Evaluation of activities of some plant leaf extract on typhoidal and non-typhoidal Salmonella isolate from selected hospitals in Bauchi, Nigeria

This study evaluated the antibacterial activity of aqueous and methanol extracts of Cymbopogan citratus, Psidium guajava and Anacadium occidentale on clinical isolates of typhoidal and non typhoidal Salmonella. The typhoidal Salmonella isolates where S. typhi and S. paratyphi A, while non typhoidal was S. typhimurium. Well diffusion method was used. The plant extracts were tested individually and in combinations for synergistic activity. Both methanol and aqueous extracts had significant (p<0.05) in vitro activity. Anacadium occidentale was more active followed by Psidium guajava and the least active was Cymbopogan citratus with higher activity when plants extracts were combined for synergism. The Minimum Inhibitory Concentration (MIC) of extracts on the salmonella isolates ranged from 50 mg/ml to 200 mg/ml and the Minimum Bactericidal Concentration ranged from 25 mg/ml to 200 mg/ml. From the findings, it proved that plants possess some potentials as antibiotics and can be further studied to isolate the compound that is most active and formulated as drug against the disease.


Introduction
Salmonelosis is a disease caused by salmonella species which are members of the family enterobacteriaceace. They are gram negative facultative anaerobic rods. Salmonella species are classified into serovars (serotypes) based on the lipopolysacharide (O) flagellar protein (H) and sometimes the capsular (VI) antigens. There are more than 2500 known serovars, within a serovar, there may be strains that differ in virulence [12] Salmonella is generally divided into two categories; non typhoidal and typhoidal. The non typhoidal salmonella is the most common form and is carried by both humans and animals. Most serotypes of salmonella such as Salmonella jariana and Salmanolla entiritidis cause non typhoidal salmonellosis. Salmonella typhimurium is involved in the invasive non-typoidal. [12].
The serovars responsible for typhoid fever is restricted to human beings, which is transmitted through direct contact with the feacal matter of an infected person, that is to say it's transmitted through feaco-oral route. Typhoid fever is endemic in developing and under developed world where unsanitary conditions are more likely to prevail and which can affect as many as 21.5 million people, each year. Recorded cases of typhoid fever in the developed world are mostly related to recent travel to areas where, salmonella Typhoid is endemic.
Typhoid fever symptoms appear between 8-14 days after eating contaminated food and last anywhere from 3-60 days. They include fever, weakness, lethargy abdominal pain, coughing, nose bleeding and delirium and enlarged organs. Typhoid fever is a serious illness that can result in death [8].
Herbal medicine is still the main stay of about 75-80 % of the world population, particularly, in developing countries for primary health care [11]. This is primarily because of the general belief that herbal drugs are without any side effects. Besides, they are cheap and locally available. Before scientists made impact into the research for drugs curing human infections, the traditional means of treating diseases were done by means of concoctions from plants either in single form or mixtures without knowing that the agents were used against some pathogenic micro-organism [19]. Limited knowledge about the practices of use of plant medication that is herbal medicine and lack of scientific studies of plants have led to the neglect of novel bioactive components that may bring about remarkable result in treatment of infectious diseases with little or no side effect [18].
In the last three decades, the search for natural bioactive compounds that can serve as antimicrobial agents had increased tremendously due to the increasing resistance possessed by microorganisms to synthetic antibiotic [15].
The scope of this study includes isolation of Salmonella, extraction of African lemon grass (Cymbopogan citratus) guava leaf (Psidium guajava) cashew leaf (Anacardium Occidentale) and the testing of the efficacy of these extract on the isolate.

Ethical clearance
The consent of the clinically suspected patients was sort, while the Ethics Committee of Abubakar Tafawa Balewa University teaching Hospital Bauchi gave approval for the study. Confidentiality of the subjects' identities was duly maintained.

Study area
The study area is Bauchi Local Government Area, where samples were collect from new General Hospital Bayara, Specialist Hospital and ATBU Teaching Hospital, Bauchi state is located on Lat. 10

Sample size/Sample population
Sample size is 210. Samples were collected from male, female and children. The sample size was determined using the Fisher formula for cross-sectional study [17].

Clinical samples
A total of 210 clinical samples were collected, blood and stool Samples were collected from patient with febrile or diarrheal illness attending new General Hospital Bayara, Specialist Hospital and ATBU Teaching Hospital, Bauchi on weekly basis from May to August 2016. Blood sample were collected using syringe and needle while stool samples were collected using sterile bottles, patient where served with sterile bottles to collect a small quantity of their stools.

Plants collection and identification
Plant samples collected or used are African lemon grass (Cymbopogan citratus), Cashew leaf (Anacardium Occidentale) and guava leaf (Psidium guajava). These were collected at Mbak area of Dass town a local government in Bauchi state where they are found to be growing naturally or planted and transported to ATBU herbarium for authentication with the following voucher numbers; Psidium guajava ATBUHB 2489, Anacardium Occidentale ATBUHB 2490 and Cymbopogan citratus ATBUHB 2491

Isolation, characterization and identification of Typhoidal and non-Typhoidal Salmonella
Stool sample were inoculated in a non-selective broth-selenite F enrichment broth and incubated at 37 °C for 24hrs, the pre cultured stool sample were sub cultured on deoxycholate citrate agar (DCA), salmonella shigella agar (SSA) agar and brilliant green agar (BGA) and incubated at 37 °C for 18-24 hrs where growth were detected based on their, colonial characteristic and morphological appearance on these media. Blood samples were inoculated into Terathionate broth. A minimum of blood to broth ratio of 1 in 10 mls, was maintained, and this was incubated at 37 °C and checked for signs of bacterial growth daily for up to seven days. Bottles that show signs of growth were subculture on to Brilliant Green Agar (BGA) Salmonella Shegela Agar (SSA), and Mac conkey Agar.
Blood culture broth with no bacterial growth after seven days was sub-cultured before being reported as negative result. Typical colonies of Salmonella appear as pink colonies with or without black centers. Many cultures of Salmonella produce colonies with large, glossy black centers or may appear as almost completely black colonies. [22]

Biochemical screening and serology
Sugar fermentation tests Nutrient broth cultures were prepared and used in Bijou bottles containing the basal medium and appropriately prepared. Carbohydrate (mannitol, maltose, dulcitol, sucrose and glucose) were inoculated with drop of the nutrient broth suspension of the test isolate and were loosely capped and incubated at 37 °C overnight. They were observed for change in colour from amber to red and for gas production (in the medium filled inverted Durham tube) [9,13]

Serotyping of identified Salmonella species
Colonies considered to be of Salmonella spp. were further tested for somatic (O) and flagella (H) antigens with polyvalent antisera (OXOID), [9,13].

Antibacterial sensitivity of commonly used drug for Typhoid treatment
The susceptibility testing was carried out by disc diffusion method using Mueller Hinton agar and it was tested in vitro for susceptibility to the following antibiotics (OXOID Ltd., UK) suggested by [23], Ciprofloxacin (CPX,10 µg), Amoxicillin (AMC,30 µg) and Chloramphenicol (CH,30 µg).

Plant sample preparation
The leaves and grass were washed, drained and air dried at room temperature and grinded to powder using mortar and pestle. All the powdered samples were labelled and stored in a dark polythene bags at room temperature prior utilization [21].

Extraction procedure of plants sample prepared
100 grams of each powder plant sample stored were weighed and extracted with methanol and aqueous (Water) for 3-6 hours using soxhlet extractor [7].

Preparation of inoculum
McFarland standard; 0.5 McFarland standard was prepared by 0.5 ml of 1 % Barium chloride (BaCl2) to 99.5 mls of 1 % Sulphuric acid (H2SO4). The turbidity of 0.5 McFarland standard was used for the estimation of the amount of salmonella. Colonies of Salmonella was suspended in sterilized 0.9 % sodium chloride solution (normal saline) which was compared with 0.5 McFarland solution the microbial suspension (1ml) in normal saline was added to 74 mls of sterile medium kept at 45 °C to give bacterial population density of 1.2×107 Cells/ml [16].

Preparation of stock suspension of extract
Stock preparation of plant extract was prepared by dissolving 0.4 grams of extract in to 1 ml of dimethyl sulfoxide (DMSO) for methanol extract and 1 ml sterilized distilled water was used for aqueous extract to make 400 mg/ml and stored at room temperature pending usage.

Assays for antibacterial activity of plant extracts
The antibacterial activity was carried out by well diffusion method [10]. Preparation of inoculums was obtained using McFarland standard to have inoculums density of 1.2×107 Cfu/mls, wells of 4 mm in diameter were bored on already inoculated Mueller Hilton agar plates using a sterile well borer and 60 µL of extracts was dispensed in the wells and stand for 40min. as pre-diffusion time, these were incubated at 37 °C for 18-24 hr. Diameters of zones of inhibition was determined by subtracting the diameter of the wells and recorded to the nearest millimeter.
Preliminary testing of this preparation was carried out using the stock concentration 400 mg/ml to test whether or not the plant is active. The antibacterial activity assay was done in duplicate at concentrations 400, 200, 100 50 and 25 mg/ml were the means were taken as the mean zones of inhibition (MZI). To investigate the synergistic activity of the extracts equal volume of 0.5 ml from the stock preparation of both solvents, this combination was made in twos and the three and diluted to make concentration of 400, 200, 100, 50 and 25 mg/ml.

Determination of minimum inhibitory concentration (MIC) of extract against the bacteria
This was determined by serial dilution method, where different concentration was obtained. 0.4 g of the Stock solution prepared from plant extract was dissolved in 1 ml of DMSO, making a solution of 400 mg/ml from where serial dilution of different concentrations was made 200 mg/ml, 100 mg/ml, 50 mg/ml, and 25 mg/ml. Nutrient broth was used to produce overnight growth; 0.1 ml McFarland standard inoculums was inoculated in the tubes and incubated at 37 °C for 24 hr. The tubes least turbidity (bacterial growth) was the MIC. [14]

Determination of minimum bactericidal concentration (MBC)
The above tube that showed no bacterial growth was further subculture on to Mueller Hilton agar incubated at 37 °C for 18-24 hr. The least plate that showed no bacterial growth was the MBC. [14] 5. Results and discussion

Screening for synergistic activity of extracts on bacterial isolates
The result of the combination of extract in different combinations according to Solvents used varying ranges of MZI was observed. The activity of combination of two extracts on S. typhi have MZI range from 2-23 mm Table 4, on S. Paratyphi A have MZI range from 1-19 mm Table 5, when it was tested on S. typhimurium it gave MZI range from 1-15 mm Table  6. Table 7 is a result of when the three extracts where combined together depending on solvent and tested on the isolates, MZI result ranged from 2-17.5 mm.

Comparative antibacterial activity of different plant samples used
The inhibitory activity of different plant extract at concentration 400 ml/ml showed different inhibitory patterns, Figures 1, 2 and 3. Anacadium occidantale (Ao) of methanol extract have higher activity ranging from 17-19.

Antibacterial activity of extract
The inhibitory activity of all the crude plant methanol and aqueous extract against the typhoidal and non-typhoidal Salmonella isolates with A. occidental, and P. guajava being the most active Tables 1, 2 and 3 is in conformity with other reports by [6]. This justifies the use of these plants for Therapeutic purposes. The result clearly revealed pronounced activity with methanol extract on the organism than aqueous extracts, This could be as a result of the antibacterial compounds present in the plant which may be more readily extracted with methanol than aqueous solvents. Data reports have shown that some antimicrobial agents are not extractible with aqueous solvents [5,20]. The serotype that showed more susceptibility to the extracts is the non typhoidal, S. typhimurium followed by S. Paratyphi A. and S. typhi has the least susceptibility this could be due to its resistance and virulence as studied by so many researchers. [1,2].
The high antimicrobial potential of many of these plants extracts against test organism is of obvious public health importance. Typhoid fever no doubt is frequently connected to resistance with a number of commercial antibiotics which result into complication as arthritis intestinal perforation and of course death.
The body on the other hand has been observed to accommodate plant extracts at relatively high dose, without visible side effects [3]. The stability of the natural drugs to antimicrobial resistance by the organisms is also an advantage.

Synergetic activity of extracts
The methanol and aqueous extracts where combined to observe for synergetic effect and the study revealed that the combination of the plant extract has higher antibacterial activity effects (Synergism) having a higher inhibitory effect than when used singly as shown on tables 4 ,5, 6 and 7. This clearly indicates that single plant extract could cause inhibitory effect ranging from 0 -19 mm. However, when extract was combined the inhibitory surpassed that of single plant, 0 -22 mm in combination of two tables 4, 5 and 6 and 2 -19 mm, with combination of the three plants as on table 7. The synergetic effect may be due to formation of certain complexes which becomes more effective in the inhibition on the organism, similar conclusions were drawn by [10,24].

Minimum inhibitory concentration (MIC) of extracts
A result of MIC for various species of salmonella subjected to the different plant extracts is presented on Table 8. The MIC of (Ao) on S. typhi with methanol extract was 50 mg/ml and aqueous extract 100 mg/ml, on S. paratypi A., both extracts was 50 mg/ml, on S. typhimurium was 100 mg/ml with methanol extract and aqueous extract was 50 mg/ml.
The MIC of (Pg) with methanol extract on S. tyhi was 50 mg/ml and aqueous extract 100 mg/ml, on S. paratyphi A., was 50 mg/ml for both solvents extract, on S. typhimurium methanol extract was 100 mg/ml and aqueous 200 mg/ml.
The MIC of (Cc) was 200 mg/ml for both solvent extract for the three salmonella isolates.

Minimum bactericidal concentration (MBC)
Result of MBC is as shown on Table 9. The MBC of (Ao) of both solvent extracts on the isolates was 25 mg/ml except for S. typhimurium on aqueous extract which is 50 mg/ml. The MBC of (Pg) extract of both solvents on S. typi and S. typhimurium was 50 mg/ml and on S. paratyphi A. was 25 mg/ml. The MBC of (Cc) extract of both solvents on S. typhi was 50 mg/ml, on S. paratyphi A. was100 mg/ml with methanol extract with and 200 mg/ml with aqueous extract while on S. typhimurium had MBC 100 mg/ml with methanol extract and 200 mg/ml with aqueous extract.

Conclusion
Result obtained from this study indicates that the leaves of Psidium guajava, Anacadium occidentale and Cymbopogan citratus (grass) inhibited the medically important Bacteria-Salmonella species. This proves that the plant possesses some potential as alternative sources of antimicrobial substances.

Recommendation
Since herbal products are better in combinations as revealed by this studies, further work is required to carry out a further study into possible combinations of these extracts that would give an enhanced and exploitable activity by synergistic or additive reasons in a single therapeutic formulation. This will offer an advantage of using lower but effective therapeutic dosage of natural product as compared to the dose of synthetic substance that would be required to achieve similar therapeutic effect.

Compliance with ethical standards
In compliance with the ethical standard the consent of the clinically suspected patients was sort, while the Ethics Committee of Abubakar Tafawa Balewa University teaching Hospital Bauchi gave approval for the research. Confidentiality of the subjects' identities was duly maintained. Attached is a copy of the ethical approval and consent form.