Antibiogramic Efficacy of Cymbopogan citratus, Ocimum gratissimum, and Kalanchoe pinnata Methanolic and Aqueous Extracts on Colistin-Resistant Non-Clinical Isolates of Gram-negative Bacteria

Authors

DOI:

https://doi.org/10.56919/usci.2432.011

Keywords:

Antibiotics resistance, Colistin, Susceptibility, Medicinal plants, Phytochemicals

Abstract

Study’s Excerpt/Novelty

  • This study represents the first comprehensive assessment of the antibacterial effects of methanolic and aqueous extracts from Ocimum gratissimum, Kalanchoe pinnata, and Cymbopogon citratus against colistin-resistant Gram-negative bacteria.
  • Our work uniquely identifies and quantifies the active phytochemical components, such as flavonoids and tannins, within these extracts.
  • Additionally, the differential antibacterial activities observed, particularly the effectiveness of the aqueous extract of Ocimum gratissimum against Providencia stuarti, Enterococcus dispar, and Escherichia coli, underscore the potential of these extracts as alternative therapeutic agents in combating antibiotic-resistant infections.

Full Abstract

Antibiotic resistance is a significant global public health concern, with recent reports indicating that Gram-negative bacteria are developing resistance to colistin, a last-resort antibiotic.  This study aimed to assess the antibacterial effects of methanolic and aqueous leaf extracts from Ocimum gratissimum, Kalanchoe pinnata, and Cymbopogan citratus medicinal plants against colistin-resistant bacteria.  The extracts were obtained through maceration, followed by qualitative phytochemical analysis (including alkaloids, flavonoids, glycosides, saponins, sterols, phenolic compounds, and tannins) and quantitative evaluation of alkaloids, flavonoids, and tannins.  Furthermore, antibacterial properties were tested against twelve colistin-resistant Gram-negative bacterial strains using the disc diffusion method.  Qualitative analysis revealed the presence of flavonoids, proteins, carbohydrates, and tannins in all leaf extracts, while amino acids, fixed oil, and fats were absent.  The highest flavonoid content was found in the methanolic extract of C. citratus (153 ± 2.7µg/ml) and the lowest in the aqueous extract of K. pinnata (0.0 ± 0.0µg/ml).  Tannin levels were highest in the methanolic extract of O. gratissimum (282.4 ± 13.5µg/ml) and lowest in the aqueous extract of C. citratus (27.1 ± 5.7µg/ml).  Phenolic compound concentrations ranged from 136.9 ± 58.0µg/ml (methanolic extract of O. gratissimum) to 14.5 ± 2.9µg/ml (aqueous extract of K. pinnata).  The antibacterial activity of the methanolic extracts of O. gratissimum, K. pinnata, and C. citratus exhibited varied results, with all isolates resistant to all concentrations of the leaf extracts.  However, the aqueous extract of Ocimum gratissimum inhibited Providencia stuarti at 250mg, while Enterococcus dispar and Escherichia coli were sensitive to the aqueous leaf extracts of Ocimum gratissimum across all concentrations.  These findings indicate increasing resistance of Gram-negative bacteria to colistin and various concentrations of the methanolic and aqueous extracts of the three medicinal plants.

References

Abaji, A.A. and Omoruyi, I.M. (2022). Preliminary antibiogramic studies and phenotypic detection of colistin-resistant Pseudomonas species isolated from poultry birds in Benin City. Samuel Adegboyega University Science and Technology Journal. 7(1):1-12.

Abubakar, A. R. and Haque, M. (2020). Preparation of medicinal plants: Basic extraction and fractionation procedures for experimental purposes. In Journal of Pharmacy and Bio-Allied Sciences, Vol. 12, Issue 1, pp. 1 – 10. Wolters Kluwer Medknow Publications. https://doi.org/10.4103/jpbs.JPBS_175_19

Adebolu, T.T. and Oladimeji, S.A. (2015). Antimicrobial activity of leaf extracts of Ocimum gratissimum on selected diarrhoea causing bacteria in SouthWestern Nigeria. African Journal of Biotechnology, 4(7): 682-684. https://doi.org/10.5897/AJB2005.000-3126

Adwan, G. and Mhanna, M. (2008). Synergistic Effects of Plant Extracts and Antibiotics on Staphylococcus aureus Strains Isolated from Clinical Specimens. Middle-East Journal of Scientific Research, 3: 134-139. https://idosi.org/mejsr/mejsr3(3)/5.pdf

Ajiboye, A.A., Fadimu, O.Y., Ajiboye, M.D., Agboola, D.A., Adelaja, A.B. and Bem, A.A. (2014). Phytochemical and Nutritional Constituents of Some Common Vegetables in South-West, Nigeria. Global Journal of Science Frontier Research, 14(3): 49-51.

Akinmoladun, A.C., Ibukun, E.O., Afor, E., Obuotor, E.M. and Farombi, E.O. (2007). Phytochemical constituent and antioxidant activity of extract from the leaves of Ocimum gratissimum. Scientific Research and Essay, 2(5): 163-166.

Amengialue, O. O., Edobor, O. and Egharevba, A. P. (2013). Antibacterial activity of extracts of ocimum gratissimum on bacteria associated with diarrhoea. Bayero Journal of Pure and Applied Sciences, 6(2): 143 – 145. https://doi.org/10.4314/bajopas.v6i2.30

Asaolu, M.F., Oyeyemi, O.A. and Olaniokun, J.O. (2009). Chemical compositions, phytochemical constituents, and in vitro biological activity of various extracts of Cymbopogon citratus. Pakistan Journal of Nutrition, 8(12): 1920-1922. https://doi.org/10.3923/pjn.2009.1920.1922

Baron, S., Hadjadj, L., Rolain, J. and Olaitan, A.O. (2016). Molecular mechanisms of polymyxin resistance: knowns and unknowns. International Journal of Antimicrobial Agents, 48(6): 583-591. https://doi.org/10.1016/j.ijantimicag.2016.06.023

Biswas, S., Brunel, J., Dubus, J., Reynaud-Gauert, M. and Rolain, J. (2012). Colistin: an update on the antibiotics of the 21st century. Expert Review of Anti-infective Therapy, 10(8): 917-34. https://doi.org/10.1586/eri.12.78

Brisse, S., Passet, V. and Grimont, P.A.D. (2014). Description of Klebsiella quasipneumoniae sp. nov., isolated from human infections, with two subspecies, Klebsiella quasipneumoniae subsp. quasipneumoniae subsp. nov. and Klebsiella quasipneumoniae subsp. similipneumoniae subsp. nov., and demonstration that Klebsiella singaporensis is a junior heterotypic synonym of Klebsiella variicola. International Journal of Systematic and Evolutionary Microbiology, 64: 3146–3152. https://doi.org/10.1099/ijs.0.062737-0

Cassir, N., Rolain, J. and Brouqui, P. (2014). A new strategy to fight antimicrobial resistance: the revival of old antibiotics. Frontiers in Microbiology, 5: 551. https://doi.org/10.3389/fmicb.2014.00551

Chanthaboury, M., Choonharuangdej, S., Shrestha, B. and Srithavaj, T. (2022). Antimicrobial Properties of Ocimum Species: An In Vitro Study. Journal of International Society for Preventive and Community Dentistry, 12(6): 596-602. https://doi.org/10.4103/jispcd.JISPCD_155_22

Chetia J, Upadhyaya S and Saikia, LR, 2014. Phytochemical analysis, antioxidant and antimicrobial activity, and nutrient content analysis of Ocimum gratissimum linn. from Dibrugarh, N. E. India. International Journal of Pharmaceutical Science Review and Research, 40: 229-235

CI, K.C. and Indira, G. (2016). Quantitative estimation of total phenolic, flavonoids, tannin and chlorophyll content of leaves of Strobilanthes kunthiana (Neelakurinji). Journal of Medicinal Plants, 4: 282-286.

Cryer, M., Lane, K., Greer, M., Cates, R., Burt, S., Andrus, M., Zou, J., Rogers, P., Hansen, M. D. H., Burgado, J., Satheshkumar, P. S., Day, C. W., Smee, D. F. and Johnson, F. B. (2017). Isolation and identification of compounds from Kalanchoe pinnata having human alphaherpesvirus and vaccinia virus antiviral activity. Pharmaceutical Biology, 55(1): 1586 –1591. https://doi.org/10.1080/13880209.2017.131090 7

Del Bianco, F., Morotti, M., Pedna, M.F., Farabegoli, P. and Sambri, V. (2018). Microbiological surveillance of plasmid mediated colistin resistance in human Enterobacteriaceae isolates in Romagna (Northern Italy): August 2016–July 2017, International Journal of Infectious Diseases, 69:96-98. https://doi.org/10.1016/j.ijid.2018.02.006

Gallardo-Godoy, A., Muldoon, C., Becker, B., Elliott, A.G., Lash, L.H., Huang, J.X., Butler, M.S., Pelingo, R., Kavanagh, A.M., Ramu, S., Phetsang, W., Blaskovich, M.A.T. and Cooper, M.A. (2016). Activity and predicted nephrotoxicity of synthetic antibiotics based on polymyxin B. Journal of Medicinal Chemistry, 59(3): 1068-77. https://doi.org/10.1021/acs.jmedchem.5b01593

Gashe, F. and Zeleke, G. (2017). Antimicrobial activities of Vernonia amygdalina Del and Prunus africana extracts against multidrug-resistant clinical strains. Research Journal of Medicinal Plants, 11: 142–147. https://doi.org/10.3923/rjmp.2017.142.147

Hancock, R.E.W. and Speert, D.P. (2000). Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and impact on treatment. Drug Resistance Updates, 3: 247-255. https://doi.org/10.1054/drup.2000.0152

Hao, P.M. and Quoc, L.P.T. (2024). Chemical profile and antimicrobial activity of Ocimum gratissimum L. essential oil from Dak Lak province, Vietnam. Journal of Plant Biotechnology, 51:50-54. https://doi.org/10.5010/JPB.2024.51.005.050.

Hasan, Z.Y.M., Al-Halbosiy, M.M.F., Al-Lihaibi, R.K. and Al-Nauimi, A.H. (2022). Antimicrobial of lemongrass (Cymbopogon citratus L.) volatile oil and cytotoxic effects against L20B and MCF-7cell lines. Biodiversitas, 23(10): 5298-5301. https://doi.org/10.13057/biodiv/d231039

Hossain, M.A., AL-Raqmi, K.A., AL-Mijizy, Z.H., Weli, A.M. and Al-Riyami, Q. (2013). Study of total phenol, flavonoids contents and phytochemical screening of various leaves crude extracts of locally grown Thymus vulgaris. Asian Pacific Journal of Tropical Biomedicine, 3(9):705-710. https://doi.org/10.1016/S2221-1691(13)60142-2

Lawal, O.A., Ogundajo, A.L., Avoseh, N.O. and Ogunwande, I.A. (2017). Cymbopogon citratus. In Medicinal Spices and Vegetables from Africa. Kuete, V. (Ed.). Academic Press, Elsevier, United Kingdom. Pp.692. https://doi.org/10.1016/B978-0-12-809286-6.00018-2

Lee, J., Lee, Y., Lee, K., Thomas, T., Riley, V. and Kim, H. (2014). The changes of PCR ribotype and antimicrobial resistance of Clostridium difficile in a tertiary care hospital over 10 years. Journal of Medical Microbiology, 63:819–823. https://doi.org/10.1099/jmm.0.072082-0

Li, D., Yu, T., Zhang, Y., Yang, M., Li, Z., Liu, M. and Qi, R. (2010). Antibiotic resistance characteristics of environmental bacteria from an oxytetracycline production wastewater treatment plant and the receiving river. Applied and Environmental Microbiology, 76(11): 3444-3451. https://doi.org/10.1128/AEM.02964-09

Liu, Y. and Liu, J. (2018). Monitoring colistin resistance in food animals: An urgent threat. Expert Review of Anti-infective Therapy, 16(6): 443-446. https://doi.org/10.1080/14787210.2018.1481749

Liu, Y., Wang, Y., Walsh, T.R., Yi, L., Zhang, R., Spencer, J., Doi, Y., Tian, G., Dong, B., Huang, X., Yu, L., Gu, D., Ren, H., Chen, X., Lv, L., He, D., Zhou, H., Liang, Z., Liu, J. and Shen, J. (2016). Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. The Lancet, Infectious Diseases, 16(2): 161-168. https://doi.org/10.1016/S1473-3099(15)00424-7

Loi, M., Paciolla, C., Logrieco, A. F., and Mulè, G. (2020). Plant Bioactive Compounds in Pre - and Postharvest Management for Aflatoxins Reduction. In Frontiers in Microbiology. Vol. 11, p. 243. Frontiers Media S.A. https://doi.org/10.3389/fmicb.2020.0024 3

Lomovskaya, O., Zgurskaya, H.I., Totrov, M. and Watkins, W.J. (2007). Waltzing transporters and ‘the dance macabre’ between humans and bacteria. National Review in Drug Discovery, 6: 56-65. https://doi.org/10.1038/nrd2200

Mabika, B.M., Mounioko, F., Mboumba, L.W., Souza, A. and Yala, J.F. (2020). Phenotypic characterization of the resistance of Salmonella – Shigella isolates to colistin and detection of mcr1/2 genes. Journal of Applied Biosciences, 156: 16132 – 16138. https://doi.org/10.35759/JABs.156.6

Macdonald, I.O., Oludare, A.S. and Olabiyi, A. (2010). Phytotoxic and anti-microbial activities of flavonoids in Ocimum gratissimum, Life Science Journal, 7(3): 45-48.

Moussaoui, F. and Alaoui, T. (2016) Evaluation of Antibacterial Activity and Synergistic Effect between Antibiotic and the Essential Oils of Some Medicinal Plants. Asian Pacific Journal of Tropical Biomedicine, 6, 32-37. https://doi.org/10.1016/j.apjtb.2015.09.024

Nakamura, C.V., Ueda-Nakamura, T., Bando, E., Melo, A.F., Cortez, D.A., Filho, B.P. (1999). Antibacterial activity of Ocimum gratissimum L. essential oil. Memorias do Instituto Oswaldo Cruz, 94(5):675-8. https://doi.org/10.1590/S0074-02761999000500022

Ohadoma, S.C., Amazu, L.U., Enye, J.C. and Okolo, C.E. (2015). Comparative analysis of therapeutic benefits of Ocimum gratissimum and Vernonia amygdalina. European Journal of Pharmaceutical and Medical Research, 2(6): 29-32.

Okwu D.E. and Nnamdi F.U. (2011). Two Novel Flavonoids from Bryophyllum pinnatum and Their Antimicrobial Activity. Pharmacy and Respiratory Journal, 3, 1-10.

Omoruyi, I.M. and Emefo, O.T. (2012). In vitro evaluation of the antibiogramic properties of Myristica fragrance on food-borne pathogens. Malaysian Journal of Microbiology, 8(4): 213-218. https://doi.org/10.21161/mjm.42312

Omoruyi, I.M. and Ojubiaja, S.E. (2022). Antibiogram and virulence gene detection in Escherichia coli and Vibrio species isolated from market dumpsites in Edo South Senatorial District, Nigeria. Science World Journal, 17(1): 17-25. https://doi.org/10.4314/njb.v40i1.5

Omoruyi, I.M., Ovia, P.P., Ahmad-Dirisu, Z. and Osemwowa, E. (2023). Plasmid-borne mobilizable colistin-resistant gene (MCR-1) detection and multidrug-resistant bacteria isolated from abattoir environment. Nigerian Journal of Biotechnology, 40(1): 43-55.

Ordonez, A.A.L., Gomez, J.D. and Vattuone, M.A. (2006). Antioxidant activities of Sechium edule (Jacq.) Swartz extracts. Food Chemistry, 97(3):.452-458. https://doi.org/10.1016/j.foodchem.2005.05.024

Panche, A.N., Diwan, A.D. and Chandra, S.R. (2016). Flavonoids: An overview. Journal of Nutritional Science, 5: 1-15. https://doi.org/10.1017/jns.2016.41

Pattewar, S.V., Patil, D.N. and Dahikar, S.B. (2013). Antimicrobial potential of extract from leaves of Kalanchoe pinnata. International Journal of Pharmaceutical Science Research, 4: 4577-4580.

Poirel, L., Jayol, A. and Nordmann, P. (2017). Polymyxins: Antibacterial activity, susceptibility testing, and resistance mechanisms encoded by plasmids or chromosomes. Clinical Microbiology Reviews, 30(2): 557-596. https://doi.org/10.1128/CMR.00064-16

Schweitzer, B., Balázs, V.L., Molnár, S., Szögi-Tatár, B., Böszörményi, A., Palkovics, T., Horváth, G. and Schneider, G. (2022). Antibacterial effect of Lemongrass (Cymbopogoncitratus) against the aetiological agents of pitted keratolyis. Molecules, 27(4):1423. https://doi.org/10.3390/molecules27041423

Sharma, K., Guleria, S. and Razdan, V.K. (2020). Green synthesis of silver nanoparticles using Ocimum gratissimum leaf extract: characterization, antimicrobial activity, and toxicity analysis. Journal of Plant Biochemistry and Biotechnology. https://doi.org/10.1007/s13562-019-00522-2(0123456789().,-volV)(0123456789(). ,- volV)

Sharma, R., Sharma, C.L. and Kapoor, B. (2005). Antibacterial resistance: Current problems and possible solutions. Indian Journal of Medical Sciences, 59: 120-129. https://doi.org/10.4103/0019-5359.15091

Silva, J.C., Pereira, R.L.S., de Freitas, T.S., Rocha, J.E., Macedo, N.S., Nonato, C.F.A., Linhares, M.L., Tavares, D.S.A., da Cunha, F.A.B., Coutinho, H.D.M., de Lima, S.G., Pereira-Junior, F.N., Maia, F.P.A., Neto, I.C.P., Rodrigues, F.F.G. and Santos, G.J.G. (2022). Evaluation of antibacterial and toxicological activities of essential oil of Ocimum gratissimum L. and its major constituent eugenol, Food Bioscience, 50, Part B, 102128. https://doi.org/10.1016/j.fbio.2022.102128

Singleton, V.L., Orthofer, R. and Lamuela-Raventós, R.M., (1999). Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. In Methods in enzymology. Vol. 299, pp. 152-178. Academic press. https://doi.org/10.1016/S0076-6879(99)99017-1

Sofowora, A. (1993). Phytochemical Screening of Medicinal Plants and Traditional Medicine in Africa Edition. Spectrum Books Ltd., Nigeria, 150-156.

Subramaniam, G., Yew, X.Y. and Sivasamugham, L.A. (2020), Antibacterial activity of Cymbopogon citratus against clinically important bacteria, South African Journal of Chemical Engineering, 34: 26-30, https://doi.org/10.1016/j.sajce.2020.05.010.

Tiwari, P., Kumar, B., Kaur, M., Kaur, G., and Kaur, H. (2011). Phytochemical screening and extraction: A review. Internationale Pharmaceutica Sciencia, 1(1): 98-106.

Trease, G.E. and Evans, W.C. (1989) Pharmacognosy. 11th Edition, Bailliere Tindall, London, 45-50.

Walsh, T.R., and Wu, Y. (2016) China bans colistin as a feed additive for animals. Lancet Infectious Diseases, 16: 1085. https://doi.org/10.1016/S1473-3099(16)30329-2

Wang, R., Dorp, L.V., Shaw, L.P., Bradley, P., Wang, O., Wang, X., Jin, L., Zhang, Q., Liu, Y., Rieux, A., Rieux, A., Dorai-Schneiders, T., Weinert, L.A., Iqbal, Z., Didelot, X. and Balloux, F. (2018). The global distribution and spread of the mobilized colistin resistance gene mcr-1. Nature Communications, 9: 1179. https://doi.org/10.1038/s41467-018-03205-z

Yala, J.F., Mabika, R., Bisseye, C. and Kenguele, H. (2016). Phenotypic and genotypic characterization of extended-spectrum beta-lactamases producing enterobacteriaceae (ESBLE) in patients attending Omar Bongo Ondimba Military Hospital at Libreville (Gabon). Current Research in Microbiology and Biotechnology, 4: 944-949.

Yala, J.F., Mabika, R.M., Mounioko, F., Minko, O.Z., Lepengue, A.N. and Souza, A. (2020). In Vitro Antibacterial Activity of an Aqueous Extracts of the Tephrosia Vogelii Hook.f Combined To Imipenem on E. coli Strains. Scholars Academic Journal of Pharmacy, 9(1): 36-45. https://doi.org/10.36347/sajp.2020.v09i01.008

Yang, R., Wang, L.Q. and Liu, Y. (2014). Antitumor activities of Widely-used Chinese Herb—Licorice. Chinese Herbal Medicine, 6: 274–281. https://doi.org/10.1016/S1674-6384(14)60042-3

Zakharchenko, N. S., Belous, A. S., Biryukova, Y. K., Medvedeva, O. A., Belyakova, A. V. and Masgutova, G. A., Trubnikova, E. V., Buryanov, Y. I. and Lebedeva, A. A. (2017). Immunomodulating and revascularizing activity of Kalanchoe pinnata synergise with fungicide activity of biogenic peptide cecropin P1. Journal of Immunology Research, https://doi.org/10.1155/2017/3940743.

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2024-06-11

How to Cite

Adodo, E., Ahamioje, D., Ibitoye, Y. I., & Omoruyi, I. M. (2024). Antibiogramic Efficacy of Cymbopogan citratus, Ocimum gratissimum, and Kalanchoe pinnata Methanolic and Aqueous Extracts on Colistin-Resistant Non-Clinical Isolates of Gram-negative Bacteria. UMYU Scientifica, 3(2), 102–113. https://doi.org/10.56919/usci.2432.011