Determination of Antioxidant and Anticancer Activities and Exopolysaccharides of Metabolites Synthesized by Probiotic Levilactobacillus brevis Strains Newly Isolated from Breast Milk

Year 2025, Volume: 9 Issue: 2, 123 - 132

Yusuf Alan , Metin Ertaş

https://doi.org/10.31594/commagene.1660124

Abstract

In our study, the probiotic, exopolysaccharide, metabolite contents, antioxidant activities, and anticancer activities of metabolites on HT-29 and Caco-2 cell lines of four Levilactobacillus brevis (YAAS1, YAAS2, YAAS3, and YAAS4) strains isolated from breast milk in Türkiye and identified by MALDI-TOF MS were investigated. It was determined that the strains exhibited strong probiotic character by surviving in pH+pepsin, bile and pancreatin medium; although, they decreased depending on time and concentration. The strains synthesized the most acetic acid but could not synthesize tartaric, pyruvic, malic, and fumaric acids. It was determined qualitatively and quantitatively that the strains synthesized compounds similarly in terms of exopolysaccharide amount. The strains exhibited lower DPPH activity than standard antioxidants. However, ABTS activities were similar to standard antioxidants. Bacterial metabolites induced notable anticancer effects in the HT-29 cell line at low concentrations, while eliciting comparable effects in the Caco-2 cell line at high concentrations. When all the results were considered in general, it was determined that the strains showed identical properties to each other but L. brevis YAAS4 strain exhibited a better character structure. Given the scarcity of studies on Lactobacillus brevis strains newly isolated from human breast milk, this research significantly advances current understanding. Characterizing the specific properties identified in our study through future research will be crucial for determining the potential of these isolates as effective probiotics for industrial and health applications.

Keywords

Levilactobacillus brevis , MALDI-TOF MS , HT-29 , Caco-2

Ethical Statement

Ethical approval for this study was obtained from the Ethics Committee of Van Eğitim ve Araştırma Hastanesi for Clinical Research (28.09.2017, No: 2017/7).

Anne Sütünden Yeni İzole Edilen Probiyotik Levilactobacillus brevis Suşlarının Sentezlediği Metabolitlerin Antioksidan ve Antikanser Aktiviteleri ile Ekzopolisakkarit Tayini

Abstract

Çalışmamızda, Türkiye'deki anne sütünden yeni izole edilen ve MALDI-TOF MS ile tanımlanan dört Levilactobacillus brevis (YAAS1, YAAS2, YAAS3 ve YAAS4) suşunun probiyotik, ekzopolisakkarit, metabolit içerikleri, antioksidan aktiviteleri ve metabolitlerinin HT-29 ve Caco-2 hücre hatları üzerindeki antikanser aktiviteleri araştırıldı. Suşların pH+pepsin, safra ve pankreatin ortamında canlılığını sürdürerek güçlü probiyotik karakter sergilediği, ancak bu özelliğinin zamana ve konsantrasyona bağlı olarak azaldığı belirlendi. Suşlar en fazla asetik asit sentezlediği, ancak tartarik, pirüvik, malik ve fumarik asitleri sentezlemedikleri belirlendi. Ekzopolisakkarit miktarı bakımından suşlar birbirlerine benzer şekilde sentezlediği nitel ve nicel olarak belirlendi. Suşlar standart antioksidanlardan daha düşük DPPH aktivitesi gösterdi. Ancak ABTS aktiviteleri standart antioksidanlara benzerdi. Bakteriyel metabolitler düşük konsantrasyonlarda HT-29 hücre hattına karşı iyi antikanser etki gösterirken, yüksek konsantrasyonlarda Caco-2 hücre hattına karşı benzer etki gösterdi. Tüm sonuçlar genel olarak değerlendirildiğinde suşların birbirlerine benzer özellikler gösterdiği, ancak L. brevis YAAS4 suşunun daha iyi bir karakter yapısı sergilediği belirlendi. Sonuç olarak insan sütünden yeni izole edilen L. brevis suşları üzerinde sınırlı sayıda çalışma olması nedeniyle sonuçlarımız literatüre önemli katkılar sunacaktır. Aynı zamanda L. brevis suşlarımızın sergilediği özellikler dikkate alınarak yapılacak yeni araştırmalar ile iyi karakterdeki suşların endüstri ve sağlık alanında iyi bir probiyotik bakteri kaynağı olmasına katkı sağlayacaktır.

Keywords

Levilactobacillus brevis , MALDI-TOF MS , HT-29 , Caco-2

Ethical Statement

Bu çalışmanın etik onayı, Van Eğitim ve Araştırma Hastanesi Klinik Araştırmalar Etik Kurulu'ndan alınmıştır (28.09.2017, No: 2017/7).

References

• Alameri, F., Tarique, M., Osaili, T., Obaid, R., Abdalla, A., Masad, R., & Ayyash, M. (2022). Lactic acid bacteria isolated from fresh vegetable products: potential probiotic and postbiotic characteristics including immunomodulatory effects. Microorganisms, 10(2), 389. https://doi.org/10.3390/microorganisms10020389
• Alan, Y. (2024). Chemical changes of potential probiotic Lactiplantibacillus plantarum and Lactobacillus pentosus starter cultures in natural Gemlik type black olive fermentation. Food Chemistry, 434, 137472. https://doi.org/10.1016/j.foodchem.2023.137472
• Alan, Y., Savcı, A., Koçpınar, E.F., & Ertaş, M. (2022). Postbiotic metabolites, antioxidant and anticancer activities of probiotic Leuconostoc pseudomesenteroides strains in natural pickles. Archives of Microbiology, 204(9), 571. https://doi.org/10.1007/s00203-022-03180-6
• Alan, Y., Keskin, A.O., & Sönmez, M. (2025). Probiotic and functional characterization of newly isolated Lactiplantibacillus plantarum strains from human breast milk and proliferative inhibition potential of metabolites. Enzyme and Microbial Technology, 182, 110545. https://doi.org/10.1016/j.enzmictec.2024.110545
• Albesharat, R., Ehrmann, M.A., Korakli, M., Yazaji, S., & Vogel, R. F. (2011). Phenotypic and genotypic analyses of lactic acid bacteria in local fermented food, breast milk and faeces of mothers and their babies. Systematic and Applied Microbiology, 34(2), 148-155. https://doi.org/10.1016/j.syapm.2010.12.001
• Angelin, J., & Kavitha, M. (2020). Exopolysaccharides from probiotic bacteria and their health potential. International journal of biological macromolecules, 162, 853-865. https://doi.org/10.1016/j.ijbiomac.2020.06.190
• Anjum, J., Nazir, S., Tariq, M., Barrett, K., & Zaidi, A. (2020). Lactobacillus commensals autochthonous to human milk have the hallmarks of potent probiotics. Microbiology, 166, 966-980. https://doi.org/10.1099/mic.0.000966
• Assaf, A.M., Haddadin, R.N., Aldouri, N.A., Alabbassi, R., Mashallah, S., Mohammad, M., & Bustanji, Y. (2013). Anti-cancer, anti-inflammatory and anti-microbial activities of plant extracts used against hematological tumors in traditional medicine of Jordan. Journal of Ethnopharmacology, 145(3), 728-736.
• Asenova, A., Hristova, H., Ivanova, S., Miteva, V., Zhivkova, I., Stefanova, K., & Rasheva, I. (2024). Identification and Characterization of Human Breast Milk and Infant Fecal Cultivable Lactobacilli Isolated in Bulgaria: A Pilot Study. Microorganisms, 12(9), 1839. https://doi.org/10.3390/microorganisms12091839
• Axel, C., Brosnan, B., Zannini, E., Peyer, L.C., Furey, A., Coffey, A., & Arendt, E.K. (2016). Antifungal activities of three different Lactobacillus species and their production of antifungal carboxylic acids in wheat sourdough. Applied microbiology and biotechnology, 100, 1701-1711. https://doi.org/10.1007/s00253-015-7051-x
• Barcenilla, C., Ducic, M., López, M., Prieto, M., & Álvarez-Ordóñez, A. (2022). Application of lactic acid bacteria for the biopreservation of meat products: A systematic review. Meat Science, 183, 108661. https://doi.org/10.1016/j.meatsci.2021.108661
• Choi, S.J., Yang, S.Y., & Yoon, K.S. (2021). Lactic acid bacteria starter in combination with sodium chloride controls pathogenic Escherichia coli (EPEC, ETEC, and EHEC) in kimchi. Food Microbiology, 100, 103868. https://doi.org/10.1016/j.fm.2021.103868
• Christensen, G.D., Simpson, W.A., Younger, J.J., Baddour, L.M., Barrett, F.F., Melton, D.M., & Beachey, E.H. (1985). Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices. Journal of Clinical Microbiology, 22(6), 996-1006. https://doi.org/10.1128/jcm.22.6.996-1006.1985
• Das, D., & Goyal, A. (2015). Antioxidant activity and γ-aminobutyric acid (GABA) producing ability of probiotic Lactobacillus plantarum DM5 isolated from Marcha of Sikkim. LWT-food Science and Technology, 61(1), 263-268. https://doi.org/10.1016/j.lwt.2014.11.013
• Diguță, C.F., Nițoi, G.D., Matei, F., Luță, G., & Cornea, C.P. (2020). The biotechnological potential of Pediococcus spp. isolated from Kombucha microbial consortium. Foods, 9(12), 1780. https://doi.org/10.3390/foods9121780
• Ding, M., Qi, C., Yang, Z., Jiang, S., Bi, Y., & Lai, J. (2019). Geographical location specific composition of cultured microbiota and Lactobacillus occurrence in human breast milk in China. Food & function, 10(2), 554–564. https://doi.org/10.1039/C8FO02182A
• DuBois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.T., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical chemistry, 28(3), 350-356.
• Duraisamy, S., Husain, F., Balakrishnan, S., Sathyan, A., Subramani, P., Chidambaram, P., & Kumarasamy, A. (2022). Phenotypic assessment of probiotic and bacteriocinogenic efficacy of indigenous LAB strains from human breast milk. Current Issues in Molecular Biology, 44(2), 731-749. https://doi.org/10.3390/cimb44020051
• Dušková, M., Šedo, O., Kšicová, K., Zdráhal, Z., & Karpíšková, R. (2012). Identification of lactobacilli isolated from food by genotypic methods and MALDI-TOF MS. International journal of food microbiology, 159(2), 107-114. https://doi.org/10.1016/j.ijfoodmicro.2012.07.029
• FAO/WHO (2023). Food and Agriculture Organization of the United Nations/World Health Organization Health and Nutritional Properties of Probiotics in Food including Powder Milk with Live Lactic Acid Bacteria. Retrieved from: http://pc.ilele.hk/public/pdf/20190225/bd3689dfc2fd663bb36def1b672ce0a4.pdf
• Freeman, D.J., Falkiner, F.R., & Keane, C.T. (1989). New method for detecting slime production by coagulase negative staphylococci. Journal of clinical pathology, 42(8), 872-874. https://doi.org/10.1136/jcp.42.8.872
• Han, Q., Kong, B., Chen, Q., Sun, F., & Zhang, H. (2017). In vitro comparison of probiotic properties of lactic acid bacteria isolated from Harbin dry sausages and selected probiotics. Journal of Functional Foods, 32, 391-400. https://doi.org/10.1016/j.jff.2017.03.020
• Hemarajata, P., & Versalovic, J. (2013). Effects of probiotics on gut microbiota: mechanisms of intestinal immunomodulation and neuromodulation. Therapeutic advances in gastroenterology, 6(1), 39-51. https://doi.org/10.1177/1756283X12459294
• Jamaly, N., Benjouad, A., & Bouksaim, M. (2011). Probiotic potential of Lactobacillus strains isolated from known popular traditional Moroccan dairy products. British Microbiology Research Journal, 1(4), 79-94. https://doi.org/0.9734/BMRJ/2011/438
• Jeurink, P.V., Van Bergenhenegouwen, J., Jiménez, E., Knippels, L.M.J., Fernández, L., Garssen, J., & Martín, R. (2013). Human milk: a source of more life than we imagine. Beneficial microbes, 4(1), 17-30. https://doi.org/10.3920/BM2012.0040
• Jost, T., Lacroix, C., Braegger, C.P., Rochat, F., & Chassard, C. (2014). Vertical mother–neonate transfer of maternal gut bacteria via breastfeeding. Environmental microbiology, 16(9), 2891-2904. https://doi.org/10.1111/1462-2920.12238
• Kao, Y.T., Liu, Y.S., & Shyu, Y.T. (2007). Identification of Lactobacillus spp. in probiotic products by real-time PCR and melting curve analysis. Food Research International, 40(1), 71-79. https://doi.org/10.1016/j.foodres.2006.07.018
• Khagwal, N., Sharma, P.K., & Sharma, D.C. (2014). Screening and evaluation of Lactobacillus spp. for the development of potential probiotics. African Journal of Microbiology Research, 8(15), 1573-1579. https://doi.org/10.5897/AJMR2013.6138
• Kim, H.S., Jeong, S.G., Ham, J.S., Chae, H.S., Lee, J.M., & Ahn, C.N. (2006). Antioxidative and probiotic properties of Lactobacillus gasseri NLRI-312 isolated from Korean infant feces. Asian-australasian journal of animal sciences, 19(9), 1335-1341. https://doi.org/10.5713/ajas.2006.1335
• Kim, H., Kim, J.S., Kim, Y., Jeong, Y., Kim, J.E., & Paek, N.S. (2020). Antioxidant and probiotic properties of lactobacilli and bifidobacteria of human origins. Biotechnology and Bioprocess Engineering, 25, 421-430. https://doi.org/10.1007/s12257-020-0147-x
• Kim, Y., Lee, D., Kim, D., Cho, J., Yang, J., Chung, M., & Ha, N. (2008). Inhibition of proliferation in colon cancer cell lines and harmful enzyme activity of colon bacteria by Bifidobacterium adolescentis SPM0212. Archives of Pharmacal Research, 31, 468-473. https://doi.org/10.1007/s12272-001-1180-y
• Lara-Villoslada, F., Olivares, M., Sierra, S., Rodríguez, J.M., Boza, J., & Xaus, J. (2007). Beneficial effects of probiotic bacteria isolated from breast milk. British Journal of Nutrition, 98(1), 96-100. https://doi.org/10.1017/S0007114507832910
• Mandal, S., Hati, S., Puniya, A.K., Khamrui, K., & Singh, K. (2014). Enhancement of survival of alginate‐encapsulated Lactobacillus casei NCDC 298. Journal of the Science of Food and Agriculture, 94(10), 1994-2001. https://doi.org/10.1002/jsfa.6514
• Maragkoudakis, P.A., Zoumpopoulou, G., Miaris, C., Kalantzopoulos, G., Pot, B., & Tsakalidou, E. (2006). Probiotic potential of Lactobacillus strains isolated from dairy products. International Dairy Journal, 16(3), 189-199. https://doi.org/10.1016/j.idairyj.2005.02.009
• Marshall, V.M., & Rawson, H.L. (1999). Effects of exopolysaccharide‐producing strains of thermophilic lactic acid bacteria on the texture of stirred yoghurt. International Journal of Food Science & Technology, 34(2), 137-143. https://doi.org/10.1046/j.1365-2621.1999.00245.x
• Martín, R., Langa, S., Reviriego, C., Jimínez, E., Marín, M. L., Xaus, J., & Rodríguez, J.M. (2003). Human milk is a source of lactic acid bacteria for the infant gut. The Journal of pediatrics, 143(6), 754-758. https://doi.org/10.1016/j.jpeds.2003.09.028
• Mojibi, P., Tafvizi, F., & Torbati, M.B. (2018). Cell-bound exopolysaccharide extract from indigenous probiotic bacteria induce apoptosis in HT–29 cell-line. Iranian Journal of Pathology, 14(1), 41. https://doi.org/10.30699/ijp.14.1.41
• Murphy, K., Curley, D., O’Callaghan, T.F., O’Shea, C.A., Dempsey, E.M., O’Toole, P.W., & Stanton, C. (2017). The composition of human milk and infant faecal microbiota over the first three months of life: a pilot study. Scientific reports, 7(1), 40597. https://doi.org/10.1038/srep40597
• Murugu, J., & Narayanan, R. (2024). Production, purification, and characterization of a novel exopolysaccharide from probiotic Lactobacillus amylovorus: MTCC 8129. Indian Journal of Microbiology, 64(3), 1355-1365. https://doi.org/10.1007/s12088-024-01346-y
• Nacef, M., Chevalier, M., Chollet, S., Drider, D., & Flahaut, C. (2017). MALDI-TOF mass spectrometry for the identification of lactic acid bacteria isolated from a French cheese: The Maroilles. International journal of food microbiology, 247, 2-8. https://doi.org/10.1016/j.ijfoodmicro.2016.07.005
• Noureen, S., Riaz, A., Arshad, M., & Arshad, N. (2019). In vitro selection and in vivo confirmation of the antioxidant ability of Lactobacillus brevis MG000874. Journal of applied microbiology, 126(4), 1221-1232. https://doi.org/10.1111/jam.14189
• Peter, S.B., Qiao, Z., Godspower, H.N., Omedi, J.O., Zhang, X., Xu, M., & Rao, Z. (2023). Antioxidant and biotechnological potential of Pediococcus pentosaceus RZ01 and Lacticaseibacillus paracasei RZ02 in a millet-based fermented substrate. Systems Microbiology and Biomanufacturing, 3(4), 571-584. https://doi.org/10.1007/s43393-022-00126-3
• Pino, A., Bartolo, E., Caggia, C., Cianci, A., & Randazzo, C.L. (2019). Detection of vaginal lactobacilli as probiotic candidates. Scientific Reports, 9(1), 3355. https://doi.org/10.1038/s41598-019-40304-3
• Pourjafar, H., Ansari, F., Sadeghi, A., Samakkhah, S.A., & Jafari, S.M. (2023). Functional and health-promoting properties of probiotics’ exopolysaccharides; isolation, characterization, and applications in the food industry. Critical Reviews in Food Science and Nutrition, 63(26), 8194-8225. https://doi.org/10.1080/10408398.2022.2047883
• Rajoka, M.S.R., Mehwish, H.M., Siddiq, M., Haobin, Z., Zhu, J., Yan, L., & Shi, J. (2017). Identification, characterization, and probiotic potential of Lactobacillus rhamnosus isolated from human milk. LWT-food Science and Technology, 84, 271-280. https://doi.org/10.1016/j.lwt.2017.05.055
• Rodríguez-Pastén, A., Pérez-Hernández, N., Añorve-Morga, J., Jiménez-Alvarado, R., Cariño-Cortés, R., Sosa-Lozada, T., & Fernández-Martínez, E. (2022). The activity of prebiotics and probiotics in hepatogastrointestinal disorders and diseases associated with metabolic syndrome. International Journal of Molecular Sciences, 23(13), 7229. https://doi.org/10.3390/ijms23137229
• Sahadeva, R.P.K., Leong, S.F., Chua, K.H., Tan, C.H., Chan, H.Y., Tong, E. V., & Chan, H.K. (2011). Survival of commercial probiotic strains to pH and bile. International Food Research Journal, 18(4), 1515-1522. https://doi.org/10.1111/j.1472-765X.2005.01744.x
• Savcı, A., Koçpınar, E.F., Alan, Y., & Kurşat, M. (2020). Antioxidant, antimicrobial, and DNA protection activities of some Tanacetum species and phenolic richness in their ethanolic extracts. International Food Research Journal, 27(1). https://doi.org/10.26656/fr.2017.01.060
• Son, S.H., Yang, S.J., Jeon, H.L., Yu, H.S., Lee, N.K., Park, Y.S., & Paik, H.D. (2018). Antioxidant and immunostimulatory effect of potential probiotic Lactobacillus paraplantarum SC61 isolated from Korean traditional fermented food, jangajji. Microbial Pathogenesis, 125, 486-492. https://doi.org/10.1016/j.micpath.2018.10.018
• Stepanović, S., Vuković, D., Hola, V., Bonaventura, G.D., Djukić, S., Ćirković, I., & Ruzicka, F. (2007). Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci. Apmis, 115(8), 891-899. https://doi.org/10.1111/j.1600-0463.2007.apm_630.x
• Surayot, U., Wang, J., Seesuriyachan, P., Kuntiya, A., Tabarsa, M., Lee, Y., & You, S. (2014). Exopolysaccharides from lactic acid bacteria: structural analysis, molecular weight effect on immunomodulation. International Journal of Biological Macromolecules, 68, 233-240. https://doi.org/10.1016/j.ijbiomac.2014.05.005
• Torino, M.I., Taranto, M.P., Sesma, F., & De Valdez, G.F. (2001). Heterofermentative pattern and exopolysaccharide production by Lactobacillus helveticus ATCC 15807 in response to environmental pH. Journal of Applied Microbiology, 91(5), 846-852. https://doi.org/10.1046/j.1365-2672.2001.01450.x
• Treven, P., Mahnič, A., Rupnik, M., Golob, M., Pirš, T., Matijašić, B.B., & Lorbeg, P.M. (2019). Evaluation of human milk microbiota by 16S rRNA gene next-generation sequencing (NGS) and cultivation/MALDI-TOF mass spectrometry identification. Frontiers in microbiology, 10, 2612. https://doi.org/10.3389/fmicb.2019.02612
• Uysul, A., Kurt, Ş., Soylu, S., Soylu, E.M., & Kara, M. (2019). Yaprağı yenen sebzelerdeki mikroorganizma türlerinin MALDI-TOF MS (Matris destekli lazer desorpsiyon/iyonizasyon uçuş süresi kütle spektrometresi) tekniği kullanılarak tanılanması. Yuzuncu Yıl University Journal of Agricultural Sciences, 29(4), 595-603. https://doi.org/10.29133/yyutbd.627850
• Vougiouklaki, D., Tsironi, T., Papaparaskevas, J., Halvatsiotis, P., & Houhoula, D. (2022). Characterization of Lacticaseibacillus rhamnosus, Levilactobacillus brevis and Lactiplantibacillus plantarum metabolites and evaluation of their antimicrobial activity against food pathogens. Applied Sciences, 12(2), 660. https://doi.org/10.3390/app12020660
• Wegh, C.A., Geerlings, S.Y., Knol, J., Roeselers, G., & Belzer, C. (2019). Postbiotics and their potential applications in early life nutrition and beyond. International journal of molecular sciences, 20(19), 4673. https://doi.org/10.3390/ijms20194673
• Yang, S.J., Kim, K.T., Kim, T.Y., & Paik, H.D. (2020). Probiotic properties and antioxidant activities of Pediococcus pentosaceus SC28 and Levilactobacillus brevis KU15151 in fermented black gamju. Foods, 9(9), 1154. https://doi.org/10.3390/foods9091154
• Yasmin, I., Saeed, M., Khan, W.A., Khaliq, A., Chughtai, M.F.J., Iqbal, R., & Tanweer, S. (2020). In vitro probiotic potential and safety evaluation (hemolytic, cytotoxic activity) of Bifidobacterium strains isolated from raw camel milk. Microorganisms, 8(3), 354. https://doi.org/10.3390/microorganisms8030354
• Zhang, X., Mushajiang, S., Luo, B., Tian, F., Ni, Y., & Yan, W. (2020a). The composition and concordance of Lactobacillus populations of infant gut and the corresponding breast-milk and maternal gut. Frontiers in Microbiology, 11, 597911. https://doi.org/10.3389/fmicb.2020.597911
• Zhang, L., Zhao, B., Liu, C.J., & Yang, E. (2020b). Optimization of biosynthesis conditions for the production of exopolysaccharides by Lactobacillus plantarum SP8 and the exopolysaccharides antioxidant activity test, Indian Journal of Microbiology, 60(3) 334-345. https://doi.org/10.1007/s12088-020-00865-8
• Zhao, W., Liu, Y., Latta, M., Ma, W., Wu, Z., & Chen, P. (2019). Probiotics database: A potential source of fermented foods. International Journal of Food Properties, 22(1), 198-217. https://doi.org/10.1080/10942912.2019.1579737
• Zheng, J., Wittouck, S., Salvetti, E., Franz, C.M.A.P., Harris, H.M.B., Mattarelli, P., O’Toole, P.W., Pot, B., Vandamme, P., Knapen, H.J.F.G., & Walter, J. (2020). A taxonomic note on the genus Lactobacillus and proposal of Lactiplantibacillus gen. nov., Ligilactobacillus gen. nov., Latilactobacillus gen. nov., Limosilactobacillus gen. nov., Lentilactobacillus gen. nov., Levilactobacillus gen. nov., Pediococcus pentosaceus gen. nov., etc. International Journal of Systematic and Evolutionary Microbiology, 70(4), 2782–2858. https://doi.org/10.1099/ijsem.0.004107