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

Abstract

The threat of antibiotic resistance and the antecedent evolution of harmless microbes into superbugs is an epidemic of global concern. This study assessed the in-vitro herb to herb antibacterial activity of acetone, methanol, and water extracts of Ximenia caffra Sond. Myrothamnus flabellifolius Welw. Allium sativum L, and Cinnamomum verum J.Presl. against multidrug-resistant (MDR) mastitogenic Staphylococcus aureus and Klebsiella pneumoniae. Their minimum inhibitory concentration (MIC), minimal bactericidal concentration (MBC), and zone of inhibition against S. aureus and K. pneumoniae were measured using a microplate serial dilution technique and agar-well diffusion method. All the acetone and methanol plant extracts showed antimicrobial activity against MDR isolates tested. The MIC for acetone and methanol extracts against S. aureus and K. pneumoniae ranged from 6–25 mg/mL and decreased in the combined herbs from 1 mg/mL to 13 mg/mL. The MBC for acetone and methanol extracts against the tested bacteria ranged from 6–50 mg/mL. The acetone and methanol extracts had inhibition zones against S. aureus and K. pneumoniae ranging from 14–22 mm. The plant extracts showed a synergistic effect when combined with Gentamycin and Lincomycin as the zone of inhibitions was increased and ranged from 25–29 mm. Findings indicate that all the extracts had a dose-dependent inhibitory effect on the growth of multidrug-resistant S. aureus and K. pneumoniae. The study’s findings further reinforce the importance of these extracts in traditional veterinary and healthcare practices. As a result, this study concludes that antibiotics and plant extracts can be administered concurrently to treat mastitis caused mostly by S. aureus and K. pneumoniae.

Keywords:

• herb-herb combinations
• antimicrobial activity
• multidrug-resistance mastitis-causing bacteria
• Staphylococcus aureus
• Klebsiella pneumoniae

Public interest statement

Antibiotic resistance and the antecedent evolution of harmless microbes into superbugs are serious threat to public health, with significant rates of mortality and morbidity. Among other microbes, resistance has been found among mastitis pathogens (for example S. aureus and K. pneumoniae). As a result, in low income settings there has been a shift in the use of conventional drugs in households, public spaces, and livestock production. Instead of using antibiotics, plants have been identified as alternatives with important sources of active pharmaceutical ingredients (APIs).This article therefore provides a comprehensive analysis of the effectiveness of herbs (either alone or in combination) against the most frequent multidrug-resistant mastitogenic bacteria (S. aureus and K. pneumoniae) in Zimbabwe.

1. Introduction

Mastitis affects ovarian follicular responses in cows, which reduces fertility and causes dramatic changes in the flavor, color, and smell of milk. As a result, mastitis is economically significant in dairy herds. The most prevalent etiological agents of mastitis in Zimbabwe’s Midlands province are Staphylococcus aureus and Klebsiella pneumonia, although numerous other bacterial species are involved and these include Streptococcus agalactiae, Escherichia coli, Micrococcus pyogenes, Streptococcus dysgalactiae, Corynebacterium pyogenes, Streptococcus uberis, Citrobacter freundii, Proteus mirabilis (Gufe et al., 2021). Antibiotics are widely used to treat mastitis following bacterial identification and antibiotic susceptibility testing. The development and spread of antibiotic resistance among mastitis-causing bacteria, on the other hand, make mastitis treatment difficult. The golden age of new antibiotics came to an end in the 1960s. Since then, the growth of resistant bacteria has outpaced the drug development process (Cheesman et al., 2017), creating a global human health concern (Awouafack et al., 2013). Furthermore, the prevalence of Multidrug Drug Resistance (MDR) bacteria (also known as superbugs) has grown dramatically in recent decades due to widespread antimicrobial overuse (Cheesman et al., 2017; Gufe et al., 2021; Souza et al., 2019). Antibiotics used as animal feed additives appear to promote the emergence of antibiotic-resistant bacteria (Chattopadhyay, 2014). However, reducing antibiotic use may contribute to an increase in the prevalence of mastitis. This consequently calls for antibiotic susceptibility testing profiling before treatment of mastitis (Gufe et al., 2021).

Antibiotic resistance has been associated with clinical isolates of S. aureus (the primary source of infectious mastitis) and K. pneumoniae (the source of environmental mastitis). Resistance has been found among mastitis pathogens, including S. aureus which developed resistance to penicillin rapidly after its discovery (Cheesman et al., 2017). Recently, the prevalence of penicillin resistance has reached 88% in Tanzania (Ndahetuye et al., 2019), presenting concerns in mastitis therapy. In another recent study carried out in the Midlands province of Zimbabwe, it was revealed that S. aureus and K. pneumoniae were the most prevalent antibiotic-resistant mastitis-causing bacteria (Gufe et al., 2021). As a result, it is vital to seek innovative treatments that are better, cheaper, and have mild or no side effects, especially in developing countries. It has long been established that several natural plant and plant-derived substances are used to treat ailments (Al Bshabshe et al., 2020; Farooqui et al., 2015). Scientific evidence supports the idea that many plants have a high potential for producing a wide variety of secondary metabolites, which are the source of plant-derived antimicrobial compounds (PDAms) (Farooqui et al., 2015). Herbal remedies, such as single herb, and herb-to-herb combinations are used in many civilizations.

Traditionally, X. caffra leaves as well as roots have been used in the treatment of fever, infertility, diarrhea, and wounds (De Wet et al., 2012). X. caffra root and leaf extracts have been reported to be antifungal and antibacterial (Mulaudzi et al., 2011; Munodawafa et al., 2013). Several bioactive compounds have been identified from leaf extracts of X. caffra and these include flavonoids, glycosides, and gallic acid (Maroyi, 2016). Additionally, M. flabellifolius extracts have been found to contain bioactive compounds including alkaloids, steroids, polyphenols, terpenoids, anthocyanins, triterpenes, cardiac glucosides, flavonoids, saponins, and tannins (Brar et al., 2018), and these compounds have been linked to antiviral, antioxidant, antidiabetic and antimicrobial activities (Nantapo & Marume, 2022). The presence of bioactive compounds with significant therapeutic activities has contributed to the use of M. flabellifolius ethnomedicine and nutritional additive for animals. Generally, this plant has been used in animals for growth enhancement and health (Cheikhyoussef et al., 2015; Nantapo & Marume, 2022).

According to a report by Rahman (2003), Allium species boost the immune system and reduce ailments like diabetes and heart disease. Through a variety of clinical researches, the effectiveness of Allium sativum L. against bacterial and fungal infections has been reported (Rahman, 2003). The herb is traditionally used to cure earaches, stomach problems, and lung conditions (Badal et al., 2019). According to Al-Snafi (2013), A. sativum contains phytochemicals that have sulfur, such as vinyldithiins, thiosulfinates (allicin), ajoenes, and diallyl trisulfide. Due to the presence of a considerable quantity of allicin, A. sativum extracts have been shown to exhibit strong antibiotic activity against bacterial species such as Streptococcus mutans, Escherichia coli, Staphylococcus aureus, and Klebsiella aerogenes (Kuda et al., 2004; Wallock-Richards et al., 2014). Generally, many extracts of A. sativum were reported to be effective against pathogenic bacteria species (El-Saber et al., 2020). Pharmacologically, Cinnamomum verum extracts are active against diabetes, bacterial species, and cancer. This is attributed to a number of bioactive compounds associated with the extracts including eugenol, cinnamyl acetate, cinnamaldehyde, camphor and copane. The plant extracts are traditionally used to treat bronchitis, asthma, diarrhea, inflammation headache, and heart diseases (Singh et al., 2021). Antimicrobial activity of C. verum has been reported against many microbes. For example, exracts of C. verum were reported to be active against bacteria such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Bacillus cereus (Gende et al., 2008) and fungal species including Penicillium roqueforti, Candida lipolytica and Zygosaccharomyces rouxii (Goñi et al., 2009; Matan et al., 2006; Parthasarathy & Thombare, 2013).

Information on the use of X. caffra, M. flabellifolius, A. sativum, and C. verum against multidrug-resistant mastitogenic bacteria in Zimbabwe is limited. However, combining these herbs appears to boost antimicrobial activity since bioactive components increase with the number of herbs used (Che et al., 2013). Understanding the anti-staphylococcal and anti-klebsiella capabilities of individual herbs or synergistic combinations of herbs may have a substantial impact on cattle productivity and human health. As a result, the current study focuses on the effectiveness of herbs (either alone or in combination) against the most frequent multidrug-resistant mastitogenic bacteria (S. aureus and K. pneumoniae) in Zimbabwe.

2. Materials and methods

2.1. Preparation of plant extracts

Ximenia caffra (sour plum/Munhengeni) and Myrothamnus flabellifolius (Mupfandichimuka) were collected from Harare Botanical gardens. Allium sativum (garlic) and Cinnamomum verum (Cinnamon) were bought in Harare supermarkets. The plants were identified, authenticated, and certified by state-registered Botanists. The three plant materials were carefully washed using sterile distilled water to remove dirt and other foreign matters, oven-dried for 72 h at 60 ± 2 °C, and placed in a biosafety cabinet until completely dry. Dried leaves were then milled into fine powders stored in paper bags at room temperature. Cinnamon was bought already in powder form. For extraction, each plant’s fine powder was weighed and subjected to the three solvents, acetone, methanol, and distilled water. One gram of each plant extract was added to 10 ml of the solvent and shaken for 15 minutes. The obtained extracts (methanol (M.E.), acetone (A.E.), and distilled water (D.E.)) were strained using a cotton plug to remove the big solid plant particles and then filtered using a Whatman No.1 filter paper. After filtration, the extraction solvent was left to evaporate using a rotary vacuum evaporator, and the final dried extract was kept in vials in a refrigerator at −20 °C before use.

2.2. Bacterial strains

Clinical isolates of S. aureus and K. pneumoniae for this investigation were obtained from Central Veterinary Laboratories in Harare, and these isolates have previously been isolated and identified in our prior work (Gufe et al., 2021). The multi-drug resistance of the bacterial isolates was assessed using the Kirby-Bauer agar disc diffusion technique on Mueller—Hinton agar, and the findings were interpreted using the Clinical and Laboratory Standards Institute guidelines (CLSI; Hombach et al., 2013). MDR S. aureus and K. pneumoniae were classed as resistant to at least three antibiotic classes and were used to evaluate the efficiency of the herb-herb combination. For antimicrobial activity testing in this investigation, one S. aureus strain and one K. pneumoniae strain that indicated resistance to the most antibiotics tested were chosen. These strains of bacterial species were obtained from 540 isolates screened in our previous study (Gufe et al., 2021).

2.3. Plant extracts-agar well diffusion method

Agar well diffusion assay on Mueller Hinton Agar (MHA) was used as directed by (Irshad et al., 2012). One thousand microlitres of inoculums (approximately 1.5 × 10⁸ bacterial cells/ml) were pipetted into well-labelled plates. Twenty millilitres of autoclaved and cooled MHA were poured into the plates with bacterial suspension. The mixture was mixed by shaking and allowed to solidify, and 5 mm wells were made into the agar using sterile pasture pipettes. An amount of 100 mg stock of individual crude plant extract and the combined stock was prepared by dissolving 100 mg of dried plant extracts in 1 ml of distilled water. One hundred microliters of each plant extract were pipetted into each well, 100 µL of 0.5 mg/ml of Gentamycin (positive control) pipetted into one of the wells, while 100 µL of distilled water (negative control) was pipetted into one of the wells. The plates were incubated at 37 ± 2℃ for 24 h, and the diameter of the zone of bacterial inhibition around each well was measured (Farooqui et al., 2015; Irshad et al., 2012).

2.4. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

An antibacterial agent’s minimum inhibitory concentration (MIC) is defined as the lowest concentration at which no growth of microorganisms is observed after 24 hours of incubation (Mostafa et al., 2018). In contrast, the minimum bactericidal concentration (MBC) is the lowest concentration at which no growth of bacteria is observed (Debalke et al., 2018). The MIC of the plant extracts was determined according to the method described by (Dzoyem et al., 2014; Eloff, 2019), with a few adjustments employing 96 well microtitre plates containing 100 µL of water in each well. Serial dilutions of 100 µL of each dissolved plant extract (50 mg/mL) and the combined extracts were added (for each bacterial plate) to the first rows. The same was done for positive and negative controls, during which 100 µL of 2.5 mg/ml dissolved gentamycin was added in triplicate to the chosen last row. The bacterial suspensions of 100 µL in the growth medium were added to all wells except the last column, which is a positive control. Wells that received bacterial samples, growth medium, and distilled water were used as the negative control. The plates were incubated at 37 ± 2℃ for 24 h. Each row received 100 microliters of a 0.2 mg/mL solution of iodonitrotetrazolium chloride (INT), and then the plate was re-incubated for at least 30 mins to guarantee proper colour development. INT is a dehydrogenase activity detecting reagent that is metabolically active bacteria transform into a highly coloured red-purple formazan. A clear solution showed that growth was being inhibited. According to set-up ratios, pipetting known quantities of the four separate dissolved plant extracts was used to create herb-herb combinations. This value was used to calculate the extract’s MIC. The extracts used for MIC determination were dissolved in either acetone or methanol. For MBC determination, an equal volume of various concentrations of each extract and nutrient agar was mixed in microtubes to make up to 0.5 mL solution. The organism suspensions were added to each tube and incubated aerobically at 37ºC for 24 h. After incubation, the suspensions were subcultured on nutrient agar and further incubated for 24 h to determine the MBC. The lowest dilution that yielded no single bacteria colony was taken as the minimum bactericidal concentration. MBC/MIC ratios were calculated using MBC/MIC (Eloff, 1998).

2.5. Fractional inhibitory concentration index (FICI) calculation and interpretation

The calculation of fractional inhibitory concentration index (FICI) was based on MIC of plant extracts and the FICIs were determined utilising the formula: FICI = ΣFIC = FIC (plant extract 1) + FIC (plant extract 2), Where: FIC (extract) = MIC of extract in combination/MIC of extract alone (Haroun et al, 2016). The interactions were classified as indicated in Table 1.

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

2.6. Data Analysis

The data was recorded and analyzed using Microsoft Excel®. The mean and standard deviation descriptive data were extracted and evaluated using tables. The herb-to-herb combinations were investigated using one-way variance analysis (ANOVA).

3. Results and Discussion

3.1. Antimicrobial susceptibility testing of isolated bacteria

The multi-drug resistance (resistant to three or more antibiotic classes) mastitogenic bacteria were retrieved from storage, and their antimicrobial susceptibility pattern was re-determined (Table 2). K. pneumoniae was found to be MDR resistant after developing resistance to antibiotics such as Ertapenem 10 µg, Vancomycin 30 µg, Amikacin 30 µg, Ampicillin 10 µg, Penicillin G 10 µg, Ceftriaxone 30 µg, Kanamycin 30 µg, Neomycin 10 µg, Cloxacillin 5 µg, and Lincomycin 15 µg while remaining susceptible to Gentamycin. Vancomycin, Amikamycin, Neomycin, and Gentamycin were effective against S. aureus, while the other antibiotics tested were ineffective. Antimicrobial resistance is becoming more recognized as a global animal and human health risk that necessitates a well-coordinated One Health policy. MDR bacteria are becoming more widespread, making mastitis treatment and control more difficult (Gufe et al., 2021). These MDR bacteria jeopardize the effectiveness of several currently available and cheap antimicrobials on the market, particularly in underprivileged communities. For these reasons, scientists seek innovative antimicrobials from medicinal plants to combat the growing threat of dangerous MDR bacteria.

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.

3.2. Antimicrobial properties of extracts against MDR mastitis-causing bacteria

At 30 mg/ml concentrations, the antibacterial properties of the plant extracts were tested, and all of the plant extracts showed some level of antimicrobial activity against S. aureus and K. pneumoniae. In addition, all extracts were antibacterial against multidrug-resistant mastitis-causing bacteria, S. aureus, and K. pneumoniae. Notably, distilled water plant extracts’ antimicrobial activity was generally low, as indicated by the inhibition zones in Table 3. Acetone extracts of M. flabellifolius, A. sativum, and C. verum resulted in relatively wide inhibition zones against S. aureus (21 mm, 17 mm, and 20 mm, respectively). Leaf methanol extracts of X. caffra showed a broader zone of inhibition against S. aureus than K. pneumoniae.

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.

Similarly, methanol and acetone leaf extracts of A. sativum and C. verum inhibited the growth of S. aureus more than K. pneumoniae. Gentamicin was a positive control treatment and showed large inhibition zones (21 and 20 mm) against S. aureus and K. pneumoniae. The highest antibacterial activity was recorded for methanol extract of X. caffra against S. aureus and acetone extract of M. flabellifolius against K. pneumoniae. Generally, acetone extracts of M. flabellifolius, A. sativum, and C. verum and methanol extract of X. caffra exhibited stronger antimicrobial potency. This study showed that all the extracts from all medicinal plants studied provided a glimpse of future new drug discoveries to cure and control mastitis caused by MDR pathogenic bacteria (Tables 3, 4, 5, 6, 7 and 8).

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).

Gentamycin (positive control) was chosen for this investigation based on the results shown in Table 2, which reveal that all isolates of K.pneumoniae and S. aureus are susceptible to Gentamycin. Gentamycin is now being used to treat mastitis with modest effectiveness in Zimbabwe. Plant extracts and Gentamycin synergized against MDR S. aureus and K. pneumoniae (Table 4). When plant extracts were mixed with Gentamycin, the zone of inhibition increased (Tables 3 and 4). The synergistic action of plant extracts and Gentamycin demonstrated a high potential for plant-antibiotic combinations for treatment, even though they have not been thoroughly explored. As a result, further research is needed to identify novel compounds. They should be examined in vivo for toxicity after extraction and before being employed in novel therapeutic therapies.

Furthermore, because people have utilized these plants for numerous generations and animals, their toxicity may be minimal. Communal farmers have traditionally utilized herbs to treat cattle diseases such as mastitis. Consequently, our findings highlight the necessity of employing plant extracts in conjunction with antibiotics to manage MDR mastitis-causing bacteria, which are becoming a concern to animal udder health. Additionally, these extracts were efficacious against MDR bacteria at low concentrations, reducing the possibility of negative repercussions. There is evidence that current antibacterial medicines can be improved by partnering with plant-derived substances. For example, the efficacy of β-lactams coupled with bioactive compounds produced from various fruits, vegetables, and grains against -lactam-resistant strains of bacteria is considerably enhanced (Phitaktim et al., 2016; Sakagami et al., 2005; Siriwong et al., 2016). The capacity of plant chemicals to “re-purpose” conventional antibiotics to treat microbial illnesses may substantially influence the fight against MDR bacteria (Nascimento et al., 200). A different research found that extracting the leaves of Berberis aetnensis lowered the MIC of ciprofloxacin against E. coli, S. aureus, and P. aeruginosa (Nascimento et al., 200). In addition, an MDR strain of P. aeruginosa was discovered to respond favorably to antibiotics and extracts of jambolana, thyme, clove, and pomegranate (Nascimento et al., 200). Similarly, clove-tetracycline and clove-ampicillin combinations showed increased antibacterial action against Proteus spp. and K. pneumoniae, respectively (Nascimento et al., 200). Additionally, when used in combination with other antibiotics, crude extracts of several other plants showed noticeably reduced Minimum inhibitory concentration against drug-resistant P. aeruginosa and clinical isolates of methicillin-resistant S. aureus (Adwan et al., 2010). In addition to offering hope in the fight against MDR pathogens, these synergistic studies also hint at the potential for better and more effective treatment.

When it was discovered that Lincomycin was less effective against S. aureus and K. pneumoniae, plant extracts were combined with these antimicrobial (Lincomycin), and synergism was examined. Table 5 shows that the addition of plant extracts to lincomycin increased the zone of inhibition against S. aureus and K. pneumoniae. Overall, the results of this experiment indicated that plant extracts can enhance the antibacterial activity of antibiotics against S. aureus and K. pneumoniae. These data corroborate with previous studies suggesting that certain plant extracts can increase the antibacterial activity of antibiotics in vitro (Betoni et al., 2006; Esimone et al., 2006).

The MBC of methanol plant extracts was significantly different across all extraction media (p < 0.001). Acetone extracts of A. sativum, C.verum, and M. flabellifolius (47 mg/mL, 23 mg/mL, and 13 mg/mL, respectively) had relatively lower MBCs than other extraction methods of the same herbs (Table 5). However, methanol extracts of X. caffra recorded the lowest MBC (13 mg/mL) compared to this herb’s acetone and distilled water extracts. The MIC of methanol extracts are significantly different (p < 0.001), with the lowest MIC of X.caffra (6 mg/mL) and the highest of A. sativum (25 mg/mL). Acetone extracts MICs show significant differences (p < 0.001), and the lowest was recorded for M. flabellifolius (6 mg/mL) and the highest of 25 mg/mL for A. sativum and C.verum.

The MBC of methanol and acetone plant extracts differed significantly (p < 0.001). However, methanol extracts of A. sativum and M. flabellifolius had the same MBC of 6 mg/mL. The MBCs of X.caffra and C.verum were the same (13 mg/mL) and significantly higher (p < 0.001) than the MBCs of A. sativum and M. flabellifolius. Acetone extracts of all herbs had significantly different MBCs (p < 0.001), with the highest of 25 mg/mL for A. sativum and the lowest of 6 mg/mL for M. flabellifolius. The MIC of methanol extracts for M. flabellifolius (13 mg/mL) was significantly higher (p < 0.001) than for A. sativum, X.caffra, and C.verum (6 mg/mL). Acetone extracts of M. flabellifolius had significantly lower (p < 0.001) MIC (6 mg/mL) when compared with A. sativum, X.caffra, and C. verum.

All the plant extracts were bactericidal as the MBC/MIC ratios were less than 4. When compared to S. aureus (MIC = 6–25 mg/mL), K. pneumoniae, a Gram-negative bacteria, may have been the most sensitive (MIC = 6–13 mg/mL) (Table 2). The lowest MICs were observed for methanol extracts of X. caffra, C. verum, A. sativum, and acetone M. flabellifolius (6 mg/mL). These extracts are highly potent against K. pneumoniae and S. aureus, respectively. The highest MICs (25 mg/mL) were recorded for C. verum acetone, and A. sativum methanol extracts against S. aureus. The disparity in susceptibility between K. pneumoniae and S. aureus might be attributed to differences in the cell wall structure. The cell wall of S. aureus comprises 70–100 layers of peptidoglycans (Naz & Bano, 2012; Tekwu et al., 2012). Peptidoglycan comprises two polysaccharides, N-acetyl-glucosamine and N-acetyl-muramic acid, joined together by peptide side chains and cross bridges. As an explanation, this is undoubtedly an understatement, and additional mechanisms are most likely involved.

The antibacterial effects of the plant extracts shown in Tables 3 and 6 may be due to bioactive compounds found in these plants, as previously documented. Given the presence of many bioactive compounds such as flavonoids, glycosides, and gallic acid, X. caffra root and leaf extracts have been found to be antifungal and antibacterial (Maroyi, 2016; Mulaudzi et al., 2011; Munodawafa et al., 2013). Accordingly, M. flabellifolius extracts have been shown to include alkaloids, steroids, polyphenols, terpenoids, anthocyanins, triterpenes, cardiac glucosides, flavonoids, saponins, and tannins, all of which have been connected to the plant’s antimicrobial action (Brar et al., 2018; Cheikhyoussef et al., 2015; Nantapo & Marume, 2022). Allium species, notably A. sativum L., have been shown to exhibit antibacterial properties (Al-Snafi, 2013; Badal et al., 2019). This report agrees with our findings, which Wallock-Richards et al. (2014) attributed to the presence of allicin. Our findings are comparable with those of Kuda et al. (2004), who found that A. sativum extracts have high antibiotic action against Staphylococcus aureus and Klebsiella aerogenes, among other bacterial species. Cinnamomum verum extracts were found to be effective against Staphylococcus aureus and Klebsiella aerogenes. This might be due to bioactive chemicals found in plant extracts such as eugenol, cinnamyl acetate, cinnamaldehyde, camphor, and copane (Singh et al., 2021). Extracts of Cinnamomum verum have been shown to be effective against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Bacillus cereus (Gende et al., 2008).

Some of the MICs of herb combinations were not significantly different. Generally, the MICs of herb combinations indicated significant differences (p < 0.001), with a few showing no significant difference (Tables 7 and 8). All Herb combinations with A (X. caffra acetone) resulted in an indifferent herb interaction (Table 7). Most of the herb combinations (67%) were indifferent against S. aureus (14/21) (Table 7). Only four herb combinations (20%) had an additive effect, four herb combinations (20%) had a synergistic interaction, and none were antagonistic. In contrast, most herb combinations (48%) were synergistic against K. pneumoniae (10/21) (Table 8). Eight herb combinations (36%) had an indifferent effect, three herb combinations (14%) had an additive interaction, and none of the herb combinations was antagonistic against K. pneumoniae (Table 8).

4. Conclusion

According to our findings, all of the multidrug-resistant bacteria were sensitive to Gentamycin but resistant to Lincomycin. Furthermore, the results showed that plant extracts in acetone and methanol had a good antibacterial potential against MDR mastitis-causing S. aureus and K. pneumoniae. The plant extracts and the two antibiotics were combined and exhibited a significant level of synergism. Combining antibiotics with plant extracts has a synergistic effect against resistant bacteria, as evidenced by an increase in the zone of inhibition, opening up new options for treating bacterial mastitis. This action permits the antibiotic to be utilized in situations where it was previously ineffective during therapeutic therapy. As a consequence, this study suggests that it is possible to utilize these drugs and plant extracts concurrently to treat mastitis caused mostly by S. aureus and K. pneumoniae. It would be interesting to explore the extracts’ mode of action, phytochemical composition, and toxicity, and then extend it to additional resistant mastitis-causing bacteria and fungi. More scientific research on previously unknown herbal medicines used to treat mastitis, as well as in-depth phytochemical screening and in-vivo effects of anti-mastitis herbs, are required.

Acknowledgments

The authors thank Central Veterinary Laboratories for the laboratory space, reagents, and bacteria used in this study.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Notes on contributors

Claudious Gufe

Claudious Gufe is a PhD candidate at the King Mongkut’s University of Technology Thonburi in Bangkok, Thailand, and a veterinary laboratory scientist.

Tanaka Nicola Mugabe

Tanaka Nicola Mugabe Is student at Chinhoyi University of Technology, Chinhoyi, Zimbabwe. She has interest in environmental and animal biotechnology.

Zakio Makuvara

Zakio Makuvara He holds Masters of Science in Applied Microbiology and Biotechnology from National University of Science and Technology in Zimbabwe. PhD student at the University of South Africa, working on antimalarial plant based medicines.

Jerikias Marumure

Jerikias Marumure Completed his Masters of Science in Applied Microbiology and Biotechnology from National University of Science and Technology in Zimbabwe. PhD student at University of South Africa, working on antibacterial resistance and the efficacy of antidiarrheal plants against cholera.

Mbonjani Benard

Mbonjani Bernard Is a Laboratory Assistant at Central Veterinary Laboratory in Harare, Zimbabwe. He has interests in animal health management, microbiology, including the isolation, identification, and susceptibility testing of microorganisms.