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 microflora 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].
1.1 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 flies [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 [9–11].
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 [13–15]. 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) affirmed 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 gram-positive 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 [23–25]. Nevertheless, a contradiction occurred in the findings 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.
1.3 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 fire 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 flexneri, 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 Gram-positive 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, confirms 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.
1.4 MICROBIAL RESISTANCE
The development and widespread use of antibiotics must rank as the most remarkable of all medical advances made in the 20th century. Overconfident 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 [33–35]. 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 first 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 identified 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].
[40] has reported S. typhi resistance to many antibiotics on the market. Antimicrobial susceptibility testing was performed on all serovar typhi isolates by using the Kirby-Bauer disk diffusion method for ampicillin, chloramphenicol, tetracycline, trimethoprim, gentamicin, amoxicillin, ciprofloxacin and ceftriaxone. It was found that S.typhi was resistant to chloramphenicol (73%), trimethoprim (71%), ampicillin /amoxicillin (70%) tetracycline (64%), gentamicin (46%) and amoxicillin/clavulanic acid (24%) but susceptible to ciprofloxacin and ceftriaxone.
[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 significant at P = 0.05 when compared with hot and cold water extracts. Amongst the commercial antibiotics examined, it was concluded that ciprofloxacin had the highest zone of growth inhibition of 9.0 mm; Ofloxacin (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 fluoroquinolones susceptibility criteria and a nalidixic acid screening test in Salmonella enterica serovar Typhi and Paratyphi A. All the isolates were found susceptible to ciprofloxacin and ofloxacin. 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.
1.5 COMBINATION CHEMOTHERAPY
The development of resistance to first-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.