Transcript 2: Antimicrobial activity of crude extract isolates from the strain B3

ANTIFOULING ACTIVITY OF BACTERIA ASSOCIATED
WITH THE SURFACE OF MARINE INVERTEBRATES
AND SEAWEEDS
By
Gayathri.R
Reg.No:09MBT08
Under the guidance of
Dr. S. SATHEESH
Lecturer
International Centre for
Nanobiotechnology
Manonmaniam Sundaranar University
Rajakkamangalam – 629 502
Kanyakumari District
Tamil Nadu, India
• Introduction,
• Objective,
• Materials and methods,
• Results,
• Discussion,
• Summary &
• References.
# Biofilm are architecturally complex communities of microorganisms
in which the cells are held together by an extracellular matrix called
“slime”, typically containing exopolysaccharides ( EPS ), proteins,
and even nucleic acids.
# This process of undesirable accumulation of microorganisms,
plants, animals and algae, most often in aquatic environment is
called biofouling.
# Impacts on biodiversity, habitats or ecological processes,
# Economic impacts and
# Impacts on health ( humans, plants and animals ).
Anti – fouling is the process of removing or preventing
the accumulation of microorganisms.
# Preventing the initial settlement of fouling species
by repelling or killing them,
# Preventing the development of settled organisms
by killing them, inhibiting their growth or reducing their adhesion
ability and
# Removing the fouling through cleaning.
# Physical methods,
# Chemical methods,
# Mechanical cleaning,
# Heat treatment,
# Water velocity,
# Enzymes as antifoulants,
# Coatings methods,
# Non – coating methods and
# Natural Product Antifoulants ( NPA ).
# To screen the crude extract of the bacterial strain isolated
from the marine living sources against biofilm bacteria.
# To observe the inhibitory activity of the crude extract by
adhesion assay,
# To prepare a bacterial extract mixd antifouling coatings for
biofilm development assay,
# To study the anti – settlement activity of the crude extract
against gastropods and
# To characterize the crude extract by TLC, HPLC and NMR.
# Isolation of bacteria from marine samples,
# Biochemical characterization of the isolated bacterial strains,
# Gram’s staining,
# Motility Test,
# Indole production Test,
# Methyl-Red Test ,
# Voges-Proskauer Test,
# Citrate Utilization Test,
# Starch hydrolysis,
# Urea hydrolysis,
# Catalase Test and
# Gelatin Hydrolysis Test.
# Preparation of crude bacterial extract,
# Antibacterial assay against biofilm bacteria,
# Influence of crude bacterial extract on the EPS production in
biofilm bacteria,
# Influence of crude bacterial extract on the adhesion of biofilm
bacteria onto the surface,
# Biofilm development assay,
# Gastropods settlement assay and
# Characterization of crude extract by TLC, HPLC and NMR.
Strain A3 - Gastropods II
Strain B3 - Sea cucumber II ( 2 )
Strain C2 - Seaweeds
Strain D - Polychaetes
Strain E - Sea lilly
Table : 1 - Biochemical and physiological characteristics of the five marine
surface associated bacterial isolates used in the present study
Sl.No.
Biochemical and
Physiological tests
Organisms
Strain A3
Strain B3
Strain C2
Strain D
Strain E
1.
Gram staining
-
+
+
+
+
2.
Morphology
rod
cocci
cluster
cocci
cocci
3.
Motility
non motile
non motile
non motile
non motile
non motile
4
Gelatin Hydrolysis
+
-
-
+
-
5.
Starch Hydrolysis
-
+
-
+
-
6.
Catalase Activity
+
+
+
+
+
7.
Citrate Utilization
+
+
-
-
+
8.
Indole production
-
-
-
-
-
9.
Methyl Red
+
+
+
+
+
10.
Voges Proskauer
+
+
-
-
-
11.
Glucose
-
-
-
+
+
12.
Fructose
-
-
-
+
+
13.
Lactose
-
-
-
-
-
14.
Sorbitol
+
-
-
+
-
15.
Urease
Acid
+
+
+
+
+
Alkaline
-
-
-
-
-
Acid
+
+
+
+
+
Alkaline
+
+
-
-
+
Gas
-
-
-
+
+
16.
Triple Sugar Iron (TSI)
Table : 2 - Antimicrobial activity of crude bacterial extract
produced by different bacterial strains against target bacteria
Target Bacteria
S.No.
Organisms
Bacillus sp
E.coli
Klebsiella sp
V.harveyi
V.parahaemolyt
icus
1.
Strain A3
-
+
-
-
+
2.
Strain B3
+
-
-
+
-
3.
Strain C2
+
-
+
-
-
4.
Strain D
+
-
+
+
-
5.
Strain E
-
-
-
+
-
Table: 3 - Carbohydrate concentration of crude bacterial extract
produced by five bacterial strains
Concentrations
S.No.
Bacterial Strains
50 µg
100 µg
1.
A3
2.7944
2.5988
2.
B3
3.128
2.794
3.
C2
3.236
2.667
4.
D
3.571
2.392
5.
E
3.571
2.706
Table : 4 - Protein concentration of crude bacterial extract
produced by five bacterial strains
Concentrations
S.No.
Bacterial Strains
50 µg
100 µg
1.
A3
0.611
0.5775
2.
B3
0.479
0.573
3.
C2
0.405
0.42
4.
D
0.61
0.575
5.
E
0.490
0.460
Table : 5 – Gastropod Settlement Assay
S.No.
Strains
Patella sp
Babylonia sp
1.
A3
+
+
2.
B3
+
+
3.
C2
+
+
4.
D
-
+
5.
E
-
+
Table : 6 - Rf values of compounds observed in TLC
S.No.
Isolated Strains
Rf Values (cm)
1.
Strain A3
0.8421
2.
Strain B3
0.6111
3.
Strain C2
0.7282
4.
Strain D
0.8
5.
Strain E
0.8877
Figure – 1: Antimicrobial activity of crude extract isolates from the
strain A3 against target bacteria
Vibrio parahaemolyticus
Figure – 2: Antimicrobial activity of crude extract isolates from the
strain B3 against target bacteria
Bacillus sp
Vibrio harveyi
Figure – 3: Antimicrobial activity of crude extract isolates from the
strain C2 against target bacteria
Bacillus sp
Klebsiella sp
Figure – 4: Antimicrobial activity of crude extract isolates from the
strain D against target bacteria
Bacillus sp
Klebsiella sp
V.harveyi
Figure – 5: Adhesion assay using crude extract of the strain E
( number of bacterial cells adhered on glass coupons )
Figure – 6 : Biofilm development assay using crude extract mixed coated coupons.
Strain D
Strain A3
Figure– 7 : Gastropod settlement assay using crude extract
Patella sp
Babylonia sp
Figure– 8 : Thin layer chromatogram of the crude extract isolated from the
strains A3, B3, C2, D and E.
Strains A3
Strain B3
Strain C2
Strain D
Strain E
Figure – 9 : HPLC spectrum of crude extract isolated from the strain A3
Figure – 10 : HPLC spectrum of crude extract isolated from the strain B3
Figure – 11 : HPLC spectrum of crude extract isolated from the strain D
Figure – 12 : HPLC spectrum of crude extract isolated from the strain E
Fig – 13 : 1H NMR spectrum of the crude extract isolated from the strain D
Fig – 14 : 13C NMR spectrum of the crude extract isolated from the strain D
# Natural products and their synthetic analogs exhibiting
anaesthetic, repellant and settlement inhibition properties,
but non – toxic to the non – target organisms, are preferred as
potential antifouling agents.
# Sessile, soft – bodied marine organisms maintaining a clean
surface were identified as possible sources of natural product
antifoulants ( NPA ).
# The present study was carried out to assess the antifouling
activity of surface associated bacteria.
# Five bacterial strains ( Bacillus sp, Klebsiella sp, E.coli,
V.harveyi and V.parahemolyticus ) were used as target
organisms for the screening.
# Of 19 surface associated bacteria isolated, five bacterial
extract were found to possess inhibitory activity against target
bacteria.
# These five bacterial extract were also found to possess
inhibitory activity against the settlement of gastropods and one
bacterial extract from sealilly was found to possess inhibitory
activity against an adhesion of bacteria onto the surfaces.
# The bacterial extract was characterized by TLC, HPLC and
NMR.
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