SYNERGISTIC AND ANTAGONISTIC EFFECTS ON ANTIMICROBIAL PROPERTIES OF ORGANIC AND AQUEOUS EXTRACTS OF BACILLUS CLAUSII KP10 IN COMBINATION WITH CONVENTIONAL ANTIBIOTICS

S. Kabeer and Z. Mushtaq*

Bioactive Molecules Research Lab (BMRL), Department of Biochemistry, University of Agriculture, Faisalabad, Pakistan, 38040

*Corresponding Email: zahidmushtaquaf@uaf.edu.pk

ABSTRACT

 Due to emergence of multidrug resistance, alternative approaches are practiced and needed in conventional antimicrobial therapies. Drug combination therapies have been used like synergism for better results with decreased drug dosage to avoid toxicity than monotherapy, but results can be additive and antagonistic depending on their combined effect. The present project was therefore designed to assess the effects of already reported bioactive fractions of B.clausii KP10 with each other and with antibiotics in synergistic manner to combat microbes.

Different solvents were used for the extraction and fractionations. Antimicrobial activities were performed against different available bacterial and fungal strains. In our experiments crude methanolic extract (CME), n-hexane fraction (n-HF), chloroform (CLF), ethyl acetate (EAF), Methanol Soluble (MSF) and water soluble fractions (WSF) were obtained and then combinations of each were prepared with (1:1, v/v) each other and with reported antibacterial and antifungal drugs streptomycin (STM) and Terbinafine (TER) separately (1:1:1). Single extract with drug combination was also prepared (1:1). Maximum zone of inhibition was shown by combination of n-HEF:MSF (24 mm) against S. aureus. CLF:WSF:STM showed maximum ZOI (37.2 mm) against E. coli. WSF: STM showed maximum ZOI (33 mm) against S. aureus. Maximum antifungal activity was shown by CME:WSF:TER (26 mm) and EAF:TER with maximum ZOI (17 mm) against Fusarium solani. n-HEF:EAF:TER showed maximum ZOI (18 mm) against Aspergillus niger. Combination of extracts with each other usually showed antagonistic behavior but extracts with standard drugs have shown synergy which could be a better approach in the development of antimicrobials therapies.

https://doi.org/10.36899/JAPS.2023.1.0603
Published first online September 20, 2022

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INTRODUCTION

Infectious diseases caused by bacteria and fungi affect millions of people worldwide.The condition is getting worst day-by-day due to rapid evolution of multidrug-resistant microbes, bacteria have a multitude of mechanisms by which they can rapidly acquire resistance (Buroni et al., 2019).Therefore, new approaches are needed to combat infective microbes and overcome their microbial resistance (Martins et al., 2020). Gram positive bacteria such as Staphylococcus aureus are mainly responsible for toxic shock syndrome, post-operative wound infections, endocarditis, pneumonia, food poisoning and osteomyelitis (Benayacheet al., 2001). Bacillussubtilis cause anthrax infections to human (La Jeon et al., 2012). Gram negative bacteria such as Pasteurella multocida are often associated with chronic as well as acute infections in both animals and humans (Harper et al., 2006). In intestine of human Escherichia coli is present and causes the infection of lower urinary tract, septicaemia or coleocystis. (Benhassaini et al., 2003). Fungi can also cause many diseases to plants and humans (Panackal et al., 2006). For example, A. niger cause invasive diseases associated with otomycosis and other infections to human (Araiza et al., 2006), A. niger also caused Black mold disease on certain fruits and vegetables such as onions, grapes and peanuts (Moghtader, 2013). Fusarium solaniis the main causative agent in 37%–50% of fungal keratitis cases. Onychomycosis is another human infection with a high mortality rate caused by Fusarium fungi (Monod and Mehul, 2019), also caused localized infections in skin and other parts of body (Gupta et al., 2000).

Resistance of multidrug in the human pathogenic microbes has been developed as a result of indiscriminate usage of antimicrobial drugs that are mostly used to treat infectious diseases. Resistance of antibiotic is caused by a multi-factorial reasons, including the specific nature of relationship of the microbes to antibiotics & also usage of antimicrobial agent, host characteristics and environmental factors (Xieet al., 2009). This alarming situation has convinced scientists to find out new antimicrobial agents from different sources as novel antimicrobial chemotherapeutic agents. One of the leading approach is to control these infectious diseases without side effects is use of some specific types of bacteria which are capable of producing bioactive compounds that can help to cure diseases such as probiotics (Xieet al., 2009). Bacteria produce biologically active compounds against other bacteria and fungi that acts against the specific physiological conditions of a diseased body as antimicrobial agents. Bacillus genus is well known for producing the bioactive compounds that work as antimicrobial agents. Compounds like bacteriocins and bacteriocin like inhibitory compounds are synthesized ribosomally antimicrobial peptides which are formed by different bacteria which are mostly effective against species that are closely related too (Riley and Wertz 2002; Cherif et al., 2003;Abriouelet al., 2011).

In clinical practice, antimicrobial combination therapy is one of the leading novel advances to combat resistance of microbes (Lambert, 2000; Hemaiswarya et al., 2008; Van Vuurenet al., 2009). Combination therapy has stimulated renewed interest in recent years with major safety concerns. For example, combinations of gentamicin and chloramphenicol could be enhanced by use of the plant materials against Methicillin resistant Staphylococcus aureus (MRSA) (Darwish et al., 2002; Lu and Di, 2020).  The most common reasons of combination therapy usage are to reduce the emergence of resistance in strains to minimize toxicity by lowering the dose of toxic drug, treat poly-microbial infection and also increase killing or inhibition of resistance in organisms to appropriate doses of single antimicrobial compound (Rahal, 1978; King et al., 1981).

Therefore, the purpose of present work was to determine the antimicrobial effects of already reported bioactive fractions of B. clausii KP10 with each other and in combination with standard drugs to find out the possible synergistic or antagonistic effects of the combination for the development an improved antimicrobial therapy.

MATERIALS AND METHODS

Experimental work was conducted in August 2019 in Bioactive Molecules Research Laboratory (BMRL), Department of Biochemistry, University of Agriculture, Faisalabad, Pakistan.

Culturing of bacteria and extraction: Bacillus clausii KP10 isolated previously by our research group (Erum et al., 2017), that was used in this experiment as source of bioactive extracts. Nutrient agar and broth (MERCK) were used for the growth of bacterial cultures (Muller et al., 2016). The medium of the pH was maintained at 10 with 0.1 N HCl / NaOH before sterilization. The medium was autoclaved at 121 °C for 20 minutes at 15psi pressure. Bacterial culture was grown at the 40 °C for 24 hours for sporulation (Erum et al., 2017). Then cell mass of bacteria was obtained when centrifuged at 11963 ×g for 10 minutes. Bioactive fractions were obtained. For extraction of bioactive compounds different organic (n-hexane, chloroform, ethyl acetate and methanol) and aqueous solvents were used as described in literature (Nighat and Mushtaq, 2019; Nisa, 2011). Finally, carefully layers were separated then dried weighed and dissolved in Dimethyl sulfoxide and stored at 4 °C for bioactivities.

Working and stock solutions preparation: In distilled water streptomycin was dissolved and terbinafine was solubilized in Dimethyl sulfoxide to make 10 mg/mL final concentration and then stored at the 4 °C until use (Liu et al., 2015). For positive control streptomycin and terbinafine was also used with concentration of 50 mg/mL for antimicrobial activities. Combinations of extracts were prepared as follows 1:1 and 1:1:1 v/v as shown in the table 1.

Antimicrobial assay: Antimicrobial assays of extracts of each combination were checked against certain gram-negative bacteria (Pasteurellamultocida and Escherichia coli), gram-positive bacteria (Staphylococcusaureus and Bacillus subtilis) and also two fungal strains (Fusarium solani and Aspergillus niger) using well-diffusion method (Zaidan et al., 2005; Ahmad and Aqil, 2007).

Statistical analysis: The data was analyzed by one-way analysis of variance (ANOVA) and Tukey HSD’s multiple range test (p≤0.05) using the SPSS software to test significance of differences among mean values of samples for different combinations regarding bacterial and fungal strains.

Table 1. Combinations for the Evaluation of Synergistic and Antagonistic interaction of selected antibiotics with Bacillus clausii KP10 extracts/fractions.

Sample #	Combination I (1:1)	Combination II (1:1:1)	Combination III (1:1)
1	CME:n-HEF	CME:n-HEF:+ve drugs	CME:+ve drugs
2	CME:CLF	CME:CLF:+ve drugs	n-HEF:+ve drugs
3	CME:EAF	CME:EAF:+ve drugs	CLF:+ve drugs
4	CME:MSF	CME:MSF:+ve drugs	EAF:+ve drugs
5	CME:WSF	CME:WSF:+ve drugs	MSF:+ve drugs
6	n-HEF:CLF	n-HEF:CLF:+ve drugs	WSF:+ve drugs
7	n-HEF:EAF	n-HEF:EAF:+ve drugs
8	n-HEF:MSF	n-HEF:MSF:+ve drugs
9	n-HEF:WSF	n-HEF:WSF:+ve drugs
10	CLF:EAF	CLF:EAF:+ve drugs
11	CLF:MSF	CLF:MSF:+ve drugs
12	CLF:WSF	CLF:WSF:+ve drugs
13	EAF:MSF	EAF:MSF:+ve drugs
14	EAF:WSF	EAF:WSF:+ve drugs
15	MSF:WSF	MSF:WSF:+ve drugs

CME:Crude Methanolic Extract, n-HF:n-Hexane Fraction, CF:Chloroform Fraction, EAF:Ethyl acetate Fraction, MSF:Methanol Soluble Fraction, WSF:Water Soluble Fraction, +ve drugs= streptomycin (STM), Terbinafine (TER).

Antimicrobial activities were performed by well diffusion method with the help of using above mentioned combinations (table1).

RESULTS AND DISCUSSION

Combination drug therapy has been shown to delay the emergence of microbial resistance and could also produce beneficial effects in treatment of microbial infections. Synergism of drug with bioactive microbial extracts and with known antibiotics is a modern concept and it could be advantageous (additive or synergistic interaction) or may be deleterious (toxic or antagonistic outcome) (Gibbon, 2004). Bioactive compounds can be good option when used concurrently with the standard drugs, where they enhance the activity of the drug (Aiyegoro and Okoh, 2009). Pharmacological benefits were recorded in way when one drug is involved to clear infection from one part of the body while other drug clear it from different site (Williamson, 2001).

Drug interactions with microbial extracts have gained remarkable scientific interest (Ocampo et al., 2014; Yilancioglu et al., 2014). Therefore, antimicrobial assays of bioactive fractions of B. clausii KP10 with each other and with standard drugs were analyzed. Following fractions were obtained from the Bacillus clausii KP10 by using solvents such as methanol extract, n-hexane fraction, chloroform fraction, ethyl acetate fraction, methanol soluble fraction and water soluble fraction. Yields were 50, 100, 110, 113, 100 and 90 mg. All obtained extracts were then further diluted in the DMSO to a specific concentration.

According to our previous data published bioactive extracts of Bacillus clausii KP10 were bioactive in nature and effective against Escherichia coli, Pasteurella multocida, Bacillussubtilis andStaphylococcus aureus, but most effective against P.multocida. So they can be used as antibacterial and antifungal agents (Nighat and Mushtaq, 2019; Nighat et al., 2020). Bacillusclausii KP10 bioactive fractions have great potential as antimicrobials against different tested microorganisms. The results obtained from combination I as mentioned in materials and methods are summarized in Table 2 and Figure 1. CME:CLF showed maximum ZOI (11 mm) against P. multocida, CME:EAF showed maximum ZOI (22 mm) against E. coli and n-HEF:MSF showed highest antibacterial assay against the S. aureus with maximum ZOI (24 mm) recorded. Results of combination I shows that the interactions between organic and aqueous extracts were mostly antagonistic, when used with each other against selected bacterial and fungus strains. Antagonism occurs when one drug hinders another drug's effect (Johansen et al., 2000). Similar studies in literature showed that the combination of Ethyl acetate:Chloroform (1:1), Water:Methanol (1:1) and Methanol:Acetone (1:1) extracts of Trichophyton, Microsporum and Epidermophyton genera exhibited no synergistic activity with each other and showed antagonistic behavior in combination against all tested microorganisms (Koroishi et al., 2008). The results give clear evidence that antimicrobial assay of organic and aqueous extracts of B. clausii KP10 is reduced when used in combination, but when the same extract of B. clausii KP10 used concurrently with standard drug streptomycin (STM) they enhance the activity of the drug.

Table 2. Antimicrobial activities of combination I.

*Results are expressed as mean (ZOI)±S.D.

Antibacterial activities of combination II and III

In our experiments we checked both combinations single extract of B. clausii with antibiotic and also combined extract of B. clausii KP10 with standard antibiotic streptomycin as shown in combination II and III. We conclude that the combined effects with streptomycin was almost equal in general behavior in both combinations. Combinations showed promising synergistic results and antagonistic interactions were not observed with streptomycin. These results were further confirmed by measuring the zone of inhibition (Figure: 2a, 2b and 3) which showed that the highest synergism was observed in the combination of CLF:WSF:STM (1:1:1) against E. coli maximum inhibition zone were recorded (37 mm). CLF:STM showed maximum ZOI (34 mm) against E. coli, WSF:STM showed maximum ZOI (33 mm), CLF:MSF:STM maximum ZOI (37 mm) against B. subtilis, WSF:STM showed maximum ZOI (34 mm) against S. aureus, CLF:STM showed maximum ZOI (31 mm), EAF:MSF:STM showed maximum ZOI (31 mm) against P. multocida.

 Oliveira et al., (2011) studied synergistic effect of tetracycline, norfloxacin and erythromycin with ethanol extracts of Mangifera indica L. peel against the selected Staphylococcus aureus strains. Separate extracts did not have any beneficial antibacterial activities but when these extracts combined with antibiotics, significant synergistic effect was observed.

Antifungal activities of combination II and III

Results obtained from single extract of B. clausii against Fusarium solani no antifungal activity was observed and also combination I exhibited no interaction against tested fungi aresummarized in table 2. But when the same combinations were used with standard Antifungal drug (1:1:1) in combination II and (1:1) in combination III as mentioned in materials and methodsthe activity was enhanced and showed synergistic effects in (1:1:1) mostly against Fusarium solani (CME:WSF:TER) showed maximum inhibition zone (26 mm) EAF:TER showed maximum ZOI (17 mm) against Fusarium solani. In some combinations we observed both results minor synergy in (CLF:TER) showed maximum ZOI (14 mm) but mostly additive/antagonistic effects against A. niger (Table 3) and (Fig 4a and Fig 4b). Advantages over monotherapy include increased fungal killing potency decreased resistant strains and reduced dose-related toxicity of the antifungal drugs (Vitale et al., 2005). Overall, our results proved that drug combinations that inhibit growth of one fungus but it may not be always suitable for other due to the potential differences towards drug combination and type of strains too.

Literature revealed that the combination of fluconazole with crude extracts of green tea showed no significant difference in zones of inhibition. Whereas crude extracts showed diminished activity with combination of fluconazole than with fluconazole alone activity. Result shows combination of crude extracts of green tea with conventional antifungal fluconazole showed antagonism along with minor synergy (Erolls et al., 2015).

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