Antibacterial effect of Thymus sp. and Boswellia sp. extracts on Streptococcus pneumoniae and Klebsiella pneumoniae isolates

The antimicrobial activity of essential oils of Boswellia and thyme (Boswellia sp., and Thyme sp.) was evaluated against 20 clinical isolates of Streptococcus pneumoniae and 5 isolates of Klebsiella pneumoniae. Essential oils were prepared using methanol and water (1:1) with HPLC technique. Antimicrobial activity and minimum inhibitory concentration (MIC) were measured using disk diffusion method against 20 isolates of S. pneumoniae and 5 isolates of K. pneumoniae isolated from different patients. Flavonoids and phenolic compounds are the main constituents of Boswellia and thyme which may have the antimicrobial activity. Boswellia extract was more efficient than thyme extracts; 60% of S. pneumoniae isolates and one K. pneumoniae isolate were sensitive to Boswellia extract, 30% of S. pneumoniae isolates were sensitive to thyme extract, and no effect on K. pneumoniae clinical isolates was observed. Inhibition zones ranged from 1-12 mm with thyme extract, while Boswellia extracts showed 2 to 30 mm diameters of inhibition zone. This study is significant due to the widespread problem of microbial drug resistance and the need for natural antibiotic to fight diseases.


INTRODUCTION
Based on the World Health Organization (WHO), pneumonia disease is considered as a common serious illness that threatens health, especially in developing countries.According to WHO, 15% of the total death of children is due to pneumonia (WHO, 2015).Pneumonia disease caused by Klebsiella pneumoniae is the major cause of infants and patients injury in hospitals intensive care units (WHO, 2014).
Medicinal plants and their essential oils always had choice of use for different purposes such as treatment of some diseases all over the world; therefore it has high economic value (Joshi et al., 2011).Boswellia is one of the most effective medicinal plants.Genus Boswellia, belonging to Burseraceae family includes both trees and shrubs; a family that contains 20 species widespread in the dry regions (Hussain et al., 2013).Frankincense is the oligomer resin that is extracted from tapping in the inner bark of the trees.Boswellia essential oils and oligomer have medicinal properties such as immune enhancing, antibacterial, anti-inflammatory, wound healing and anticancer properties (Shareef, 2011).Gum is used traditional as a diuretic and for the treatment of diarrhea, dysentery, cardiac disease, cough, hemorrhage, dyspnea, polyuria, urinary troubles, piles, ulcers and burns (Ismail et al., 2014).Most of the research on Boswellia focused on the effectiveness of its extracts on memory regions of the brain (Farshchi et al., 2010).Thyme sp. is also one of the medicinal plants.Genus Thymus belonging to the family Lamiaceae consists of 928 species, native to Europe, and grows in the Mediterranean basin and northern Europe, as well as other parts of the world such as Asia, South America, and Australia.Thyme is also used in traditional medicine for the treatment of coughs, upper respiratory infections, acute and chronic bronchitis, whooping cough; also in cases of respiratory system disorders due to its antitussive, antioxidant, anti-inflammatory, and antimicrobial properties.Thyme is employed for external use as a mouth wash in gargles to treat laryngitis.Recent data suggest that the essential oil of thyme is one of the best choices to fight inflammations, with its main components being carvacrol and thymol (Nabavi et al., 2015).Thymol and carvacrol are phenolic compounds with strong antifungal and therapeutic properties (Lakis et al., 2012).Thymol compound reduces bacterial resistance to some antibiotics such as penicillin (Nabavi et al., 2015); however, many studies indicated the antimicrobial activity of Boswellia and thyme extracts, especially against K. pneumonia (Javed et al., 2015).Ismail et al. (2014) found that K. pneumoniae was sensitive against high concentrations of frankincense extracts of Boswellia serrata whereas investigations on Cinnamon and frankincense essential oil for their antibacterial activity against pathogenic bacterial isolates, showed that K. pneumoniae was less sensitive to cinnamon giving negative results with frankincense; however, frankincense essential oil showed good antimicrobial activity against the other tested bacteria (Shareef, 2011).Fournomiti et al. (2015) investigated the antimicrobial activity of some medicinal plant extracts: oregano (Origanum vulgare), sage (Salvia officinalis), and thyme (Thymus vulgaris) against three clinical bacteria isolates (Escherichia coli, Klebsiella oxytoca, K. pneumoniae), and the results showed that K. pneumoniae was the second most sensitive strain to thyme and oregano essential oils were the most efficient.Sienkiewicz et al. (2012) screened the antimicrobial activity of thyme essential oil against clinical multidrug resistant strains of Staphylococcus, Enterococcus, Escherichia and Pseudomonas, with the results indicating that thyme essential oil strongly inhibited the growth of the tested clinical strains.Recently, Benmoussa et al. (2016) used four methods for extraction of the volatile compounds from Tunisian T. vulgaris leaves.The results showed that T. vulgaris essential oils had antibacterial effect against multi-drug resistant bacteria, and solventfree microwave extraction method was efficient giving high antimicrobial activity which was attributed to 17 volatiles compounds, and high monoterpense hydrocarbons content.Thus, it was postulated that solvent-free microwave extraction (SFME) is the best alternative method of essential oils extraction.Moreover, Sakkas et al. (2016) studied the efficacy of five essential oils (Basil, chamomile blue, origanum, thyme, and tea tree oil) on 3 g negative and positive clinical isolates (Acinetobacter baumannii, E. coli, K. pneumoniae and P. aeruginosa) using broth macrodilution method; antibacterial activity against this multi-drug resistant isolates was poor.
The aim of this study was to investigate the antibacterial activity of Boswellia sp. and Thyme sp.extracts on Pneumonia clinical isolates.

Sample collection and essential oils extraction
Thyme leaves and Boswellia gums were collected from local markets in Dammam, Saudi Arabia, dried and ground into powder.30 g of ground leaves or powdered gum was dissolved in 100 ml of 96% ethanol; similarly, 30 g of ground leaves or powdered gum was extracted in 100 ml of distilled water, and the last group of extraction was prepared in 100 ml of 1:1 water and 96% ethanol mixture.Extracts were placed three days at room temperature.Thereafter, oils were extracted by rotary evaporation at 60°C for 24 h for ethanol evaporation.The extracts were filtered using disposable membrane bacterial filters to get sterile extracts.

Bacterial strains
Standard strains of Pneumoniae were used; Streptococcus pneumoniae ATCC and K. pneumoniae ATCC.In addition, 20 clinical isolates of S. pneumoniae and 5 of K. pneumoniae were isolated from different patient's materials.Standard strains of E. coli ATCC, Staphylococcus aureus and Pseudomonas aeruginosa strains were obtained from Laboratory of Microbiology of King Fahad Hospital in Khobar City.

Screening of antibacterial effects
This study used well diffusion assay technique (Chung et al., 1998); and 0.1 ml of overnight bacterial culture strain was inoculated into trypticose soy broth.Overnight culture inoculum was spread over blood agar by using L-shape spreader and sterile swaps.Holes were made by using sterile 5 mm diameter cork porer; 25 µl of different extracts were added in each hole, and was inoculated with a loopful of the test organism previously diluted to 0.5 McFarland turbidity standards seeded on the medium.Plates were then incubated at 37°C for 24 h; sterile water and solvents were used as control along with a combination of thyme and Boswellia (1:1), and diameter of inhibition zone was estimated in mm.

Minimum inhibitory concentration assay
Bacterial isolates showed susceptibility to the extracts, their antibacterial response was determined using extracts at dilutions 0.25, 0.5, 0.75 µg/ml and sterile solvent as control treatment.
Minimum inhibitory concentration (MIC) was determined as described by Sienkiewicz et al. (2012), and was read in µ/ml after overnight incubation at 37°C.All experiments were made in replicate.

Chemical analysis
Chemical analysis of thyme and frankincense was performed using (HPLC) chromatography and Agilent device (1100HPLC).Phenolic compounds were estimated at 280 nm wavelength, and flavonoid analysis carried out using the following condition: degazer autosampler, qauternery pumb and column cabin at 35ºC; the fragmentation column was zorbox ODS of 5 μm 4.6 × 250 mm dimensions.The flow rate of the mobile phase was 1 ml/min as described by Pascale et al. (1999) and Pirjoet et al. (2000).Zielinski and Kozlowsk (2000) proposed that higher extraction yield were achieved with water compared to water/ethanol mixture and ethanol alone.This can be explained on the basis that proteins and carbohydrates are more soluble in water than in ethanol and aqueous mixtures of ethanol.

Results presented in
Table 2 shows the results of antibacterial activity of Boswellia and thymus extracts on pneumoniae isolates and standard ATCC isolates, using well diffusion and their MIC.S. pneumoniae (ATCC), was used as control isolates, and was more sensitive to Boswellia extracts than K. pneumoniae (ATCC) reference strain.K. pneumoniae (ATCC) gave 8 and 7 mm zones of inhibition with Boswellia and thyme extracts, respectively.Results indicate that the inhibition zone ranged from 2 to 30 mm; 12 S. pneumoniae isolates were sensitive to Boswellia extracts, which equals 60% of the tested isolates.MIC of four S. pneumoniae isolates was 0.75 µg/ml, and five isolates had 0.5 µg/ml MIC.30% from the tested bacterial isolates were resistant to Boswellia extract.However, six isolates of S. pneumoniae (represent 30% of isolates) were sensitive to thymus extracts and the rest were resistant.Inhibition zones of S. pneumonia isolates ranges from 1 to 12 mm, and MIC values of the sensitive isolates were 0.25 µg/ml for 3 isolates and 0.75 µg/ml for 2 isolates, with one isolate having 0.5 µg/ml MIC.Results of Table 2 also show that six S. pneumoniae isolates (30% of isolates) were sensitive to Boswellia and thyme (1:1) mixed extract, and gave inhibition zones ranging from 2 to 7 mm; whereas isolates numbers 4 and 8 were more sensitive to Boswellia extracts, and the rest of tested isolates were resistant.No effect of thymus extracts on any of K. pneumoniae clinical isolates was observed.Results indicate that Boswellia extract has higher antimicrobial activity than thyme extract.
Al-Saidi et al. ( 2012) reported on the antibacterial activity of omanilubans (oleo-gum resins of Boswellia) essential oils against both Gram-positive and Gram negative.Results of this investigation indicated that Boswellia volatile oils were more active slightly against Gram positive than Gram negative bacteria.These results are in accordance with those of other investigators (Al-Saidi et al., 2012).The resistance of some of the Gram-negative bacteria could be due to the more hydrophilic outer membrane containing lipopolysaccharide (LPS), and the outer membrane acting as a penetration barrier for macromolecules and hydrophobic compounds (Nikaido, 1996).Raja et al. (2011) reported that the lack of antibacterial activity of active compounds of Boswellia sp.against Gramnegative bacteria might be attributed due to the presence of lipophilic outer membrane in Gram-negative, which composed primarily of lipopolysaccharide molecules and forms hydrophilic permeability barrier providing protection against the effects of highly hydrophobic compounds.This is probably the explanation of the resistance of Gram-negative bacteria to lipophilic acetyl-keto-βboswellia acid (AKBA).Sienkiewicz et al. (2012) concluded that thyme oil has strong antibacterial  properties against many pathogenic bacteria, with this activity due to the high content of phenolic compounds with antibacterial properties, such as thymol and carvacol, which constitute more than 40% of the oil ingredients.Similarly, Al-bayati (2008) reported that combinations of two essential oils of T. vulgaris and Pimpinella anisum possessed high inhibitory activities on K. pneumoniae and other tested bacteria through synergistic action.These results are different from our results, since most K. pneumoniae and S. pneumoniae strains were resistant against mixed extracts of Boswell and thyme.This could be due to the limited number of tested bacterial isolates.Similarly, Javed et al. (2015) studied the antibacterial activity of Boswellia sacra and Nigella sativa essential oils and found that B. sacra essential oil gave the lowest zone of inhibition against K. pneumoniae (13 mm / 2.5 µg/ml).These results agree with results of this research, since the application of Boswellia extract recorded 12 mm inhibition zones against K. pneumoniae.Application of mixture of two essential oils of B. sacra and N. sativa gave high zone of inhibition (23 mm) against K. pneumoniae; however, the results here indicate that mixed extracts of Boswellia and thyme gave 17 mm zone of inhibition against S. pneumoniae (isolate number 8).Moreover, no susceptibility was reported against K. pneumoniae clinical isolates.Similarly, Al-bayati et al. (2008) reported that K. pneumoniae was resistant to methanol extract of T. vulgaris and P. anisum.It has frequently been reported that Gram positive bacteria were more sensitive to the essential oil than Gram negative bacteria, which could be due to the presence of hydrophilic outer membrane that blocks the penetration of hydrophobic essential oil into target cell membrane.The antibacterial activities of methanol extract of T. vulgaris essential oil could be associated with the presence of phenolic compounds like carvacrol, thymol, γ-terpinene and p-cymene, which have antibacterial properties (Essawi and Sour, 2000).Similarly, Nazzaro et al. (2013) reported that rod-shape bacteria are generally sensitive for essential oils with antibacterial properties, but in this study, most K. pneumoniae isolates which are rod-shaped showed resistant response against the tested extracts.Many bioactive compounds such as thymol, carvacol, flavoinds, biphenyls and aliphatic phenols are found in thymus species (Nadia et al., 2013) while Sakkas et al. (2016) noted that the inhibitory effects of thyme are due to thymol, p-cymene and linalool compounds.Most of the antimicrobial activity in Thymus essential oil appears to be associated with phenolic compounds (thymol and carvacol); however, in some cases an elevated concentration in these components does not necessarily increase the antimicrobial properties (Rota et al., 2008).This result could explain the differential antibacterial activity of Boswellia and thyme extract, since thyme extract was efficient at low rate of bioactive concentration compared with thyme extract.The chemical composition of essential oil in plants of genus Thyme is species and chemotype dependent (Nabavi et al., 2015).This result may explain why thyme extract did not show strong antimicrobial activity despite the high rate of antibacterial properties.
Results of Tables 3 and 4 show the flavonoid and phenolic compounds concentrations in thyme and frankincense using HPLC chemical analysis methods.Results showed that phenol and flavonoid concentrations in thyme are higher than in frankincense (2485.6 and 509.2 ppm phenol content) and (3963.6 and 55.6 ppm flavonoid), respectively.Despite the higher concentrations of flavonoid and phenolic compounds in thyme than those of Boswellia (Tables 3 and 4), Boswellia showed higher antimicrobial activity than Thyme, except K. pneumoniae isolate No. 3 which was sensitive.Results also show that the application of aqueous and alcohol extracts of thyme and Boswellia gave high inhibitory action compared to aqueous extracts, which may be related to increased  -Silva et al., 2013).The inhibitory action of these compounds against the bacteria cell depends on their concentrations and composition.The target of these compounds is degradation of the cell wall, increased membrane fluidity and inhibition of a membraneembedded enzyme (Nazzaro et al., 2013).Phenolics compounds can penetrate into the phospholipids layer of the bacterial cell wall, bind to proteins and block their normal functions.Because of their lipophilic nature, essential oils and their compounds can influence the percentage of unsaturated fatty acids and their structure (Burt and Reinders, 2003).Tables 3 and 4 indicate that essential oils of thyme and Boswellia contain acidic components such as benzoic acid (254.3ppm in thyme and 102.9 ppm in Boswellia), e-vanilli acid (47.9 ppm in thyme and 147.7 ppm in Boswellia), allagic acid (164.7 ppm in thyme and 49.9 ppm in Boswellia).These acids can change membrane proteins then block their normal functions (Nazaro et al., 2013).Similarly, essential oils are rich in phenolic compounds that can affect structure and unsaturated fatty acids of bacteria cell wall (Sakkas et al., 2016).Results also show that thyme and Boswellia contain flavonoid compounds such as hispertin (38.5 ppm in thyme and 12.1 ppm in Boswellia), narengin (975.9 ppm in thyme and 7.5 ppm in Boswellia).Flavonoid compounds hydroxyl group bind to amino group of cell proteins, leading to formation of non-dissolved proteins, an action that consequently leads to cell death (Nazzaro et al., 2013).

Conclusion
The results of this study demonstrate the effectiveness of Thyme sp. and Boswellia sp.extracts against pneumoniae clinical isolates.S. pneumoniae showed more sensitivity to Boswellia extracts than K. pneumoniae.Thus, further studies is required for new compounds from natural sources that have antimicrobial activity against different pathogenic bacteria especially gram negative and multi drug resistant bacteria.

Table 1 .
Screening for the best solvent of essential oils using inhibition zone (mm) method on pneumonia standard isolates (ATCC).
Boswellia gave the highest antibacterial activity than water extracts.However, Costal et al. (2012) reported that the highest extraction yield for Thymus lotocephalus was achieved with water than water ethanol mixture.
Hasson et al. (2011)Thymus and Boswellia extracts from ethanol water mixture (1:1) gave the highest antibacterial effect against the tested bacteria (S. pneumoniae and K. pneumoniae) as measured by diameter of inhibition zone.These results are not in accordance with the findings ofHasson et al. (2011), in which methanolic extracts of

Table 2 .
Antibacterial effect of Boswellia and Thymus extracts on pneumoniae isolates by well diffusion and MIC method.