Antimicrobial Activity of Some Common Fruits in Bangladesh against Six Different Pathogenic Bacteria

In late time, the interest for the restorative plants is expanding in both creating and created nations because of developing acknowledgment of common items as being non-poisonous, without reactions, effortlessly accessible, and of reasonable costs. Conventional medication, being a huge component in the social patrimony, still remains the fundamental plan of action for a vast dominant part of individuals for treating medical issues like cancer and so on [1]. Roughly 80% of the populace utilizes it as a cure. To manage the advancement of wellbeing and treating illnesses generally use of bioactive organic build-ups has profound roots in man's history and is all-encompassing which is increasing more prominent consideration and prominence in numerous locales of the world [2, 3]. Plants and plant items are utilized in treating jungle fever, looseness of the bowels, consumption, gonorrhoea, stomach issue, and different irresistible sicknesses. Endeavours of researchers in building up biogenic fixings with promising antimicrobial movement are yielding productive outcomes step by step [4]. Regular antimicrobials contain dynamic mixes of bioactive mixes to battle against the obstruction of microbes and save probiotic species in this way, they are alluring choices in numerous malady models [5,6]. The citrus and non-citrus natural products are such a potential specialist where non-citrus organic products incorporate banana (Musa sharpen), pomegranate (Punicagranatum), sapodilla (Manilkarazapota), apple (Malusdomestica), pineapple (Ananascomosus), cucumber (Cucumissativus), guava (Psidiumguajava) and so forth and the citrus organic products incorporate lime (Citrus aurantiifolia), pomelo (Citrus maxima), kaffir lime (Citrus hystrix) et cetera. Several health benefits of citrus fruits have been described in part to Vitamin C, which is  the most common vitamin found in fruits and vegetables [7]. A plethora of Citrus species are having potential antimicrobial activity followed by different secondary metabolites or phytochemicals [8]. These phytochemicals are believed to be responsible for wide range of protective health benefits including anti oxidative, anti-infl ammatory, antitumor, and antimicrobial activities [9,10]. Pineapples are the most recent and weirdest weapon to develop in the worldwide fight against superbugs which are anticipated to slaughter up to 10 million individuals per year by 2050. Moreover, potential enzyme named Bromelain, obtained from pineapple, is a strong anti-enter pathogenic agent that showed most efficacies against several pathogenic bacteria. Diminishing effectiveness and opposition of pathogens to antimicrobial medications made the inquiry of another antimicrobial operator as a vital system for the foundation of elective treatments is troublesome dealing with contaminations. E.coli, Enteropathogenic E. coli, Enterotoxigenic E. coli, S. aureus, V. parahaemolyticus, and Salmonella spp. are the real reasons for intestinal diseases. Pineapples, Lime, Kaffir lime, and Pomelo are heavenly tropical natural products that have been praised for quite a long time for their particular and exceptional taste, as well as for their marvelous medical advantages. Because of the report of expanding advancements of medication obstruction in human pathogen and in addition unfortunate symptoms of certain antimicrobial specialists, it is important to scan for new operators which are better, less expensive, and without reaction for treating irresistible sicknesses particularly in creating healthy nations. On the other hand as the wellbeing parts of substance or manufactured sustenance added substances are being scrutinized, the interest for normally happening additives has been expanded around the world [11]. The investigation of novel antimicrobial specialists from regular assets comprehensive of plant or plant-based items (flavor or herbs) and others has been utilized chiefly to cure disease, nourishment security, and sustenance preservation reason since numerous years back [12,13]. Overall aimless employments of anti-infection agents to control pathogenic microscopic organisms have prompted the spread of the safe strains of microorganisms making treatment of bacterial diseases by these medications troublesome. In this way, researchers are looking for safe elective treatments against these hazardous and even perilous pathogens. Plants create numerous phytochemicals having antimicrobial properties. These might be utilized as normal medications which are for the most part non-toxic and more affordable than the manufactured medications. In this investigation, four distinctive antimicrobial characteristics contain assets, for example, non- citrus natural products incorporate pineapple (Ananascomosus), and the citrus organic product incorporates lime (Citrus aurantiifolia), pomelo (Citrus maxima), kaffir lime (Citrus hystrix) were broken down for antimicrobial adequacy. Finally, in this endeavor, we tried to discover the antimicrobial properties of the juices of some basic eatable Citrus and non-citrus organic products against six pathogenic microbes.

Cultures of six pathogenic bacteria namely- Gram-positive S. aureus and other is Gram-negative E. coli, Enteropathogenic E. Coli (EPEC), Enterotoxigenic E. Coli (ETEC), V. parahaemolyticus, Salmonella spp. were obtained from the institutional microbiology laboratory and were stored in a refrigerator. Antimicrobial properties of fresh fruit juices were evaluated by agar well diffusion method. Briefly, petriplates containing nutrient medium composed of peptone, beef extract, sodium chloride, agar and distilled water were prepared for the evaluation of antimicrobial activities of fruit juices. 100µl of inoculum of each bacterium under our study was uniformly spreaded on the nutrient agar lawn in separate petriplates with the help of a sterilized glass spreader and the plates were then incubated for 30 minutes in the room temperature inside a laminar hood. Following this, with the help of a sterile cork, wells having the diameter of 6mm were bored in the plates. 50 µl of fresh  crude and diluted fruit juice samples, distilled water and antibiotic disc erythromycin & penicillin were poured separately into the wells. Distilled water and antibiotic acted as negative and positive controls respectively. After 10 minutes of incubation at room temperature all the plates were transferred to an incubator and kept inside it at 37°C for 24 hours. Following the incubation, plates were checked for the presence of zones of inhibition (ZOI). Diameters of the observed ZOIs were measured in mm and the results were recorded by taking photographs of the ZOIs on the plates.

Fruit juices are valuable and readily available resources for primary health care and complementary health care system, undoubtedly fruit juices containing substances of medicinal value that have yet to be discovered, though large number of fruit juices are constantly being screened for their antimicrobial effect, these fruits may prove to be a rich source of compounds with possible antimicrobial activities. In the present study agar well diffusion method was used here in order to get antimicrobial activity of fresh citrus and non-citrus fruit juice against six bacterial strains among one is Gram-positive S. aureus and others are Gram-negative E. coli, Enteropathogenic E. coli (EPEC), Enterotoxigenic E. coli (ETEC), V. parahaemolyticus, Salmonella spp. Also MIC measurement was done to know the minimum inhibitory concentration of fruit juices by OD measurement using UV. Spectrophotometer. The non-citrus fruit include banana (Musa acuminate), pomegranate (Punicagranatum), sapodilla (Manilkarazapota), apple (Malusdomestica), pineapple (Ananascomosus), cucumber (Cucumissativus), and guava (Psidiumguajava) whereas, only pineapple can show antimicrobial activity. The citrus fruit include lime (Citrus aurantiifolia), pomelo (Citrus maxima), kaffir lime (Citrus hystrix) whereas, all showed antimicrobial activity. Results of our study showed that juices of four fruits exhibited inhibitory actions against the Gram-positive S. aureus and other is Gram-negative E. coli, Enteropathogenic E. coli (EPEC), Enterotoxigenic E. coli (ETEC), V. parahaemolyticus, Salmonella spp. Table no.1-4 represented the antimicrobial activity of various concentrations (100%, 75%, 50%, and 25 %.) of fresh Lime, Kaffir lime, Pineapple and Pomelo fruit juices respectively. The minimum inhibitory concentration (MIC) of various concentration of Lime, Kaffir lime, Pineapple and Pomelo juices based on OD measurement (mean±sd) represented in Table 5-8. The zones of inhibition against various selected pathogens were measured in mm (mean±sd). Zone of inhibition were seen against all species confirming the antimicrobial activity of fresh Lime, Kaffir lime, Pineapple and Pomelo juices (Figures 1-4). The fresh crude lime fruit juice produced the highest antimicrobial activity against S. aureus (21.33±1.155mm) followed by V. parahaemolyticus (20.33±0.577mm), E. coli (18.00±1.00mm), EPEC    (18.333±0.577    mm),    ETEC    (17.667±0.577mm), & Salmonella spp. (14.00±1.00 mm) (Table 1). The fresh crude kaffir lime fruit juice produced the highest antimicrobial activity against EPEC    (23.00±1.00mm)    followed    by    V.   parahaemolyticus (19.33±1.154mm),     ETEC      (16.667±0.577mm),     S.      aureus (19.67±0.577mm) & E.coli (16.667±1.528 mm) (Table 2).

The fresh crude pineapple fruit juice produced the highest antimicrobial activity against EPEC (18.00±1.00 mm) followed  by E. coli (16.33±0.577 mm), & ETEC (11.33±1.527 mm) (Table 3). The fresh crude pomelo fruit juice produced the highest antimicrobial activity against EPEC (15.667±0.577mm) (Table- 4). Table 1-4 show the diameters (in mm) of zones of inhibitions (ZOI) created in the agar plate by crude (100%) and diluted (75%), (50%), (25%) juices of three Citrus and one non Citrus fruits respectively. It appeared from our study that one Citrus fruit namely –lime exhibited inhibitory actions against all the bacteria tested in our study. Pineapple also exhibited inhibitory action against as many as three bacterial species except for S. aureus, V. parahaemolyticus, Salmonella spp. Kaffir lime, another Citrus fruit exhibited its inhibitory activities against as many as five bacterial species except against Salmonella. The fresh citrus fruit juices & pineapple juices were subjected to get the MIC against selected pathogens

The MIC observed by OD measurement, whereas 75% concentration of lime juice showed as minimum inhibitory concentration (MIC) against E. coli (0.083±0.0159), EPEC (0.0447±0.0136), V. parahaemolyticus (0.181±0.0108),  S. aureus (0.169±0.0145), Salmonella spp. (0.2530±0.0135), and 50% concentration of lime juice against ETEC (0.0553±0.0138) respectively (Table 5). The MIC observed at 75% concentration of pomelo juice against EPEC (0.2410±0.0105) (Table 8). The MIC observed at 25% concentration of kaffir lime juice against ETEC (0.0303±0.0182), 50% concentration of kaffir lime juice against E. coli (0.064±0.0125), and S. aureus (0.0760±0.0207), 75% concentration of kaffir lime juice against EPEC (0.0957±0.0156), and V. parahaemolyticus (0.1570±0.0125) (Table 6). The MIC observed at 25% concentration of pineapple juice against EPEC (0.2453±0.0145), 50% concentration of pineapple juice against ETEC (0.2503±0.0165), 75% concentration of pineapple juice against E. coli (0.0247±0.0181) (Tables 7,8). From the above observation we can see that, Pineapple juice, showed appreciable inhibitory potential both in crude and in diluted form against E. coli, EPEC & ETEC but failed to inhibit other three bacteria such as V. parahaemolyticus, Salmonella spp. & S. aureus. Lime juice, however, showed appreciable inhibitory potential both in crude and in diluted form against E. coli, EPEC, ETEC, V. parahaemolyticus, Salmonella spp. & S. aureus. Whereas, Pomelo juice, showed appreciable inhibitory potential both in crude and in diluted form against EPEC only. Kaffir lime juice; however, showed appreciable inhibitory potential both in crude and in diluted form against E. coli, EPEC, ETEC, V. parahaemolyticus & S. aureus but failed to inhibit Salmonella spp. (Figures 5-6).

Herbal medicines are a valuable and readily available resources for primary health care and complementary health care system, undoubtedly the plant kingdom still holds many species of plants containing substances of medicinal value that have yet to be discovered, though large number of plants and their fruits are constantly being screened for their antimicrobial effect, these may prove to be a rich source of compounds with possible antimicrobial activities, but more pharmacological investigations are necessary. Overall, in terms of extent of the increasing ZOIs,  it appears that Citrus fruits exhibited better inhibitory activities against pathogens than non-Citrus fruits. The non-citrus fruit include banana (Musa acuminate), pomegranate (Punicagranatum), sapodilla (Manilkarazapota), apple (Malusdomestica), pineapple (Ananascomosus), cucumber (Cucumissativus), guava (Psidiumguajava) whereas, only pineapple showed antimicrobial activity [14]. The citrus fruit include lime (Citrus aurantiifolia), pomelo (Citrus maxima), kaffir lime (Citrus hystrix) whereas, all showed antimicrobial activity. Enteropathogenic E. coli (EPEC) is only organism that shows sensitive activity against four fruit juices such as three citrus fruits including lime, kaffir lime, pomelo & one non citrus fruit juice including pineapple. The fresh crude lime fruit juice produced the highest antimicrobial activity against S. aureus (21.33±1.155 mm), followed by V. parahaemolyticus (20.33±0.577 mm), E. coli    (18.00±1.00    mm),    EPEC    (18.333±0.577mm),    ETEC (17.667±0.577mm), & Salmonella spp. (14.00±1.00mm) [15]. The fresh crude kaffir lime fruit juice produced the highest antimicrobial activity against EPEC (23.00±1.00mm) followed by

V. parahaemolyticus (19.33±1.154mm)ETEC (16.667±0.577mm), S. aureus (19.67±0.577mm) & E. coli (16.667±1.528mm) [16,17]. The fresh crude pineapple fruit juice produced the highest antimicrobial activity against different pathogenic bacteria including EPEC (18.00±1.00mm) followed by E. coli (16.33±0.577mm) & ETEC (11.33±1.527mm). The fresh crude pomelo fruit juice produced the highest antimicrobial activity against EPEC (15.667±0.577mm) only. This study supports the use of these fresh fruit juices of Pineapple, Pomelo, Lime, and Kaffir lime not only as dietary supplements but also as the agent to prevent or control the bacterial infections. Investigation of the antimicrobial activity of limejuice has been investigated and limejuice has been found to have high antimicrobial activity [18]. In this study it is observed that the potency of  lime  fruit  is  enhanced  by  the type  of  solvent  used indicating that there are some active ingredients in lime which have high antimicrobial effect. Lime juice was found very effective against V. parahaemolyticus. Similar results have been reported by (WHO, 2002), tested four strains of V. parahaemolyticus, E. coli, Salmonella spp. The result of antimicrobial susceptibility assay and the MIC assays showed promising evidence for the antimicrobial activity of fresh Pineapple, Pomelo, Lime, and Kaffir lime juices against bacterial pathogens. The fresh citrus fruit juices & pineapple juices were subjected to get the MIC against selected pathogens. The MIC observed at 75% concentration of lime juice against E. coli, EPEC, V. parahaemolyticus, S. aureus and Salmonella spp. 50% concentration of lime juice against ETEC [19]. The MIC observed at 75% concentration of pomelo juice against EPEC. The MIC observed at 50% concentration of kaffir lime juice against E. coli, 75% concentration of kaffir lime juice against EPEC, 25% concentration of kaffir lime juice against ETEC, 75% concentration of kaffir lime juice against V.  parahaemolyticus and 50% concentration of kaffir lime juice  against  S. aureus.  The MIC observed at 75% concentration of concentration of pineapple juice against ETEC. The antimicrobial activity of lime juice and standard antibiotic (Erythromycin) against EPEC whereas, the lime juice showed maximum antimicrobial activity than antibiotic, here the negative control was distilled water, which showed no antimicrobial activity. Hereafter, same findings found whereas, lime juice showed most potential antimicrobial activity even than penicillin against E. coli. Therefore, these fruit juices could be seen as a good source for useful alternative medicine.