Pathogenic bacteria increase the incidence of resistant strains worldwide, and thus become an important cause of mortality and morbidity around the world [1,2].

Nosocomial and community-acquired infections are both caused by emerging resistant strains to antimicrobials [2]. The lowest concentration of antibiotic that inhibits obvious growth of bacteria after overnight incubation is considered as MIC [3]. The lowest inhibitory concentration could be either mainly bacteriostatic or bactericidal. Bacteriostatic effect of antimicrobial needs the help of human defense system. If human defense system is not satisfactory or has already been impaired due to infection such as meningitis and endocarditis, the existing infection will relapse after stopping bacteriostatic dose. This circumstance requires bactericidal dose [4].

In contrast, bacteriostatic dose is not preferable for the treatment of streptococcal infections and C. gangrene as the cidal dose causes the release of internal toxin from dying cells which could be dangerous for human [5]. Therefore, treatment of infectious diseases is becoming more difficult due to emerging resistance and low susceptibility of strains to antibiotics [6].

Medicinal plants contain compounds that are capable of providing health benefits by initiating certain physiological actions in the body [2]. According to an estimation made by the World Health Organization (WHO), around 80% of people in developing countries prefer medicinal plants than drugs for their simple health problems [7]. Different parts of medicinal plants are used as raw drugs, and demonstrated numerous medicinal properties [8]. Euphorbiaceae is the fourth major family of the angiosperms, containing more than 300 genera and almost 7,500 species. Plants that belong to Euphorbiaceae

family are used as food and in the treatment of infectious diseases such as diarrhoea, dysentery, and skin infections [9]. B. angulatais a species that belongs to the family Euphorbiaceae [10], native to Borneo island of Malaysia. The soft whitish part of this fruit (berry) is edible, while the red part (skin) is sour, and usually cooked by the rural communities [11]. The antimicrobial activity of genus of Euphorbia, which belongs to the family Euphorbiaceae, has been carried out against many bacteria. Ethanol, acetone, and aqueous extracts of Euphorbia fruticosa and methanol extracts of Euphorbia macroclada demonstrated antimicrobial effect against

S. aureus [12]. The antimicrobial activity of B. angulata fruit extracts is not previously tested. Therefore, the aims of this study are to: investigate the bacteriostatic and bactericidal effects of B. angulate (whole fruit, fruit skin, and berry) fruit extracts on a number of human pathogenic Gram-positive bacteria, and Gram-negative bacteria, evaluate different methods for screening antimicrobial property of B. angulate using, agar well diffusion, and micro dilution methods, and compare bacteriostatic and bactericidal effects of different extracts from different parts of the fruit using different solvents.

Plant material

B. angulate fruits were purchased from Bau, Sarawak, Malaysia in 2013. The fruits were wrapped in papers and placed in a box, before being transferred via airmail. The fruit was identified by Mr. Kamaruddin Salleh (Forest Research Institute of Malaysia). The collected fruits were later stored at -30°C at the Department of Nutrition Kulliyyah of Allied Health Science IIUM, Kuantan.

Extraction method

The three parts of the B. angulate fruit (whole fruit, fruit skin and berry) were freeze dried, and reduced to a coarse powder. All the three parts of the fruit were then extracted using three solvents (methanol, ethanol and water) in a shaker incubator for 24 h. Next, the extracts were centrifuged for 15 min at 4,000 rpm and filtered (whatman No. 55 filter paper) before the solvents were evaporated using rotary evaporator. The obtained different extracts including methanol extract of whole fruit (MW), methanol extract of fruit skin (MS), methanol extract of fruit berry (MB), ethanol extract of whole fruit (EW), ethanol extract of fruit skin (ES), ethanol extract of fruit berry (EB), aqueous extract of whole fruit (DW), aqueous extract of fruit skin (DS), aqueous extract of fruit berry (DB) were used against listed pathogens.

Tested microorganisms

Microorganisms used in this study were S. pneumoniae, S. epidermidis, K. pneumoniae, and P. aeruginosa. All patients’ isolates used in this study were obtained from diagnostic microbiology lab USM Kelantan. Then, the collected microorganisms were cultivated in non-selective media (screened human donor blood), then cultured overnight at 37°C.All these microorganisms were identified by standard laboratory methods including Gram staining, catalase test, optochin test and biochemical tests (Indole, TSI, urea and motility tests) for Gram negative isolates.

Antimicrobial assays

The antimicrobial activity of each extract of B. angulate fruit with different concentration was tested in vitro against the listed isolates. Inoculum was prepared from overnight cultures of different cultured microorganisms (0.5 McFarland). A broad spectrum antibiotic (Ciproboy-200 infusion 100 μL/mL dH2O) was used as a positive control and DMSO was used as a negative control throughout the study

Shortly, after inoculum preparation (15-20 min) 100 μL of the adjusted inoculum (0.5 McFarland) was transferred to the surface of Müller-Hinton agar (MHA) plate and spread uniformly using a sterilized bent glass rod spreader, Nextinoculated medium (MHA) was left for 15 min at room temperature to allow the agar to absorb the inoculum. Then, seven holes with 6 mm diameter were made using sterilized tips. Then, 100 μL of organic extract from each part of the fruit (whole fruit, fruit skin and berry) with different concentrations (1,000, 500, 250, 125, 62.0, 31.0 mg/mL) was introduced into each hole. Same volume of DMSO and diluted ciprobay-200 infusions were also loaded as a negative and positive control. All plates were incubated at 37°C with upside down position [13,14]. The results were obtained by measuring the zone diameter using calipers. MIC was defined as the lowest concentration that shows a zone of inhibition around the well after 24 h or 48 h incubation at 37°C. All experiments were carried out three times.

Microdilution Method

Microdilution method was carried out on low susceptible microorganisms (S. epidermidis and K. pneumoniae) according to Klancnik [15]. Antimicrobial activity of plant extracts was determined using sterile 96-well plate. In this method, all wells were filled with 120 μL of broth. Subsequently fruit extracts (whole fruit, fruit skin and berry) with the same volume was added to the first wells of first row containing nutrient broth. Then the plant extract was serially diluted to create a concentration of 500, 250, 125, 62.0, 31.0, 15.6, 7.5 and 3.75 mg/mL. Later on, 50 μL of adjusted inoculum to 0.5 McFarland was introduced into each well. Negative control wells were prepared from Mueller-Hinton broth (MHB) and bacterial suspension only, while positive control wells were prepared from MHB and diluted broad spectrum antibiotic (ciproboy-200). The plate was then shaken for 1 min prior to transfer it to incubator for 24 h at 37°C. The final dilution of the plant extracts that maintained its inhibitory effect resulting in 85% growth of isolates was recorded as MIC value of the extract. The MBC of extract was determined by sub culturing each well on MHA and further incubation for 24 h at 37°C. The highest diluted well that yielded no growth or 95% growth on MHA was taken as MBC. Then the respiratory activity of cultured microorganisms into 96 well plate was determined after introducing 10 μL of TTC (2-p-iodophenyl-3-pnitrophenyl-5-phenyl tetrazolium chloride, 20 mg/mL dH₂O) into each well, and growth of bacteria was then observed after 30 min incubation of plate in a dark place.

Statistical analysis

The experiments were carried out in triplicate, and the statistical analysis for comparisons was done by GraphPad® Prism V.6.04. The results were expressed as mean ±SD. A difference was considered statistically significant if P<0.05.

Different parts of medicinal plants have various medicinal properties. Parts such as roots, stem, leaves, flowers, and fruit are used traditionally as raw drug [8]. The medicinal properties of B. angulate are mostly deposited in the skin of the fruit. In this study, the antimicrobial activity of the extract against listed bacteria carried out using agar well diffusion and micro dilution methods. In terms of diameter of the zone of inhibition, all extracts showed active inhibitory effect against the tested bacteria. S. pneumoniae was highly susceptible among all the four isolates. Result showed maximum inhibition with the ethanol extract of fruit skin (37 ± 1 mm), followed by methanol extract of fruit skin (33 ± 1 mm) and ethanol extract of fruit berry (30 ± 1 mm) against S. pneumoniae at the concentration of 1,000 mg/mL. The potential effect of the skin extract of BA may be due toflavonoid in the fruit skin [11]. Whole extract of the fruit, using methanol, ethanol and aqueous was less effective than observed antimicrobial activity in the fruit skin at the minimum concentration (31 mg/mL), this was found to be highest in DB extract of the fruit (14.3 ± 0.5 mm) at the concentration of 31 mg/mL against S. pneumoniae (Table 1). This antimicrobial activity of aqueous extract of the fruit may be due to dissolving fruit components in aqueous solution [16]. In another study, the antimicrobial effect of apricot juice against different Gram-positive bacteria, as well Gram negative bacteria was stated that the large zone of inhibition by aqueous extract of apricot observed against P. mirabilis with a 12 mm inhibition zone [17]. Antimicrobial activity of EW against S. epidermidis showed the same inhibition (28 ± 1 mm) zone to MW against S. pneumonia at the highest concentration (1,000 mg/mL) on agar well diffusion (Tables 2 and 3). MS was highly active (15 ± 1.0 mm) against P. aeruginosa at the highest concentration (1,000 mg/mL) (Tables 4-10), while the minimum zone (6.8 ± 1.0 mm) was shown by aqueous extract of whole fruit on agar well diffusion method (Figure 1). The activities of aqueous extract of fruit skin and fruit berry were not observed at the concentration of less than 500 mg/mL. This low activity of extracts at the lowest concentration may be due to less permeability in the outer membrane of P. aeroginosa, which is one of the important causes of resistance of this bacterium to antibiotics. This permeability is 10–100 folds lower in P. aeruginosa than some other bacteria such as E. coli. Other than that, in this bacterium, antibiotic is transported to the outer membrane of bacterial cell through efflux pump, which is located in membrane of P. aeruginosa, thus causing intrinsic resistance [18] (Figure 2). In this study, the minimum concentration in microdilution was absorbed at the concentration of 7.8 mg/mL in MW, MS, ES, and DS on microdilution method against S. epidermidis and K. pneumoniae. Similarly, a study on ethanol, methanol, and aqueous extracts of Dasmodium gangeticum, Nelumbo nucifera, Canabis, as well as white sesame, and black sesame was carried out using agar well diffusion method. The study shows maximum inhibitory zone in aqueous extract of these plants [19]. In microdilution method bacterial growth in 96 wells showed antimicrobial activity of different extracts, which were further determined using triphenyltetrazolium chloride ri phenyl. Result of microdilution method is recommended as this is the best alternative test that also combines with ATP measurement. This method is cheap, less time-consuming, and designed to screen many bacteria and plant extract together [12]. Moreover, in this study, the results of different parts of extracts against different bacteria on microdilution method also were different. Aqueous extract of skin at the concentration of 15.6 mg/ mL was acted as a bactericidal against K. pneumoniae on microdilution method, followed by MS at the concentration of 31 mg/mL against S. epidermidis. The extracts from B. angulata showed potential effect on Gram-positive bacteria and Gram-negative bacteria tested in this study. Mixed results were found, mostly depending on the methods, parts of fruit, and solvents used. The ES was the most sensitive fruit extract using microdilution method against S. epidermidis and K. pneumoniae.

Table 1: Mean and SD of aqueous extracts of against S. pneumonae on agar well diffusion method (mm).

Table 2: Mean and SD of methanol extracts against S. pneumonae on agar well diffusion method (mm).

Table 3: Mean and SD of ethanol extracts against S. epidermidis on agar well diffusion method (mm).

Table 4: Mean and SD of ethanol extracts against S. pneumonae on agar well diffusion method (mm).

Table 5: Mean and SD of methanol extracts against S. epidermidis on agar well diffusion method (mm).

Table 6: Mean and SD of aqueous extracts against S. epidermidis on agar well diffusion method (mm).

Table 7: Mean and SD of methanol extracts against K. pneumoniae on agar well diffusion method (mm).

Table 8: Mean and SD of ethanol extracts against K. pneumoniae on agar well diffusion method (mm).

Table 9: Mean and SD of aqueous extracts against K. pneumoniae on agar well diffusion method (mm).

Table 10: Mean and SD of methanol extracts against P. aeruginosa on agar well diffusion method (mm).

Table 11: Mean and SD of ethanol extracts against P. aeruginosa on agar well diffusion method (mm).

Table 12: MIC and MBC of extracts against S. epidermidis and K. pneumonia.

Figure 1: Aqueous extract of whole fruit against P. aeruginosa on agar well diffusion method.

Figure 2: ethanol extract of whole fruit against P. aeruginosa, (I), methanol extract of whole fruit, fruit skin and fruit berry against S. pneumoniae, (II), aqueous extract of fruit skin against S. pneumoniae and ethanol extract of fruit skin against S. epidermidis (III).

Authors acknowledge the whole laboratory staff of biomedical science Allied Health Science IIUM Kuantan and laboratory staff of microbiology Lab University of Sains Malaysia.