In vitro antimicrobial activities of Saudi honeys originating from Ziziphus spina‐christi L. and Acacia gerrardii Benth. trees

Abstract Honeys originating from Sidr (Ziziphus spina‐christi L.) and Talh (Acacia gerrardii Benth.) trees in Saudi Arabia exhibited substantial antimicrobial activity against pathogenic gram‐positive bacteria (Bacillus cereus, Staphylococcus aureus), gram‐negative bacteria (Escherichia coli, Salmonella enteritidis), and a dermatophytic fungus (Trichophyton mentagrophytes). The diameter of zones of inhibition represents the level of antimicrobial potency of the honey samples. Precisely, Talh honey showed significantly higher antibacterial activity against all tested bacteria than Sidr honey. The antifungal activity of Talh and Sidr honey types was significantly at par against a dermatophytic fungus. The water‐diluted honey types (33% w/v) significantly induced a rise in the antimicrobial activity from that of the natural nondiluted honeys. Microbial strains displayed differential sensitivity; gram‐positive bacteria were more sensitive and presented larger inhibition zones than gram‐negative bacteria and the fungus. The sensitivity was highest in B. cereus and S. aureus, followed by T. mentagrophytes, E. coli, and S. enteritidis. The antimicrobial activity of water‐diluted honeys (Sidr and Talh) was high than that of broad‐spectrum antibacterial antibiotics (tetracycline and chloramphenicol) against bacterial strains, but these honeys were relativity less potent than antifungal antibiotics (flucoral and mycosat) against a fungal strain. Our findings indicate the antimicrobial potential of Saudi honeys to be considered in honey standards, and their therapeutic use as medical‐grade honeys needs further investigations.

In Saudi Arabia, honey consumption is gradually increasing, as honey is a principle ingredient in foods and in folk medicines (Al-Ghamdi & Adgaba, 2015;Alqarni, 2011;Alqarni et al., 2016). Many locally produced and imported honeys are available in the Saudi F I G U R E 1 Location sites for honey collection in Saudi Arabia. Asterisks indicate the regions from where the honey samples were collected market. Sidr honey and Talh honey are two major honey types in Saudi Arabia and the Arabian Peninsula. These honeys are locally named with reference to their floral nectar source. Talh honey is produced from Acacia gerrardii Benth. trees and Sidr honey from Ziziphus spina-christi L. (Adgaba et al., 2017;Al-Ghamdi, 2007;Al-Khalifa & Al-Arify, 1999;Alqarni et al., 2016). Ziziphus and Acacia are the most common plants of economic importance in Saudi Arabia and are the major floral sources of high-valued expensive honeys (Alqarni, Hassan, & Owayss, 2015;Alqarni, 2015).
Our study aimed to evaluate the antimicrobial potential of the most preferred honeys in Saudi Arabia, Sidr and Talh honeys, against pathogenic bacterial and fungal strains. Their potential antimicrobial activity was also equated with that of antibiotics commonly used against the targeted microbial strains. This research pursuing the antimicrobial potential of honey types will be helpful in treating the pathogenic microorganisms threatening the public health and changing antibiotics into last-resort drugs. three repeats. After running a triplicate measurement of antimicrobial activity, the mean value of these three repeats was calculated.

| Honey Samples
The codes and regional data of these unifloral honeys are presented in Table 1. Two forms of honey samples, natural (nondiluted crude honey) and water-diluted honey (33% w/v) (Elbanna et al., 2014), were used for the examination of their potential antimicrobial action.

| Microbial Strains
The microbial pathogenic strains of two gram-positive bacte-

| Assessment of Antibacterial Activity
Antimicrobial activities of each honey type (Sidr and Talh) were assessed using the well-diffusion bioassay technique (Elbanna et al., 2014). Sterilized Muller-Hinton or potato dextrose agar media (Oxoid) were poured into sterilized petri dishes, left to solidify at room temperature (25 ± 1°C), and swabbed with fresh bacterial or fungal strain cultures. Wells at the center of agar plates were made using a sterile cork borer (9 mm diameter) and filled with 300 µl of natural honey or water-diluted honey (33% w/v). To give honey enough time for diffusion, all plates were placed in a refrigerator (~5°C) for 2 hr and then incubated at 37°C for 24 hr (for bacteria) and at 28°C for 48-72 hr (for the fungus). The potential antimicrobial activities of honey treatments were expressed by measuring the diameter (mm) of a clear (inhibition) zone of each well, with distilled water taken as a control. In separate experiments, the antimicrobial activity of two broad-spectrum antibacterial (tetracycline and chloramphenicol) and two antifungal (flucoral and mycosat) antibiotics (Mast Diagnostic GmbH, Germany) were assessed against their respective microbial strains using the agar disk diffusion method and measuring the clear zone diameter (mm) of each disk (EFSA, 2012).

| Statistical Analysis
The mean antimicrobial activity of Sidr and Talh honey samples against each tested microbial strain was measured. The data were analyzed using analysis of variance (ANOVA) under a complete randomized design after testing for homogeneity of error variances according to the procedure defined by Gomez and Gomez (1984).
InfoStat software (Rienzo et al., 2016) was used for the statistical analysis. Statistical means were compared for significant differences at p ≤ .05 using the least significant difference (LSD) test.

| Antimicrobial Activity of Honeys
In vitro antimicrobial activities of the most common unifloral honey types in Saudi Arabia (Sidr honey (SH) and Talh honey (TH) were evaluated against pathogenic strains of gram-positive bacteria (B. cereus, S. aureus), gram-negative bacteria (E. coli, S. enteritidis), and dermatophyte fungi (T. mentagrophytes). Natural and water-diluted (33% w/v) forms of SH and TH were used for testing their potential antimicrobial activity. The data revealed that SH and TH honey types have significant differential antimicrobial potentialities against the

TA B L E 2 Diameter of inhibition zone
indicating the antimicrobial activity of Sidr and Talh honey samples against pathogenic gram-positive (G + ) bacteria, gram-negative (G -) bacteria, and a dermatophyte fungus tested microbial strains. The microbial strains were significantly inhibited as measured in terms of their zone of inhibition (ZOI), and a large ZOI reflects a high sensitivity of tested microbial strains. No microbial strains were resistant to any of the honey types.
The microbial strains presented differential sensitivity to the honey types. Gram-positive (G + ) bacteria were more sensitive to both honey types (SH and TH), with significantly higher ZOI values than those of gram-positive (G -) bacteria and fungi ( Antimicrobial activities of honeys were significantly amplified when natural honeys were diluted with water (33% w/v). A comparison of the antimicrobial activities of individual honey types, that is, natural SH versus water-diluted SH ( Figure 2a) and natural TH versus water-diluted TH ( Figure 2b) showed significantly higher inhibition in water-diluted honeys against all tested G + and Gbacterial strains, and fungal strains.

F I G U R E 2
Comparison of the antimicrobial activity of natural versus diluted honeys: (a) Sidr (b) Talh. Water-diluted honey has significantly higher antimicrobial activity than natural honey. The asterisks indicate the significant difference between the graph bars F I G U R E 3 Comparison of the antimicrobial activity of Sidr and Talh honeys: (a) natural honey (b) water-diluted honey. Talh honeys have higher antimicrobial activity than Sidr honey. The asterisks indicate the significant difference between the graph bars TH displayed higher antimicrobial activity than SH against G + and Gbacteria but not against the fungal strain, where both honey types were significantly similar ( Figure 3). The comparison of the antimicrobial activities between natural SH and natural TH (Figure 3a), and between water-diluted SH and water-diluted TH ( Figure 3b) revealed that each form of TH was more effective than the respective form of SH against a single microbial strain. Figure 4 displayed the antimicrobial activity of the tested honeys with zone of microbial growth inhibition on the cultures of tested microbial strains.

| Antimicrobial Activity of Antibiotics
The disk diffusion test for antibiotics evaluated the antimicrobial activity of two antibacterial (tetracycline and chloramphenicol) and two antifungal (flucoral and mycosat) antibiotics against their respective microbial strains. Our results indicated a significant difference among the antimicrobial activities of the tested antibiotics.
For antibacterial antibiotics, the largest ZOI was recorded for S. Of the antifungal antibiotics, mycosat showed significantly higher antimicrobial action against T. mentagrophytes than flucoral ( Figure 5).

| Comparison Among Antimicrobial Action of Honey and Antibiotics
It is apparent from the data analysis that the high antimicrobial activity (larger ZOIs) shown by bacterial strains particularly with water-diluted SH (Figure 6a) and water-diluted TH (Figure 6b) is significantly greater than that of the tested broad-spectrum antibacterial antibiotics (tetracycline and chloramphenicol). S. enteritidis (gram-negative bacteria) treated with water-diluted SH showed exception where ZOIs values were significantly lower than chloramphenicol but significantly at par with tetracycline ( Figure 6a). Nevertheless, antifungal antibiotics exhibited significantly higher antimicrobial activity against the fungal strain than the tested water-diluted SH and TH honeys ( Figure 7).

| Antimicrobial activity of honeys
Saudi Sidr honey (SH) and Talh honey (TH) displayed substantial antimicrobial activities against tested pathogenic microbial strains.
These primary findings strengthened the idea for using Saudi honeys as potential alternative broad-spectrum strategy to treat bacterial and fungal infections. Use of various types of honeys due to its antimicrobial effects has been published in numerous studies (Bradshaw, 2011;Israili, 2014  TA B L E 4 Antimicrobial activities of antifungal antibiotics against the fungal strain Antibiotics (Antifungal)
SH and TH presented lethal bactericidal and fungicidal effects because no further change in the inhibition zone was detected even after ten days. Al-Nahari et al. (2015) evaluated that the antimicrobial effect of Manuka honey (L. scoparium) was more evident than that of Seder and Nigella sativa honey against both antibiotic (imipenem)-resistant and antibiotic-sensitive bacteria (P. aeruginosa).

F I G U R E 5
Comparison between antibiotics for their antimicrobial activity against single microbial strains.
The asterisks indicate the significant differences between the antibiotics F I G U R E 6 Comparison of antimicrobial activities of antibacterial antibiotics with water-diluted SH (a) and water-diluted TH (b). The common letters on bars indicate no significant difference Manuka honey was bactericidal, but Seder and N. sativa honeys were only bacteriostatic. In contrast, SH was completely bactericidal against our tested bacterial strains.
Saudi honeys showed dose-dependent antibacterial activity: Sidr (Z. spina-christi) and Dharm (Lavandula dentata) were more potent at high concentrations (50%-80% w/v) against E. coli, Proteus mirabilis, S. aureus, Shigella flexneri, and S. epidermidis than Majra honey (Hypoestes forskaolii) (Ghramh, Khan, & Alshehri, 2018). In contrast, only one concentration of water-diluted honey (33% w/v) was adopted from Elbanna et al. (2014) and substantially inhibited the tested microbial strains. Exploring the antimicrobial activity with a series of honey dilutions could be a potential future investigation to determine the dose dependency (if any).
Water-diluted (33% w/v) honeys revealed an elevated antimicrobial activity as compared to nondiluted honeys. An enzymatic reaction of glucose oxidase is being active in water-honey medium.
Hydrogen peroxide is produced when glucose oxidase oxidizes glucose to gluconic acid (Mandal & Mandal, 2011). Synthesis of hydrogen peroxide in water-diluted honeys could be the potential reason for elevated antimicrobial activity. This also explains why nectar (in plant or in bee stomach or in unripe honey) is not infected with microbes. The dilutions of honey between 30% and 50% (v/v) led to maximum levels of accumulated hydrogen peroxide (Bang, Buntting, & Molan, 2003), and the dilution range was similar to our tested honey dilution concentrations (33% w/v). However, the antimicrobial activity of honey is extremely complex and might be due to the involvement of multiple compounds and several nonperoxide components that are also reported to contribute to the unique antibacterial activity of honey, such as physico-chemical properties, osmotic pressure, acidic pH, and nonperoxide phytochemical components, including antioxidants and antimicrobial peptides (Ayaad, Shaker, & Almuhnaa, 2009;Brudzynski, 2006;Halawani & Shohayeb, 2011;Kwakman & Zaat, 2012;Mavric, Wittmann, Barth, & Henle, 2008;Molan, 1992;Molan & Russell, 1988;Simon et al., 2009). Elbanna et al. (2014) attributed the antimicrobial activity of three unifloral Egyptian honeys (~88%) to nonperoxide agents, whereas hydrogen peroxide contributed less (~12%) to the tested honeys. In contrast, some scientists reported a fourfold decline in the antimicrobial activity of honey upon dilution (Adeleke, Onakoya, & Alli, 2002;Olaitan, Adeleke, & Ola, 2007), possibly due the presence of catalase in water that neutralized the hydrogen peroxide (Szweda, 2017). Due to the presence of numerous compounds in honey, bacterial resistance is less likely to be developed in honey-treated bacteria (Carnwath, Graham, Reynolds, & Pollock, 2014;Machado De-Melo et al., 2018), favoring the use of honeys against microbial infections.

| Antimicrobial activity of antibiotics
In the present study, broad-spectrum antibacterial (tetracycline and chloramphenicol) and antifungal (flucoral and mycosat) antibiotics were also effective against their respective microbes.
Interestingly, the antibacterial activity of water-diluted SH and TH was greater than that of the tested antibacterial antibiotics. These findings should be considered as indicative rather than conclusive, as varied doses and two different testing methods were used for evaluation of antimicrobial activity. Karayil, Deshpande, and Koppikar (1998) and Elbanna et al. (2014) found that water-diluted honey inhibited the growth of certain pathogenic bacteria relatively more than some antibiotics. Although the tested antibiotics and bacterial strains were different from those in our study, the elevated effectiveness of water-diluted honey over tested antibiotics is in consistent with our findings.
Agbagwa and Frank-Peterside (2010) found better antibacterial activity for SH than for imipenem (antibiotic) against a pathogenic Gbacterium (P. aeruginosa) and partially supported our results regarding superior antibacterial activity of SH compared with tested antibiotics. Nigerian honey samples (40% v/v) gave better antimicrobial activity against P. aeruginosa, S. aureus, E. coli, and K.
pneumoniae than four antibiotics, namely amoxicillin, streptomycin, ceftriaxone, and erythromycin (Braide et al., 2012). Based on the published reports in literature (Israili, 2014;Liu et al., 2018), it is likely predictable that the use of honey in combination with antibiotics could synergize the antimicrobial activity. Müller et al. (2013) found a synergistic effect between Medihoney and rifampicin antibiotic on S. aureus but not between Manuka honey and rifampicin. Thus, further investigations with different honeys and common broad-spectrum antibiotics may unveil their F I G U R E 7 Comparison between antifungal antibiotics and water-diluted honeys (SH and TH) for their antimicrobial activity against Trichophyton mentagrophytes. The common letters on the bars indicate no significant difference synergism against microbes to establish their parallel use as an effective antimicrobial therapy.

| Honey as a promising therapeutic alternative to antimicrobial agents
Honey is traditionally used as therapeutic agent against skin infections and wounds caused by microbial pathogens (Israili, 2014;Liu et al., 2014;McLoone et al., 2016). Our results presented the potent antimicrobial prosperities of SH and TH against skin infection causing bacterial agents and dermatologically important filamentous fungi. These findings suggest the prospective use of Saudi honeys in the clinical treatments of different microbial infections. The antimicrobial activity of honey could be due to its various contents such as high sugar, total phenolic compounds and hydrogen peroxide levels.
Furthermore, the bactericidal mechanisms of these content may include DNA degrading activity, interruption of cell division, alteration in the cell morphology and general loss of structural integrity of the microbial cell (Israili, 2014;Johnston, McBride, Dahiya, Owusu, & Nigam, 2018). The microbes may not develop resistance against honey in the same way as they develop for other commonly used antimicrobial agents. These features may make the honey a promising alternative to the commonly used antibiotics.

| CON CLUS ION
Conclusively, Sidr and Talh honey samples have significant antimicrobial potential against gram-positive and gram-negative bacteria and dermatophytic fungi regardless of the sample origin. Talh honey was more potent against tested microbial strains than Sidr honey. Water dilution of honeys elevated the antimicrobial activity above that of natural nondiluted honeys. Microbial strains showed differential sensitivity, and G + bacteria were more sensitive than Gbacteria and fungi. The in vitro antimicrobial activity of honeys was comparable with that of common broad-spectrum antibacterial antibiotics. Our findings are indicative of the potential antimicrobial quality of Saudi honeys considered in honey standards, and further investigations are necessary to standardize the Sidr and Talh honeys for their therapeutic applications as medical-grade honeys.

ACK N OWLED G M ENTS
This research work was supported by the Deanship of Scientific Research, King Saud University, Riyadh, Saudi Arabia through research group project RG-1440-189.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L S TATEM ENT
This study does not involve any human or animal testing. Informed Consent: Consent was obtained from all study participants for its submission in this journal.