num-
bers of viable cells (Table II) while a reduction of a mere 0.94–1.96
log
10
CFU was observed at pH 6.5–6.6.
a
In which only 50 % of the initial glucose
was utilized within a 1-d incubation inter-
val.
b
Including branched isomers.
c
Stearic, oleic and linoleic acids.
d
Particles of wheat straw, 10 g/L.
e
Control values (i.e. without particles) in
parentheses.
f
Subcultured 10
×
in a medium containing
C
8
- or C
10
-acid at IC
50
/3.
Table II. Cell concentration of E. coli (c, 1/mL)
a
determined in strains CCM 3954 and CCM 4225 by plating after
a 30-min incubation with C
8
- and C
10
-acids at different pH
Treated cultures Control cultures
Strain
pH log c fatty acid
b
pH log c
CCM 3954 5.53 9.67 ± 0.09 C
8
6.52 8.37 ± 0.18*
5.20 <2*
C
10
6.49 8.63 ± 0.33*
5.18 6.35 ± 0.32*
CCM 4225 5.35 9.60 ± 0.33 C
8
6.60 7.64 ± 0.32*
5.21 2.56 ± 0.48*
C
10
6.54 8.42 ± 0.08*
5.19 <2*
a
Surviving after treatment with fatty acids; means of triplicates ±SD.
b
g/L.
*Significantly different from the control values (p < 0.01). Cell concentration <100/mL was considered as 100/mL
in statistical calculations.
DISCUSSION
Caprylic and capric acids, but not other acids prevented utilization of glucose in the cultures of
2 strains of E. coli. Unsaturated C
18
-fatty acids and saturated C
2
–C
6
- and C
12
–C
18
-fatty acids did not
influence substrate utilization.
The inhibitory effect of SCFA on E. coli reported by Prohászka (1980) and Wallace et al. (1989)
was not observed in this study with our strains. Caproic acid, and to a lesser extent also valeric acid, how-
ever, decreased cell yield expressed as turbidity of cultures. Both Prohászka (1980) and Wallace et al.
(1989) found that inhibitory effect of SCFA on E. coli was pH-dependent and ceased in the neutral pH
range. In our study the pH in cultures supplemented with SCFA fell from 6.71 at the beginning of the incu-
bation to 5.55 at its end. The SCFA were thus present in the cultures mostly in their active, nondissociated
form, except for the initial period. Therefore, also the activity of medium-chain fatty acids (C
8
, C
10
)
depends on pH, being much higher in an acid environment. It is noteworthy that a certain bactericidal acti-
vity of C
8
- and C
10
-acids exists at pH near 7. This has not been the case for formic and lactic acid, which
allowed growth of E. coli in the neutral pH range (Dibner and Buttin 2002). The acid milieu itself was not
Table I. Inhibitory concentration (g/L)
a
of C
2
–C
18
fatty acids against E. coli
strains CCM 3954 and CCM 4225 grown on glucose
Fatty acid CCM 3954 CCM 4225
C
2
, C
3
, C
4
b
, C
5
b
, C
6
>5 >5
C
8
0.45 0.30
C
10
2.03 1.25
C
12
, C
14
, C
16
, C
18
c
>5 >5
Growth in the presence of solid particles
d,e
C
8
1.72 (0.82) 1.83 (0.85)
C
10
2.57 (1.98) 5.00 (1.90)
Growth of adapted
e,f
strains
C
8
0.92 (0.63) 1.20 (0.47)
C
10
>5. (2.00) 2.90 (1.92)
734 M. MAROUNEK et al. Vol. 48
bactericidal as concentrations of viable bacteria taken from control cultures (pH 5.53 and 5.35) were >1/pL
(i.e. >10
9
/mL; Table II). Enterobacteriaceae while traveling through the gastrointestinal tract must endure
extremely low pH in the stomach. E. coli, salmonellas and shigellas possess specific survival mechanisms
that include amino-acid decarboxylases and shock proteins that prevent or repair macromolecular damage
(reviewed by Bearson et al. 1997). Apparently, medium-chain fatty acids are capable of overcoming this
barrier.
Several factors can influence the antimicro-
bial activity of fatty acids. Galbraith et al. (1971)
showed that calcium ions added in excess as CaCl
2
reversed the antimicrobial effects of lauric (C
12
)
and linoleic (C
18:2
) acid. Fatty acids differed in
the extent to which they reacted with cations to
form insoluble salts. Increasing chain length
increased the extent of insoluble calcium soap
formation (Jenkins and Palmquist 1982). In this
study CaCl
2
added in excess counteracted capric
acid, but not caprylic acid, apparently due to the
low affinity of the latter acid for Ca
2+
-ions. Har-
foot et al. (1975) and Maczulak et al. (1981) sug-
gested that the presence of a particulate phase in
a milieu reduced the inhibitory effect of fatty
acids on bacterial growth. This has also been
shown in Table I (middle). Food particles pro-
vide a site for the adsorption of fatty acids, which
would otherwise adsorb onto the bacteria and
inhibit bacterial metabolism and growth. As expec-
ted, an increase of the resistance of E. coli strains
was observed after their serial transfer in a me-
dium containing a subinhibitory concentration of
caprylic and capric acid.
Sprong et al. (2001) considered the bacteri-
cidal activity of lipids as biologically significant
when a reduction to >0.5 log
10
CFU/mL was
observed. Both caprylic and capric acid meet this
criterion. These acids were identified as the antimicrobial compounds of the stomach contents of suckling
rabbits (Cañas-Rodriguez and Smith 1966). Our previous work (Marounek et al. 2002) indicated a signifi-
cant antimicrobial activity of caprylic and capric acid against bacteria of the animal digestive tract. The results
presented here showed that these acids were active also against E. coli and may thus enhance the resistance
to certain types of gastrointestinal infections.
This work was supported by grant no. QF3134 of the Czech National Agency for Agricultural Research (NAZV).
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... There are conflicting reports about the relative antimicrobial efficacy of different MCOA. Only caprylic acid (C8:0), and, to a lesser extent, capric acid (C10:0) have shown significant antimicrobial activity against two strains of Escherichia coli in one study (Marounek et al., 2003). However, in another study, both lauric acid (C12:0) and caprylic acid (C8:0) had the ability to damage the cytoplasmic structure of bacteria (Skrivanová et al., 2006). ...
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Colibacillosis in Indonesia until now still appears frequently, so the case of colibacillosis laying hens cannot reach the peak of egg production; the egg production period is delayed and easily infected with other diseases. The purpose of this research is that the acidifier-dextrose combination is expected to be able to suppress the development of Avian Pathogenic Escherichia coli (APEC) bacteria in laying hens so that, in the end, the case of colibacillosis can be controlled in Indonesia. A total of 240 heads of laying hens were divided into 6 treatments and each consisted of 40 replications. The results of this research state that a combination of acidifier-dextrose can increase Hen Day Production (p
... The beneficial effect of lower pH on the minimum bactericidal concentrations (MBC) of medium-to long-chain FA has been reported for L. monocytogenes (22). For the Gram-negative bacterium Escherichia coli, however, a chain-length-dependent effect of pH was observed when tested against C2 to C18 fatty acids (50). Higher efficacy at lower pH was observed for C8 and C10 but not for C2 to C6 or C12 to C18 FA. ...
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... The use of citric acid creates an acidic pH of 3.5 -4.0 in the intestinal tract which inhibits the replication of Escherichia coli [5]. According to [13] stated that there are two acid mechanisms in inhibiting bacterial development, namely the undissociated form of citric acid will directly enter the lipid membrane of gram-negative bacteria and the ability of hydrogen-free protons directly from dissociated citric acid which can reduce pH thereby creating conditions acid, the condition negatively affects the presence of pathogenic bacteria. Organic acids that enter the cytoplasm which have neutral pH conditions inhibit bacterial growth by inhibiting the oxidative phosphorilation process and increasing energy consumption. ...
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