INTRODUCTION:

Urinary tract infections (UTIs) are the most commonly infections which causes diseases in many countries¹. Those are caused by many types of pathogens, known as uro-pathogens, with special virulence factor which facilitates their viability in urinary tract². The bacterial resistant patterns have been varied by geographical location as well as by time so periodically antibiotic-resistant testing is always in need³.

Pathogens always varies depending on age, sex, and hospitalization⁴. Escherichia coli (E.coli) is defined as a pathogen with higher prevalence especially in healthcare-associated UTIs, followed by other Enterobacteriaceae spp.⁵.

It is a successful competitor at this crowded site, and can cause a broad spectrum of infections with the help of some virulence factors⁶, besides its antibiotic resistance (AR) mechanisms such as the production of extended-spectrum β-lactamases (ESBL).

Uropathogenic E.coli strains (UPEC) can create a specific biofilm⁷, which is defined as the irreversible attachment to the surface. This mode of growth makes E.coli one of the major causes of persistent UTIs⁸. High antimicrobial concentrations are required to suppress organisms growing in those biofilms, as AR can increase 1000 fold^9–11.

Honey is the natural sweet substance from nectar or from the secretion of living parts of plants or excretions of plants, which honey bees collect¹². Many years ago, honey was used as an agent for the treatment of disease. Aristotle described pale honey as being ‘‘good for sore eyes and wounds”¹³. It was already found that honey has the most potent inhibitory effect on bacteria. This is due to, it is non-toxic and not produce any adverse effects¹⁴.

The present study was designed to study resistance pattern, biofilm formation among uropathogenic E.coli, and evaluate the use of different types of Egyptian honey as an antibacterial agent.

MATERIAL AND METHODS:

Bacterial collection and identification:

Bacterial strains (n=17) were kindly collected from Kasr Alaini Hospital, Egypt. All of them were collected on both Cled and MacConkey agar. Isolates were defined as E.coli according to routine biochemical identification¹⁵, then they were stored on glycerol broth and stored at -80 freezers until been used.

Antibiotic sensitivity test:

Antimicrobial susceptibility of the E. coli isolates was determined by the disk diffusion as recommended by CLSI¹⁶. The commercial antibiotics used: imipenem (10µg), cefotaxime (30µg), cefoxitin (30µg), gentamicin (10µg), amikacin (30µg), ciprofloxacin (5µg), levofloxacin (5µg), ceftazidime (30μg), and ampicillin (10µg). Results were interpreted according to CLSI criteria.

ESBL detection test by combination disk method:

ESBL confirmatory test was done to all isolates using combination disk method (Bio-Rad, France) as recommended by CLSI¹⁷ and include cefotaxime (30μg), cefotaxime/clavulanic acid (30/10μg), ceftazidime (30 μg), and ceftazidime/clavulanic acid (30/10μg). Klebsiella pneumoniae standard strain ATCC 700603 was used as a positive control.

Biofilm formation assay:

Evaluation of biofilm formation by E.coli was performed according to the method described by Mohamed, et al.¹⁸ using Tissue Culture Plate (TCP) method with some modifications. Wells containing sterile media were used as negative control. By using STAT FAX 2100 microplate reader, the optical density (O.D) was measured at 630nm. The cut-off value (ODc) is defined as three standard deviations (SD) above the mean OD of the negative control, that is, sample’s ODc = average OD of negative control +(3*SD of negative control). After comparing the O.D of biofilm to the control and according to the readings, the isolates were classified as follows: O.D ≤ O.Dc no biofilm producer, O.Dc <O.D ≤ 2×O.Dc weak biofilm, 2×O.Dc <O.D ≤ 4×O.Dc moderate and 4×O.Dc <O.D strong biofilm as described.

Preparation of honey samples:

Four Egyptian honey types (citrus, camphor, marjoram, and black seed honey) were purchased from the stores of Ministry of Agriculture, Giza, Egypt. Hundred percent pure honey (100% v/v) was obtained after filtration. To get 50% honey solutions (v/v), 0.5ml of honey was diluted in 0.5ml sterilized distilled water¹⁹.

Screening of the antibacterial activity of honey:

a. Agar well diffusion method (Zone of Inhibition Evaluation):

The activity of each honey type was evaluated by measuring the zone of inhibition using the agar well diffusion assay¹². The adjusted bacterial culture (0.5 McFarland) was spread on separate sterile solidified Muller Hinton agar (MHA) plate using a disposable sterile cotton swab. Each plate was punched to make four wells (6mm diameter) by using a sterile cork borer at different sites of the plates. 100μl of each honey type were pipetted into the wells in assay plates. Plates were incubated overnight at 37°C. Inhibition zones were observed, and the diameter was measured.

b. Microbroth dilution method:

The activity of honey was confirmed using the microdilution method. Briefly, 100μl of honey concentrations ranged from 50% to 0.1% were placed in 96 microtiter plate was used, except for the control wells, 100μl sterile nutrient broth instead of honey. Each well was inoculated with 100μl of 0.5 McFarland E.coli prepared culture. The whole procedure was repeated for all the organisms tested to each of the honey types. Plates were then incubated at 37°C for 24 h and observed by visual inspections for the presence and absence of growth (turbidity). The lowest concentration of honey that inhibited the growth of E.coli is defined as MIC¹².

Statistical analysis:

Data were expressed as means, and standard deviations (SD) were presented as error bars.

RESULTS AND DISCUSSION:

E. coli is a commonly found of in human/animal gastrointestinal tract²⁰, at which UPEC strains are the most prevalent causes of UTIs worldwide ²¹. Recently, E.coli was reported as the most predominant uro-pathogen in studies from different countries, showing different predominance percentages. Jha, et al. reported that E. coli was the most common uropathogen in the study (36%)². Higher predominance percentages of E. coli were observed by 37.9%,61.1%, 60.15% in Nigeria, Bahrain, India respectively^22–24

In this study, 17 E. coli strains were collected and the antibiotic susceptibility test was performed (Figure 1). High AR rates among E. coli strains toward ampicillin (100%) followed by cephalosporins such as ceftazidime and cefotaxime at which the resistance rates were 88% and 77% respectively. On the other hand, imipenem was the most active antibiotic at which the susceptibility was 94%. The AR is growing alarmingly worldwide, especially organisms such as E. coli and Klebsiella²². According to our results, all E. coli strains were resistant to ampicillin, which comes in line with that recently reported in other studies^25–27. Marked resistance was observed by Lawhale, et al to doxycycline, quinolones, and cephalosporins ²³. Resistances to ampicillin, nalidixic acid and ciprofloxacin were also observed⁵. Most E. coli isolates were resistant to cefepime (100%) and cephalothin (74%) and highly susceptible to imipenem, vancomycin, and doxycycline by 100% percent as reported by Delpech et al,³.

Figure (1): Resistance percentages among E. coli strains toward different antibiotics.

Combination disk method was used to detect ESBL production. Out of 17 isolates, 11 isolates (64.7%) were ESBL producers while only 6 isolates (35.3%) were non-ESBL producers. Recently, different types of ESBL producing Enterobacteriaceae have emerged as serious nosocomial pathogens throughout world²⁸. ESBL-mediated antibiotic resistance is the rapidly growing mode of resistance observed in most of the clinically relevant Gram-negative pathogens²⁹. In addition to the study of Sanmugam, et al. reported that 80-90% of E.coli isolates showed resistance to the cephalosporin group of drugs³⁰, Baaity, et al. showed that 26% of isolates were ESBL producers by using the conﬁrmatory double-disc synergy diffusion test³¹.

Biofilm formation assay results were presented in Figure (2). 70.6% of the strains were found to be biofilm formers at which 17.7% of them were categorized as moderate biofilm formers and 52.9% were categorized as weak biofilm formers. Only 5 isolates (29.4%) were non-biofilm formers (Figure 3). Biofilms, dense matrix-encapsulated population that was attached to the surfaces ^32,33, is associated with the chronic nature of infection with their inherent resistance to antibiotic ^34,35. As recently reported, its known that biofilm formation ability in E.coli found to enhance its colonization leading to increase the rate of UTIs, makes it very difficult to treat as they exhibit strong AR profile ⁹. In our study, 70.6% of the strains had the ability to form a biofilm. The ability for biofilm formation was recorded in 76.5% of the UPEC isolates⁸. By comparing biofilm and non-biofilm producers, biofilm producers showed maximum AR ⁹.

Figure (2): Optical density values of biofilm formation assay among E.coli strains at 630 nm. Cutoff value (O.Dc) = 0.322. Standard deviations were presented by error bars.

Figure (3): Quantification of biofilm formation percentage according to control cutoff value (O.Dc).

Concentration of 50% Egyptian honey types (Citrus, Camphor, Marjoram, and Black seed) was prepared, and antibacterial activity using well diffusion method was assessed. Higher inhibition zones were detected by citrus honey which was chosen for further antibacterial testing by micro-broth method (Table 1). 50% citrus honey concentration was defined as MIC for 3 isolates, and a concentration of 12.5% was defined as MIC for 1 isolate (Table 1).

Table (1): Antibacterial activity of honey using well diffusion and microbroth dilution methods.

Iso.	Inhibition zones diameter of (50%) of honey types (cm) ± SD*			MIC of citrus honey by microbroth dilution method
Iso.	Citrus	Camphor	Marjoram	Black seed
E1	-	-	-	-	˃ 50%
E2	-	-	-	-	˃ 50%
E3	-	-	-	-	˃ 50%
E4	3 ± 0	2.8 ± 0	2.7 ± 0	2.8 ± 0	50%
E5	-	-	-	-	˃ 50%
E6	-	-	-	-	˃ 50%
E7	-	-	-	-	˃ 50%
E8	-	-	-	-	˃ 50%
E9	-	-	-	-	˃ 50%
E10	-	-	-	-	˃ 50%
E11	4 ± 0	3.2 ± 0	2.6 ± 0	3.2 ± 0	12.5%
E12	-	-	-	-	˃ 50%
E13	-	-	-	-	˃ 50%
E14	3.2 ± 0	3 ± 0	3.2 ± 0	3 ± 0	50%
E15	3.7 ± 0	3.7 ± 0	2.7 ± 0	3.2 ± 0	50%
E16	-	-	-	-	˃ 50%
E17	-	-	-	-	˃ 50%

*SD: Standard deviation

Many factors have been shown to contribute to the antibacterial activity of honey³⁶. Egyptian Clover Honey was reported to have strong bactericidal effect on planktonic cells of Proteus mirabilis, as it completely blocked swarming motility and inhibited biofilm formation³⁷. In addition, an in vitro antibacterial activity of Syrian honey against P. aeruginosa, suggested as an alternative topical choice for wound infections³⁸. This antibacterial activity due to the high sugar content of honey is beneficial as it draws water out of bacterial cells³⁹.

On the other hand, Sam et al. observed that at the highest concentration, for all Pseudomonas strains no inhibition occurred ⁴⁰. During our research, different antimicrobial activities of honey types were observed, these difference in antimicrobial potency among honey type can be more than 100-fold, depending on its geographical, seasonal and botanical source as well as harvesting, processing and storage conditions ^41,42

CONCLUSION:

This study showed the high AR rates of uropathogenic E.coli toward the tested antibiotics, besides its ESBL production and biofilm formation ability. Egyptian citrus honey at a concentration of 50% and 12.5% had the ability to inhibit E.coli isolates (23.53%), acting as an antibacterial agent against such AR pathogenic strains.

CONFLICT OF INTEREST:

The authors declare that there are no conflicts of interests regarding the publication of this article.

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Received on 10.10.2019 Modified on 07.12.2019

Research J. Pharm. and Tech. 2020; 13(8):3720-3724.

DOI: 10.5958/0974-360X.2020.00658.7