Molecular identification, characterization, and antagonistic activity profiling of Bacillus cereus LOCK 1002 along with the in-silico analysis of its presumptive bacteriocins

Samarth Islam; Mithila Farjana; Muhammad Ramiz Uddin; Sharmin Akter; Anika Jabin; Hazika Tuz-Zohura Nafisa; Siam Siraji; A K M Helal Morshed; Fahmida Hoque Rimti; Zannatul Naim; Mohiuddin Sakib; Pallab Sarker; Sabiha Naznin; H M Iftekhar Alam; Tanzila Ismail Ema; Mahbuba Siddiquy; Mohammad Habibur Rahman

doi:10.5455/javar.2022.i635

Molecular identification, characterization, and antagonistic activity profiling of Bacillus cereus LOCK 1002 along with the in-silico analysis of its presumptive bacteriocins

J Adv Vet Anim Res. 2022 Dec 31;9(4):663-675. doi: 10.5455/javar.2022.i635. eCollection 2022 Dec.

Authors

Samarth Islam^{1

2}, Mithila Farjana^{3

2}, Muhammad Ramiz Uddin^{3

2}, Sharmin Akter^{4

2}, Anika Jabin^{1

2}, Hazika Tuz-Zohura Nafisa⁵, Siam Siraji¹, A K M Helal Morshed^{6

2}, Fahmida Hoque Rimti^{7

2}, Zannatul Naim⁸, Mohiuddin Sakib¹, Pallab Sarker⁹, Sabiha Naznin¹⁰, H M Iftekhar Alam¹¹, Tanzila Ismail Ema^{1

2}, Mahbuba Siddiquy¹², Mohammad Habibur Rahman^{13

2}

Affiliations

¹ Department of Biochemistry and Microbiology, North South University, Dhaka, Bangladesh.
² Immunoinformatics and Vaccinomics Research Unit, RPG Interface Lab, Jashore, Bangladesh.
³ Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK, USA.
⁴ Department of Biology, Indiana State University, Terre Haute, IN, USA.
⁵ Department of Microbiology, University of Dhaka, Dhaka, Bangladesh.
⁶ Pathology and Pathophysiology Major, Academy of Medical Science, Zhengzhou University, Zhengzhou, China.
⁷ Bachelor of Medicine and Bachelor of Surgery, Chittagong Medical College, Chattogram, Bangladesh.
⁸ Department of Animal Production and Management, Sher-e-Bangla Agricultural University, Dhaka, Bangladesh.
⁹ Department of Medicine, Sher-E-Bangla Medical College Hospital, Dhaka, Bangladesh.
¹⁰ Department of Biomedical Engineering, Military Institute of Science and Technology, Dhaka, Bangladesh.
¹¹ Division of Validation, BEXIMCO Pharma Ltd., Dhaka, Bangladesh.
¹² State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China.
¹³ Vaccinology Lab, Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh, Bangladesh.

Abstract

Objectives: This research aimed to isolate, identify, and characterize a new strain of Bacillus cereus through different molecular biology approaches so that it could be further studied for therapeutic purposes against selective enteric pathogens.

Materials and methods: Pure isolates of B. cereus were prepared from buffalo yogurt samples in REMBA medium. Initially, the morphological, physiological, and biochemical properties were studied accordingly. Following the tests, the molecular identification for the strain identification was conducted through plasmid DNA extraction, PCR, agarose gel electrophoresis, and 16S rRNA sequencing up to 1.37 kb. Afterward, the antibiotic sensitivity [Epsilometer test (E-Test)] and antifungal activity were tested considering different concentrations. Being classified from the aforementioned tests, a comprehensive antimicrobial activity test was conducted using the cell-free-supernatant (CFS) of the test strain against selective enteric pathogens in humans in vitro. Besides, the different clusters of genes were identified and characterized for understanding the presumptive bacteriocins present in the CFS of the strain in silico, where molecular string properties were calculated. Finally, the evolutionary relationship among diversified bacteriocins synthesized by different Bacillus strains was studied to predict the CFS-containing bacteriocins of the new strain.

Results: Purified isolates of B. cereus were Gram-positive rods and showed significant tolerance (p < 0.0001) to different concentrations of pH, phenol, bile salt, and NaCl. 16S rRNA revealed the strain as LOCK 1002, which was strongly sensitive to all the antibiotics used and resistant to selective antifungal agents. The CFS of B. cereus LOCK 1002 was found to be a very promising antagonist to all the enteric pathogens used in the culture condition. Two gene clusters were predicted to be interconnected and responsible for different presumptive bacteriocins.

Conclusion: The newly identified LOCK 1002 can be a very potent strain of B. cereus in use as an antimicrobial agent for having different bacteriocin coding gene clusters.

Keywords: 16S rRNA analysis; B. cereus LOCK 1002; PEMBA medium; antimicrobial activity profiling of B. cereus; bacteriocins, genetic clustering; cerein 7B.