Anti-bacterial Effect of Caffeine on Escherichia coli and Pseudomonas fluorescens

Anti-bacterial Effect of Caffeine on Escherichia coli and Pseudomonas fluorescens
Anti-bacterial Effect of Caffeine on Escherichia coli
and Pseudomonas fluorescens
Almira Ramanavièienë1,
The aim of this study was to investigate the direct effect of caffeine on
Viktoras Mostovojus2,
Gram-negative bacteria cultures. For this purpose we have selected Escheri-
Irina Bachmatova3,
chia coli as widely distributed in the environment and within human intesti-
nes, and Pseudomonas fluorescens as commonly found in a wide range of
Arûnas Ramanavièius 2,4
terrestrial and aquatic habitats.
Our investigations showed that caffeine affects the growth of E. coli DH5?
1 Laboratory of Ecological
and Ps. fluorescens 5443 bacteria. At high a caffeine concentration (1%) in
Immunology, Institute of
the culture medium the growth of both bacteria was inhibited. The optical
Immunology of Vilnius University,
density of E. coli and Ps. fluorescens cultures decreased 2.7 and 3.1 times
Molëtø pl. 29,
(p < 0.001), respectively, in comparison with the control samples, where these
LT-2021 Vilnius, Lithuania
bacteria were growing in the culture medium without any caffeine. Caffeine
2 Department of Analytical and
concentration decrease influenced the growth of bacteria in a different way.
Environmental Chemistry,
The growth of E. coli was inhibited more significantly as compared to Ps.
Vilnius University,
fluorescens at caffeine concentrations 0.1-0.5%.
Naugarduko 22,
Different concentrations of E. coli DH5? and Ps. fluorescens grew on a
LT-2006 Vilnius, Lithuania
solid medium without or with 1% of caffeine. It was shown that caffeine
3 Department of Molecular
present in the solid medium suppressed the growth of E. coli and Ps. fluo-
Microbiology and Biotechnology,
rescens bacteria 6.4 and 5.3 times (p < 0.05), respectively.
Our findings show an antibacterial effect of caffeine. In addition to it, the
Institute of Biochemistry,
sensitivity of different bacteria to caffeine can vary greatly depending on
Mokslininkø 12,
caffeine concentration. Further, E.coli bacteria are more sensitive to caffeine
LT-2600 Vilnius, Lithuania
in comparison to Ps. fluorescens bacteria.
4 Sector of Immunoanalysis and
Informatics, Institute of
Key words: caffeine, E. coli, Ps. fluorescens
Immunology of
Vilnius University,
Molëtø pl. 29,
LT-2021 Vilnius, Lithuania
INTRODUCTION
pathways involved in the cellular response to DNA
damage. It reduces DNA damage-induced cell cycle
Caffeine (1,3,7-trimethylxanthine) at submillimolar
arrested in G , S, and G /M, abolishing the G /M
concentrations exerts a wide variety of physiological
1
2
2
checkpoint by inhibiting ATM (ataxia-telangiectasia-
effects on different organisms (1). Caffeine has long
mutated) kinase activity (3, 4). Caffeine potentiates
been known to have numerous actions (2), including
the lethal effects of ionizing radiation, which could
(i) inhibition of phosphodiesterases, thereby increas-
be useful for cancer therapy (5). It also plays an
ing intracellular cAMP; (ii) direct effects on intra-
important role in the development of immune resis-
cellular calcium concentrations; (iii) indirect effects
tance against bacterial invaders by increasing the
on intracellular calcium concentrations via membra-
concentration of some immunocompetent cells and
ne hyperpolarization; and (iv) antagonism of adeno-
reinforcing the activity of lysozyme (6, 7). The an-
sine receptors. Caffeine also influences multiple
timicrobial activity of lysozyme is known to be di-
rected against certain Gram-positive bacteria and to
a lesser degree in vitro against Gram-negative bac-
Address corespondence to: A. Ramanavièienë, Institu-
teria (8, 9).
te of Immunology of Vilnius University, Molëtø pl. 29,
To expand our knowledge about the influence of
LT-2021 Vilnius, Lithuania. E-mail: almyra@imi.lt
caffeine on cells, some immune parameters and an-
ISSN 1392?0138. A c t a m e d i c a L i t u a n i c a . 2003. T. 10, Nr. 4
&#

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Almira Ramanavièienë, Viktoras Mostovojus, Irina Bachmatova, Arûnas Ramanavièius
timicrobial activity of lysozyme, we decided to in-
cose, 2% agar (control plates) medium and on the
vestigate the direct effect of caffeine on Gram-ne-
same solid medium with 1% of caffeine (experi-
gative bacteria culture. For this purpose we have
mental plates). For this experiment different dilu-
selected Escherichia coli DH5? as a model strain
tions of E. coli and Ps. fluorescens bacteria (10, 100,
frequently used in microbiological experiments, and
1000, 10000, 100000, 1000000) were prepared in phy-
Pseudomonas fluorescens 5443 for comparison and
siological solution, and 20 µl of each bacteria solu-
estimation of caffeine effect.
tion was poured into control and experimental pla-
E. coli bacteria are widely distributed in the en-
tes. Bacteria were growing at 30 °C for 48 hours.
vironment. The presence of E. coli and other spe-
Afterwards the plates were visually estimated and
cies of bacteria within our intestines is necessary
bacteria colonies counted. The pictures were taken
for us to develop and operate properly and to re-
by an Olympus C2020Z digital camera.
main healthy, as along with other species of bacte-
The data obtained in all tests were compared
ria, E. coli provide us with many necessary vitamins
with the control. Sigma Plot for Windows (8th de-
(10). However, some of these different strains of
mo version) Student?s t-test was used for determi-
bacteria (there may be several within given species)
nation of the significance (p < 0.05) and evaluation
can be harmful to human (11).
of the experiments.
Ps. fluorescens encompasses a diverse group of
bacteria that are commonly found in a wide range
RESULTS
of terrestrial and aquatic habitats. There are over
200 species of Pseudomonas bacteria. Some of them
Our investigations show that caffeine affects E. coli
are pathogenic to plants, animals and human (12).
DH5? and Ps. fluorescens 5443 strains. At a high
However, only three species are known to be patho-
caffeine concentration in the culture medium (1%)
genic for man; they are resistant to most antibiotics
both bacteria were growing at a comparable rate.
(13). Their main targets are immunocompromised
In the flask with E. coli, the optical density decre-
individuals, burn victims, and individuals on respira-
ased 2.7, while in the flask with Ps. fluorescens 3.1
tors or with indwelling catheters.
times (p < 0.001) in comparison with the control.
The aim of this study was to investigate the di-
The growth of Ps. fluorescens was practically not
rect effect of caffeine on bacteria (E. coli and Ps.
influenced by lower than 1% caffeine concentrations,
fluorescens). Our finding could be taken into ac-
the optical density increased at 0.5% caffeine up to
count in medical practice.
3.54 OD units and remained very similar at lower
caffeine concentrations if compared with control me-
MATERIALS AND METHODS
dia (Fig. 1). At 1% and 0.5% caffeine concentra-
tions, the optical density of E. coli was very similar
E. coli DH5? and Ps. fluorescens 5443 bacteria
and approximately 2.7 times lower (p < 0.01) if
strains were obtained from the Institute of Bioche-
compared with control samples. We also observed
mistry. For estimation and confirmation of
the results, bacteria were grown in a liquid
and on a solid medium. Two different esti-
5
mation methods were used and the experi-
Pseudom
P
onas f
seudomonas luorescens
fluorescens
Esherichia col
Escherichia i
coli
ment were repeated three times. These bac-
teria were growing in Nutrient Broth (NB)
4
culture medium.
To start the growth, 2 ml of overnight
3
***
cultured E. coli and Ps. fluorescens stocks
0 nm
were added to 100 ml of NB medium con-
60
taining 0.12 % of glucose with 0.01%, 0.1%,
OD
2
0.5%, 1% or without (in control flask) caf-
**
* *
feine (Sigma, St. Louis, USA), respectively.
The bacteria were aerobically cultured in
1
shaking flasks closed with cotton stoppers on
a rotary shaker at 30 °C for 24 hours. Op-
0
tical density (OD) measurements at 600 nm
0
0.01
0.1
0.5
1
were used to monitor the concentration of
Concentration of caffeine, %
bacteria.
Fig. 1. E. coli and Ps. fluorescens concentration dependence upon
The same bacteria strains also were grow-
different caffeine concentration in the culture medium. * p < 0.001,
ing on a solid NB containing 0.12% of glu-
** p < 0.01, *** p < 0.05 compared to the control samples
&$

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Anti-bacterial Effect of Caffeine on Escherichia coli and Pseudomonas fluorescens
that the concentration and optical density of E. coli
A
bacteria increased if caffeine concentration decreas-
ed, and at 0.01% caffeine in culture medium the
optical density became similar to the control (Fig. 1).
Different amounts of E. coli and Ps. fluorescens
were grown on solid medium without or with 1% of
caffeine. The largest and distinct bacteria colony was
observed at a 100000 time dilution. The visual estima-
tion and bacteria colony counting were performed at
this dilution. In Fig. 2 we can see a smaller amount of
E. coli bacteria colony on a solid medium with caffei-
ne (plate B) in comparison with the control (plate A).
In control plates we calculated 402 ± 75 bacteria co-
lonies and in experimental plates with 1% of caffeine
B
63 ± 19 bacteria colonies. Thereby, caffeine in the
solid medium suppresses the growth of bacteria colo-
nies 6.4 times (p < 0.05).
Ps. fluorescens bacteria grow better on a solid
medium with caffeine than E. coli, but in compari-
son with the control we observed a decreased
amount of bacteria colonies (Fig. 2). In control pla-
tes (plate C) we counted 1708 ± 205 bacteria co-
lonies and in experimental plates with 1% of caffei-
ne (plate D) 324 ± 32 bacteria colonies. Hence,
caffeine in the solid medium suppressed bacterial
growth 5.27 times (p < 0.05).
C
DISCUSSION
Caffeine plays an important role in the develop-
ment of immune resistance against bacterial inva-
ders by increasing the concentration of some immu-
nocompetent cells and reinforcing the lysozyme ac-
tivity (6, 7). The results of this work demonstrate
that caffeine has a direct antibacterial effect. We
observed that the same caffeine concentration af-
fects the growth of E. coli and Ps. fluorescens bac-
teria in a different way. Our findings show that E.
coli bacteria are more sensitive to caffeine than Ps.
fluorescens. A statistically significant caffeine effect
D
on E. coli was observed at a 0.1% caffeine concen-
tration; below this concentration caffeine had almost
no effect on the bacterial growth, whereas a statis-
tically significant effect of caffeine on Ps. fluores-
cens bacteria colony growth was observed only at a
1% concentration of caffeine. These data agree with
the results published by other authors that depen-
ding on the concentration caffeine has a different
effect not only on mammalian organisms (14, 15),
but also on different bacteria. Rocha GM et al. (16)
found that caffeine alone up to 0.02% had a low
effect on mouse renal inner medullary collected duct
cells. The results of other authors show that at caf-
Fig. 2. E. coli growing on solid medium without caffeine
feine concentrations from 0.01 to 0.2% the cytoto-
(A) and with 1% of caffeine (B). Ps. fluorescens growing
xic effect of ionizing radiation and other physical or
on solid medium without caffeine (C) and with 1% of
chemical agents is strongly enhanced (4, 5).
caffeine (D)
&%

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Almira Ramanavièienë, Viktoras Mostovojus, Irina Bachmatova, Arûnas Ramanavièius
The antibacterial effect of caffeine can have a
their basis character. J Applied Bacteriol 1992; 72:
positive or a negative impact on human health. Caf-
180?7.
feine destroys the beneficial bacteria in the colon
9. Cottagnoud P, Tomasz A. Triggering of pneumococcal
(17, 18). On the other hand, if some pathogenic
autolysis by lysozyme. J Inf Dis 1993; 167: 684?90.
bacteria prevail in the organism, caffeine intake can
10. http://people.ku.edu/%7Ejbrown/ecoli.html
11. Renwick SA, Wilson JB, Clarke RC, Lior H, Borczyck
have a positive and curative effect.
AA, Spilka J et al. Evidence of direct transmission of
E. coli O157:H7 infection between calves and a hu-
CONCLUSIONS
man. J Inf Dis 1993; 168: 792?3.
12. Brundza K. Mikrobiologija. Vilnius: Mintis. 1969.
Our findings show a clear antibacterial effect of caf-
13. Hacène H, Daoudi-Hamdad F, Bhatnagar T, Baratti
feine. The sensitivity of different bacteria to caffei-
JC, Lefebvre G. H107, a new aminoglycoside anti-
ne can vary greatly depending on caffeine concen-
Pseudomonas antibiotic produced by a new strain of
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could be taken into account in medical practice.
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ACKNOWLEDGMENT
in rats blocks a subsequent nicotine-conditioned taste
avoidance in a one-bottle, but not a two-bottle test.
This work was financially supported by Lithuanian
Pharmacol Biochem Beh 2001; 70: 279?89.
State Science and Studies Foundation project No.
16. Rocha GM, Michea LF, Peters EM, Kirby M, Xu Y,
Ferguson DR, Burg MB. Direct toxicity of nonsteroi-
C 03047 and COST Program Project No. 853.
dal antiinflammatory drugs for renal medullary cells.
Proc Natl Acad Sci USA 2001; 98: 5317?22.
Received 7 July 2003
17. ttp://www.californiaantiaging.com/download/Septem-
Accepted 19 September 2003
ber%20newsletter%20-%20CHI.doc
18. http://www.webnat.com/education/cllntestinalSystem.htm
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