Abstract Background: The aim of this study is to evaluate the antibacterial activity of seven Algerian honey samples on the growth, viability and biofilm formation of clinical strains of Pseudomonas aeruginosa isolated from infected wounds. Material and methods: The antibacterial activity of honey was determined by both well assay and microdilution assay. The effect of honey samples on the viability of P.aeruginosa was studied by the time-kill assay. The antibiofilm effect was performed by the assessment of the inhibitory and the eradication effects of biofilm in a 96-well microplate. Results: The results revealed that Algerian honey exhibit a high level of antibacterial activity against P. aeruginosa isolated from infected wounds. The inhibitory diameters of tested honey are ranging from 14.97 ± 3.88 mm to 27.98 ± 3.19 mm. However, MIC values are in the range of 10 to 40. The MIC50 varied from 7.88 % (w/v) for honey sample 5 to 18.5 % (w/v) for honey sample 7, the MIC90 is in the range of 18.78 to 38.35 % (w/v). The MBCs values are ranging from 22.43±6.62 % (w/v) to 39.51±8.35 % (w/v), and the MBC/MIC ratios are less than 2, this indicated that Algerian honey displayed bactericidal activity on P.aeruginosa strains. The study of time-kill assay has shown that honey samples (1, 2, 3, 4, and 7) destroy P.aeruginosa after 24h of incubation; honey samples 5 and 6 destroy P.aeruginosa after 21h of incubation. The antibiofilm effect revealed that all tested honey are effective to inhibit and to eradicate the biofilm with percentage varied from 54.32% to 97.48% and from 40.21% to 87.65% respectively. Conclusion: the results obtained suggest that Algerian honey is effective as an alternative treatment against multidrug-resistant P.aeruginosa, indeed honey can play an important role to treat and to prevent the formation of biofilm in wounds.
Keywords: antibacterial activity, alternative treatment, honey, Pseudomonas aeruginosa, wound infections. 1. Introduction In infected wounds, activated leukocytes release cytolytic enzymes, free radicals, oxygen, and inflammatory mediators, leading to an imbalance between local pathological factors and the integrity of immune defenses 1. This promotes the colonization of wounds by pathogenic bacteria, especially Pseudomonas aeruginosa, which has become an important cause of morbidity and mortality in recent years, particularly in immunocompromised patients
1,2. The pathogenicity of this bacterium is largely caused by multiple bacterial virulence factors such as flagella, pili, adhesion factors, alginate, exotoxin A, ex-enzyme S, elastases, alkaline protease, and thermolabile phospholipase C and siderophores. These factors induce damage to the epithelial cell wall and changes in cell physiology and function . Moreover, P. aeruginosa is characterized by genetic flexibility enabling it to develop resistance to antimicrobial agents, because it has a number of inherent antibiotic-resistance mechanisms . This causes the emergence of multidrug-resistant P.aeruginosa in the hospital environment, which is associated with high-risk and long-stay patients, more expensive hospitalization and increased mortality 6. Recently, the prevalence of the emergence of multidrug-resistant P. aeruginosa has attracted particular attention from medical professionals in Algeria 7–9, particularly in the hospital environment where, if not controlled, it becomes a life-threatening situation 1,10,11. It has been found that the most important factor that makes P. aeruginosa tolerant to antimicrobial agents is its ability to form biofilms; these are surface-attached microbial communities of bacteria enclosed in a protective polysaccharide matrix with characteristic architecture and phenotypic and biochemical properties distinct from their free-swimming, planktonic counterparts 12. The extracellular matrix is thought to hold these communities together as well as contribute to bacterial persistence at infection sites by protecting against the host immune system and antimicrobial stresses. Recently, it has been emphasized that bacterial biofilm plays an important role in chronic wounds, particularly in the context of the prolongation of the inflammatory phase of repair. They interfere with the natural healing process by acting as a mechanical barrier, which prevents reepithelialization, stimulating a chronic state of inflammation and providing protection against endogenous and exogenous antimicrobial agents. Since biofilm makes the treatment of infected wounds ineffective, which increases the hospital stay and makes their management very costly, the need for new antimicrobial agents has increased. Great attention is paid to natural products, among which honey is described as a very effective means against several types of infections, particularly, wound infections . Antibacterial activity of honey is associated with a combination of several components such as hyperosmolarity, acidic propriety, hydrogen peroxide, and other factors include lysozyme, phenolic acids and flavonoids . Antibacterial activity of honey is a useful choice for the treatment of various wounds. It is effective in removing microbial contamination, reducing the wound area and stimulating wound healing . In addition, honey is able to maintain a wound in a healthy state through the wound debridement process and to increase collagen synthesis Nonetheless, the quality of honey varied based on the geographical floral origin, season, environmental factors, and storage conditions . Algeria is an African country which occupies the center of the continent; it is characterized by very diverse forest ecosystems and an important variation in climate that varied from the Mediterranean to the Saharan type. These conditions offer production of honey of good quality and very diversified.
However, very few studies have been conducted on Algerian honey, and no published data exist on the antimicrobial activity of most of the Algerian honey. Therefore, in the current study, we aimed to evaluate the antibacterial and antibiofilm effects of seven Algerian honey types against forty clinical strains of P.aeruginosa isolated from infected wounds and the standard strain P.aeruginosa PAO1. 2. Material and method 2.1. Tested Honey Seven honey samples from different floral types were obtained from bee farms in different geographical regions of north-eastern Algeria (figure 1). Honey samples were directly collected in sterile dark glass bottles. Each honey sample was first filtered with a sterile filter of 0.22 µm pore sizes (Millipore, Nunc, Paramus, NJ, USA) to remove bacterial contaminations and stored at 2-8°C until used. The following honey dilutions were prepared in sterile distilled water: 2.5%, 5%, 10%, 20%, 40%, 80% (w/v) and undiluted honey. Figure 1: Geographical locations of honey harvesting sites 2.2. Tested bacterial strains In this study forty clinical strains of P.aeruginosa were used, these strains were isolated from infected wounds from two hospitals (Ibn Sina and Ibn Rochd Hospitals, Annaba, Algeria). The identification was done by using microbiological and biochemical methods including Gram staining, oxidase, and catalase tests, growth in King A and King B medium, analytical profile index (API) 20NE (Biomerieux, Paris, France). The choice of bacterial strains was made according to their antibiotic resistance profile, only strains that have shown multidrug resistance are selected. In addition, a standard strain P. aeruginosa PA01 was used as the positive control. An inoculum of each tested bacteria of approximately 106 colony-forming units (CFU)/mL was prepared in nutrient broth (Difco, MD, USA). 2.3. Antibacterial activity Well diffusion assay Wells diffusion assay was performed according to Molan et al. (1988). Wells of 6 mm of diameter are prepared in Mueller Hinton agar (Difco, MD, USA) plates. The agar plate surface is inoculated by spreading a volume of the microbial inoculum over the entire agar surface, 100 µL of tested honey is introduced into the well. A well filled with sterile water served as control. Plates were incubated at 37°C for 24 h. Honey diffuses in the agar medium and inhibits the growth of the microbial strain tested. The antibacterial activity of the samples was compared on the basis of the radius of a clear inhibition zone around the wells 24,25. Broth microdilution assay Broth microdilution assay is performed according to Boorn et al. (2010) by using sterile 96-well round-bottomed polystyrene microtitre plates (Fisher Scientific, UK). A volume of 100 µL of inoculum of tested strain is added to 100 µL of honey at different concentrations (from 2.5 to 100%) in each well. Control wells containing only broth (negative control) or only bacteria and broth (positive control) were also constructed. The plates are incubated at 37◦C for 24 hours. Minimum inhibitory concentration (MIC) is the lowest concentration of honey which results in 100% inhibition of growth of the tested bacteria. For each honey types, the MIC50% and MIC90% were calculated using the geometric formula: MIC50=MICa+((n-a)(MICb-MICa)/b Where: MICa: MIC of the highest cumulative percentage below 50%, MICb: MIC of the lowest cumulative percentage above 50%. n: 50% of the number of bacteria tested. a: number of bacteria in the group at MICa b: number of the bacteria in the group at MICb Minimum bactericidal concentration (MBC) was determined by cultivating a volume of 10µL of all wells that have not shown growth. The Petri dishes were incubated at 37°C for 24 hours. The MBC was the lowest concentration of honey that destroyed 99.9% of bacteria. For each honey sample, the MBC/MIC ratio was determined and used to classify honey samples as bacteriostatic or bactericidal honey 26. Time-kill analysis The time-kill assay is performed to evaluate the effect of honey on the growth and viability of P.aeruginosa. One tube of diluted honey 40 % (w/v) at a volume of 2 mL was inoculated with 0.02 mL of bacterial inoculum at approximately 106 CFU/mL. Another tube of inoculated nutrient broth without honey was used as control. The tubes were incubated in dark at 37 °C with constant shaking at 200 rpm. Bacterial growth was quantified every three hours. Broth aliquots were collected, cultivated on agar nutrient medium and incubated for 24 hours at 37°C to determine the total CFU/mL. Honey samples were considered bactericidal when a decrease in CFU compared with the initial inocula (control without honey) 26. Antibiofilm assay The antibiofilm activity of honey samples against the three strains of P.aeruginosa which have a strongly adherent profile was assessed according to Horniackova et al. (2017)27. Fresh tryptone soya broth (Difco, MD, USA) was added to an overnight culture of P. aeruginosa to adjust its turbidity to achieve a cell density of 106 CFU/mL. The wells of sterile 96-well polystyrene microplates were inoculated with 100 μL of the bacterial suspension and 100µL of honey at 40% (w/v). A well inoculated with bacterial suspension was used as a positive control, another well filled with tryptone soya broth was used as a negative control. After incubation for 24 hours at 37°C, the wells were gently aspirated and then washed thrice with sterile phosphate-buffered saline (a mixture of 80 g NaCl, 2 g KCl, 14.4 g Na2HPO4 , and 2.4g KH2PO4 in 800mL of distilled water at pH 7.2) to remove any non-adherent cells. To fix the adherent cell, aliquots of 200 μl of methanol (99%) were added and left for 20 min. The wells were stained with 200 μl crystal violet (2%) 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 ethanol (99%) and the optical densities of the stained adherent biofilms were read at a wavelength of 550 nm. The test was repeated three times, and the average optical densities (OD) were calculated. The percentage of biofilm mass reduction was calculated according to this formula: Percentage of biofilm mass reduction (%)= (OD positive control –OD treatment)/(OD positive control)×100 To assess the biofilm eradicating activities of honey samples, wells with established biofilms were aseptically washed three times with phosphate-buffered saline to remove planktonic bacteria, and 200 μL of honey at 40% (w/v) was added. After 24 hours of incubation at 37°C, the honey solutions were removed and each well was washed three times with sterile phosphate-buffered saline. The adherent biofilms were quantified spectrophotometrically. The percentages of biofilm eradication were calculated 28,29. 2.4. Statistical analysis The results obtained were analyzed by GraphPad Prism version 7.00 for Windows, (GraphPad Software, La Jolla California USA). The statistical analysis was determined by one-way analysis of variance (ANOVA) followed by a post hoc Tukey test to estimate the differences in antibacterial activity between honey samples. 3. Results Well diffusion assay The results of the inhibitory diameters of tested honey are reported in figure 2. The Algerian honey has shown a good antibacterial effect on P.aeruginosa strains, the averages of inhibitory diameters are in the range between 14.97±3.88 mm to 27.98 ± 3.19 mm. There are no significant differences between antibacterial activities of honey samples 1, 2 and 3 (P=0.053), but there are highly significant differences between antibacterial activities of other honey samples.
Figure 2: Inhibitory diameters (± S.D mm) of seven Algerian honey samples against P.aeruginosa strains isolated from wound infections. There are no significant differences between means that have the same letter (P = 0,053), but there are very highly significant differences between means that have different letters (P 0.05) between honey samples which have the same letters but there are very highly significant differences (P <0.0001) between honey samples which have different letters.
Table 4: Percentage of eradication of biofilm (%) by honey samples against P.aeruginosa strains Tested strains Percentage of eradication of biofilm (%) Honey 1 Honey 2 Honey 3 Honey 4 Honey 5 Honey 6 Honey 7 P.aeruginosa 1 42.42 a 41.57 a 58.22 b 56.69 a 77.85 b 40.21 a 81.07 c P.aeruginosa 2 43.52 a 41.06 a 54.67 50.88 b 64.22 b 40.87 a 87.65 c 4. Discussion To the best of our knowledge, this is the first study that reported the antibacterial and antibiofilm effects of Algerian honey against multidrug-resistant P.aeruginosa isolated from infected wounds. The inhibitory diameters reported in figure 3 and the MIC values in table 1 showed that Algerian honey samples have good antibacterial activity against P.aeruginosa. The low MIC50 and MIC90 values which ranged from 7.88 to 18.5 and from 18.78 to 38.35 % (w/v) demonstrated that Algerian honey is effective against P.aeruginosa from infected wounds. Agbagwa et al. (2010) have reported that Nigerian honey has smaller inhibitory diameters than Algerian honey (3-9 mm)30. Indeed, Deb Mandal and Mandal, (2011) have reported that Indian honey has comparables MIC values ranged from 8.25 to 25 % (v/v) 16. Also, Shenoy has reported that honey has MIC values on P.aeruginosa ranged from 15 to 25 % (v/v) 31.
However, Henriques has reported that the MIC of Manuka honey is 9.5%32. It has been clearly demonstrated previously that all honey samples haven’t the same antibacterial activity 33,34. The difference in antibacterial activity of Algerian honey is related to the difference in geographical and botanical localities, which varies with the plant source. Algerian botanical flora is very well known for its diversity; this contributes to producing a good quality of honey. The MBC values are ranging from 22.44 (± 6.62) for honey sample 5 to 39.51 (± 8.35) % (w/v) for honey 3. Indeed, MBC/MIC ratios are determined; these ratios are used to define the bactericidal or bacteriostatic effect of an antibacterial agent. If the MBC/MIC ratio is greater than to 4; the antibacterial agent has a bacteriostatic effect, however, if the MBC/MIC ratio is less than or equal to 4; the antimicrobial agent has a bactericidal effect 35. Indeed, we can suggest that all tested honey have a bactericidal effect because the MBC/MIC ratios were less than 4. In order to verify the impact of honey on the viability of P.aeruginosa, a time-kill curve was conducted. As shown in figure 3, the most strains (73.17 %) are inhibited after 9 hours of growth with honey and the honey samples have destroyed all tested strains in the first twenty-four-hour of incubation with the dilution 40% (w/v). As reported by Molan, 1992, the bactericidal effect of honey is dependent on the time of honey action and the bacterial species; it varies from several to 40 hours 36.
Moreover, the concentration of honey also plays an important role. Our results are in agreement with those of Wilkinson and Cavanagh, 2005, they have studied the efficacy of kinds of honey on P.aeruginosa and they have shown that all honey samples had a bactericidal effect. Jantakee and Tragoolpua, 2015 have also reported that honey at concentrations from 5 to 50% has been found to be bactericidal. Furthermore, Shenoy and al., 2012 have reported that honey at 50 % (w/v) destroys P.aeruginosa strains in 24 hours . As shown in table 3 and table 4, the evaluation of the antibiofilm effect of honey demonstrated that all honey types reduced significantly the formation of biofilm and remove between 40.21 % to 87.65% of biofilms produced by P.aeruginosa strains. These results are in agreement with those of Alandejani et al. (2009) and with those of Abbas, (2014) against P.eruginosa . It can be deduced from these results that honey is a good treatment for biofilms formed in infected wounds, in particular, those of P. aeruginosa. However, little information is available on the active components in honey responsible for the antibiofilm properties. It has been suggested that honey could prevent the development of biofilm either by interfering with bacterial adherence to a surface or by inhibiting the growth of attached cells in the early biofilm stage or by alteration of extracellular polymeric substances matrix. Majtan et al. (2014) have recently shown that methylglyoxal accounts for the antibiofilm activity of Manuka honey 40. In addition, the bactericidal activity of honey depended on a combination of several factors that can act synergistically to prevent the development of bacterial biofilms in wounds. Since the structure and characteristics of bacteria in biofilm differ significantly to the planktonic forms which are metabolically active . The treatment of infections in wounds must prevent systemic bacterial invasion, sepsis and biofilm formation . Honey can ensure effective treatment, not only by reducing microbial load and avoiding microbial contamination and biofilm formation but also by revitalizing the host\’s immune defenses.
In this study, we demonstrate the efficacy of seven Algerian honey in the treatment of infected wounds caused by multidrug-resistant P. aeruginosa. The antibacterial and antibiofilm effects of honey without risk of resistance or side effects, combined with healing and anti-inflammatory effects, could improve the treatment of infected wounds. Further study on the active substances in honey and the molecular mechanism of the antibacterial and the antibiofilm effects of honey are needed.