Antibacterial potential components of Bacillus species and antibiotics residues in branded and unbranded honey samples from Nigeria

Honey is a sweet viscous liquid produced by honey bee, Apis mellifera from the nectar of plants. Honey is a natural product that has been used from ancient times till now as food and for medicinal purpose. This study was carried out to determine the mode of action of Bacillus species and antibiotics residues in branded and unbranded honey samples from Nigeria. Bacilli spp. count was carried out by initially heating diluted honey samples in water bath set at 80°C for 15 min, while total bacterial count was carried out using the pour plate method. Antibacterial activity of identified Bacillus spp on Micrococcus was determined using well-in agar method while the mode of action was carried out by reporter assay method. Detection of tetracycline, gentamycin and streptomycin was analyzed using high performance liquid chromatography (HPLC) column oven L-2300 and Column intensil ODS-3C18 (250 x 4.6 mm). Honey samples (2 g) were extracted for HPLC by deprotenizing using acetonitrile and methanol with flow rate of 1 mL/min and RID detector was used to detect antibiotic residue. Bacilli from honey were characterized physiologically, morphologically and biochemically, they were tentatively identified as Bacilli licheniformis, Bacilli subtilis, Bacilli coagulans, Bacilli cirulans, Bacilli pumilis and Bacilli badius. The most prevalent Bacillus spp. were B. licheniformis and B. subtilis. Total bacteria count for branded honey ranged from 2.2 x 10 2 to 5.5 x 10 3 cfu/g, while Bacilli count ranged from nil to 6.2 x 10 2 cfu/g. For unbranded honey samples, total bacteria count ranged from 7.0 x 10 3 to 3.5 x 10 2


INTRODUCTION
Honey is a sweet viscous liquid produced by honey bee, Apis mellifera from the nectar of plants (EC, 2001).
Honey is made up of starch: 1%, triaccharides: 2%, maltose: 7%, water: 17%, glucose: 31% and fructose: 38% (Vanhanen et al., 2011;National Honey Board, 2008).Honey consumption in developing countries such as China, Argentina, India, Brazil and Egypt is estimated to be 0.1 to 0.2 kg per capita.Developed countries consume generally higher amounts (Bogdanov, 2009).Product of honey fermentation are honey vinegar, honey beer and alcoholic beverages.It is also used for cosmetic products such as lotions, ointments, creams, perfumes and so on.The quality of honey produced in recent years have decreased, this might be due to infection of bees by microorganisms, resulting in the death of bees and also the quest for wealth has led to the use of antibiotics to increase the yield of honey production.Others add sweeteners to increase the volume of honey, thus reducing the quality and its antimicrobial capacity (Crosby and Alfred, 2004).Branded honey is processed and well packaged, they usually have a brand name and somestimes NAFDAC number as in the case of Nigeria.Some branded honey might have been analysed by been exposed to high level of heat, pollens are often filtered off and pasteurized thus making the product appealing to the eyes.Most of them lack some basic health benefits and the origin cannot be detected.Unbranded honey do not have brand names and they are usually the local products.They are raw honey from the farm harvested by local farmers who may or may not be into bee farming.The handling of unbranded honey is usually poor and does not look presentable but have heath benefits.
The extensive use of antibiotics in beekeeping has led to an accumulation of antibiotic residues in honey, thereby leading to decreased quality and difficulty in marketing (Tillotson et al., 2006;Sapna and Nimisha 2010;Orianne et al., 2013;Reybroeck et al., 2012;Mahmoudi et al., 2014).Antibiotics are used in beekeeping as growth promoters and to treat diseases.Research has shown the presence of antibiotics residue in honey.For example, oxytetracycline and chloramphenicol residues have been found to be above the regulatory standards in honey (Saridaki-Papakonstadinou et al., 2006;Ortelli et al., 2004); other antibiotics such as erythromycin, lincomycin, monensin, streptomycin and enrofloxacin have also been detected in honey (Imad et al., 2013).Antibiotic residues have a relatively long half-life and may have direct toxic eff ects on consumers and also result in drug resistant organisms.The microbes found in honey and honeycomb are bacteria, molds and yeast; they come from the bees, nectar or from external sources.Honey composition has an effect on the growth and survival of many species of microorganisms by bacteriostatic or bactericidal, based on this, it is expected that honey should contain small number and limited variety of microorganisms (Snowdon and Oliver, 1996;Snowdon, 1999;Olaitan et al., 2007).Studies carried out has shown that the intestine of bees contains 1% yeast, 27% Gram-positive bacteria (Bacillus, Bacteridium, Streptococcus and Clostridium spp.) and 70% Gramnegative bacteria (Achromobacter, Citrobacter, Enterobacter, Erwinia, Escherichia coli, Flavobacterium, Klebsiella, Proteus and Pseudomonas).
Few pathogens have been found in honey (Snowdon andOliver, 1996, Snowdon, 1999;Evans and Armstrong, 2006).Bacillus spp.are the most predominant microorganism in honey; they possess spores and have the ability to withstand the characteristic nature of honey (Nevas et al., 2006).A study carried out by Esawy et al. (2011Esawy et al. ( , 2012) ) reported a novel Bacillus subtilis in honey isolate as a new source of very important enzymes such as levansucrase, dextranase and lipase.Bacillus strain Mori 2 has been isolated from honey.It has the ability to synthesize surfactin and also has anti-Paenibacillus larvae activity.B. subtilis Subsp.Subtilis Mori 2 was isolated from honey sample in Morillos Argentina (Sabate et al., 2012).The present study aimed to dentify the predominant Bacilli spp. in branded and unbranded honey samples from Nigeria and their ability to produce components that can inhibit pathogenic organisms.The study also investigated the presence of antibiotics residue in the selected honey samples in order to advice if the components can be used in bee farming rather than antibiotics because honey is consumed.

Honey samples
Eighteen honey samples obtained from Nigeria were used for the study.Seven of them were branded honey obtained from supermarket in Rivers State, Nigeria and the remaining 11 were unbranded which were obtained from the local retailers at various locations in Nigeria.

Enumeration and identification of Bacilli spp.
Ten grams of each honey sample was weighed and diluted in 90 ml of sterile saline water.Two (2) milliliters of diluted honey samples were transferred in duplicates into sterile test tubes.One set of the tubes was heated in water bath set at 80°C for 15 min for Bacilli count.The second set of tubes was not heated for total count.Tenfold dilutions was carried out and 1 of 10 2 -10 6 dilutions were prepared and used for pour plate on Nutrient Agar (HiMedia).Plates were incubated aerobically at 37°C for 24 to 48 h.Morphological characteristics such as the shape, colour and elevation were examined.The number of colony forming unit (cfu/g) was recorded by counting the number of colonies on each plate ranging from 30 to 300.Representative dorminant colonies were isolated and purified by streaking several times on Nutrient Agar.*Corresponding author.E-mail: obakpororo.agbagwa@uniport.edu.ngAuthor(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Stock cultures were maintained in slants for further study, isolates were identified using the Bergey's Manual.

Taxonomic identity
Gram staining of 24 h grown culture (16 ± 2 h at 37°C) of Bacillus isolates was carried using standard procedure (Cappuccino and Sherman, 2002).Prepared cultures were examined by phase contrast microscopy (DP50 digital camera, Olympus Optical Co. Ltd., Tokyo, Japan).Scanning electron microscopy (SEM) was carried to further identify the cell morphology and isolates purity.Freshly grown isolates were harvested and fixed by 0.3% glutaldehyde, dehydrated and embedded using the method described by McDougall et al. (1994).Motility was also carried out using semi-solid agar medium in a test tune.The isolates were inoculated with a straight wire making a single stab down the center of the tube to about half the depth of the medium and incubated at 37°C.
Catalase test was carried out using a loopful of overnight grown culture.Cultures were smeared on a clean glass slide and 3% H2O2 was added and allowed to react.The presence of effervescence was recorded as catalase positive (Vanderzant ,1992).Starch hydrolysis was carried out by streaking overnight culture on starch agar plates (1% starch in NA) and incubated at 37°C for 24 to 48 h.After incubation, plates were flooded with iodine solution for 15 to 3 min examined for the formation of clear zone.
Gelatin hydrolysis, lecithin test, casein hydrolysis and hemolytic effect were carried out using the the methods of Sorokulova et al. (2008).Voges-Proskauer (VP) test on Bacillus isolates was carried out using the methods of Cappuccino and Sherman (2002).
Physiological tests on the Bacillus isolates were carried out on LB broth with different pH (2.0, 4.0, 6.5, 8.0 and 10.0) which was prepared by adjusting the medium by using 0.1 N NaOH/HCl (Cappuccino and Sherman, 2002).After sterilization by autoclaving, the aliquots of sterile LB broth (5 ml) with different pH values were inoculated with 50 µL (10 5 to 10 6 CFU/ml) of freshly grown (16 ± 2 h) Bacillus isolates, this was ensured by using Mc Farland Standard.The aliquots were then incubated at 37°C for 24 to 48 h and observed for growth which was indicated by turbidity change.
Growth at different temperature was carried out, LB broth (5 ml) were inoculated with 50 µL (10 5 to 10 6 CFU/ml) of freshly grown Bacillus isolates and incubated at different temperatures (4, 15, 25, 35, 45 50 and 55°C) for 24 to 48 h.After incubation, the tubes were observed for growth by turbidity change.Growth at different salt concentrations was carried out on LB broth tubes prepared with different salt (NaCl) concentrations (4, 5, 6.5 and 8%) were inoculated with 50 μL (10 5 to 10 6 CFU/ml) of freshly grown Bacillus isolates and incubated at 37°C for 24 to 48 h.After incubation, growth observation was made based on the turbidity change using Mc Farland standard.

Enzyme and antibiotics assay
Alpha-amylase activity was assayed by the dinitrosalicylic acid (DNS) procedure described by Bernfeld (1955) using 1% soluble starch.One milliliter of the enzyme was incubated for 15 min at 55°C with 1 mL of 1% soluble starch in 0.1 M phosphate buffer.The reaction was stopped by addition of 1 mL DNS and heated in boiling water for 10 min and allowed to cool in running tap for 5 min.Absorbance was read at 540 nm.One unit of α-amylase activity was defined as the amount of enzyme required to produce 1 µmole glucose equivalent per minute under the assay conditions.

Antibacterial activity and detection of mode of action (MOA)
The inhibitory activity of the Bacillus isolates was determined using well diffusion method of 18 -24 h neutralized cell-free supernatant on indicator strain Micrococcus luteus ATCC 9341 (Devi and Halami, 2011).Cell wall stress response reporter strain B. subtilis BSF 2470 for lipid II interfering antibiotics was used in this study.Chromogenic plate assay as described by Burkard et al. (2007) and Mascher et al. (2008) was followed for detecting MOA of the Bacillus isolates.A vertical streak of reporter strain was made on LB agar plates supplemented with X-Gal, and then the Bacillus isolates were streaked horizontal to the vertical line.The plates were incubated for 24 to 36 h at 37°C and blue coloration of the reporter strain was observed at the inter-junction, which indicates the inhibitory MOA of the tested culture on cell wall.The culture, B. subtilis ATCC 6633 was used as a positive control and a negative control was also used.

Estimation of antibiotics by HPLC
Honey samples were extracted by deproteinizing chemical procedure using acetonitrile (ACN).Two grams (2 g) of honey sample was placed into 10 mL test tube and shaken intensively with 3 mL ACN for 1 min.The mixture was centrifuged for 15 min at 5000 rpm.Supernatant was collected and dried under nitrogen gas.The residue was re-dissolved in 3 mL of methanol, filtered through 0.45 µm and 10 µl was injected into HPLC system.The determination of antibiotic residue in honey samples was carried out according to the method of Shafqat et al. ( 2012), Ashad et al. (2012), Imdad et al. (2013).HPLC (Schimad Zu Japan) was used to detect the presence of tetracycline, streptomycin and erythromycin in the samples.Antibiotics were obtained from Hi-Media (India).Separation was achieved using Column oven L-2300 and column Intersil ODS -3 C18 (250 x 4.6 mm).Dimension of stationary phase was 5 µ, pump range accuracy of flow was 1 ml/min and RID detector was used in the detection of antibiotic residue.All the solvents were filtered through 0.45 µm membrane filter.Antibiotics detection was performed using different mixture of an aqueous mobile phase A (60%) and acidified water and organic mobile phase B (40%), methanol and ACN with flow rate of 1 mL/min.

Enumeration of total microbial and Bacilli count
Total count and total Bacilli counts was carried out in branded and unbranded honey samples, total count for branded honey ranged from 2.2 × 10 2 to 5.5 × 10 3 cfu/g, while Bacilli count ranged from nil to 6.2 × 10 2 cfu/g.Total count for unbranded honey samples ranged from 3.5 × 10 2 to 7.0 × 10 3 , cfu/g while Bacilli count ranged from 5 × 10 1 to 1.6 × 10 3 cfu/g (Table 1).According to the Resolution MERCOSUL GMC, number 15/94 technical rules for identity fixation and honey quality approved by ordinance number 367 from September 4 th 1997, honey may contain a maximum number of 100 cfu/g.The results obtained from total and Bacilli count indicate the presence of outstanding microbial load in the unbranded honey samples which could be attributed to the unprocessed nature of the product.Bacilli count was found in most of the honey samples except for one (LP) unbranded honey.The presence of Bacillus in honey shows that they can survive in honey in dormant state; hence they are sometimes called friendly bacteria because they may not necessarily be harmful.

Taxonomic identification of Bacillus spp.
Twenty-six isolates were selected from the 18 honey samples for further identification.All the isolates selected were found to be Gram-positive rod shape.Twenty-four of the isolates were catalase positive and negative for RW3 and SJ3.Most of the isolates were motile except RW1, RW2 and EH2.Isolates were confirmed as Bacillus spp.by their heat tolerance, Gram staining, catalase reaction and other biochemical test were carried out.
Electron microscopic studies of the selected Bacillus isolates showed differences in cell morphology and cell width (Plate 1A and B).The rod shape and size also varied in the different isolates.Twenty-six Bacillus isolates were cultured in LB broth at pH 2, 4, 6.5 and 10.Most of the isolates were able to grow at pH 8 and 10, while none of the 26 isolates grew at pH 4. Isolate AD1, TW2, TC1 and TC2 did not grow in pH 2. The range of temperature growth was between 30 and 45°C, while isolates UD1, SJ2, EH2, RW1, RW2 and TC1 & 2 grew at 50°C.Most of the isolates did not grow at temperature 55°C except isolate UD1, UD2, SJ2, TW1 and TW2.

Antibacterial activity and mode of action
Identified Bacillus spp.were screened for antibacterial activity against the indicator, Micrococcus luteus ATCC 9341.Four out of 26 isolates showed inhibitory effect against Micrococcus luteus ATCC 9341 (Plate 1C and D).The four isolates were further screened to know their mode of action (MOA).The antibacterial substances produced by the different Bacillus isolated from honey samples differed in their MOA.The reporter strain that effectively detected MOA of antibacterial substances of B. subtilis BSF 2470 was used to sense the lipid 11 interfering antibiotics via RS two component response regulator system which results in change in colour (Mascher et al., 2004;Devi and Halami, 2001).When subjected to MOA, isolate SF2 is on the cell wall (Plate 1).Isolate EH1, RW2 and TW1 antibacterial activity may be due to some other molecules that the bacteria exported.Thus, Bacillus spp.from honey samples which showed antibacterial activity could be used to treat bee diseases instead of use of antibiotics.This shows that some Bacillus in honey are friendly, thus the therapeutic properties of honey.The current ban of the use of antibiotics as therapeutic agents against bee infections has led to research for natural alternatives.The components produced by these Bacillus spp.from honey and bees can be further researched on and applied in science (Alberori et al., 2016).The bioactive metabolites of these species can be characterized to determine their mechanism of action behind their antibacterial activity and therapeutic characteristics.A study carried out by Olofsson et al. (2014) showed that LAB symbionts are source of unknown factors that contribute to honey properties and they were able to inhibit wound pathogens such as MRSA and VRE among others.Their study confirmed that honey contained myraid active compounds that can be applied against human infections due to resistant microorganisms.

Enzyme assay
Results obtained from starch hydrolysis showed that 90% of the isolates could hydrolyze starch except SJ1, 2, 3 and PR1 & PR2.Hemolytic activity was seen in most of the isolates which suggests that they are pathogenic strains except for isolate UD2, SF 1, 2, 3, TW2 and RF1.Isolates UD1, 2, 3, OG1, 2, SF1, 2, 3 and RC1 were unable to hydrolyze phospholipids on egg yolk plates, thus they can be considered nonpathogenic.The isolates were identifined using the methods Bergy's Manual of Systematic Bacteriology (Sneath et al., 1959).The isolates from honey samples were tentatively identified as B. licheniformis, B. subtilis, B. coagulans, B. cirulan and B. pumilis. Sherwani et al. (2013) identified B. polymyxa, B. pumilis, B. cereus and B. megaterium.Out of the 26 Bacilli isolates, 15 were able to produce alpha amylase enzyme.One unit (U/mL) of α-amylase is defined as the amount of protein (α-amylase) required to liberate 1 µ mole (0.27 mg equivalence) of reducing sugar (Dglucose) from starch/min under the assay condition.Amylase ranged from 1.803 to 4.104 µ/mole (Table 2).The production of amylase by Bacillus strains from honey samples shows that Bacillus from honey could be new sources of very important enzymes that could be used in the food industry.Recent studies confirmed that Bacillus spp.from honey can be used for production of important enzymes such as levansucrase and other important enzymes (Esawy et al., 2011(Esawy et al., , 2012;;Safty, 2011).

Antibiotics detection
The branded and unbranded honey samples were evaluated for the presence of tetracycline, gentamycin and streptomycin.The detection of tetracycline in the honey samples was found to be among the highest and most frequent in occurrence than all the other antibiotics tested as shown in Table 1.This could be attributed to   (2013) where gentamycin was not detected in all the honey samples tested.The presence of antibiotics in honey has become alarming due to the broad use of antibiotics for the treatment of bee diseases.There is ban currently in some countires on the use of antibiotics to treat bee infections in honey production; the presence of these residues in honey after being consumed for a long time can cause health challenge.It is necessary to check the importation of honey into Nigeria.The US, some years ago, banned the importation of honey from China and Asian countries into US due to the presence of illegal animal antibiotics, sweetners used; they were mislabeled, and heavy metals were found in those honey.Animal antibiotics found in honey such as chloranphenicol can cause cacinogenicity, serious fetal reaction, aplasic anemia and other health related issues (Shafqat et al., 2012).

Conclusion
Bacillus spp.are prevalent in honey samples due to the microbial flora of the honey bee themselves, pollen or external sources.This study revealed that certain Bacillus spp.from honey contains certain antimicrobial components that can be used to inhibit the growth of pathogenic organisms such as Micrococcus letus.Further studies can be carried out on this aspect of the work to determine what these components are.The Bacillus spp.also exhibited the production of enzymes so they can be novel sources of bioactive compounds that could be used in apiculture.However, the presence of antibiotics residue in honey beyond the maximum residual limit is of great concern since that could pose serious health challange and also increase drug resitantance.Honey imported into the country should be properly screened to know the source and its quality.It is advisable that the stipulated guidelines by WHO and individual countries for honey production and antibiotics usage in beekeeping should be adhered strictly.Honey should be produced

Table 1 .
Total mesophilic bacterial count, total Bacilli count and antibiotics detected in honey samples.

Table 2 .
Taxonomic identity of Bacilli isolate and alpha amylase production.