www.elsevier.com/locate/ijfoodmicro

International Journal of Food Mic

Characterization of the bactericidal effect of dietary sphingosine

and its activity under intestinal conditions

Sam Possemiers a, John Van Camp b, Selin Bolca a, Willy Verstraete a,*

aLaboratory of Microbial Ecology and Technology (LabMET), Ghent University, Coupure Links 653, B-9000 Ghent, BelgiumbLaboratory of Food Science and Human Nutrition, Ghent University, Belgium

Received 10 November 2004; received in revised form 12 May 2005; accepted 12 May 2005

Abstract

Sphingosine is known as a natural antimicrobial agent, protecting the human skin from bacterial colonization and possibly

affecting the intestinal microbial community after ingestion. In this study we further investigated the antibacterial spectrum of

dietary d-eythro-sphingosine in saline towards three intestinal pathogens and to the health promoting lactobacilli and

bifidobacteria. The degree of bactericidal effect was studied using plate counts and Live/Dead analysis combined with flow

cytometry. To assess activity under complex intestinal conditions, sphingosine was dosed to the Simulator of the Human

Intestinal Microbial Ecosystem (SHIME) for a period of 11 days. Finally, we tried to elucidate the factors influencing the

activity and the mode of action of sphingosine. In all performed experiments, high correlation occurred between plate counts

and Live/Dead analysis. In saline a strong antibacterial effect was seen to all tested species, Gram-negative and Gram-positive,

and sphingosine not selectively acted against pathogens, as health promoting bacteria were also affected. Under simulated

intestinal conditions however, no shifts in bacterial concentrations were detected. Experiments with individual medium

components thought that the effect of sphingosine is very easily neutralized by BSA, stearic acid and surfactants. Based on

our results, d-erythro-sphingosine would only be active when protonated and its mode of action would imply electrostatic

attraction to the bacteria and disruption of membrane integrity. In conclusion, the application of sphingosine is limited to

specific environments, as activity was very sensitive to inhibition. Yet, because of its broad spectrum membrane disrupting

activity, it could be very useful under controlled conditions.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Sphingosine; Antibacterial; Mode of action; SHIME; Intestine; Sphingolipids

0168-1605/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.ijfoodmicro.2005.05.007

* Corresponding author. Tel.: +32 9 264 59 76; fax: +32 9 264 62

48.

E-mail address: Willy.Verstraete@UGent.be (W. Verstraete).

1. Introduction

Sphingolipids are estimated to constitute 0.01 to

0.02% of the human diet with a yearly intake per

capita of about 115 g, with dairy products being the

primary source of uptake (Vesper et al., 1999).

robiology 105 (2005) 59–70

S. Possemiers et al. / International Journal of Food Microbiology 105 (2005) 59–7060

Despite this low consumption, sphingolipids and their

metabolites, ceramide and sphingosine (SPH), are

highly bioactive molecules with multiple beneficial

effects on human health, e.g. cancer inhibition

(Schmelz et al., 2000) and the inhibition of cholesterol

absorption (Eckhardt et al., 2001; Jiang et al., 2001).

Another possible effect would be the protective capa-

cities of sphingolipids against bacterial toxins and

infection by bacteria or viruses. Food sphingolipids

can compete for cellular binding sites on the intestinal

mucosa (Bibel et al., 1992b; Fantini et al., 1997).

Because microbial adherence is often the first step

in infection (Ofek et al., 2003), this competition

could protect against food borne pathogens. Infants

consuming milk supplemented with gangliosides, a

specific group of sphingolipids, had significantly

lower E. coli and higher Bifidobacterium sp. counts

in their faeces (Rueda et al., 1998). Beside this, SPH

could also have a direct bactericidal effect. Free SPH

acts as a natural antimicrobial barrier of the human

skin (Arikawa et al., 2002; Bibel et al., 1993, 1995)

and has been shown to strongly decrease the concen-

tration of several food borne pathogens at low con-

centration (Sprong et al., 2001, 2002).

d-erythro-sphingosine (SPH) is the most prevalent

sphingoid base in mammalian tissues and dairy pro-

ducts. The amino group of this component is usually

substituted with long-chain fatty acids to produce

ceramides. The addition of a polar phosphocholine

produces sphingomyelin, the main sphingolipid pre-

sent in foods (Vesper et al., 1999). During intestinal

passage, sphingomyelin is partially digested to cera-

mide and SPH by human sphingomyelinases and

ceramidases (Liu et al., 2000; Lundgren et al., 2001;

Nilsson and Duan, 1999) and colon bacteria could

also be able to transform sphingolipids (Nyberg et

al., 1997). Therefore, SPH can be present in the

large intestine and exert bactericidal effects and

protect against intestinal pathogens. Sprong et al.

(2001) showed that 25 AM SPH tested in saline,

minimally gave a 4 log reduction of the Escherichia

coli, Salmonella enteritidis, Campylobacter jejuni,

and Listeria monocytogenes concentrations, whereas

sphingomyelin and ceramide showed no antibacterial

effects. However, the colon harbours up to 1014

microorganisms from approximately 400 different

species, which strongly influence human health (Mac-

Farlane and Cummings, 1999). If SPH does not act

selectively towards pathogens, but also kills beneficial

intestinal bacteria like lactobacilli and bifidobacteria,

this could negatively influence human health.

Up to now, bactericidal activity of SPH has only

been tested in saline (Sprong et al., 2001). Under

intestinal conditions however, the environment is

very complex with changing conditions in the different

parts of the colon (Macfarlane et al., 1992). It is known

that environmental conditions strongly influence the

efficacy of an antibacterial component (Bessems,

1998). Another important factor influencing activity

towards different bacterial species is the mode of

action of SPH. Next to a possible detergent activity,

interference with a protein-kinase system analogous to

mammalian cells is proposed (Bibel et al., 1992a), but

no clear evidence in this direction was obtained.

Therefore, bactericidal activity of SPH further has to

be characterised in terms of its mode of action and

activity under the physiological conditions of the intes-

tine. In this work, selectivity of action towards three

pathogenic strains, being E. coli, Enterobacter aero-

genes BE1 and Clostridium perfringens, was tested by

comparing results with experiments using lactobacilli

and bifidobacteria. This was done using plate counts as

well as with Live/Dead analysis combined with flow

cytometry, allowing rapid screening of the degree of

bactericidal effect. In addition, a modification of the

Simulator of the Human Intestinal Microbial Ecosys-

tem (SHIME) (De Boever et al., 2000; Molly et al.,

1993) was used to assess the efficacy under more

relevant conditions and the influence of different

environmental conditions. This way, a more clear

view of the mode of action and activity of dietary

SPH as a natural antibacterial agent was obtained.

2. Materials and methods

2.1. Bacterial strains and growth conditions

As examples of pathogenic strains, a multidrug

resistant clinical isolate E. coli ESBL112 (LMG

22096), E. aerogenes BE1 (LMG 22092) (De Gheldre

et al., 2001) and C. perfringens (LMG 11264) were

used to investigate the spectrum of bactericidal activ-

ity of SPH and Lactobacillus acidophilus (LMG

13550), Lactobacillus amylovorus (LMG 18197),

Bifidobacterium longum (LMG 11047) and Bifido-

S. Possemiers et al. / International Journal of Food Microbiology 105 (2005) 59–70 61

bacterium bifidum (LMG 11582) were used as exam-

ples of health promoting strains. All strains were

obtained from the Belgian Coordinated Cultures of

Microorganisms (BCCMk/LMG, Ghent, Belgium).

The culture conditions of the different strains are

described in Table 1. Aerobic experiments with liquid

cultures were performed in test tubes, whereas anae-

robic liquid incubations were performed in flasks,

sealed with rubber tops and anaerobiosis was obtained

by flushing the flasks with N2 during 20 cycles of 2

min 700 mbar overpressure and 900 mbar underpres-

sure. Anaerobic plate incubations were performed in

jars, containing AnaeroGenk bags (Oxoid, Hamp-

shire, UK) to remove oxygen.

2.2. Antimicrobial susceptibility testing

2.2.1. In vitro assay

d-erythro-sphingosine was obtained from AvantiRPolar lipids, Inc. (Alabaster, Alabama). Stock solu-

tions (4 mM) were prepared in absolute ethanol and

stored at �20 8C. Bactericidal activity was tested in

saline (8.5 g/l NaCl) at pH 7. Incubates contained

3.8% (v/v) ethanol but this did not affect bactericidal

activity of SPH (data not shown).

The activity of SPH was tested in triplicate at

concentrations of 0, 25, 50, 100 and 150 AM. Bacter-

ial cultures were tenfold diluted in saline to avoid

matrix effects. 300 Al diluted culture (F106 CFU/

ml) was then transferred to an eppendorf tube contain-

ing 662 Al saline. Finally, SPH stock solution was

added together with absolute EtOH so that to every

tube a total volume of 38 Al was dosed. Samples were

vortexed and incubated for 2 h at 37 8C under aerobic

or anaerobic conditions, depending on the growth

conditions for the different strains (Table 1).

Table 1

Bacterial strains tested for susceptibility to sphingosine (SPH), the cultu

Bacteria Broth

E. coli Luria-Bertani (A)

E. aerogenes BE1

C. perfringens Reinforced Clostridial Medium (AN)

L. acidophilus De Man Rogosa Sharpe (AN)

L. amylovorus

B. bifidum De Man Rogosa Sharpe+0.5 g/l L-cystein-HCl

B. longum

Cultures were incubated aerobically (A) or anaerobically after flushing w

2.2.2. Plate counts

After incubation, tenfold dilution series of the dif-

ferent samples were prepared in saline and plated in

triplicate. Plates were incubated at 37 8C under con-

ditions as described in Table 1. Results were presented

as mean logarithmic decrease in bacterial concentra-

tion in function of the concentration of SPH. To

achieve this, logarithmic bacterial concentrations

were subtracted from the concentration in the control

samples without SPH. Mean logarithmic decrease in

bacterial concentration and standard deviation were

calculated for the different incubates and plotted.

2.2.3. Live/Dead analysis

For Live/Dead analysis, a Cyan flow cytometer

(DakoCytomation, Glostrup, Denmark) was used,

equipped with a 50 mW solid state blue Sapphire

laser (488 nm) as the excitation light source. The

Live/DeadR BacLightk bacterial viability kit (L-

13152, Molecular Probes, Eugene, Oregon) was

applied for staining the bacteria. A stock solution of

the SYTO 9 and propidium iodide nucleic acid stains

was prepared by diluting both stains in 5 ml of filter

sterilized sheath fluid (DakoCytomation, Glostrup,

Denmark). From this, a staining solution was made

by diluting the stock solution 1/10.

After incubation, the same samples as used for

plating were tenfold diluted in filter sterilised saline.

Aliquots of 500 Al cell suspensions and 500 Al stain-ing solution were mixed and incubated for 20 min at

room temperature in the dark prior to analysis. Live

(+injured) and dead stained cells were discriminated

based on a FL1 (530/40 nm) vs. FL3 (613/20 nm)

excitation plot. Results were presented as mean loga-

rithmic decrease in function of the concentration of

SPH. For this, the ratio of live to dead cells was

re conditions for liquid cultures and media used for plate counts

Agar Time (h)

Luria-Bertani (A) 24

Tryptose Sulfite Cycloserine agar (AN) 72

Rogosa agar (AN) 72

(AN) Raffinose-Bifidobacterium (AN) 24

ith N2 (AN).

S. Possemiers et al. / International Journal of Food Microbiology 105 (2005) 59–7062

calculated and divided by the ratio of the control

sample without SPH. After logarithmic transforma-

tion, mean logarithmic decrease in Live/Dead ratio

and standard deviation were calculated for the differ-

ent incubates and plotted.

2.3. Simulator of the human intestinal microbial

ecosystem (SHIME)

The reactor setup was based on the adult SHIME

(De Boever et al., 2000; Molly et al., 1993) with this

difference that only the first colon reactor was used.

The SHIME was designed to maintain a microbial

community that is representative for that in the

human intestine. In this setup, the SHIME consisted

of three reactors that simulate the stomach and duo-

denum (reactor 1), jejunum and ileum (reactor 2) and

the ascending colon (reactor 3). Reactor design, inocu-

lum preparation, reactor startup and feeding scheme

were previously described by De Boever et al. (2000).

After a microbial community stabilization period of 3

weeks, the content of reactor 3 was split in two sepa-

rate reactors (250 ml), the experiment and control

reactor, to ensure identical microbial communities at

the beginning of the experiment. For a period of 11

days, SPH (AvantiR Polar Lipids, Alabaster, Alabama,

USA) diluted in 100% ethanol was daily added to the

experiment reactor in a final concentration of 50 AMand an equal amount of 100% ethanol was added to the

control reactor. On days 1, 3, 6, 8, 10 and 11 samples

were taken from the experiment and control reactor

and aerobic mesophilic bacteria, anaerobic mesophilic

bacteria, coliforms, Staphylococcus spp., Enterococ-

cus spp. and Clostridium spp. were plated (Table 2).

2.3.1. Effect of pH

To evaluate the influence of pH on bactericidal

activity of SPH, experiments were conducted with

Table 2

Analysed bacterial groups from the SHIME microbial community, to

Bacterial group Medium

Aerobic mesophilic bacteria Brain Heart Infusion agar

Anaerobic mesophilic bacteria Brain Heart Infusion agar+

Coliforms Mc. Conkey agar

Staphylococcus spp. Mannitol Salt agar

Enterococcus spp. Enterococcus agar

Clostridium spp. Tryptose Sulfite Cycloserin

E. coli (F106 CFU/ml) in buffered saline at a con-

centration of 25 AM SPH. With a 0.1 M phosphate, a

0.1 M BIS-TRIS and a 0.1 M TRIS buffer, pH was

respectively set to 5.5, 6, 7 and 8; to pH 5.5, 6 and 7

and to pH 7.5, 8 and 9. After 2 h of incubation, the

logarithmic effect of SPH was calculated as the

inverse value of the logarithmic decrease, as described

above, obtained by plating and Live/Dead analysis.

2.3.2. Effect of different medium components

To evaluate whether the medium in which the

experiment is conducted, influences the degree of

bactericidal capacity of SPH, an experiment was con-

ducted with E. coli (F106 CFU/ml) in Luria-Bertani

broth at a concentration of 25 AM SPH.

Saline containing different concentrations of glu-

cose, lactose, Tween 20, bovine serum albumin, stea-

ric acid or sphingomyelin was used for experiments to

examine the effect of individual medium components.

Sphingomyelin was obtained from AvantiR Polar

lipids, Inc. (Alabaster, Alabama), the other com-

pounds were purchased from Sigma-Aldrich (St.

Louis, MO). E. coli (F106 CFU/ml) was used as

test organism at a concentration of 25 AM SPH.

After 2 h incubation, the logarithmic effect of SPH

was calculated as the inverse value of the logarithmic

decrease, as described above, obtained by plating and

Live/Dead analysis.

2.3.3. Haemolytic activity

To 165 Al phosphate-buffered saline, 5 Al stock

solution (4 mM in absolute EtOH) of SPH or sphin-

gomyelin diluted in 100% ethanol was added in a final

concentration of 5, 10, 25, 50, 100, 200, 400, 600 and

800 AM SPH or sphingomyelin. After incubation for 1

min at 37 8C in a shaking water bath, 30 Al of redblood cells of sheep origin (10% suspension, ICN,

Brussels, Belgium) were added. Simultaneously, red

gether with the isolation media and incubation conditions used

Condition Time (h)

Aerobic 24

0.5 g l�1 cystein Anaerobic 72

Aerobic 24

Aerobic 48

Aerobic 48

e agar Anaerobic 72

S. Possemiers et al. / International Journal of Food Microbiology 105 (2005) 59–70 63

blood cells were incubated in phosphate-buffered sal-

ine (0% lysis) and in double-distilled water (100%

lysis). To take possible effects of ethanol into account,

5 Al 100% ethanol was added to the latter samples.

Samples were centrifuged for 1 min at 10,000�g

after incubation for 2 h at 37 8C. The supernatant

was diluted two times in phosphate-buffered saline

and percentage haemolysis was determined by mea-

suring the absorption at 540 nm with a Biokinetics

EL312e multi-well reader (Bio-Tek Instruments Eur-

ope, Spijkenisse, Nederland) (Van Der Meer et al.,

1991). Dose activity curves were generated for doses

of membrane lipid (abscissa) versus haemolytic activ-

ity (ordinate). The data were fitted by a 4 parametric

logistic model using the Marquardt-Levenberg algo-

rithm (SigmaPlot 8.0, Systat Software Inc., Rich-

mond, California). The IC50 value was obtained

from the parameters of the fitted function.

y ¼ minþ max�min

1þ 10tlogIC50�xbhillslope:

In this equation, y represents the haemolyitic activ-

ity (%) and x represents the logarithm of the concen-

tration membrane lipid (AM). Parameter min equals

Fig. 1. Bacterial susceptibility in saline to increasing concentrations of sph

Dead analysis combined with flow cytometry (2 and 4).

the baseline of 0% haemolysis, max is the plateau of

100% activity. Parameter IC50 gives the transition

center. The hillslope determines the slope of the

curve at the transition center.

2.4. Statistical analysis

All values are reported as meanF standard error of

the mean (nmin=3). To investigate whether the addi-

tion of individual medium components and pH

changes significantly influenced the bactericidal activ-

ity of SPH, an independent Student’s t-test was per-

formed ( p b0.05).

3. Results

3.1. Bactericidal effect in saline

Three intestinal pathogens (E. coli, E. aerogenes

BE1 and C. perfringens) and four health promoting

species (L. acidophilus, L. amylovorus, B. longum

and B. bifidum) were tested for susceptibility to

SPH (Fig. 1), in order to evaluate the host range of

ingosine (SPH) as monitored using plate counts (1 and 3) and Live/

Fig. 2. Bactericidal activity of sphingosine (SPH) towards E. coli in

saline (E) and Luria-Bertani broth (n) as monitored using plate

counts and Live/Dead analysis combined with flow cytometry.

S. Possemiers et al. / International Journal of Food Microbiology 105 (2005) 59–7064

the antibacterial agent and the selectivity towards

pathogens. Plate counts and Live/Dead analysis gave

similar results. All tested strains were strongly

reduced (N4 log units) at 150 AM. In graph 3, the

apparent difference between the tested lactobacilli and

bifidobacteria is due to a difference in detection limit

rather than a difference in sensitivity. Lactobacilli are

spread on top of the plates (100 Al/plate), giving a

detection limit of 100 CFU/ml; for bifidobacteria

pouring plates are used (1 ml/plate) with a detection

limit of 10 CFU/ml. At 25 AM E. coli was reduced the

most and L. acidophilus the least, but in general no

real differences in the effect of SPH were noted. This

means SPH was active against Gram-positive as well

as Gram-negative bacteria, to pathogens as well as

health promoting bacteria. At equivalent concentra-

tions, neither ceramide nor sphingomyelin exerted

antibacterial activity against the tested species (data

not shown).

3.2. Antibacterial effect under complex conditions

When the bactericidal effect of SPH on E. coli in

saline was compared with the activity in Luria-Bertani

broth (Fig. 2), inhibition of the activity clearly

occurred in Luria-Bertani broth. While a 4 log reduc-

tion was seen in saline at 25 AM using plate counts as

well as Live/Dead analysis, almost no decrease in

bacterial concentration occurred in Luria-Bertani

broth.

To assess the antibacterial activity of SPH in com-

plex environments like the intestinal tract, a final con-

centration of 50 AM SPH was dosed to the experiment

reactor of the modified SHIME system for a period of

11 days. However, no difference in the evolution of the

concentrations of the aerobic mesophilic bacteria,

anaerobic mesophilic bacteria, coliforms, Staphylo-

coccus spp., Enterococcus spp. and Clostridium spp.

were detected between the experiment and control

reactor, indicating that SPH exerted no bactericidal

activity under simulated intestinal conditions.

3.3. Influence of individual medium components

To assess the origin of this inhibitory effect of

environmental conditions, the influence of specific

medium components was investigated. Different con-

centrations of individual medium components were

added to saline and the experiment was repeated (Fig.

3). Even at a concentration of 25 g/l, glucose and

lactose did not inhibit the capacity of SPH to decrease

bacterial concentrations, indicating that medium car-

bohydrates do not cause the noted matrix effects.

Proteins on the other hand, interact with SPH, when

supplied as bovine serum albumin (BSA), and dis-

played a concentration dependent inhibitory effect. At

5 g/l, BSA already decreased the effect by 50% and at

25 g/l no activity of SPH was seen anymore. Sphin-

gomyelin, a membrane lipid and containing the SPH

backbone, also influenced the activity of SPH at low

concentration. At 120 AM sphingomyelin, SPH only

showed a minor activity. Stearic acid and the surfac-

tant Tween 20 finally, showed no inhibitory effect

until a threshold concentration of respectively 14

AM and 0.4 mM was reached.

Live/Dead analysis proved to be a good alternative

to plate counts to monitor the antibacterial effect of

SPH. Both techniques showed the same trends

describing the effect of individual medium compo-

nents. This was confirmed after calculating the overall

correlation between the two techniques for these

Fig. 3. Effect of individual medium components on the antibacterial effect of 25 AM sphingosine (SPH) towards E. coli, detected with plate

counts (black) and Live/Dead analysis (dashed). Data are presented as log effect, being the logarithmic decrease in bacterial concentration

(plating) or Live/Dead ratio (Live/Dead analysis). Significant effects are presented with *p b0.05, difference compared with control.

S. Possemiers et al. / International Journal of Food Microbiology 105 (2005) 59–70 65

experiments. Correlation ranged between 0.83 and

0.89, showing good similarity.

3.4. Influence of abiotic environmental conditions

Because abiotic environmental conditions can also

influence the degree of antibacterial activity, the influ-

ence of oxygen presence, temperature and pH was

evaluated. Experiments performed under aerobic or

anaerobic conditions with 25 AM SPH, either with

aerobically or anaerobically grown E. coli cultures,

gave no significant differences ( p N0.05; data not

shown) which indicates little or no influence of oxy-

gen presence. Temperature however had a major influ-

ence on the activity of SPH. When the in vitro assay

was performed at 4 8C, no significant decrease in

bacterial concentration was noted ( p N0.05; data not

shown). This means that SPH was not active at this

temperature.

Buffered saline, set at different pH values, was

used to investigate the influence of pH on the anti-

bacterial activity of SPH. E. coli was used as test

0

20

40

60

80

100

5 10 25 50 100 200 400 600 800

Concentration ( M)

% ly

sis

SPH

SM

Fig. 5. Haemolytic activity of sphingosine (SPH) and sphingomye-

lin in phosphate-buffered saline after 2 h incubation at 37 8C. ForSPH, a 50% inhibitory concentration (IC50) of 174 AM was calcu-

lated, while for sphingomyelin the IC50 was never reached within

the range of tested concentrations.

S. Possemiers et al. / International Journal of Food Microbiology 105 (2005) 59–7066

organism and was incubated at 37 8C for 2 h with 25

AM SPH (Fig. 4). Using BIS-TRIS and TRIS buffers,

high and constant bactericidal activity was seen at the

normal pH range of the large intestine (5.5–7), but at

pH 8 and 9 the effect rapidly decreased with one log

unit per pH unit. However, when a phosphate buffer

was used to set the pH from 5.5 to 8, almost no

activity of SPH was seen in comparison with the

other buffers, indicating a negative effect of phosphate

on the antibacterial activity of SPH.

3.5. Haemolytic activity of sphingosine

To investigate whether the antibacterial activity of

SPH is based on membrane disruption, the haemolytic

activity of different concentrations of SPH and sphin-

gomyelin was tested in saline under the same incuba-

tion conditions as the in vitro assay to monitor

bactericidal activity. For 2 h, red blood cells were

Live/Dead

0

1

2

3

4

5

5,5 6 9pH

Lo

g e

ffec

t

BIS-TRIS

TRIS

Phosphate

Plating

0

1

2

3

4

5

Lo

g e

ffec

t

**

**

7 8

5,5 6 9pH7 8

BIS-TRIS

TRIS

Phosphate

Fig. 4. Plate counts and Live/Dead analysis of the activity of 25 AMSPH towards E. coli in pH-buffered saline using a BIS-TRIS, TRIS

or phosphate buffer. Data are presented as log effect, being the

logarithmic decrease in bacterial concentration (plating) or Live/

Dead ratio (Live/Dead analysis). With the phosphate buffer only

very small effects were noted. Using the TRIS-based buffers, a

significant decrease in effect was seen at high pH (*p b0.05,

difference compared with saline at pH 7).

incubated with increasing concentrations of SPH or

sphingomyelin (Fig. 5). The baseline of 0% lysis and

the maximum of 100% lysis were set using PBS buffer

and double distilled water respectively, with 5 Alethanol added as control. While SPH induced strong

haemolysis at increasing concentrations, sphingomye-

lin showed only minor activity at very high concentra-

tion. As calculated with SigmaPlot 8.0, the IC50 of

SPH was 174 AM. While 800 AM SPH induced 100%

lysis, sphingomyelin did not even give 20% lysis at

this concentration and the IC50 was never reached.

4. Discussion

The intestinal microbial community plays a signif-

icant role in processes as food digestion, bioconver-

sion of endogenous or exogenous compounds,

immunomodulation, and prevention from infection

by intestinal pathogens (Gibson and Roberfroid,

1995; Hart et al., 2002). Therefore, strategies improv-

ing the composition towards beneficial bacteria have

gained much attention. One way of reaching this,

could be preventing the colonisation of the gut by

harmful bacteria like food pathogens. Sprong et al.

(2001, 2002) proposed bovine fat milk components to

be important for this purpose. Based on the strong

bactericidal activity towards pathogens in saline, they

stated that sphingolipids like SPH may enhance the

resistance to certain food-borne intestinal infections.

This strong activity was in good correlation with our

Fig. 6. Structure of sphingosine (SPH). For bactericidal activity

SPH has to be positively charged.

S. Possemiers et al. / International Journal of Food Microbiology 105 (2005) 59–70 67

results. In saline, all tested strains, Gram-positives and

Gram-negatives, were very susceptible to SPH after a

short incubation period. However, when tested under

more realistic matrix conditions, no bactericidal activ-

ity was detected. Tests with individual medium com-

ponents showed that SPH is very sensitive to

inhibition and therefore not active in complex

matrices like in the intestine. Based on the combina-

tion of our results, the mode of action of SPH would

include electrostatic attraction and disruption of the

bacterial membrane.

The first reports on the importance of SPH as an

antimicrobial component originate from skin research.

Based on in vitro and in vivo research, Bibel et al.

(1992a, 1993) stated that SPH could have an impor-

tant barrier function protecting the human skin from

colonisation by pathogens. SPH would act as a broad

spectrum antimicrobial agent with antibacterial as

well as antifungal activity. Antibacterial activity was

mainly restricted to Gram-positive bacteria, with no

effect towards E. coli (Bibel et al., 1992b). Because

sphingolipids are present in many foods, including

soy and dairy products (Ahn and Schroeder, 2002;

Molkentin, 2000), and because intestinal metabolism

of sphingomyelin towards SPH has been shown in

vitro and in vivo (Liu et al., 2000; Nyberg et al.,

1997), SPH could also be important to protect against

intestinal colonisation by pathogens. Therefore,

Sprong et al. (2001) investigated the susceptibility

of a number of food pathogens to increasing concen-

trations of SPH. Yet, in contrast with the results of

Bibel et al. (1992a), Gram-positive as well as Gram-

negative bacteria, including E. coli, were sensitive to

low concentrations. Our results are in good concor-

dance with the results obtained by Sprong et al.

(2001), with a similar decrease in concentration of

E. coli when tested in saline at 25 AM SPH (F4 log

CFU/ml). This strain even was the most sensitive of

all tested strains. Any differences in species sensitivity

may be due to differences in cell wall composition,

like the outer membrane, because SPH needs to reach

the cytoplasmic membrane to exert its activity (Gia-

cometti et al., 1999; Ishikawa et al., 2002). In general,

in these experiments only small differences in sensi-

tivity occurred, with no selectivity towards Gram-

positive or Gram-negative bacteria. Important to

notice is that SPH was also active against health

promoting bacteria like lactobacilli and bifidobacteria.

Bibel et al. (1992a) formulated the hypothesis that

a protein-kinase system analogous to mammalian cells

is involved in the antibacterial activity of SPH. This

was based on the fact that they observed calcium

dependency, which could imply enzyme mediation.

However, neither in our experiments nor in the experi-

ments of Sprong et al. (2001), any necessity of cal-

cium was detected. On the contrary, the following

arguments indicate the mode of action is primary

based on disruption of cell membrane integrity.

Firstly, the activity of SPH rapidly decreases above

pH 8 (Fig. 4). Because the amino group of SPH has a

pKa of 7.99, with deprotonation at higher pH, the pH

effect indicates this group has to be protonated for

activity (Fig. 6). Being positively charged, SPH shows

high structural similarity with quaternary ammonium

compounds (QACs). The mode of action of the latter

is based on adsorption to the cell surface, diffusion

through the cell wall and disruption of the cytoplasmic

membrane. This probably leads to secondary leakage

of metabolites and enzymes, killing the bacteria (Mer-

ianos, 2001; Paulus, 1993). Next to QACs, high

structural similarity occurs with cationic peptides,

which also act on bacterial membranes (Giacometti

et al., 1999). Secondly, in this research we used the

Live/DeadR BacLight bacterial viability kit which has

been extensively used to study different topics, such

as permeabilization of cheese starters using mutano-

lysin (Bunthof et al., 2001), disinfectant activity of

quaternary ammonium components (Langsrud and

Sundheim, 1996) and antibiotics like phosphomycin

(Jacobsen et al., 1997). All compounds applied in

these studies have in common that their mode of

action implies membrane disruption and the fact that

the kit was shown to be efficient to study the activity

of SPH, could imply a similar mode of action. Thirdly,

SPH exhibited a very rapid antimicrobial effect. When

E. coli was incubated with 25 AM SPH for 20 min,

already a 3.5 log reduction was noted (data not

shown), which indicated a rapid attack of the bacterial

,

S. Possemiers et al. / International Journal of Food Microbiology 105 (2005) 59–7068

integrity rather than influencing an enzymatic system.

This conclusion was also stated for QACs (Ahlstrom

et al., 1999; Russell et al., 1999) and cationic peptides

(Giacometti et al., 1999). Finally, SPH has a high

surface activity. To test membrane damaging activity,

a haemolysis test is often performed (Gandhi and

Cherian, 2000; Soderlind et al., 2003; Takechi et al.,

2003). Any lysis of the erythrocytes used in this test

can be attributed to membrane damaging effects

because these cells lack organelles and metabolic

systems (De Boever and Verstraete, 1999). The low

IC50 of SPH (174 AM) in comparison with for

instance bile salts (IC50~1–5 mM) (De Boever and

Verstraete, 1999) here also suggests membrane dis-

rupting activity. These arguments lead to the hypoth-

esis that the positively charged SPH is attracted to the

bacterial cell, which is negatively charged at physio-

logical pH, by electrostatic attraction and hydrophobic

interactions and after reaching the cytoplasmic mem-

brane, the long hydrophobic chain penetrates into the

lipid bilayer, forming channels. The reduced activity

at 4 8C can then be due to the lower membrane

fluidity and the concomitant inability to perforate

the lipid bilayer.

Despite the strong activity of SPH in saline, the

hypothesis as proposed by Sprong et al. (2001), being

a bactericidal effect of SPH in the human intestine,

does not seem to hold. When tested in a matrix

simulating the intestinal conditions, no effect of 50

AM SPH was detected, which suggests a lack of

activity under intestinal conditions. Experiments

with individual medium components showed a very

high sensitivity of SPH to the presence of matrix

components. This is also seen for other antibacterial

compounds like QACs, and based on the structure and

the proposed mode of action of SPH, most of this

inhibition can be explained. BSA molecules are glob-

ular proteins with hydrophilic and hydrophobic

domains, having a net negative charge at pH 7. In

case of QACs, a rapid interaction with the hydrophilic

regions occurs, followed by hydrophobic interactions

of the tail structure (Ahlstrom et al., 1999; Merianos,

2001). The same interaction could happen between

SPH and BSA or in general with negatively charged

structures, inhibiting the attraction to the bacteria and

subsequent bactericidal activity. The importance of

this effect is confirmed by the fact that, almost no

activity of SPH was seen using a negatively charged

phosphate buffer, whereas in a positively charged

buffer the activity only decreased above pH 8. The

latter could also be an explanation for the fact that

Bibel et al. (1992a) observed a necessity of Ca2+. This

ion could neutralize the negative charges of the pep-

tone buffer and thereby reduce the inhibitory effects

rather than the fact it would be necessary for the

bactericidal activity of SPH. Non-ionic and anionic

surfactants are known to have antagonistic effects on

the activity of cationic surface active agents, when

dosed in concentrations higher than the critical micelle

concentration (CMC), by inclusion of active ingredi-

ents in the micelles or by complex formation (Paulus,

1993). As the CMC of Tween 20 is about 0.06 mM,

this explains the strong inhibitive effect from a con-

centration of 0.08 mM. Stearate also forms micelles

with negatively charged surfaces, thereby attracting

SPH. Again, the effect starts above the CMC of

stearate (F6 AM) (Cawthern et al., 1997). Finally,

negatively charged phospholipids like sphingomyelin

are known to inhibit the activity of QACs (Merianos,

2001), which was also the case for SPH.

As it is now accepted that foods like dairy products

contain a number of health promoting components,

there is an increasing interest in the relation between

nutrition and human health (Molkentin, 2000; Wal-

zem et al., 2002). In this context, sphingolipids are

very important, having a number of positive effects

like the prevention of cancer and hypercholesterole-

mia and the inhibition of bacterial adhesion (Vesper et

al., 1999). Because SPH has been shown to exert

strong and rapid antimicrobial effects towards a

large variety of species, it could act as a natural

antimicrobial agent. As no activity was seen in the

complex chemical matrix of the SHIME, the combi-

nation of the factors interacting with SPH, as men-

tioned above, limit the application of SPH as an active

antimicrobial compound in the human intestine, espe-

cially because colonic concentrations of SPH are

shown to be in the range of only a few AM (Nyberg

et al., 1997). However, many arguments favor further

research on this topic. For instance, the potential for

SPH as topical antibiotic should be further explored as

antibacterial activity on the human skin has been

shown. SPH could also be very useful as protective

coating material on body implants or as preservative

in defined preparations like pharmaceuticals. Further-

more, in this research we tested the most commonly

S. Possemiers et al. / International Journal of Food Microbiology 105 (2005) 59–70 69

present SPH in dairy products, but over 60 different

backbones are known, such as phytosphingosine or

sphinganine (Karlsson, 1970). Their little structural

differences could lead to different activities or stabi-

lity. Therefore, the applicability of sphingosines as

antibacterial agents needs further investigation.

Acknowledgements

This work was supported by a PhD grant (aspirant)

for Sam Possemiers from the Fund for Scientific

Research-Flanders (Fonds voor Wetenschappelijk

Onderzoek (FWO) Vlaanderen).

The authors gratefully thank Els Jolie for technical

assistance and Nico Boon, Karel Decroos, Tom Van

de Wiele and Kristof Verthe for critically reviewing

this manuscript. Special thanks go to Hans Nelis for

intellectual assistance about surface active agents.

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