J Islamabad Med Dental Coll 2021 200 O p e n A c c e s s Antimicrobial Activity by Solvents Extracted from Ocimum Basilicum Herb Against Multidrug Resistant Gram-Negative Rods Amna Ikram1, Sidrah Saleem2, Muhammad Imran3, Ayesha Ghazal4 1Scholar, Microbiology, University of Health Sciences, Lahore 2Head of Department, Department of Microbiology, University of Health Sciences, Lahore 3Assistant Professor, Department of Microbiology, University of Health Sciences, Lahore 4Lecturer, Department of Microbiology, University of Health Sciences, Lahore A B S T R A C T Background: Failure of treatment with antibiotics occurs due to increase in number of multidrug resistant gram- negative bacteria, worldwide. The objective of this study was to find out the antimicrobial activity of crude ethanolic extract and its further three fractions by Ocimum basilicum leaves against multi drug resistant gram-negative rods. Material and Methods: This descriptive study was conducted in the Department of Microbiology, University of Health Sciences, Lahore from 1stJuly 2016 to 30thJune 2017. A total of 80 multidrug resistant gram-negative rods were included in this study. Agar dilution method was performed to determine minimum inhibitory concentration of crude ethanolic extract and different fractions i.e., n-hexane, chloroform and ethyl acetate of Ocimum basilicum leaves against multidrug resistant gram-negative rods i.e., extended spectrum beta lactamases and carbapenemase producers. Multi-inoculater was used for inoculation. Result: The mean MICs of crude ethanolic extract, n-hexane fraction, chloroform fraction, and ethyl acetate fraction of Ocimum basilicum against ESBLs were 100.0±8.00, 168.13±8.00, 176.88±8.00 and 41.75±8.00 respectively. Similarly, the mean MICs of crude ethanolic extract, n-hexane fraction, chloroform fraction, and ethyl acetate fraction of Ocimum basilicum against carbapenemase producers were 77.50±8.00, 113.75±8.00, 132.50±8.00 and 29.50±8.00 respectively. Conclusion: Ethyl acetate fraction and crude ethanolic extract from leaves of Ocimum basilicum showed good antibacterial effectiveness against ESBLs and carbapenem resistant organisms than other fractions. This finding may also promote the effective use of O. basilicum herb and its components in modern medicine. Key words: Multi-drug Resistance, Minimum Inhibitory Concentration (MIC), Ocimum bassilicum Authors’ Contribution: 1Conception; Literature research; manuscript design and drafting; 2, Critical analysis and manuscript review; 3 Data analysis; 4Manuscript Editing. Correspondence: Amna Ikram Email: amnaikramm@gmail.com Article info: Received: February 1, 2021 Accepted: December 13, 2021 Cite this article. Ikram A, Saleem S, Imran M, Ghazal A. Antimicrobial Activity by Solvents Extracted from Ocimum Basilicum Herb Against Multidrug Resistant Gram-Negative Rods. J Islamabad Med Dental Coll. 2021; 10(4): Funding Source: Nil Conflict of Interest: N I n t r o d u c t i o n Failure of treatment with antibiotics occurs due to increase in number of MDR bacteria worldwide.1One of the most significant problems in hospital settings is multidrug resistant gram- negative rods (MDR-GNR), which includes MDR Pseudomonas aeruginosa, Acinetobacter baumannii, carbapenemase-producing Klebsiella pneumonia and extended-spectrum-beta- lactamase (ESBL) producing Escherichia coli.2 Medicinal plants are a wealthy source of antimicrobial components. A wide range of medicinal plant extracts were used to treat several O R I G I N A L A R T I C L E J Islamabad Med Dental Coll 2021 201 infections before the progression of western medicine, due to their possible antimicrobial activity.3 In Pakistan, 80% of the people (mostly rural) depend upon the traditional medicines.4 Ocimum basilicum is a common pharmaceutical plant that is traditionally used for the treatment of various diseases worldwide.4 It is a cultured plant widespread in the tropics of Africa and Asia.5 It belongs to the family Lamiaceace and it is the most abundant of the genus Ocimum.6O.basilicum is a culinary herb, commonly called basil, also called sweet basil. Arial parts which include leaves and flower of O. basilicum are conventionally used as galactagogue, carminative, digestive, stomachic, aromatic, antispasmodic and tonic agent.7 Ethanolic, methanolic, hexane and watery extracts of O.basilicum were assessed in a previous study and out of all these extracts of O.basilicum, hexane extract showed the strongest spectrum of antimicrobial activity.8Extract of leaves of basil show antimicrobial activity against human dental plaque pathogens and reduced Streptcoccus mutans and Lactobacillus acidophilus colony count.9 The present study was conducted to find out the antimicrobial activity of O. basilicum herb against MDR-GNR. This study will provide an adequate knowledge about possible advantage of this natural therapeutic product. This study may also promote the effective use of O. basilicum herb and its components in modern medicine. M e t h o d o l o g y This descriptive study was conducted in the Department of Microbiology, University of Health Sciences, Lahore over a period of 1 year, from 1stJuly 2016 to 30thJune 2017. WHO calculator was used for the calculation of sample size with anticipated population proportion to be 70%, significance level 95%, and margin of error 10%. 10 Eighty (80) multidrug resistant gram-negative rods including 40 ESBLs and 40 carbapenem resistant gram-negative rods were collected conveniently from microbiology section of CMH Lahore, Jinnah Hospital Lahore and Post Graduate Medical Institute (PGMI) Lahore. They were re-confirmed at the department of Microbiology, University of Health Sciences Lahore on the basis of morphology, cultural characteristics and biochemical identifications. All carbapenemase producing gram negative rods were reconfirmed for meropenem resistance. Modified Hodge test (MHT) was performed to confirm the organisms for carbapenemase production. To reconfirm ESBLs producing organisms, double disk diffusion test was performed. The results were interpreted according to clinical laboratory standard institute guidelines (2017).11Organisms sensitive to cephalosporins were excluded (because we used ESBLs which are resistant to cephalosporin), confirmed MDR strains were preserved in 16% (v/v) glycerol in brain heart infusion and stored in refrigerator at -70°C till use. Ethical approval was taken from Ethical Review Committee of institute. Ocimum basilicum (Basil) leaves were collected from local nursery of Lahore. Plant was confirmed by taxonomist who worked at Botany Department, University of the Punjab (Lahore). After collection, the leaves were washed first with tap water and then with sterilized distilled water and air dried at room temperature.12Dried leaves of the plant were ground by using electric grinder. Two kilogram of powdered material was soaked in ten liter of ethanol for two weeks. After two weeks, whatmann (1) filter paper was used for filtration. As a result, ethanolic extract of O. basilicum was obtained which was dried under vacuum in a rotary evaporator. The ethanolic extract was concentrated by using a rotary evaporator at temperature 40°C.13 Crude ethanolic extract of O. basilicum was further partitioned according to increasing polarity into n- hexane, chloroform and ethyl acetate fraction. Separating funnel was used for further successive partitioning by three solvents in order of increasing polarity, first by n-hexane, second by chloroform J Islamabad Med Dental Coll 2021 202 and third by ethyl acetate. The layers that were formed in a flask were separated. Rotary evaporator was used to evaporate respective solvent and fractions of O. basilicum dried. Air tight containers were used to store all solvent extracts at 4°C till further investigation.14 Agar dilution method was used for the determination of MIC. The stock solution was prepared using the extract and fractions to be tested. DMSO (Dimethyl sulfoxide) (universal solvent having no antibacterial activity) was used for the preparation of stock.15 The organisms were streaked on nutrient agar medium. After incubation at 37°C for 24h, 4 or 5 pure colonies were selected. They were shifted in a sterile normal saline tube and carefully rotated. Prepared suspension was equal to the 0.5 McFarland’s standards. Further, diluted this suspension in 1:10 sterile normal saline. Inoculums in this dilution had 106CFU/ml concentration. Within 15min of preparation, this suspension was used.16 Multi inoculator was used for inoculation of the prepared extract plates. After inoculation, plates were incubated at 35°C for 24 hours. After 24 hours of incubation, the plates were seen for any kind of growth. Already identified by PCR, ESBL producing strain and Modified hodge test, positive strains were used as positive control for ESBLs and carbapenemase producing organisms respectively from Microbiology department of University of Health Science Lahore. DMSO incorporated in plate containing mueller-hinton agar was used as negative control.2 All statistical analysis was done by using SPSS software (version 17.0, SPSS Inc). All MICs results of crude extract and different solvent fractions of Ocimum basilicum leaves were expressed as mean ± S.D. For comparison of mean among MICs of crude extract and different solvent fractions of Ocimum basilicum leaves, ANOVA test was applied followed by Post hoc tukey test. R e s u l t Out of 40 carbapenemase producing gram negative rods, there were 22(55%) Acinetobacter baumanii, and 18(45%) Pseudomonas aeroginos. Similarly out of 40 ESBL producing organisms, there were 16(35%) Klebsiella pneumoniae and 24(65%) Escherichia coli. Minimum inhibitory concentration (MIC) of crude extract and three different fractions of Ocimum basilicum leaves against MDR gram negative rods is shown in (Table I). Mean MICs of crude extract and different fractions of O.basilicum leaves against multidrug resistant gram negative rods is shown in (Table II). The minimum inhibitory concentration of extract and all fractions of Ocimum basilicum leaves against cabapenemase producing organisms and ESBLs are shown in (Figure I, II). ANOVA test was applied to compare mean MICs of crude extract and different solvent fractions of Ocimum basilicum leaves, for multiple comparison post hoc tukey test was applied. All the results were statistically significant (Table II). Table I: Minimum inhibitory concentration (MIC) of crude extract and three different fractions of Ocimum basilicum leaves against MDR gram negative rods. Organisms Crude ethanolic extract n-hexane fraction Chloroform fraction Ethyl acetate fraction ESBLs (n=40) 75mg/ ml 100m g/ml 125m g/ml 125m g/ml 150m g/ml 175m g/ml 175m g/ml 200mg/ml 40mg/ ml 50mg/ml 2 36 2 2 7 31 37 3 31 9 Carbapen em (n=40) Crude ethanolic extract n-hexane fraction Chloroform fraction Ethylcetate fraction 75mg/ ml 100mg/ml 75mg /ml 100m g/ml 125m g/ml 150m g/ml 100m g/ml 125m g/ml 150mg /ml 30mg/ml 36 4 7 11 15 7 7 13 19 40 J Islamabad Med Dental Coll 2021 203 Analysis of the mean MICs of crude extract and different fractions of O.basilicum leaves against multidrug resistant gram negative rods show significant difference. Figure 1: Comparative analysis of Mean MICs against Carbapenemase Procedures Figure 2: Comparative analysis of Mean MICs against ESBL procedures Ethyl acetate fraction has least value of MIC and shows greatest antibacterial activity against carbapenemase producing organisms and ESBLs followed by crude ethanolic extract, n-hexane fraction and then chloroform fraction. D i s c u s s i o n Phytochemical active compounds like flavonoids, tannins, alkaloids and phenolic compounds in O.basilicum plant extract are potent inhibitors of microbial growth.17-18In the present study, crude ethanolic extract and its further three fractions i-e n-hexane, chloroform and ethyl acetate were used for evaluating its antimicrobial activity. Out of these fractions, ethyl acetate fraction exhibited best antimicrobial activity against MDR-GNR. Ethyl acetate fraction of basil leaves contain polyphenolics, flavonoids and alkaloids.19The highest antimicrobial action of ethyl acetate fraction could be due to these bioactive compounds.17These findings support the idea that ethyl acetate fraction of O.basilicum leaves could be used as antimicrobial with broad-spectrum antimicrobial properties. Crude ethanolic extract showed second best antimicrobial activity. N-hexane and chloroform fractions showed less activity as compared to the fraction of ethyl acetate. Ad khalil 201320 in his study discussed that O.basilicum ethanolic extract contain compounds which have strong antibacterial activity against gram positive (Staph aureus) and gram negative organisms (E.coli). It is known that ethanol is a highly polar solvent which is able to extract phytochemicals efficiently; greater number of active constituents could have been produced by extraction of ethanol which are responsible for good antibacterial activity. Abubutain I 201921 used gas chromatography coupled with mass spectrometry (GC/MS) to Table II: Mean MICs of crude extract and different fractions of O.basilicumleaves against multidrug resistant gram negative rods Organism Crude ethanolic extract N-hexane fraction Chloroform fraction Ethyl acetate fraction P- value MIC against Carbapenemase producers 77.50±8.00 113.75±8.00 132.50±8.00 29.50±8.00 <0.05 * MIC agaisnt ESBL producers 100±8.00 168.13±8.00 176.88±8.00 41.75±8.00 J Islamabad Med Dental Coll 2021 204 analyze ethanolic extract of O.basilicum, results of his study showed that extract contains phenols, terpene, esters, steroids and fatty acids. These compounds showed antimicrobial activity against some gram positive, gram negative and fungal stains that were used in his study. Issazadeh k 201217also reported that crude ethanolic extract exhibited antibacterial activities more than the aqueous extract. This is consistent with findings of this study. N-hexane fraction obtained from crude ethanolic extract of O.basilicum herb exhibited a high range of MIC against multidrug resistant gram negative rods. In a previous research, Patil D 7 reported that n-hexane extract of O.basilicum showed antimicrobial activity against bacterial (gram positive and gram negative) and fungal strains that were used in their study. The results revealed that hexane extract had broad-spectrum activity followed by ethanolic extract against all tested bacteria. Bilal A. 201222also reported that hexane extract of O.basilicum has strong, broad spectrum antimicrobial and anticandidal activity than ethanolic extract. In contrast, the results obtained from current study showed that crude ethanolic extract had strong antibacterial activity than n- hexane fraction. Variations between these results could be due to MDR strains used in current study. Tabassum S 201612 reported the effect of hexane extract of O.basilicum on selected bacterial strains. Staphylococcus aureus, Pseudomonas aeruginosa, E coli, Proteus mirabilis and Klebsiella pneumoniae were included in their study. The results of their study revealed that antimicrobial activity of crude ethanolic and hexane extracts for gram negative bacteria was much higher than gram positive bacteria. In current study, no gram-positive bacteria were used so, in future a study could be designed against MDR-gram positive bacteria. Useful phytochemicals of O. basilicum can be a great scope for future researches. Moreover, isolation and purification of pure compounds should be carried out. HPLC analysis should be done to identify active compounds, because a variety of multifunctional compounds is present in the extract and fractions of Ocimum basilicum, which can be used for the production of novel antibiotics. Chloroform fraction obtained from crude ethanolic extract of O.basilicum herb exhibited high range of MIC against multidrug resistant gram negative rods than all other fractions. Aruna K 201523 showed the effect of O.basilicum leaf extracts against ESBL and MBL producing uropathogens. Methanol, Ethanol, Chloroform, Acetone and Butanol extracts of O.basilicum were analyzed against these organisms. Chloroform extract showed best strong and broad- spectrum antibacterial activity against tested strains than others. The difference in results might be due to difference in extraction technique e.g, the plant extract used in the current study being fractionated from crude, whereas others used pure active chloroform extract. Further GC-MS analysis of the chloroform extract of O.basilicum also done by ArunaK 201523 showed that eugenol and estragole are major contributors. These compounds express good antibacterial activity in the chloroform extract of O.basilicum. Gebrehiwot H 201613 also reported that major constituent of chloroform extract of leaves of O. basilicum was estragole (38.22%). Estragole is a potent phytochemical compound which has the ability to inhibit all tested bacteria and fungi. 13 It can be assumed that the extracts or its components affect some key processes in the organism’s growth. Antimicrobial activity of the different extracts of O.basilicum could be due to some active components (flavonoids, alkaloids, estragole, phenolics, tannins, linalools) which have the ability to combine with extra cellular and soluble protein and to make a complex with bacterial cell wall disrupting membrane of microbes.24 J Islamabad Med Dental Coll 2021 205 C o n c l u s i o n Ocimum basilicum crude ethanolic extract and different solvent fractions had potent antibacterial activity against all tested strains of ESBLs and carbapenemase producing gram negative rods. O.basilicum can be used as alternative antimicrobial drug. The medicinal value of O.basilicum extract might be due to natural bioactive phytochemicals. Acknowledgement We are thankful to University of Health Sciences, Lahore for full technical and financial support to perform this research activity. R e f e r e n c e s 1. Azimi T, Maham S, Fallah F, Azimi L, Gholinejad Z. Evaluating the antimicrobial resistance patterns among major bacterial pathogens isolated from clinical specimens taken from patients in Mofid Children’s Hospital, Tehran, Iran: 2013–2018. Infect drug resist: 2019, 12:2089. Doi: 10.2147/IDR.S215329 2. Jean SS, Gould IM, Lee WS, Hsueh PR. New drugs for multidrug-resistant gram-negative organisms: time for stewardship. Drugs. 2019;79(7):705-14. Doi.10.1007/s40265-019-01112-1 3. Helal IM, El-Bessoumy A, Al-Bataineh E, Joseph MR, Rajagopalan P, Chandramoorthy HC, Ben Hadj Ahmed S. Antimicrobial efficiency of essential oils from traditional medicinal plants of asir region, saudi arabia, over drug resistant isolates. BioMed research international. 2019. Doi.10.1155/2019/8928306 4. Jamal QA, Munir SH, Sherwani SK, Sualeh MO, Jabeen UZ, Malik M, Hussain MU. Antibacterial activity of two medicinal plants: Withania somnifera and Curcuma longa. European Academic Research. 2013;1:1335-45. 5. Snoussi M, Dehmani A, Noumi E, Flamini G, Papetti A. Chemical composition and antibiofilm activity of Petroselinum crispum and Ocimum basilicum essential oils against Vibrio spp. strains. Microbial pathogenesis. 2016; 90:13-21. Doi.10.1016/j.micpath.2015.11.004 6. Balakrishnan P, Ramalingam P, Nagarasan S. A comprehensive review on Ocimum basilicum. Journal of Natural Remedies. 2018;8(3):71-85. Doi.10.18311/jnr/2018/21324 7. Shahrajabian MH, Sun W, Cheng Q. Chemical components and pharmacological benefits of Basil (Ocimum basilicum): a review. International Journal of Food Properties. 2020;23(1):1961-70. Doi.10.1080/10942912.2020.1828456 8. Patil DD, Mhaske DK, Wadhawa GC. Antibacterial and Antioxidant study of Ocimum basilicum Labiatae (sweet basil). J. Adv. Pharm. Educ. Res. 2011;2:104- 12. ISSN 2249-3379 9. Eswar P, Devaraj CG. The Effect of Chewing Fresh Ocimum basilicum Leaves on Caries Microorganisms in Human Dental Plaque. Advances in Human Biology. 2015;5(3):56. 10. Sun Y, Li M, Chen L, Chen H, Yu X, Ye J, Zhang Y, Ma C, Zhou T. Prevalence and molecular characterization of carbapenemase-producing gram- negative bacteria from a university hospital in China. Infectious Diseases. 2016 ;48(2):138-46. Doi.10.3109/23744235.2015.1094822 11. Clinical & Laboratory Standard Institute. Performance Standard for Antimicrobial Disk Suseptability Tests: Twenty seventh information supplement M100S, 27th ed. CLSI, Wayne, PA,USA,2017. ISSN 2162-2914 12. Tabassum S, Amin F, Erum S, Javed H, Kazmi F, Nisar MF, Ullah I, Murtaza I, Ashraf M. Effect of Hexane and Ethanol Extracts of Ten Basil Genotypes on the Growth of Selected Bacterial Strains. International Journal of Agriculture & Biology. 2016;18(4). 735-40. Doi:10.17957/IJAB/15.0157 13. Gebrehiwot H, Bachetti RK, Dekebo A. Chemical composition and antimicrobial activities of leaves of sweet basil (Ocimum basilicum L.) herb. Int. J. Basic Clin. Pharmacol. 2015;4(5):869-75. Doi.10.18203/2319-2003.ijbcp20150858 14. Emran TB, Rahman MA, Uddin MM, Rahman MM, Uddin MZ, Dash R,et al. Effects of organic extracts and their different fractions of five Bangladeshi plants on in vitro thrombolysis. BMC complementary and alternative medicine. 2015;15(1):1-8. Doi. 10.1186/s12906-015-0643-2 15. Hakim ML, Kusdarwati R. The Effectiveness of Extracts Basil Leaves (Ocimum sanctum Linn) against Saprolegnia sp. by in Vitro. IOP Conf. Ser.: Earth Environ. Sci.2017;5(1): 012010. IOP Publishing. Doi:10.1088/1755-1315/55/1/012010 16. Silva VA, Sousa JP, Guerra FQ, Pessôa HL, Freitas AF, Alves LB, et al. Antibacterial activity of Ocimum basilicum essential oil and linalool on bacterial isolates of clinical importance. International Journal of Pharmacognosy and Phytochemical Research. 2015;7(6):1066-71. ISSN: 0975-4873 17. Issazadeh K, MAJID KP, Massiha A, Bidarigh S, Giahi M, ZULFAGAR MP. Analysis of the Phytochemical J Islamabad Med Dental Coll 2021 206 18. Contents and Anti-microbial Activity of Ocimum basilicum L. IJMCM.IR. 2012;2(1). 141-47. 19. Nsele NW. Assessment of the antibacterial activity of Artemisia afra, Erythrina lysistemon and Psidium guajava (Doctoral dissertation). 2013 20. Sholichah AR. Phytochemical screening and antioxidant activity of ethanolic extract and ethyl acetate fraction from basil leaf (Ocimum basilicum L.) by DPPH radical scavenging method. InIOP Conference Series: Materials Science and Engineering 2017 Nov 1 (Vol. 259, No. 1, p. 012008). IOP Publishing. Doi:10.1088/1757- 899X/259/1/012008 21. Khalil A. Antimicrobial activity of ethanolic extracts of Ocimum basilicum leaf from Saudi Arabia. Biotechnology. 2013;12(1):61-4. Doi: 10.3923/biotech.2013.61.64 22. Ababutain IM. Antimicrobial activity and gas chromatography-mass spectrometry (GC-MS) analysis of Saudi Arabian Ocimum basilicum leaves extracts. J Pure Appl Microbiol. 2019;13(2):823-33. Doi.org/10.22207/JPAM.13.2.17 23. Bilal A, Jahan N, Ahmed A, Bilal SN, Habib S, Hajra S. Phytochemical and pharmacological studies on Ocimum basilicum Linn-A review. IJCRR. 2012;4(23):73-83. ISSN: 0975-5241 24. Aruna K, Gore M, Tariq M. Synergistic Interaction of Ocimum basilicum extracts with antibiotic against β- lactamase producing uropathogens. Intr. J. Adv. Pharm. Res: 2015; 6:177-184. 25. Adam ZA, Omer AA. Antibacterial activity of Ocimum basilicum (Rehan) leaf extract against bacterial pathogens in Sudan. American Journal of Research Communication. 2015;3(8):94-9. ISSN: 2325-4076