Characterization and Antibacterial Activity of Bacillus subtilis MK-4 Isolated from Southern Area of Pakistan

Author's: Sajid Iqbal, Hazir Rahman, Farida Begum, Imran Sajid, Muhammad Qasim
Authors' Affiliations
Article Type: Research Article     Published: Dec. 29, 2019 Pages: 41-50
DOI:        Views 112       Downloads0


Antibacterial molecules are generally considered as secondary metabolites produced by bacteria during the stationary phase of their growth, which can kill or inhibit the growth of other bacteria. Nowadays, the unsystematic use of antibiotics has resulted in resistant bacteria. Investigation of new antibacterial metabolites and the identification of unexplored antibacterial exhibiting bacteria are necessary. In this study, the bacterial isolate MK-4 was obtained from the soil of the local habitat (Karak, Pakistan). The isolate MK-4 was preliminarily screened for antibacterial activity against a set of Gram-positive as well as Gram-negative bacterial isolates. Antibacterial activity was evaluated against 9 ATCC bacterial strains including Staphylococcus aureus (29213), Staphylococcus epidermidis (12228), Escherichia coli (25922), Salmonella typhimurium (14028), Shigella flexneri (12022), Streptoccocus pneumonia (6305), Pseudomonas aeruginosa (27853), Klebsiella pneumoniae (13889) and Vibrio cholerae (9459) and 4 clinical multidrug-resistant (A. buemannii, S. aureus, E. coli and P. aeruginosa). Antibacterial activity was measured as zone of inhibition (ZOI) in mm. Identification of bacterial isolate B. subtilis MK-4 was based on 16S rRNA gene sequencing apart from biochemical and morphological characteristics. The isolate was further optimized for growth as well as for antibacterial metabolites production at different pH, temperature and incubation time. The isolate MK-4 showed maximum growth at 30°C, maximum antibacterial activity at 37°C. MK-4 exhibited maximum growth and antibacterial activity at pH 8 after 48 hours incubation time.


Soil bacteria, Antibacterial activity, optimization, Bacillus, Pakistan.


Iqbal, S., Rahman, H., Begum, F., Sajid, I., Qasim, M., 2019. Characterization and Antibacterial Activity of Bacillus subtilis MK-4 Isolated from Southern Area of Pakistan. Int. J. Mol. Microbiol., 2(3): 41-50.


Agaba, P., Tumukunde, J., Tindimwebwa, J.V.B., Kwizera, A., 2017. Nosocomial bacterial infections and their antimicrobial susceptibility patterns among patients in Ugandan intensive care units: a cross sectional study. BMC Res. Notes., 10(1), p.349.

Al-Ajlani, M.M., Hasnain, S., 2010. Bacteria exhibiting antimicrobial activities; screening for antibiotics and the associated genetic studies. In Open Conf. Proceed. J.,  1: 230-238).

Al-Ajlani, M.M., Sheikh, M.A., Ahmad, Z., Hasnain, S., 2007. Production of surfactin from Bacillus subtilis MZ-7 grown on pharmamedia commercial medium. Microbial Cell Factor., 6(1): 17.

Alsohiby, F.A.A., Yahya, S., Humaid, A.A., 2016. Screening of Soil Isolates of Bacteria for Antagonistic Activity against Plant Pathogenic Fungi. PSM Microbiol., 01(1): 05-09.

Boucher, H.W., 2010. Challenges in anti-infective development in the era of bad bugs, no drugs: a regulatory perspective using the example of bloodstream infection as an indication. Clin. Infec. Dis., 50(Supplement_1): pp.S4-S9.

Caulier, S., Nannan, C., Gillis, A., Licciardi, F., Bragard, C., Mahillon, J., 2019. Overview of the antimicrobial compounds produced by members of the Bacillus subtilis group. Front. Microbiol., 10: 302.

Donadio, S., Maffioli, S., Monciardini, P., Sosio, M., Jabes, D., 2010. Antibiotic discovery in the twenty-first century: current trends and future perspectives. The J. Antibiot., 63(8): p.423.

Felnagle, E.A., Rondon, M.R., Berti, A.D., Crosby, H.A. and Thomas, M.G., 2007. Identification of the biosynthetic gene cluster and an additional gene for resistance to the antituberculosis drug capreomycin. Appl. Environ. Microbiol., 73(13): 4162-4170.

Ghribi, D., Abdelkefi-Mesrati, L., Mnif, I., Kammoun, R., Ayadi, I., Saadaoui, I., Maktouf, S., Chaabouni-Ellouze, S., 2012. Investigation of antimicrobial activity and statistical optimization of Bacillus subtilis SPB1 biosurfactant production in solid-state fermentation. BioMed Res. Int., 2012.

Goldenberger, D., Perschil, I., Ritzler, M. and Altwegg, M., 1995. A simple” universal” DNA extraction procedure using SDS and proteinase K is compatible with direct PCR amplification. Genome Res., 4(6): 368-370.

Holt, J.G., Krieg, N.R., Sneath, P.H., Staley, J.T., Williams, S.T., 1994. Bergey’s manual of determinative bacteriology. 9th. Baltimor: William & Wilkins.

Iqbal, M.N., Ali, S., Anjum, A.A., Muhammad, K., Ali, M.A., Wang, S., Khan, W.A., Khan, I., Muhammad, A., Mahmood, A., Irfan, M., Ahmad, A., Ashraf, A., Hussain, F., 2016. Microbiological Risk Assessment of Packed Fruit Juices and Antibacterial Activity of Preservatives against Bacterial Isolates. Pak. J. Zool., 48(6): 1695-1703.

Iqbal, M.N, Ashraf, A., 2019. Withania somnifera: Can it be a Therapeutic Alternative for Microbial Diseases in an Era of Progressive Antibiotic Resist- ance? Int. J. Nanotechnol. Allied Sci., 3(1): 16-18.

Iqbal, M.N, Ashraf, A., 2018. Ceftazidime Resistant Bacteria in Clinical Samples: Do We Need New Antibiotics? Int. J. Molec. Microbiol., 1(2): 16-18.

Karbalaei-Heidari, H.R., Ziaee, A.A., Schaller, J.,  Amoozegar, M.A., 2007. Purification and characterization of an extracellular haloalkaline protease produced by the moderately halophilic bacterium, Salinivibrio sp. strain AF-2004. Enzyme Microbial Technol., 40(2): 266-272.

Maleki, H., Dehnad, A., Hanifian, S., Khani, S., 2013. Isolation and molecular identification of Streptomyces spp. with antibacterial activity from northwest of Iran. BioImpacts: BI, 3(3): 129.

Nasfi, Z., Busch, H., Kehraus, S., Linares-Otoya, L., König, G.M., Schäberle, T.F., Bachoual, R., 2018. Soil Bacteria Isolated From Tunisian Arid Areas Show Promising Antimicrobial Activities Against Gram-Negatives. Fronti. Microbiol., 9: 2742.

Ravot, G., Masson, J. M., Lefèvre, F., 2006. 34 applications of extremophiles: the industrial screening of extremophiles for valuable biomolecules. In Methods in Microbiol.,  35: 785-813). Academic Press.

Sánchez, L.A., Gómez, F.F., Delgado, O.D., 2009. Cold-adapted microorganisms as a source of new antimicrobials. Extremo., 13(1): 111-120.

Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Molec. Biol. Evol., 30(12): 2725-2729.

Usta, A., Demirkan, E., 2013. The effect of growth parameters on the antibiotic activity and sporulation in Bacillus spp. isolated from soil. The J. Microbiol., Biotechnol. Food Scie., 2(5): 2310.

Yun, T.Y., Feng, R.J., Zhou, D.B., Pan, Y.Y., Chen, Y.F., Wang, F., Yin, L.Y., Zhang, Y.D., Xie, J.H., 2018. Optimization of fermentation conditions through response surface methodology for enhanced antibacterial metabolite production by Streptomyces sp. 1-14 from cassava rhizosphere. PloS one., 13(11):  e0206497.

Yunus, F.N., Khalid, Z.Z., Rashid, F., Ashraf, A., Iqbal, M.N., Hussain, F., 2016. Isolation and Screening of Antibiotic producing Bacteria from Soil in Lahore City. PSM Microbiol., 01(1): 01-04.

Yunus, F.N., Saeed, H., Rashid, F., Iqbal, M.N., Ashraf, A., 2017. Isolation and Identification of Esterase Producing Bacillus subtilis from Soil. PSM Microbiol., 2(2): 24-28.

Related Content