Propolis-Sahara honeys preparation exhibits antibacterial and anti-biofilm activity against bacterial biofims formed on urinary catheters

Abstract

Objective: To evaluate the antibacterial effect of Sahara honeys (SHs) against bacterial
biofilms formed on urinary catheters in combination with propolis-Sahara honeys (P-SHs).
Methods: Three clinical isolates were subjected to biofilm detection methods. The antibacterial
and anti-biofilm activity for SHs and P-SHs were determined using agar well diffusion and the
percentage of biofilm inhibition (PBI) methods.
Results: The PBI for Gram-positive bacteria [Staphylococcus aureus (S. aureus)] was in the
range of 16%–47%, while PBI for Gram-negative bacteria [Pseudomonas aeruginosa and
Escherichia coli (E. coli)] were in range of 17%–57% and 16%–65%, respectively. The
highest PBI (65%) was produced by SH2 only on E. coli. In agar well diffusion assay, zones
of inhibition ranged from 11–20 mm (S. aureus), 12–16 mm (Pseudomonas aeruginosa) and
11–19 mm (E. coli). The highest inhibition (20 mm) was produced by SH1 only on S. aureus.
In addition, the treatment of SHs and P-SHs catheters with a polymicrobial biofilms reduced
biofilm formation after 48 h exposure period.
Conclussions: SHs and P-SHs applied as a natural agent can be used as a prophylactic agent to
prevent the formation of in vitro biofilm.

Full text

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Microbiological research doi: ©2016 by the Asian Pacific Journal of Tropical Disease. All rights reserved.
Propolis-Sahara honeys preparation exhibits antibacterial and anti-biofilm activity against bacterial
biolms formed on urinary catheters
Saad Aissat1,2, Moussa Ahmed1,2*, Noureddine Djebli2
1Institute of Veterinary Sciences, University Ibn Khaldoun Tiaret, Tiaret, Algeria
2Pharmacognosy and Api-Phytotherapy Research Laboratory, Mostaganem University, Mostaganem, Algeria
Asian Pac J Trop Dis 2016; 6(11):
Asian Pacific Journal of Tropical Disease
journal homepage: www.elsevier.com/locate/apjtd
*Corresponding author: Moussa Ahmed, Pharmacognosy and Api-Phytotherapy
Research Laboratory, Mostaganem University, Mostaganem, Algeria.
Tel: +213 65234059
E-mail: moussa7014@yahoo.fr
Foundation Project: Supported by Project CNEPRU, Department of Biology,
University-Abdelhamid IBN Badis-Mostaganem, Algeria (Grant No. F02220120001).
The journal implements double-blind peer review practiced by specially invited
international editorial board members.
1. Introduction
A number of previous studies have shown the urinary tract
colonization and infection caused by Staphylococcus aureus
(S. aureus), Pseudomonas aeruginosa (P. aeruginosa) and
Escherichia coli (E. coli) in patients with indwelling urinary
catheters[1,2]. Al-Mathkhury et al.[3] demonstrated the Gram-
negative opportunistic P. aeruginosa common colonization of
urinary catheters and biofilm development on them. Several
factors may contribute for the pathogenicity of bacterial
biofilm formation, including the production of extracellular
compounds (E. coli: flagellum; S. aureus: lipopolysaccharides,
exopolysaccharide; P. aeruginosa: flagella and pili), production
of resistant “persister cells”, surface adherence and biofilm
formation[4-6]. The adhesion of bacteria to a surface depends
on various factors (nutrient levels, pH changes, desiccation,
ultraviolet radiation and osmotic stress)[7,8]. More recently, some
substances showing antibacterial properties, such as nitrous
oxide chlorhexidine, nitrofurazone and gentian violet, have
been used to modify the surface of urinary catheters[9]. But the
biofilms are notoriously difficult to eradicate. In addition to
the difficulty of treating biofilms with conventional antibiotics,
recently alternative treatments are playing their role in the
treatment of biofilms.
The antimicrobial activities of bee products, such as honey and
propolis, have been researched over recent years as alternatives
for new therapeutic agents for the treatment of bacterial biofilm
infections[10,11]. Algerian honey [Sahara honey (SH)] was
reported to inhibit the growth of S. aureus, P. aeruginosa and E.
coli[12,13]. Today, no information is available about the effects
ARTICLE INFO ABSTRACT
Objective: To evaluate the antibacterial effect of Sahara honeys (SHs) against bacterial
biofilms formed on urinary catheters in combination with propolis-Sahara honeys (P-SHs).
Methods: Three clinical isolates were subjected to biofilm detection methods. The antibacterial
and anti-biofilm activity for SHs and P-SHs were determined using agar well diffusion and the
percentage of biofilm inhibition (PBI) methods.
Results: The PBI for Gram-positive bacteria [Staphylococcus aureus (S. aureus)] was in the
range of 16%–47%, while PBI for Gram-negative bacteria [Pseudomonas aeruginosa and
Escherichia coli (E. coli)] were in range of 17%–57% and 16%–65%, respectively. The
highest PBI (65%) was produced by SH2 only on E. coli. In agar well diffusion assay, zones
of inhibition ranged from 11–20 mm (S. aureus), 12–16 mm (Pseudomonas aeruginosa) and
11–19 mm (E. coli). The highest inhibition (20 mm) was produced by SH1 only on S. aureus.
In addition, the treatment of SHs and P-SHs catheters with a polymicrobial biofilms reduced
biofilm formation after 48 h exposure period.
Conclussions: SHs and P-SHs applied as a natural agent can be used as a prophylactic agent to
prevent the formation of in vitro biofilm.
Contents lists available at ScienceDirect
Article history:
Received 15 Aug 2016
Received in revised form 25 Aug, 2nd
revised form 29 Aug, 3rd revised form
7 Sep 2016
Accepted 15 Sep 2016
Available online 30 Sep 2016
Keywords:
Antibacterial
Anti-biofilm
Propolis
Sahara honey

Saad Aissat et al./Asian Pac J Trop Dis 2016; 6(11): 931
of SHs on biofilms. Therefore, this study was performed to
investigate the role of SHs at different concentrations alone or in
combination with propolis-Sahara honeys (P-SHs) on biofilms.
We also investigated the effects of P-SHs on biofilms for the first
time.
2. Materials and methods
2.1. Honey and propolis samples
The present study was carried out on raw SH of different floral
origins, namely, Euphorbe (Euphorbia spp.) and Sidr honey. The
propolis used in this study was obtained from Southern Algeria.
2.2. Preparation of propolis solutions
The propolis was cold-macerated to make an extract with olive
oil (20 g of brute propolis/2 mL of olive oil). The mixture was
heated at 50 °C for 15 min before microbiological testing.
2.3. Preparation of honey with olive oil – propolis
The mixture was stirred gently with a spatula until
homogeneous gel was formed. The mixture was heated at 50
°C for 15 min. For a microbiological test of a mixture of honey,
20 g of propolis was made, where the honey was added in a
concentration of 25%, 50% or 100%.
2.4. Bacterial isolates and growth media
The catheters were removed from patients and then cut under
aseptic conditions using a sterile scalpel. The catheter was
carefully and aseptically cut. Three discs were placed on the
surface of Chapman, MacConkey and King A agar plates. Colony
formation was monitored by examining plates after 48 h of
incubation.
2.5. Antibacterial susceptibility testing
In this study, two different assays were performed to evaluate
the antibacterial potential of the honey samples: agar-well
diffusion (AWD ) and percentage of biofilm inhibition (PBI).
2.5.1. AWD
Antibacterial studies have been evaluated by the method
of AWD by Moussa et al.[13]. Briefly, agar plates (90 mm)
were containing 20 mL of nutrient agar at 37 °C for 24 h and
adjusted by diluting fresh cultures to a turbidity equivalent to 0.5
McFarland scale (approximately 2 × 108 colony-forming unit/
mL). An 8 mm diameter well was cut into the agar and 100 μL
of undiluted, and 25% and 50% honey solution (w/v) prepared
in sterile distilled water was aliquoted into the well. The controls
were set up with equivalent quantities of water. After incubation,
the diameters of the inhibition zones were measured.
2.5.2. PBI
The method adopted was described by Akujobi and Njoku
with little modification[14]. Briefly, 0.2 mL of 0.5 McFarland
standardised culture was added to 4 mL of the test (SHs and
P-SHs). Concentration in a test tube while inoculation of 4
mL of nutrient broth with 0.2 mL of the cell suspension was
served as the control. The optical density (OD) was determined
in a spectrophotometer at 620 nm prior to incubation (T0)
and recorded after the cultures were incubated for 24 h in the
dark at 37 °C. The OD was determined at T0 and again after
24 h of incubation at 620 nm. The OD for each replicate at T0
was subtracted from the OD for each replicate after 24 h of
incubation. The PBI was calculated using the following formula:
PBI% = [(OD control – OD experimental)/OD control] × 100
OD = absorbance at 620 nm.
2.5.3. Biofilm response to SHs and P-SHs
The bacterial anti-adhesive activity of the SHs and P-SHs
against bacterial biofilms was qualitatively evaluated by the
following method (Table 1).
Table 1
Exposure of SHs and P-SHs treatment on bacterial biofilm.
Tube Experiment I Treatment after 24 h Experiment II Incubation
Tube 1 Nutrient broth
+ catheter
Negative control Nutrient broth + catheter 48 h
bacterial (single and mixed)
Tube 2 Nutrient broth
+ catheter
SHs (25%, 50% and
100%)
Nutrient broth + catheter 48 h
bacterial (single and mixed)
Tube 3 Nutrient broth
+ catheter
Propolis Nutrient broth + catheter 48 h
bacterial (single and mixed)
Tube 4 Nutrient broth
+ catheter
P-SHs (25%, 50%
and 100%)
Nutrient broth + catheter 48 h
bacterial (single and mixed)
Tube 1: Sterile catheter segments were immersed in 5 mL sterile culture tubes
nutrient broth and incubated at 37 °C for 24 h (Experiment I), and after 24 h,
the tubes were inoculated with 100 μL of bacterial inoculum (2 × 108 cells/
mL) and incubated at 37 °C for 48 h (Experiment II); Tube 2: Sterile catheter
segments were immersed in 5 mL sterile culture tubes nutrient broth + SHs
(25%, 50% and 100%) and incubated at 37 °C for 24 h (Experiment I), and
after 24 h, the tubes were inoculated with 100 μL of bacterial inoculum (2 ×
108 cells/mL) and incubated at 37 °C for 48 h (Experiment II); Tube 3: Sterile
catheter segments were immersed in 5 mL sterile culture tubes nutrient broth
+ propolis and incubated at 37 °C for 24 h (Experiment I), and after 24 h,
the tubes were inoculated with 100 μL of bacterial inoculum (2 × 108 cells/
mL) and incubated at 37 °C for 48 h (Experiment II); Tube 4: Sterile catheter
segments were immersed in 5 mL sterile culture tubes nutrient broth + P-SHs
at (25%, 50% and 100%) and incubated at 37 °C for 24 h (Experiment I), and
after 24 h, the tubes were inoculated with 100 μL of bacterial inoculum (2 ×
108 cells/mL) and incubated at 37 °C for 48 h (Experiment II).

Saad Aissat et al./Asian Pac J Trop Dis 2016; 6(11):
932
3. Results
3.1. Antibacterial activity
Figures 1–3 show the PBI data for the bee product tested
against Gram-negative and Gram-positive bacteria. PBI for
Gram-positive bacteria (S. aureus) were in the range of 16%–
47%, while they were 17%–57% and 16%–65% for Gram-
negative bacteria P. aeruginosa and E. coli, respectively. The
highest PBI (65%) was produced by SH2 only on E. coli.
100% 50% 25%
SH2
PBI (%)
SH1
Concentration of SHs
25
20
15
10
5
0
Figure 1. Growth inhibitory activity of the SHs against S. aureus.
Figure 2. Growth inhibitory activity of the SHs against P. aeruginosa.
100% 50% 25%
SH1
PBI (%)
SH2
Concentration of SHs
60
50
40
30
20
10
0
Figure 3. Growth inhibitory activity of the SHs against E. coli.
80
70
60
50
40
30
20
10
0
PBI (%)
100% 50% 25%
SH1 SH2
Concentration of SHs
In AW D assays, zones of inhibition ranged from 11–20 mm
S. aureus, 12–16 mm P. aeruginosa and 11–19 mm E. coli. The
highest inhibition (20 mm) was produced by SH1 only on S.
aureus (Table 2).
The results of the synergistic effect between SHs and P-SHs
are given in Figures 4–6, respectively. In combination with
P-SHs, the PBI ranged from 16% to 97% against S.aureus, 31%
to 87% against P. aeruginosa and 22% to 67% against E. coli,
respectively. The highest PBI (97%) was produced by SH2 in
combination with propolis on S.aureus (Table 2).
Table 2
Mean zones of inhibition values of the honey samples against bacterial
tested determined by AWD.
Concentrations S. aureus P. aeruginosa E. coli
100% 50% 25% 100% 50% 25% 100% 50% 25%
SH1 20 15 13 16 14 13 17 12 11
SH2 18 13 11 15 14 12 19 13 11
P-SH1 17 12 11 17 11 9 16 13 11
P-SH2 18 13 11 19 15 12 18 12 11
Negative control - - - - - - - - -
90
80
70
60
50
40
30
20
10
0
PBI (%)
100% 50% 25%
P-SH1 P-SH2
Concentration of P-SH1 and P-SH2
Figure 4. Growth inhibitory activity of the P-SHs against S. aureus.
In AW D assays, zones of inhibition ranged from 22 to 18
mm against S. aureus, 31–87 mm and 22–67 mm against P.
aeruginosa and E. coli, respectively. The highest concentration
required was 100% to simultaneously inhibit all bacteria tested
(Table 2).
3.2. Effect of SHs and P-SHs on biofilm-forming
bacterial growth
In a second series of experiments, the disruption of preformed
biofilms (48-h growth in the absence of SHs and P-SHs) by
addition of SHs and P-SHs at various concentrations for 48 h.
The SHs and P-SHs inhibited biofilm formation on catheters
(Table 3). This suggested that SHs, propolis and P-SHs have a
better ability to prevent biofilm formation.

Saad Aissat et al./Asian Pac J Trop Dis 2016; 6(11): 933
3. Discussion
Biofilm-producing bacteria are intrinsically resistant to
antimicrobial agents, which is a main cause of the pathogenesis
of catheter infection[15,16], and the susceptibility of bacteria
biofilms to the current therapeutic agents remains low. Currently,
researches are focused on the development of anti-biofilm agents
that are nontoxic, as it is believed that such molecules will not
lead to future drug resistance[17]. Researches aiming at new
anti-biofilm originating mainly from bee products have long
been revered for their healing.The anti-biofilm properties of
bee products as a natural antibiotic agent have been extensively
studied. Strong antibacterial activities of propolis and honey
against both Gram-positive and Gram-negative bacteria have been
reported[11,18]. In addition, several investigators examined the
effects of honey and propolis on biofilms. Campeau and Patel[10]
reported that Manuka honey has a synergistic interaction with
vancomycin against S. aureus biofilms and an additive interaction
with gentamicin against P. aeruginosa biofilms. In addition,
Jenkins and Cooper[19] reported that Manuka honey-exposed
planktonic S. aureus cells were enlarged and had more septa.
Cooper et al.[20] reported that Manuka honey at concentrations
below 10% (w/v) promoted the growth of established biofilms
of S. aureus. Also, Alandejani et al.[21] studied S. aureus and P.
aeruginosa biofilms but only evaluated 50% Manuka and Sidr
honey. The therapeutic effects of the propolis and honey are
well known. Several aspects of this use indicate that they also
have functions such as antibacterial and anti-biofilm proprieties.
Biological activities of honey and propolis are mainly attributed
to the phenolic compounds such as flavonoids[22]. It has recently
been reported that several flavonoids reduced biofilm formation
in Vibrio harveyi and E. coli O157:H7[23]. Also, various honeys
were investigated for the presence of nitrite/nitrate, the stable
nitric oxide metabolites[24]. Barraud et al.[25] observed a decrease
in biofilm biomass and an increase in planktonic biomass at
low concentrations of nitric oxide donors. In addition, honey
is a saturated or supersaturated solution of carbohydrates of
glucose and fructose[26]. Previous work by Dusane et al.[27] has
reported that the lauroyl glucose after 48 h of incubation with
P. aeruginosa and Pseudomonas aureofaciens resulted in 51%
and 57% of the disruption of preformed biofilms, respectively.
Carvacrol (thymol isomer) is present in the essential oil of
Algerian propolis (4.47%)[28]. Antibacterial effect of carvacrol
and its isomer thymol against E. coli, P. aeruginosa and
Enterococcus faecalis have been proved[29]. Several studies have
examined the effect of various types of antimicrobial treatment
in controlling biofilm formation on these devices [central
venous catheters, mechanical heart valves and urinary (Foley)
catheters][30]. To our knowledge, this is the first time these
novel anti-biofilm agents (SHs and P-SHs) are reported on the
tested organisms. SHs, propolis and P-SHs exhibited excellent
antibacterial and anti-adhesive properties against S. aureus, P.
aeruginosa and E. coli, which was evidenced in our adhesion
based assays.
Conflict of interest statement
We declare that we have no conflict of interest.
Table 3
Effects of SHs and propolis on single and mixed microbial growth and biofilm formation after 48 h of incubation.
Type of isolates S. aureus single P. aeruginosa single E. coli single Microbial cultures mixed
Nutrient broth + catheter (negative control) BF (++) BF (++) BF (++) BF (++++)
Nutrient broth + catheter + SHs BF (--) BF (--) BF (--) BF (--)
Nutrient broth + catheter + propolis BF (--) BF (--) BF (--) BF (--)
Nutrient broth + catheter+ SHs + propolis BF (--) BF (--) BF (--) BF (--)
BF: Biofilm formation; ++: Adhesion; --: No adhesion; ++++: ?.
100
90
80
70
60
50
40
30
20
10
0
PBI (%)
100% 50% 25%
P-SH1 P-SH2
Concentration of P-SH1 and P-SHs
Figure 5. Growth inhibitory activity of the P-SHs against P. aeruginosa. Figure 6. Growth inhibitory activity of the P-SHs against E. coli.
100% 50% 25%
P-SH1
PBI (%)
P-SH2
Concentration of P-SH1 and P-SH2
80
70
60
50
40
30
20
10
0

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934
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