In-vitro assessment of the efficacy of herb-herb combinations against multidrug-resistant mastitis-causing bacteria: Staphylococcus aureus and Klebsiella pneumoniae

Figures & data

Table 1. Plant extracts interaction classification

Class	Interaction	FICI
1	Synergistic	≤0.5
2	Additive	0.5–1.0
3	Indifferent	≥1.0–≤4.0
4	Antagonistic	>4.0

Table 2. Resistance percentages for the S.Aureus and K. pneumoniae against tested antibiotics

Isolate	No. tested	Antibiotics
		Pen	Sxt	Ery	Gen	Neo	Kan	Clo	Erta	Ceft	Amp	Ami	Van	Tet	Lin
K. pneumoniae	29	100	0	7	0	100	7	7	7	7	100	100	100	69	100
S. aureus	129	67	40	48	0	0	47	16	0	69	47	0	0	86	100

Key: Pen-Penicillin, Sxt-Sulphamethoxazole, Ery-Erythromycin, Gen-Gentamycin, Neo-Neomycin, Kan-Kanamycin, Clo-Cloxacillin, Erta-Ertapenem, Ceft-Ceftriaxone, Amp-Ampicillin, Ami-Amikacin, Van-Vancomycin, Tet-Tetracycline, and Lin-Lincomycin.

Table 3. Antibacterial activity of the crude plant extracts (30 mg/ml) on S. aureus and K. pneumoniae

Treatments	S. aureus			K. pneumoniae
	Acetone	Methanol	Distilled water	Acetone	Methanol	Distilled water
X. caffra	20 ± 0.1	22 ± 0.2	0.00	16 ± 0.1	20 ± 0.4	0.00
M. flabellifolius	21 ± 0.1	18 ± 0.6	2 ± 0.2	22 ± 0.1	18 ± 0.1	0.00
A. sativum	17 ± 0.6	14 ± 0.3	0.00	14 ± 0.1	15 ± 0.3	0.00
C. verum	18 ± 0.2	15 ± 0.1	2 ± 0.1	18 ± 0.2	16 ± 0.5	0.00
Methanol	0.00	0.00	0.00	0.00	0.00	0.00
Acetone	0.00	0.00	0.00	0.00	0.00	0.00
Distilled water	0.00	0.00	0.00	0.00	0.00	0.00
Gentamycin (30µg/mL)	21 ± 0.4			20 ± 0.1
Lincomycin (15µg/mL)	0			0

Inhibition zones of the plant extracts were shown as means of three replicates (n = 3) ± standard deviation.

Table 4. In vitro effect of plant extracts combined with Gentamycin (G, 30 µg/mL) on S. aureus and K. pneumoniae

Treatments	S. aureus		K. pneumoniae
	Acetone	Methanol	Acetone	Methanol
X. caffra + G	29 ± 0.3	29 ± 0.2	29 ± 0.1	29 ± 0.1
M. flabellifolius +G	28 ± 0.1	27 ± 0.3	27 ± 0.1	27 ± 0.2
A. sativum +G	27 ± 0.5	26 ± 0.1	28 ± 0.1	26 ± 0.2
C. verum + G	27 ± 0.2	26 ± 0.1	26 ± 0.2	25 ± 0.1

Inhibition zones of the plant extracts were shown as means of three replicates (n = 3) ± standard deviation.

Table 5. In vitro effect of plant extracts combined with Lincomycin (Lin, 15 µg/mL) on S. aureus and K. pneumoniae

Treatments	S. aureus		K. pneumoniae
	Acetone	Methanol	Acetone	Methanol
X. caffra + L	24 ± 0.2	27 ± 0.1	22 ± 0.2	26 ± 0.2
M. flabellifolius +L	23 ± 0.2	26 ± 0.2	26 ± 0.2	24 ± 0.1
A. sativum +L	21 ± 0.1	20 ± 0.2	22 ± 0.3	22 ± 0.1
C. verum + L	23 ± 0.1	21 ± 0.1	25 ± 0.1	25 ± 0.3

Inhibition zones of the plant extracts were shown as means of three replicates (n = 3) ± standard deviation

Table 6. Antibacterial activity (MIC and MBC in mg/ml and MBC/MIC) of the methanol, acetone and distilled water herb extracts on S. aureus and K. pneumoniae

Extract	S. aureus									K. pneumoniae
	MBC (mg/mL)			MIC (mg/mL)			MBC/MIC			MBC (mg/mL)			MIC (mg/mL)			MBC/MIC
	M	A	D	M	A	D	M	A	D	M	A	D	M	A	D	M	A	D
A. sativum	50^a	47^a	R	25^a	25^a	R	2^a	2^a	2^a	13^a	25^a	R	6^b	12^a	R	2^a	2^a	-
C. verum	24^b	23^b	R	13^b	25^a	R	2^a	1^b	2^b	6^b	12^b	R	6^b	13^a	R	1^b	1^b	-
M. flabellifolius	22^bc	13^c	R	11^c	6^c	R	2^a	2^a	2^b	13^a	6^c	R	13^a	6^b	R	1^b	1^b	-
X. caffra	13^c	21^b	R	6^d	11^b	R	2^a	2^a	2^b	6^b	13^b	R	6^b	13^a	R	1^b	1^b	-
CV%	18.8	11.7	2.3	5.2	5.2	0.7	9.3	8.8	2.2	1.2	3.2	-	0.6	0.8	-	1.3	2.3	-
LSD at 5%	0.09	0.06	0.01	0.01	0.02	0.001	0.33	0.28	0.11	0.002	0.01	-	0.00	0.002	-	0.03	0.05	-
p-value	<0.001	<0.001	<0.001	<0.001	<0.001	0.76	0.81	<0.001	<0.001	<0.001	<0.001	-	<0.001	<0.001	-	<0.001	<0.001	-

M-methanol, A-acetone, D- distilled water, MBC- Minimum Bactericidal Concentration, MIC- Minimum Inhibitory Concentration, Different letters in the same column reflect statistically significant differences.

Table 7. Ficis of S. aureus for herb extracts combinations

Herb combination	MIC herb combinations (mg/mL)	MIC 1 (mg/mL)	FIC 1	MIC 2 (mg/mL)	FIC 2	FICI	Type of interaction
A+C	11^d	11	1.00	25	0.44	1.4	Indifferent
A+D	12^d	11	1.09	13	0.92	2.0	Indifferent
A+E	12^d	11	1.09	25	0.48	1.6	Indifferent
A+F	11^e	11	1.0	25	0.44	1.4	Indifferent
A+G	6^i	11	0.55	6	1	1.6	Indifferent
A+H	6^j	11	0.55	11	0.55	1.1	Indifferent
B+C	12^c	6	2	25	0.48	2.5	Indifferent
B+D	8^g	6	1.33	13	0.62	2.0	Indifferent
B+E	6^i	6	1	13	0.46	1.5	Indifferent
B+F	7^h	6	1.17	25	0.28	1.5	Additive
B+G	2^n	6	0.33	6	0.33	0.7	Additive
B+H	2^n	6	0.33	11	0.18	0.5	Synergistic
C+E	13^b	25	0.52	13	1.0	1.5	Indifferent
C+F	13^b	25	0.52	25	0.52	1.0	Indifferent
C+G	13^c	25	0.52	6	2.17	2.7	Indifferent
C+H	3^m	25	0.12	11	0.27	0.4	Synergistic
D+E	14^a	13	1.08	13	1.08	2.2	Indifferent
D+F	9^f	13	0.69	25	0.36	1.1	Indifferent
D+G	3^k	13	0.23	6	0.50	0.7	Additive
D+H	3^l	13	0.23	11	0.27	0.5	Synergistic
E+G	3^kl	25	0.12	6	0.50	0.6	Additive
E+H	3	25	0.12	11	0.27	0.4	Synergistic
CV%	1.4	-	-	-	-	-	-
LSD at 5%	1.844	-	-	-	-	-	-
P value	<.001	-	-	-	-	-	-

Symbols: A-acetone extract from X. caffra; B-methanol extract from X. caffra; C-acetone extract from C. verum; D-methanol extract from C. verum; E-acetone extract from A. sativum; F-methanol extract from A. sativum; G-acetone extract from M. flabellifolius; H-methanol extract from M. flabellifolius, MIC- Minimum Inhibitory Concentration, FIC-fractional inhibitory concentration, FICI-fractional inhibitory concentration index, Different letters in the same column reflect statistically significant differences (p<0.05).

Table 8. Ficis of K. pneumoniae for herb extracts combinations

Herb combinations	MIC herb combinations (mg/mL)	MIC 1 (mg/mL)	FIC 1	MIC 2 (mg/mL)	FIC 2	FICI	Type of interaction
A+C	6^c	13	0.46	13	0.46	1.0	Indifferent
A+D	7^b	13	0.53	6	1.17	1.7	Indifferent
A+E	6^c	13	0.46	6	1	1.5	Indifferent
A+F	6^d	13	0.46	6	1	1.5	Indifferent
A+G	1^l	13	0.08	6	0.17	0.3	Synergistic
A+H	1^lm	13	0.08	13	0.08	0.2	Synergistic
B+C	3^g	6	0.50	13	0.23	0.7	Additive
B+D	4^f	6	0.67	6	0.67	1.3	Indifferent
B+E	3^g	6	0.50	6	0.50	1.0	Indifferent
B+F	3^h	6	0.50	6	0.50	1.0	Additive
B+G	3^hi	6	0.50	6	0.50	1.0	Additive
B+H	3^i	6	0.50	13	0.23	0.7	Additive
C+E	2^j	13	0.15	6	0.33	0.5	Synergistic
C+F	2^j	13	0.15	6	0.33	0.5	Synergistic
C+G	1^m	13	0.08	6	0.17	0.3	Synergistic
C+H	1^m	13	0.08	13	0.08	0.2	Synergistic
D+E	8^a	6	1.33	6	1.33	2.7	Indifferent
D+F	6^e	6	1	6	1	2.0	Indifferent
D+G	1^k	6	0.17	6	0.17	0.3	Synergistic
D+H	1^m	6	0.17	13	0.08	0.3	Synergistic
E+G	1^m	13	0.08	6	0.17	0.3	Synergistic
E+H	1^m	13	0.08	13	0.08	0.2	Synergistic
CV%	3.10	-	-	-	-	-	-
LSD at 5%	1.64	-	-	-	-	-	-
P-value	<.001	-	-	-	-	-	-

Symbols: A-acetone extract from X. caffra; B-methanol extract from X. caffra; C-acetone extract from C. verum; D-methanol extract from C. verum; E-acetone extract from A. sativum; F-methanol extract from A. sativum; G-acetone extract from M. flabellifolius; H-methanol extract from M. flabellifolius, MIC- Minimum Inhibitory Concentration, FIC-fractional inhibitory concentration, FICI-fractional inhibitory concentration index, Different letters in the same column reflect statistically significant differences (p<0.05).