Antibacterial, Anti-swarming and Antibiofilm Activities of Local Egyptian Clover Honey Against Proteus Mirabilis Isolated from Diabetic Foot Infection

 

Hisham A. Abbas*

Department of Microbiology and Immunology-Faculty of Pharmacy-Zagazig University- Zagazig- Egypt

*Corresponding Author E-mail: h_abdelmonem@yahoo.com

 

 

ABSTRACT:

Diabetic foot infection is a serious complication of diabetes that can lead to amputation of lower extremities. Proteus mirabilis is common in diabetic foot infections. These infections are problematic in treatment due to high resistance to antibiotics and biofilm formation. This study investigated the antimicrobial, anti-swarming and antibiofilm activities of local Egyptian clover honey against a clinical Proteus mirabilis isolate from diabetic foot ulcer. Honey is one of the oldest remedies for wound infections. Honey at a concentration of 40% was bactericidal to the planktonic cells of Proteus mirabilis. Swarming and biofilm formation are correlated. At 1/2 MIC of honey, it completely blocked swarming of Proteus mirabilis on 1.5% Luria-Bertani (LB) agar and inhibited biofilm formation by a percentage of 85.86±3.41. This study suggests the use of clover honey as an alternative therapy for treatment of diabetic foot infections caused by Proteus mirabilis due to combined antibacterial, anti-swarming and antibiofilm activities.

 

KEYWORDS: Anti-swarming, antibiofilm, clover honey, Proteus mirabilis, Diabetic foot infection.

 

 


INTRODUCTION:

Diabetic foot infection is a common complication of diabetes. Neuropathy and peripheral vascular problems contribute to this complication. About 15% of diabetic patients are expected to suffer from diabetic foot infections during their life which may lead to gangrene and amputation1-3.  The etiology of diabetic foot infection is complex and polymicrobial infection is common. Proteus spp are among the common bacteria that infect diabetic foot ulcers4-6.

 

Swarming of Proteus is a characteristic phenomenon, which enables these bacteria to colonize surfaces and invade the host tissues7. In swarming, the short vegetative cells differentiate into long hyperflagellated swarmer cells which can migrate across solid surfaces8,9. Flgellar swarming motility is related to biofilm formation10. 

 

Biofilms are adaptive survival lifestyle of bacteria in which they are anchored to a surface and housed within a matrix composed of polysaccharides, proteins and DNA11,12. Biofilms are common in chronic wound infections such as diabetic foot infections13. Biofilm infections are very difficult to treat due to the high resistance of biofilm cells to the action of antimicrobial agents and to their ability to escape the immune system14,15. Alternatives to antibiotic therapy are thus necessary to combat multi-resistant bacteria in chronic wounds such as diabetic foot ulcers. Honey is one of these alternatives16.

 

Honey is known from the ancient times as a therapy for infected wounds especially those that do not respond to conventional therapies, such as diabetic ulcers17,18. In addition to its antimicrobial activity, honey is a natural cheap product that does not interfere with wound healing17,18. This study aimed to investigate the correlated anti-swarming and antibiofilm activities of honey against Proteus mirabilis isolated from diabetic foot ulcer.      

 

MATERIALS AND METHODS:

Media and chemicals:

Tryptone soya broth was the product of Oxoid (Hampshire, UK). Luria-Bertani (LB) agar and LB broth was purchased from Lab M Limited (Lancashire, United Kingdom). Mueller Hinton broth and agar were obtained from Oxoid, Hampshire, England. Other chemicals were of pharmaceutical grade.

 

Honey sample:

Clover honey was obtained from Isis Company, Egypt and was kept in dark container at room temperature. To ensure its sterility, a loopful of honey was added to blood agar plate and incubated for 24h at 37 ΊC. Absence of growth indicated its sterility.

 

Bacterial strains:

A clinical isolate of Proteus mirabilis isolated from diabetic foot ulcer was obtained from the stock culture of the Department of Microbiology and Immunology, Faculty of Pharmacy, Zagazig University.

 

Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC):

The minimum inhibitory concentration (MIC) of clover honey was determined by the broth microdilution method according to Clinical Laboratory and Standards Institute Guidelines19. Proteus mirabilis from overnight culture was suspended in sterile saline to achieve a turbidity equivalent to that of 0.5 McFarland standard and then diluted with sterile saline to have a cell density of 106 CFU/ml. Fifty μl aliquots of the diluted bacterial suspension in Mueller-Hinton broth were added to the wells of a microtiter plate containing 50 μl of twice the concentrations of clover honey. The plates were incubated at 37 ΊC for 20 h, and the MIC was calculated as the lowest concentration of clover honey that inhibited the visible growth in the wells. For determination of the minimum bactericidal concentration (MBC), 10μl of broth from the wells with no growth were transferred to plates of Mueller Hinton agar. After incubation of the plates 24h at 37°C, MBC was calculated as the lowest concentration that could cause 99.99% reduction in growth as seen by no visible growth to less than five colonies. The bactericidal activity of clover honey was investigated by comparing MBC to MIC. If MBC/MIC ≤ 4, honey is considered bactericidal.

 

Inhibition of swarming:

The anti-swarming effect of clover honey was investigated by the modified method of Hay et al.20 Five μl from an overnight culture of Proteus mirabilis were delivered onto the center of the surface of dried LB swarming agar (1.5%) plates with sub-inhibitory concentration of clover honey (20%). Following overnight incubation of LB plates at 37ΊC, the swarming zones diameters were measured in mm. Moreover, sections of LB swarming agar in the presence and absence of clover honey were aseptically cut from the centre of the colony that contains vegetative cells and from the edge of the colony that contains swarmer cells. The bacteria were washed from the cut agar pieces with phosphate buffered saline, simple stained with safranine and examined under the oil immersions lens.

 

Assessment of biofilm production of Proteus mirabilis strains:

The biofilm formation by Proteus mirabilis was assessed according to Stepanovic et al.21 with some modifications. Fresh tryptone soya broth (TSB) was added to an overnight culture of Proteus mirabilis isolate to adjust its turbidity to achieve a cell density of 1 Χ 106 CFU/ml. The wells of sterile 96-well polystyrene microplates with rounded bottom were inoculated with aliquots of 200 ΅l of the adjusted bacterial suspension and the plates were incubated for 24 h at 37°C. The wells were gently aspirated and then washed thrice with sterile phosphate buffered saline (PBS, pH 7.2) to remove any non-adherent cells. To fix the adherent cell, aliquots of 200 μl of 99% methanol were added and left for 20 min. The wells were stained with 200 μl crystal violet (1%) for 20 min and the unbound dye was removed under running distilled water and dried in air. The bound dye was eluted by adding 160 μl of 95% ethanol and the optical densities of the stained adherent biofilms were read with a microplate reader at a wavelength of 490 nm. The test was repeated three times, and the average optical densities were calculated. The cut-off OD (ODc) that corresponds to three times standard deviations above the mean OD of the negative control was calculated and the biofilm formation capacity was assessed as non-biofilm forming (OD ≤ ODc), weak biofilm forming (OD > ODc, but ≤ 2x ODc), moderate biofilm forming (OD>2x ODc, but ≤ 4x ODc), or strong biofilm forming (OD> 4x ODc).

 

Inhibition of biofilm formation:

The biofilm inhibiting activity of sub-inhibitory concentration of clover honey was studied by following the protocol previously described for assessment of biofilm production21 by adding aliquots of 100 ΅l of the prepared bacterial suspension to the wells, to which aliquots of 100 ΅l of clover honey were added to have a final concentration of 20% honey. The optical densities of the stained adherent biofilms in the presence and absence of clover honey were measured using a microplate reader at a wavelength of 490 nm and the percentage of inhibition of biofilm formation was calculated.

 

RESULTS:

Antibacterial activity of clover honey:

Clover honey exerted antibacterial activity against Proteus mirabilis. It was found to inhibit and kill Proteus mirabilis at a concentration of 40%. Clover honey exerted bactericidal activity since MBC/MIC is equivalent to 1.

 

Inhibition of swarming activity of Proteus mirabilis:

The anti-swarming activity of sub-inhibitory concentration of clover honey (20%) was investigated. Clover honey could completely inhibit swarming motility of Proteus mirabilis (Figures 1and 2).


 

Figure 1. Blocking of swarming motility by clover honey (20%). Control LB agar plate (A) showing swarming motility. LB agar plate with clover honey (B) showing no swarming.

 

Figure 2. Simple stained Proteus mirabilis isolate from LB swarming agar plates with 20% honey and without honey examined under oil immersion lens (magnification X 1000), V, vegetative cells from colony centers and S, swarming cells from colony edges. In the presence of clover honey, swarming cells were more or less similar to vegetative cells.

 

 


Inhibition of biofilm formation:

According to the criteria of Stepanovic et al. 21, Proteus mirabilis isolate was found to be strong biofilm forming. Sub-inhibitory concentration of clover honey (20%) inhibited biofilm formation to a percentage of 85.86±3.41.

 

DISCUSSION:

The clinical use of honey in treating wound infections, that was known thousands of years ago, was rediscovered in modern medicine22. This use stems from the antibacterial activity and wound healing effect. The healing activity of honey may be attributed to the maintenance of a moist wound environment that facilitates healing, high viscosity that presents a shield against infection, its mild acidity and hydrogen peroxide content that enable wound healing23. Honey has a broad spectrum antibacterial activity. It was reported to have activity against common bacteria in diabetic foot ulcers; namely Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pnuemoniae, Escherichia coli and Proteus mirabilis24-26. The mechanisms of antibacterial activity include low water content, high osmolarity and low pH in addition to hydrogen peroxide and non-peroxide phytochemical components of honey27. Persistent wounds such as diabetic foot ulcers are problematic to treat due to biofilm nature of infection28. Honey was found to treat wound infections resistant to antibiotics[16]. Moreover, honey was reported to have antibiofilm activity29.

Swarming of Proteus mirabilis is linked to host tissues invasion and biofilm formation7,10,30,31. To interfere with tissue invasion and biofilm formation, swarming inhibitors may be beneficial.

 

Proteus mirabilis isolate was found to be strong biofilm forming. Clover honey showed complete anti-swarming activity at 20%.

 

Various blockers of swarming motility of Proteus mirabilis were identified such as p-nitrophenyl glycerol (PNPG) and resveratrol32. However, the effect of both compounds on inhibition of biofilm formation was not previously studied.

 

To correlate anti-swarming activity of clover honey with its antibiofilm activity, the effect of sub-MIC of clover honey on biofilm formation was investigated and significant inhibition of biofilm formation was achieved. Similar correlated anti-swarming and antibiofilm activities of sub-MIC of salicylic acid against pseudomonas aeruginosa was reported by Chow et al.33 and this correlation was interpreted by inhibition of bacterial motility needed for biofilm formation.

 

Honey was reported to inhibit quorum sensing34. Quorum sensing controls swarming and biofilm formation35. The anti-swarming and antibiofilm activities of honey may be due to quorum sensing inhibition.

In summary, clover honey is recommended for the treatment of diabetic foot ulcers caused by Proteus mirabilis. The underlying reasons are interference with swarming, tissue invasion and biofilm formation.

 

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Received on 19.07.2013          Accepted on 01.08.2013        

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Asian J. Pharm. Res. 3(3): July-Sept. 2013; Page 114-117