PR2 RRL Outline (1)



Impacts of E. Coli

Effect on Society

Effect on the Environment

Effect on Human Health

Antibacterial Properties of Honey

Antibacterial Properties of Oregano

Extraction Method of Oregano and Honey



Synergistic Effect of Certain Substances

Antibacterial Assay

Chapter 2

Review of Related Literature

Impacts of E. Coli

Escherichia coli, also known as E. coli, is a common bacterium found in our environment, our food sources, and our water source as well. The commonness of an issue may often be attributed to triviality; however, E.

coli is not to be taken lightly. The bacteria can cause a variety of diseases including pneumonia, neonatal meningitis, and urinary tract infection (Madappa & Go, 2019).

E. coli are microorganisms found within the environment, foods, and intestines of individuals and animals. E. coli are a large and various cluster of microorganisms. Though most strains of E. coli are harmless, others will make you sick. Some types of E.

coli will cause symptoms, whereas others cause tract infections, respiratory disease, and different diseases. (CDC, 2019)

Antibacterial Properties of Honey

Honey contributes to wound healing and acceleration and management of wound infection. Honey can inhibit each gram positive as well as gram negative bacterium. Chemical and physical properties of honey contribute to the impact of healing, that is considered a special tonic against dangerous clinical microorganisms. Basically, honey could be a mixture containing invert sugars, organic acids, minerals, aromatic acids and waxes etc. while enzymes and hormones are a number of unstable compounds found in honey.

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(Khalil et al., 2013)

Honey has been used for treatment of various diseases ranging from 2000 years in the past. Pure honey has shown bactericidal activity for several infectious organisms. The utilization of honey as a drugs has continued into the present day medicine. Most of antimicrobial studies against infective bacterium were investigated on honey created by the honeybee. However, the scientific investigation worked on the honey made by stingless bee (Trigona iridipennis) is simply restricted on propolis of the honey. In Ethiopia, honey created by T. iridipennis is nationally used for ancient treatment, like metastasis ailments, surface infections and different diseases in line with treatments conducted with the use of honeybee honey. T. Iridipennis honey is also effective to treat completely different infections at low concentrations (Andualem, 2013).

Antibacterial Properties of Oregano

Oregano, one type of liplike herb plant that has been well-known for an extended time as a well-liked remedy, it may be a terribly versatile plant. It had been recorded that herb compactum, herb minutiflorum, and sweet marjoram exhibit antifungal activity, medicinal drug activity, and antimicrobial activity. Therefore, its potential therapeutic roles like diaphoretic, carminative, spasmolytic, antiseptic, and tonic properties are recognized.

In China, oregano has also been widely used as a feed additive. Its versatile antibacterial properties, and rapid effectiveness have made it a favorable choice among other feed additives. Now, oregano is being researched in vitro and in vivo around the world; however, there is still a lack of information on oregano essential oil. Its antibacterial mechanism is said to have come from its principal components— thymol and carvacrol (Si et al., 2008).

The herb is usually found and distributed in the Mediterranean and Asia. Its whole grass can extract aromatic oil that is known as oregano essential oil (OEO). As previously mentioned, the main components of the essential oil are carvacrol and thymol. They are known for their strong biological activities that range from anti-inflammatory to antibacterial properties. Cattelan et al. (2013) found that OEO can inhibit the growth of a variety of bacteria due to its broad spectrum of antibacterial properties. As cited by Nostro et al. (2007), another study by Lambert et al. (2001) showed that OEO increased the permeability of S. aureus and P. aeruginosa cell membranes, hence, making it weaker. Therefore, OEO can be used as an effective natural antibacterial agent. (Cui et al., 2019)

Extraction Method of Oregano and Honey


Honey and oregano are different substances that call for different extraction methods. There are various options to choose from when it comes to the extraction methods for oregano or honey. In the research conducted by Mandal et al. (2010), honey was prepared and purified through the use of a sterile cheesecloth. They then autoclaved the honey at 121°C for 15 minutes. After they streaked the honey on blood agar and nutrient plates, it was incubated at 35°C for 24 hours to check for its microbial purity. Finally, it was stored at 4°C.

Oregano, however, requires a much more elaborate extraction method since essential oils are relatively more difficult to obtain. Based on the research entitled, “Antibacterial effect of oregano essential oil alone and in combination with antibiotics against extended-spectrum ?-lactamase-producing Escherichia coli” (2008), the oregano essential oil was acquired through 2 hours of hydrodistillation using a modified clevenger type apparatus. This process involves the plant material being doused in water and subjected to heat. This causes the sacs that hold the plant essential oil to rupture. Once the oil gathers with the water, it will be ready for easy acquisition (Dilworth et al., 2017).



Based on the research Mandal et al. (2010) conducted, they used 12 sterile culture tubes with Molten nutrient agar (20 mL in each) and autoclaved for 15 minutes at 121°C. With the agar, the tubes were held in a water bath with 55°C temperature to add different concentrations of honey at 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700 and 800 µL, which is also equivalent to 0.25%, 0.50%, 0.75%, 1.00%, 1.25%, 1.50 %, 1.75%, 2.00 2.50% 3.00% 3.50% 4.00% (v/v), respectively.

For the experiment that Rezvani, Niakan, Kamalinejad, and Hamze (2017) conducted they made 5 different mixtures of honey and cinnamon by incorporating 1%–5% w/w of cinnamon extract into 99%– 95% w/w of honey respectively. each of the given mixtures were considered as the first solution was diluted into seven serially two-fold dilutions (from 1:2 to 1:128 v/v). Eight mixtures with varying concentrations were tested. Nonetheless, it should be indicated that the honey extract was diluted into distilled water.

Andualem (2013) performed an experiment, the honey he used was acquired from Lie Armacheho, Southwest Ethiopia called the T. iridipennis honey. To remove debris the honey was aseptically sieved with a disinfected mesh. Pure distilled water was used to dilute the honey with the following concentrations;12.5, 25.0 and 50.0% (v/v).

Another experiment was conducted by Nwankwo, Osaro-Matthew, and Ekpe (2015) they diluted their honey with pure distilled water to concentrations between 25% (v/v) to 100% (v/v) (i.e 1ml of honey to 4mls of distilled water). Lime extract was also diluted to the concentrationsbetween 25% (v/v) to 100%(v/v) (i.e 1ml of lime to 4mls of distilled water). The combination of Lime and Honey was diluted at 10:50, 20:50, 30:50, 40:50, (Using sterile distilled water 40%, 30%, 20%, 10% respectively to make it up to 100% (v/v) concentrations, 50:50v/v concentration).

Ghramh, Khan, and Alshehri (2019) used two different concentrations of honey sample on their experiment. The two honey samples were prepared by making use of a pure distilled water to test the antibacterial activity. In a 50 mL beaker (Karter Scientific, USA) 10 grams of each honey was weighed in by using an electrical balance (Shimadzu Corporation, Japan) after that 16 and 10 mL of water was added to make the concentrations to be 80% and 60% (w/v) respectively. The calculations used the formula: C1 V1 = C2 V2, for the different concentrations of water and honey quantities.


According to the study of Rodriguez et al. (2017) the emulsions of OVEO was prepared in BHIBB with concentrations in the range of (80e0.312 mL/mL) using Tween 80 (1%, v/v; Sigmae Aldrich, USA) as an emulsifier.

Cui et al. (2019) also conducted research about Oregano. The OEO that they used was diluted into a NB medium containing 104?5 CFU / mL MRSA (0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL and 0.8 mg / mL). After that it was situated in a shaker for 48 hours. As the medium remained clear it shows that the value of MIC was at the lowest concentration of OEO. The bacterial suspension was streaked on a solid medium which was containing an essential oil (not less than the MIC). The lowest concentration of essential oil required to kill MRSA is called the MBC value.

Mahfouf et al. (2016) used oregano to test the antioxidant and antibacterial properties against E.coli. The radical scavenging ability of oregano essential oil was described in with minor modification when it was tested by DPPH radical scavenging assay. Four concentrations of 0.125, 0.25, 0.5, and 1 mg/ml of the essential oil were prepared in methanol based on the pre examination. The solution of DPPH was made by dissolving 2.4 mg of DPPH in 100 ml of methanol then 100 ?l of oregano essential oil was added to 2 mL of DPPH solution.

Synergistic Effect of Certain Substances

On several occasions, substances are fused or synergised to create an even more effective antibacterial. To properly evaluate these mixtures, they may be used against a wide array of bacteria. In a research by Khalil et al. (2013), the combination of honey and basil was used against 13 different pathogenic bacteria— these being Klebsiella pneumonia, Pseudomonas aeroginosa, Escherichia coli, Salmonella typhi, Salmonella typhimirium, Xanthomonas campestris, Enterococcus faecalis faecalis, Bacillus subtilis, Staphylococcus aureus, Clostridium perfringens type C, Clostridium perfringens type D, Clostridium uvoei. As stated by Khalil et al. (2013), honey has already proven its contribution to healing of wounds and prevention of infections, and basil has shown effectiveness against cerebral strokes, hypertension, diabetes, lipid disorders, alcohol intoxication, anxiety, cardio-vascular diseases, headaches, nerve pain, cough, cold, migraines, menstrual cramps, and sinusitis. They were substances with properties worth examining and hence were used to create a valuable research.

Rezvani et al. (2017), on the other hand, studied the synergistic antibacterial effect of honey and cinnamon. They used the mixture and tested it against Streptococcus mutans. The component cinnamon was chosen due to it already being highly effective against Streptococcus mutans (Rezvani et al., 2017). Along with honey, cinnamon was only expected to show an enhanced antibacterial activity.

In the field of food microbiology, Cattelan et al. (2018) was able to test a synergy of oregano essential oil and salt. They looked into its antibacterial effect on E. coli present in salad dressings. This research aimed to analyze not only the antimicrobial properties of the combination but also its interaction with various food components (Cattelan et al., 2018).

A study on the combined effects of honey and ginger has also been conducted (Omoya and Akharaiyi, 2012). Ginger was chosen mainly because of its medicinal values relating to blood flow and inflammation. It was then synergized with honey to further enhance its properties. It was tested against Escherichia coli, Salmonella typhi, Pseudomonas aeruginosa, Bacillus cereus, Staphylococcus aureus, and Klebsiella pneumoniae (Omoya and Akharaiyi, 2012).

Lime has also been fused with honey before in a research by Nwankwo et al. (2015). Lime extracts display a high antimicrobial activity; however, the antimicrobial effects of several of its active components can only be released once used in conjunction with a specific solvent (Nwankwo et al., 2015). Hence, honey was able to act as a solvent for the lime extract, generating intriguing results for the research.

Antibacterial Assay

One way of evaluating the antibacterial activity of a sample is through the agar well diffusion assay. This method involves creating a small hole or well on the surface of the agar. The substance to be tested is then put into the well. This is usually used when assessing the antimicrobial properties of plants or microbial extracts (Balouiri et al., 2016).

This process was performed by Khalil et al (2013). Constant lawns were created using a bent spreader and sterilized cotton swabs were used to remove excess substance. A 6mm sterilized borer was needed for them to make wells in the nutrient and a test solution of 50 µL was introduced to each well. The discs they used were standard Gentamycin (Sigma-Aldrich, St. Louis, MO, USA) discs (10 µg) prepared as a positive control while the negative control was the sterile dH2O. Using a Vernier caliper, the diameter of the zone of inhibition was measured in “mm”.

Another and more common way of examining antibacterial activity is through the disc diffusion method. It is the official method used in many clinical microbiology laboratories for routine antimicrobial susceptibility testing (Balouri et al. (2016).

In a research executed by Banjara et al. (2012), the disc diffusion method was used. Whatmann filter paper (no. 41) were cut out into discs with 6mm diameters. After being autoclaved, they were impregnated with 1mL of the different concentrations of the substance they used. The saturated discs were put at each petri dish with the bacterial lawns. Then, they incubated them at 37°C for 24 hours.

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CATEGORY POINTS RATING (Encircle or mark to indicate score) REMARKS

4 6 8 10

1. Relevance of the RRL components to the research topic The RRL components is not related to the research topic The RRL components is slightly related to the research topic The RRL components is somewhat related to the research topic The RRL components is significantly related to the research topic

4 6 8 10

2. Organization and structure of the RRL components to the research topic No organization, sequencing, or structure. Weakly organized with no logical sequencing or structure. Well organized, but demonstrates illogical sequencing or structure. Well organized, demonstrates logical sequencing and structure.




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Balouiri M, Sadiki M, Ibnsouda SK. 2016. Methods for in vitro evaluating antimicrobial activity: A review. Journal of Pharmaceutical Analysis. 6:71–79.

Banjara RA, Jadhav SK, Bhoite SA. 2012. Antibacterial activity of di-2-ethylaniline phosphate screened by paper disc diffusion method. Journal of Applied Pharmaceutical Science. 2:230–233.

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PR2 RRL Outline (1). (2019, Dec 04). Retrieved from

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