In Vitro Antibacterial Activity of Several Plant Extracts and Oils against Some Gram-Negative Bacteria.

BACKGROUND: Medicinal plants are considered new resources for producing agents that could act as alternatives to antibiotics in the treatment of antibiotic-resistant bacteria. The aim of this study was to evaluate the antibacterial activity of 28 plant extracts and oils against four Gram-negative bacterial species.
METHODS: Experimental, in vitro, evaluation of the activities of 28 plant extracts and oils as well as some antibiotics against E. coli O157:H7, Yersinia enterocolitica O9, Proteus spp., and Klebsiella pneumoniae was performed. The activity against 15 isolates of each bacterium was determined by disc diffusion method at a concentration of 5%. Microdilution susceptibility assay was used in order to determine the minimal inhibitory concentrations (MICs) of the plant extracts, oils, and antibiotics.
RESULTS: Among the evaluated herbs, only Origanum syriacum L., Thymus syriacus Boiss., Syzygium aromaticum L., Juniperus foetidissima Wild, Allium sativum L., Myristica fragrans Houtt, and Cinnamomum zeylanicum L. essential oils and Laurus nobilis L. plant extract showed anti-bacterial activity. The MIC50 values of these products against the Gram-negative organisms varied from 1.5 (Proteus spp. and K. pneumoniae( and 6.25 µl/ml (Yersinia enterocolitica O9 ) to 12.5 µl/ml (E. coli O:157).
CONCLUSION: Among the studied essential oils, O. syriacum L., T. syriacus Boiss., C. zeylanicum L., and S. aromaticum L. essential oils were the most effective. Moreover, Cephalosporin and Ciprofloxacin were the most effective antibiotics against almost all the studied bacteria. Therefore, O. syriacum L., T. syriacus Boiss., C. zeylanicum L., and S. aromaticum L. could act as bactericidal agents against Gram-negative bacteria.

Introduction

Medicinal and aromatic plants are used on a large scale in medicine against drug-resistant bacteria, which are considered one of the most important reasons for the lack of success of treatment in infectious diseases. Medicinal plants are the major sources of new medicines and may constitute an alternative to the usual drugs.[1]

Aromatic oils are used in many industries, including food preservation,[2] pharmacy, and medicine.[3],[4] They are expected to form new sources of antimicrobial drugs, especially against bacteria.[5] The antibacterial effectiveness of aromatic oils has been divided into a good, medium, or bad.[6],[7] These oils can also produce some defense products against several natural enemies.[8] In addition, and in order to continue their natural growth and development, aromatic oils may produce some secondary metabolites in response to some external stress.[9]

The extracts and oils of 28 plants used in this work have been traditionally employed by people for various purposes in different parts of the world. Cinnamomum zeylanicum essential oil has antibacterial and antifungal activities[10] as well as anti-diabetic properties;[11] Citrus limon and Rosmarinus officinalis L. essential oils possess antioxidant properties;[12],[13] Citrus aurantium has immunological effects in humans;[14] Eucalyptus globulus oil has good antimicrobial activities;[15],[16] Thymus pannonicus essential oil has an excellent effect against E. coli O157:H7;[17] light thyme essential oil inhibits the growth of E. coli O157:H7 in foods;[18] Brillantaisia lamium extract exhibits antibacterial and antifungal effects against Staphylococcus aureus, Enterococcus faecalis, Candida tropicalis, and Cryptococcus neoformans;[19] and finally Crinum purpurascens herb extract has antimicrobial activities against Salmonella paratyphi A and B.[20] Traditionally, many plant extracts and oils are used as medicinal plants in Syria for many purposes, particularly for respiratory and gastrointestinal disorders.

The aim of this study was to screen the in vitro antibacterial activity of 28 plant extracts and oils against some Gram-negative bacteria, including: E. coli O157:H7, Yersinia enterocolitica O9, Proteus spp., and Klebsiella pneumoniae.

Materials and Methods

Microorganisms and Growth Conditions

Fifteen local isolates of E. coli O157:H7, Y. enterocolitica O9, Proteus spp., and K. pneumoniae were grown for 24-48 h in 2YT agar (peptone, 16 g/liter; yeast extract, 10 g/liter; NaCl, 5 g/liter; agar, 13 g/liter [Difco, BD, Spars, MD]). The bacteria were suspended in a sterile phosphate-buffered saline (PBS). Bacteria abundance in the PBS was monitored by recording the optical density (OD) at 590 nm.[21] The exact doses were assessed retrospectively by viable counts on 2YT agar plates.

Plant Samples Collection

Rosmarinus officinalis L., Origanum syriacum L., Thymus syriacus Boiss., Salvia palaestina Benth., Mentha piperita L., and Lavandula stoechas L. (Lamiaceae); Citrus aurantium L. and Citrus medica L. (Rutaceae); Syzygium aromaticum L., Myrtus communis L., and Eucalyptus camaldulensis Dehnh. (Myrtaceae); Cinnamomum zeylanicum L. and Laurus nobilis L. (Lauraceae); Juniperus foetidissima Wild (Cupressaceae); Pelargonium roseum L. (Geraniaceae); Scilla maritima Squill and Allium sativum L. (Liliaceae); Pinus halepensis Miller. (Pinaceae); Artemisia herba-alba Asso. (Compositae); Anabasis haussknechtii Boiss. (Chenopodiaceae); Crataegus aronia L. (Rosaceae); Mercurialis annua L. (Euphorbiaceae); Matthiola crassifolia Boiss. (Brassicaceae); Myristica fragrans Houtt. (Myristicaceae); Brassica nigra Koch. (Cruciferae); Coriandrum sativum L. (Apiaceae); Zingiber officinale Rosc. (Zingiberaceae); and Achillea fragrantissima Forssk. (Asteraceae) samples were collected during the flowering season from different regions in Syria between March and July 2010, or purchased from local markets (table 1). The samples were cleaned from any strange plants, dust, or any other contaminants.

Table 1

Plants and their families, collection sites, and parts used

Scientific name	Plant family	Collection site	Altitude (m)	Collection time	Extracted part	Extract or oil
Rosmarinus officinalis L.	Lamiaceae	Latakia	300	June	Aerial parts	Oil
Origanum syriacum L.	Lamiaceae	Kafr Nobol-Idlib	446	July	Aerial parts	Oil
Thymus syriacus Boiss.	Lamiaceae	Alsoja Mountain-Damascus	840	July	Aerial parts	Oil
Salvia palaestina Benth.	Lamiaceae	Alyarmouk Valley-Konaitera	800	June	Aerial parts	Oil
Mentha piperita,. L.	Lamiaceae	Latakia	300	June	Aerial parts	Oil
Lavandula stoechas L.	Lamiaceae	Tartous	300	June	Aerial parts	Oil
Citrus aurantium L.	Rutaceae	Latakia	300	April	Flowers	Oil
Citrus medica L.	Rutaceae	Latakia	300	April	Flowers	Oil
Syzygium aromaticum L.	Myrtaceae	Market			Flowers	Oil
Myrtus communis L.	Myrtaceae	Latakia	300	June	Leaves	Extract
Eucalyptus camaldulensis Dehnh.	Myrtaceae	Tartous	300	June	Flowering branches	Oil
Cinnamomum zeylanicum L.	Lauraceae	Market			Barks	Oil
Laurus nobilis L.	Lauraceae	Latakia	300	July	Leaves	Extract
Juniperus foetidissima Wild	Cupressaceae	Dobaya-Damascus	800	June	Leaves	Oil
Pelargonium roseum L.	Geraniaceae	Kodsaya-Damascus	916	May	Aerial parts	Extract
Scilla maritime Squill.	Liliaceae	Tartous	300	March	Bulbs	Extract
Allium sativum L.	Liliaceae	Market			Bulbs	Oil
Pinus halepensis Miller.	Pinaceae	Dobaya-Damascus	900	May	Leaves	Extract
Artemisia herba-alba Asso.	Compositae	Alsoja Mountain-Damascus	840	March	Aerial parts	Extract
Anabasis haussknechtii Boiss.	Chenopodiaceae	Alkariatain-Homs	500	March	Aerial parts	Extract
Crataegus aronia L.	Rosaceae	Alkonaitera	1100	April	Flowering branches	Extract
Mercurialis annua L.	Euphorbiaceae	Kasab-Latakia	800	March	Aerial parts	Extract
Matthiola crassifolia Boiss.	Brassicaceae	Latakia	10	March	Aerial parts	Extract
Myristica fragrans Houtt.	Myristicaceae	Market			Fruit	Oil
Brassica nigra Koch.	Cruciferae	Market			Seeds	Oil
Coriandrum sativum L.	Apiaceae	Market			Seeds	Oil
Zingiber officinale Rosc.	Zingiberaceae	Market			Rhizome	Oil
Achillea fragrantissima Forssk.	Asteraceae	Palmyra	405	July	Aerial parts	Oil

Essential Oil Extraction

Essential oils from fresh, clean, weighed aerial parts, flowers, leaf fruits, barks, seeds, rhizomes, and bulbs (table 1) extracted by hydro-steam distillation using the Clevenger apparatus were collected and stored in sterile vials.[22] Briefly, 100 to 150 g of each plant was introduced in the distillation flask (1 L), which was connected to a steam generator via a glass tube and to a condenser to retrieve the oil. This was recovered in a funnel tube. Aromatic molecules of the essential oils were released from the plant material and evaporated into hot steam. The hot steam forced the plant material to release the essential oil without burning the plant material itself. Then, steam containing the essential oil was passed through a cooling system in order to condense the steam. The steam was applied for 3 h. After settling the recovered mixture, essential oil was withdrawn. The supernatant essential oil was filtered through anhydrous Na2SO4 to dry the yielded essential oil. Afterward, the essential oil was collected in tightened vials and stored in a refrigerator. For the antimicrobial activity test, several dilutions of the oils were done using dimethyl sulfoxide (DMSO).

Preparation of Ethanolic Extracts

Successive solvent extraction was performed for some plants (table 1). Leaves and bulbs were washed, air dried for 7-8 days, and ground into powder before they were placed into the flask of the Soxhlet apparatus for extraction using ethanol with increasing order of polarity to extract the phytoconstituents separately at 20ºC for 3-4 h. (The ethanol used was HPLC grade obtained from Sigma-Aldrich, Germany.) Whatman No.1 filter papers were then applied to filter the extracts. After that, reduced pressure was applied to evaporate and dry the filtrates, which were stored at -20ºC in labeled, sterile, screw-capped bottles.

Antibacterial Susceptibility Assay

Muller-Hinton Broth (MHB, Merck) medium was used to grow the test isolates for 22 h at 37°C. Final bacterial numbers were standardized to 1×10 6 CFU/ml. A total of 0.1 ml of bacterial suspension was poured on each plate, containing Muller-Hinton Agar (MHA, Merck). The lawn culture was prepared by sterile cotton swab and allowed to remain in contact for 1 min. Thereafter, a 5% concentration of each plant extracts was prepared. The sterile filter paper discs (6-mm diameter) were placed on the lawn cultures, and 24 h after incubation at 37°C, the inhibition zone was measured in mm.

Antibiotics Minimum Inhibitory Concentration Determination

In order to estimate the antibiotics susceptibility, the well broth microdilution method was used with 96-well plates (TPP, Switzerland). The antibiotics were diluted twofold in LB broth® (Acumedia, Michigan, USA), and the wells were inoculated with 1×10 6 CFU of bacteria (in a 0.2 ml final volume). The incubation period was 24 h at 37°C. The lowest concentration that inhibited 50% of visual growth was recorded and interpreted as the MIC50. The MIC testing was performed according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI).[23] The range of the concentrations assayed for each antibiotic was 0.064 to 128 μg/ml. The absorbance was determined at 590 nm (Thermo-Lab Systems Reader, Finland). All the tests were performed in triplicate and then averaged. The investigated antibiotics were Ciprofloxacin, Levofloxacin, Ofloxacin, Sparfloxacin, Ceftazidime, Ceftriaxone, and Cefotaxime. Positive control was done without adding any antibiotics.

Plants Extracts and Oils Minimum Inhibitory Concentration Determination

The microdilution broth susceptibility assay was used.[24] Three replicates of the serial dilutions of each essential oil were prepared in LB broth medium in 96-well microtiter plates, using a range of concentrations for each essential oil from 0.75 to 50 µl/ml. Next, 100 μl of freshly grown bacteria, standardized until a bacterial number of 1×10 6 CFU/ml in LB broth was achieved, was added to each well. Positive and negative controls were also done. The plate was incubated with shaking for 24 h at 37˚C. The lowest concentration that inhibited 50% of visual growth was recorded and interpreted as the MIC50.

Statistical Analysis

Optimal concentrations for the most effective essential oils and plant extracts were estimated by Probit Analysis (SPSS Inc. 2010; Finney, 1971). Minimum concentrations to achieve 50% inhibition of the various bacteria (MIC50) were considered significantly different if their 95% confidential limits did not overlap.

Results

Table 2 demonstrates that O. syriacum. L., T. syriacus, S. aromaticum, C. zeylanicum, L. nobilis L., J. foetidissima, A. sativum L., and M. fragrans Houtt. had good antibacterial activities against the Gram-negative bacteria, whereas the rest of the studied extracts were ineffective.

Table 2

Number of Gram-negative isolates susceptible to each plant extract

	Number of isolates susceptible to plant extracts
	E. coli O157:H7	Y. enterocolitica O9	Proteus spp	K. pneumoniae
Rosmarinus officinalis L.	1	2	2	2
Origanum syriacum L.	12	12	13	12
Thymus syriacus Boiss.	12	15	15	11
Salvia palaestina Benth.	0	0	0	0
Mentha piperita. L.	1	0	2	1
Lavandula stoechas L.	3	3	5	6
Citrus aurantium L.	1	0	0	0
Citrus medica L.	1	1	0	0
Syzygium aromaticum L.	9	14	13	14
Myrtus communis L.	0	3	2	3
Eucalyptus camaldulensis Dehnh.	1	2	2	2
Cinnamomum zeylanicum L.	14	15	15	13
Laurus nobilis L.	14	13	13	15
Juniperus foetidissima Wild	11	11	12	13
Pelargonium roseum L.	2	2	3	5
Scilla maritime Squill.	2	1	1	2
Allium sativum L.	14	15	15	15
Pinus halepensis Miller.	0	0	0	0
Artemisia herba-alba Asso.	0	0	0	0
Anabasis haussknechtii Boiss.	0	0	0	0
Crataegus aronia L.	1	0	0	0
Mercurialis annua L.	0	0	1	0
Matthiola crassifolia Boiss.	3	4	2	3
Myristica fragrans Houtt.	13	13	13	12
Brassica nigra Koch.	0	0	0	0
Coriandrum sativum L.	3	3	2	0
Zingiber officinale Rosc.	3	3	4	5
Achillea fragrantissima Forssk.	0	0	0	0

The MIC50 values for these plant extracts and oils were 12.5, 12.5, 25, 12.5, 12.5, 25, 12.5, and 6.25 µl/ml, respectively, against E. coli O157:H7; and 1.5, 6.25, 6.25, 6.25, 6.25, 25, 6.25, and 12.5 µl/ml, respectively, against Y. enterocolitica O9; and 1.5, 3.125, 1.5, 1.5, 3.125, 12.5, 3.125, and 12.5 µl/ml, respectively, against Proteus spp.; and 6.25, 3.125, 1.5, 3.125, 6.25, 12.5, 6.25, and 6.25 µl/ml, respectively, against K. pneumoniae (table 3).

Table 3

Minimum inhibitory concentrations (MICs) for the selected essential oils and extracts against some Gram-negative bacteria

	MIC50 and MIC90 of plant extracts (µl/ml)
	E. coli O157:H7		Y. enterocolitica O9		Proteus spp		K. pneumoniae
	MIC50	MIC90	MIC50	MIC90	MIC50	MIC90	MIC50	MIC90
Origanum syriacum.	12.5	NA	1.5	12.5	1.5	12.5	6.25	NA
Thymus syriacus. Boiss.	12.5	NA	6.25	25	3.125	25	3.125	NA
Syzygium aromaticum	25	50	6.25	25	1.5	50	1.5	25
Cinnamomum zeylanicum	12.5	25	6.25	25	1.5	25	3.125	NA
Laurus nobilis L.	12.5	NA	6.25	50	3.125	50	6.25	12.5
Juniperus foetidissima Wild	25	50	25	50	12.5	50	12.5	25
Allium sativum L.	12.5	25	6.25	50	3.125	50	6.25	50
Myristica fragrans Houtt	6.25	50	12.5	50	12.5	NA	6.25	NA

In contrast, when studying the optimal concentrations that could inhibit 50% of the bacterial isolates, the X 2 values were not significant (P>0.05) for all the studied concentrations, indicating adequate fit of the Probit regression models (table 4).

Table 4

Optimal inhibitory concentrations of the selected essential oils and extracts against some Gram-negative bacteria

Bacteria	Plant	MIC50 ( µl/ml)	X 2	Significance
E. coli	O. syriacum. L.	9.48	1.33	0.932
	T. syriacus. Boiss.	7.85	2.42	0.788
	S. aromaticum	16.11	2.8	0.732
	C. zeylanicum	9.48	1.33	0.932
	L .nobilis L.	20.43	6.32	0.276
	J. foetidissima Wild	21.82	2.98	0.703
	A. sativum L	8.41	1.71	0.888
	M. fragrans Houtt.	7.91	3.01	0.699
Y. enterocolitica	O. syriacum. L.	1.59	1.36	0.929
	T. syriacus. Boiss.	5.76	0.69	0.983
	S. aromaticum	5.22	1.28	0.937
	C. zeylanicum	5.76	0.69	0.983
	L .nobilis L.	4.30	2.99	0.702
	J. foetidissima Wild	12.57	1.87	0.867
	A. sativum L	5.52	2.05	0.842
	M. fragrans Houtt.	7.00	0.63	0.986
Proteus spp.	O. syriacum. L.	1.12	1.93	0.859
	T. syriacus. Boiss.	4.68	3.72	0.591
	S. aromaticum	2.21	4.92	0.426
	C. zeylanicum	1.35	1.73	0.885
	L .nobilis L.	4.68	3.72	0.591
	J. foetidissima Wild	6.98	0.78	0.978
	A. sativum L.	4.68	3.72	0.591
	M. fragrans Houtt.	6.03	0.63	0.986
K. pneumoniae	O. syriacum. L.	5.20	1.38)	0.927
	T. syriacus. Boiss.	3.03	3.58	0.612
	S. aromaticum	1.33	1.79	0.877
	C. zeylanicum	2.97	4.91	0.427
	L .nobilis L.	3.51	1.20	0.954
	J. foetidissima Wild	9.81	5.22	0.390
	A. sativum L.	8.75	3.86	0.570
	M. fragrans Houtt.	12.4	6.53	0.258

Table 5 also shows that Ceftazidime, Cefotaxime, and Ciprofloxacin were the most effective antibiotics against E. coli O157:H7 (MIC50= 0.25, 0.5, and 2 µg/ml, respectively). Moreover, Ceftazidime and Ciprofloxacin were the most effective antibiotics against Y. enterocolitica O9 (MIC50= 0.25 and 0.5 µg/ml, respectively) and against Proteus spp. (MIC50= 4 and 2 µg/ml, respectively) and Ceftriaxone, Cefotaxime, and Ciprofloxacin were the most effective antibiotics against K. pneumoniae (MIC50= 0.25, 0.25, and 0.5 µg/ml, respectively).

Table 5

Minimum inhibitory concentrations (MICs) of some antibiotics against Gram-negative bacteria

	MIC50 and MIC90 of some antibiotics (µg/ml)
	E. coli O157:H7		Y. enterocolitica O9		Proteus spp		K. pneumoniae
	MIC50	MIC90	MIC50	MIC90	MIC50	MIC90	MIC50	MIC90
Ciprofloxacin	2	64	0.5	NA	2	NA	0.5	NA
Levofloxacin	4	NA	4	NA	4	NA	4	NA
Ofloxacin	4	NA	2	64	2	NA	4	NA
Sparfloxacin	NA	NA	32	NA	32	NA	32	NA
Ceftazidime	0.25	NA	0.25	64	4	NA	4	NA
Ceftriaxone	32	NA	32	NA	32	NA	0.25	NA
Cefotaxime	0.5	NA	8	NA	64	NA	0.25	NA

Discussion

Because of their safety and low cost as well as their impact on a large number of microbes,[25]medicinal plants may have the ability to treat bacterial resistance to many types of antibiotics. The antimicrobial effects of aromatic oils extracted from a large number of plants have been evaluated and reviewed,[26],[27] and the mechanisms that enable the natural ingredients of herbs and spices to resist microbes have been discussed.[28] The results show that these mechanisms vary greatly depending on the components of the essential oil.[29],[30]

In the present study, the efficacy of some plant extracts and oils was determined, quantitatively, by measuring the diameter of the inhibition zones around the discs (table 2). Only O. syriacum. L., T. syriacus Boiss., S. aromaticum L., C. zeylanicum L., L. nobilis L., J. foetidissima Wild, A. sativum L., and M. fragrans Houtt. extracts inhibited the growth of the tested bacteria. In addition, O. syriacum. L., T. syriacus Boiss., S. aromaticum L., and C. zeylanicum L. essential oils were the most effective, and their MIC50 values varied from 1.5 µl/ml to 25 µl/ml against various kinds of bacteria. Because the values of minimum bactericidal concentration (MBC) and MIC are usually very similar,[31] it can be logically assumed that the above-mentioned plant extracts and oils have a bactericidal effect on Gram-negative bacteria, especially against Proteus spp. and K. pneumoniae.

The Probit Analysis (table 4) revealed that the minimum concentrations of the essential oils that could inhibit 50% of the various bacteria were T. syriacus Boiss. for E. coli O157H7 (7.85 µl/ml), O. syriacum. L. for Proteus spp. and Y. enterocolitica (1.12 and 1.59 µl/ml, respectively), and S. aromaticum for K. pneumoniae (1.33 µl/ml).

Ooi et al.[32] reported that Cinnamomum verum shows excellent activities against E. coli and Proteus vulgaris. Preuss et al.[33] found that origanum essential oil proves cidal to E. coli and K. pneumoniae. In addition, Barbosa et al.[34] found that the MIC90 of Origanum vulgare essential oil is 0.46% (v/v) against E. coli. López et al.[35] found that 8-10% (v/v) concentrations of Origanum vulgare essential oil can completely inhibit the growth of E. coli and other Gram-negative bacteria. Elsewhere, Mkaddem et al.[36] reported that Mentha essential oils are very active against K. pneumoniae bacteria, whereas they are less effective against E. coli. Furthermore, Mentha longifolia oil is thought to exhibit an antimicrobial activity against some Gram-positive bacteria such as Streptococcus mutans and Staphylococcus aureus, but without affecting Pseudomonas aeruginosa.[37]

Since the antibacterial effectiveness of medicinal plants varies dramatically depending on the phytochemical characteristics of plant families and subfamilies, it is not surprising to note the difference in this efficacy even when using samples taken from the same plant, but from two different regions.[38] Our results reveal that the cephalosporins were the most effective antibiotics against almost all the studied bacteria, and only Ciprofloxacin, one of the fluoroquinolones group, was effective against these bacteria.

Conclusion

O. syriacum. L., T. syriacus Boiss., S. aromaticum L., C. zeylanicum L., J. foetidissima Wild, A. sativum L., and M. fragrans Houtt. oils and L. nobilis L. extract were the most effective plant extracts against the Gram-negative bacteria studied in this work. These plant extracts could be a potential source of new antibacterial agents.

Further and more specific studies, in vivo, are recommended to determine the efficacy of these essential oils in the treatment of gram-negative bacterial infections.