Indian Journal of Experimental Biology

Vol. 48, April 2010, pp. 407-414

Antibacterial effects of goat and chicken heart tissues against
human pathogenic bacteria
M Sundaramoorthy &

T S Saravanan
*

Department of Biotechnology, ARJ College of Engineering & Technology, Mannargudi, India 614 001
and
PG & Research Department of Zoology, Jamal Mohamed College, Tiruchirappalli, India, 620 020
Received 14 May 2009; revised 17 November 2009

The crude buffer (Tris Buffer Saline-I) extracts of muscles, liver, kidney and heart of goat and chicken (White leghorn)
were screened against 16 clinical isolates. Among the five tissues, the heart tissue of each animal showed significant
bactericidal activities on many isolates. The acid extracted crude proteins of both heart tissues also showed significant
antibacterial activities against many bacterial isolates. The crude proteins of goat heart tissues displayed strong bactericidal
activities against Salmonella paratyphi A and Salmonella typhimurium (MIC: 16 g/ml) whereas the crude proteins of
chicken heart tissues displayed strong bactericidal activities against Escherichia coli ATCC and Pseudomonas aeruginosa at
16 and 63 g/ml concentrations respectively. The peptides of low molecular weight (< 30 kDa) were also separated from the
acid extracted crude proteins of goat and chicken heart tissues by SDS-PAGE after staining with silver nitrate solution.
Keywords: Antibacterial, Bactericidal, Chicken, Goat, Heart Tissue, Pathogenic bacteria
The emergence of multiple drug resistant bacteria has
created alarming clinical situations in the treatment of
infectious diseases. This has rekindled interest in the
therapeutic use of certain natural products along with
modern medicines. Human beings, since time
immemorial have been using herbs and plant products
as medicine for developing immunity or resistance
against cold, fever and pain. The antimicrobial
properties of different plants have been reported
1-7
.
Recent research has been focused on finding out
natural antibiotics from various tissues of
invertebrates and vertebrates since the immune
system of multi-cellular organisms includes peptides
that protect them from a broad range of microbes.
Meister et al.
8
and Gallo and Huttner
9
have reported
that the higher organisms up-regulate their production
level of native antimicrobial peptides (AMP) upon
injury or bacterial infection. Antimicrobial peptides
are either constitutive or inducible, serve as crucible
components in the innate host defenses and represent
a potential source of useful natural antibiotics for
pharmaceutical applications
10,11
and are widely
distributed from plants and lower invertebrates to
higher animals including human beings and tend to be
found in those parts of animals that are most likely to
come into contact with pathogens from the
environment
12
.
These are mostly low molecular weight (< 30 kDa)
cationic antimicrobial peptides including defensins
and cathelicidins. AMPs hold significant potential
activities as therapeutic agents for prophylaxis and
treatment of infections, promotion of wound healing
and immune modulation
13
. According to Brogden
14
, a
number of antimicrobial peptides have been reported
in many tissues and cell types in a variety of plant and
animal species including cattle and birds. However,
there is paucity of information regarding
antimicrobial proteins/peptides of consumable
animals like goat and chicken tissues.
Therefore, in the present study, an attempt has been
made to screen the tissue extracts of muscles, liver,
kidney and heart of goat and chicken for their
antibacterial effects against certain selective human
pathogenic bacteria like Escherichia coli,
Staphylococcus aureus, Proteus mirabilis, Klebsiella
pneumonia, Pseudomonas aeruginosa and Salmonella
sps. and to substantiate the presence antibacterial
proteins/peptides in the tissues showing bactericidal
properties to focus our idea to generate natural
antimicrobial proteins/peptides in bulk at cheaper cost
from the goat and chicken like domestic animals.

*Correspondent author
Mobile: 919894421487
E-mail: drtssaravanan50@yahoo.com; thiruver@gmail.com
INDIAN J EXP BIOL, APRIL 2010


408
Materials and Methods
The antibacterial effects of the different tissue
extracts of goat (Capra hircus) and chicken (Gallus
gallus) were tested in triplicates against the bacterial
isolates by agar dilution method
15
. This method is an
established technique and is convenient for testing a
number of isolates simultaneously.
Preparation of tissue extractsThe muscles, liver,
kidney and heart tissues of goat and chicken were
collected from a local slaughter house at
Tiruchirappalli. The tissues were washed with water
and then with TBS- I buffer. After washing, the
tissues were homogenized using the same buffer
individually at a concentration of 100 mg wet wt/ml.
The extracts were collected by centrifugation at
12,000 rpm and physically sterilized by membrane
filter (0.45 m pore size) and stored in sealed
vials at 20
o
C until use.
Collection of bacterial isolatesThe bacterial
isolates like E. coli, S. aureus, P. mirabilis, K.
pneumoniae, S. typhi, S. paratyphi A, S.
typhimurium and P. aerugenosa, along with E .coli
ATCC and P. aerugenosa ATCC strains were
collected from Microbiology Laboratory, KAPV
Medical College, Tiruchirappalli. The isolates were
also confirmed by gram staining and standard
biochemical tests. Then they were maintained in
semisolid nutrient agar medium.
Preparation of culture platesThe Muller Hinton
Agar (MHA) was prepared by boiling 3.8 g MHA in
100 ml distilled water. Eighteen ml of this media was
aliquoted in screw capped bottles to a required
number and autoclaved at 121
o
C for 20 min and then
allowed to cool approximately to 50
o
C. Tissue
extracts (2 ml) was mixed with the medium at sterile
condition. Immediately the medium containing tissue
extract was plated and allowed to solidify. Then the
plates were stored at 4
o
C until inoculation.
Antibacterial assayThe organisms from the
semisolid media were sub-cultured for mid
logarithmic phase. The growths were suspended in the
1% peptone water and the turbidity was adjusted to
1.5 10
7
CFU/ml using 0.5 Mc Farland standard
(prepared by mixing 0.05 ml of 1.175% BaCl
2
and
9.95 ml of 1% H
2
SO
4
to make a total volume of
10 ml). They were further diluted to 20 times with the
1% peptone water. Finally the diluted organisms were
inoculated by spotting (16 at a time in a single plate)
on to the respective plates using a device like Steer
replicator. The inoculated plates were incubated at
37C overnight, and the growth inhibition of the
organisms by the tissue extracts was determined and
photographed. The absence of bacterial colonies in
any spotted place indicated the growth inhibition of
the respective bacteria by the tissue extract. Thus the
tissues showing antibacterial activity were identified.
Preparation of crude antimicrobial proteinsThe
crude antimicrobial proteins were prepared from the
tissues (heart) showing antibacterial activities by acid
extraction as described by Matutte et al
14
. The other
aliquots of the goat and chicken heart tissues collected
earlier were cut into small pieces on wet ice. The
pieces were washed with water and then with TBS-I
buffer repeatedly. The cleaned tissues were
immediately placed in liquid nitrogen, and after 24 h,
the frozen tissues were pulverized with a mortar and
pestle. The pulverized powder was placed in a boiling
10% (v/v) acetic acid for 10 min. The solution was
allowed to cool to room temperature and centrifuged
at 12,000 rpm for 30 min at 10
o
C. The supernatant
containing the crude proteins was concentrated by
lyophilization and stored at -20
o
C until use.
Minimum inhibitory concentrations (MICs
of crude antimicrobial proteins (microdilution
method)The MICs of the crude proteins of goat and
chicken heart were determined against seven clinical
isolates (E. coli ATCC, P. mirabilis, S. aureus,
P. aeruginosa ATCC, S. typhi, S. paratyphi A and
S. typhimurium) which were sensitive to the buffer
extracts of the heart tissues. This test was performed
in a 96 micro well plate as described by Zhu et al.
17
.
The isolates were sub-cultured on the Muller Hinton
Agar (MHA) plates. The individual colonies were
suspended in 1% sterile peptone water and the
turbidity of the suspension was adjusted to 2 10
6
Colony Forming Units (CFU). A stock solution of
crude protein was prepared at a concentration of
2000 g/ml in phosphate buffer saline (pH 7). From
this stock solution, two fold serial dilutions of the
proteins were made with 1% peptone water so as to
make the highest concentration of 500 g/ml and the
lowest concentration of 16 g/ml in the appropriate
rows of micro well plate. Then aliquots of 100 l of
the bacterial suspension were added in 100 l protein
solution. After 24 h of incubation at 37C, the
bacterial growth was assessed by measuring the
absorbance at 620 nm using ELISA auto reader
(RT2100C). A positive control (growth inhibition
with classical combined antibiotics; cephataxime and
chloromphenicol at a concentration of 5 mg/ml, each)
and a negative control (bacterial growth without
SUNDARAMOORTHY & SARAVANAN: ANTIBACTERIAL EFFECTS OF TISSUE EXTRACTS AGAINST BACTERIA


409
antibiotics or proteins) were also performed along
with to set the base lines. The MIC of the proteins is
defined as the minimum protein concentration that
inhibits the bacterial growth significantly.
SDS-PAGE analysis of buffer and crude proteins of
heart tissuesFrom the buffer extracts and crude
proteins, 20 l of each heart tissue was subjected to
SDS-poly acrylamide gel electrophoresis on 15%
mini gels according to the standard protocols
18
. The
samples were heated for 3 min at 100C in sample
buffer (25% 1 M tris-HCl, pH 6.8; 4% SDS; 2%
-mercaptoethanol and 5% glycerol) and then were
run along with mid-range marker proteins. The gels
were fixed and the bands were visualized after silver
staining using UV lamp. Finally the molecular
weights of the bands were determined using
Gel.doc.system.

Results
Among the different tissues of goat and chicken
screened for their antibacterial activities, only the
heart tissues of the both animals showed activities
against most of the organisms (E. coli, S. aureus.,
P. mirabilis, P. aeruginosa of pus and urine samples,
Salmonella sps., of blood sample along with E. coli
and P. aeruginosa ATTC standard strains).
Antibacterial activities of goat tissuesThe goat
heart tissue extract inhibited the growth of four
organisms, E. coli 527/PC, P. mirabilis 159/UC,
S. typhimurium 144/BC and P. aeruginosa ATCC
where as the other tissues did not show bactericidal
activities on any of the organisms mentioned above
(Table 1, Fig. 1).
Antibacterial activities of chicken tissuesThe
chicken heart tissue showed bactericidal activity
against eight bacterial isolates viz. E. coli 337/UC,
E. coli 527/PC, P. mirabilis 105/PC, P. mirabilis
159/UC, S. typhi 31/BC, S. typhi 40/BC, S.
typhimurium 144/BC and P. aeruginosa ATCC
(Table 1, Fig. 2). There were no antibacterial
activities for other tissues.
MICs of crude proteinsTo determine the MIC,
the organisms were allowed to grow with different
concentrations of the crude antimicrobial proteins.
After the specific incubation period, the absorbance of
the turbidity was measured, which was corresponding
to the growth of the respective bacterial isolate. The
absorbance of the test wells were also compared with
positive (presence of growth) and negative (growth
inhibition by known antibiotics) controls.
Goat: The MIC of the goat heart crude proteins was
found to be 250 g/ml for four bacterial isolates
(E. coli ATCC, P. mirabilis, P. aeruginosa ATCC and
S. thphi) while it was found to be 16 g/ml for two
isolates, S. paratyphi A and S. tyhimurium
(Table 2, Fig. 3a).
Table 1Antibacterial activity of goat and chicken tissues against
different human pathogenic bacteria
Sr.No Bacterial
isolates


Tissue type
Muscles Liver Kidney Heart
1 E. coli ATCC
25922
G + + + +
C + + + +
2 E. coli 337/UC G + + + +
C + + + -
3 E. coli 527/PC G + + + -
C + + + -
4 S. aureus
489/UC
G + + + +
C + + + +
5 S. aureus
398/PC
G + + + +
C + + + +
6 P. mirabilis
526/PC
G + + + +
C + + + +
7 P. mirabilis
105/PC
G + + + +
C + + + -
8 P. mirabilis
159/UC
G + + + -
C + + + -
9 K. pneumoniae
471/UC
G + + + +
C + + + +
10 K. pneumoniae
146 /BC
G + + + +
C + + + +
11 S. typhi 31/BC G + + + +
C + + + -
12 S. typhi 40/BC G + + + +
C + + + -
13 S. paratyphi A
32/BC
G + + + +
C + + + +
14 S. typhimurium
(144 /BC)
G + + + -
C + + + -
15 P. aeruginosa
ATCC 27853
G + + + -
C + + + -
16 P. aeruginosa
528/UC
G + + + +
C + + + +
G = Goat
C = Chicken
(+) = Growth of bacteria;
(- ) = Inhibition of growth
INDIAN J EXP BIOL, APRIL 2010


410
Chicken: The MIC of chicken heart crude proteins
was found to be 250 g/ml for the isolates, S. aureus,
S. thphi and S. tyhimurium (Table 2, Fig. 3b). The
growth of other two organisms, P. mirabilis and
S. paratyphi A was inhibited at 125 g/ml
concentration where as the MICs were found to be
16 and 63 g/ml for E. coli ATCC and P. aeruginosa
ATCC respectively. Thus this protein has strong
antibacterial activity against E. coli ATCC.
SDS-PAGE analysisThe low molecular weight
peptides below 30 kDa were also identified from the
both buffer extracts and acid extracts (crude proteins)
of goat and chicken heart tissues by SDS-PAGE after
the silver staining (Fig. 4). Five bands of 12, 14, 16,
18 and 25 kDa and four bands of 12, 14, 16 and
25 kDa peptides were observed in the goat and
chicken heart tissues respectively along with the some
higher molecular weight protein bands (>30 kDa) in


Fig. 1 Antibacterial activity of goat tissues against different human pathogenic bacteria 1. E. coli ATCC 25922; 2. E. coli 337/UC; 3.
E. coli 527/PC; 4. S. aureus 489/UC; 5. S. aureus 398/PC; 6. P. mirabilis 526/PC; 7. P. mirabilis 105/PC; 8. P. mirabilis 159/UC; 9.
K. pneumonia 471/UC; 10. K. pneumonia 146 /BC; 11. S. typhi 31/BC; 12. S. typhi 40/BC; 13. S. paratyphi A 32/BC; 14.
S. typhimurium 144 /BC; 15. P. aeruginosa ATCC 27853 and 16. P. aeruginosa 528/UC.

SUNDARAMOORTHY & SARAVANAN: ANTIBACTERIAL EFFECTS OF TISSUE EXTRACTS AGAINST BACTERIA


411
the both buffer and acid extracts. Further, the number
of bands was found to be reduced in the acid extracts
than the buffer extracts which suggest that the partial
purification of low molecular weight proteins by acid
extraction.

Discussion
In this study, among the different tissues tested,
only the heart tissues of both goat and chicken
showed antibacterial activities on E. coli, S. aureus.,
P. mirabilis., P. aeruginosa., S. typhi and
S. typhimurium with some tissue specific variations
and this suggests the presence of certain antibacterial
agents in their heart tissues. The acid extract (crude
proteins) of goat and chicken heart tissues exhibited a
broad spectrum of antibacterial activity for both gram-
positive and gram-negative bacteria of human clinical
samples. The goat heart crude proteins showed
bactericidal activity against six gram-negative isolates
(E. coli ATCC, P. mirabilis, P. aeruginosa ATCC,
S. thphi, S. paratyphi A and S. tyhimurium) whereas
the chicken heart crude protein showed antibacterial
activity against all the same gram-negative bacteria
and with a gram-positive bacteria, S. aureus. The

Fig. 2 Antibacterial activity of chicken tissues against different human pathogenic bacteria

INDIAN J EXP BIOL, APRIL 2010


412
crude proteins of these heart tissues also displayed
potent antibacterial activities against some isolates
like S. paratyphi A and S. typhimurium and E. coli
ATTC even at the lowest concentration of 16 g/ml.
Li et al.
19
have also reported antimicrobial activity in
the acid extract of liver of white leghorn hens (Gallus
gallus). In fact these bactericidal substances may be
the low molecular weight antimicrobial proteins or
Table 2 MICs of goat and chicken heart crude proteins against different human pathogenic bacterial growth
Concentration of crude proteins (g/ml) versus absorptions
of bacterial growth
Bacterial isolates
500 250 125 63 32 16
Positive control
(growth inhibition
with common
antibiotics)
Negative
control(100% growth
with out peptide and
antibiotics)
G 0.997 1.004 1.181 1.154 1.112 1.111 0.539 1.016
E.coli ATCC 25922
C 0.649 0.674 0.672 0.718 0.774 0.849 0.372 0.909
G 0.910 0.978 1.006 1.090 1.055 1.075 0.553 1.048
P. mirabilis
C 0.646 0.673 0.714 0.741 0.724 0.714 0.434 0.731
G 1.083 1.113 1.177 1.120 1.166 1.322 0.662 1.021
S.aureus
C 0.672 0.738 0.785 0.775 0.755 0.809 0.452 0.741
G 0.805 0.810 0.859 0.880 0.904 0.866 0.302 0.854 P. aeruginosa
ATCC 27853 C 0.539 0.548 0.593 0.601 0.651 0.661 0.364 0.621
G 0.960 0.982 1.099 1.140 1.137 1.146 0.355 1.063
S. typhi
C 0.581 0.611 0.700 0.817 0.905 0.753 0.319 0.625
G 1.119 1.167 1.137 1.105 0.965 1.135 0.437 1.386
S. paratyphi A
C 0.880 0.912 0.964 1.016 1.005 0.935 0.345 0.987
G 1.024 0.933 1.065 0.933 0.906 1.172 0.356 1.223
S. typhimurium
C 0.750 0.735 0.828 0.856 0.825 0.869 0.308 0.805
G = Goat; C = Chicken



Fig. 3 MICs of (a) goat and (b) chicken heart crude proteins against different human pathogenic bacteria

SUNDARAMOORTHY & SARAVANAN: ANTIBACTERIAL EFFECTS OF TISSUE EXTRACTS AGAINST BACTERIA


413
peptides. The SDS-PAGE analysis of the buffer and
acid extracts of the heart tissues also revealed the
presence of protein bands below 30 kDa (Fig. 4).
There are number of evidences for the presence of
antimicrobial peptides in the vertebrate tissues. Both
lymphoid and nonlymphoid tissues (such as cells of the
mucosal surfaces of the respiratory or gastrointestinal
tract) as well as dermal glands of vertebrates produce
antimicrobial peptides
20
. The granular glands of frog
skin were shown to store secretory granules that
contain microbicidal peptides. Some of the most
studied multi-membered families of antimicrobial
peptides include the bombinins were isolated from the
European frog Bombinavariegate
21-23
and the
magainins from the African frog Xenopus laevis
24
.
Com et al.
25
have reported the alpha and beta defensins
in the testis and epididymis of rat and mouse. SMAP of
sheep myeloid cells was reported by Bagella et al.
26
.
Xiao et al.
27
have also reported the fowlicidin
antimicrobial peptides from chicken. Since the goat
and chicken heart tissues displayed potent and broad
spectrum of antibacterial effects against various human
pathogenic bacteria, the present communication will
lead to construct a foundation for the development of
natural antibiotics from the hearts of consumable
animals like goat and chicken.

Acknowledgement
Thanks are due to Prof. S. Dhanapul, Department
of Microbiology, KAPV Govt. Medical College,
Tiruchirappalli, for the test organisms and permission
to carry out the work in his laboratory.

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Fig. 4 SDS-PAGE analysis of goat and chicken heart tissues
[Lane 1, Molecular weight Markers. Goat heart proteins: Lane 2,
Buffer tissue extract; Lane 3, Crude proteins of acid extract.
Chicken heart proteins: Lane 4, Buffer tissue extract; Lane 5,
Crude proteins of acid extract.]
INDIAN J EXP BIOL, APRIL 2010


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