Page 6 - IJA2014.Vol.4No.01

International Journal of Aquaculture, 2014, Vol.4, No.01
http://ija.sophiapublisher.com
3
Before performing the assay, the broth was shaken
well to get a uniform solution of the broth culture.
Antibacterial activities of 29 LAB isolated were
checked against several pathogens isolated from fish
(4), shrimp (5) and human (20). The tested pathogens
consisted of: Aeromonas hydrophila, Vibrio harveyi,
Pseudomonas aeruginosa, Vibrio metschnikovii,
Vibrio coralliilyticus, Vibrio fluvialis, Vibrio cholerae,
Vibrio parahaemolyticus, Vibrio mimicus, Vibrio
hollisae, Bacillus cereus, Campylobacter jejuni,
Escherichia coli and of Salmonella. The species of
Aeromonas and Pseudomonas are part of the culture
collections in our lab. The members of genus Vibrio
were isolated from water samples from a shrimp farm
at Edavanakkad of Ernakulam district (Kerala, India)
and were identified in our laboratory following the
protocol of Noguerola & Blanch (2008). The
Salmonella serotypes used in the present study were
provided by the WHO as part of a memorandum under
the Global Salmonella Surveillance Programme.
Inhibition zones around each LAB isolate was noted
after overnight incubation at 37℃. Sensitivity of a
pathogen to the LAB isolates was defined by the
absence of visible growth of the test organism around
the disc or the well containing the sterile culture broth.
Inhibition zone less than 10 mm was considered as
negative (-), 10-14 mm as moderate (+) and larger
than 15 mm as good (++).
1.4 Molecular identification and construction of the
phylogenic tree
The 14 LAB isolates displaying highest antibacterial
activity against the tested pathogens was screened by
the 16S rRNA genes as described by (RingØ et al.,
2006). All sequences were analyzed and edited in
BioEdit and blasted against the sequences available in
GenBank. Searches for sequence similarity were
performed in GenBank data library with the help of
BLAST programme. The sequences were then
imported into BioEdit Sequence Alignment Editor,
where in CLUSTAL W Multiple Alignment (Hitachi
Software Engineering Co.) was used to assemble and
align them. Phylogenetic tree was constructed by
using the Neighbor- Joining method, the Bootstrap
value being 1000.
1.5 Nucleotide sequence accession numbers
The nucleotide sequences for the 16S rRNA analyses
described in this report have been submitted to the
GenBank under the accession no. KJ156315 (LAB
155), KJ156316 (LAB 156), KJ156317 (LAB 158),
KJ156318 (LAB 159), KJ156319 (LAB 160),
KJ156320 (LAB 161), KJ156321 (LAB 168),
KJ156322 (LAB 176), KJ156323 (LAB 177),
KJ156324 (LAB 179), KJ156325 (LAB 180),
KJ156326 (LAB 229), KJ156327 (LAB 240) and
KJ156328 (LAB 243). Isolate number are given in
brackets.
2 Results and Discussion
2.1 Antibacterial activity
Antibacterial activity of LAB isolates from the gut of
M. cephalus against various fish, shrimp and human
pathogens is given in Tables 1 and 2. LAB is known to
exert their antimicrobial property by the individual or
joint production of organic acids, H2O2 and/or by the
production of different classes of bacteriocins
(Klaenhammer, 1993; Vandenbergh, 1993; RingØ et
al., 2005; Tiwari et al., 2008). As untreated culture
broths were used for the antibiogram in the present
study, the antibacterial property cannot be attributed to
any particular component. LAB are known to be more
effective against the Gram-positive pathogens rather
than the Gram-negatives (Stoffels et al., 1992; Abee et
al., 1995; Rodriguez et al., 2005; RingØ, 2008).
However, in the current study antibacterial activities
were noted against the Gram-negative pathogens
encountered in aquaculture such as: Vibrio harveyi,
Vibrio parahaemolyticus, Aeromonas hydrophila and
Pseudomonas aeruginosa. LAB isolates tested in the
present study also showed good antibacterial activity
against different serovars of Salmonella (Table 2). In
the present study it was observed that gut associated
LAB displayed antibacterial activity against one or a
number of Gram-negatives tested. Isolates belonging
to the Lactobacillus genus were noticed to possess
antimicrobial activity against both fish and human
pathogens. The most promising gut bacteria with
respect to inhibition; Lactobacillus casei (LAB 156)
inhibited growth of 25 out of the 29 pathogenic
bacteria strains tested.