In-vitro Antimicrobial Activity of the Combined Effect of Kalanchoe Crenata and Vernonia Amygdalina on Salmonella Species


 IntroductionThe major breakthrough in the treatment of pathogenic diseases was the unearthing of naturally occurring antipathogenic agents or antibiotics. There have been upsurges in antibiotic-resistant strains of clinically important pathogens, which made way to the emergence of new-fangled bacterial strains that are multi-resistant. The major aim of scientists is to develop new antibiotics or other therapeutic strategies at a pace greater than that at which bacteria are developing resistance. Development of resistance to first-line antimicrobial therapies made way to recommendations for combination therapies for the treatment of some infections and some of this form of chemotherapy seems to be very successful.ObjectivesThis research was carried out the determine the effect of Kalanchoe crenata extract on salmonella Tyhi load. It was carried out to also assess the potency of the extract of Vernonia amygdalina on Salmonella typhi and also to ascertain the effect of the combined extract of Kalanchoe crenata and Vernonia amygdalina on salmonella typhi.MethodIn this research, Salmonella typhi was exposed to a crude extract of Kalanchoe crenata and Vernonia amygdalina and also the combination of the two extracts. Agar wells diffusion method was employed.ResultsThe combined effect was not sensitive to the Salmonella strain. The Salmonella strain was resistant to V. amygdalina than to K. crenata. K. cranata had the strongest activity against S. typhi with its highest zone of growth inhibition of 20 mm and lowest zone of inhibition of 7 mm while V. amygdalina produced consistent zone of growth inhibition of 5–6 mm; The combined effect produced a zone inhibition diameter only at the 100 mg/ml with zone of inhibition value of 14 mm. The subsequent lower concentrations did not show any activity against the microbes. At P-value = 0.05 two-way ANOVA statistics exhibited significant difference amongst the effects produced by the different extracts, though there were no substantial differences in the effects produced by the various concentrations.ConclusionThe salmonella strain was resistant to V. amygdalina than to K. crenata. At P-value = 0.05 there was a substantial difference in the sensitivity of the bacteria to the different extracts.


Introduction
Microorganism normally referred to as microbes are a very important group of organisms in the environment. Various groups of these microbes exist; bacteria, virus and fungi are examples of major groups of microbes that exist [1]. These individual groups have some peculiar characteristics and properties and hence they tend to undergo different life activities to survive [2]. However, individuals in the same group usually have similar life activities but with few variations among members in the group [3]. When these microorganisms get into the human body and are able to overwhelm the immune system and also the normal resident micro ora then they cause infectious diseases [4]. These bacteria responsible for many of the current infectious diseases are therefore referred to as the infectious agent (pathogens) [5].

TYPHOID FEVER
Typhoid fever is a bacterial infection that affects people globally and is caused by the bacterium Salmonella typhi. The disease can be transmitted through the bacterial contamination of water, milk, food, fruits and vegetables. Healthy carriers of the infection and even contaminated food handlers can transmit the disease to healthy individuals. The bacteria can also be carried from feces to food by ies [6]. The World Health Organization estimated an annual rate of about 12.6 million infections with about 600,000 possible deaths [7].
Poor food hygiene, poverty and inadequate supply of clean water has resulted in the increase in typhoid infections and acute gastroenteritis in African [8]. In recent years multidrug resistance to the typhoid infection has increased worldwide [9][10][11].
Due to the increase in resistance to the treatment of typhoid fever by antibiotics, herbal preparations are gaining popularity in both rural and urban areas in Africa for the treatment of the disease. These medicinal herbal preparations have less side effects compared to the chemical agents [12]. This work was carried out to investigate the combined effect of kalanchoe crenata and Vernonia amygdalina on salmonella typhi.
1.2 VERNONIA AMYGDALINA V. amygdalina, which is commonly known as the bitter leaf, is one of the plants mostly exploited in West Africa [13]. Studies have proven many therapeutic properties of phytochemicals present in the plant [13][14][15]. The antimicrobial activities of V. amygdalina on E. coli, Salmonella and Shigella sp [16,17] have been experimented. The result revealed that the organisms were not as active except for Shigella sp which showed considerable sensitivity [17]. It was further proven that the growth of gram positive bacterium Staphylococcus aureus and the gram negative bacterium E. coli have been strongly inhibited by the aqueous extracts of the leaves [18].
The entirety of the plant is pharmacologically useful [19]. Both the roots and leaves are utilized in phytomedicine for the treatment of kidney diseases, fever, stomach discomfort and hiccups, among others [20]. Bukar et al., (2013) a rmed that the sensitivity of V. amygdalina was more towards gram positive bacteria compared to the of gram-negative bacteria; However, other researchers revealed that the activity of V. amygdalina on gram-negative bacteria was comparable to its effect towards the grampositive species [18,21]. For example, methanol extract of V. amygdalina affected not only the growth of gram-positive bacteria such as B. subtilis, B. cereus, M. kristinae, B. pumilus, S. aureus and E. cloacae but exhibited potency against gram-negative bacteria which include Proteus vulgaris, Klebsiella pneumoniae, Shigella dysenteriae, Pseudomonas aeruginosa, and E. coli [22,23]. Extract of ethanol from the plant also exhibited an antibacterial effect on both gram-positive (Clostridium sporogenes, Staphylococcus pyogenes and S. aureus) as well as the gram-negative (E. coli and Salmonella typhi) bacteria [23][24][25]. Nevertheless, a contradiction occurred in the ndings regarding the activity of the ethanol extract obtained from V. amygdalina. (Ogbulie et al., 2007) showed that the best solvent and the technique to give the optimal antibacterial effect of V. amygdalina are ethanol and Soxhlet extractions. On the other hand, despite the inhibitory effect of V. amygdalina on S. aureus, [26] showed that this extract could not inhibit the methicillin-resistant (MRSA UELSHB 102, UELSHB) and methicillin-sensitive (MRSA NCTC 6571) strains of the bacteria while chloroform, water and blended extract of V. amygdalina leaves exhibited low inhibitory effect on its growth.

KALANCHOE CRENATA
The external applications of K. crenata are the same as those of Bryophyllum pinnatum [27]. The juice obtained by squeezing the leaves that have been passed over re slightly is most commonly used for the treatment of headache, general debility, dysentery, smallpox and convulsion. One or two drops of the leaf juice are dropped into the ear for earaches. A poultice of the leaves is applied over wounds and sores. The leaves can be boiled in water and the extract is given as a sedative for asthma and palpitation [27]. Similarly, the leave extract mixed with honey and salt serves as a remedy for chronic cough. Also, dried leaves extract is applied to the infected wound [28].
The treatment of rheumatism, as well as stiff joint in East Africa, is done by slightly heating the leaves and rubbing it over the human body [29]. Solvent type as well as the preparation technique utilized strongly affect the antipathogenic (antimicrobial) potency of plants [26,30,31]. On the basis of this background, in-vitro antimicrobial activities of the extracts B. pinnatum and K. crenata from various solvents were tested against clinically important pathogens in the work done by [27]. [27] have reported that K. crenata extracts have unequal effects on tested organisms which includes Gram-negative E. coli ATCC 25922, E. coli, Pseudomonas aeruginosa, Salmonella paratyphi, Klebsiella pneumoniae, Shigella exneri, Citrobacter spp, Gram-positive organisms S. aureus ATCC 25213, Bacillus subtilis, S. aureus, Enterococcus faecalis, and a fungus Candida albicans excluding "Omidun" extract of K. crenata which revealed no substantial activity. It was further suggested by Aibinu et al., that extracts of K. crenata showed broad spectrum in their activities and that extracts from the squeezed leaves of K. crenata proved to be the most active; it exhibited improved antimicrobial activity compared to leaves from B. pinnatum prepared using the same method. The effect of K. Crenata has manifested against the Grampositive as well as the Gram-negative organisms with its activity greatly manifested on the Gram-negative organisms [27]. It was also reported that palm wine extracted from K. crenata showed higher activity on the E. coli organisms tested, Citrobacter spp and Salmonella paratyphi and further explain that utilization of palm-wine as a solvent release some amount of active elements which shows high activity against enteric organisms. The different antimicrobial potency exhibited by the plant extract dissolved in different solvents as revealed by various researchers, con rms the traditional use of the plant in the treatment of microbial infections such as sore, dysentery, ear infections, abscesses as well as wound infections. In the work reported by [27] stated that aqueous extracts, as well as methanol extraction of dried leaves of K. crenata, exhibited moderate antibacterial activities.

MICROBIAL RESISTANCE
The development and widespread use of antibiotics must rank as the most remarkable of all medical advances made in the 20th century. Overcon dent assertions that infectious diseases would soon be a thing of the past, however, the threat of resistant strains casts a shadow over all the past achievements of antibiotics [32]. Recently, there is the upsurge of antibiotic-resistant strains of important clinical pathogens, which led to the development of new bacterial species that are multi-resistant [33][34][35]. Mortality and morbidity have suddenly increased, owing to the high cost coupled with non-availability of new generation antibiotics [36]. There is, therefore, the need to unravel other sources with proven antimicrobial potency. This paved way for researchers to delve into more effective antimicrobial agents of plant origin, with much emphasis to discover active ingredient with a potential effect on infectious microbes to synthesize new antimicrobial drugs [37,38]. The major aim of scientists now must be to develop new antibiotics or other therapeutic strategies at a pace greater than that at which bacteria are developing resistance [39]. In 2000, the Food and Drugs Administration approved a new synthetic agent shown to be effective against both MRSA and vancomycin-resistant Enterococcus faecalis. Linezolid (Zyvox), which works by blocking the initiation of protein synthesis, belongs to a new class of antibiotics called oxazolidinones [39]. It is the rst new anti-MRSA compound to be introduced for more than 40 years. Another approach to countering resistant forms is to identify and target the mechanism by which the bacteria combat antibiotic therapy [39]. A team at Rockefeller University in New York have identi ed two genes that enable resistant forms to rebuild their cell walls after antibiotic treatment. Therefore targeting these genes, they hope to restore the potency of a cell wall inhibitor such as penicillin [39].
[41] also collected S. typhi samples from the University College Hospital, Ibadan, Nigeria and exposed to ten standard different antibiotics and also to crude extract of Phyllanthus amarus and Paraquetina nigrescent. Ethanolic extracts of P. amarus had the strongest activity against S. typhi with 8.0 mm zone of growth inhibition followed by hot water (4.7 mm) and cold water (3.8 mm) with statistically signi cant at P = 0.05 when compared with hot and cold water extracts. Amongst the commercial antibiotics examined, it was concluded that cipro oxacin had the highest zone of growth inhibition of 9.0 mm; O oxacin (6.0 mm) Amoxicillin, (4.0 mm) while other antibiotics had no effect on test organism. Unfortunately, the resistance of S. typhi strains to all of these antibiotics is becoming more common globally. As such, appropriate treatment varies with the geographic distribution of resistant strains [41].
[42] evaluated the current uoroquinolones susceptibility criteria and a nalidixic acid screening test in Salmonella enterica serovar Typhi and Paratyphi A. All the isolates were found susceptible to cipro oxacin and o oxacin. However, some research has proven that the combined effects of two drugs have shown more antimicrobial potency than the effects of the two when applied separately. That is, an antibiotic which not having much effect when combined with another will produce an effect greater than the separate effects of each drug.

COMBINATION CHEMOTHERAPY
The development of resistance to rst-line antimicrobial therapies has led to recommendations for combination therapies for the treatment of some infections [43]. The accomplishment of combination therapies in recent times has been revealed by researchers, owing to their application in the treatment of ailment. However, few reports have been given on the in vitro actions that result from various combination of these drugs. For example, an assessment of the in vitro action of azithromycin combined with gentamicin showed growth inhibition which warranted a clinical trial of this combination in treating gonorrhoea infections [43].
Base on this idea some bacteria that used to be monoresistant to certain antibiotics are now being susceptible to this same antibiotic combined with another [43].
In this experiment, the separate effect produced by V. amygdalina and K. crenata is being experimented and then compared to the combined effect produced by the two extracts with an initial hypothesis made that bacteria strain would be sensitive to the various extracts.

PLANT MATERIALS
The leaves of V. amygdalina were collected from the University of Cape Coast (UCC) Science Botanical Garden at Amamoma and were authenticated at the Cape Coast University's herbarium by Mr. Otoo.

PREPARATION OF EXTRACTS
The method (Mother Tincture) devised by Jean-Michael (1994) was employed for the extraction. The leaves of the plant were air-dried for 2 days and then nally dried in an oven at 45 o C for 3 days as described by [44]. The dried leaves were then blended using an electric blender and the powder stored in a sterile bottle at room temperature [45]. Fifty grams (50) g of the powdered plant leaves were weighed and dissolved in 450 mL 70% ethanol. The mixture was then held in an airtight container, kept in a cool dark place for 3 days and then ltered using a sterile lter paper. The ltrate retrieved was concentrated by evaporation using a water bath at 97 o C and then stored in sterile bottles until it is needed.

BACTERIA SPECIES
Strains of Salmonella species were acquired from the Microbiology Laboratory of Centre for Plant Medicine Research, Mampong in the Eastern Region of Ghana.
Consent was sought from the laboraory technician, that the strains were not going to be used on animals or humans for the experimental research but was going to be an in-vitro experiment, therefore no ethical clearance was needed.

PREPARATION OF EXTRACT CONCENTRATIONS
Thousand milligrams (1000 mg) each of the V. amygdalina and K. crenata extracts were weighed using weighing balance and together dissolved into 20 mL of 2% DMSO in a clean and well-dried container.
Another 2000 mg each of the extracts was weighed and dissolved in 20 mL each of 2% DMSO in separate containers. For each of the stocks prepared (100 mg/ml), half of the volume (10 mL) was taken and serially diluted to get concentrations of 50 mg/mL, 25 mg/mL, and 12.25 mg/mL into separate containers. The containers with their contents were covered and stored in the refrigerator until they were needed for use [46].

PHYTOCHEMICAL SCREENING
The phytochemical constituents of both V. amygdalina and K. crenata ethanolic extracts were determined using methods described by [47].

PREPARATION OF INOCULUM
The pure culture of the organisms from the cotton swab was plated out on Salmonella-Shigella agar and incubated at 37 0 C for 24 hours. After incubation, the colony of the organisms was taken and inoculated into 7 mL of peptone water in test tubes and shook vigorously so as to obtain homogeneity of the solution as described by [17].

MICROBIAL SENSITIVITY TEST
Agar well diffusion method as described by [44] was employed using Salmonella-Shigella agar. Agar plates were prepared according to the manufacturer's speci cation aseptically to a thickness of 5-6 mm. The agar was then left to solidify and the plates then upturned to prevent condensate from coming into contact with the agar surface. To ensure sterility, the plates were incubated at 37 o C for 24 hours. The prepared inoculum of the bacteria species was inoculated onto the prepared media by dipping cotton swab into the inoculum and wiping it on the surface of the media. The inoculated agar plates with the lids covered were allowed to dry at room temperature. Thereafter, sterile pipette tips (5.0 mm diameter) were used to punch wells in the seeded Salmonella-Shigella agar. The agar plugs were removed with a amed and cooled inoculating loop. Into the separate well was poured different concentrations of the various plant's extracts and the solvent blank (2% DMSO). Standard antibiotic (Cipro oxacin) which salmonella is known to be sensitive to was used as a positive control [42]. The experiment for each extract and organism was repeated in triplicates so in all 12 plates were prepared. The samples were incubated at 37 o C for 24 hours. The diameter of zones of inhibition on the plates was measured using a transparent meter rule and recorded. The measured zones of inhibition of the different extracts were compared. The zones of inhibition created by the various quantities (concentrations) of the extracts also were compared [17]. Antimicrobial actions of the extracts acquired from V. amygdalina as well as K. crenata leaves and a mixture of the two plant extracts, using 70% ethanol on the test organisms are listed in Table 3. The screening of antimicrobial activity of extracts was assayed in vitro by the agar diffusion method and using Cipro oxacin as a positive control drug in all samples.

Results showing the phytochemical screening of Vernonia amygdalina
The antimicrobial effect was determined by taking the diameter of zone of inhibition recorded. The average and standard error mean (SEM) of the zone of inhibition for each concentration from each of the triplicates were calculated and recorded in Table 3. The values are mean from ZOI of three replicates ± standard error mean The K. crenata extracts was found to be most potent antimicrobial agent with its highest ZOI of 20 mm at 100 mg/ml and lowest ZOI of 7 mm at 12.25 mg/ml; comparing with the V. amygdalina extract and the combined extracts it is found the K. crenata is still the extract that produced the highest ZOI at their lowest concentrations.

Discussion
The antimicrobial action was determined by taking the diameter of the zone of inhibition and recorded. The K. crenata extracts was found to be most potent antimicrobial agent with its highest zone of inhibition of 20 mm at 100 mg/ml and lowest zone of inhibition of 7 mm at 12.25 mg/ml ( Fig. 1.0); comparing with the V. amygdalina extract and the combined extracts it is found the K. crenata is still the extract that produced the highest zone of inhibition even at their lowest concentrations ( Table 1) The microbes showed a slight high degree of resistance against the V. amygdalina more than against the K. crenata; From Table 1 it can be seen that at different concentrations V. amygdalina had a lower zone of inhibition than K. crenata.
The combined effect produced a zone inhibition diameter only at the 100 mg/ml with a zone of inhibition value of 14 mm. The subsequent lower concentrations did not show any activity against the microbes even though their corresponding concentrations for K. crenata showed activity with decreasing effect directly proportional to the decrease in concentration. This shows that V. amygdalina and K. cranata together may produce an antagonistic effect. At lower concentrations (< 100 mg/ml based on this research) there was no activity. However, at higher concentrations (≥ 100 mg/ml) there was an activity with a 14 mm inhibition zone. This result may be due to the fact that the two plants extract when combined produce an antagonistic effect (especially at lower doses of each extract in the mixture). Therefore the activity showed at the 100 mg/ml concentration may be due to the fact that one of the plants is more potent than the other so as there is increase in concentrations of the combined and as such, the relative increase in the individual concentrations the more potent moiety (in this case K. crenata) of the mixture produced its effect but with a lesser activity; in this research K. crenata is the one that contributed more of the effect in the combined extract (zone of inhibition: 14 mm) since at all concentrations it had higher activity than V. amygdalina in their separate forms; therefore when comparing the zone of inhibition of K. crenata and ''K. crenata in mixture'' (combined extract) it is seen that at 100 mg/ml the zone of inhibition produced by K. crenata was reduced from 20 mm to 14 mm (Table 1) in the effect produced by ''K. crenata in mixture'' (combined extract). Note that K. crenata at the same 100 mg/ml concentration produced more activity singly than when it was combined with V. amygdalina.
The standard antibiotic (5 µg Cipro oxacin) produced a zone of inhibition of 26 mm. This result is in line with the standard zone of inhibition range provided by the Clinical Laboratory Standard Institute (CLSI). According to CLSI reference (ZOI ≤ 15 mm is resistance, ZOI ≥ 21 mm implies sensitivity to Cipro). [40] also reported a zone of inhibition of 25.8125 ± 1.875 mm for Salmonella typhi sensitive to Cipro oxacin. However [41], reported that cipro oxacin had a zone of growth inhibition of 9.0 mm for S. typhi, this which implies resistance but the nding was reported as sensitivity.
The phytochemical screening of K. crenata extract revealed the presence of anthraquinones which was absent in V. amygdalina.
Statistical analysis from the two-way ANOVA at P-value of 0.05 exhibited a signi cant difference between the effects produced by the different extracts, though there were no signi cant differences in the effects produced by the concentrations. For the concentrations, the calculated F-ratio, F (3,6) = 3.194 and P-value = 0.1052. The P-value was observed to be greater than 0.05 after calculation, which implies there is no signi cant difference among the effects produced by the different concentrations.
For the extracts the calculated F-ratio, F (2,6) = 5.995 and P-value = 0.0371. The P-value obtained after calculation was less than 0.05 which implies there exist signi cant difference among the effects produced by the different extracts.

Conclusion
The combined effect was not sensitive to the Salmonella strain. However, K. cranata was sensitive and it is the extract that produced the maximum effects at all the concentrations prepared whereas V. amygdalina was also sensitive but with an almost constant zone of inhibition at all concentrations. It can also be concluded that the salmonella strain was resistant to V. amygdalina than to K. crenata. At Pvalue = 0.05 there was a substantial difference in the sensitivity of the bacteria to the different extracts.

Recommendation
K. crenata has shown potency for antimicrobial activity than V. amygdalina however further works must be done on the V. amygdalina and K. crenata to test for the actual phytochemicals that are producing the antimicrobial effects. The test should be repeated using gram-positive microbes especially to compare the combined effect to that of this research and also using higher concentrations of the combined effect.