Diabetic foot infections are common in diabetic patients¹. Diabetic foot ulcers are considered a serious complication of diabetes as it increases the risk of amputation². Proteus spp are common in diabetic foot ulcers^3,4. The swarming motility of P. mirabilis contributes to its ability to invade the tissues⁵. In swarming, P. mirabilis cells differentiate from vegetative short, motile cells with a few flagella into multinucleate aseptate swarmer cells with length that may reach 40 times the length of vegetative cell^6,7.Motility of bacteria plays an important role in colonization of a surface and the spreading of bacteria across that surface.

As a result, the formation of surface adherent sessile communities of microbial cells that are enclosed in a matrix, and known as biofilms, is affected by bacterial motility^8,9. Biofilms are highly resistant to antimicrobial therapy^8,10,11. Biofilm formation is common in chronic wounds such as diabetic foot ulcer¹². Bacteria can exhibit different types of motility. Bacteria may colonize a surface by a flagella-mediated swimming toward the surface and attaching by means of type IV pili and flagella^8,9,13-15.

Ambroxol is a commonly used mucolytic and expectorant in patients with asthma and chronic bronchitis¹⁶. In addition, it was reported to have antioxidant and anti-inflammatory activities¹⁷. Ambroxol is antiadhesive and it can prevent the adherence of P. aeruginosa to cultured mammalian cells or detach the adherent bacterial cell from the mammalian cells¹⁸. Furthermore, Ambroxol can interfere with biofilm formation as a result of its ability to inhibit adhesion, quorum sensing and biofilm matrix production¹⁹. It is of importance to study the effect of ambroxol on bacterial motility and biofilm formation.

The present study investigates the inhibition of swarming and swimming motilities of Proteus mirabilis, blocking of biofilm formation and eradication of pre-formed biofilms by ambroxol as a strategy to treat diabetic foot infections caused by Proteus mirabilis.

MATERIALS AND METHODS:

Media and chemicals

Macconkey’s agar, nutrient agar, tryptone soya broth and Mueller Hinton broth were the products of Oxoid (Hampshire, UK). Luria-Bertani (LB) agar and LB broth were obtained from Difco (France) and triple sugar iron (TSI) agar was purchased from Lab M Limited (Lancashire, United Kingdom.). Ambroxol hydrochloride and Dimethyl sulphoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, USA). Other chemicals were of pharmaceutical grade.

Bacterial strains

Five clinical isolates of Proteus mirabilis obtained from diabetic foot ulcers from patients admitted to the Surgery Department in Zagazig University Hospitals were used in this study. The isolates were identified by morphology, gram staining and biochemical reactions according to koneman et al.²⁰.

Determination of minimum inhibitory concentration

The minimum inhibitory concentration (MIC) of ambroxol was determined by the broth microdilution method according to Clinical Laboratory and Standards Institute Guidelines (CLSI)²¹.Bacterial inocula were prepared and standardized to have a turbidity matching that of 0.5 McFarland standard. Sterile saline was used to dilute the bacterial suspensions to achieve a cell density approximating 10⁶ CFU/ml. To the wells of a microtiter plate with 50 μl of twice the concentrations of ambroxol, 50 μl aliquots of the bacterial suspensions in Mueller-Hinton broth were added. After incubation of the plates at 37 ºC for 20 h, the MIC was calculated as the lowest concentration of ambroxol that showed no visible growth in the wells.

Inhibition of swarming and swimming

To determine the effect of ambroxol on swimming and swarming, the modified method of Hay et al.²² was used. For swarming assay, overnight culture of Proteus mirabilis was prepared and 5 μl from this culture was inoculated on the center of the surface of dried LB swarming agar (1.5%) plates containing different sub-inhibitory concentrations of ambroxol (0.6, 0.7, 0.8, 0.9 mg/ml). After overnight incubation of the plates at 37ºC, the swarming zones diameters were measured in mm. In order to differentiate swarming, sections of agar from swarming assay plates with and without ambroxol were cut under aseptic conditions. The sections were cut from the centre of the colony which contains vegetative cells and from the edge of the colony with swarmer cells. After removal of the bacteria from the cut agar pieces with phosphate buffered saline, they were simple stained with crystal violet and examined under the oil immersions lens.

For swimming assay, the overnight Proteus mirabilis cultures were stabbed into the centre of the dried LB swimming agar (0.4%) with ambroxol (0.6, 0.7, 0.8, 0.9 mg/ml). After overnight incubation of the plates at 37ºC, the swimming zones diameters were measured in mm. Controls plates for swarming and swimming assays were also prepared and inoculated in the same way.

Assessment of biofilm production of Proteus mirabilis strains

For determination of biofilm production by Proteus mirabilis strains, the modified method of Stepanovic et al.²³ was used. Overnight cultures of Proteus mirabilis isolates were prepared, diluted with fresh tryptone soya broth (TSB), and adjusted to a cell density of 1 × 10⁶ CFU/ml. To the wells of sterile 96-well polystyrene microplates with rounded bottom, aliquots of 200 µl of the adjusted bacterial suspension were inoculated. After incubation for 24 h at 37°C, the contents of the wells were gently aspirated and the wells were then washed three times with sterile phosphate buffered saline (PBS, pH 7.2). The adherent cells were fixed with 200 μl of 99% methanol for 20 min and then stained with 200 μl crystal violet (1%) for 20 min. The excess dye was then removed under running distilled water, and then the plates were left to air dry. The bound dye was extracted by the addition of 160 μl of 95% ethanol and the optical densities of the stained adherent films were read with a microplate reader at a wavelength of 490 nm. The test was repeated three times, and the mean optical densities were calculated. The cut-off OD (ODc) was defined as three times standard deviations above the mean OD of the negative control. According to the criteria of Stepanovic et al.²³, the test isolates were categorized into four groups; non-biofilm forming (OD ≤ ODc), weak biofilm forming (OD > ODc, but ≤ 2x ODc), moderate biofilm forming (OD>2x ODc, but ≤ 4x ODc), and strong biofilm forming (OD> 4x ODc).

Inhibition of biofilm formation

To study the effect of ambroxol on biofilm formation, the same procedure described for assessment of biofilm production²³ was followed but instead of adding 200 µl of bacterial suspensions to the microtiter plate, aliquots of 100 µl of the prepared bacterial suspension were added to the wells of sterile 96-well polystyrene microplate containing 100 µl of the different concentrations of ambroxol. After measuring the optical densities of the stained adherent biofilms in the presence and absence of ambroxol with a microplate reader at a wavelength of 490 nm, the percentage of inhibition of biofilm formation was calculated.

Eradication of pre-formed biofilms

To the microtiter plate wells with pre-formed biofilms, 200 µl of different sub-MICs of ambroxol were added and the plates were incubated for 24 h at 37°C. The wells were stained with crystal violet as in procedure of biofilm production assessment and the optical density was measured at 490 nm. The percentage of biofilm eradication was calculated.

Statistical analysis

One way ANOVA test (Newman-Keuls Multiple Comparison Test), P<0.05 was used to detect the significant effects of sub-MICs of on swarming, swimming, biofilm inhibition and biofilm removal.

RESULTS:

Identification of Proteus mirabilis isolates

Proteus mirabilis isolates were identified as Gram-negative rods. They produced lactose non-fermenting colonies on Macconkey’s agar and showed swarming on nutrient agar. They produced hydrogen sulphide from triple sugar iron agar and were urease positive and indole fermentation negative.

Inhibition of swarming and swimming activities of Proteus mirabilis

Ambroxol at sub-inhibitory concentrations inhibited swarming activities of Proteus mirabilis isolates on LB agar plates in a dose-dependent manner. At a concentration of 0.9 mg/ml, ambroxol could completely inhibit swarming motilities (Figures 1& 2). The effect of ambroxol on cell morphology was also investigated (Figure 3). The vegetative cells from the colony centers were short cells, while the swarmer cells from the colony edges appeared elongated. In the presence of 0.9 mg/ml of ambroxol, the swarmer cells were shorter and more or less similar to vegetative cells; an observation which further indicates the swarming behavior inhibition by ambroxol.

DISCUSSION:

Diabetic foot infection (DFI) is a serious complication affecting about 15% of diabetic patients at some stage of their life and can lead to amputation of lower extremities^24,25. Neuropathy and reduced blood supply to the lower extremities are risk factors for diabetic foot infections^26,27. Proteus mirabilis is a common etiologic agent of opportunistic infections including wound infection. Proteus mirabilis can colonize and infect the human host^28,29.

Swarming enhances the ability of Proteus mirabilis to invade the host tissues. Moreover, swarming is necessary for biofilm formation in Proteus mirabilis. Flagella-driven swarming motility is necessary for surface adhesion in Proteus mirabilis. After adhesion of abiotic or biotic surface, P. mirabilis colonizes the surfaces and then biofilms are formed. Biofilms protect P. mirabilis from the immunity of the host and antibiotic treatment^30,31,5,32. As a result, agents which can block swarming are likely to interfere with tissue invasion and biofilm formation.

The bacteria that cause DFIs may be present as a biofilm; a community of microbial sessile cells that are attached to a surface and housed within a matrix of extracellular polymeric substances¹². Biofilm formation is common in chronic wounds such as diabetic foot ulcer (DFU)³³. The chronic DFU might also involve microbial biofilms which are not easily eradicated by conventional antibiotic therapy. As a consequence, anti-biofilm agents can be of value in treating diabetic foot infections.

In this study, the biofilm forming ability of Proteus mirabilis isolates was investigated and the tested isolates were strong biofilm forming. Ambroxol was reported to have antibiofilm activity against P. aeruginosa. Thus, Abbas et al.³⁴ reported that ambroxol could inhibit biofilm formation at concentrations ranging between 1.875 and 7.5 mg/ml. Lu et al.¹⁹ found that ambroxol at 1.87-3.75 mg/ml can interfere with the formation and maturation of biofilms of P. aeruginosa; it exhibited antiadherent activity to abiotic surfaces and quorum sensing inhibiting activity.

To the best of my knowledge, the activity of ambroxol against Proteus mirabilis motility and biofilms was not determined. Sub-inhibitory concentrations of ambroxol could inhibit swarming and swimming motilities. This effect was concentration-dependent. Ambroxol showed complete anti-swarming and anti-swimming activities at 0.9 mg/ml. Ambroxol could reduce swarming by 53.74%-94.52% and 78.75%-99.51% at 0.6 mg/ml and 0.7 mg/ml, respectively. Furthermore, swarming was completely inhibited at 0.8 mg/ml in 3 isolates and by 97.51%-98.88% in 2 isolates and was completely blocked in all isolates at 0.9 mg/ml. Moreover, swimming motility was reduced by 18.56%-63.5%, 30.22%-85.12% and 81.37%-97.28% at concentrations of 0.6 mg/ml, 0.7 mg/ml and 0.8 mg/ml, respectively. Complete blocking of swimming was also achieved at 0.9mg/ml.

Different compounds were found to inhibit swarming of Proteus mirabilis such as p-nitrophenyl glycerol (PNPG) and resveratrol, but their effect on biofilms was not investigated. Wang et al.³⁵ reported that resveratrol inhibited P. mirabilis swarming in a dose-dependent manner; it significantly inhibited swarming at 15 µg/ml and completely inhibited swarming at 60 µg/ml. Moreover, the anti-swarming effect of PNPG was reported by Liaw et al³⁶.

The blocking of motility may be linked to the prevention of adhesion to tissues and the subsequent biofilm formation. To investigate this possibility, the antibiofilm effect of sub-MICs of ambroxol was evaluated and it was noticed that ambroxol could inhibit biofilm formation and to remove established biofilms in a dose-dependent manner. Regarding the inhibition of biofilm formation, the magnitude of inhibition ranged between 73.95% and 81%, 80.8% and 83.15% at concentration of 0.6 mg/ml and 0.7 mg/ml, respectively. Moreover, ambroxol at 0.8 mg/ml reduced biofilm formation by 83%-90.1%, while at 0.9 mg/ml, biofilm formation was reduced by 90.25%-100%. Similarly, eradication of established biofilms was found to be concentration-dependent. Biofilms were removed by 44.53%-64.02%, 48.45%-72.48% and 57.24%-76.67% at concentrations of 0.6 mg/ml, 0.7 mg/ml and 0.8 mg/ml, respectively. At concentration of 0.9 mg/ml, ambroxol could eradicate pre-formed biofilms by 78.38%-83.77%.

Chow et al.³⁷ found that sub-inhibitory concentrations of salicylic acid could significantly reduce flagella-mediated swarming motility and biofilm formation in pseudomonas aeruginosa in a dose-dependent manner and they suggested that the biofilm inhibiting activity was due to suppression of bacterial motility that is required for biofilm formation.

The proposed mechanism of inhibition of swarming and biofilm formation of ambroxol is the interference with quorum sensing. Ambroxol was reported as a quorum sensing inhibitor in Pseudomonas aeruginosa.¹⁹ Swarming is regulated by quorum sensing as a flagella-driven movement of swarmer cells to spread over a surface and to form a biofilm³⁸.

In conclusion, ambroxol is a new agent for the treatment of diabetic foot ulcers caused by Proteus mirabilis due to its ability to interfere with its ability to invade the tissues of diabetic foot and to form biofilms.

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Received on 21.07.2013 Accepted on 01.08.2013

Asian J. Pharm. Tech. 2013; Vol. 3: Issue 3, Pg 109-116