1. health and fitness
2. disease

Egyptian Journal for Aquaculture Vol.2 No.2 2012
In vitro the effects of Lactobacillus acidophilus cell free extract
and crab haemolymph serum as antagonizing Aeromonas
hydrophila and Vibrio alginolyticus
Mohammed E. Enany1, Mohamed E. Abou El-Atta2, and Mohamed M.
El.Tantawy2
1- Microbiology Dept., Faculty of Veterinary Medicine, Suez Canal University.
2- Fish Health Dept., Central laboratory for aquaculture, Abbassa, Abou Hammad,
Sharkia, Egypt.
Abstract
This study aimed to provide the in vitro effects of Lactobacillus
acidophilus cell free extract and blue crab haemolymph serum for
antagonizing growth of Aeromonas hydrophila and Vibrio
alginolyticus isolated from diseased blue crab in comparison with
florfenicol. Lactobacillus acidophilus was isolated from cattle milk
whey. It was found that Lactobacillus acidophilus cell free extract
and blue crab haemolymph serum had antibacterial effect against A.
hydrophila and V. alginolyticus. The inhibition zones of bacterial
growth were 12 and 16 mm in Lactobacillus acidophilus cell free
extract discs; 15 and 12 mm in crab haemolymph serum discs and 25
and 22 mm in Florfenicol discs with A. hydrophila and V.
alginolyticus, respectively, with a critical evaluation of results
obtained.
Key words: Lactobacillus acidophilus,
alginolyticus, crab, antibacterial.
Introduction
Diseases of fish are major
problems for the fish farming
industry, which currently is the fastest
growing
food-protein
producing
sector with an annual increase of
approximately 9%. Among those,
bacterial infections are considered as
the major cause of mortality in fish
hatcheries (Grisez and Ollevier,
1995). Attention focused on the
marine animals, production increased
in the later seasons as it plays a role in
environmental balance in marine
aquaria. Crustaceans, such as crabs,
shrimp and lobster, provide a high
quality protein; also, it contains
Aeromonas
hydrophila,
Vibrio
Omega 3 fatty acids that afford
potential health benefits (Wickins
and lee, 2002). Bacterial strains were
identical to Vibrio cholera. V.
vulnificus, V. parahaemolyticus and
others were routinely isolated from the
haemolymph and external carapace of
blue crab (Davis and Sizemore,
1982).
The prophylactic and therapeutic
controls of the bacterial diseases
mostly begin in vitro, their application
are based on the oral administration of
antibiotics or by injection in narrow
scale. However, such treatments may
cause excess lost of drugs, and the
development of resistant bacterial
63
In vitro the effects of Lactobacillus acidophilus cell free extract and crab haemolymph serum as
antagonizing Aeromonas hydrophila and Vibrio alginolyticus
strains (Aoki et al., 1985), yield
residues in the animal and introduce
potential hazard to public health and
to the environment. Furthermore, the
normal microbial flora in the digestive
tract, which is beneficial to fish, may
be killed or inhibited due to oral
chemotherapy (Sugita et al., 1991).
Therefore,
the
probiotics
or
haemolymph serum may be one of the
factors of solutions. This work aimed
to study the antibacterial activity of
Lactobacillus acidophilus cell free
extract and crab haemolymph serum
for antagonizing A. hydrophila and V.
alginolyticus bacteria in comparison
with the antibiotic Florfenicol in vitro
and the inhibition zone measured in
mm.
Materials and methods
Isolation
of
Lactobacillus
acidophilus from Cattle milk
Lactobacillus acidophilus was
isolated from cattle milk whey
according to (Savadogo et al., 2004)
with some modifications. Cattle milk
was collected from the animal under
aseptic condition in previously
sterilized bottles and kept at room
temperature till formation of crud,
sieved and collected in sterile test
tubes and incubated at 26oC for 72
hrs, since whey a suitable medium for
the growth of its content of lactic acid
bacteria. The isolates were platted out
onto a specific broth medium, MRS
(De Man, Rogosa and Sharpe, 1960)
then streaked on MRS agar to obtain
separate colonies; the pure colonies
were kept on slope of MRS medium
in 4oC for identification according to
(Bergey et al., 2004).
Preparation
of
Lactobacillus
acidophilus cell free extract
64
Propagation
was
performed
according to Ajitha, et al. (2004).
Bacteria were grown aseptically in 10
ml of nutrient broth for 24 hrs at room
temperature (28 ± 2°C). Five ml of
log phase culture was then transferred
under aseptic conditions into 250 ml
of MRS broth and placed on a rotary
shaker at 150 rpm for 24 hrs at 28 ±
2°C. The bacterial strain was
harvested by centrifuging at 10,000
rpm under aseptic conditions for 15
min at 4°C. The cell free extract was
saved, filtered and sterilized through
Celtron filters of 0.2 mm pore size
(Merck). PH adjusted to 6.8 by means
of one mole NaOH. The cell free
extract was stored at -70oC till use.
Blue crab haemolymph serum
Haemolymph was collected from
blue crabs according to Noga et al.
(1996 a&b) by inserting a 22 G
needle attached to a 3 ml syringe into
the arthrodial membrane of the
swimming leg and gently aspirating
into the syringe. The collecting
haemolymph kept in Eppindorf tubes
at 4oC till clot completed. Frozen
haemolymph was kept at room
temperature to allow homogenize and
break up the clot. It centrifuged at
50,000 rpm for 20 min. The
haemolymph serum was pooled and
stored at -70oC until use.
Bacterial strains
Aeromonas hydrophilia and Vibrio
alginolyticus were previously isolated
from diseased blue crab. A swap from
TSA (Treptic Soya agar) slants of
bacterial strains under test (A.
hydrophilia and V. alginolyticus,) were
inoculated in nutrient broth (tube for
each strain) and incubated at 28-30oC
overnight then subculture on TSA
Enany et al.,
medium supplemented with 0.5%
sodium chloride.
acidophilus isolated from Cattle milk
was shown in Table (1), photo (1).
Antibacterial activity
The
results
revealed
that
Lactobacillus acidophilus cell free
extract and blue crab haemolymph
serum had antibacterial effect against
Aeromonas hydrophila and Vibrio
alginolyticus. This was obvious by a
clear inhibition zone with no growth
of bacteria around the test discs. This
inhibition zones measured in mm and
found as 12 and 16 mm in
Lactobacillus acidophilus cell free
extract discs; 15 and 12 mm in blue
crab haemolymph serum discs, and 25
and 22 in Florfenicol discs,
respectively, while normal saline
impregnated discs showed bacterial
growth in the entire circumference of
TSA around the disc as shown in table
(2) and photo (2)
Agar disc diffusion method of
Bauer et al (1996) was used for the
assessment of antibacterial activity.
Filter papers discs of 4 mm diameter
were kept in aluminum foil placed in
screw caped glass bottle and sterilized
in autoclave; filter papers take three
symbols (L, H and C) as abbreviations
to Lactobacillus acidophilus cell free
extract, blue crab haemolymph serum
and control, while Florfenicol discs
took the symbol (F). The sterilized
filter paper discs with symbol (L)
were impregnated with sufficient
amount of Lactobacillus acidophilus
cell free extract over night, discs with
symbol (H) were impregnated with
sufficient amount of blue crab
haemolymph serum for 2 h, and discs
with symbol C were impregnated with
sufficient amount of sterile saline,
(negative control). All discs were air
dried under aseptic precautions.
Florfenicol discs, Ffc 30 µ g was also
used (positive control). Agar plates
were prepared, one plate seeded with
A. hydrophilia and the second with V.
alginolyticus.
Under
aseptic
conditions, on the surface of each
plate, four discs were gently fixed (L,
H, Ffc and C) and were incubated at
28-30oC overnight and the results
were recorded. Antibacterial activity
was expressed in terms of diameter of
inhibition zone in mm. Inhibition was
indicated by the absence of bacterial
growth around the test discs and were
obvious around the sterile disc.
Results
The biophysical and biochemical
characteristics
of
Lactobacillus
Discussion
Probiotics are simply means “for
life”, originating from the Greek
words “pro” and “bios” (Gismondo et
al., 1999). Today probiotics are quite
commonplace in health promoting
“functional foods” for humans, as
well as therapeutic, prophylactic and
growth supplements in animal
production
and
human
health
(Mombelli and Gismondo, 2000;
Ouwehand et al., 2002; Sullivan and
Nord, 2002; Senok et al., 2005).
The trial for controlling the
pathogenic
microorganisms
was
carried out by the use of safe, cheap,
available, with no any harmful effect
if it compared to that produced due to
the chemotherapeutics use of drugs, it
is the probiotic bacteria. By disc
diffusion inhibition it was found that
the inhibition zone diameter of lactic
acid
bacteria
(Lactobacillus
65
In vitro the effects of Lactobacillus acidophilus cell free extract and crab haemolymph serum as
antagonizing Aeromonas hydrophila and Vibrio alginolyticus
acidophilus) isolated from cattle milk
whey was found 12 and 16 mm
against Aeromonas hydrophila and
Vibrio alginolyticus, respectively. We
conclude
that
Lactobacillus
acidophilus was effective against the
isolated microorganisms, first, by the
antagonism to pathogens that shown
in vitro. Second, by competition with
pathogens for nutrients or for
adhesion sites and this agree with the
results performed by Ajitha et al.
(2004) who studied the effect of
Lactic acid bacteria (LAB) in vitro on
some bacterial pathogens and cell free
extracts of four strains of Lactic acid
bacteria (LAB) viz. Lactobacillus
acidophilus, Streptococcus cremoris,
Lactobacillus bulgaricus –56 and
Lactobacillus bulgaricus –57 found to
be inhibitor for growths of Vibrio
alginolyticus in nutrient broth.
Antagonism of LAB against Vibrio
alginolyticus confirmed by streak
plating and suppression of growth of
Vibrio was obtained. Shafiqur
Rahman et al. (2009) studied the
antibacterial activity of probiotic
bacteria in vitro by the well diffusion
assay where the cell-free extracts of 3day old cultures of candidate
probionts were added in pre-formed
wells, dug in Mueller-Hinton agar
media and previously swabbed with
target pathogens, revealed that a good
number of probionts challenged the
pathogens successfully as evidenced
by the production of clear zones of
inhibition against the growth of the
target pathogens. Probionts isolated
from Basal medium that selects the
growth of Bacillus were found most
efficient against Vibrios as they
yielded 20-30 mm zones of inhibition.
Nogami and Maeda (1992) rendered
the antibacterial activity to the
66
production of extracellular products
from these probiotic bacteria as rapid
acidification through the production
of organic acids, mainly lactic acid. In
addition, their production of acetic
acid, ethanol, aroma compounds,
bacteriocins, exopolysaccharides, and
several enzymes is of importance in
Lactic acid bacteria (LAB), De Vuyst
et al. (2004). Probiotics in aquaculture
have been shown to have several
modes
of
action:
competitive
exclusion of pathogenic bacteria
through the production of inhibitory
compounds; improvement of water
quality; enhancement of immune
response of host species and
enhancement of nutrition of host
species through the production of
supplemental
digestive
enzymes
(Thampson et al., 1999 and
Verschuere et al., 2000). Inhibition
of Vibrio by the cultures such as L.
acidophilus, obtained in this study is
in agreement with those obtained with
LAB culture filtrate and by LAB in
mixed culture against A. salmonicida
(Gildberg et al., 1995). The
haemolymph antibacterial activity as
results of some material produced
inside the humeral cells and released
outside the cells as a defense
mechanism against the invading
microorganisms and its non specific in
action, the same results were obtained
by Noga et al. (1996 a&b).
In general, crabs do not have a
specific immune response (i.e.
antibody or true lymphocytes) and
instead rely on relatively non-specific
broad-spectrum defenses such as
phagocytosis,
encapsulation
and
nonspecific
defensive
molecules
(Fries,
1984).
The
defense
mechanisms of crustaceans depend
completely on the innate immune
Enany et al.,
system that is activated when
pathogen-associated
molecular
patterns are recognized by soluble cell
surface host proteins such as lectins
that were antimicrobial, clotting and
pattern recognition proteins which in
turn activate cellular or humoral
effectors mechanisms to destroy
invading pathogens (Vazquez et al.,
2009). In crustaceans, circulating
hemocytes play significant roles in the
innate immune response, including
release of non self-recognition
proteins,
clotting
proteins,
antimicrobial
peptides
and
prophenoloxidase (So¨derha¨ll and
Cerenius 1998; Terwilliger, 1999).
Haemolymph of the brachyuran
crustacean
Callinectes
sapidus
possesses bactericidal activity which
is highly inhibitory to Gram-negative
bacteria cultured from blue crab
carapace,
including
Aeromonas
hydrophila, Vibrio parahemolyticus,
V. alginolyticus and V. vulnificus.
Several strains of Escherichia coli
were also susceptible, no lysozymelike
activity
detected.
The
antibacterial activity appeared to be
confined to the haemocytes. Initial
investigations revealed that this
antibacterial
activity
was
proteinaceous
(inactivated
by
proteolysis) and was found mainly
within the hemocytes (Noga et al.,
1996 a &b).
Conclusion: It was found that
chemotherapeutic materials has more
potent antibacterial activity than
natural materials since the control
positive disc of Florfenicol was wider
in diameter of inhibition against
Aeromonas hydrophila and Vibrio
alginolyticus ,while crab haemolymph
serum antibacterial activities need
more for studying as separation of its
constituents
by
electric
chromatography and study each band
in a separate manner and decide
which band would be more effective
against several pathogenic bacteria
then a biochemical analysis must be
done to stand on its chemical
composition before their use in a
commercial. The use of probiotic
materials as cell free supernatants of
some bacteria as Lactobacillus
acidophilus is easily to be obtained in
a manufactured product and is safe for
human and aquaculture as well as it
does not play any role in mutations
and resistance produced by the misuse
of the whole bacterial cells.
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69
In vitro the effects of Lactobacillus acidophilus cell free extract and crab haemolymph serum as
antagonizing Aeromonas hydrophila and Vibrio alginolyticus
Table (1): Physical and biochemical characteristics of Lactobacillus acidophilus
isolated from Cattle milk.
Items
Shape and arrangement
Gram staining
Motility
Growth at 10oC
Growth at 45oC
Catalase test
Growth in 4% NaCl
Growth in 6.5% NaCl
0.3% methylene blue
Reaction
Rods, pairs chains
Gram positive
Non motile
+
+
+
+
Items
Fructose
Glycerol
Sucrose
Maltose
Lactose
Mannitol
Glucose
Galactose
Reaction
+
+
+
+
+
+
Photo (1): Lactobacillus acidophilus Gram-positive reaction and bacilli shape from researching
microscope using the oil-emersion lens (X= 1000).
70
Enany et al.,
Table (2): Inhibition zones (mm) diameters due to antibacterial activity of blue crab
haemolymph serum, Lactobacillus acidophilus cell free extract and
Florfenicol against A. hydrophila and V. anguillyseptica on TSA.
Disc type
Flofenicol disc
Haemolymph serum disc
Lactobacillus acidophilus
Control (sterile saline)
Inhibition zones diameter (mm)
Aeromonas hydrophila Vibrio alginolyticus
25
22
15
12
12
16
0
0
Photo (2): In vitro antibacterial activity of Lactobacillus acidophilus cell free extract (L) and blue crab
haemolymph serum (H) against Aeromonas hydrophila and Vibrio alginolyticus comprised with
Florfenecol (F). C=control negative, A h= A. hydrophila and V a=V. anguillyseptica.
71
‫‪In vitro the effects of Lactobacillus acidophilus cell free extract and crab haemolymph serum as‬‬
‫‪antagonizing Aeromonas hydrophila and Vibrio alginolyticus‬‬
‫استخدام مستخلص الالكتوباسيلس اسيدوفيلس الخالى من الخاليا و سيرم هيموليمف‬
‫الكابوريا ضد االيروموناس هيدروفيال و الفيبريوالجينوليتكس معمليا‬
‫محمد السيد عنانى‪ ، 1‬محمد السيد أبو العطا‪ ، 2‬محمد مصطفى الطنطاوى‬
‫‪2‬‬
‫‪ - 1‬قسم البكتريولوجى – كلية الطب البيطرى ‪ -‬جامعة قناة السويس‪.‬‬
‫‪ - 2‬قسم صحة ورعاية األسماك – المعمل المركزى لبحوث الثروة السمكية ‪ -‬العباسة‬
‫الملخص العربى‬
‫تهدف هذه الدراسة الى توفر استخدام البروبيوتك فى المعمل الستعداء بعض‬
‫المعزولة من الكابوريا المريضة‪ .‬لقد وجد أن مسضتخل‬
‫مسضبباا االمضرال البكتيريضة‬
‫الككتوباسضيلس اسضيدوفيلس المعزولضة مضن شضرل ألبضان‬
‫الجاموس و الخالى من الخكيا و سيرم هيموليمف الكابوريا له نشاط مضاد لبكتريا االيرومونضاس هيضدروفيك و‬
‫الفيبريوالجينضضوليتكس وتضضم قيضضاس قطضضر منطقضضة التثبضضي‬
‫بضضالمم وكان ض ‪ 12‬و‪ 11‬مضضم ف ض‬
‫الككتوباسيلس اسيدوفيلس الخالى من الخكيا‪11،‬و‪ 12‬مم فى اقرا‬
‫فى اقرا‬
‫أقضضرا‬
‫مسضضتخل‬
‫سضيرم هيموليمضف الكابوريضا و‪21‬و‪ 22‬مضم‬
‫الفلورفينيكول على التوال مع اجراء تقييم للنتائج الت تم الحصول عليها‪.‬‬
‫‪72‬‬