Antibacterial Potential of Jatropha sp. Latex against Multidrug-Resistant Bacteria

Objective This study was aimed to evaluate the antibacterial activity of the latex of three species members of Jatropha (J. curcas, J. gossypilofia Linn., and J. multifida) against methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum beta-lactamase- (ESBL-) producing Escherichia coli and ESBL-producing Klebsiella pneumonia, carbapenemase-resistant Enterobacteriaceae (CRE)-E. coli, K. pneumoniae-carbapenemase (KPC), and carbapenemase-resistant Pseudomonas aeruginosa (CRPA). Method The antibacterial activities were calculated based on the inhibition zones using the Mueller–Hinton agar diffusion method, minimum inhibitory concentration (MIC) using Mueller–Hinton broth in a microdilution method, and minimum bactericidal concentration (MBC) using blood agar plate. Results The latex of Jatropha showed antibacterial activities against the MRSA and CRPA. All latex of Jatropha appeared to have the antibacterial activities against MRSA and CRPA in the diffusion method (20.4–23.7 mm and 12–15 mm), MIC (0.19–6.25%, and 25%), and MBC (0.39–12.5% and 50%). Phytochemical screening of latex indicated the presence of flavonoids. Conclusions The latex of J. curcas, J. gossypilofia Linn., and J. multifida has the potential to be developed as antibacterial agents, especially against MRSA and CRPA strain, but further in vivo research and discovery of the mode of its action are required to shed the light on the effects.


Introduction
Bacterial resistance to antibiotics has become a serious problem in the world, including in Indonesia. is is a factor in the high mortality rate and continues to increase every year [1]. Inappropriate use of antibiotics is one of the causes of antibiotic resistance [2]. e emergence of multidrugresistant (MDR) bacteria causes treatment ineffectiveness [3]. is has a devastating effect on patients suffering from MDR bacterial infections, increasing the treatment costs, which become increasingly high and difficult to afford and causing prolonged illness and even death [4]. One alternative to overcome these problems is to use natural ingredients such as materials from fungi [5] and plants [6], one of which is Jatropha.
Our study aimed to investigate the antibacterial activities of Jatropha latex against MDR bacteria. In this study, we used three species of Jatropha plants to measure the antibacterial activities against MDR bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli, ESBL-producing Klebsiella pneumonia, carbapenemase-resistant Enterobacteriaceae (CRE)-E. coli, K. pneumoniae-carbapenemase (KPC), and carbapenemase-resistant Pseudomonas aeruginosa (CRPA). ree types of latex used in this study were Jatropha curcas, Jatropha gossypilofia Linn., and Jatropha multifida.

Bacterial Preparation.
e tested organisms were subcultured BAP for 24 h at (35 ± 2)°C. e colonies were inoculated in a normal saline solution.

Agar Well Diffusion Assay.
e antibacterial activities of latex were evaluated using agar well diffusion assay [7,8]. In this method, 100 μL of each test organism, which was equivalent to a 0.5 McFarland standard, was inoculated on the MHA. en, it was spread on to the surface of the agar using a sterilized glass spreader. After 10 minutes of inoculation, the wells were prepared using a sterilized steel cork borer (1 cm in diameter). Wells were made on each plate, out of which three wells were loaded with each latex. Each test was done in triplicate. All the plates were then incubated aerobically at 35 ± 2°C for 16-20 h. e antibacterial activities were evaluated by measuring the diameters of zones of inhibition (mm) against the test organism.

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC).
e MIC of Jatropha latex was determined using Mueller-Hinton broth microdilution [9]. MIC determination was performed by a serial dilution technique using 12-well microtiter plates. MHB (100 μL) was placed into the well/plate and Jatropha latex (100 μL) in the dilution series. 10 μL bacterial cell suspensions were placed in each well/plate. Microplates were incubated aerobically at 35 ± 2°C for 16-20 h. e lowest concentrations without visible growth completely inhibited the bacteria (MIC) [10]. e MBC was defined as the lowest concentration of the extract that did not allow any growth [10]. e MBC of Jatropha latex was determined following the methods described by [11] with slight modifications. Wells were subcultured using a 10 μL inoculating loop on to a 5% sheep BAP at (35 ± 2)°C for 16-20 h incubation.

Phytochemical Screening.
e Jatropha latex was screened for the presence of different classes of secondary metabolites, including alkaloids and flavonoids, using previously described methods [12].

Agar Well Diffusion Assay.
e antibacterial activities of three Jatropha species were examined in vitro using the diffusion and dilution of six MDR bacteria. Figure 1 shows that all latex exhibited inhibition zones against MRSA and CRPA. J. curcas showed a greater inhibitory zone diameter of 23.7 mm against MRSA and 15 mm against CRPA (Table 1).

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC).
e antibacterial activities of the Jatropha latex (J. curcas, J. gossypilofia Linn., and J. multifida) were assayed in vitro by the agar microdilution method against MRSA and CRPA. Table 2 demonstrates that among the three Jatropha latex, J. multifida exhibited the smallest value of MIC and MBC against MRSA (0.19% and 0.39%). Figures 2-4 show that J. curcas and J. gossypilofia Linn. had the same activities against MRSA. Table 2 displays that all latex exhibited the same value of MIC and MBC against CRPA (25% and 50%). Figures 5-7 exhibit that J. curcas, J. gossypilofia Linn., and J. multifida had the potential to be developed as antibacterial agents for MRSA and CRPA strains.

Phytochemical Analysis.
e secondary metabolites are presented in Table 3. Flavonoids are present in all Jatropha latex.

Discussion
e research for new antibacterial agents from a natural or organic source has become a very important endeavor, considering the escalating levels of antibiotic resistance. One of the efforts in this research is focused on the use of Jatropha latex, which is widely available. Traditional medicine has been practiced worldwide, including in Indonesia, for centuries. e agar well diffusion and microdilution methods were used in the present study due to their routine use as a quantitative method in clinical laboratories. In the agar well diffusion method, 100 μL of each MDR organism, equal to a 0.5 McFarland standard, was inoculated on the MHA. e wells were prepared using a sterilized teel cork borer (1 cm diameter). Wells were made on each plate, out of which three wells were loaded with each of latex. All the plates were then incubated at 35 ± 2°C for 16-20 h. Antibacterial activities were evaluated by measuring the diameters of zones of inhibition (mm) against the MDR organism. In microdilution method, susceptibility test in 12 well microtiter plates contained various concentrations (50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.19, 0.09, 0.04, and 0.02%) of Jatropha latex. en, the standardized numbers of MDR bacteria were inoculated into the wells of the microtiter plates and incubated at 35 ± 2°C for 16-20 h.
e MIC value was observed as the lowest concentration where no viability was detected in the wells after incubation. Wells in MIC assays were subcultured using a 10 μL inoculating loop onto a BAP at 35 ± 2°C for 16-20 h incubation. e MBC was defined as the lowest concentration of the extract that did not permit any growth. e latex of Jatropha showed antibacterial activities against the MRSA and CRPA. All latex of Jatropha showed the   of Jatropha neopauciflora Pax demonstrated inhibition against S. aureus. In another study, the latex of Jatropha curcas displayed potent antimicrobial activity against Pseudomonas aeruginosa [14]. Phytochemical screening of latex showed the     presence of flavonoids. ese flavonoid compounds have been reported to be used by plants for protection against bacteria and are responsible for antimicrobial activity [15]. e latex of J. curcas, J. gossypilofia Linn., and J. multifida has the potential to be developed as anti-MDR-bacterial agents, especially against MRSA and CRPA strain, but further in vivo research is still needed to explicate on the effects.

Conclusion
e latex of J. curcas, J. gossypilofia Linn., and J. multifida is prospective for development as an antibacterial agent, especially against MRSA and CRPA strain, but advanced in vivo research and discovery of the mode of its action are necessary to throw light upon the effects.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.