Antimicrobial properties of skin mucus from four freshwater cultivable Fishes (Catla catla, Hypophthalmichthys molitrix, Labeo rohita and Ctenopharyngodon idella)

Abstract

The fishes are living in the medium rich in pathogenic microbes. The mucus secreted by the skin of fish showed more antimicrobial properties. The mucus collected from the two exotic fishes and two indigenous fishes were tested against the five pathogenic bacteria (Klebsiella pneumonia, Vibrio cholerae, Salmonella typhi, Escherichia coli and Pseudomonas aeruginosa) and five pathogenic fungi namely (Mucor globosus, Rhizopus arrhizus, Candida albicans, Aspergillus flavus and Aspergillus niger). The fishes are living in media rich in pathogenic microbes which secrete substances against them. The mucus secreted by the skin of fish showed more antimicrobial properties. More antibacterial and antifungal activity were observed in an indigenous fishes (Catla catla and Labeo rohita) than exotic fishes (Hypophthalmichthys molitrix and Ctenopharyngodon idella).

Full text

African Journal of Microbiology Research Vol. 6(24), pp. 5110-5120, 28 June, 2012
Available online at http://www.academicjournals.org/AJMR
DOI: 10.5897/AJMR11.532
ISSN 1996-0808 ©2012 Academic Journals
Full Length Research Paper
Antimicrobial properties of skin mucus from four
freshwater cultivable Fishes
(Catla catla, Hypophthalmichthys molitrix, Labeo rohita
and Ctenopharyngodon idella)
Balasubramanian S., Baby Rani P., Arul Prakash A., Prakash M.*, Senthilraja P.
and Gunasekaran G.
Department of Zoology, Annamalai University, Annamalai Nagar, Chidambaram-608002, TN, India.
Accepted 24 August, 2011
The fishes are living in the medium rich in pathogenic microbes. The mucus secreted by the skin of
fish showed more antimicrobial properties. The mucus collected from the two exotic fishes and two
indigenous fishes were tested against the five pathogenic bacteria (Klebsiella pneumonia, Vibrio
cholerae, Salmonella typhi, Escherichia coli and Pseudomonas aeruginosa) and five pathogenic fungi
namely (Mucor globosus, Rhizopus arrhizus, Candida albicans, Aspergillus flavus and Aspergillus
niger). The fishes are living in media rich in pathogenic microbes which secrete substances against
them. The mucus secreted by the skin of fish showed more antimicrobial properties. More antibacterial
and antifungal activity were observed in an indigenous fishes (Catla catla and Labeo rohita) than exotic
fishes (Hypophthalmichthys molitrix and Ctenopharyngodon idella).
Key words: Antibacterial, antifungal, fish mucus, exotic, indigenous fish.
INTRODUCTION
Chemicals from nature have been a part of human
civilization ever since our early ancestor’s began
exploiting natural compounds to improve and enrich their
own lives (Agosta, 1996). A major part of these chemicals
come from animals. Indeed, animals are therapeutic
arsenals that have been playing significant roles in the
healing processes, magic rituals, and religious practices
of peoples (Costa and Marques, 2000). All living
organisms including fish coexist with a wide range of
pathogenic and non- pathogenic microorganisms and
therefore, posses complex defense mechanisms which
contribute to their survival. One mechanism is the innate
immune system that combats pathogens from the
moment of their first contact (kimbrell and Beutler, 2001).
The specific immunity including antibody and specific
cell-mediated responses are significantly less diverse
*Corresponding author. E-mail: dnaprakash@gmail.com.
than those of higher (Ellis, 1974; Manning, 1998).
The development of resistance by a pathogen to many
of the commonly used antibiotics provides an impetus for
further attempts to search for new antimicrobial agents,
which overcome the problems of resistance and side
effects. Action must be taken to reduce this problem such
as controlling the use of antibiotics, carrying out research
to investigate drugs from natural sources. Drugs that can
either inhibit the growth of pathogen or kill them and have
no or least toxicity to the host cell are considered for
developing new antimicrobial drugs. It is well known that
the global trade in animal based medicinal products
accounts for billions of dollars per year (Kunin and
Lawton, 1996). Unlike conventional antibiotics, which are
synthesized enzymatically by microorganisms, are
encoded by a distinct gene (AMP) and made from an
mRNA template. The continuous use of antibiotics has
resulted in multi resistant bacterial strains all over the
world (Mainous and Pomeroy, 2001). Consequently,
there is an urgent need to search for alternatives

to synthetic antibiotics. In spite of modern improvements
in chemotherapeutic techniques, infectious diseases are
still an increasingly important public health issue (WHO,
2002). It has been estimated that in 2000, at least two
million people died from diarrhoeal disease worldwide
(WHO, 2002). Still there is a need for new methods of
reducing or eliminating pathogens, possibly in
combination with existing methods (Leistner, 1978). In
the aquatic environment, fish are in constant interaction
with a wide range of pathogenic and non-pathogenic
microorganisms (Subramanian et al., 2007).
Fish live in a challenging environment facing so many
problems. The microbes play a major role in affecting the
fish health. They escape from such an environment by
producing some substances on the dermal layer (Mucus).
The epidermal mucus produced primarily by epidermal
goblet or mucus cells are composed mainly of water and
gel forming macromolecules including mucins and other
glycoproteins (Shephard, 1993). The composition and
rate of mucus secretion has been observed to change in
response to microbial exposure or to environmental
perturbation such as hyperosmolarity and acidity
(Agarwal et al., 1979; Zuchelkowski et al., 1981; Ellis,
2001). The mucus substance secreted from the surface
of fish performs a number of functions including disease
resistance, respiration, ionic and osmotic regulation,
locomotion, reproduction, communication, feeding and
nest building (Ingram, 1980; Fletcher, 1978). Despite an
intimate contact with high concentrations of pathogens
(bacteria and viruses) in their environment, the fish can
still maintain a healthy system under normal conditions.
This could be attributed to a complex system of innate
defense mechanisms within themselves, particularly the
products of broad spectrum-antimicrobial compound.
Many researchers have proved that the mucus
substances are good resistant to invading pathogens
(Ingram, 1980; Fletcher, 1978; Austin and Mcintosh,
1988; Fouz et al., 1990).
Fish mucus (slime layer) is the first physical barrier that
inhibits entry of microbes from an environment into fish. It
acts as a chemical barrier containing enzymes and
antibodies which can kill invading disease causing
organisms (Rottmann et al., 1992). A fatty acid
compositional study of the flesh of Haruan (Channa
striatus) revealed those unusually high arachidonic acids,
but almost no eicosapentaenoic acids, which were
hypothesized to be actively involved in initiating tissue
wound repair (Mat Jais et al., 1994). Antimicrobial activity
in mucus has been demonstrated in several fish species
(Austin and Mcintosh, 1988), yet this activity seems to
vary from one fish species to the other and can be
specific towards certain bacteria (Noga et al., 1995).
When we are reviewing the literature among the fresh
water fishes the studies are available mostly on cold
water fishes.
Studies are available on C. striatus, Cyprinus carpio
(Cole et al., 1997) and Etheostoma crossopterum (Knouft
Balasubramanian et al. 5111
et al., 2003). Though studies are available on the
microbicidal activities of fish mucus they are pertaining
only against bacteria except a single study against fungi
(Hellio et al., 2002). Antimicrobial activity was
demonstrated in Channa punctatus and Cirrhinus mrigala
(Kuppulakshmi et al., 2008). Antimicrobial activity of skin
and intestinal mucus of five different freshwater fish
Channa species was studied by Dhanraj et al. (2009).
Apart from these no studies are available on the mucus
of the cultivable fresh water fishes like Catla catla, Labeo
rohita (Indigenous fishes), Hypophthalmichthys molitrix
and Ctenopharyngodon idella (Exotic fishes).
Hence it was decided to evaluate the bactericidal and
fungicidal properties of surface and column feeders
namely C. catla, H. molitrix, Labeo rohita and
Ctenopharyngodon idella. In the present investigation few
microbial species of bacteria such as, Klebsiella
pneumonia, Vibrio cholerae, Salmonella typhi,
Escherichia coli, Pesudomonas aeruginosa and fungi,
Mucur globosus, Rhizopus arrhizus, Candida albicans,
Aspergillus flavus and Aspergillus niger were selected.
MATERIALS AND METHODS
Collection of mucus
The healthy live fishes approximately 6 months old, weigh about
500 gms of each C. catla, L. rohita, H. molitrix C. idella were
purchased from near by fish farm in Pinnalur, Cuddalore District,
Tamil Nadu. Mucus was carefully scraped from the dorsal surface
of the body using a sterile spatula. Mucus was not collected in the
ventral side to avoid intestinal and sperm contamination. The
collected fish mucus was stored at 4ºC for further use.
Preparation of mucus sample for the antibacterial and
antifungal studies.
The mucus samples were collected aseptically from the fish and
thoroughly mixed with equal quantity of sterilized physiological
saline (0.85% NaCl) and centrifuged at 5000 rpm for 15 min, the
supernatant was used for the antimicrobial studies and kept at 4°C
until use.
A thin layer of molten agar (Muller Hinton Agar) was dispensed in
petriplates of 10 × 10 cm and was labled properly. Triplicates were
maintained for each strain. In the same way for fungal studies PDA
medium was dispensed in petriplates for different strains of fungi in
triplicates and the plates were marked.
Inoculation of bacterial strains
The microbial strains were collected from the Balaji High-tech
Laboratory in Manjakuppam, Cuddalore district, Tamil Nadu.
In vitro antibacterial assay was carried out by disc diffusion
technique (Bauer et al., 1996). Whatman No.1 filter paper discs
with 4 mm diameter were impregnated with known amount (10 µl)
of test sample of fish mucus and a standard antibiotic disc. At room
temperature (37ºC) the bacterial plates were incubated for 24 h.
The fungal plates were incubated at 30ºC for 3 to 5 days for
antifungal activity. The results were recorded by measuring the
zones of growth inhibition surrounding the disc. Clear inhibition
zones around the discs were expressed in terms of diameter of

5112 Afr. J. Microbiol. Res.
Table 1. Antibacterial activity of skin mucus from Catla and Silver carp.
S/N
Name of the Bacterial Pathogens
Zone of inhibition (in mm)
Control (Ciproflaxin)
(in mm)
Catla
Silver carp
1
K. pneumonia
25
22
24
2
V. cholerae
21
20
22
3
S. typhi
32
15
28
4
E. coli
23
16
22
5
P. aeruginosa
29
22
32
Table 2. Antibacterial activity of skin mucus from Rohu and Grass carp.
S/N
Name of the Bacterial Pathogens
Zone of Inhibition (in mm)
Control
(Ciproflaxin) (in mm)
Rohu
Grass carp
1
K. pneumonia
24
7
24
2
V. cholerae
21
7
22
3
S. typhi
14
12
28
4
E. coli
21
17
22
5
P. aeruginosa
19
15
32
zone of inhibition and were measured in mm using cm scale,
recorded and the average were tabulated.
Antimicrobial assay
The spectrum of antimicrobial activity was studied using five
different strains of human pathogenic bacteria and five species of
fungal pathogens. One antibiotic agent Ciproflaxin for pathogenic
bacteria and Ketoconazole for pathogenic fungi were used as
control.
RESULTS
Antimicrobial effect of the mucus of surface feeder and
column feeder freshwater fishes namely, C. catla, H.
molitrix (Surface feeder), L. rohita (Column feeder), C.
idella were tested against, pathogenic bacteria viz, K.
pneumonia, V. cholerae, S. typhi, E. coli, P. aeruginosa
and five pathogenic fungi viz, Mucor globosus, Rhizopus
arrhizus, Candida albicans, Aspergillus flavus,
Aspergillus niger. The activity was measured in terms of
zone of inhibition in mm.
Antibacterial effect of mucus from surface feeders
and column feeders
The inhibition effects of mucus of C. catla, H. molitrix
against five pathogenic bacterial strains are given in
Table 1 and the zone of inhibition by the mucus of L.
rohita, C. idella are given in Table 2. The zone of
inhibition values of mucus were compared with control
(Ciproflaxin) and the observed values are tabulated in
Tables 1 and 2, respectively.
The mucus of C. catla showed more effect in controlling
the growth of gram-negative bacteria Salmonella typhi
with an inhibition zone of 32 mm in diameter which is
more than the control (Figure 3). Next to S. typhi, the
mucus of C. catla showed a better effect on P.
aeruginosa having an inhibition zone of 29 mm in
diameter (Figure 5). That was followed by the K.
pneumonia with an inhibition zone of 25 mm in diameter
(Figure 1). Among the five gram-negative bacteria tested
V. cholerae and E. coli showed very less sensitivity to the
mucus of C. catla with an inhibition zone of 21 and 23
mm in diameter (Figures 2 and 4).
The mucus of H. molitrix showed more effect in
controlling the growth of K. pneumonia and P. aeruginosa
with an inhibition zone of 22 and 22 mm in diameter
(Figures 1 and 5). Moderate effect was observed in
controlling the growth of V. cholerae with a zone of
inhibition is 20 mm in diameter (Figure 3). S. typhi (15
mm) and E. coli (16 mm) showed very less sensitivity to
the mucus of H. molitrix (Figures 3 and 4).
Whereas the mucus of L. rohita showed a strong effect
in controlling the growth of K. pneumonia with an
inhibition zone of 24 mm diameter (Figure 1). V. cholerae
and E. coli showed a better effect in the mucus of H.
molitrix with an inhibition zone of 21 mm in diameter
(Figures 2 and 4). Among the five bacteria tested S.
typhi and P. aeruginosa showed less sensitivity to the
mucus with an inhibition zone is 14 mm and 19 mm in
diameter (Figures 3 and 5).
The mucus of C. idella showed more effect in
controlling the growth of E. coli with an inhibition zone of
17 mm diameter (Figure 4). The moderate effect was
observed in controlling the growth of S. typhi (12 mm)
and P. aeruginosa (15 mm) by the mucus of C. idella.

Balasubramanian et al. 5113
Figure 1. K. pneumonia antibacterial activity of fish skin mucus.
Figure 2. Vibrio cholera Antibacterial activity of fish skin mucus.
Among these, the K. pneumonia and V. cholerae showed
very less sensitivity to the mucus of C. idella with an
inhibition zone of 7 mm diameter (Figures 1 and 2).
The antibacterial activity of control Ciproflaxin showed
maximum activity in three bacteria and some bacteria it
showed less activity than the mucus sample.
Antifungal effect of mucus
The effect of mucus from C. catla, H. molitrix against five
pathogenic fungal strains are given in Table 3 and the
zone of inhibition by the mucus of L. rohita, C. idella are
given in Table 4. The zone of inhibition values of control
(Ketoconazole) are tabulated in Tables 3 and 4,
respectively. The mucus of C. catla showed a maximum
effect in controlling the growth of A. flavus with an
inhibition zone of 17 mm diameter which is less than the
control (19 mm) (Figure 9). Next to this, the Mucor
globosus (16 mm) and R. arrhizus (16 mm) have more
zone of inhibition (Figure 6 and Figure 7). Whereas as
the mucus of C. catla has less effect in controlling the
growth of C. albicans (14 mm) and A. niger (9 mm)
(Figures 8 and 10). Likewise the mucus collected from H.
molitrix has highest effect in controlling the growth of A.
flavus with an inhibition zone is 17 mm (Figure 9). On the
Figure 2. Vibrio cholerae

5114 Afr. J. Microbiol. Res.
Figure 3. Salmonella typhi Antibacterial activity of fish
skin mucus.
Figure 4. Escherichia coli Antibacterial activity of fish
skin mucus.
contrary there is no effect in controlling the growth of
Rhizopus sp and C. albicans. (Figures 7 and 8). The
mucus of H. molitrix shows the moderate effect in
controlling the growth of Mucor globosus (14 mm) and A.
niger (8 mm) (Figures 6 and 10).
L. rohita a column feeder showed more effect in
controlling the growth of A. flavus (17 mm). The mucus
of L. rohita has very less sensitivity against the growth of
M. globosus and R. arrhizus (14 mm) (Figures 6 and 7).
But the mucus of L. rohita has the better effect in
controlling the growth of C. albicans (15 mm) and A. niger
(15 mm) (Figures 8 and 10).
The mucus of C. idella shows the highest activity
against A. flavus with an inhibition zone of 16 mm in
diameter (Figure 9). Next to A. flavus the mucus shows
better effect in controlling the growth of M. globosus (15
mm), R. arrhizus (15 mm) and C. albicans (13 mm) in
diameter (Figures 6, 7 and 8). But it failed to control the
growth of A. niger (Figure 10).
The antifungal activity of control Ketoconazole showed
a variety of activity against M. globosus (16 mm), R.
arrhizus (15 mm), C. albicans (17 mm), A. flavus (19 mm)
Figure 3. Salmonella typhi
Figure 4. Escherichia coli

Balasubramanian et al. 5115
Figure 5. Pseudomonas aeruginosa Antibacterial
activity of fish skin mucus. Co – Control, C – Catla, R
– Rohu, S - Silver carp, G - Grass carp.
Table 3. Antifungal activity of skin mucus from Catla and Silver carp.
SNo
Name of the Fungal Pathogens
Zone of Inhibition (in mm)
Control
(Ketoconazole) (in mm)
Catla
Silver carp
1
M. globosus
16mm
14
16
2
R. arrhizus
16
-----
15
3
C. albicans
14
-----
17
4
A. flavus
17
17
19
5
A. niger
9
8
11
Table 4. Antifungal activity of skin mucus from Rohu and Grass carp.
SN
Name of the Fungal Pathogens
Zone of Inhibition (in mm)
Control
(Ketoconazole) (in mm)
Rohu
Grass carp
1
M. globosus
14
15
16
2
R. arrhizus
14
15
15
3
C. albicans
15
13
17
4
A. flavus
17
16
19
5
A. niger
15
----
11
and A. niger (11 mm), respectively.
DISCUSSION
The epithelial surfaces of fish, such as the skin, gills and
the alimentary tract provide first contact with potential
pathogens. The biological interface between fish and
their aqueous environment consists of a mucus layer
composing of biochemically-diverse secretions from
epidermal and epithelial cells (Ellis, 1999). This layer is
thought to act as a lubricant to have a mechanical
protective function, to be involved in osmoregulation and
play a possible role in immune system of fish. Fish tissue
and body fluids contain naturally occurring proteins or
glycoproteins of non-immunoglobulin nature that react
with a diverse array of environmental antigens and may
confer an undefined degree of natural immunity to fish.
Antimicrobial peptides are among the earliest developed
molecular effectors of innate immunity and are significant
Figure 5. Pseudomonas aeruginosa

5116 Afr. J. Microbiol. Res.
Figure 6. Mucor globosus Co – Control, C – Catla, R – Rohu,
S - Silver carp, G - Grass carp.
Figure 7. Rhizopus arrhizus Co – Control, C – Catla, R –
Rohu, S - Silver carp, G - Grass carp.
in the first line of host defense response of diverse
species.
Most antimicrobial peptides found through out the
animal and plant kingdom are small, functionally
specialized peptides (Boman, 1995). Several
endogenous peptides with antimicrobial activity from fish,
especially from the skin and skin mucus are reported
(Park et al., 1997). Endogenous peptides play an
important role in fish defense, possess broad spectrum of
antimicrobial activity against bacteria, yeast and fungi.
The epidermic and the epithelial mucus secretions act as
biological barriers between fish and the potential
Figure 6. Mucor globosus
Figure 7. Rhizopus arrhizus

Balasubramanian et al. 5117
Figure 8. Candida albicans Co – Control, C – Catla, R –
Rohu, S - Silver carp, G - Grass carp.
Figure 9. Aspergillus flavus Co – Control, C – Catla, R – Rohu,
S - Silver carp, G - Grass carp.
pathogens of their environment (Shephard, 1993). Group
of researchers suggest that the epidermal mucus acts as
a first line of defense against pathogens and therefore
may offer a potential source of novel antimicrobial
compounds (Ellis, 2001; Fouz et al., 1990; Grinde et al.,
1988; Nagashima et al., 2001; Sarmasik, 2002).
The mucus producing cells in epidermal and epithelial
layers had been reported to differ between fish species
and therefore could influence the mucus composition.
Furthermore, the biochemical substances of mucus have
been showed to differ depending on the ecological and
physiological condition (Subramanian et al., 2008). In the
present study also the mucus secreted by fishes are
having strong resistance to the microbes. The mucus
collected from all the four fishes show vary activity
against the tested bacteria.
Figure 8. Candida albicans
Figure 9. Aspergillus flavus

5118 Afr. J. Microbiol. Res.
Figure 10. Aspergillus niger Co – Control, C – Catla, R –
Rohu, S - Silver carp, G - Grass carp.
Amphipathic α-helical peptides, such as dermaseptin,
ceratotoxin and magainin bind with anionic
phospholipids-rich membranes and dissolve them like
detergents (Pouny et al., 1992; Shai, 1995). These
peptides are known to exert action by binding to the
surface of the microbial membranes and causing a lysis
of the intracellular contents. Our present study was also
supported by the above studies in showing the
antibacterial activity.
Fish contain serum and cellular interferon which
possess anti-viral proteins, enzymes- inhibitors (e.g. α-
macroglobulin and other β-globulins) that inhibit the extra
cellular proteases secreted by pathogens (Alexander and
Ingram, 1992). They added that number of relatively
specific lytic molecules, like hydrolase enzymes
(Lysozyme, Chinase and Chitobiase) act on fungi and
bacteria. Fish also contain lectins possess antifungal and
antibacterial activities. Mucus contain several proteases
(serine proteases, cysteine proteases, metalloproteases
and trypsin (like proteases) having strong antibacterial
activity (Fast et al., 2002). The mechanism by which
antimicrobial substance kill microbes are still unclear, but
it is currently thought that different peptides employ
different strategies. These include the fatal depolarization
of the cell membrane (Westerhoff et al., 1989), the
formation of pores and subsequent leakage of the cell
contents (Yang et al., 2000) or the damaging of critical
intracellular targets after internalization of the peptide
(Kargol et al., 2001).
The antimicrobial substance present in the mucus may
function either in the cytoplasm against intracellular
pathogens or extracellularly through release to mucosal
surfaces after infection-induced cell lysis or apoptosis.
Few antimicrobial agents structurally identified in the
mucus of bony fishes are proteins. It has been proposed
that these compounds bind to and essentially dissolve
cellular membrane (Ebran et al., 1999; Zasloff, 2002).
The data of present study indicate that the antimicrobial
activity of the fish mucus may be due to the presence of
the above said substances. The mode of action of
mucus is yet to be determined but studies have proposed
various killing mechanisms for fish derived AMPs such as
cytoplasmic membrane disruption, pore or channel
formation (Syvitski et al., 2005) and inhibition of cell wall
and nucleic acid synthesis (Partzykat et al., 2002;
Brogden, 2005).
In the present study, variation in their antimicrobial
activity was observed among the fish mucus. This may
be due to the variation in the relative levels of lysozyme,
alkaline phosphatase, cathepsin B and proteases of the
epidermal mucus of all fish species (Subramanian et al.,
2007).
Both the indigenous and exotic fish species have the
activity against the bacterial pathogens, whereas some
fungal pathogens were not controlled by the mucus of
exotic fishes. But the mucus of indigenous fishes controls
the tested fungal pathogens. Native fish species
(indigenous) thrives better in prevalent conditions in
controlling the mosquitoes than exotic fishes (Chandra et
al., 2008). Falling in line with the above observation, the
indigenous fish species such as C. catla and L. rohita
show higher antimicrobial activity than that of the exotic
fish species such as H. molitrix and C. idella. This is the
first report on the antimicrobial activity of skin mucus of
Figure 10. Aspergillus niger

cultivable indigenous fishes of India. Moreover the mucus
of fish possesses antimicrobial agents which could be
used to formulate new drugs for the therapy of infectious
diseases caused by pathogenic and opportunistic
microorganisms. These properties of mucus suggest that
it may be beneficial in aquaculture and human health-
related applications. Further studies are needed to isolate
the bioactive compounds (antimicrobial substances) from
the mucus of these cultivable fish species and the
mechanism of antimicrobial action.
AKNOWLEDGEMENTS
Authors thank the authorities of Annamalai University,
and the Head of the Department of Zoology for providing
the facilities to carry out this study.
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