Sains Malaysiana 54(7)(2025): 1713-1723

http://doi.org/10.17576/jsm-2025-5407-06

 

Pencerapan dan Analisis Fenomena Pembunuhan Klonal Escherichia coli

atas Medium Pertumbuhan Minimum Tertakrif Berbanding dengan Medium Kaya

(Observation and Analysis of Escherichia coli Clonal Killing Phenomenon on Defined Minimal Growth Medium Compared to Rich Medium)

 

FOO PEI YA¹, NAZALAN NAJIMUDIN², PRAVIN KUMRAN NYANASEGRAN³ & NG CHYAN LEONG^3,*

 

¹Department of Biological Sciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia

²School of Biological Sciences, Universiti Sains Malaysia, 11700 Gelugor, Penang, Malaysia

³Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia

 

Diserahkan: 9 September 2024/Diterima: 23 Mei 2025

 

Abstrak

Bakteria menggunakan komunikasi kimia yang rumit yang melibatkan tingkah laku kerjasama dan persaingan demi kelangsungan hidup dalam menghadapi keadaan yang mencabar. Kajian terdahulu telah melaporkan bahawa dalam persekitaran kekurangan nutrien, bakteria menunjukkan tingkah laku radikal, termasuk rembesan sebatian antimikrob. Sebatian ini tidak hanya memberi kesan kepada strain bakteria yang berkaitan tetapi juga koloni klon, membawa kepada fenomena yang dikenali sebagai pembunuhan klon. Namun, fenomena pembunuhan klon pada medium minimum masih kurang dikaji. Penyelidikan ini mengkaji persaingan antara dua koloni klon Escherichia coli ATCC 25922 (EC 25922) pada media kompleks (LB) dan media minimum (M9). Seterusnya, fenomena pembunuhan klon diperhatikan dan dianalisis. Hasil menunjukkan bahawa koloni yang menghadap ke luar (EC-Out) menunjukkan kadar pertumbuhan yang lebih tinggi daripada koloni yang menghadap ke dalam (EC-In). Pada kedua-dua agar media LB dan M9, koloni klon EC 25922 menunjukkan kesan penindasan oleh koloni bersebelahan dan menghasilkan zon perencatan antara dua koloni. Pembentukan zon perencatan berlaku lebih cepat pada agar LB berbanding agar M9 dan mungkin disebabkan oleh faktor yang dirembeskan yang menghalang penggabungan koloni. Pembentukan zon perencatan juga bergantung pada jarak pemisahan awal antara koloni klon. Strain EC 25922 yang sama tidak dapat tumbuh apabila diinokulasikan dalam zon perencatan, menunjukkan kehadiran perencat persaingan. Penemuan ini mencadangkan bahawa sintesis faktor antimikrob serta ketersediaan nutrien mungkin mempengaruhi perkembangan biofilem dan corak pertumbuhan populasi bakteria klon di dalamnya. Pemahaman dalam pembunuhan klon boleh membantu penemuan antibiotik baharu dan meningkatkan pengetahuan mengenai daya tahan bakteria dalam keadaan yang buruk.

Kata kunci: Escherichia coli ATCC 25922; pembunuhan klon; perencatan pertumbuhan; sebatian antimikrob; tekanan nutrien

 

Abstract

Bacteria employ intricate chemical communication involving both cooperative and competitive behaviours for survival when living in harsh conditions. Previous studies have reported that in a nutrient deprivation environment, bacteria exhibit radical behaviours, including antimicrobial compound secretion. These compounds affect not only related bacterial strains but also sibling clonal cells, leading to the phenomenon known as clonal killing. However, the clonal killing phenomenon on minimal medium remains underexplored. This study investigates competition between two Escherichia coli ATCC 25922 (EC 25922) clonal colonies on rich media (LB) and minimal media (M9). Subsequently, the clonal killing phenomenon was observed and analysed. The results indicated that colonies facing outward (EC-Out) exhibited higher growth rates than colonies facing inward (EC-In). On both LB and M9 media, EC 25922 clonal colonies demonstrated suppression exerted by the neighbouring colony and produced a zone of inhibition between the two colonies. This zone of inhibition formed more rapidly on LB agar than M9 agar and is possibly due to secreted factor(s) that prevent colonies from merging. The inhibition zone formation also depended on the initial separation distance between the clonal colonies. The same strain of EC 25922 was unable to grow when inoculated within the zone of inhibition, indicating the presence of a competitive inhibitor. These findings suggest that the synthesis of antimicrobial factors and nutrient availability may affect biofilm development and the growth pattern of clonal bacterial population inside them. Understanding clonal killing could help find new antibiotics and improve our knowledge of bacterial resilience under unfavourable conditions.

Keywords: Antimicrobial compounds; clonal killing; Escherichia coli ATCC 25922; growth inhibition; nutrient stress

 

RUJUKAN

Adler, J. 1973. A method for measuring chemotaxis and use of the method to determine optimum conditions for chemotaxis by Escherichia coli. Journal of General Microbiology 74(1): 77-91.

Adler, J. 1966. Chemotaxis in bacteria. Science 153(3737): 708-716.

Alves, C.S., Melo, M.N., Franquelim, H.G., Ferre, R., Planas, M., Feliu, L., Bardají, E., Kowalczyk, W., Andreu, D., Santos, N.C., Fernandes, M.X. & Castanho, M.A.R.B. 2010. Escherichia coli cell surface perturbation and disruption induced by antimicrobial peptides BP100 and pepR. Journal of Biological Chemistry 285(36): 27536-27544.

Baev, M.V., Baev, D., Radek, A.J. & Campbell, J.W. 2006. Growth of Escherichia coli MG1655 on LB medium: Monitoring utilization of sugars, alcohols, and organic acids with transcriptional microarrays. Applied Microbiology and Biotechnology 71(3): 310-316.

Baquero, F., Lanza, V.F., Baquero, M.R., del Campo, R. & Bravo-Vázquez, D.A. 2019. Microcins in Enterobacteriaceae: Peptide antimicrobials in the eco-active intestinal chemosphere. Frontiers in Microbiology 10: 2261.

Be’er, A. & Ariel, G. 2019. A statistical physics view of swarming bacteria. Movement Ecology 7(1): 9.

Be’er, A., Zhang, H.P., Florin, E.L., Payne, S.M., Ben-Jacob, E. & Swinney, H.L. 2009. Deadly competition between sibling bacterial colonies. Proceedings of the National Academy of Sciences 106(2): 428-433.

Budič, M., Rijavec, M., Petkovšek, Ž. & Žgur-Bertok, D. 2011. Escherichia coli bacteriocins: Antimicrobial efficacy and prevalence among isolates from patients with bacteraemia. PLoS ONE 6(12): e28769.

Cascales, E., Buchanan, S.K., Duché, D., Kleanthous, C., Lloubès, R., Postle, K., Riley, M., Slatin, S. & Cavard, D. 2007. Colicin biology. Microbiology and Molecular Biology Reviews 71(1): 158-229.

Corning, P.A. 2022. A systems theory of biological evolution. Biosystems 214: 104630.

dos Santos, A.L.S., Galdino, A.C.M., de Mello, T.P., de Souza Ramos, L., Branquinha, M.H., Bolognese, A.M., Columbano Neto, J. & Roudbary, M. 2018. What are the advantages of living in a community? A microbial biofilm perspective! Memórias do Instituto Oswaldo Cruz 113(9): e180212.

Elbing, K.L. & Brent, R. 2019. Recipes and tools for culture of Escherichia coli. Current Protocols in Molecular Biology 125(1): e83.

Flemming, H.C., Wingender, J., Szewzyk, U., Steinberg, P., Rice, S.A. & Kjelleberg, S. 2016. Biofilms: An emergent form of bacterial life. Nature Reviews Microbiology 14(9): 563-575.

Gibbs, K.A., Urbanowski, M.L. & Greenberg, E.P. 2008. Genetic determinants of self identity and social recognition in bacteria. Science 321(5886): 256-259.

Grafen, A. 1986. Natural selection, kin selection and group selection. Behavioural Ecology: An Evolutionary Approach.

Hall-Stoodley, L., Costerton, J.W. & Stoodley, P. 2004. Bacterial biofilms: from the natural environment to infectious diseases. Nature Reviews Microbiology 2(2): 95-108.

Mavridou, D.A.I., Gonzalez, D., Kim, W., West, S.A. & Foster, K.R. 2018. Bacteria use collective behavior to generate diverse combat strategies. Current Biology 28(3): 345-355.e4.

Ng, F.L. 2019. Clonal interaction between Escherichia coli ATCC 25922 colonies. PhD Thesis. Universiti Sains Malaysia.

Niehus, R., Oliveira, N.M., Li, A., Fletcher, A.G. & Foster, K.R. 2021. The evolution of strategy in bacterial warfare via the regulation of bacteriocins and antibiotics. eLife 10: e69756.

Paul, R., Ghosh, T., Tang, T. & Kumar, A. 2019. Rivalry in Bacillus subtilis colonies: Enemy or family? Soft Matter 15(27): 5400-5411.

Platt, T.G. & Bever, J.D. 2009. Kin competition and the evolution of cooperation. Trends in Ecology & Evolution 24(7): 370-377.

Portell, X., Pot, V., Garnier, P., Otten, W. & Baveye, P.C. 2018. Microscale heterogeneity of the spatial distribution of organic matter can promote bacterial biodiversity in soils: Insights from computer simulations. Frontiers in Microbiology 9: 1583.

Prigent-Combaret, C., Vidal, O., Dorel, C. & Lejeune, P. 1999. Abiotic surface sensing and biofilm-dependent regulation of gene expression in Escherichia coli. Journal of Bacteriology 181(19): 5993-6002.

Ratiu, I.A., Ligor, T., Bocos-Bintintan, V., Al-Suod, H., Kowalkowski, T., Rafińska, K. & Buszewski, B. 2017. The effect of growth medium on an Escherichia coli pathway mirrored into GC/MS profiles. Journal of Breath Research 11(3): 036012.

Schneider, C.A., Rasband, W.S. & Eliceiri, K.W. 2012. NIH Image to ImageJ: 25 years of image analysis. Nature Methods 9(7): 671-675.

Serra, D.O., Richter, A.M. & Hengge, R. 2013. Cellulose as an architectural element in spatially structured Escherichia coli biofilms. Journal of Bacteriology 195(24): 5540-5554.

Sezonov, G., Joseleau-Petit, D. & D’Ari, R. 2007. Escherichia coli physiology in Luria-Bertani broth. Journal of Bacteriology 189(23): 8746-8749.

Soares, A., Gomes, L.C., Monteiro, G.A. & Mergulhão, F.J. 2021. The influence of nutrient medium composition on Escherichia coli biofilm development and heterologous protein expression. Applied Sciences 11(18): 8667.

Sperotto, G., Stasiak, L.G., Godoi, J.P.M.G., Gabiatti, N.C. & De Souza, S.S. 2021. A review of culture media for bacterial cellulose production: Complex, chemically defined and minimal media modulations. Cellulose 28(5): 2649-2673.

Tso, W.W. & Adler, J. 1974. Negative chemotaxis in Escherichia coli. Journal of Bacteriology 118(2): 560-576.

Valero, P., Giannakis, S., Mosteo, R., Ormad, M.P. & Pulgarin, C. 2017. Comparative effect of growth media on the monitoring of E. coli inactivation and regrowth after solar and photo-Fenton treatment. Chemical Engineering Journal 313: 109-120.

Varik, V., Oliveira, S.R.A., Hauryliuk, V. & Tenson, T. 2016. Composition of the outgrowth medium modulates wake-up kinetics and ampicillin sensitivity of stringent and relaxed Escherichia coli. Scientific Reports 6(1): 22308.

Wade, M.J. 1985. Soft selection, hard selection, kin selection, and group selection. The American Naturalist 125(1): 61-73.

Wang, C.H. & Koch, A.L. 1978. Constancy of growth on simple and complex media. Journal of Bacteriology 136(3): 969-975.

Williams, P. 2007. Quorum sensing, communication and cross-kingdom signalling in the bacterial world. Microbiology 153(12): 3923-3938.

Xavier, J.B. & Foster, K.R. 2007. Cooperation and conflict in microbial biofilms. Proceedings of the National Academy of Sciences 104(3): 876-881.

Zhao, X., Yu, Z. & Ding, T. 2020. Quorum-sensing regulation of antimicrobial resistance in bacteria. Microorganisms 8(3): 425.

 

*Pengarang untuk surat-menyurat; email: clng@ukm.edu.my