Time-kill Study of Ethyl Acetate Extract of Stinging Nettle on Bacillus subtilis subsp. spizizenii ATCC CRM-6633 Strain NRS 231

Aims: This work investigated the antibacterial activity of selected ethyl acetate extract of Urtica dioica against Bacillus subtilis subsp. spizizenii ATCC® CRM-6633™ Strain NRS 231 (subtilin producer) based on the time-kill approach. Also for further study, the effects of the ethyl acetate extract on B. subtilis cells was studied by performing Scanning Electron Microscope (SEM). Study Design: Prospective Methodology: to our previous result, ethyl acetate extract was selected to be more potent against Bacillus subtilis subsp. spizizenii ATCC® CRM-6633™ Strain NRS 231 based on its inhibition zone diameter. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of ethyl acetate extract against tested bacteria were performed and time-kill study as well as using SEM for further study on structural degeneration of the selected bacterial cells were performed. Results: The MIC and MBC of the ethyl acetate extract against tested bacteria were found at 8.33 and 16.67 mg/mL from broth micro-dilution assay, respectively. The SEM observations gave ideas on the effects of ethyl acetate extract of U. dioica on the growth of B. subtilis subsp. spizizenii ATCC® CRM-6633™ Strain NRS 231. However, the results from SEM suggested that the antibacterial action was due to the internal shrinkage of the cells which collapsed finally. The time-kill approach, determined the kill rate of ethyl acetate extract of U. dioica against B. subtilis subsp. spizizenii ATCC® CRM-6633™ Strain NRS 231 and significantly inhibited the cell growth and possessed bacteriostatic activity at lower concentration (8.33 mg/mL). Conclusion: This study showed the potential of U. dioica extract as alternative therapy against bacterial infection.


INTRODUCTION
The prevalence of resistant bacteria in the community and hospitals cause severe problems for treatment of patients [1]. The demand for new antimicrobial drugs is higher than last decades because of the appearance of multi-drug resistance in common bacteria [2]. Natural products have been a rich source of antibacterial drugs for many decades, but investments in this area have decreased in the last two decades [3]. Researchers found literally thousands of phytochemical from plants as safe and generally effective alternatives with less adverse impact in recent years [4]. Also Bacillus subtilis subsp. spizizenii ATCC ® CRM-6633 ™ Strain NRS 231 produces small quantity of unsuccinylated subtilin. Subtilin (from lanbiotic) is lanthioninecontaining peptides which showed antimicrobial like pheromone-like autoinducing activity [5].
Stinging nettle (Urtica dioica L.) from Urticaceae family has been used for a long period of time as a medicinal herb in many part of the world especially in Middle East [6,7]. However, this plant is annual and perennial herb which is recognized with its stinging hair, and is used to treat gastritis in Turkish folk medicine [8], and in Iran [9,10]. Besides, it is used to treat rheumatic pain and for common colds and coughs [11].
The present investigation showed the effect of ethyl acetate extract of U. dioica against B. subtilis subsp. spizizenii ATCC ® CRM-6633 ™ Strain NRS 231 which previously was selected as one of our effective and potent crude extract based on its high antimicrobial and antioxidant activities [12,13] at ultra-structural level through SEM observation at 8.33 mg/mL concentration with in vitro time killing. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) values of the extract against B. subtilis subsp. spizizenii ATCC ® CRM-6633 ™ Strain NRS 231 were also performed and the time-killed curved as well as structural degeneration of these bacterial cells were studied. The aim of this present study to investigate the antibacterial effect of ethyl acetate extract of Urtica dioica followed by effect of subtilin to reach bactericidal effect (synergistic effect) against Bacillus subtilis subsp. spizizenii ATCC ® CRM-6633 ™ Strain NRS 231 based on the time-kill approach to determine the kill rate of this extract against selected bacteria as well as structural degeneration of bacterial cells were monitored and studied.

Preparation of the Extract
The leaves of U. dioica were collected from Salmanshahr city in Mazandaran province in Iran in August 2007. The Voucher specimens were deposited by the code of 6725-TEH at the Herbarium of the University of Tehran (Iran) in 2010.
The leaves were washed, rinsed and dried under sunlight before ground to powder form. The dried samples were grinded into powder form. Then the residue of the sample was allowed to dry off before extraction. The Soxhlet apparatus was selected for the method of extraction by following solvents from non-polar to polar [14]. These solvents were namely; hexane, chloroform, ethyl acetate and methanol extract. The dried ethyl acetate extract was weighed and then kept in 4°C. The ethyl acetate was selected due to its high antioxidant and antimicrobial activity.

MIC and MBC Determination
The broth micro-dilution assay was conducted for this study to detect minimum inhibitory concentration (MIC). The bacterial extract at twofold dilution was prepared with sterile medium.  Also the test was performed in triplicate [16,17]. Viable cells were calculated every 4 h for 48 h to give exact cfu/mL, meanwhile kill curves were plotted with time against the logarithm of the viable cell count. In order to accurately, each experiment was carried out twice on separate occasions. Generation time is following by formula by Todar (2012)

Preparation of bacteria cells for Scanning Electron Microscope (SEM)
For the preparation of bacterial cells as a sample for SEM analysis, 1 mL aliquots of bacterial cell suspension (1×10 5 cell/mL) were seeded on Nutrient Agar plates from the culture bottles under sterile conditions. The bacteria cell suspensions were taken at the late exponential growth phase and were incubated at 37°C

RESULTS AND DISCUSSION
Based on various different studies, some medicinal plants might indeed be capable sources of new antibacterial agents even against some antibiotic-resistant strains [19,20]. The antimicrobial activity of U. dioica may due to the presence the activity of 5-hydroxytryptamine (5-HT) [21,22]. Lass-Flörl et al. [23] and Perkhofer et al. [24] found antifungal activity of 5-HT (serotonin) against Candida and Aspergillus species, respectively. Hence, serotonin may affect growth inhibition by disrupting cell membrane synthesis.
Ethyl acetate extract of U. dioica showed bacteriostatic effect against B. subtilis subsp. spizizenii ATCC ® CRM-6633 ™ Strain NRS 231. Based on the graph (Fig. 1), it is expected that the increase in concentration up to 4 times MIC, 8 times MIC and above would cause bigger log cycle reduction and therefore drop the extract near the bactericidal effect. A quantity of biologically active compounds can saturate more target sites and cause rapid bactericidal action against the bacteria [25]. The MBC value was found at 16.67 mg/mL.
The time-kill study was performed over a period of 48 h with bacteria being exposed to ½MIC, MIC and 2MIC values. In this research, a time-kill assay was carried out against B. subtilis subsp. spizizenii ATCC 6633 with ethyl acetate extract of U. dioica to determine whether this extract had antibactericidal activity. Fig. 1 shows the viable cells of B. subtilis subsp. spizizenii ATCC ® CRM-6633 ™ Strain NRS 231 were reduced by 5.57 log 10 CFU/mL within 16 h after exposure to MIC and 2 times the MIC of ethyl acetate extract of U. dioica. Although, control (without ethyl acetate extract) showed a 0.82 log 10 increase in CFU/mL by 12 hours. Bacterial cultures were monitored for up to 48 h, and no regrowth was observed. At control and ½MIC, ethyl acetate extract did not reduce bacterial CFU during first 24 hour after incubation. At MIC and 2MIC, ethyl acetate extract showed a 1 log 10 CFU/mL decrease in colony counts after 12 h but at MIC (8.33 mg/mL) with a subsequent minor regrowth of 0.14 log 10 CFU/mL from 16 h to 24 h. However, the bactericidal effect was achieved at MIC and 2MIC after 16h by this extract. The results showed that the addition of the extract definitely affected the growth of the tested bacteria. The higher concentration of the extract added, the severe inhibition to the cells occurred.
The results for confirmation of the MBC value by spread plating is shown in Table 1       At 24 hours of exposure to the extract (Fig. 2 C), the cells became swollen and altered in its shaped. The shape of the cells became irregular and values in shape and sizes. Some of the cells were shrunken and collapsed. In fact, some of the cells were necrotic. AT 36 hours of exposure (Fig. 2 D) the cells became shrunken and seem to lose their structure and their ability to function normally and eventually unable to resume their growth. The cells became lysed and crumpled revealing that the severity of damages occurred to the cell. The cells showed significant morphological changes with the formation of collapsed cells. At least, only dead cells were observed and this condition was beyond repair.
From our previous study, we found out that this extract showed moderate total phenolic and flavonoid contents at 31.75±0.59 GAE/g extract and 36.19±2.37 QE/g extract, respectively [12]. However, the antimicrobial mode of action is related to the phenolic compounds. The relationship between phenolics compounds and their antimicrobial activity has been reported. Also flavonoids as potent and major therapeutics has been well distinguished [26] and some researchers such as Cushnie and Lamb [27] found that flavonoids can related to damage of bacterial membrane. In this case, flavonoids would help an increase in the permeability of the inner bacterial membrane including loss of membrane potential. There is no general consensus on the actual mechanisms underlying flavonoids antibacterial activities [28].
Some studies also mentioned that oxidized phenols inhibit metabolic enzymes causing an inactivation of the reproductive activity of the cell [29]. Phenolic compounds interact with membrane function through electron transport, protein and nucleic acid synthesis, enzyme activity and nutrient uptake. It seems that chelatinion of transition reactive metals ions such as iron and copper, by them reduces bioavailability for bacterial growth [30]. Some studies revealed that the Gram positive bacteria are more sensitive than Gram negative bacteria.
Cáceres et al. [31] found that Urera baccifera from same family exhibited no antimicrobial activity against some pathogenic microorganisms. Their study on U. dioica extracts showed same results on tested microorganisms. Dulger and Gonuz [32] were tested 80% ethanol of U. dioica extract on some tested bacteria but extract did not show any inhibition zone. In other study by Steenkamp et al. [33] on U. urens (Urticaceae family), they found this plant no possess antimicrobial activity which their results supports other studies [34,32]. However, Janssen and Scheffer [35] found that Urtica dioica had noticeable antibacterial activity against Staphylococcus aureus. Our previous results demonstrated that the ethyl acetate and hexane extract exhibited highest inhibition against some pathogenic bacteria such as B. subtilis, B. cereus, MRSA and Vibrio parahaemolyticus [13]. The mode of action of the ethyl acetate extract against B. subtilis was not studied, but it could be assumed that the effects of the ethyl acetate extract of U. dioica was exerted on the outer membrane of the cell wall which then changed the membrane structure and permeability of the cell. According to Zao et al. [36], this alteration of the membrane structure may be referred to the breakage of the hydrogen bonds which functions in maintaining the hardness of the membrane. Further study is needed for the isolation and identification of bioactive compounds including the in vivo evaluation of antimicrobial activity, along with toxicity experiments, present in this extract before it is used for marketing in the making of pharmaceutical drugs.

CONCLUSION
From the above results, it can be concluded that the ethyl acetate extract derived from U. dioica leaves and young stems could be considered as potential antimicrobial agent with its possible applications in pharmaceutical industries.