Identification of a New Compound (4-Fluoro-2-Trifluoromethyl Imidazole) Extracted from a New Halophilic Bacillus aquimaris Strain Persiangulf TA2 Isolated from the Northern Persian Gulf with Broad-Spectrum Antimicrobial Effect

Document Type : Research Paper


1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.

2 Department of Microbiology, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.

3 Department of Biology, Ahvaz Branch, Islamic Azad University, Ahvaz, Iran.

4 Department of Fisheries, Ahvaz Branch, Islamic Azad University, Ahvaz, Iran.


Background: The unique ecosystem of the Persian Gulf has made it a rich source of natural antimicrobial compounds 
produced by various microorganisms, especially bacteria, which can be used in the treatment of infectious diseases, 
especially those of drug-resistant microbes.
Objectives: This study aimed to identify antimicrobial compounds in the bacteria isolated from the northern region of the 
Persian Gulf in Abadan (Chavibdeh port), Iran, for the first time. 
Materials and Methods: Sampling was performed in the fall on November 15, 2019, from 10 different stations (water 
and sediment samples). The secondary metabolites of all isolates were extracted, and their antimicrobial effects were 
investigated. 16S ribosomal ribonucleic acid sequencing was used for the identification of the strains that showed the 
best inhibition against selected pathogens, and growth conditions were optimized for them. A fermentation medium 
in a volume of 5000 mL was prepared to produce the antimicrobial compound by the superior strain. The extracted 
antimicrobial compounds were identified using the gas chromatography-mass spectrometry technique. Minimum 
inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined for the superior strain. 
The effects of salinity, pH, and temperature on the production of antimicrobial compounds were determined by measuring 
the inhibitory region (mm) of methicillin-resistant Staphylococcus aureus (MRSA).
Results: Four new strains with antimicrobial properties (i.e., Halomonas sp. strain Persiangulf TA1, Bacillus aquimaris
strain Persiangulf TA2, Salinicoccus roseus strain Persiangulf TA4, and Exiguobacterium profundum strain Persiangulf 
TA9) were identified. The optimum growth temperatures were determined at 37-30, 37, and 40 °C for TA1 and TA2, TA4, 
and TA9 strains, respectively. The optimum pH values for the four strains were 7, 6-7, 7.5, and 6.5-7.5, respectively. The 
optimal salt concentrations for the four strains were 15%, 2.5-5%, 7.5%, and 5%, respectively. The ethyl acetate extract 
of strain Persiangulf TA2 showed extensive antimicrobial activity against human pathogens (75%) and MRSA. The most 
abundant compound identified in TA2 extract was the new compound 4-fluoro-2-trifluoromethyl imidazole. The MBC 
and MIC for the ethyl acetate extract of strain TA2 were 20 and 5 mg. mL-1 (Staphylococcus aureus), 40 and 20 mg. mL-1
(MRSA, Escherichia coli, and Enterococcus faecalis), 40 and 10 mg. mL-1 Acinetobacter baumannii), and 80 and 40 mg. 
mL-1 (Staphylococcus epidermidis, Shigella sp., Bacillus cereus, and Klebsiella pneumoniae), respectively. The optimal 
conditions for antibiotic production by TA2 strain were 5% salt concentration, pH of 7, and temperature of 35 °C. 
Conclusion: Newly detected natural compounds in TA2 strain due to superior antimicrobial activity even against MRSA 
strain can be clinically valuable in pharmacy and treatment.


Main Subjects

1. Nathan C, Cars O. Antibiotic resistance—problems, progress, 
and prospects. N Engl J Med. 2014;371(19):1761-1763. doi: 
10.1056/NEJMp14080402. Peterson JW. Bacterial pathogenesis. Medical Microbiology. 
4th edition: University of Texas Medical Branch at Galveston; 
3. de Kraker ME, Stewardson AJ, Harbarth S. Will 10 million 
people die a year due to antimicrobial resistance by 2050?. 
PLoS Med. 2016;13(11):e1002184. doi: 10.1371/journal.pmed. 
4. Gootz TD. The global problem of antibiotic resistance. Crit Rev 
Immunol. 2010;30(1). doi: 10.1615/CritRevImmunol.v30.i1.60
5. Mondol MAM, Shin HJ, Islam MT. Diversity of secondary metabolites from marine Bacillus species: chemistry and biological activity. Mar Drugs. 2013;11(8):2846-2872. doi: 10.3390/
6. Nikaido H. Multidrug resistance in bacteria. Annu Rev Biochem. 
2009;78:119-146. doi: 10.1146/annurev.biochem.78.082907.145923
7. Poosarla A, Krishna RM. Isolation of potent antibiotic producing Actinomycetes from marine sediments of Andaman and 
Nicobar Marine Islands. J Microbiol Antimicrob. 2013;5(1):6-
12. doi: 10.5897/JMA11.075
8. Attimarad SL, Ediga GN, Karigar AA, Karadi R, Chandrashekhar N, Shivanna C. Screening, isolation and purification of antibacterial agents from marine actinomycetes. Int Curr Pharm 
J. 2012;1(12):394-402. doi: 10.3329/icpj.v1i12.12448 
9. Al-Zereini W. Natural products from marine bacteria (Doctoral dissertation, Kaiserslautern, Techn. Univ., Diss., 2006).
10. Burja AM, Banaigs B, Abou-Mansour E, Burgessd JG, Wrighta 
PC. Marine cyanobacteria- a prolific source of natural products. Tetrahedron. 2001;57:9347-9377. doi: 10.1016/s0040-4020 
11. Bhatnagar I, Kim S-K. Pharmacologically prospective antibiotic agents and their sources: a marine microbial perspective. 
Environ Toxicol Pharmacol. 2012;34(3):631-643. doi: 10.1016/j.
12. Ravikumar S, Krishnakumar S, Inbaneson SJ, Gnanadesigan M. 
Antagonistic activity of marine actinomycetes from Arabian 
Sea coast. Arch Appl Sci Res. 2010;2(6):273-280.
13. Jensen P, Fenical W. Marine bacterial diversity as a resource for 
novel microbial products. J Ind Microbiol. 1996;17(5):346-351. 
doi: 10.1007/BF01574765
14. Valentine DL. Adaptations to energy stress dictate the ecology 
and evolution of the Archaea. Nat Rev Microbiol. 2007;5(4):316-
323. doi: 10.1038/nrmicro1619
15. Desriac F, Defer D, Bourgougnon N, Brillet B, Le Chevalier P, 
Fleury Y. Bacteriocin as weapons in the marine animal-associated bacteria warfare: inventory and potential applications 
as an aquaculture probiotic. Mar Drugs. 2010;8(4):1153-1177. 
doi: 10.3390/md8041153
16. Agah H, Elskens M, Fatemi SMR, Owfi F, Baeyens W, Leermakers M. Mercury speciation in the Persian Gulf sediments. 
Environ Monit Assess. 2009;157(1-4):363-373. doi: 10.1007/
17. Darabpour E, Ardakani MR, Motamedi H, Ronagh MT. 
Isolation of a potent antibiotic producer bacterium, especially against MRSA, from northern region of the Persian 
Gulf. Bosnian J Basic Med Sci. 2012;12(2):108. doi: 10.17305/
18. Mozafari H, & Raeis A.S. The impact of physical factors (wind 
and sea surface temperature) on the geopolitics of the Persian 
Gulf. J Plotical Int Res. 2009; 1(3):163-186.
19. Jayadev A, Lekshmi M. Screening and Isolation of Protease 
producing Marine Bacteria. Emergent Life Sci Res. 2016;2:73-
76. doi: 
20. Yeon S-H, Jeong W-J, Park J-S. The diversity of culturable organotrophic bacteria from local solar salterns. J Microbiol. 
21. Asthana RK, Tripathi MK, Srivastava A, Singh AP, Singh SP, 
Nath G, et al. Isolation and identification of a new antibacterial 
entity from the Antarctic Cyanobacterium Nostoc CCC 537. J 
Appl Phycol. 2009;21(1):81. doi: 10.1007/s10811-008-9328-2
22. Mahato N, Sinha M, Sharma K, Koteswararao R, Cho MH. Modern Extraction and Purification Techniques for Obtaining High 
Purity Food-Grade Bioactive Compounds and Value-Added 
Co-Products from Citrus Wastes. Foods. 2019;8(11):523. doi: 
23. Darabpour E, Ardakani MR, Motamedi H, Ghezelbash G, 
Ronagh MT. Isolation of an antibiotic producer Pseudomonas 
sp. from the Persian Gulf. Asian Pac J Trop Med. 2010;3(4):318-
321. doi: 10.1016/S1995-7645(10)60077-6
24. Xiong H, Qi S, Xu Y, Miao L, Qian P-Y. Antibiotic and antifouling compound production by the marine-derived fungus 
Cladosporium sp. F14. J Hydro-Environ Res. 2009;2(4):264-270. 
doi: 10.1016/j.jher.2008.12.002
25. Vos P, Garrity G, Jones D, Krieg NR, Ludwig W, Rainey FA, et 
al. Bergey’s manual of systematic bacteriology: Volume 3: The 
Firmicutes. Springer Sci Business Media; 2011. doi: 10.1007/
26. Simair AA, Khushk I, Qureshi AS, Bhutto MA, Chaudhry HA, 
Ansari KA, et al. Amylase production from thermophilic Bacillus sp. BCC 021-50 isolated from a marine environment. 
Fermentation. 2017;3(2):25. doi: 10.3390/fermentation3020025
27. Puntambekar AN & Dake MS. Isolation, purification , and 
optimization of thermophilic ana alkaliphilic protease origination from hot water spring bacteria. Asian J Pharm Clin Res. 
2017;10(9):284-291. doi: 10.22159/ajpcr.2017.v10i9.19717
28. Ilesanmi OI, Adekunle AE, Omolaiye JA, Olorode EM, Ogunkanmi AL. Isolation, optimization and molecular characterization of lipase producing bacteria from contaminated soil. Sci 
Afr. 2020;8:e00279. doi: 10.1016/j.sciaf.2020.e00279
29. Khan IA, Jahan P, Hasan Q, Rao P. Genetic confirmation of 
T2DM meta-analysis variants studied in gestational diabetes mellitus in an Indian population. Diabetes Metab Syndr. 
2019;13(1):688-694. doi: 10.1016/j.dsx.2018.11.035
30. Anantha PS, Deventhiran M, Saravanan P, Anand D, Rajarajan S. A comparative GC-MS analysis of bacterial secondary 
metabolites of Pseudomonas species. The Pharma Innovation. 
2016; 5(4, Part B):84.
31. Darabpour E, Ardakani MR, Motamedi H, Ronagh MT. Isolation of a broad spectrum antibiotic producer bacterium, Pseudoalteromonas piscicida PG-02, from the Persian Gulf. Bangladesh J Pharmacol. 2011;6(2):74-83. doi: 10.3329/bjp.v6i2.8592
32. Vijayan K, Singh IB, Jayaprakash N, Alavandi S, Pai SS, Preetha R, et al. A brackishwater isolate of Pseudomonas PS-102, 
a potential antagonistic bacterium against pathogenic vibrios 
in penaeid and non-penaeid rearing systems. Aquaculture. 
2006;251(2-4):192-200. doi: 10.1016/j.aquaculture.2005.10.010
33. Goel N, Fatima SW, Kumar S, Sinha R, Khare SK. Antimicrobial resistance in biofilms: Exploring marine actinobacteria as 
a potential source of antibiotics and biofilm inhibitors. Biotechnol Rep. 2021; 30:e00613. doi: 10.1016/j.btre.2021.e00613
34. Mondol M, Shin H, Islam M. Diversity of secondary metabolites from marine Bacillus species: chemistry and biological activity. Mar Drugs. 2013;11(8):2846-2872. doi: 10.3390/35. Chau KM, Van Quyen D, Fraser JM, Smith AT, Van TTH, 
Moore RJ. Broad spectrum antimicrobial activities from 
spore-forming bacteria isolated from the Vietnam Sea. PeerJ. 
2020;8:e10117. doi: 10.7717/peerj.10117
36. Liu Y, Teng K, Wang T, Dong E, Zhang M, Tao Y, et al. Antimicrobial Bacillus velezensis HC6: production of three kinds of lipopeptides and biocontrol potential in maize. J Appl Microbiol. 
2020;128(1):242-254. doi: 10.1111/jam.14459
37. Feliatra F, Batubara UM, Nurulita Y, Lukistyowati I, Setiaji J. 
The potentials of secondary metabolites from Bacillus cereus SN7 and Vagococcus fluvialis CT21 against fish pathogenic 
bacteria. Microb Pathog. 2021; 158:105062. doi: 10.1016/j.micpath.2021.105062
38. Prieto ML, O’Sullivan L, Tan SP, McLoughlin P, Hughes H, 
O’Connor PM, et al. Assessment of the bacteriocinogenic potential of marine bacteria reveals lichenicidin production by 
seaweed-derived Bacillus spp. Mar Drugs. 2012;10(10):2280-
2299. doi: 10.3390/md10102280
39. Siefert JL, Larios-Sanz M, Nakamura LK, Slepecky RA, Paul JH, 
Moore ER, et al. Phylogeny of marine Bacillus isolates from the 
Gulf of Mexico. Curr Microbiol. 2000;41(2):84-88. doi: 10.1007/
40. Cherian T, Yalla SK, Mohanraju R. Antimicrobial potential 
of methanolic extract of Bacillus aquimaris isolated from the 
marine waters of Burmanallah coast, South Andaman. Int 
J Bio-Pharma Res 2019;8(12):2806-2813. doi: 10.21746/ijbpr.2019.8.12.1
41. Chu J, Wang Y, Zhao B, Zhang X-m, Liu K, Mao L, et al. Isolation and identification of new antibacterial compounds from 
Bacillus pumilus. Appl Microbiol Biotechnol. 2019;103(20):8375-
8381. doi: 10.1007/s00253-019-10083-y
42. Odekina PA, Agbo MO, Omeje EO. Antimicrobial and Antioxidant Activities of Novel Marine Bacteria (Bacillus 2011SOCCUF3) Isolated from Marine Sponge (Spongia officinalis).
Ulum-i Daroei. 2020;26(1):82-87. doi: 10.34172/PS.2019.59
43. Harounabadi S. The survey of molecular and antimicrobial 
activity of isolated bacteria from the Caspian Sea. Iran J Med
Microbiol . 2016;10(16-23). 
44. Norouzi H, Khorasgani MR, Danesh A. Anti-MRSA activity of a bioactive compound produced by a marine Streptomyces and its optimization using statistical experimental 
design. Iran J Basic Med Sci. 2019;22(9):1073. doi: 10.22038/
45. Darabpour E, Ardakani MR, Motamedi H, Ronagh MTJ,. 
Isolation of a broad spectrum antibiotic producer bacterium, 
Pseudoalteromonas piscicida PG-02, from the Persian Gulf. 
Bangladesh J Pharmacol. 2011;6(2):74-83. doi:  10.3329/bjp.
46. Baharudin MMA-a, Ngalimat MS, Mohd Shariff F, Balia Yusof 
ZN, Karim M, Baharum SN, et al. Antimicrobial activities of 
Bacillus velezensis strains isolated from stingless bee products 
against methicillin-resistant Staphylococcus aureus. PloS One. 
2021;16(5):e0251514. doi:10.1371/journal.pone.0251514 
47. Graça AP, Bondoso J, Gaspar H, Xavier JR, Monteiro MC, de 
la Cruz M, et al. Antimicrobial activity of heterotrophic bacterial communities from the marine sponge Erylus discophorus 
(Astrophorida, Geodiidae). PLoS One. 2013;8(11):e78992. doi: 
48. Fariq A, Yasmin A, Jamil M. Production, characterization and 
antimicrobial activities of bio-pigments by Aquisalibacillus 
elongatus MB592, Salinicoccus sesuvii MB597, and Halomonas 
aquamarina MB598 isolated from Khewra Salt Range, Pakistan. Extremophiles. 2019;23(4):435-449. doi: 10.1007/s00792-
49. Srilekha V, Krishna G, Srinivas VS, Charya MS. Antimicrobial 
evaluation of bioactive pigment from Salinicoccus sp isolated 
from Nellore sea coast. Int J Biotechnol Biochem. 2017;13:211-
50. Bibi F, Naseer MI, Azhar EI. Assessing the diversity of bacterial 
communities from marine sponges and their bioactive compounds. Saudi J Biol Sci. 2021;28(5):2747-2754. doi: 10.1016/j.
51. Velmurugan S, Raman K, Viji VT, Donio M, Jenifer JA, Babu 
MM, et al. Screening and characterization of antimicrobial secondary metabolites from Halomonas salifodinae MPM-TC and 
its in vivo antiviral influence on Indian white shrimp Fenneropenaeus indicus against WSSV challenge. J King Saud Univ, Sci. 
2013;25(3):181-190. doi: 10.1016/j.jksus.2013.03.002
52. Debashish G, Malay S, Barindra S, Joydeep M. Marine enzymes. Mar Biotechnol. 2005;1:189-218. doi: 10.1007/b135785
53. Siwach A, Verma PKJBc. Synthesis and therapeutic potential of 
imidazole containing compounds. BMC Chem. 2021;15(1):1-
69. doi: 10.1186/s13065-020-00730-1
54. Graz M, Hunt A, Jamie H, Grant G, Milne P. Antimicrobial activity of selected cyclic dipeptides. Die Pharmazie. 1999; 
55. Ström K, Sjögren J, Broberg A, Schnürer J. Lactobacillus plantarum MiLAB 393 produces the antifungal cyclic dipeptides 
cyclo (L-Phe-L-Pro) and cyclo (L-Phe-trans-4-OH-L-Pro) and 
3-phenyllactic acid. Appl Environ Microbiol. 2002;68(9):4322-
4327. doi: 10.1128/AEM.68.9.4322-4327.2002
56. Tan LT-H, Chan K-G, Chan CK, Khan TM, Lee L-H, Goh 
B-H. Antioxidative potential of a Streptomyces sp. MUM292 
isolated from mangrove soil. BioMed Res Int. 2018;2018. doi: 
57. Takaya Y, Furukawa T, Miura S, Akutagawa T, Hotta Y, Ishikawa N, et al. Antioxidant constituents in distillation residue 
of Awamori spirits. J Agric Food Chem. 2007;55(1):75-79. doi: 
58. Ser H-L, Palanisamy UD, Yin W-F, Abd Malek SN, Chan K-G, 
Goh B-H, et al. Presence of antioxidative agent, Pyrrolo [1, 2-a] 
pyrazine-1, 4-dione, hexahydro-in newly isolated Streptomyces 
mangrovisoli sp. nov. Frontiers In Microbiology. 2015;6:854. doi: 
59. Rajivgandhi GN, Ramachandran G, Kanisha CC, Li J-L, Yin L, 
Manoharan N, et al. Anti-biofilm compound of 1, 4-diaza-2, 
5-dioxo-3-isobutyl bicyclo [4.3. 0] nonane from marine Nocardiopsis sp. DMS 2 (MH900226) against biofilm forming K. 
pneumoniae. J King Saud Univ, Sci. 2020;32 (8): 3495-3502. doi: