In Vitro Anti-Staphylococcal Activity of Alkaloids from the Leaves of Callistemon Rigidus

Multidrug resistant Staphylococcus aureus poses a severe global threat worldwide due to their prevalence, genomic plasticity and limited therapeutic options being refractory to most antibiotic classes. This necessitates the discovery of new antistaphylococcal interventions. Callistemon rigidus R.Br. (Myrtaceae) has been found to possess antibacterial potential against clinical isolates. Thied me study was a isolate and evaluate the antibacterial alkaloids from the leaves of Callistemon rigidus and assess their in vitro anti-staphylococcal potential. Alkaloid isolation was carried out by modified method for plant alkaloid extraction. The in vitro anti-staphylococcal potential of the alkaloid bioactive fraction was assessed by using micro broth dilution and plate count assay methods. Pus and wound isolates had MIC 50 of 80 μg/mL and IC 50 of 27.22 μg/mL respectively. Burn isolates showed MIC 50 of 320 μg/mL and 13.57 μg/mL respectively. Urine and vaginal isolates exhibited a MIC 50 of 80 μg/mL and IC 50 of 13.15 μg/mL respectively. Cefixime had MIC 50 values was 320 μg/mL, 160 μg/mL and 160 μg/mL for pus and wound, burn, urine and vaginal isolates indicating the refractory behaviour when compared to alkaloid bioactive fraction from the leaves of Callistemon. The alkaloid bioactive fraction exhibits immense activity compared to standard antibiotic Cefixime. This work is the first report of alkaloids and their antimicrobial activity from Callistemon rigidus leaves. The results suggest further isolation of individual alkaloids from alkaloid bioactive fraction and assessment their anti-staphylococcal activity as leads for development of anti-staphylococcal drugs.


INTRODUCTION
M ultidrug resistant Staphylococcus aureus has created a critical situation for the clinicians due to their refractory behaviour against the current armamentarium of antimicrobials.Methicillin resistant Staphylococcus aureus (MRSA) infections complicate the therapy in the immunocompromised, the elderly, the infants and patients in surgical and burn units (Saxena and Gomber, 2010).Hence there is a need to develop therapeutic alternatives to overcome the refractory behaviour in treating chronic infections caused by these multidrug resistant pathogenic microbes.Plant extracts possess a variety of specialized chemicals and biochemicals as a response to various environmental stresses like competition for nutrients, space, allelopathic interactions and pathogenesis (Saxena and Kumar, 2000).These specialized chemicals and biochemicals are largely underexplored and possess antimicrobial properties which may be directly or indirectly used in development of new drugs to overcome the multidrug resistant pathogenic microbes.
Phytochemical investigations previously have revealed aromatic nature of the plant owing to the higher content of α-pinene, 1, 8-cineol and α-terpineol (Jirovitz et al., 1997).The crude leaf extract possesses higher concentration of alkaloids whose antimicrobial properties have not been reported in the literature so far.In the present study we report the isolation of alkaloids from leaves of Callistemon rigidus and their assessment for anti-staphylococcal potential using a test panel of reference and clinical isolates.

Leaf Collection
Fresh and healthy leaves (no visible contamination) of Callistemon rigidus were collected in month of October from the Thapar University campus by Ms. Charu Gomber and confirmed with the herbarium sample deposited at the Department of Biotechnology and Environmental Sciences, numbered as #7 San 03.Samples were thoroughly washed under running water and air-dried.The fresh weight was noted, and the samples were subjected to drying at 37°C and pulverised into a fine powder.

Isolation of crude alkaloid extracts
Microwave assisted extraction was employed for direct extraction of the alkaloids from the dried pulverized leaves of Callistemon rigidus by cellular destruction initially and then steeping with 5% acetic acid at 120 rpm, 28 °C for 2 h.The insoluble material was removed by filtration and the aqueous phase was washed with dichloromethane (CH 2 Cl 2 ) so as to remove chlorophylls followed by extraction with ethyl acetate for removal of fats.The aqueous phase was further taken up for basification with sodium carbonate as it exhibited the antimicrobial activity.It was re-extracted with dichloromethane to obtain organic and aqueous fractions which were tested for antimicrobial activity (Hadi and Bremner, 2001) (Fig. 1).Subsequently phytochemical tests using Dragendroff's, Mayer's and Marquis Reagents were carried out to confirm the presence of alkaloids.

Evaluation of antimicrobial activity of crude Alkaloid Bioactive Fraction (ABF)
The bioactivity of the crude alkaloid bioactive fraction was evaluated by in vitro microbroth dilution method using 96-well microtitre plate and plate count assay.

Test Panel of Microorganisms
The test panel comprised of reference and clinical isolates of Staphylococcus aureus.The resistance patterns of these isolates have been predetermined and have been grouped as VRSA, VISA, MRSA and MARSA (Table 1).The maintenance medium of these cultures was Trypticase Soya Agar and these were activated on cation adjusted Mueller Hinton (MH) broth 18-24 h prior to the test.

In vitro microbroth dilution assay
The in vitro microbroth dilution assay using 96 well microtitre plate and 3-(4, 5-dimethyl-2-thiazolyl -5-Diphenyl-2H)-tetrazolium bromide (MTT) assay was performed to establish to MIC and MIC 50 of the alkaloid bioactive fraction (O' Shea et al., 2009).Since visual MIC of each organism varies in the test panel of microorganisms being tested, MIC 50 is the term used for the minimum MIC which is inhibiting 50% of the microbes in the test panel.The alkaloid bioactive fraction (ABF) was evaluated between 640-10 μg/mL.50 μl of the bacterial suspension in saline was added to 125 μl of MH broth to achieve a final bacterial cell concentration of 10 5 cells in the test and control wells on the titre plate.Subsequently the plates were incubated for 2.5 hours at 37°C after which 25 μl of the test extract at different concentrations was added.These were then incubated for 24 hours at 37°C.After 24 hours 20 μl of 0.02% MTT was added to each well.The MIC value was taken as the lowest concentration of the ABF where no colour change occurred.The assay was performed in triplicates.Cefixime served as a positive control.

Plate count assay
The surface plate count assay was used to estimate the viable counts of the test panel bacteria at MIC concentration of ABF (Miles and Mishra, 1938;Slack and Wheldon, 1978).Briefly 10 μl aliquots were withdrawn using a sterile tip   2,4,6,8,10,22,24 h from the 96-well titre plates and placed as a single drop on MH agar plates divided into number of sectors and observed for the growth of the bacteria as pinhead colonies without the presence of any confluence after incubation of 12-18 h at 37 °C.

Statistical analysis
All the data presented in the tables and figures are Mean ± SD values of triplicate readings.Further Graph pad Prism ver.5.1 has been used to assess the difference in IC 50 values in different groups of test microbe's viz.puswound, burn and urine-vaginal isolates by one way ANOVA and Bonferroni's multiple comparison test.

RESULTS
The minimal inhibitory concentration (MIC) of ABF as observed by in vitro micro broth dilution assay using visual method using MTT ranged between 10-640 μg/mL for Pus and wound isolates.The MIC 50 of these isolates was 80 μg/mL.Burn isolates exhibited a MIC range of 20-640 μg/mL with a MIC 50 of 320 μg / mL.Vaginal and Urine isolates exhibited MIC range of 80-640 μg/ mL with MIC 50 of 80μg/mL.The MIC for the reference strain was 80 μg/mL.The positive control Cefexime exhibited a MIC in the range of 40-320 μg/mL (Table 2).A non-significant difference between average MIC values of ABF and Cefixime was observed in different groups of test isolates.
The viable count reduction by plate assay indicated that at MIC concentrations the ABF induced a bactericidal action in SauA4 and SauG2.Bacteriostatic activity of ABF at MIC concentration was exhibited by SauG3 and SauG10.Rest pus and wound isolates exhibited a reduction in viable count when compared to the control (Fig 2).
Among the burn isolates both Sau G15 and Sau G17 exhibited reduction in viable counts by 1 log at MIC concentration of the ABF when compared to initial inoculums count indicating a bactericidal action (Fig. 3).
Sau G7 and Sau G11 were the only among urine and vaginal isolates exhibiting bactericidal activity at MIC concentrations of ABF.There was 2-log and 3-log reduction in the viable count in Sau G7 and Sau G11 respectively when compared to the initial inoculum count (Fig. 4) IC 50 range of ABF for pus and wound isolates was between 3.422-189.9μg/ ml.Sau A4 exhibited the least IC 50 of 3.42 μg/ml.The burn isolates exhibited IC 50 in range of 6.413-80.9μg/ml.Sau G16 exhibited the least IC 50 of 6.4 μg/ ml.The urine and vaginal isolates were having an IC 50 in the range of 4.21-   3).
A non-significant difference was found in the average IC 50 values of pus-wound, burn and urine-vaginal isolates by one way ANOVA at p<0.05.Similarly Bonferroni's multiple comparison tests also indicated a nonsignificant difference in the three groups of test isolates.

DISCUSSION
Plant alkaloids are known to possess anti-cancer and anti-bacterial activities.Alkaloid rich fractions from leaves of Prosopis julifora exhibited a MIC of 50 μg/ml against Staphylococcus aureus (Singh et al., 2011).Alkaloid extract from stem bark of Mahonia manipurensis exhibited an antibacterial activity against Bacillus cereus and Enterococcus faecalis however Staphylococcus aureus was not tested by them (Pfoze et al., 2011).Garba and Okeniyi (2012) have evaluated the total alkaloid extracts from Jatropha curcas, Calotropis procera, Mangifera indica, Carica papaya and Psidium guajava and found that the possessed a broad spectrum antibacterial activity.All the alkaloids from leaves of Elaeagnus mollis had obvious anti-microbial effect on Bacillus   (Patrice et al., 2007).Alkaloid from Sida acuta leaves exhibit potential antibacterial activity in the range of 16 -400 μg/ml (Karou et al., 2005).Our results also exhibit a potential activity of the alkaloid bioactive fraction from Callistemon rigidus with MIC 50 ranging between 80-320 μg/ml and IC 50 values ranging between 3.422-189.9μg/ml.The presence of alkaloids possessing anti-bacterial activity in Callistemon rigidus is being reported for the very first time.Further separation of individual alkaloids from the alkaloid bioactive fraction and their assessment against the same test panel of clinical, multidrug resistant Staphylococci would help in finding out relevant alkaloids which could be developed into drugs.Further synergistic activities of these alkaloids would also be helpful in developing formulations in development personal care and hygiene products to prevent transmission of Staphylococcus in the community and hospital settings.

CONCLUSION
The alkaloid bioactive fraction from Callistemon rigidus was as efficient as evident from its MIC 50 and IC 50 values against the test panel isolates.The ABF was having potential anti-Staphylococcal activity and was efficacious as Cefixime which is a pure antimicrobial.Hence further separation of individual alkaloids from the ABF would reduce the MIC 50 and IC 50 value than currently observed to overcome the refractory strains of Staphylococcus aureus.

Figure 2 :Figure 3 :
Figure 2: Kill pattern of pus and wound isolates by alkaloid bioactive fraction at MIC

Figure 4 :
Figure 4: Kill pattern of urine and vaginal isolates by bioactive alkaloid fraction at MIC

Table 2 .
Minimal Inhibitory Concentration (MIC) of ABF against test isolates by in vitro microbroth dilution assay.from the test samples containing MIC concentrations of ABF at different time intervals viz.
a -Non-significant difference in mean MIC values of ABF and Cefixime by unpaired t-test micropipette