Plants have been of great importance from ancient times for various reasons. Plants are known to act as a vital source of natural products which may be utilized in the synthesis of new drugs to protect human beings from different pathogenic infections [1Nascimento GGF, Locatelli J, Freitas PC, Silva GL. Antibacterial activity of plant extracts and phytochemicals on antibiotic-resistant bacteria. Braz J Microbiol 2000; 31: 247-56.
[http://dx.doi.org/10.1590/S1517-83822000000400003] ]. Consumption of fruit juices plays a beneficial role in the maintenance of good health and prevention from several ailments. Citrus fruit juice is commonly consumed by humans due to its nutritional value and special flavor. The positive health benefits of juice have been ascribed in part to the presence of vitamin C (ascorbic acid) in plenty [2Boudries H, Madani K, Touati N, Souagui S, Medouni S, Chibane M. Pulp antioxidant activities, mineral contents and juice nutritional properties of Algerian Clementine Cultivars and Mandarin. Afr J Biotechnol 2012; 11: 4258-67.
[http://dx.doi.org/10.5897/AJB11.2943] , 3Rekha C, Poornima G, Manasa M, et al. Ascorbic acid, total phenol content and antioxidant activity of fresh juices of four ripe and unripe citrus fruits. Chem Sci Trans 2012; 1: 303-10.
[http://dx.doi.org/10.7598/cst2012.182] ]. Also, citrus fruits have been reported to encompass numerous bioactive compounds including phenolics, flavonoids, vitamins, and essential oils. These bioactive compounds are believed to contain plenty of phytochemicals exhibiting antioxidative, anti-inflammatory, antitumor, and antimicrobial properties [4Aruoma OI, Landes B, Ramful-Baboolall D, et al. Functional benefits of citrus fruits in the management of diabetes. Prev Med 2012; 54(Suppl.): S12-6.
[http://dx.doi.org/10.1016/j.ypmed.2012.02.012] [PMID: 22373887] -8Pejin B, Savic A, Sokovic M, et al. Further in vitro evaluation of antiradical and antimicrobial activities of phytol. Nat Prod Res 2014; 28(6): 372-6.
[http://dx.doi.org/10.1080/14786419.2013.869692] [PMID: 24422895] ]. In addition, lemon juice (both from the fresh fruit and juice concentrates) has been reported to contain a significant amount of citric and ascorbic acid content, which is responsible for its acidic behavior. Acid content of any solution is determined by its pH. Among the all citrus fruits, lemon is most acidic with pH 2.30. The acid value of fruits decreases as the fruit ages [9Penniston KL, Nakada SY, Holmes RP, Assimos DG. Quantitative assessment of citric acid in lemon juice, lime juice, and commercially-available fruit juice products. J Endourol 2008; 22(3): 567-70.
[http://dx.doi.org/10.1089/end.2007.0304] [PMID: 18290732] ].

Bacterial species have the genetic ability to transmit and acquire resistance against drugs. Hence, the application of plant products may prove to be a viable option. In the present study, we have evaluated the efficacy of C. limon fruit juice against the five pathogenic microbes such as Salmonella typhi (S. typhi) or Salmonella enterica typhi, which is an obligate parasite that survives only in the human body and is susceptible to various antibiotics. This is a Gram-negative, facultative anaerobic bacterium belonging to the family Enterobacteriaceae. Genus Salmonella is a rod-shaped Gram-negative bacterium responsible for causing typhoid fever. Presently, there are over 2,500 identified serotypes of Salmonella. The bacterial species can be controlled and killed by pasteurization. They are sensitive to low pH (4.5 or below). The bacterial cell division is halted at an Aw of 0.94, in combination with a pH of 5.5 [10Giannella RA. Salmonella 1996., 11Girma G. A Review on Shigella Dysenteriae and Salmonella Typhi: Implications for Food Handlers. Food Science and Quality Management 2015; 39: 55-66.].

Neisseria gonorrhoeae (N. gonorrhoeae), also known as gonococci, is a Gram-negative diplococcus bacterium. It was isolated by Albert Neisser. The species are facultatively intracellular and are found in pairs. It is responsible for causing the sexually transmitted disease called gonorrhea. Gonococcal infection is a major health problem; nearly sixty million cases are reported worldwide annually. The highest number of cases have been reported from developing countries including India. More than 300,000 cases are reported from United States in Centers for Disease Control and Prevention. The site of infection of gonococcus (exclusive human pathogen) is urogenital epithelia. However, in men and women urethra and the uterine cervix are the initial sites for gonococcal infection. It has been studied that this organism remains intracellularly located during infection in humans. Antibiotic treatment is provided initially for the gonococcal infection [12Edwards JL, Apicella MA. The molecular mechanisms used by neisseria gonorrhoeae to initiate infection differ between men and women. Clinical Microbiology Reviews 2004; 965-81., 13Hill SA, Masters TL, Wachter J. Gonorrhea - an evolving disease of the new millennium. Microb Cell 2016; 3(9): 371-89.
[http://dx.doi.org/10.15698/mic2016.09.524] [PMID: 28357376] ].

Citrobactera Gram-negative bacterium belongs to the family Enterobacteriaceae. Species of Citrobacter have a capacity to convert tryptophan to indole. The species C. amalonaticus, C. koseri, and C. freundii can use citrate as a sole carbon source. Citrobacter species are differentiated by their ability to build up uranium from phosphate complexes. These bacteria are found abundantly in the surroundings with soil sewage, fresh water and can also be found in human intestine. They do not cause serious diseases but can be a reason for a few infections like sepsis, meningitis and urinary tract infection. The bacteria have been reported to cause bacteremia, osteomyelitis, endophthalmitis, suppurative arthritis, endocarditis, and intra-abdominal infections, particularly in neonates and immune-compromised hosts [14Ranjan KP, Ranjan N. Citrobacter: An emerging health care associated urinary pathogen. Urol Ann 2013; 5(4): 313-4.
[PMID: 24311922] , 15Gopal SD, Raj S. Incidence of citrobacter urinary tract infection in type 2 diabetes and its relationship to glycemic control. International Journal of Contemporary Medical Research 2017; 4: 60-3.].

Shigella flexneri (S. flexneri) is a rod shaped, non-spore forming, non-motile bacterium. It causes shigellosis, an acute bloody diarrhea. Shigella flexneri is the most common cause of the endemic form of shigellosis, resulting into death of a large number of people. Shigella flexneri has become a major cause of health risk in both the under-developed and the developing countries. It was recognized by Shiga (1890) that one species of Shigella may cause bacillary dysentery. The bacterial species were isolated from a shigellosis patient in 1984 in China. Less number of organisms from 10 to 100 can produce disease. It is a Gram-negative bacterium; It initiates infection by interaction with cells and causes intense inflammation in the colonic and rectal epithelium. The mode of infection is direct contact with an infected person or with contaminated food and water [16Zaidi MB, Estrada-García T. Shigella: A highly virulent and elusive pathogen. Curr Trop Med Rep 2014; 1(2): 81-7.
[http://dx.doi.org/10.1007/s40475-014-0019-6] [PMID: 25110633] ].

Staphylococcus epidermidis (S. epidermidis) belongs to the species of genus Staphylococcus and it is a Gram-positive bacterium. S. epidermidis is a facultative anaerobic bacterium. It is present in normal human flora (skin and mucosal flora). The patients with weak immune system are more susceptible to infection or otherwise, it is known as less pathogenic. The infection caused by S. epidermidis is known as nosocomial infection because it is acquired by hospital equipment or health care facility. It colonizes on the skin surface of human beings. Additionally, the abundant prevalence of S. epidermidis is on the human skin. This high incidence is mainly due to the extraordinary capacity of S. epidermidis to stick to the surfaces of indwelling medical devices during device insertion and formation of multilayered agglomerations called biofilms [17Cheung GY, Rigby K, Wang R, et al. Staphylococcus epidermidis strategies to avoid killing by human neutrophils. PLoS Pathog 2010; 6(10)e1001133
[http://dx.doi.org/10.1371/journal.ppat.1001133] [PMID: 20949069] , 18Namvar AE, Bastarahang S, Abbasi N, et al. Clinical characteristics of Staphylococcus epidermidis: A systematic review. GMS Hyg Infect Control 2014; 9(3): Doc23.
[http://dx.doi.org/10.3205/dgkh000243] [PMID: 25285267] ].

The problem of resistance of microorganisms to antimicrobial drugs is one of the world's current challenges. The advent of some new naturally bioactive components from plants or plant-based products has been of interest to many researchers. Hence, a great deal of attention has been paid to the antibacterial activities of C. limon juice as a potential and promising source of pharmaceutical agents. Due to the rapid increase of antibiotic resistance, plants that have been used as medicines over hundreds of years, offer as an obvious choice for study. It is interesting to determine whether their traditional uses are supported by actual pharmacological effects. On the other hand, plant-based antimicrobials are attractive as they are often devoid of many side effects associated with synthetic antimicrobials. The aim of this study is to detect presence of different phytochemicals in the juice of C. limon, and to evaluate its antibacterial activity against different bacterial isolates. The search for new antimicrobial compounds from natural sources is, thus, an ongoing process.

In order to characterize the presence of some phytochemicals in C. limon juice, the relevant qualitative tests were performed. The results showed that the C. limon juice extract was rich in antioxidant phytochemicals such as alkaloids, flavonoids, phenols, quinines, terpenoids and carbohydrates. The flavonoids are known to show antioxidant activity through scavenging or chelating mode of actions. Therefore, in the present investigation, the total flavonoid content present in the extract was estimated using aluminum chloride colorimetric method. In C. lemon fruit juice extract, the flavonoid content was found to be 19.5 ± 1.15 mg/g. The total phenolic content of C. limon juice was estimated to be 7.5 ± 0.56 mg/g.

A systematic examination of in vitro antibacterial activity of the aqueous preparation C. limon showed inhibitory effect on the bacterial growth. The results of antimicrobial activity of different concentrations of lemon fruit juice against the bacterial species using both the positive and negative controls are presented in Table 1. The antibacterial efficacy of C. limon fruit juice was evaluated by using disc diffusion method as described in Materials and Methods. Among the tested bacterial species, S. flexneri displayed maximum zone of inhibition (15.2 ± 0.17 mm) at 1000 µg/disc concentration followed by S. epidermidis (13.4 ± 0.38 mm). At 700 µg/disc concentration, C. species and S. typhi exhibited zones of inhibition of 13.4 ± 0.32 mm and 9.7 ± 0.17 mm, respectively. The diameter of zone of clearance for standard drug, ampicillin, against different bacterial species used was found to be in the range from 14 mm to 16 mm which shows broad-spectrum range of the drug. However, N. gonorrhoeae was found to display resistance against C. limon fruit juice, but it showed sensitivity to ampicillin displaying a zone of inhibition of 14 ± 0.26 mm. The layout of this experiment on a LB agar plate is shown in Fig. (1).

C. limon is an important medicinal plant of the family Rutaceae. The alkaloids isolated from different parts of this plant such as flower, juice, leaves, peel stem, and root have been shown to display their antibacterial potential against clinically important pathogenic bacterial species [26Kawaii S, Tomono Y, Katase E, et al. Quantitative study of flavonoids in leaves of citrus plants. J Agric Food Chem 2000; 48(9): 3865-71.
[http://dx.doi.org/10.1021/jf000100o] [PMID: 10995283] ]. Citrus flavonoids have a broad spectrum of biological activity including antibacterial, antifungal, anti-diabetic, anticancer and antiviral activities [27Ortun˜o A, Ba’idez A, Go’mez P, et al. Citrus paradisi and Citrus sinensis flavonoids: Their influence in the defence mechanism against Penicillium digitatum Food Chem 2006; 98: 351-8.
[http://dx.doi.org/10.1016/j.foodchem.2005.06.017] ]. Limonoids obtained from C. limon are known to exhibit substantial antibacterial and antifungal activities. The extracts of citrus fruits e.g. lemon, orange and grape fruit are among the most studied natural antimicrobials for food processing because of their strong inhibitory potential of the growth of bacteria [28Corbo MR, Speranza B, Filippone A, et al. Study on the synergic effect of natural compounds on the microbial quality decay of packed fish hamburger. Int J Food Microbiol 2008; 127(3): 261-7.
[http://dx.doi.org/10.1016/j.ijfoodmicro.2008.07.014] [PMID: 18804302] ]. The results reveal the presence of alkaloids, flavonoids, steroids, terpenoids, saponins, cardiac glycosides, and reducing sugars in the juice concentrates responsible for its potential against tested bacterial species. The phytochemicals from lemon have been reported to exhibit antibacterial activity. These phytochemicals are citral [29Shi C, Song K, Zhang X, et al. Antimicrobial activity and possible mechanism of action of citral against cronobacter sakazakii PLoS One 2016; 11(7)e0159006
[http://dx.doi.org/10.1371/journal.pone.0159006] [PMID: 27415761] ], carveol, carvone, citronellol, and citronellal [30Lopez-Romero JC, González-Ríos H, Borges A, Simões M. Antibacterial effects and mode of action of selected essential oils Components against Escherichia coli and Staphylococcus aureus Evid Based Complement Alternat Med 2015; 2015795435
[http://dx.doi.org/10.1155/2015/795435] [PMID: 26221178] ], Linalool and linalyl acetate [31Hossain S, Heo H, De Silva BCJ, Wimalasena SHMP, Pathirana HNKS, Heo GJ. Antibacterial activity of essential oil from lavender (Lavandula angustifolia) against pet turtle-borne pathogenic bacteria. Lab Anim Res 2017; 33(3): 195-201.
[http://dx.doi.org/10.5625/lar.2017.33.3.195] [PMID: 29046693] ], α-pinene and β-pinene [32Rivas da Silva AC, Lopes PM, Barros de Azevedo MM, Costa DC, Alviano CS, Alviano DS. Biological activities of α-pinene and β-pinene enantiomers. Molecules 2012; 17(6): 6305-16.
[http://dx.doi.org/10.3390/molecules17066305] [PMID: 22634841] ].

Table 1
Effect of different concentrations of C. limon juice on some bacterial species.

Fig. (1) One of the representative plates showing antimicrobial efficacy of C. limon fruit juice against S. flexneri. S (in centre) and B (on top) represents presence of standard drug i.e. ampicillin (25µg, positive control) and DDW (negative control), respectively. The sections in the plate marked as 2, 3, 4 and 5μl denote the concentrations of C. limon juice to be 100, 200, 300 and 500µg/disc, respectively.

Earlier studies have demonstrated that some phytochemicals such as essential oils, protopine and corydaline alkaloids, lactons, polyacetylene, acyclic sesquiterpenes, hypericin and pseudohyperic present in the C. limon extract are effective in arresting the growth of various bacteria [33Keles OS, Bakirel AT, Alpinar K. Screening of some Turkish plants for antibacterial activity. Turk J Vet Anim Sci 2001; 25: 559-65.-35Johann S, Oliveira VL, Pizzolatti MG, et al. Antimicrobial activity of wax and hexane extracts from Citrus spp. peels MemInst Oswaldo Cruz 2007; 102: 681-5.]. Ghasemi et al. (2009) have indicated that citrus varieties are considered as rich sources of secondary metabolites with the ability to produce a broad spectrum of biological activities [36Ghasemi K, Ghasemi Y, Ebrahimzadeh MA. Antioxidant activity, phenol and flavonoid contents of 13 citrus species peels and tissues. Pak J Pharm Sci 2009; 22(3): 277-81.
[PMID: 19553174] ]. Giuseppe et al. (2007) have reported the presence of limonoids in Citrus species, which could be considered responsible for activity against many clinically isolated bacterial strains [37Giuseppe G, Davide B, Claudia G, Ugo L, Corrado C. Flavonoid composition of citrus juices. Molecules 2007; 12: 1641-73.
[http://dx.doi.org/10.3390/12081641] [PMID: 17960080] ]. Ekawati and Darmanto (2019) have demonstrated lemon juice as a potential antibacterial agent against diarrhea-causing pathogen [38Ekawati ER, Darmanto W. Lemon (Citrus limon) Juice Has Antibacterial Potential against Diarrhea-Causing Pathogen. , Earth and Environmental Science 217 2019.012023
[http://dx.doi.org/10.1088/1755-1315/217/1/012023] ].

The phenolic and flavonoid contents of any extract are responsible for its antioxidant behavior. Our findings suggest that C. limon fruit juice contains a significant amount of total phenols and flavonoids. Xi et al. (2017) have mentioned the total phenolic and flavonoid contents in two different cultivars such as pangdelusaningmeng and beijingningmeng of lemon; the values for total phenolics were 0.47 ± 0.01 and 0.38 ± 0.01; and for total flavonoids 0.44 ± 0.01 and 0.33 ± 0.00 µg/g, respectively [39Xi W, Lu J, Qun J, Jiao B. Characterization of phenolic profile and antioxidant capacity of different fruit part from lemon (Citrus limon Burm.) cultivars. J Food Sci Technol 2017; 54(5): 1108-18.
[http://dx.doi.org/10.1007/s13197-017-2544-5] [PMID: 28416860] ].

In an in vitro anti-microbial study of C. limon fruit peels, the results similar to that of ours have been discussed (Kumar et al., 2011) using disc diffusion method. They have used extracts prepared in different solvents e.g. acetone, ethanol, ethyl acetate, petroleum ether, water and analyzed the phytochemical constituents present in each of them. The acetone extracts of C. limon peel have been found to exhibit significant antibacterial activities against Escherichia coli, Bacillus subtilis, Klebsiella pneumonia, Salmonella typhi and Staphylococcus aureus. The C. limon fruit peel extracts have also been found effective against some gastrointestinal pathogens such as Shigella Spp. and E. coli strains [40Kumar KA, Narayani M, Subanthini A. Antimicrobial activity and phytochemical analysis of citrus fruit peels -Utilization of fruit waste. Int J Eng Sci Technol 2011; 3: 5414-21.]. The present investigation has revealed the antibacterial efficacy of C. limon juice against Salmonella typhi, Neisseria gonorrhoeae, Citrobacter species, Shigella flexneri and Staphylococcus epidermidis. Among the tested bacterial species, Shigella flexneri displayed maximum inhibition followed by the other microbes such as Staphylococcus epidermidis, Citrobacter species and Salmonella typhi.

The effects of natural and concentrated lemon juice, fresh and dehydrated lemon peel extracts, and essential oils, have been examined against V. cholerae O1 biotype Eltor serotype Inaba tox+ [41de Castillo MC, de Allori CG, de Gutierrez RC, et al. Bactericidal activity of lemon juice and lemon derivatives against Vibrio cholerae. Biol Pharm Bull 2000; 23(10): 1235-8.
[http://dx.doi.org/10.1248/bpb.23.1235] [PMID: 11041258] ]. A complete inhibition for V. cholerae was recorded by using concentrated lemon juice and essential oils at all studied dilutions (diluted to 10^-2, and 2 x 10^-3) and different exposure periods (5, 15 and 30 min.). The extract of fresh and dehydrated lemon peel was found to moderately inhibit the expansion of V. cholera. It was moderately inhibited by using fresh and dehydrated lemon peel extracts. Lemon juice (freshly squeezed) displayed complete inhibition of V. cholerae after 5 min of exposure to 108 CFU/ml by using disc diffusion method.

In these experiments, the peel extract of C. limon was shade dried and powdered. This material was used to prepare extracts in cold water, hot water, acetone, ethyl acetate, methanol, and ethanol. These extracts have been evaluated for their antibacterial and antifungal activities against Salmonella typhimurium, Trichophyton mentagrophytes, Micrococcus aureus, Pseudomonas aeruginosa, Microsporum canis and Candida albicans using agar well diffusion method in vitro. Except the hexane extract, all the extracts of lemon registered their antimicrobial activities against the tested bacterial pathogens. The methanol and acetone extracts have been found to display maximum zone of inhibition i.e. 18 mm.

In addition, lemon juice has been found to inhibit the growth of several fungal species [42Al-Snafi AE. Nutritional value and pharmacological importance of citrus species grown in Iraq. J Pharm (Cairo) 2016; 6: 76-108.]. These workers have shown that the fungal pathogens demonstrated a zone of inhibition of 18 mm only in methanolic extract of C. limon. They have also studied the potential inhibitory effect of C. limon oil on lipophilic, yeast-like fungus Malassezia furfur by disc diffusion and micro-dilution methods. The diameter of inhibition zone was found to be 50 mm which were greater than inhibition zone of reference antibiotics such as for gentamycin 16.5 mm and for streptomycin 17 mm. The antibacterial activity of crude extracts (aqueous and ethanolic) of C. limon fruits has been determined against four wound isolates of pathogens. These isolates such as Staphylococcus sp, Pseudomonas sp, Escherichia coli and Klebsiella sp have been observed with inhibition zones 20, 18, 20 and 15 mm for aqueous extract and 15, 20, 11, and 10 mm for ethanolic extract, respectively, using in vitro disc diffusion method [42Al-Snafi AE. Nutritional value and pharmacological importance of citrus species grown in Iraq. J Pharm (Cairo) 2016; 6: 76-108.]. These microbes were found to be coupled with a variety of pathogenesis to the organisms. The abovementioned extracts might diminish the risk of microbes mediated pathogenesis after administration in humans.

The results of the present study demonstrate that aqueous extract of the fruit of C. limon contained the phytochemicals such as alkaloids, flavonoids, phenols, quinines, terpenoids and carbohydrate. The extract was found to possess promising antimicrobial activity against several bacterial species. It appears that the use of lemon extract as an antibacterial agent would be suitable for the development of cost-effective, safe and efficient novel drugs active against several pathogenic multidrug-resistant microorganisms in the future. It could be used as a natural antimicrobial and also represents a useful therapeutic supplement.