Figure 3 shows the distribution of the E. coli isolates according to fruit type. A total of 40 isolates were obtained and according to fruit type, water melon with 16 (40%) isolates had the highest occurrence of E. coli followed by fruit salad with 11 (27.5%). The distribution of the isolates from other types of fruits was 2 (5%), 5 (12.5%) and 6 (12.5%) for pineapple, orange and apple, respectively. Table 1 shows the distribution of the E. coli isolates according to locations they were purchased.
From Table 1, the highest number of E. coli isolates was observed from Bogobiri with a total of 19 (47.5%) followed by Watt with 9 (22.5%) isolates. The location with the least amount of E. coli was Akim with 7 (17.5%) isolates.
Table 1
Distribution of the E. coli isolates according to location
Location
|
Fruit type
|
Total (%)
|
Watermelon
|
Pineapple
|
Oranges
|
Apple
|
Fruit Salad
|
Akim
|
2
|
-
|
-
|
1
|
4
|
7 (17.5)
|
Bogobiri
|
10
|
2
|
3
|
2
|
2
|
19 (47.5)
|
Marian
|
2
|
-
|
-
|
1
|
2
|
5 (12.5)
|
Watt
|
2
|
-
|
3
|
1
|
3
|
9 (22.5)
|
Total
|
16
|
2
|
6
|
5
|
11
|
40 (100.0)
|
Figure 4 shows the GC-MS chromatogram (abundance versus retention time) of the bioactive compounds from T. tetraptera. The bioactive compounds are represented by the various peaks. The peaks were 26, representing a total of 26 bioactive compounds. Table 2. The table shows the retention time (min) of the 28 bioactive compounds, their molecular weights, peak areas (%) and development (min). The retention time ranged from 8.274 to 24.710 mins for compounds methone and beta-Caryophyllene. On the other hand, the molecular weights of the various compounds ranged from 16.04 to 478.7 g/mole for methone and carpaine, respectively. Peak area shows that lupeol was the most abundant with a peak area of 21.71 while eugenol had the least peak area with a value of 1.13.
Table 2
GC-MS output of the T. tetraptera
Compounds
|
R.T (min)
|
MW (g/mol)
|
Peak area (%)
|
Dev (min)
|
Methone
|
8.274
|
16.04
|
7.86
|
36
|
Lemonene
|
8.839
|
136.24
|
4.21
|
51
|
Octanol
|
9.421
|
130.228
|
2.3
|
74
|
Methanol
|
9.718
|
156.27
|
9.42
|
62
|
Dihydrocarvone
|
10.352
|
152.23
|
2.74
|
42
|
Carvone
|
10.894
|
150.22
|
1.82
|
69
|
Beta-bourbonene
|
12.251
|
204.35
|
8.15
|
19
|
Methyl-10-methylheptadecanoate
|
12.758
|
298.00
|
1.64
|
52
|
Alpha-copaene
|
12.962
|
204.00
|
3.71
|
63
|
Campesterol
|
14.313
|
401.01
|
2.53
|
28
|
Piperitone
|
14.802
|
152.23
|
5.49
|
39
|
Beta-pinene
|
15.428
|
136.23
|
7.82
|
47
|
Lupenon
|
15.820
|
424.00
|
2.64
|
51
|
Beta amyrene
|
16.326
|
410.00
|
1.22
|
58
|
Terpinen-4-ol
|
16.711
|
154.25
|
3.43
|
74
|
Lupeol
|
18.336
|
426.00
|
21.71
|
39
|
3-Carene
|
18.758
|
136.24
|
1.62
|
42
|
Butanedioly dihydrazide
|
20.213
|
112.00
|
2.44
|
72
|
Alpha-pinene
|
20.801
|
136.23
|
8.79
|
68
|
Eugenol
|
21.369
|
164.2
|
1.13
|
53
|
Sabinene
|
21.741
|
136.23
|
6.57
|
31
|
15-Hydroxypentadecanoic
|
22.303
|
258.38
|
3.86
|
29
|
Carpaine
|
22.718
|
478.7
|
4.14
|
72
|
T-Cadinol
|
23.352
|
222.27
|
2.72
|
54
|
Alpha-ocimene
|
23.694
|
136.24
|
6.63
|
18
|
Olean-12-en-3-0ne
|
23.857
|
424.00
|
1.77
|
24
|
n-Hexadecanoic acid
|
24.293
|
256.00
|
3.42
|
36
|
Beta-caryophyllene
|
24.710
|
204.36
|
0.86
|
41
|
Table 3 shows the summary of the antimicrobial sensitivity profile of the aqueous and ethanolic extracts of T. tetraptera. Among the isolates that were sensitive, ethanolic extract was better than aqueous extract at all concentrations used. At 100%, 38 and 28 isolates, representing 95% and 70.0% were sensitive while at 50% of the extract concentration, 34 and 19 isolates were sensitive, representing 85.0% and 47.5% of the total isolates for ethanolic and aqueous extracts. As the concentration decreased, the number of isolates that were sensitive decreased while those that showed resistance increased. At concentrations of 6.25 and 3.125%, the number of isolates that were resistant to the aqueous and ethanolic extracts were 34 and 17 isolates, respectively. Comparatively, trimethoprim showed better results than the extracts used at the all concentrations (100 mg/l to 3.125 mg/l) used in this study.
Table 3
Antimicrobial sensitivity profile of the extracts and trimethoprim
Extracts and control
|
100 (%)
|
50 (%)
|
25 (%)
|
12.5 (%)
|
6.25 (%)
|
3.125 (%)
|
Sensitive
|
|
|
|
|
|
|
Aqueous extract
|
28 (70.0)
|
19 (47.5)
|
14 (35.0)
|
11 (27.5)
|
8 (20.0)
|
4 (10.0)
|
Ethanolic extract
|
38 (95.0)
|
34 (85.0)
|
28 (70.0)
|
27 (67.5)
|
20 (50.0)
|
19 (47.5)
|
Trimethoprim (mg/l)
|
40 (100.0)
|
39 (97.5)
|
38 (95.0)
|
33 (82.5)
|
29 (72.5)
|
17 (42.5)
|
Resistant
|
|
|
|
|
|
|
Aqueous extract
|
10 (25.0)
|
16 (40.0)
|
24 (60.0)
|
26 (65.0)
|
31(77.5)
|
34 (85.0)
|
Ethanolic extract
|
2 (5.0)
|
6 (15.0)
|
10 (25.0)
|
12 (30.0)
|
18 (45.0)
|
17 (42.5)
|
Trimethoprim (mg/l)
|
0 (0.0)
|
1 (2.5)
|
2 (5.0)
|
6 (15.0)
|
10 (25.0)
|
20 (50.0)
|
Intermediate
|
|
|
|
|
|
|
Aqueous extract
|
2 (5.0)
|
5 (12.5)
|
2 (5.0)
|
3 (7.5)
|
1 (2.5)
|
2 (5.0)
|
Ethanolic extract
|
0 (0.0)
|
0 (0.0)
|
2 (5.0)
|
1 (2.5)
|
2 (5.0)
|
4 (10.0)
|
Trimethoprim (mg/l)
|
0 (0.0)
|
0 (0.0)
|
0 (0.0)
|
1 (2.5)
|
1 (2.5)
|
3 (7.5)
|
Key: ≤10 mm = Resistant, 11–15 mm = Intermediate and ≥ 16 mm = Sensitive.
Table 4 shows the predicted ADMET properties for ligands that meet Lipinski ROF. Adsorption was evaluated using water solubility, intestinal absorption, skin permeability, P-glycoprotein substrate, P-glycoprotein I and II inhibitors. Water solubility values of the study bioactive compounds ranged from − 2.25 to 5.477 while that of trimethoprim was − 2.744 and it was within the range of our study ligands. Intestinal absorption for the various ligands ranged from 91.485 to 95.257 and was higher than that of trimethoprim which was 71.787. Skin permeability for the ligands ranged from − 1.063 to -2.715 and − 2.735 for trimethoprim. A total of 11 ligands were not substrates P-glycoprotein while ligands II and X as well trimethoprim were substrates. All the ligands as well trimethoprim were not inhibitors P-glycoprotein I and II inhibitors. Distribution was evaluated using VDss (human), fraction unbound (human), BBB and CNS permeabilities. From the predicted result presented in Table 4, the VDss values ranged from − 0.574 to 0.812 for the ligands and 0.954 for the control drug. Fraction unbound values ranged from 0.0104 to 0.514 for the ligands and 0.554 for the control drug. For BBB and CNS permeabilities, the values for the ligands ranged from 0.084 to 0.792 and − 2.977 to -1.763, respectively while for trimethoprim, the respective values were − 1.225 and − 3.273 for BBB and CNS permeabilities. The predicted parameters for metabolism were CYP2D6 and CYP3A4 substrates and CYP1A2, CYP2C19, CYP2C9, CYP2D6 and CYP3A4 inhibitors. From the predicted result presented in Table 4, none of the ligands as well as trimethoprim were substrates to CYP2D6 while to CYP3A4, 11 ligands were not its substrates but ligands 10, 13 and trimethoprim were substrates. All the ligands as well as the trimethoprim were not inhibitors to CYP2C19, CYP2C9, CYP2D6 and CYP3A4; however, ligand VIII was predicted to inhibit CYP1A2. The predicted excretion parameter showed that total clearance ranged from 0.191 to 1.86 for the ligands while it was 0.98 for trimethoprim. All the ligands and the control drugs were not substrates to renal OCT2. Ames’s toxicity profiling of the ligands and trimethoprim showed that all the ligands apart from VIII and trimethoprim could be carcinogenic. On the other hand, all the ligands did not indicate hepatoxicity concerns while trimethoprim was predicted to be hepatoxic. Furthermore, all the ligands as well as trimethoprim were not inhibitors to hERG I and II.
Figures 5a and 5b show the summary of the various predicted targets for the various ligands and trimethoprim. The result showed various targets that ranged from 6 to 10 targets for the various bioactive compounds while for trimethoprim, the number of targets were 7. Figure 6 shows a summary of the top predicted targets for the various ligands and trimethoprim. The top targets were family A G protein coupled receptors, fatty acid binding family, oxidoreductase, enzyme and lyase for the ligands while for trimethoprim, it was kinase. The most frequent target was nuclear receptor that was the most frequent target in six of the 13 bioactive compounds.
Table 4
Predicted ADMET properties for ligands that meet Lipinski ROF
ADMET
|
Predicted values
|
Absorption
|
I
|
II
|
III
|
IV
|
V
|
VI
|
VII
|
VIII
|
IX
|
X
|
XI
|
XII
|
XIII
|
XIV
|
Water solubility (log mol/L)
|
-2.668
|
-3.568
|
-2.556
|
-2.217
|
-2.385
|
-2.586
|
-2.296
|
-2.25
|
-4.31
|
-4.724
|
-4.073
|
-4.472
|
-5.477
|
-2.744
|
Caco2 permeability (log Papp in 10− 6 cm/s)
|
1.229
|
1.401
|
1.471
|
1.376
|
1.412
|
1.134
|
1.502
|
1.559
|
1.438
|
0.849
|
1.479
|
1.411
|
1.565
|
0.038
|
Intestinal absorption (human)
|
97.324
|
95.898
|
92.552
|
95.257
|
97.555
|
97.957
|
94.014
|
92.041
|
91.485
|
91.891
|
94.296
|
95.155
|
92.51
|
71.787
|
Skin Permeability (log Kp)
|
-1.872
|
-1.721
|
-1.531
|
-1.919
|
-2.131
|
-1.915
|
-2.182
|
-2.207
|
-2.714
|
-2.782
|
-1.923
|
-1.063
|
-2.715
|
-2.735
|
P-glycoprotein substrate
|
No
|
Yes
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Yes
|
No
|
No
|
No
|
Yes
|
P-glycoprotein I inhibitor
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
P-glycoprotein II inhibitor
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Distribution
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
VDss (human) (log L/kg)
|
0.174
|
0.396
|
0.183
|
0.137
|
0.182
|
0.176
|
0.21
|
0.24
|
-0.771
|
0.812
|
0.42
|
0.364
|
-0.574
|
0.954
|
Fraction unbound (human)
|
0.458
|
0.48
|
0.488
|
0.453
|
0.526
|
0.47
|
0.514
|
0.251
|
0.237
|
0.378
|
0.28
|
0.389
|
0.104
|
0.554
|
BBB permeability
|
0.607
|
0.732
|
0.648
|
0.584
|
0.585
|
0.6
|
0.563
|
0.374
|
-0.318
|
-0.351
|
0.596
|
0.792
|
-0.084
|
-1.225
|
CNS permeability
|
-2.155
|
-2.37
|
-2.174
|
-2.119
|
-2.478
|
-2.169
|
-2.473
|
-2.007
|
-2.977
|
-2.948
|
-2.151
|
-1.912
|
-1.763
|
-3.273
|
Metabolism
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
CYP2D6 substrate
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
CYP3A4 substrate
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Yes
|
No
|
No
|
Yes
|
No
|
CYP1A2 inhibitior
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Yes
|
No
|
No
|
No
|
No
|
No
|
No
|
CYP2C19 inhibitior
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
CYP2C9 inhibitior
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
CYP2D6 inhibitior
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
CYP3A4 inhibitior
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Excretion
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Total Clearance
|
0.244
|
0.213
|
1.558
|
1.182
|
0.273
|
0.191
|
1.269
|
0.282
|
1.752
|
0.856
|
1.085
|
0.462
|
1.86
|
0.98
|
Renal OCT2 substrate
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Toxicity
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
AMES toxicity
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Yes
|
No
|
No
|
No
|
No
|
No
|
Yes
|
Max. tolerated dose (human)
|
0.821
|
0.777
|
0.723
|
0.94
|
0.773
|
0.843
|
0.857
|
1.024
|
-0.922
|
-0.685
|
0.343
|
0.736
|
-0.713
|
0.01
|
hERG I inhibitor
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
hERG II inhibitor
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Oral Rat Acute Toxicity (LD50)
|
1.796
|
1.88
|
1.679
|
1.946
|
1.87
|
1.777
|
1.811
|
2.118
|
1.69
|
2.968
|
1.918
|
1.702
|
1.449
|
2.936
|
Oral Rat Chronic Toxicity (LOAEL)
|
2.028
|
2.336
|
1.981
|
2.017
|
1.98
|
2.068
|
2.02
|
2.049
|
2.939
|
-1.167
|
1.475
|
2.414
|
3.109
|
1.135
|
Hepatotoxicity
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
No
|
Yes
|
Skin Sensitisation
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes
|
Yes
|
No
|
Yes
|
No
|
Yes
|
No
|
T.Pyriformis toxicity
|
0.42
|
0.579
|
0.207
|
0.333
|
0.419
|
0.439
|
0.189
|
0.3
|
0.907
|
0.285
|
1.49
|
0.924
|
0.865
|
0.301
|
Minnow toxicity
|
1.205
|
1.203
|
1.193
|
1.29
|
1.402
|
1.23
|
1.545
|
1.702
|
0.085
|
1.127
|
0.743
|
0.676
|
-0.956
|
2.499
|
Key: I = Methone, II = Lemonene, III = Octanol, IV = Menthol, V = Dihydrocarvone, VI = Piperitone, VII = Terpinene-4-ol, VIII = Eugenol, IX = 15-Hydroxypentadecanoic acid, X = Carpaine, XI = T-Cadinol, XII = Alpha-ocimene and XIII = hexadecanoic acid.
Figure 7 shows the various interactions between the various ligands and trimethoprim against of dihydropteroate synthase of E. coli. The various docking poses shows the various bond lengths and the amino acid residues that were involved in the interactions between the ligands and the enzymes. Table 5 shows the various amino acids involved in the interactions and their docking scores. The amino acid residues and their various docking scores and values ranged from − 4.0 to -5.3 kcal/mole and this was lower than that of trimethoprim that returned a docking score of -6.5 kcal/mole. However, one of the ligands, carpaine returned a positive docking score of + 44.3 kcal/mole, indicating an unfavorable docking. The interacting amino acid residues varied from one ligand to the other. As shown in Table 5, the most common amino acid residue among the bioactive compounds was isoleucine 117 followed by threonine 62 as both residues were found to be involved in eight and six complexes, respectively. The amino acids also varied in terms of number. Trimethoprim complexed with 9 amino acids and this was the highest while the bioactive compounds or test ligands complexed with 2 to 7 amino acid residues. However, carpaine complexed with a total of 8 amino acids.
Table 5
Amino residues involved in binding of the various bioactive compounds against dihydropteroate synthase
Bioactive compounds
|
Amino acid residues
|
Docking/Vina scores (kcal/mole)
|
1
|
Lys 221 and Ile 117
|
-4.8
|
2
|
Arg63 and Ile 117
|
-4.4
|
3
|
Gly217, Phe190, Ile117 and Asp 185
|
-4.0
|
4
|
Asn22 and Ile117
|
-4.5
|
5
|
Arg225, Thr62 and Met 139
|
-4.7
|
6
|
Thr 62, Ile117 and Met 139
|
-4.8
|
7
|
Thr62, His257 and Arg63
|
-4.8
|
8
|
Thr 62, Arg 225, Ile117, Phe190 and Lys221
|
-5.1
|
9
|
Thr62, Asn115
|
-5.3
|
10
|
Ser61, Lys221, Asn22, Met139, Asp105, Asn115, Ile117 and Asp96
|
44.3
|
11
|
Lys221 and Phe190
|
-4.4
|
12
|
His257, Ile20, Met139 and Phe190
|
-4.4
|
13
|
Thr62, Met139, Ile117, Phe190, Lys221, Arg63 and His63
|
-4.8
|
Trimethoprim (2,4-diamino-5-(3′,4′,5′-trimethoxybenzyl) pyrimidine)
|
Asn22, Asp96, Met139, Arg225, Glu 60, Ile20, His257, Lys221 and Asn 115.
|
-6.5
|
Key: I = Methone, II = Lemonene, III = Octanol, IV = Menthol, V = Dihydrocarvone, VI = Piperitone, VII = terpinene-4-ol, VIII = eugenol, IX = 15-hydroxypentadecanoic acid, X = carpaine, XI = T-cadinol, XII = alpha-ocimene and XIII = hexadecanoic acid and XIV = Trimethoprim |