Screening for Antibacterial Properties of Three Medicinal Plants Against Salmonella SP. Isolates Obtained From Broiler Carcass in Indonesia

Antibacterial properties of three medicinal plants used widely amongst the native Indonesians, i.e.,sirih (Piper betel), garlic (Allium sativum) and jinten hitam (Nigella sativa) were screened and evaluatedagainst Salmonella sp. isolates. For this purpose, the three plants' powder, aqueous and ethanol extractswere prepared. The tests' results demonstrated the three plants' anti Salmonella sp. activities. In thisconjunction, the aqueous extract of Allium sativum, the ethanol extracts of Nigella sativa, and Piperbetel, consecutively produced 17 mm, 15 mm and 13 mm diameter of bacterial growth inhibition zones.As a comparison, sensitivity tests of three commercial antibiotics, i.e., chloramphenicol, tetracycline,and gentamycin on Salmonella sp. isolates produced 19.7-27.3 mm growth inhibition zones. Garlichaving antimicrobial potential was comparable to the commercial antibiotics, can be used as adecontaminant against Salmonella sp. to maintain the quality of the broiler carcasses and thereforeprolonging the carcass shelf-life.


INTRODUCTION
The spread of multi-drug resistant pathogens is one of the most serious threats to successful treatment of microbial diseases. Down the ages, herbs and spices have evoked interest as sources of natural products for their potential uses as alternative remedies to heal many infectious diseases (Parekh et al., 2005), including gastroenteritis. In relation to this, Amieva (2005) reported that Salmonella infection was responsible for 10 to 15% of acute gastroenteritis cases.
Basically, Salmonella organisms are gram negative rods that belong to the family of enterobacteriaceae. This group contains the etiologic agents of food-borne salmonelosis as well as the agents that cause typhoid and paratyphoid fever (Jay, 1996). Eggs, poultry, meat, meat products and chocolates are the most common sources of food borne salmonellosis (Amieva, 2005). Salmonellosis is the most prevalent food borne disease in many countries worldwide (D'Aoust, 1997). Chicken products are widely acknowledged to be a significant reservoir for Salmonella and have frequently been incriminated as a source of Salmonella contamination (Mulder 1999;Baeumler et al., 2000).
The development of antimicrobial resistance among pathogenic bacteria has emerged as a major public health concern. This has led to an intensive discussion concerning the prudent use of antimicrobial agents, especially in veterinary medicine, nutrition and agriculture (Caprioli et al., 2000). The utilization of antimicrobial substances has played an important role in animal husbandry, since they are used in prophylaxis, treatment and growth promotion.
Overall, the largest quantities of antimicrobials are used as regular supplements for prophylaxis or growth promotion in the feed of animal herds and poultry flocks. These results in the exposure of a large number of farm animals, irrespective of their health, to frequently sub therapeutic concentrations of antimicrobials (Dupont and Steele, 1987;Franco et al., 1990). Furthermore, antibiotics given to animals and their closely related compounds used in human therapy have been exerting selective pressure on their target bacteria for decades (Witte, 1998), and therefore, can generate a reservoir of antimicrobial resistant bacteria (Endtz et al., 1991, Smith et al., 2002. The presence of antimicrobial-resistant bacteria in animal origin human food stuffs threaten the efficacy of human drugs if the antimicrobial-resistant bacteria or the antimicrobial-resistance genes become incorporated into the human bacterial populations (Smith et al., 2002). The use of agricultural antibiotic increases the frequency of antibiotic resistant zoonotic pathogens such as Salmonella sp. (Smith et al., 2002). Most antimicrobialresistant Salmonella sp. infections are acquired from eating contaminated foods from animal (Angulo et al., 2000).
The husbandry practices used in the poultry industry and the widespread use of medicated feeds in broiler and layer operations made poultry a major reservoir of antimicrobial-resistant Salmonella sp. (D'Aoust et al., 1992). Resistance in Salmonella sp. limits the therapeutic options available to veterinarians and physicians in the treatment of certain human cases of salmonellosis (Witte, 1998). Saad et al. (2007) reported that Salmonella sp. was found about 17.53% in the broiler chicken carcasses sold in local markets and 7.25% in the chicken carcasses released from slaughterhouse.
In this conjunction, this study assessed the potentials of antimicrobial activities of three medicinal plants, i.e.: Sirih (Piper betle), Garlic (Allium sativum) and Jinten Hitam (Nigella sativa) that are widely used amongst native Indonesians to cure various health disturbances. Powder, aqueous and ethanol extracts of the plant materials were prepared and evaluated against isolates of Salmonella sp.
Firstly, Allium sativum or garlic, a member of the Liliaceae family, is one of the cultivated medicinal plants in Indonesia. It is commonly used in many cultures as a seasoning. According to the US Food and Drug Administration survey, garlic is the second most utilized supplement amongst the Echinacheae (Timbo et al., 2006). The genus Allium includes garlic, scallions, onions, and leek. They contain sulfur compounds which are medicinally active. The majority of reported medicinal effects of garlic appear to come from the sulfur containing compounds, high trace mineral content, and enzymes. The types of sulfur found in whole garlic mostly are Salkylcysteine sulfoxide and the γ-glutamyl-Salkylcysteine. The most abundant sulfur compound in garlic is alicin (S-alkylcysteine sulfoxide) (Bongiorno et al., 2008).
Secondly is Nigella sativa Linn. (Black cumin), is an herbaceous plant that has been used for centuries to treat various ailments, including infectious disease. The seeds are commonly used in recipes in Asian countries (Ali and Blunden, 2003;Randawa and Al-Ghamdi, 2002). Thirdly is Piper betel Linn. It is one of the important medicinal plants in different regions. It belongs to the family of Piperaceae. Piper betel leaves extract is reported to contain a large number of bioactive molecules like polyphenol, alkaloids, steroid, saponin, and tannin. The genus Piper (Piperaceae) has been distributed in tropical and subtropical regions of the world (Shalini et al., 2012). Besides, Piper betel is cultivated in India, Srilanka, Malaysia, Indonesia, Philippines (Parmer et al., 1997).
The objectives of this study were to screen and to evaluate the antibacterial properties of three medicinal plants used widely amongst the native Indonesian, i.e., Sirih (Piper betel), Garlic (Allium sativum) and Jinten Hitam (Nigella sativa) against Salmonella sp. Isolates.

Extracting the Antimicrobial Plants Ingredients
The dried plant materials (Piper betle leaves, Nigela sativa seeds and Alium sativum bulbs) were finely ground into powder. Except for the Allium sativum powder, ethanol was then added to the two other plants' powder. The mixtures of Piper betle and the Nigela sativa powder and alcohol were shaken for one hour to homogenize the solution. The agitation was required to accelerate the dilution of the two plants' active substances in the ethanol solvent. Those mixtures were kept for 24 hours. Each of the crude plant extract was filtered by paper filter. Then each of the obtained ethanol solution contained the plant active substances was poured into a florentine tube. The tube was then placed in a rotary evaporator to evaporate the ethanol solvent at 40°C at 140-160 rpm and at 15-20 lbs of pressure.
Sterile aquadest was added to each obtained extract to make four concentrations of the plant extract, i.e. 50, 25, 12.5 and 6.25 percents. Then, 50 micro liters of each concentration was dropped at a sterile paper disc. Each disc was then laid on a Meuller Hinton agar medium that previously had been inoculated with the bacterial isolate and then was incubated for 24 hours at 37°C.
The bacterial growth inhibition zones were observed and measured. The size or the diameter of the growth inhibition zones indicated the effectiveness of the plant substances (the aqueous extract of Allium sativum, and the ethanol extracts of Piper betle, and Nigella sativa) in controlling the bacterial infection.

Culture Collection
Isolates of Salmonella sp. were obtained from broiler carcass samples collected from some local markets in Bogor, West Java, Indonesia. These specimens were then used for bacterial verification. The bacterial specimens were brought to the Indonesia Research Center for Veterinary Science (IRCVS) laboratory in Bogor. They were cultivated in blood agar media plates. The inoculated blood agar plates were incubated for 24 hours at 17°C. The bacterial isolates grown in the nutrient agar plates were identified by employing the Cowan and Steel methods (2003).

Antibacterial Effect of the Medicinal Plant
Susceptibility of Salmonella sp. was determined by agar disc diffusion method using Mueller Hinton agar. Blank discs (Oxoid) filled with each concentration of the plant solutions were laid at the agar media containing 0.1 ml of Salmonella sp. 10 6 cfu/ml equal to 0.5 McFarland Standard. After 24 hours of incubation at 37 o C, the bacterial inhibition zones were measured and recorded.

Antibiotics Susceptibility Test
Susceptibility to different antibiotics was tested using the Mueller Hinton agar media. In this case, various commercial antibiotics discs i.e.: Chloramphenicol 30 µg, Tetracycline 30 µg, and Gentamycin 10 µg were employed. The antibacterial activity was carried out by employing 0.1 ml solution containing 10 6 cfu/ml Salmonella sp., that was inoculated onto the agar media by spreading them over the media's surface. The antibiotics discs were laid at the cultured media (Bauer et al., 1966). The susceptibility of the Salmonella sp. isolates to these antibiotics was recorded after 24 hour incubation at 37 o C.

Experimental Design and Data Analysis
There were two main factors observed in this in vitro study. The first factor was the type of medicinal plants. This factor had three levels i.e.: the Piper betle, the Allium sativum, and the Nigella sativa. The second factor was the concentration of each medicinal plant substance. This factor had four levels, i.e.: 50, 25, 12.5 and 6.25 percents. In this conjunction, as a comparison, commercial antibiotics were also tested against Salmonella sp. isolates. In this case, antibiotic discs successively containing 30 µg Chloramphenicol, 30 µg Tetracycline and 10 µg Gentamycin were employed in the tests. The observed dependent variable of this investigation was the diameter of the bacterial growth inhibition zones produced by the medical plant extracts and the antibiotics.
The analysis of variance was used to analyze data regarding the diameter of the bacterial growth inhibition zones. Additionally, the Duncan's Multiple Range Test procedure was also employed to determine the differences amongst the means of the bacterial growth inhibition zone diameters (Walpole, 1968). Table 1 pointed out that the main effect of type of medicinal plant tested in this study on the bacterial growth inhibition zones was significant. Subsequently, Table 1 revealed that Allium sativum, one of the three medicinal plants tested, produced the greatest effect on the bacterial growth inhibition zones, followed by Nigella sativa and Piper betle.

Result of analysis of variance presented in
Further analysis in Table 2 displayed the main effect of the second factor, i.e.: the concentration of the medicinal plant substance on the bacterial growth inhibition zone. This effect was also significant. Then, Table 2 disclosed also that overall, the 50% concentrations of the three medicinal plant substance tested, produced the largest effect on the bacterial growth inhibition zones, followed by the 25%, the 12.5%, and the 6.25% concentrations.
In this conjunction, garlic that has a broad spectrum of antimicrobial activity could destroy many species of bacteria, virus, parasites, prozoa, and fungi. Additionally, Singh and Shukla (1984) pointed out that garlic is active against strains of bacteria that are highly resistant to antibiotics.
Further observation specifies that garlic is also active against the following bacteria: Shigella dysenteriae, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Streptococcus spp., Salmonella spp., and Proteus mirabilis.
Eventually, the crude extracts and the essential oil of the Nigela sativa seeds possess antibacterial activity against several bacteria (Ali et al., 2001;Halwani et al., 1999). The oil shows pronounced dose dependent antibacterial activity which is more against Gram positive than Gram negative bacteria. Among the Gram negative bacteria that is sensitive to the test is Pseudomonas aeruginosa (Salman et al., 2008). In contrast to the findings of earlier studies, it turns out that Salmonella sp. -the pathogen bacteria tested in this study is sensitive to Nigella sativa.
Additionally, Piper betel leaves are also reported to contain volatile oil, such as betel phenol and chavicol, tannin, sugar, and vitamin C. Moreover, Betel's leaves possess some other activities like antidiabetic, antiulcer, antitumor (Majumdar et al., 2003) and antimicrobial (Majumdar et al., 2002;Shalini et al., 2012). Thus, the findings of these earlier researches are in accordance with the findings of this study, i.e., disclosing the antimicrobial activities of the three commonly used medicinal plants amongst the native Indonesian against the pathogenic bacteria, Salmonella sp. Table 3 disclosed the combined effects of the type of medicinal plants (Allium sativum, Piper betle, and Nigella sativa) and the concentration of the medicinal plant substance on Salmonella sp. growth inhibition zones. These interaction effects were also significant. Further, Table 3 pointed out that Allium sativum produced the largest growth inhibition zones, followed by Nigella sativa and Piper betle at all concentrations. These bacterial growth inhibition zones differed significantly. In this conjunction, Table  3 remarkably demonstrated that of the three medicinal plants tested in this study, Allium sativum at 50% concentration produced the largest bacterial growth inhibition zones.
Additional antibacterial tests were accomplished with commercial antibiotics including chloramphenicol, tetracycline, and gentamicyn. The results are presented in Table 4. All three antibiotics employed were susceptible to bacteria Salmonella sp., isolated from broiler chicken carcasses. Susceptibility of Salmonella sp. to antibiotics chloramphenicol, tetracycline and gentamycin were characterized by the size of the bacterial growth inhibition zones of 27.3, 22.3, and 19.7 mm respectively. As a matter of fact, garlic has been used for thousands of years as food and medicine in many cultures (Braun and Cohen, 2005). Garlic is well known for its anti-infective properties against a wide range of microorganisms (Iwalokun et al., 2004). Garlic can be eaten raw as well as cooked. However, it is more often cooked to enhance flavor as well as to add nutritional benefits. One study mentioned that garlic could be used to extend the shelf-life of livestock origin food products.
On the other hand, the observed Salmonella sp.'s growth inhibition zone elicited by chloramphenicol, tetracycline, and gentamycin indicated that isolate's susceptibilities (Table 4). Yet, the present study demonstrated the largest potency of Allium sativum's antimicrobial effect against Salmonella sp. isolate. This pointed out that garlic had more effective susceptibility potency than Nigela sativa and Piper betel did. Yabaya et al. (2010)

CONCLUSION
The three widely used medicinal plants amongst native Indonesian evaluated in this study, i.e.: Piper betle, Allium sativum, and Nigella sativa had bactericidal effects on Salmonella sp. The higher the concentration of the medicinal plant substances, the larger the diameter of the bacterial growth inhibition zones produced. Of the three plants tested, Allium sativum persistently produced the most effect on Salmonella sp., the gram negative pathogenic bacteria at all concentrations.