{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,18]],"date-time":"2026-03-18T06:11:36Z","timestamp":1773814296148,"version":"3.50.1"},"reference-count":50,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2021,3,10]],"date-time":"2021-03-10T00:00:00Z","timestamp":1615334400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Bridging Grant","award":["304\/PPSP\/6316187"],"award-info":[{"award-number":["304\/PPSP\/6316187"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJERPH"],"abstract":"<jats:p>Despite modern medicine, there is an increasing trend for cases of the bacterial infection leptospirosis, and this has led to the exploration of alternative medicines from various sources including plants. The aim of this study was to investigate the in vitro anti-leptospiral activity of Phyllanthus amarus extracts alone and combined with penicillin G, ceftriaxone, and doxycycline. Antimicrobial susceptibility testing was performed using the microdilution broth technique upon methanol extract (ME), aqueous extract (AE), and antibiotics against the Leptospira interrogans serovars Australis, Bataviae, Canicola, and Javanica, to determine minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs). The results were analyzed using an ELISA microplate reader combined with microscopic analysis. Synergy testing using a checkerboard assay was performed to determine the fractional inhibitory concentration index values of extracts combined with antibiotics against leptospires. Scanning electron microscopy (SEM) was used to investigate morphological changes of leptospires caused by potential anti-leptospiral agents alone and combined with antibiotics. The MICs and MBCs for P. amarus extracts ranged from 100 to 400 \u00b5g\/mL for AEs and from 400 to 800 \u00b5g\/mL for MEs. Penicillin G was the most effective anti-leptospiral drug, with MICs and MBCs ranging from &lt;0.01 to 0.78 and &lt;0.01 to 3.13 \u00b5g\/mL, respectively, followed by ceftriaxone, with both MICs and MBCs ranging from 0.05 to 0.78 \u00b5g\/mL, and doxycycline, with MICs and MBCs ranging from 0.39 to 3.13 \u00b5g\/mL and 12.5 to 25 \u00b5g\/mL, respectively. Combinations of P. amarus extracts and antibiotics did not show synergistic effects on all tested Leptospira serovars, with some combinations demonstrating antagonistic effects. SEM analysis, however, showed distorted Leptospira surfaces. P. amarus AE performed better anti-leptospiral activity than P. amarus ME. The morphological effects of P. amarus extract alone and its combination with antibiotic on Leptospira cells revealed promising anti-leptospiral properties.<\/jats:p>","DOI":"10.3390\/ijerph18062834","type":"journal-article","created":{"date-parts":[[2021,3,10]],"date-time":"2021-03-10T13:27:20Z","timestamp":1615382840000},"page":"2834","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["In Vitro Anti-Leptospiral Activity of Phyllanthus amarus Extracts and Their Combinations with Antibiotics"],"prefix":"10.3390","volume":"18","author":[{"given":"Che Ain Munirah","family":"Ismail","sequence":"first","affiliation":[{"name":"Department of Medical Microbiology &amp; Parasitology, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian, Kelantan 16150, Malaysia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3344-1518","authenticated-orcid":false,"given":"Zakuan Zainy","family":"Deris","sequence":"additional","affiliation":[{"name":"Department of Medical Microbiology &amp; Parasitology, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian, Kelantan 16150, Malaysia"},{"name":"Hospital Universiti Sains Malaysia, Universiti Sains Malaysia Kampus Kesihatan, Jalan Raja Perempuan Zainab 2, Kota Bharu, Kelantan 16150, Malaysia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5392-4707","authenticated-orcid":false,"given":"Ruzilawati Abu","family":"Bakar","sequence":"additional","affiliation":[{"name":"Hospital Universiti Sains Malaysia, Universiti Sains Malaysia Kampus Kesihatan, Jalan Raja Perempuan Zainab 2, Kota Bharu, Kelantan 16150, Malaysia"},{"name":"Department of Pharmacology, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian, Kelantan 16150, Malaysia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9458-2288","authenticated-orcid":false,"given":"Nabilah","family":"Ismail","sequence":"additional","affiliation":[{"name":"Department of Medical Microbiology &amp; Parasitology, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian, Kelantan 16150, Malaysia"},{"name":"Hospital Universiti Sains Malaysia, Universiti Sains Malaysia Kampus Kesihatan, Jalan Raja Perempuan Zainab 2, Kota Bharu, Kelantan 16150, Malaysia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,3,10]]},"reference":[{"key":"ref_1","first-page":"1","article-title":"Leptospirosis: A re-emerging infection","volume":"33","author":"Lim","year":"2011","journal-title":"Malays. J. Pathol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"323","DOI":"10.1007\/s10389-014-0611-1","article-title":"Risk factors associated with rural and urban epidemics of leptospirosis in Tiruchirappalli district of Tamilnadu, India","volume":"22","author":"Prabhakaran","year":"2014","journal-title":"J. Public Health"},{"key":"ref_3","first-page":"65","article-title":"Leptospirosis in humans","volume":"387","author":"Haake","year":"2015","journal-title":"Curr. Top. Microbiol. Immunol."},{"key":"ref_4","unstructured":"World Health Organization (2009). Leptospirosis: Fact Sheet, Regional Office for South-East Asia."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"296","DOI":"10.1128\/CMR.14.2.296-326.2001","article-title":"Leptospirosis","volume":"14","author":"Levett","year":"2001","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Costa, F., Hagan, J.E., Calcagno, J., Kane, M., Torgerson, P., Martinez-Silveira, M.S., Stein, C., Abela-Ridder, B., and Ko, A.I. (2015). Global morbidity and mortality of leptospirosis: A Systematic Review. PLOS Negl. Trop. Dis., 9.","DOI":"10.1371\/journal.pntd.0003898"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/s00408-010-9273-0","article-title":"Clinical and imaging manifestations of hemorrhagic pulmonary leptospirosis: A state-of-the-art review","volume":"189","author":"Marchiori","year":"2011","journal-title":"Lung"},{"key":"ref_8","unstructured":"Yi-Wei, T., Liu, D., Schwartzman, J., Sussman, M., and Poxton, I. (2015). Leptospira and leptospirosis. Molecular Medical Microbiology, Academic Press."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"412","DOI":"10.1016\/j.foodcont.2014.05.047","article-title":"Natural products as antimicrobial agents","volume":"46","author":"Gyawali","year":"2014","journal-title":"Food Control"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"40","DOI":"10.3923\/ijp.2011.40.45","article-title":"Overview pharmacognostic properties of Phyllanthus Amarus Linn","volume":"7","author":"Joseph","year":"2011","journal-title":"Int. J. Pharmacol."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Sarin, B., Verma, N., Mart\u00edn, J.P., and Mohanty, A. (2014). An overview of important ethnomedicinal herbs of Phyllanthus species: Present status and future prospects. Sci. World J., 2014.","DOI":"10.1155\/2014\/839172"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1","DOI":"10.15406\/icpjl.2019.07.00191","article-title":"Antibacterial effects of Phyllantus amarus on urinary tract pathogens","volume":"7","author":"Oladosu","year":"2019","journal-title":"Int. Clin. Pathol. J."},{"key":"ref_13","first-page":"294","article-title":"Antimicrobial activity of solvent extracts of Ocimum sanctum, Azadirachta indica and Phyllanthus amarus against clinical pathogens","volume":"8","author":"Bharathi","year":"2014","journal-title":"Glob. J. Pharmacol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"323","DOI":"10.1080\/14786410600650404","article-title":"Antimicrobial potentiality of Phyllanthus amarus against drug resistant pathogens","volume":"20","author":"Mazumder","year":"2006","journal-title":"Nat. Prod. Res."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"378","DOI":"10.4103\/0974-8490.159577","article-title":"Preclinical screening of Phyllanthus amarus ethanolic extract for its analgesic and antimicrobial activity","volume":"7","author":"Bhat","year":"2015","journal-title":"Pharmacogn. Res."},{"key":"ref_16","first-page":"1231","article-title":"Screening of Phyllanthus amarus, Acalypha indica and Datura metel for its antimicrobial activity against selected pathogens","volume":"3","author":"Sekar","year":"2012","journal-title":"Int. J. Pharm. Biol. Arch."},{"key":"ref_17","first-page":"215","article-title":"Antimicrobial effect of Phyllanthus amarus and Parquetina nigrescens on Salmonella typhi","volume":"11","author":"Oluwafemi","year":"2008","journal-title":"Afr. J. Biomed. Res."},{"key":"ref_18","unstructured":"Chandan, S., Umesha, S., and Balamurugan, V. (2012). Antileptospiral, antioxidant and dna damaging properties of Eclipta alba and Phyllanthus amarus. Open Access Sci. Rep., 1."},{"key":"ref_19","first-page":"1","article-title":"Optimization of soxhlet extraction and physicochemical analysis of crop oil from seed kernel of Feun kase (Thevetia peruviana)","volume":"1911","author":"Suwari","year":"2017","journal-title":"AIP Conf. Proc."},{"key":"ref_20","unstructured":"Azwanida, N.N. (2015). A review on the extraction methods use in medicinal plants, principle, strength and limitation. Med. Aromat. Plants, 4."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"685","DOI":"10.4269\/ajtmh.2004.71.685","article-title":"Susceptibility of Leptospira serovars to antimalarial agents","volume":"71","author":"Murray","year":"2004","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"ref_22","unstructured":"Raoult, D. (1993). Methodology of antibiotics testing for intracellular pathogens. Antimicrobial Agents and Intracellular Pathogens, CRC Press, Inc."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"European Committee for Antimicrobial Susceptibility Testing of the European Society of Clinical Microbiology Infectious Diseases (2003). Determination of minimum inhibitory concentrations (mics) of antibacterial agents by broth dilution. Clin. Microbiol. Infect., 9, ix\u2013xv.","DOI":"10.1046\/j.1469-0691.2003.00790.x"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1168","DOI":"10.1055\/s-0030-1250736","article-title":"Plant-based antimicrobial studies\u2013methods and approaches to study the interaction between natural products","volume":"77","author":"Viljoen","year":"2011","journal-title":"Planta Med."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Nightingale, C.H., Ambrose, P.G., Drusano, G.L., and Murakawa, T. (2007). The predictive value of laboratory tests for efficacy of antibiotic combination therapy. Antimicrobial Pharmacodynamics in Theory and Clinical Practice, Informa Healthcare USA, Inc.","DOI":"10.3109\/9781420017137-8"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"10","DOI":"10.4314\/bajopas.v4i1.2","article-title":"Preliminary phytochemical screening and antimicrobial activities of some medicinal plants used in Ebiraland","volume":"4","author":"Alli","year":"2011","journal-title":"Bayero J. Pure Appl. Sci."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"50","DOI":"10.4103\/0973-8258.97129","article-title":"Determination of antimicrobial potentialities of different solvent extracts of the medicinal plant: Phyllanthus amarus Schum. and Thonn","volume":"6","author":"Sen","year":"2012","journal-title":"Int. J. Gree Pharm."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"497","DOI":"10.1016\/j.foodres.2011.09.007","article-title":"Factors influencing levels of phytochemicals in selected fruit and vegetables during pre-and post-harvest food processing operations","volume":"50","author":"Tiwari","year":"2013","journal-title":"Food Res. Int."},{"key":"ref_29","first-page":"198","article-title":"Antimicrobial activity of Senna alata and Phyllanthus amarus","volume":"7","author":"Ogunjobi","year":"2013","journal-title":"Glob. J. Pharmacol."},{"key":"ref_30","first-page":"1","article-title":"Preliminary phytochemical investigation and antibacterial activity of Phyllanthus amarus Schum & Thorn","volume":"27","author":"Dhandapani","year":"2007","journal-title":"Anc. Sci. Life"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"238","DOI":"10.4314\/bajopas.v10i1.35","article-title":"Chemical constituents and antimicrobial properties of Phyllanthus amarus (Schum & Thonn)","volume":"10","author":"Zubair","year":"2017","journal-title":"Bayero J. Pure Appl. Sci."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"S371","DOI":"10.4103\/pm.pm_10_20","article-title":"Potential antileptospiral constituents from Phyllanthus amarus","volume":"16","author":"Shirahatti","year":"2020","journal-title":"Pharmacogn. Mag."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"241","DOI":"10.1007\/s11101-018-9591-z","article-title":"Comprehensive review of antimicrobial activities of plant flavonoids","volume":"18","author":"Bartoszewski","year":"2019","journal-title":"Phytochem. Rev."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/s12038-019-9868-4","article-title":"Elucidating the effect of anti-biofilm activity of bioactive compounds extracted from plants","volume":"44","author":"Lahiri","year":"2019","journal-title":"J. Biosci."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Poletto, M. (2018). Lignin as Natural Antioxidant Capacity. Lignin\u2014Trends and Application, IntechOpen.","DOI":"10.5772\/intechopen.68464"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Dong, S., Yang, X., Zhao, L., Zhang, F., Hou, Z., and Xue, P. (2020). Antibacterial activity and mechanism of action saponins from Chenopodium quinoa Willd. husks against foodborne pathogenic bacteria. Ind. Crops Prod., 149.","DOI":"10.1016\/j.indcrop.2020.112350"},{"key":"ref_37","first-page":"75","article-title":"Antimicrobial activity and mechanism of total saponins from Allium chinense","volume":"34","author":"Yu","year":"2013","journal-title":"Food Sci."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"976","DOI":"10.1080\/14786419.2014.964705","article-title":"Antibacterial mode of action of 1, 8-dihydroxy-anthraquinone from Porphyra haitanensis against Staphylococcus aureus","volume":"29","author":"Wei","year":"2015","journal-title":"Nat. Prod. Res."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"302","DOI":"10.3906\/vet-1510-24","article-title":"An investigation of antibacterial effects of steroids","volume":"41","author":"Otlu","year":"2017","journal-title":"Turkish J. Vet. Anim. Sci."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"6063","DOI":"10.1039\/C8FO01569D","article-title":"Health functions and structure\u2013activity relationships of natural anthraquinones from plants","volume":"9","author":"Li","year":"2018","journal-title":"Food Funct."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"693","DOI":"10.1016\/j.ijantimicag.2017.12.024","article-title":"Antibiotic susceptibilities of livestock isolates of Leptospira","volume":"51","author":"Liegeon","year":"2018","journal-title":"Int. J. Antimicrob. Agents"},{"key":"ref_42","first-page":"775","article-title":"Susceptibility of Leptospira serotypes against various antimicrobials by broth microdilution method","volume":"83","author":"Chitra","year":"2013","journal-title":"Indian J. Anim. Sci."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"2750","DOI":"10.1128\/AAC.00044-08","article-title":"Antimicrobial susceptibilities of geographically diverse clinical human isolates of Leptospira","volume":"52","author":"Ressner","year":"2008","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"124","DOI":"10.4103\/ijmm.IJMM_15_458","article-title":"Antimicrobial susceptibility of Leptospira spp. isolated from environmental, human and animal sources in Malaysia","volume":"35","author":"Benacer","year":"2017","journal-title":"Indian J. Med. Microbiol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"4002","DOI":"10.1128\/AAC.48.10.4002-4005.2004","article-title":"Determination of susceptibilities of 26 Leptospira sp. serovars to 24 antimicrobial agents by a broth microdilution technique","volume":"48","author":"Murray","year":"2004","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Seesom, W., Jaratrungtawee, A., Suksamrarn, S., Mekseepralard, C., Ratananukul, P., and Sukhumsirichart, W. (2013). Antileptospiral activity of xanthones from Garcinia mangostana and synergy of gamma-mangostin with penicillin G. BMC Complement. Altern. Med., 13.","DOI":"10.1186\/1472-6882-13-182"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"1127","DOI":"10.1128\/AAC.39.5.1127","article-title":"Effects of Penicillin, Ceftriaxone, and Doxycycline on Morphology of Borrelia burgdorferi","volume":"39","author":"Kersten","year":"1995","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"516","DOI":"10.1099\/mic.0.000027","article-title":"Morphological and biochemical features of Borrelia burgdorferi pleomorphic forms","volume":"161","author":"Herranen","year":"2015","journal-title":"Microbiology"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1038\/s41579-018-0112-2","article-title":"Types and origins of bacterial membrane vesicles","volume":"17","author":"Toyofuku","year":"2019","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"598","DOI":"10.1111\/j.1469-0691.2004.00895.x","article-title":"Lyme borreliosis: From infection to autoimmunity","volume":"10","author":"Singh","year":"2004","journal-title":"Clin. Microbiol. Infect."}],"container-title":["International Journal of Environmental Research and Public Health"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1660-4601\/18\/6\/2834\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T05:33:42Z","timestamp":1760160822000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1660-4601\/18\/6\/2834"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,3,10]]},"references-count":50,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2021,3]]}},"alternative-id":["ijerph18062834"],"URL":"https:\/\/doi.org\/10.3390\/ijerph18062834","relation":{},"ISSN":["1660-4601"],"issn-type":[{"value":"1660-4601","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,3,10]]}}}