Rapp. Comm. int. Mer Médit., 37,2004
273
SURVIVAL STUDY OF LISTERIA MONOCYTOGENES IN SEAWATER
Elmnasser N.* and Bakhrouf A.
Laboratoire d’Analyse et de Contrôle des Polluants Chimiques et Microbiologiques de l’Environnement.
Faculté de Pharmacie. Rue Avicenne. 5000 Monastir, Tunisie - *
mnassernoura@yahoo.fr
Abstract
We studied the behaviour of Listeria monocytogenesin sterile seawater. Recuperation of this bacterium on Tryptone Soya Agar shows that
it can survive in sterile seawater for a long period of time in atypical colonies. Characters permitting the differentiation of Listeria
monocytogenesand Listeria innocuaare then modified.
Key words: Listeria monocytogenes, survival, seawater, adaptation.
Introduction
Until the late 1970s, it was generally assumed that Listeria
monocytogenesdoes not survive in seawater (1). This view has been
challenged by the finding that this bacterium can form a dormant life
stage. L. monocytogenesis distributed widely in many natural
environments (2). The success of L. monocytogenesas a pathogen
seems to be related to its ability to adapt to its environment (3). The
purpose of this work was to determined the morphologicaland
metabolic modifications of L.monocytogenesgrowing in seawater.
Material and methods
Bacteria strain and cultural media
This study was carried out with Listeria monocytogenesstrain
isolated at the INSERM unity 452. Bacteria are recupered on
Tryptone Soya Broth supplemented with yeast extract (0.6 %) (TSB)
at 37°C for 24 h.
Growth agar was Tryptone Soya Agar supplemented with yeast
extract (0.6 %) (TSA), or prepared with seawater (TSAM).
Starvation
Cells were grown for 24 h at 37°C and then washed three times in
normal saline solution after centrifugation. The final pellet was
suspended in 200 ml of filtered seawater.
Survival and recuperation tests
Culturability was assayed by spread plate counts. Serially diluted
samples (0.1 ml) in sterilized seawater were spread in triplicate on
plate media. After 24 to 48 h of incubation at 37°C, Colony-Forming
Units (CFU) at appropriate dilutions were counted.
Biochemical profile of cells
The phenotypic profile was determined by Api listeria galleries.
Statistical analysis
Each point on the curves for enumeration of culturable cells
presented in this study represents an average of 3 petri dishes.
Statistical analysis was made by the Anova test carried out with the
help of Program Stat View
TM
512
+
.
Results
Quantitative variation analysis of L. monocytogenes incubated in
microcosm seawater, showed a decrease by 99 % of CFU/ml after 24h
of incubation in the seawater on TSA and TSAEM. The culturable
forms continue to decrease until five weeks.
After one month in seawater microcosm,L. monocytogenes
exhibited cultural modifications. Yellow pigmented, orange
pigmented colonies appeared. Atypical colonies were cocci-rod forms
with positive Gram, positive catalase and negative oxydase.
After one month of incubation in seawater, biochemical characters
modifications are noted in the atypical colonies after their isolation
from seawater microcosm (Table 1).
Table 1. L. monocytogenes biochemical characters before and after incu-
bation in the seawater.
DIM
: Differentiation L. innocua / L. monocytogenes;
ESC
: Esculine;
?
MAN
: ?-Mannosidase;
DARL
: D-Arbitol;
XYL
: D-Xylose;
MDG
: ?-Methyl-D-
Glucoside;
RIB
: Ribose;
G1P
: Glucose-1-Phosphate;
TAG
: D-Tagatose; ():
modified characters of atypical colonies compared with the typical
colonies.
The enzyme that allows the differentiation between L. innocua and
L. monocytogenes (DIM on Api Listeria) changed. The yellow
colonies lost their rhamnose enzyme and orange colonies lost their
mannosidase activity.
Discussion and conclusion
In this work we showed that L. monocytogenessurvives in the
seawater for a long period under cultivable forms. Subsequently, the
cells may be evolve towards viable but non cultivable forms as
recently demonstrated by Besnard et al. (4).
After one month of L. monocytogenessurvival in seawater, we have
observed yellow and orange pigmented colonies. It was shown that 60
% of marine bacteria are pigmented (5). This pigmentation varied
from yellow to red (6).
We showed that rods develop towards coccoid forms. This result
agrees with morphological modifications described also to occur in L.
monocytogenessubjected to high hydrostatic pressure (7). A
morphological changes of cells and/or colonies can be explain by
biochemical modifications of the envelopes of bacteria (8).
The results of ApiListeria tests show that the modifications in the
phenotypic profile of the stressed colonies involved essentially the key
characters of L. monocytogenesdetermination. The instability of
enzymes cause problems in the characterisation of species isolated
from the environment.
In an oligotrophic aquatic environment and at low organic matter
concentrations, L. monocytogenescan adapt to environmental stress.
This adaptation involves, biochemical and morphological
modifications generally used in taxonomy.
References
1-Aubert M., Gauthier M., and Daniel S., 1981. Les systèmes
d’information des microorganismes marins. Rev. Int. Océanogr. Méd., 60-
61: 37-106.
2-Farber J.M., and Peterkin P.I., 1991. Listeria monocytogenes, a food-
borne pathogen. Microbiol. Rev., 55: 476-511.
3-Rouquette C., T.Ripio M., Pellegrini E., M.Bolla J., L.Tascon R.,
Vasquez-Boland J.A., and Berche P., 1996. Identification of a CLpC
ATPase required for stress tolerance and in vivo survival of
L.monocytogenes. Mol. Microbiol., 21: 977-987.
4-Besnard B., Federighi M., Declerq E., Jugiau F., and Cappelier J.M.,
2002. Environmental and physico-chemical factors induce Vbnc state in
Listeria monocytogenes. Vet. Res., 33: 359-370.
5-Rheinheimer B., 1974. Aquatic microbiology. John Wiley and Sons,
London, pp. 80-82.
6-Tsyban A.V., 1971. Marine bacterioneustron. J. Oceanogr. Soc. Jap.,
27: 56-66.
7-Ritz M., Tholozan J.L., Federighi M., and Pilet M.F., 2001.
Morphological and physiological characterization of Listeria
monocytogenessubjected to high hydrostatic pressure. Appl. Environ.
Microbiol.,67: 2240-2247
8-Csonka L.N., and Hanson A.D., 1991. Prpkaryotic osmoregulation:
genetics and physiology. Ann. Rev. Microbiol., 45: 569-606.
