A total of 20 types of bacterial colonies were isolated from 15 fish gut samples and grow on MacConkeyagar media. Two types of colonies were observed one was sugar fermented and the other was non-sugar fermented. Due to the fermented process sugar fermented bacteria converted into red color while non-fermented into brown color see Fig 1,2.
Triple sugar iron test
The triple sugar iron test is based on lactose fermentation and non-lactose fermentation and H2S gas production. Six bacteria showed the altered media color that change from red to yellow color but the slant changed color and produce gas and not h2s gas. Two bacterial isolates change the butt color from red to black and slans remain the same and no h2s production is seen in table# 3, Fig 3.
Table. 3 Triple Sugar Iron test for a bacterial isolate from the gut of L. rohita
Sr. no
|
Sample
|
Butt color
|
Slant color
|
Gas
|
H2S
|
1
|
S1-A
|
Yellow
|
Yellow
|
Yes
|
No
|
2
|
S1-B
|
Yellow
|
Red
|
Yes
|
No
|
3
|
S2-1
|
Yellow
|
Yellow
|
Yes
|
No
|
4
|
S2-2
|
Red
|
Red
|
No
|
No
|
5
|
S2-3
|
Yellow
|
Yellow
|
Yes
|
No
|
6
|
S3-A
|
Red
|
Red
|
No
|
No
|
7
|
S3-B
|
Yellow
|
Yellow
|
No
|
No
|
8
|
S3-3
|
Yellow
|
Red
|
Yes
|
No
|
9
|
S3-4
|
Black
|
Red
|
Yes
|
Yes
|
10
|
S3-7
|
Black
|
Red
|
Yes
|
Yes
|
11
|
S4-1
|
Yellow
|
Yellow
|
Yes
|
No
|
12
|
S4-2
|
Red
|
Red
|
No
|
No
|
13
|
S5-A
|
Red
|
Red
|
No
|
No
|
14
|
S5-B
|
Yellow
|
Red
|
Yes
|
No
|
15
|
S5-C
|
Yellow
|
Red
|
Yes
|
No
|
16
|
H3
|
Red
|
Red
|
No
|
No
|
17
|
H4
|
Black
|
Red
|
No
|
No
|
18
|
H5
|
Black
|
Red
|
No
|
No
|
19
|
H6
|
Black
|
Red
|
Yes
|
Yes
|
20
|
H7
|
Yellow
|
Yellow
|
Yes
|
No
|
MR-VP TEST
The results indicated that 13 bacterial strains showed the MR positive while 7 showed negative. On the other hand, 9 samples showed VR positive and 11 showed VR negative see table # 4, fig # 4.
Table 4. Showed the MR VP and biofilm test for a bacterial isolate from the gut of L.rohita
Sr. no
|
Sample
|
MR
|
VP
|
Biofilm
|
1
|
S1-A
|
+ve
|
+ve
|
M
|
2
|
S1-B
|
-ve
|
+ve
|
M
|
3
|
S2-1
|
-ve
|
+ve
|
M
|
4
|
S2-2
|
-ve
|
-ve
|
S
|
5
|
S2-3
|
+ve
|
-ve
|
W
|
6
|
S3-A
|
+ve
|
-ve
|
W
|
7
|
S3-B
|
+ve
|
-ve
|
W
|
8
|
S3-3
|
+ve
|
-ve
|
M
|
9
|
S3-4
|
+ve
|
-ve
|
S
|
10
|
S3-7
|
+ve
|
+ve
|
S
|
11
|
S4-1
|
-ve
|
+ve
|
S
|
12
|
S4-2
|
-ve
|
-ve
|
M
|
13
|
S5-A
|
+ve
|
-ve
|
S
|
14
|
S5-B
|
+ve
|
+ve
|
M
|
15
|
S5-C
|
+ve
|
-ve
|
M
|
16
|
H3
|
-ve
|
+ve
|
W
|
17
|
H4
|
+ve
|
-ve
|
W
|
18
|
H5
|
+ve
|
-ve
|
M
|
19
|
H6
|
+ve
|
+ve
|
M
|
20
|
H7
|
-ve
|
+ve
|
W
|
Biofilm
the result of biofilm-based on ELISA technique S2-2, S3-4, S3-7, S4-1, S5-A, (six isolates) showed that weak biofilm and S1-A, S1-B, S2-1, S3-3, S4-2, S5-B, S5-C, H5 and H6 (eight isolates) showed the moderate Biofilm while S2-3, S3-A, S3-B, H3, H4 and H7 (six isolates) showed the strong biofilm.
Antibiotic resistance:
the result of antibiotic resistance against the different antibiotics. The results indicated that S1-A, S1-B, S2-1, S2-2, S2-3, S3-A, S3-B, S3-3, S3-4, S3-7, S4-1, H3, H5, showed the strong antibiotic resistance and S4-2, S5-A, S5-C, H4, H7 showed sensitivity while S5-B and H6 showed the intermediate against amoxicillin-clavulanic acid see in fig # 5. Same results were obtained except for S5-B which showed the resistance and no bacterial isolate strain showed the intermediate against cefadroxil see in fig # 6, table # 5.
Table.5 Antibiotic resistance of 20 isolates from the gut of Labeo rohita
Sr. no
|
Antibiotics
|
S1- A
|
S1- B
|
S2-1
|
S2-2
|
S2-3
|
S3-A
|
S3- B
|
S3-3
|
S3-4
|
S3-7
|
S4-1
|
S4-2
|
S5- A
|
S5- B
|
S5- C
|
H3
|
H4
|
H5
|
H6
|
H7
|
1
|
Amoxicillin Clavulanic acid
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
I
|
S
|
R
|
S
|
R
|
I
|
S
|
2
|
Cefadroxil
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
R
|
S
|
S
|
R
|
R
|
R
|
S
|
R
|
R
|
S
|
3
|
Cefoxitin
|
S
|
R
|
R
|
S
|
S
|
S
|
R
|
R
|
R
|
S
|
R
|
R
|
S
|
R
|
S
|
I
|
S
|
R
|
R
|
S
|
4
|
Cefotaxime
|
R
|
R
|
R
|
R
|
R
|
I
|
S
|
S
|
R
|
I
|
I
|
S
|
R
|
R
|
R
|
R
|
S
|
R
|
I
|
I
|
5
|
Ceftriaxone
|
R
|
I
|
I
|
I
|
R
|
S
|
I
|
I
|
S
|
S
|
I
|
S
|
S
|
I
|
R
|
I
|
R
|
R
|
I
|
S
|
6
|
Meropenem
|
I
|
S
|
I
|
S
|
S
|
S
|
S
|
I
|
S
|
I
|
S
|
S
|
S
|
I
|
S
|
S
|
R
|
I
|
S
|
I
|
7
|
Levofloxin
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
I
|
S
|
S
|
S
|
S
|
S
|
8
|
Ciprofloxin
|
I
|
S
|
S
|
S
|
I
|
R
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
I
|
S
|
I
|
S
|
S
|
S
|
9
|
Gentamicin
|
S
|
S
|
S
|
R
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
S
|
I
|
S
|
S
|
I
|
10
|
Amikacin
|
I
|
S
|
S
|
S
|
S
|
S
|
S
|
I
|
S
|
S
|
S
|
S
|
I
|
I
|
I
|
S
|
I
|
S
|
I
|
R
|
11
|
Polymyxin B
|
S
|
R
|
R
|
S
|
S
|
R
|
S
|
R
|
S
|
R
|
S
|
S
|
S
|
R
|
R
|
S
|
R
|
S
|
S
|
S
|
12
|
Colistin
|
S
|
S
|
S
|
S
|
S
|
R
|
S
|
S
|
S
|
S
|
R
|
S
|
S
|
S
|
S
|
S
|
R
|
R
|
R
|
S
|
13
|
Nitrofuranto in
|
R
|
S
|
R
|
S
|
I
|
R
|
R
|
R
|
S
|
R
|
R
|
R
|
R
|
S
|
I
|
R
|
R
|
R
|
I
|
R
|
DNA extraction and molecular analysis
In this study, DNA extraction was done by the organic and lysate DNA extraction method. The concentration of DNA quantifies at 260-280 nm wavelength by using a UV spectrophotometer. Amplification of 16S rRNA genes was carried out by using universal primers. PCR product of 20 isolates analyzed by 1.5% agarose gel see in fig # 7, table # 6.
Table.6 The assigned taxonomic name and Max Score of 20 bacterial strains along with GenBank accession numbers
Sr.no
|
Isolates
|
Assigned taxonomic name
|
Max
Score
|
Similarity
|
Accession
Number
|
1
|
S1-A
|
Klebsiella pneumoniae
|
968
|
95.62%
|
NR_117686.1
|
2
|
S2-B
|
Enterobacter cloacae
|
878
|
97.42%
|
NR_117679.1
|
3
|
S2-1
|
|
|
|
|
4
|
S2-2
|
|
|
|
|
5
|
S2-3
|
Pseudomonas oleovorans
|
488
|
91.49%
|
NR_115874.1
|
6
|
S3-A
|
Morganella morganii
|
1022
|
94.66%
|
NR_113580.1
|
7
|
S3-B
|
Citrobacter freundii
|
1048
|
97.26%
|
NR_113340.1
|
8
|
S3-3
|
Proteus mirabilis
|
850
|
92.23%
|
NR_113344.1
|
9
|
S3-4
|
Citrobacter braakii
|
811
|
88.67%
|
NR_028687.1
|
10
|
S3-7
|
|
|
|
|
11
|
S4-1
|
Enterobacter hormaechei
|
1122
|
97.00%
|
NR_126208.1
|
12
|
S4-2
|
Psychrobacter sanguinis
|
819
|
89.91%
|
NR_117833.1
|
13
|
S5-A
|
Shigella dysenteriae
|
1027
|
95.50%
|
NR_026332.1
|
14
|
S5-B
|
Citrobacter cronae
|
1020
|
97.50%
|
NR_170426.1
|
15
|
S5-C
|
Shigella sonnei
|
1058
|
98.66%
|
NR_104826.1
|
16
|
H3
|
Pseudomonas sihuiensis
|
893
|
91.02%
|
NR_148251.1
|
17
|
H4
|
Proteus mirabilis
|
830
|
92.29%
|
NR_113344.1
|
18
|
H5
|
Pseudomonas oleovorans
|
1081
|
97.93%
|
NR_115874.1
|
19
|
H6
|
Citrobacter cronae
|
1014
|
95.45%
|
NR_170426.1
|
20
|
H7
|
Enterobacter hormaechei
|
1011
|
98.27%
|
NR_042154.1
|
Phylogenetic tree
A comparison of 16s rRNA sequence in NCBI GenBank USING BAST program showed the similarities of 20 isolates were 88.67% to 98.27% see fig.8.