Antibacterial activity of various Syrian honey types against Pseudomonas aueruginosa

Wissam Zam¹*, Rim Harfouch², Salwa Bittar², Meray Sayegh²

¹Department of Analytical and Food Chemistry, Faculty of Pharmacy, Al-Andalus University for Medical Sciences, Tartous, Syrian Arab Republic.

²Department of Microbiology, Faculty of Pharmacy, Al-Andalus University for Medical Sciences, Tartous, Syrian Arab Republic.

*Corresponding Author E-mail: w.zam@au.edu.sy

ABSTRACT:

Pseudomonas aeruginosa is an opportunistic pathogen causing severe, acute and chronic nosocomial infections in urinary and pulmonary tracts, burns and wounds. Currently, there is an urgent and global need for alternative antimicrobial strategies to fight the continuous rise of P. aeruginosa resistance to different antibiotics. This led to a re-evaluation of the therapeutic use of ancient medicines such as honey. Different concentrations ranging from 0.02-50% of six different Syrian honeys were used in this study against resistant P. aeruginosa. The minimum inhibitory concentration was determined using spectrophotometric at 620 nm. Carduoideae flowers, black seed, anise and oak honey samples showed a MIC of 12.5%, whereas the MIC of Montana and cotton honey samples was 25%. In conclusion, Syrian honey possesses an in vitro antibacterial activity against P. aeruginosa and could be an alternative topical choice in the treatment of wound infections.

KEYWORDS: Pseudomonas aeruginosa; Syrian honey; MIC.

INTRODUCTION:

Physiologically, wound healing process is complex and promotes colonization of both Gram-positive and Gram-negative bacteria¹, particularly Pseudomonas aueruginosa. A P. aeruginosa infected wound is characterized by a significantly greater area and a delayed or prevented healing process². Additionally, P. aeruginosa has a high intrinsic and acquired antibiotic resistance³ that makes its treatment challenging⁴.

Honey is used in traditional medicine for its high nutritive value and for its antioxidant, bacteriostatic, anti-inflammatory and antimicrobial properties, as well as wound and sunburn healing effects⁵. The antimicrobial effect of honey has been reported by a number of workers it is commonly used as a base for ointments and has very successfully been applied in surgical dressings for open wounds and burns to avoid septic infections⁶. However the antimicrobial activity of honey varies markedly depending on its composition which depends on the plant sources it is derived from, the weather, soil, and other factors; therefore no two honeys are identical⁷. In general, honey is a complex product consisting mainly of monosaccharaides such as fructose and glucose, and also of a wide range of minor constituents, especially phenolic compounds⁸. Its antibacterial activity is especially due to the phytochemical component of the nectar as it was suggested that many of the medicinal properties of plants can be transmitted through honey⁹. The quality and diversity of Syrian landscape is considered a valuable and supporting resource for apiculture and the official production of honey is about 1,750 tons a year. The main Syrian honey types are derived from citrus trees, Eucalyptus globulus, anise, sunflowers, fruit trees, mountain plants such as thyme and rosemary, cotton, euphorbia and Erica.

As the potential role for honey as a topical agent to manage surgical site or wound infections is increasingly acknowledged and deeply dependent on its origin. In addition to that after reviewing the literature it has been found that no study has been carried out on antibacterial activity of Syrian honey against P. aeruginosa in vitro. So, the objective of this study was to evaluate the antimicrobial potential of various types of Syrian honey against P. aeruginosa.

MATERIALS AND METHODS:

Detection of the Minimum Inhibitory Concentration

Honey Preparation

Black seed, Carduoideae flowers, Anise (Pimpinella anisum), Oak (Quercus robur), Montana and cotton honeys were purchased directly from beekeepers and used in this study. Samples were stored at room temperature in the dark prior to testing in order to prevent photo-degradation. A serial double dilution of honey samples was prepared fresh daily prior to testing aseptically for use in MIC assay from 50% to 0.02% v/v in nutrient broth. From the 50% (v/v) honey solution, 12 serial 1:1 dilutions were made, resulting in final concentrations of; 50%, 25%, 12.5%, 6.3%, 3.1%, 1.6%, 0.8%, 0.4%, 0.2%, 0.1%, 0.04%, and 0.02%¹⁰.

Culture Preparation

Strains of P. aeruginosa were isolated from swabs collected from a wide range of infected wounds routinely submitted to the department of medical microbiology at AL-Andalus University Hospital. Isolates were identified as P. aeruginosa by standard bacteriological techniques. These cultures were maintained by subculture on Muller Hinton agar for up to seven days.

Microdilution assay

One hundred μl of 0.5 McFarland standardized bacterial suspension was added to 1900 μl of test honey, at each of the concentrations stated above. Control wells contained broth only (negative control) or bacteria and broth (positive control). Tubes were incubated in the dark at 37°C for 24 h.

Spectrophotometric assay for MIC determination

Optical density was determined in a spectrophotometer at 620 nm prior to incubation. After an overnight incubation at 37°C, the tubes were again examined for turbidity indicating the growth of the microorganisms. The lowest solution of the extract that inhibited the growth of the microorganism as detected by spectrophotometric assay was designated the minimum inhibitory concentration according to Patton et al.¹¹.

RESULTS:

Antibiotic sensitivity test indicates that P. aeruginosa isolate was resistant to Gentamycin, Cefuroxime, Amoxicillin/clavulanic acid and Spectinomycin as demonstrated in table 1. As shown in table 2, Carduoideae flowers, black seed, anise and oak honey samples were two times more effective than Montana and cotton honey samples in inhibiting P. aeruginosa in in-vitro tests.

Table 1. Antibiotic sensitivities of P. aeruginosa isolate

sensitivity	Inhibition zone diameter	Antibiotic name	Antibiotic symbol
Sensitive	24 mm	Imipenem	IMP
Sensitive	20 mm	Chloramphenicol	CHL
Sensitive	20 mm	Amikacin	AMK
Sensitive	29 mm	Levofloxacin	LEV
Sensitive	25 mm	Cefoperazone	CPZ
Sensitive	32 mm	Cefoperazone/Sulbactam	CPS
Sensitive	29 mm	Ceftazidime	CAZ
Sensitive	28 mm	Cefadroxil	CFR
Intermediate	19 mm	Ceftriaxone	CTR
Intermediate	21 mm	Cefotaxime	CTX
Intermediate	13 mm	Doxycycline	DOX
Resistant	No inhibition zone	Gentamycin	CN
Resistant	No inhibition zone	Cefuroxime	CXM
Resistant	No inhibition zone	Amoxicillin+ clavulanic acid	AUG
Resistant	No inhibition zone	Spectinomycin	SPC

Table 2: Minimum inhibitory concentrations (% vol/vol)

Honey type	Honey concentration (% vol/vol)
Honey type	50 %	25%	12.5 %	6.3%	3.1%	1.5%	0.75%
Carduoideae flowers	-	-	-	+	+	+	+
Black seed	-	-	-	+	+	+	+
Anise (Pimpinella anisum)	-	-	-	+	+	+	+
Oak (Quercus robur)	-	-	-	+	+	+	+
Montana	-	-	+	+	+	+	+
Cotton	-	-	+	+	+	+	+

DISCUSSION:

Results from several studies confirm that honeys from different countries and regions may have wide variations in their antimicrobial activity. It has been shown that honey may have antimicrobial action ranging from lesser than 3% to 50% and higher concentrations¹². Several authors also reported that the antibacterial efficacy of honey differs greatly with plant source^12,13. Honey may inhibit bacterial growth due to a number of different mechanisms such as osmotic effect, low pH, hydrogen peroxide generation and phytochemicals¹⁴. The combination of these diverse mechanisms may account for the inability of bacteria to develop resistance to honey, in contrast to the rapid induction of resistance observed with conventional single-component antibiotics^15,16. Since P. aeruginosa are recalcitrant to antibiotic therapy, the efficacy of honey to inhibit test isolates irrespective of their antibiotic sensitivity patterns has important clinical applications. This property may make honey useful in the treatment of drug-resistant infections.

When honey is applied to wounds, it will be diluted by body fluids, and if honey is to be an effective wound antibacterial agent, it must retain inhibitory activity on dilution. Our results presented in table 2 indicate that all kinds of tested honeys retained bactericidal activity in vitro after dilution with different MIC varying from 12.5% to 25% (%v/v). The variations in MIC values may likely be due, at least in part, to differences in the levels of the principle antibacterial components in the honey and hydrogen peroxide, which varies with the floral and geographic source of nectar, honey storage time and conditions and any other possible treatment that could affect it.

The MIC of Montana and Cotton honeys was similar to that obtained by Subrahmanyam¹⁷ and Nzeako and Hamdi¹⁸ who showed that P. aeruginosa strains were inhibited by diluted Jambhul honey at a concentration of 25%. Also the MIC of carduoideae flowers, black seed, anise and oak honeys was similar to that reported for Ulmo honey¹⁰ from chile and Manuka honey from New-Zealand which is the most studied type of honey with both in vitro and in vivo confirmed activity against a wide range of medically important bacteria¹⁹.

Further studies on human subjects are required in vivo to understand the efficacy of Syrian honeys in eliminating P. aeruginosa from wounds. Additionally, biofilms of P. aeruginosa may be present in chronic wound environment and the characteristic of bacteria can change; hence, future studies in this direction will pave the way in establishing the medicinal importance of Syrian honey against different forms of P. aeruginosa.

CONCLUSION:

Manuka honey is been classified as medical-grade due to its broad spectrum and potent antimicrobial activity, and there is a continuous effort in the search for honeys from other sources with enhanced antimicrobial properties. No previous study has been carried out on antibacterial activity of Syrian honey against P. aeruginosa in vitro. Our results indicated that s P. aeruginosa was inhibited at 25% or 12.5% antibacterial honey concentrations. This intriguing observation may have important clinical implications and could lead to a new approach for treating multidrug resistant P. aeruginosa infected wounds using honey of Syrian origin.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 28.04.2017 Modified on 10.05.2017

Res. J. Pharmacognosy and Phytochem. 2017; 9(2): 73-76.

DOI: 10.5958/0975-4385.2017.00013.9