INTRODUCTION:

Staphylococcus intermedius has been isolated from healthy dogs and pigeons as well as diseased dogs. Similar to Staphylococcus aureus, S. intermedius is known to carry many virulence factors. principal causative agent of mainly pyoderma, otitis following skin injuries in dogs and cats. These infections are commonly treated with antimicrobial drugs. It has been reported that the enterotoxin- and leukotoxin-producing S. intermedius strains are more prevalent in dogs than pigeons.^1,2

Under the genus "staphylococcus" there are so many species. There are three medically important species- S. aureus, S. epidermidis , S. saprophyticus. S. aureus causes suppurative diseases as well as systemic (affecting whole body) diseases. In veterinary medicine, the most important species are S. aureus, S. hyicus, and S. intermedius. They are a common and important cause of disease in animals, including abscesses, dermatitis, furunculosis, meningitis, osteomyelitis, food poisoning, and wound infections.³

S. intermedius causes cutaneous, bone, urinary tract and central nervous system infections in animal species. Genes from human strains of staphylococcus aureus is transmitted to staphylococcus intermedius in animal strains.⁴

Yarrow (Achillea millefolium) was named after Achilles, the Greek mythical figure who used it to stop the bleeding wounds of his soldiers. Decoctions have been used to treat inflammations, such as hemorrhoids, and headaches. The most medicinally active part of the plant is the flowering tops. They also have a mild stimulant effect, and have been used as a snuff. Popular in

European folk medicine, Yarrow contains flavonoids (plant-based chemicals) that increase saliva and stomach acid, helping to improve digestion. Yarrow may also relax smooth muscle in the intestine and uterus, which can relieve stomach and menstrual cramps. The flowers are used to treat various allergic mucus problems, including hay fever. The leaves encourage clotting, so it can be used fresh for nosebleeds.⁵

Indian Sarsaparilla (Botanical name: Hemidesmus indicus) is also known as Anantamul in Hindi, Nannari in Tamil. It mainly comprises saponins, tannins, hemidesmine, hemidesmol, hemidesterol, stearoptin, pregnane glycosides, β-sitosterol, indicusin, coumarin, volatile oils, triterpines, flavonoids. Various effects of HI, such as hypoglycemic , hypolipidemic , antioxidant, antithrombotic ,antiinflammatory , antiulcerogenic , hepatoprotective , renoprotective , and neutralization of viper venom. ^6-12

There is no literature work done in herbal extracts against staphylococcus intermedius so far. Hence the present study is aimed to check the bactericidal effectiveness of yarrow leaf and Indian sarasaparilla root extract against S. intermedius that commonly cause RTI in dogs and cats.

MATERIALS AND METHODS:

Plant material:

Yarrow leaf and Indian sarsaparilla root extract is obtained as a gift sample from Green Chem Herbal Extract and Formulations, Bangalore.

Test microorganism:

Bacterial pathogen [MHA-Hi media ,Mumbai]	ATCC
S. Intermedius	29663

Methodology:

Tryptic soy broth is used as a medium.

Preparation of different concentrations of herbal extracts:

The herbal extracts each 200mg were weighed aseptically into a sterile tube and dissolved in 2ml of sterile Tryptic soy Broth (TSB).From the stock solution various concentrations were prepared, viz.,0.15mg, 0.31 mg, 0.62 mg, 1.24mg, 2.5mg, 5mg, 10mg, 50mg, 100mg/100μl respectively in to wells of micro plates. The tested organism was grown in (TSB) Tryptic soy broth medium [MHA-Hi media, Mumbai] for 24hrs at 37°C and concentration was adjusted to 0.5 Mac-Farland Standard.^13-15 The different concentrations of extracts were taken in 100μl quantities in a U bottom micro culture plates. Control well received plain broth without plant extract. The plates were kept in sealed covers and incubated at 37°C overnight and growth/no growth was detected. All the tests were done in triplicate to minimize the test error.

Minimum Inhibitory Concentration (MIC):

Minimum inhibitory concentration of herbal extracts against tested microorganisms was determined by macro broth dilution method.¹⁶A series of two- fold dilution of each extract ( 0.15mg/100μl to 100mg/100μl) was made in to which 100μl of the standardized bacterial suspension containing 106 organisms was made in Tryptic soy broth as specified by National Committee for Clinical Laboratory Standards (NCCLS, 1990).¹⁷The control well received plain broth without herbal extract. The plates were incubated at 37°C for 24 hours and observed for visible growth. As the extracts were colored, MIC could not be read directly by visual methods. Hence subcultures from all the wells were made and growth/no growth is detected. Then the MBC were obtained.

Minimum Bactericidal Concentration (MBC):

The MBCs were determined by selecting wells that showed no growth. The least concentration, at which no growth was observed, was noted as the MBC.

RESULT AND DISCUSSION:

S. intermedius is rare human pathogen .it is seen in saliva of dogs and cats responsible for causing pyoderma. It has been cultured from the Gingiva of 53 of 135 (39%) healthy canines.⁴ In human beings, S. intermedius has rarely been found, even among individuals with frequent exposures to animals. In an investigation of the nasopharyngeal ﬂora of healthy veterinary staff, S.intermedius was found in only 1 of the 144 persons investigated.

It is reported to produce enterotoxin –A which causes food intoxication. Research was done in lung cancer patients who developed catheter-related bacteremia caused by S. intermedius.¹⁸ Yarrow leaf extract inhibits the growth of S. intermedius at concentration of 10mg/ml. whereas Indian sarsaparilla root ethanolic extract inhibits S. intermedius at 50mg/ml. The low values where there is growth inhibition is considered as MIC/MBC.

CONCLUSION:

Gram positive coagulase S. Intermedius shows growth inhibition in the two herbal extracts (Yarrow leaves and Indian sarsaparilla root ethanolic) tested in the study. Further studies should be carried out in application of this herb in vivo to evaluate its effectiveness in curing upper respiratory tract infections, pyoderma seen in dogs and cats and the person who is exposed to this pet animal.

Table 1: Antibacterial activity against coagulase positive staphylococcus intermedius

Bacterial pathogen	Concentration of herbal extracts (mg/ml)									MBC
S. Intermedius	0.15	0.31	0.62	1.24	2.5	5	10	50	100
Yarrow	G	G	G	G	G	G	NG	NG	NG	10 mg/ml
Indian Sarsaparilla	G	G	G	G	G	G	G	NG	NG	50mg/ml

NG =No Growth indicates high effectiveness of the extract

G = Growth indicates less effectiveness of the extract

Yarrow ethanolic extract - Inhibits the growth of S. intermedius at 10 mg/ml

Indian sarsaparilla ethanolic extract - Inhibits the growth of S. intermedius at 50 mg/ml

REFERENCES:

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2. Futagawa-Saito K, Sugiyama T, Karube S, Sakurai N, Ba-Thein W, Fukuyasu T:Prevalence and characterization of leukotoxin-producing Staphylococcus intermedius isolates from dogs and

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17. National Committee for Clinical Laboratory Standards, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard-Fifth Edition. NCCLS document M7-A5. NCCLS, Wayne, PA 19087-1898, 2000.

18. Franc Ois Vandenesch et al Catheter-Related Bacteremia Associated With Coagulase-Positive Staphylococcus intermedius Journal of Clinical Microbiology. 1995,33(9) 2508–2510.

Received on 14.10.2013 Modified on 25.10.2013

Research J. Pharm. and Tech. 7(2): Feb. 2014; Page 173-175