Purchased high profile positive and negative antibiotic disc containing Amoxicillin, Perfloxacin, Erythromycin, Septrin, Streptomycin, Ciprofloxacin, Rocephin, Zinnacef, Ampiclox, Gentamycin, Spafloxacin, Chloramphenicol, Augmentin and Tarivid each of different concentration were tested on the identified bacteria isolates by disk diffusion method (Fadila and Tajelmolk , 2016). The antibiotics used were the common and readily available in Nigerian markets.
Table 1. Susceptibility pattern of antibiotics against different microorganisms.
S/N
|
Bacteria
|
(µg)
|
Streptococcus spp
|
S.aureus
|
Salmonella Spp
|
Shigella Spp
|
E. coli
|
1
|
Amoxicillin
|
30
|
+
|
-
|
-
|
-
|
-
|
2
|
Perfloxacin
|
10
|
++
|
+++
|
-
|
++
|
+++
|
3
|
Erythromycin
|
10
|
++
|
NT
|
NT
|
NT
|
NT
|
4
|
Septrin
|
30
|
+++
|
+++
|
-
|
+
|
+
|
5
|
Streptomycin
|
30
|
+++
|
+++
|
-
|
+
|
++
|
6
|
Ciprofloxacin
|
10
|
+++
|
+++
|
-
|
+++
|
+++
|
7
|
Rocephin
|
25
|
-
|
+++
|
NT
|
NT
|
NT
|
8
|
Zinnacef
|
20
|
-
|
NT
|
NT
|
NT
|
NT
|
9
|
Ampiclox
|
20
|
-
|
NT
|
NT
|
NT
|
NT
|
10
|
Gentamycin
|
10
|
++
|
NT
|
+
|
++
|
+++
|
11
|
Spafloxacin
|
10
|
NT
|
++
|
-
|
++
|
++
|
12
|
Chloramphenicol
|
30
|
NT
|
++
|
-
|
-
|
+
|
13
|
Augmentin
|
30
|
NT
|
-
|
-
|
-
|
+
|
14
|
Tarivid
|
10
|
NT
|
-
|
-
|
+
|
+
|
# Diameter of inhibition zone: NT; Not tested, no inhibition (-); 5-15 mm (+); 16-25 mm (+ +); 26-35 mm (+ + +)
Streptococcus spp, were resistant to Rocephine, Zinnacef and Ampiclox at 20 µg, showed little activity for Amoxicillin. S. aureus showed resistance to 10 µg Tarivid, 30 µg of Amoxicillin and Augmentin. Salmonella spp, were resistant to all tested antibiotic except for Gentamycin at 10 µg. Shigella spp showed resistance to 30 µg of Amoxicillin, Chloramphenicol and Augmentin. Escherichia Coli was only resistant to Amoxicillin at 30 µg.
3.1 Antibacterial activity of Amx, AmxA, AmxM, AmxB, Ap, ApA, ApM, and ApB
The antimicrobial susceptibility test in Table 2 showed that Salmonella spp were resistant to Amx, ApB and ApM with no zone of inhibition (ZOI), slightly intermediate to Ap (ZOI 5.6±0.3), AmxB (ZOI 5.8 ± 0.6), and AmxM (ZOI 5.5±0.4), was susceptible to AmxA (ZOI 16.7±1.2), ApA (ZOI 17.4±1). Shigella spp were resistant to Amx, AmxB and AmxM with no ZOI, slightly intermediate to Ap (ZOI 5.8±0.5), ApM (ZOI 5.1±0.3), susceptible to AmxA (ZOI 15.4±0.9), ApA (ZOI 16.6±0.7).
S. aureus were resistant to Amx, Ap, AmxB, ApB with no ZOI, slightly intermediate to AmxM (ZOI 5.0±0.2), were susceptible to AmxA (ZOI 16.2±0.8), ApA (ZOI 25.9±1.2), and ApM (ZOI 16.8±0.8). E.coli were resistant to Amx, AmxB, ApB and AmxM with no ZOI, and susceptible to Ap (ZOI 10.8±1.2), AmxA (ZOI 21.3±1.2), ApA (ZOI 18.3±0.8), and ApM (ZOI 13.4±0.9)
Table 2 Zone of inhibition (mm) of resistant bacterial strains in response to Amx, AmxA, AmxM, AmxB, Ap, ApA, ApM, and ApB.
S/N
|
Bacteria
|
Amx
|
Ap
|
AmxA
|
ApA
|
AmxB
|
ApB
|
AmxM
|
ApM
|
1
|
Salmonella spp
|
0
|
5.6±0.3
|
16.7±1.2
|
17.4±1
|
5.8±0.6
|
0
|
5.5±0.4
|
0
|
2
|
Shigella spp
|
0
|
5.8±0.5
|
15.4±0.9
|
16.6±0.7
|
0
|
5.3-0.5
|
0
|
5.1±0.3
|
3
|
S. aureus
|
0
|
0
|
16.2±0.8
|
25.9±1.2
|
0
|
0
|
5.0±0.2
|
16.8±0.8
|
4
|
E. coli
|
0
|
10.8±1.2
|
21.3±1.2
|
18.3±0.8
|
0
|
0
|
0
|
13.4±0.9
|
Higher ZOI were observed in AmxA and ApA greater than their corresponding group mixtures, in all tested bacteria strains, higher ZOI of 16.8±0.8, and 13.4±0.9 were shown in ApM for S. aureus, and E. coli, still this ZOI are less compared to the corresponding ZOI of ApA which were 25.9±1.2 and 18.3±0.8 for S. aureus, and E. coli.
The increase in ZOI for acid catalysed synthesis is due to the linking of functional groups, substituents or change in ring size of possibly the non-active sites of the antibiotics and the different phytochemicals present in Calotropis Procera, fig 5 confirms a reaction in the mixture with a colour change from light green to brown. (Reusch, 2013).
No observable changes in ZOI of alkali combinatorial synthesis, this may be due to the linking of functional groups, substituents or change in ring size of possibly the non-active sites of the antibiotics, and the different phytochemicals present in Calotropis Procera, with a colour change from light green to a dark green cloudy solution (Reusch, 2013)
The heat catalysed mixture of both antibiotics were able to overcome resistance with ZOI increase from 0 to 5.5±0.4 mm for salmonella spp, 0 to 5.0±0.2 E. coli in amoxicillin and a +16.8 mm increase in S. aureus for ampicillin only, thus confirming the antimicrobial properties exhibited by the plant Calotropis procera extract (Barkha et al, 2021)
3.2 Validation of positive results
The validation of positive results obtained from the first combinatorial synthetic method as shown in Figure 5, and Table 2 were carried out using a different plant extract of Piliostigma reticulatum
(Boualam et al, 2021)
, with an additional antibiotic ( azithromycin), and an additional bacteria strain ( streptococcus spp ).
Streptococcus spp were resistant to A and B with no ZOI, susceptible to A1 (ZOI 11.3±1.5), A2 (ZOI 15.5±1.5), A3 (ZOI 22.4±1.2), B1 (ZOI 13.3±0.8), B2 (ZOI 19.7±1), and B3 (ZOI 21.2±0.9). S. aureus were resistant to B, with no ZOI, were susceptible to A (ZOI 13.4±0.9), A1 (ZOI 21.4±1.2), A2 (ZOI 19.2±0.7), A3 (ZOI 21.1±1.1), B1 (ZOI 12.2±0.9), B2 (ZOI 17.2±1.2), and B3 (ZOI 16.6±0.8). Salmonella spp were resistant to A, A1, A2, A3, B, B1, B2, and B3 with no ZOI.
successive increase were observed in Streptococcus spp with increasing volume of acid from 0.1 to 0.3 mL, while an increase in ZOI values were observed in S. aureus upon addition of 0.1 to 0.2 mL of acid, this was not observed upon addition of 0.3 mL of acid thus, showing that 0.1 mL of the acid is enough in 2 mL of antibiotic and plant extract to initiate reactions which may overcome resistance in bacteria.
The resistance of Salmonella spp to all prepared samples is due to the reduced concentration of the antibiotic by a factor of 10, because ampicillin and 1 mg/mL showed a ZOI of 5.6±0.3 in Table 2, and may also be due to the nature of phytochemicals present in Piliostigma reticulatum
(Boualam et al, 2021)
Table 3 Zone of inhibition (mm) of 3 resistant bacterial strains in response to A, A1, A2, A3, B, B1, B2 and B3
S/N
|
Bacteria
|
A
|
A1
|
A2
|
A3
|
B
|
B1
|
B2
|
B3
|
1
|
Streptococcus spp
|
0
|
11.3±1.5
|
15.5±1.5
|
22.4±1.2
|
0
|
13.5±0.8
|
19.7±1
|
21.2±.0.9
|
2
|
S. aureus
|
13.40± 0.9
|
21.4±1.2
|
19.2±0.7
|
21.1±1.1
|
0
|
12.2±0.9
|
17.2±1.2
|
16.6±0.8
|
3
|
Salmonella spp
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
This further confirms the necessity of prerequisite knowledge of the plant ethnopharmacolgical usage and also that of the resistant pathogen before any combinatorial method.
A decrease in colour intensity and smell were observed with successive addition of acid from 0.1 to 0.3 mL as shown in Figure 6, at 0.3 mL no smell were detected in the case of ampicillin, this implies that at 0.1 and 0.2 ml of acid added the antibiotic still retains some of the functional groups or substituents responsible for it characteristic smell, but on addition of another 0.1 mL of acid the smell were not detected, indicating that particular functional groups or substituents were definitely involved in conjugation or might be substituted
(Reusch, 2013)
Table 4. Comparison of acid enhanced combination with other methods for resistant bacteria inhibition
Bacteria strain
|
Antibiotic
|
Antibiotic
MIC
|
Mixture
MIC
|
Ref
|
P. chlororaphis
P. monteilii
|
Cefotaxime
Rifampicin
|
Nil
Nil
|
12.5µg/ml
0.195 µg/ml
|
Haq et al (2019)
|
E. coli
S. enteritidis
|
Ticarcilin 5 µg/ml
|
Nil
Nil
|
16.00 mm
18.33 mm
|
Fadila and Tajelmol (2016)
|
E. coli
S. aureus
|
Chloramphenicol30 µg
Tetracycline 30 µg
|
Nil
Nil
|
11.00 mm
12.00 mm
|
Eze et al (2013)
|
The former combinatorial method as shown in Table 4 is a trial based on probability approach where the combination may or may not inhibit bacterial growth with the effect being either additive, antagonistic or synergistic even with the right medicinal plants, but in acid catalyze methods at lower concentration with the right medicinal plants there is higher possibility that all combinations will be synergistic