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 amputation^1-3. The etiology of diabetic foot infection is complex and polymicrobial infection is common. Proteus spp are among the common bacteria that infect diabetic foot ulcers^4-6.

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 surfaces^8,9. 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 DNA^11,12. 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 system^14,15. 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 ulcers^17,18. In addition to its antimicrobial activity, honey is a natural cheap product that does not interfere with wound healing^17,18. 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 ºC. 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 ºC 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 37ºC, 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 × 10⁶ CFU/ml. The wells of sterile 96-well polystyrene microplates with rounded bottom were inoculated with aliquots of 200 µl 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 µl of the prepared bacterial suspension to the wells, to which aliquots of 100 µl 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).

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 mirabilis^24-26. 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 formation^7,10,30,31. 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

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