Cover letter

From: Aneela MehboobDepartment of Microbiology University of Karachi,Karachi Pakistan,Dear Editor, I would like to submit my research paper on “Synergistic Effect of the Combination of Commercial Essential Oils with Standard Antibiotics: In Vitro Evaluation” to your prestigious World Journal of Microbiology and Biotechnology. This research paper is about the antibacterial potential of commercial essential oils against food borne bacteria isolated from famous street vended foods in Karachi city. The interpretations are up to the mark and these commercially purchased oils produced remarkable antibacterial activity alone and in combinations against tested microorganisms. This study helps in future for the development of new antimicrobial agents to treat bacterial infections using medicinal plants. Communication about the paper please make an effort to remain coordinated to Miss Aneela Mehboob, at the accompanying location and email address： Aneela Mehboob, Department of Microbiology, University of Karachi, Karachi Pakistan E-mail: aneelamehboob21@outlook.com ORCID# org/0000-0002-9529-5037I uphold that this investigation is unique, has not been distributed before and is not right now being considered for production somewhere else. Much thanks for your regard for my paper. Yours earnestly, Aneela Mehboob

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Synergistic Effect of the Combination of Commercial Essential Oils with Standard

Antibiotics: In Vitro Evaluation

Aneela Mehboob1,* and Tanveer Abbas1

Antimicrobial effects of essential oil on tested food isolates

1Department of Microbiology, University of Karachi, Main University Road, Karachi, 75270

Sindh, Pakistan

*Corresponding author: Aneela Mehboob; E-mail: aneelamehboob21@outlook.com

*ORCID IDs: 0000-0002-9529-5037 (Mehboob); mobile no: 03432575437

ORCID ID: 0000-0002-1784-3077 (Abbas)

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ABSTRACT

The work was done on five commercial essential oils (EOs) of almond (Prunus dulcis), black seed (Nigella

sativa), lavender (Lavandula), lemon (Citrus Limon) and green cardamom (Elettaria cardamonum) to determine

the antibacterial activity against E. coli, S. fonticola, S. liquefaciens, C. freundii, and S. aureus isolated from

streets foods vended in Karachi. For the evaluation of antibacterial traits possessed by these essential oils, drop

agar diffusion method, determination of minimal inhibitory concentrations via agar well diffusion and macro

broth dilution method, and to check the combined effects of essential oils and standard antibiotics, disc diffusion

methods were performed. Among five of them, lemon and lavender come up with the most potent essential oils

showing the highest antibacterial activity in its neat form ranges between (9.93 ± 0.12 - 19 ± 0 mm) and (9.9 ±

0.17 - 14.83 ± 0.28 mm) zone of inhibition respectively with the exception of S. aureus. The combination of

essential oils of lemon and lavender at different concentrations possessed the highest antibacterial activity

against S. fonticola and S. aureus. Combinations of essential oils and antibiotics provided synergistic outcomes

against all the tested five bacterial strains, however antagonistic results were also found. This exploration

underpins the application of essential oils alone and in combination with antimicrobial agents to enhance the

drug sensitivity and helps in the development of new antimicrobial agents to treat bacterial infections using

medicinal plants. 

Keywords: C. limon; E.cardamonum; Lavandula; N.sativa; Synergies; Food isolates.

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INTRODUCTION

Food-related diseases are becoming one of the most hazardous worldwide problems. It is estimated that around

30 % of food poisoning cases are reported each year (Canini et al., 2013) along with 70 % of potential outbreaks

risks are associated with street foods consumption. Pakistan comes under developing countries and problems of

underdeveloped countries are way more different and difficult as compared to industrialized countries. In

context with developing countries, where poverty is the basic issue, an enormous amount of readymade foods

are processed, made and consumed by buyers at a cheaper rate (Faruque et al., 2010). Foodborne diseases are

mostly related to microorganisms such as viruses, bacteria, and parasites. Microbes are the main root of

foodborne illness and their chemicals released in the form of endo and exotoxins. Cases of hospitalization

regarding food poisoning involve bacterial diarrhea, dysentery, while viruses play a little role in it that can lead

to serious life-threatening condition (Teplitski et al., 2009).

Oils that are recovered from the plant source and can be expended orally are called palatable oils. These edible

oils have magnificent qualities. In all likelihood, utilized in lubricating factories, food, pharmaceuticals, and

therapeutic endeavors. Fundamentally, extrinsic and intrinsic components decide the physical and chemical

properties of essential oils (Alibe and Inuwa, 2012). Besides, these essential oils possess tremendous antioxidant

and insecticidal properties and play an extraordinary part in battling microorganisms (Silva et al., 2010)

Therefore, having these antimicrobial properties, essential oils are comprehensively utilized as a part of the

medicinal world and sustenance ventures as the natural remedy and preservation purpose respectively (Bakkali

et al., 2008). Compounds that are actually present in essential oils obtained from plants, spices, and herbs are the

primary constituents that can combat against microorganisms (Janssen et al., 1985; Deans and Ritchie, 1987;

Kim et al., 1995). In the ancient era, treatment of illness was done by natural herbal therapy. Today essential oils

are best known for raising the taste of foods, aroma and used in many cosmetic products for the betterment of

skin. Studies have been shown that essential oils have inborne antimicrobial traits and nowadays, natural

therapeutic remedies are more preferable over synthetic ones due to careless consumption of drugs (Di Pasqua et

al., 2005).

Due to immense resistance created by microbes through various mutational process against antimicrobial agents

causes a pressure on researcher to find out new techniques, remodel old ones or to make hybrid of new and old

methodology by using essential oils and plant extracts having remedial properties alone and in the form of

blends for the treatment of diseases (Rios and Recio, 2005; Fisher and Phillips, 2009). Therefore, the objective

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of this research was the assessment of the antimicrobial significance of essential oils against bacterial strains

recovered from the street foods of Karachi, Pakistan.

MATERIALS AND METHODS

Essential oils

Five essential oil samples of almond (Prunus dulcis), black seed (Nigella sativa), green cardamom (Elettaria

cardamonum), lavender (Lavandula), lemon and (Citrus Limon) were used in this study to evaluate their

antimicrobial potentials. Essential oils of almond and black seed were commercially purchased from local

market while essential oils of green cardamom, lavender and lemon were kindly supplied by Noor Oil depot,

Karachi, Pakistan.

Tested microorganisms

The antibacterial activity of these commercially purchased EOs was assessed against five foodborne bacteria

that recuperated from the famous local street food shops of Karachi. All these five bacterial strains including

gram-negative Escherichia coli, Citrobacter freundii, Serratia fonticola, Serratia liquefaciens, and gram-

positive Staphylococcus aureus were isolated and identified according to Bergey’s manual of determinative

biology (Holt et al., 1994) and ABIS online software (Costin and Ionut, 2017).

ANTIMICROBIAL SCREENING OF ESSENTIAL OILS

Drop agar diffusion method

Drop agar spread technique was executed to evaluate the antibacterial potentials of undiluted EOs (Hammer et

al., 1999; Hili, 2001; Cruz et al., 2007; Lopes et al., 2008). The Mueller Hinton agar plates were formerly

inoculated with test organisms with 0.5 McFarland concentration, 10 µL drop of essential oils was placed on

MHA plates and left untouched for proper dispersion at ambient temperature. After a while, plates were

incubated at 37 ± 1 °C for 24 hours. Zone of inhibition around each drop was measured in millimeters.

Agar well diffusion method

Agar well method was performed to examine the antimicrobial activity of EOs (Martins et al., 2013). By the

help of sterile swab, lawn of bacterial suspension (0.5 McFarland) was made on 90 mm Mueller Hinton agar

plates then 6 mm wells were cut and loaded with 70 µL of EOs diluted in 40 % DMSO. Well of negative

controls were also run simultaneously with 40 % DMSO (no antibacterial activity observed). Plates were then

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settled down at ambient temperature for few hours permitting the total dispersion of EOs (Das et al., 2010) and

finally incubated overnight at 37 ± 1 °C. Very next day zone of inhibition around every well was computed in

millimeters (mm). Similarly above method was repeated to analyze the combined effects of essential oils at

pure, 1000 µg/mL and 500 µg/mL concentrations.

Minimal inhibitory concentration (MIC)

To determine the antibacterial potentials of EOs, macro broth dilution method was performed to find out

minimal inhibitory concentrations (Weerakkody et al., 2010) along with some modification. Different

concentrations of EOs (1000, 500, 250, 125, 62.5, 15.625 µL) were prepared in 40 % DMSO. 1000 µL aliquots

of sterile Mueller Hinton broth dispensed in Eppendorf tubes containing various concentrations of EOs

supplemented with 10 µl bacterial suspension (0.5 McFarland concentration). Positive and negative controls run

side by side containing Mueller Hinton broth and bacterial suspension and Mueller Hinton broth with essential

oil respectively. After overnight incubation bacterial growth was observed by the presence and absence of

turbidity (Han et al., 2008).

Antibiotics

The standard antibiotics used were tetracycline (30 µg), chloramphenicol (30 µg), gentamicin (10 µg),

amoxicillin (10 µg), levofloxacin (5 µg), streptomycin (10 µg), and oxacillin (1 µg). These antibiotics were

purchased from Thermo Fischer Scientific Oxoid ltd.

Determination of the synergistic effect of antibiotics and essential oils on tested organisms

To assess the consolidated impact of both EOs and standard antimicrobial agents on tested organisms, 10 µL

drop of undiluted EOs were soaked on promptly accessible antibiotics disc, then plates were permitted to dry for

proper diffusion, incubated at 37 ± 1 °C for 24 hours to find out the zone of clearance around each circle. The

gained results were differentiated and tabulated between antibiotics + EOs and antibiotics alone by similar

strategy as disk diffusion method (Toroglu, 2007).

Scanning Electron Microscopy (SEM)

SEM was performed by JEOL from Japan (model# JSM-6380A). The samples were coated up to 300 Aº with

gold. The scanning electron microscope is placed at Centralized Science Laboratories, University of Karachi.

Samples were prepared by suspending 18 hours old cultures in saline and transferred into the microcentrifuge

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tubes containing Mueller Hinton Broth and MIC value of essential oils. After overnight incubation, 5-10 µL

crystal violet was added in microcentrifuge tubes for one minute. 3x washing was done with 70 %, 80 % and 90

% ethanol at 11,000 rpm for 10 minutes. After each successive washing, samples were discarded and air dried

for complete removal of ethanol.

Statistical analysis

All experimental analyses were done in triplicates and interpretations were presented as Mean ± SD.

RESULTS

Out of five essential oils, two essential oils namely C. limon and Lavandula showed antibacterial activity against

tested cultures (Table 1). In case of C.limon, minimal inhibitory concentration was 1000 µg/mL for E. coli and

for S. aureus, S. liquefaciens, C. freundii and S. fonticola, the MIC was 500 µg/mL. The MIC of EO of

Lavandula for S. aureus, S. liquefaciens and C.freundii was at 250 µg/mL and for E.coli and S. fonticola, the

MIC were1000 µg/mL and 500 µg/mL respectively (Fig. 1a). While P. dulcis, N. sativa, and, E. cardamonum

failed to give effective results. Antimicrobial activity was also evaluated by macro broth dilution method. All

the commercial essential oils in broth system produced competent antibacterial activity against all tested

organisms (Fig. 1b), MIC of E. cardamonum was 62.5 µg/mL against S. aureus, C. freundii, E. coli, and S.

fonticola, The MIC of P. dulcis was 62.5 µg/mL for S. aureus, C. freundii and, S. fonticola. For S. liquefaciens

and E. coli, the MIC were 250 µg/mL and 1000 µg/mL respectively. EO of N. sativa have 1000 µg/mL MIC for

C. freundii, 125 µg/ mL for S. fonticola and 62.5 µg/mL for E. coli, S. aureus and S. liquefaciens. EO of

Lavandula have 250 µg/mL MIC for E. coli, for S. aureus 125 µg/mL, and 1000 µg/mL for S. fonticola, S.

liquefaciens and C. freundii. In case of EO of C. limon, the MIC was 250 µg/mL for E. coli and S. aureus, 125

µg/mL for S. liquefaciens and 1000 µg/mL for C. freundii and S. fonticola.

Citrus.limon

EO of C. limon exhibited significant antibacterial activity against tested food isolates i.e. E. coli, S. liquefaciens,

C. freundii, and S. fonticola with the zone of inhibition between (10 ± 0 mm) and (19 ± 0mm) while no activity

was observed against S. aureus. As indicated by the acquired outcomes, the combination of essential oil of

lemon with all the tested antibiotics demonstrated antagonistic effects against S. liquefaciens and C. freundii.

The vice versa conditions were observed in the case of S. aureus, above mixes gave synergistic outcomes.

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Combination of EO of C. limon with amoxicillin showed synergistic effects against E. coli and S. fonticola

while with remaining antibiotics gave inhibitory results (Table 2).

Nigella sativa

EO of N. sativa in its neat form showed no activity against all tested microorganisms. The blending of EO of

black seed with levofloxacin, tetracycline, and gentamicin indicated inhibitory effect against E. coli while

synergistic effects were likewise seen by the mixing of essential oil of N. sativa with chloramphenicol and

amoxicillin, however, indifference was also observed with black seed + streptomycin against E. coli (Table 3).

Antagonistic activities were observed with the utilization of each of the seven antibiotics and essential oil of N.

sativa against S. fonticola and S. aureus. The application of streptomycin, levofloxacin, gentamicin, and

tetracycline with essential oil of black seed led to synergistic effects against C. freundii while inhibitory results

were observed with oxacillin and amoxicillin. Then again, the combination of levofloxacin, chloramphenicol,

tetracycline, and streptomycin with essential oil of N. sativa acted antagonistically but with the mix of N. sativa

with gentamicin, a synergistic effect was distinguished.

Lavandula

The pure form of EO of Lavandula was very effective against tested bacteria giving the zone of inhibition

between (9.9 ± 0.17 mm) and (14.83 ± 0.28 mm). While EO of lavender failed to inhibit the growth of S. aureus

(Table 4). The join up of EO of lavender with every tried antibiotic gave inhibitory effects against S. aureus, C.

freundii, S. fonticola, and S. liquefaciens. Whereas against E. coli, the synergistic impact was seen with

amoxicillin, indifference effects were spotted with tetracycline, and levofloxacin and antagonistic results were

obtained with the combination of oxacillin, chloramphenicol, gentamicin and streptomycin.

Elettaria cardamonum

EO of Elettaria cardamonum in its neat form possessed antibacterial characteristics against C. freundii as

(11.33 ± 0.5 mm) inhibition zone whereas no antibacterial potential was detected against rest of the four tested

organisms (Table 5). Mixing of EO of E. cardamonum with streptomycin, tetracycline, chloramphenicol, and

levofloxacin gave antagonistic effects against E. coli. While the gentamicin and amoxicillin application with EO

of E. cardamonum prompted to synergistic effects against E. coli. It can be seen that the saturation of the EO of

E. cardamonum with streptomycin, tetracycline, gentamicin, amoxicillin, and oxacillin gave synergistic

outcomes against S. aureus and inhibitory effects acquired with levofloxacin and chloramphenicol. Antagonistic

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effects were observed against C. freundii and S. fonticola by the fusion of all tested antibiotics with the EO of E.

cardamonum. Only one combination that was gentamicin + EO of cardamom led to the synergistic effect against

S. liquefaciens.

Prunus dulcis

Prunus dulcis in its undiluted form showed no antibacterial activity against any of the five tested bacteria.

Synergistic impacts were demonstrated with the mix of streptomycin, gentamicin, amoxicillin, and levofloxacin

with EO of Prunus dulcis whereas inhibitory effect was observed with tetracycline, and chloramphenicol,

however, no result was acquired with amoxicillin against E.coli (Table 6). While indicating inhibitory activities

with the application of tetracycline, gentamicin, chloramphenicol, streptomycin, and levofloxacin. However, no

effects were found with oxacillin and amoxicillin against S. fonticola. Phenomena of synergism were found

against S. liquefaciens when the blend of EO of Prunus dulcis and gentamicin was applied. But with the

levofloxacin, chloramphenicol, tetracycline, and streptomycin opposing results were obtained. While

indifference effect with the fusion of P. dulcis and oxacillin and amoxicillin was seen as well. Against S.

aureus, the fruitful combination of almond oil with tetracycline, oxacillin, and streptomycin and, levofloxacin

were achieved by giving synergistic results while the rest of the three tested antibiotics gave antagonistic

interpretations. The blending of EO of almond with levofloxacin, and tetracycline gave indifferent results.

Accompanying with streptomycin, gentamicin, and chloramphenicol provided synergistic effects against C.

freundii. No effect on the growth of C. freundii was observed with the application of oxacillin and amoxicillin

with Prunus dulcis.

Combination of essential oils of lemon and lavender (LLC)

EOs of lemon and lavender gave proficient outcomes in their unadulterated form against tested bacteria with the

exception of S. aureus demonstrated by drop agar diffusion technique. These two EOs were chosen to check the

combined impact by blending them at effective concentrations obtained by agar well diffusion method. The

combination of lemon and lavender EOs at an undiluted form, 500 µg/mL, and 1000 µg/mL displayed

noteworthy synergism against S. fonticola with (40.4 ± 0.50 mm), (35.06 ± 0.11 mm), and (30.33 ± 0.57 mm)

zone of inhibition respectively. While the additive result was seen against E. coli (24.8 ± 0.28 mm) and S.

liquefaciens (15 ± 0 mm) at 500 µg/mL and no effects were observed against C. freundii at all the tried

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concentrations. Though in the case of S. aureus, the synergistic effect was spotted as (15 ± 0 mm) zone of

inhibition at all the tested mix. These EOs alone failed to repress the development of S. aureus.

Scanning electron microscopy

Scanning electron microscopy images revealed the alteration in cellular morphology of bacterial culture after

treated with essential oils at the different MIC as shown in (Fig 2). Image (a) and (c) were the untreated control

image of E. coli and S. aureus respectively. Image (b) and (d) showing the tested bacterial culture image treated

with Eos responsible for bulging of cells, disruption of the cell wall and the arrangement of bacterial culture. It

was reported in several studies that constituents of EOs are partitioned into two broad range groups of terpenes,

terpenoids, flavonoids, aliphatic compounds and some hydrocarbon as well (Pichersky et al., 2006) that

supposed to harm and disturb the cell wall and membrane of microorganisms by their mechanism of action

(Faleiro, 2011). Therefore, according to the obtained results, it can be hypothesized that the mode of action of

tested Eos was the cell wall and membrane of bacteria.

DISCUSSION

Secondary metabolites or byproducts produced by microorganisms and medicinal plants act as an antimicrobial

agent against the extended range of microorganisms and these natural products are in the form of EOs for more

than 50 years serve as a blessing for us to increase the lifespan, used in food preservation, remodel the

conventional drugs. These herbs, spices and medicinal plants safe their selves from bugs and microbes by

producing essential oils (Demain, 2009). Previous studies support the effectiveness of EOs and their biological

composites containing bactericidal, fungicidal, antioxidant, antiviral and insecticidal properties (Randhawa and

Al-Ghamdi, 2002).

The EOs used in this research possessed antibacterial activity as E. cardamomum proved itself as a competent

candidate to battle against different diseases such as gastrointestinal disturbances and known to be utilized in

cooking to augment the deliciousness of food (Evans, 2002). Similarly, C. limon and N. sativa are heavily

loaded with bioactive compounds that are ever ready to combat against a wide range of bacteria (Olila and

Opuda, 2001; Prabuseenivasan et al., 2006). While the potentials of lavender have not been hidden as it is

known to refresh mind and have healing properties. Essential oil of Lavandula reportedly used in combination

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with multiple oils and produced synergistic effects against microorganisms (Lawless, 1995; Shealy, 1998; de

Rapper et al., 2013; Buckle, 2014).

All the five commercial EOs possessed antibacterial activity against food isolates recovered from local street

foods in Karachi. The efficiency of these EOs against tested bacteria was seen at different concentrations and

media. Some EOs proved their bactericidal potentials in broth system while some were active in the solid

medium. Agar well diffusion method was conducted to determine the minimal inhibitory concentrations of these

commercial EOs that inhibit or retard the visible growth of bacteria (Delaquis et al., 2002).

Expansion of drug resistance in the bacteria belonging to the Enterobacteriaceae family are accountable for

several fatal diseases in humans as well as in animals and should require legitimate watchfulness and control

over the spreading of these gram-negative pathogens. As commensal bacteria are generally harmless for an

immunocompetent person but they can transfer the resistant gene from one bacterium to another (Paterson,

2006). Researchers mainly focus on the foodborne pathogens like Salmonella spp, E.coli, Shigella spp but these

commensal bacteria cannot be neglected. It was studied by (DeFrancesco et al., 2004) that the commensal

organisms can be utilized for the purpose to check the predominance of resistant bacteria as their investigation

revealed that the commensal E.coli have the higher rate of drug resistance than same herd of MDR Salmonella

species. This is an alarming sign to handle and ameliorate the situation before it is too difficult to treat the

disease.

CONCLUSION

Essential oils of P. dulcis, N. sativa, Lavandula, Citrus. limon, and E. cardamonum exhibited remarkable

antibacterial activity against tested food isolates in both agar and broth medium independently. The combination

of these commercial essential oils with standard antibiotics increased the antibacterial efficiency of drugs. Hence

this study is beneficial for further research and making new antimicrobial agents that have great potential to kill

bacteria.

ACKNOWLEDGMENTS

The author desires to express her gratefulness and sincere gratitude to her respected supervisor Dr. Tanveer

Abbas, for his kind cooperation and valuable advice. This research did not receive any specific grant from

funding agencies in the public, commercial, or not-for-profit sectors.

CONFLICT OF INTEREST The Authors declare no conflict of interest.

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TABLES AND FIGURES

Table 1 Antibacterial activity of essential oils of green cardamom, lemon, lavender, almond and black seed by agar well diffusion method

Tested food

isolates

Lemon Lavender Black seed,

almond, &

cardamom

Concentrations

of EOs

1000

µg/mL

500

µg/mL

250

µg/mL

125, 62.5

& 15.625

µg/mL

1000

µg/mL

500

µg/mL

250

µg/mL

125,

62.5 &

15.625

µg/mL

1000 to

15.62µg/mL

S.aureus 20 ± 0.0 20 ± 0 ND ND 19.6 ± 0.34 18.9 ± 0.17 29.9 ± 0.17 ND ND

E.coli 15.5 ± 0.5 ND ND ND 29.43 ± 0.51 ND ND ND ND

S.liquefaciens 19.8 ± 0.34 20 ± 0 ND ND 19.93 ± 0.11 19.66 ± 0.28 12.67 ± 0.57 ND ND

C.freundii 20 ± 0 19.8 ± 0.34 ND ND ND ND 14.67 ± 0.5 ND ND

S.fonticola 20 ± 0 20 ± 0 ND ND 20 ± 0 20 ± 0 10 ± 0 ND NDaND= not detected

15

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351

Table 2 Determination of the combined effects of essential oil of Lemon with standard antibiotics

Food isolates Essential

oil

Standard antibiotics

Zone of inhibition in (mm)

Standard antibiotics + Essential oil

Lemon LEV OX AML C CN TE S LEV+L OX+L AML+L C+L CN+L TE+L S+L

E.coli 14.86 ±

0.23

29.5 ±

0.7

R R 24.3 ± 0.5 17 ±

0.0

26 ±

0

11.93

±

0.11

25 ± 0A 6 ±

0.0A

19.93 ±

0.11S

25 ±

0.0A

20 ± 0.0A 24.93 ±

0.11A

15 ±

0.0A

S.fonticola 10 ± 0.0 26.5 ±

0.70

R R 24.2 ± 0.4 16.3

±

0.51

28.2

±0.40

10.5

±

0.70

9.93 ±

0.11A

6 ±

0.0A

28.96 ±

0.05S

23 ±

0.0A

14.83 ±

0.28A

22.56 ±

0.49A

6 ±

0.0A

S.liquefaciens 9.93 ±

0.12

25.6 ±

0.46

R R 21.9

±0.05

14.1

±0.28

25.8

±0.28

13.1

±

0.17

22 ±

0.0A

6.93 ±

0.11A

8.93 ±

0.11A

20 ±

0.0A

18 ± 0.0A 18 ± 0.0A 15 ±

0.0A

S.aureus - 26 ± 0.0 R R 22.06 ±

0.11

22.1

±

0.23

26.1

±0.17

17.2

±

0.34

29.8 ±

0.34S

14.8 ±

0.26S

30 ± 0S 34.5 ±

0.5S

35.73 ±

0.25S

40 ± 0.0S 24.96

±

0.05S

C.freundii 19 ± 0 22 ± 0.0 R R 22.3 ± 0.5 16 ±

0.00

23.66

±

0.57

12 ±

0.00

25 ±

0.0A

10 ±

0.0A

8.93 ±

0.11A

25 ±

0.0A

16.8 ±

0.34A

22 ± 0.0A 12 ±

0.0A

aL: Lemon, R: resistant, LEV: levofloxacin OX: oxacillin, AML: amoxicillin, C: chloramphenicol, CN: gentamicin, TE: tetracycline, S: streptomycin; A:

Antagonistic; I: Indifference; S: Synergistic; (-): no growth. Values are represented as mean ± SD

16

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Table 3 Determination of the combined effects of essential oil of black seed with standard antibiotics

Food isolates Essential

oil

Standard antibiotics

Zone of inhibition in (mm)

Standard antibiotics + essential oil

Black

seed

LEV OX AML C CN TE S LEV+V OX+V AML+V C+V CN+V TE+V S+V

E.coli - 29.5 ±

0.7

R R 24.33±

0.57

17±0 26 ±

0.0

11.93±

0.11

2 .96 ±

0.05A

- 23 ± 0.0S 30 ± 0.0S 15 ± 0.0A 21.96 ±

0.0A

11.96

±

0.05I

S.fonticola - 26.5 ±

0.70

R R 24.23±

0.40

16.3±

0.51

28.2±

0.40

10.5 ±

0.70

13±0A - - -A -A 10 ± 0.0A -A

S.liquefaciens - 25.6 ±

0.46

R R 21.9±0

.05

14.1±

0.28

25.8±

0.28

13.1 ±

0.17

20±0A - - 20 ± 0A 17.9 ± 0.17S 14.9 ±

0.17A

-A

S.aureus - 26 ± 0.0 R R 22.06±

0.11

22.1±

0.23

26.1±

0.17

17.2 ±

0.34

25±0A - - -A 18.9 ± 0.17A 14.86 ±

0.23A

10 ±

0.0A

C.freundii - 22 ± 0.0 R R 22.33±

0.57

16±0 23.66

±0.57

12 ±

0.0

30.06 ±

0.11S

- - 21.9 ±

0.17A

23 ± 0.0S 25 ± 0.0S 15.06

±0.11

S

aB: Black seed, R: resistant, LEV: levofloxacin OX: oxacillin, AML: amoxicillin, C: chloramphenicol, CN: gentamicin, TE: tetracycline, S: streptomycin; A:

Antagonistic; I: Indifference; S: Synergistic; (-): no growth. Values are represented as mean ± SD

17

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Table 4 Determination of the combined effects of essential oil of Lavender with standard antibiotics

Food isolates Essential

oil

Standard antibiotics

Zone of inhibition in (mm)

Standard antibiotics + essential oil

Lavender LEV OX AML C CN TE S LEV+V OX+V AML+V C+V CN+V TE+V S+V

E.coli 13.06

± 0.11

29.5 ±

0.7

R R 24.33

± 0.57

17 ±

0.0

26 ±

0.0

11.93

± 0.11

30 ± 0.0I -A 21.9 ±

0.17S

28 ±

0.0A

25 ± 0.0A 26.1 ±

0.23I

20.1

±

0.17A

S.fonticola 9.9 ± 0.17 26.5 ±

0.70

R R 24.23

± 0.40

16.3

±

0.51

28.2

±

0.40

10.5 ±

0.70

-A -A -A 7.9 ±

0.17A

-A -A 9 ±

0.0A

S.liquefaciens 11.67 ±

0.57

25.6 ±

0.46

R R 21.9 ±

0.05

14.1

±

0.28

25.8

±

0.28

13.1 ±

0.17

18.1 ±

0.17A

-A 5 ± 0.0A 22 ±

0.0A

22.73 ± 0.46A 18 ± 0.0A 20 ±

0.0A

S.aureus - 26 ± 0 R R 22.06

± 0.11

22.1

±

0.23

26.1

±

0.17

17.2 ±

0.34

21.9 ±

0.17A

- 10 ± 0.0A 12 ±

0.0A

17.86 ± 0.23A 15 ± 0.0A 10 ±

0.0A

C.freundii 14.83 ±

0.28

22 ± 0 R R 22.33

± 0.57

16 ±

0.0

23.66

±

0.57

12 ±

0.0

25 ± 0.0A -A 10 ± 0.0A 18 ±

0.0A

18.06 ± 0.11A 21.83 ±

0.28A

11 ±

0.0A

18

aL: Lavender, R: resistant, LEV: levofloxacin OX: oxacillin, AML: amoxicillin, C: chloramphenicol, CN: gentamicin, TE: tetracycline, S: streptomycin; A:

Antagonistic; I: Indifference; S: Synergistic; (-): no growth. Values are represented as mean ± S

19

356357

Table 5 Determination of the combined effects of essential oil of Cardamom with standard antibiotics

Food isolates Essential oil Standard antibiotics

Zone of inhibition in (mm)

Standard antibiotics + Essential oil

Cardamom LEV OX AM

L

C CN TE S LEV+C OX+C AML+C C+C CN+C TE+C S+C

E.coli - 29.5

± 0.7

R R 24.3

±

0.57

17 ±

0.0

26 ±

0.0

11.93 ±

0.11

24.93 ±

0.11A

- 17.7 ±

046S

15 ± 0.17A 23 ± 0.0S 25 ± 0.0A -A

S.fonticola - 26.5

±

0.70

R R 24.23

±

0.40

16.3

±

0.51

28.2 ±

0.40

10.5 ± 0.70 10 ± 0.0A - - -A -A -A -A

S.liquefaciens - 25.6

±

0.46

R R 21.9

±

0.05

14.1

±

0.28

25.8 ±

0.28

13.1 ± 0.17 21.13 ±

0.23A

- - 14 ± 0.0A 18 ± 0.0S 17 ± 0.0A -A

S.aureus - 26 ±

0.0

R R 22.06

±

0.11

22.1

±

0.23

26.1 ±

0.17

17.2 ± 0.34 25 ± 0.0A 19.9±0

.05S

27.06 ±

0.11S

19.8 ±

0.23A

25 ± 0.0S 31.9 ±

0.17S

20 ±

0.0S

C.freundii 11.33 ± 0.57 22 ±

0.0

R R 22.33

±

0.57

16 ±

0.0

23.66 ±

0.57

12 ± 0 26.06 ±

0.11A

-A -A 26 ± 0.0A 19.1 ±

0.28A

22.9 ±

0.05A

13 ±

0.0A

20

aC: Cardamom, R: resistant, LEV: levofloxacin OX: oxacillin, AML: amoxicillin, C: chloramphenicol, CN: gentamicin, TE: tetracycline, S: streptomycin; A:

Antagonistic; I: Indifference; S: Synergistic; (-): no growth. Values are represented as mean ± SD

21

358359

Table 6 Determination of the combined effects of essential oil of Almond with standard antibiotics

Food isolates Essential

oils

Standard antibiotics

Zone of inhibition in (mm)

Standard antibiotics + Essential oil

Almond LEV OX AML C CN TE S LEV+P OX+P AML+

P

C+P CN+P TE+P S+P

E.coli - 29 ± 0.0 R R 24.33 ±

0.57

17 ± 0.0 26 ±

0.0

11.93 ±

0.11

30 ± 0.0S - 24 ±

0.0S

17.9 ±

0.05A

23 ±

0.0S

13.2 ±

0.3A

20.0 ±

0.1S

S.fonticola - 26.5 ±

0.7

R R 24.23 ±

0.40

16.3 ±

0.51

28.2 ±

0.40

10.5 ±

0.70

9 ± 0.0A - 6.1 ±

0.17S

-A -A -A -A

S.liquefaciens - 25.6 ±

0.46

R R 21.9 ± 0.05 14.1 ±

0.28

25.8 ±

0.28

13.1 ±

0.17

21 ±

0 .0A

-A 20.1 ±

0.17S

-A 14.06 ±

0.11I

-A 18 ± 0.0S

S.aureus - 26 ± 0 R R 22.06 ±

0.11

22.1 ±

0.23

26.1 ±

0.17

17.2 ±

0.34

29.06 ±

0.11S

19.1±0

.23S

34 ±

0.0S

-A -A 20 ± 0.0A 14.9 ±

0.05A

C.freundii - 22 ± 0 R R 22.33 ±

0.57

16 ± 0.0 23.66

± 0.57

12 ± 0 21.9 ±

0.17A

- 24 ±

0.0S

-A 23 ±

0.0S

13.1 ±

0.17A

18.8 ±

0.23S

aA: Almond, R: resistant, LEV: levofloxacin OX: oxacillin, AML: amoxicillin, C: chloramphenicol, CN: gentamicin, TE: tetracycline, S: streptomycin;

A:Antagonistic; I: Indifference; S: Synergistic; (-): no growth. Values are represented as mean ± SD

22

360361

Fig 1. Minimum inhibitory concentrations of commercial EOs: a) agar well diffusion method b) macro broth dilution method

23

362363

Fig 2. Scanning electron microscopic images of tested bacteria when treated with EOs at MIC. Escherichia coli (a) control (b) treated with lemon EO at 500

µg/mL. S.aureus (c) control (d) treated with lemon lavender combination at 1000 µg/mL.

24

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368

369

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370

371

372