Time-kill Curve Studies of Ampucare Against Escherichia coli, Staphylococcus
aureus, Klebsiella pneumoniae and Proteus vulgaris
Present study attempts to determine antimicrobial efficacy of Ampucare stored at different conditions by time kill curve studies against Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae and Proteus vulgaris. In all storage conditions, a rapid killing time was achieved by Ampucare. Bacterial count was less than 3 Log10 cfu mL-1 after 6 h of study in all organisms under study. No deviation in pattern of bacterial inhibition was found in all conditions of storage of Ampucare. There was no re growth reported even after exposure for longer time under influence of Ampucare. In conclusion, Ampucare has good antimicrobial activity under all storage conditions of study against E. coli, S. aureus, P. vulgaris and K. pneumoniae.
The infection of wounds in hospitals and in the community cause problems for
both the treatment of patients and infection control. The unique properties
of Ampucare, a polyherbal preparation for wound healing, are mainly due to presence
of Azadirachta indica (Neem) and Curcuma longa (Turmeric) as major
ingredients. Azadirachta indica is regarded as The Wonder Tree and Natures
Drug Store and is reported to possess antimicrobial, wound healing, antiinflammatory,
fungistatic, fungicidal, antioxidant, bactericidal and free radical scavenging
activities. It is also useful in control of ulcer infections and skin disease
infections (Fabry et al., 1996; Bandyopadhyay
et al., 2004; Subapriya and Nagini, 2005;
Jain and Bansal, 2003; Gomes et
al., 2007). Azadirachta indica originated from Sri Lanka, India
and Burma (Gomes et al., 2007). It is now grown
in Bangladesh, Cambodia, Nepal and in many other countries around the world
(Schmutterer and Asche, 1986). In Kenya it grows along
the coast and is traditionally used to treat several diseases. The Kiswahili
name for the plant is Mwarubaini which means a cure for forty diseases. Curcuma
longa is reported to have antibacterial (Ruskin, 1992;
Kundu et al., 2005), antimicrobial and antifungal
(Costa-Lotufo et al., 2002) properties useful
in wound healing (Wuthi-udomlert et al., 2000)
and diabetic impaired healing (Sidhu et al., 1998)
have been reported.
Therapeutic efficacy of many indigenous plants for several disorders has been
described by practitioners of traditional medicine in India. Azadirachta
indica is a tree which has been used for a long time in agriculture and
medicine. Azadirachta indica is an indigenous plant widely distributed
in India. Antimicrobial properties of A. indica were studied by several
authors (Sidhu et al., 1999) who reported the
antimicrobial activity of the seed oil against a variety of pathogens.
Ampucare derives its name from the word amputation i.e., intentional surgical
removal of a limb or body part, to remove diseased tissue or relieve pain. Ampucare
is indicated for treatment of diabetic leg ulcer, burns, bedsores, venous ulcer,
arterial ulcer, skin infections, traumatic wounds and post-operative wounds.
Efficacy and wound healing trend of Ampucare in patients suffering from diabetic
leg ulcer, venous ulcer, arterial ulcer, burns, cellulitis is, traumatic wounds,
post operative wounds and bedsores has also been studied and successful results
have been demonstrated. Several reports on antifungal activities of Ampucare
are available (Lund et al., 1997; Wolfe
et al., 1996).
The present study is conducted to evaluate antimicrobial activity of Ampucare under normal storage, freeze thawed and sedimented condition against Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae and Proteus vulgaris.
MATERIALS AND METHODS
The following strains obtained from Microbial Type Collection Center of
Institute of Microbial Technology, Chandigarh, India were used for the study-Escherichia
coli (MTCC No-739), Staphylococcus aureus (MTCC No-737), Proteus
vulgaris (MTCC No- 426) and Klebsiella pneumoniae (MTCC No-109).
Ampucare which contains extracts of Azadirachta indica and Curcuma linga
as major ingredients was used for the study. Ampucare was provided by manufacturer,
Venus Remedies Limited, India for the study. Ampucare was kept under following
conditions for this study:
||Ampucare-A: Stored under normal room temperature
||Ampucare-B: Frozen for 5 h at 0°C and thawed at
||Ampucare-C: Forced to sediment under centrifugation
at 5000 rpm for 1 h
Media and Reagents
Following media and reagents were used in study-Sterile water, 0.65% saline,
Phosphate buffer, Chlorine, Tryptic Soy broth, Lactose broth, DE neutralizing
broth with tween 80, Tryptic Soy agar, Sabouraud Dextrose Agar, MacConkey agar,
Mannitol salt agar and Sterile deionized water. All media were manufactured
by Hi Media, India and prepared as per manufacturers instructions.
Preparation of Inocula
The inoculum suspension were enumerated in duplicate by standard microbiological
procedures at the initiation and completion of testing appropriate dilution
are prepared and enumerated by standard microbiological procedure. To prepare
the inoculum suspension from an agar plate, the microbial growth was washed
from the agar surface with buffered phosphate dilution to make 10 fold dilutions
from 10-1 to 10-10 of the organism. An inoculum of 1.0
mL of each dilution was plated on the appropriate media. Plates were incubated
at 37°C for 48 h. Count of surviving organisms were recorded. Average triplicate
plate (3 plates from each dilution) counts were multiplied by the dilution factor
to arrive at cfu mL-1.
Time-Kill Curve Studies
One-way Analysis of Variance (ANOVA) was used to determine statistical difference
between Ampucare A, Ampucare B and Ampucare C in all organisms under study.
p<0.05 were considered statistically significant. A kill curve (log10 cfu mL-1 vs. time) was drawn for each product and linear regression analysis for each set of data was made. For each strain, time-kill bacterial kill studies were performed in Ampucare with an inoculum of 5x106 to 1x106 cfu mL-1. A flask of inoculated MH broth with no Ampucare served as a control. The surviving bacteria were counted after 0, 1, 2, 3, 4, 5 and 6 h incubation at 37°C by sub culturing 1 mL serial dilutions in to DE neutralizing broth.
Determining Time-Kill Endpoints
A bactericidal effect is defined as a 3 log decrease in the cfu mL-1
or a 99.9% kill over a specified time (Wolfe et al.,
1997). The definition of kill for this study has been used as per National
Committee for Clinical Laboratory Standards (1992) together with modifications
based on a suggestion by Handwerger and Tomasz that a kill can be determined
at 6 h. (May et al., 1998). A constant logarithmic
rate of kill has been assumed during a time-kill. A 90% kill at 6 h is equivalent
to a 99.9% kill at 24 h. In this study the kill measurement was determined by
the actual reduction in viable counts at 6 h for each isolate.
This study was conducted at Venus Medicine Research Centre, India between June to August 2008.
Time-Kill Curve Analysis
For E. coli time-kill curve analysis demonstrated bacterial killing
in 0 to 6 h. In all conditions under study, there was non significant change
in colony count in each time point. For Ampucare-A, 6.16 to 2.30 log10
cfu mL-1; for Ampucare-B 6.22 to 2.30 log10 cfu mL-1
and for Ampucare-C 6.50 to 2.30 log10 cfu mL-1 of colony
count was recorded (Fig. 1).
||Time kill curve of E. coli
||Time kill curve of S. aureus
||Time kill curve of P. vulgaris
With S. aureus time-kill curve analysis demonstrated that under all
conditions of study, there was non significant change in colony count in each
time point. For Ampucare-A, 6.16 to 2.30 log10 cfu mL-1;
for Ampucare-B 6.18 to 2.30 log10 cfu mL-1 and for Ampucare-C
6.17 to 2.30 log10 cfu mL-1 of colony count was recorded
In P. vulgaris also there was non significant change in colony count in every time point. For Ampucare-A, 6.36 to 2.70 log10 cfu mL-1; for Ampucare-B 6.33 to 2.60 log10 cfu mL-1 and for Ampucare-C 6.30 to 2.78 log10 cfu mL of colony count was recorded (Fig. 3).
||Time kill curve of K. pneumoniae
Similarly, for K. pneumoniae time-kill curve analysis demonstrated good bacterial killing in 6 h and all conditions under study, there was non significant change in colony count in each time point. In Ampucare-A, 6.41 to 2.30 log10 cfu mL-1; in Ampucare-B 6.36 to 2.60 log10 cfu mL-1 and in Ampucare-C 6.39 to 2.30 log10 cfu mL-1 of colony count was seen (Fig. 4).
There is no re growth of these organisms in the culture media under study even after exposure for longer time under influence of Ampucare.
A study of neem oil obtained from A. indica seed has been performed
(Handwerger and Tomasz, 1985). The use of plant extracts
and phytochemicals with known antimicrobial properties may have great significance
in therapeutic treatments. Use of plant extracts in the treatment regimen of
various diseases are gaining importance as antimicrobial, antibacterial, antiviral
and anti fungal activities of many plants are reported particularly against
skin diseases such as eczema, burns, ulcers and herpes (Gandhi
et al., 1988). Traumatic wounds are the most common type of wounds
and may include abrasions, contusion, incision tunneled wound lacerations, bites,
cuts and thermal wounds (Leaper, 2006). Most of patients
that visit emergency department are victims of traumatic lacerations. Burns
refer to any type of extremity of damage experienced by the skin. The causal
factor may be heat, cold, chemicals, radiation, steam, gases or electric current.
First degree burns are confined to erythema only, second degree burns involve
papillary dermis and may also involve reticular dermis layer whereas third degree
burns include charring of the skin and produce hard eschars (Singer
et al., 1997; Sevitt, 1976). These wounds
delay in the healing process by multiple microbial infections.
There are no reports of microbial efficacy studies of Ampucare, although the
activity of this therapeutic preparation has been established as clinical useful
in wound healing. There are only some reports available, which indicate that
ingredients used in Ampucare are antibacterial and antifungal in nature (Gandhi
et al., 1988). Combination of these ingredients into finished formulation
has not been studied so far. In the present study, Ampucare addition to a culture
lead to a clear reduction for the bactericidal population over a period of time.
Escherichia coli in the concentration of 1.7 Log10 cfu mL-1
is reduced to 0 Log10 cfu mL-1 with in of exposure to
Ampucare. Similarity, S. aureus also show reduction of bactericidal population
by 5 hof exposure. P. vulgaris and K. pneumoniae in this studies
show reduction in much lesser time than other organisms of the study. All conditions
of storage under this study have shown similar pattern of results. Ampucare
has shown excellent microbial killing properties in in vitro conditions.
There is not much variation in antimicrobial activity of Ampucare under normal
storage, freeze thawed and sedimented condition against Escherichia coli,
Staphylococcus aureus, Klebsiella pneumoniae and Proteus vulgaris.
Moreover, present study has also demonstrated that Ampucare has excellent
bactericidal properties which is evident by reduction in number of colonies
only in 6 h.
In conclusion, Ampucare has bactericidal properties which is expressed over a period of time in time-kill experiments. There is no impact of freezing/thawing or sedimentation of Ampucare on the pattern of time-kill curve in organisms under study.
Authors are thankful to CGM, Domestic operations of Venus Remedies Limited for providing the samples of Ampucare for this study.
Bandyopadhyay, U., K. Biswas, A. Sengupta, P. Moitra and P. Dutta et al., 2004. Clinical studies son the effect of Neem (Azadirachta indica) bark extract on gastric secretion and gastroduodenal ulcer. Life Sci., 75: 2867-2878.
Costa-Lotufo, L.V., G.M.A. Cunha, P.A.M. Farias, G.S.B. Viana and K.M.A. Cunha et al., 2002. The cytotoxic and embryotoxic effects of kaurenoic acid, a diterpene isolated from Copaifera langsdorffii oleo-resin. Toxicon, 40: 1231-1234.
CrossRef | Direct Link |
Fabry, W., P. Okemo and R. Ansorg, 1996. Activity of east Africian medicinal plants against Helicobacter pylori. Chemotherapy, 42: 315-317.
Direct Link |
Gandhi, M., R. Lal, A. Sankaranarayanan, C.K. Banerjee and P.L. Sharma, 1988. Acute toxicity study of the oil from Azadirachta indica seed (Neem oil). J. Ethnopharmacol., 23: 39-51.
Gomes, N.M., C.M. Rezende, S.P. Fontes, M.E. Matheus and P.D. Fernandes, 2007. Antinociceptive activity of Amazonian copaiba oils. J. Ethnopharmacol., 12: 486-492.
Handwerger, S. and A. Tomasz, 1985. Antibiotic tolerance among clinical isolates of bacteria. Rev. Infectious Dis., 7: 368-386.
Jain, A. and E. Bansal, 2003. Inhibition of propionibacterium acnes induced mediators at inflammation by Indian herbs. Phytomedicine, 10: 34-38.
Kundu, S., T.K. Biswas, P. Das, S. Kumar and D.K. De, 2005. Tumeric (Curcuma longa) rhizome paste and honey show similar wound healing potential: A preclinical study in rabbits. Int. J. Low Extrem Wounds., 4: 205-213.
Leaper, D.J., 2006. Traumatic and surgical wounds. British. Med. J., 332: 532-535.
Lund, B.C., M.E. Klepser, E.J. Ernst, R.E. Lewis and M.A. Pfaller, 1997. Antifungal activity of fluconazole on Candida albicans in an in vitro dynamic model. Pharmacotherapy, 17: 1094-1094.
May, J., K. Shannon, A. King and G. French, 1998. Glycopeptide tolerance in Staphylococcus aureus. J. Antimicrobial Chemotherapy, 42: 189-197.
Direct Link |
National Committee for Clinical Laboratory Standards, 1992. Methods for Determining Bactericidal Activity of Antimicrobial Agents. National Committee for Clinical Laboratory Standards, Wayne, Pa.
Ruskin, F.R., 1992. Neem a Tree for Solving Global Problems. National Academy Press, Washington, DC., USA.
Schmutterer, H. and K R.S. Asche, 1986. Natural pesticides from the Neem tree (Azadirachta indica A. Juss) and other tropical plants. Proceedings of the 3rd International Neem Tree Conference, Jul. 10-15, Nairobi, pp: 1-14.
Sevitt, S., 1979. Review of the complications of burns, their origin and importance for illness and death. J. Trauma, 19: 358-369.
Sidhu, G.S., A.K. Singh, D. Thaloor, K.K. Banaudha, G.K. Patnaik, R.C. Srimal and R.K. Maheshwari, 1998. Enhancement of wound healing by curcumin in animals. Wound Repair Regen, 6: 167-177.
Sidhu, G.S., H. Mani, J.P. Gaddipati, A.K. Singh and P. Seth et al., 1999. Curcumin enhances wound healing in streptozotocin induced diabetic rats and generally diabetic mice. Wound Repair Regen, 7: 362-374.
Singer, A.J., J.E. Hollander and J.V. Quinn, 1997. Evaluations and management of Traumatic lacerations. N. Engl. J. Med., 337: 1142-1148.
Subapriya, R. and S. Nagini, 2005. . Medicinal properties of Neem leaves: A review. Curr. Med. Chem. Anticancer Agents, 5: 149-156.
Wolfe, E.J., M.E. Klepser and M.A. Pfaller, 1996. Antifungal dynamics of amphotericin B (AMB) and fluconazole (FLU) in combination against Candida albicans. Proceedings of the Program and Abstracts of the Infectious Diseases Society of America 34th Annual Meeting, (PAIDSAAM'1996), Infectious Diseases Society of America, Alexandria, Va, pp: 49-49.
Wolfe, E.J., M.E. Klepser and M.A. Pfaller, 1997. Antifungal dynamics of amphotericin B and fluconazole in combination against Candida albicans, effect of exposure time. Pharmacotherapy, 17: 189-189.
Wuthi-udomlert, M., W. Grisanapan, O. Luanratana and W. Caichompoo, 2000. Antifungal activity Curcuma longa grown in a Thailand. Southeast Asian J. Trop. Med. Public Health, 31: 178-182.