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. 2023 Apr 4;28(7):3210. doi: 10.3390/molecules28073210

Molecular Characterization of Some Bacillus Species from Vegetables and Evaluation of Their Antimicrobial and Antibiotic Potency

Moldir Koilybayeva 1,*, Zhanserik Shynykul 2,*, Gulbaram Ustenova 1, Symbat Abzaliyeva 2, Mereke Alimzhanova 3, Akerke Amirkhanova 1, Aknur Turgumbayeva 2, Kamilya Mustafina 4, Gulnur Yeleken 1, Karlygash Raganina 1, Elmira Kapsalyamova 1
Editor: Manuel Simões
PMCID: PMC10095821  PMID: 37049972

Abstract

Numerous natural habitats, such as soil, air, fermented foods, and human stomachs, are home to different Bacillus strains. Some Bacillus strains have a distinctive predominance and are widely recognized among other microbial communities, as a result of their varied habitation and physiologically active metabolites. The present study collected vegetable products (potato, carrot, and tomato) from local markets in Almaty, Kazakhstan. The bacterial isolates were identified using biochemical and phylogenetic analyses after culturing. Our phylogenetic analysis revealed three Gram-positive bacterial isolates BSS11, BSS17, and BSS19 showing 99% nucleotide sequence similarities with Bacillus subtilis O-3, Bacillus subtilis Md1-42, and Bacillus subtilis Khozestan2. The crude extract was prepared from bacterial isolates to assess the antibiotic resistance potency and the antimicrobial potential against various targeted multidrug-resistant strains, including Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus group B, Streptococcus mutans, Candida albicans, Candida krusei, Pseudomonas aeruginosa, Shigella sonnei, Klebsiella pneumoniae, Salmonella enteritidis, Klebsiella aerogenes, Enterococcus hirae, Escherichia coli, Serratia marcescens, and Proteus vulgaris. This study found that the species that were identified have the ability to produce antibiotic chemicals. Additionally, the GC–MS analysis of three bacterial extracts revealed the presence of many antibiotic substances including phenol, benzoic acid, 1,2-benzenedicarboxylic acid and bis(2-methylpropyl), methoxyphenyl-oxime, and benzaldehyde. This work sheds light on the potential of Bacillus to be employed as an antimicrobial agent to target different multidrug-resistant bacterial strains. The results indicate that market vegetables may be a useful source of strains displaying a range of advantageous characteristics that can be used in the creation of biological antibiotics.

Keywords: antimicrobial activity, antibiotic substances, Bacillus subtilis, Bacillus subtilis, Khozestan2, bacterial isolates, GC–MS

1. Introduction

The prevalence of antibiotic resistance in bacterial strains poses a severe threat to public health and calls for urgent research into novel antibiotics or antimicrobial chemicals [1]. Numerous studies on the development of novel antibiotics from various microbe and plant strains have been published over the past few decades [2,3,4,5]. Antibiotics are chemicals that bacteria create and use in their natural environments for protection from the invasion of other bacterial species. In addition to serving as a kind of protection, these antibiotics are essential signaling molecules that allow the cells of the bacterial population to communicate with one another [6,7,8]. Their importance is enhanced when a bacterium is considered a probiotic which is administered in sufficient amounts and provides health benefits to the host. Cases of affirmed health profits of probiotics may comprise the support of immune health, gastrointestinal health, and so on. More precisely, the probiotic Bacillus subtilis has clinically demonstrated its effectiveness in dietary protein digestion [9]. Thus, it is impossible not to notice the growing number of benefits associated with the use of bacteria in various fields of activity. However, it is unfair not to consider the side effects of antibiotic compounds synthesized by bacteria. It was found that antibiotics influence food deterioration. Thus, spoilage of food is caused by a change in the chemical or physical properties of food caused by antibiotic-like substances of foodborne pathogens [10].

From the historical evidence, it is assumed that natural products are essential for the discovery and advancement of antibiotics [11]. It is imperative to investigate innovative antimicrobial substances with a high potential to eradicate or control a variety of microorganisms. Hence, one of the fundamental pillars of modern medicine is the antibiotic. Nevertheless, it is regrettable that some pathogenic strains render commonly-used antibiotics ineffective, and there is a requirement for novel antibiotics to take their place [12,13]. Microorganisms that may create bioactive secondary metabolites have unique structural features and biological activities. These bioactive compounds are produced by a few types of microflora found in vegetables and are employed as antibiotics. Several other notable types of investigation have also been reported to identify bacteria from vegetables with new antimicrobial agents [14,15]. Multidrug-resistant infections are more hazardous than infections caused by bacterial pathogens that are not resistant to multiple drugs because public health practitioners have recently had tremendous difficulty in treating these organisms.

Particularly, the prevalence of resistance developed in bacterial pathogens functions as a secondary infection in a number of life-threatening disorders, such as cancer, surgical procedures, transplantation, etc., and affects the effectiveness of contemporary treatments in treating these conditions [16,17]. It is evident that there are very few therapeutic drugs available to successfully treat these infections given the rapid evolution of multidrug-resistant strains due to the availability of relatively few effective treatments [18,19]. In order to evaluate the antibacterial properties against the majority of common human diseases, this study concentrated on extracting possible bacterial species from vegetable sources (potato, carrot, and tomato) and creating a crude extract from isolated bacterial strains. The main goal of this study is to provide insights into bacterial isolate composition and to provide a connection with the antimicrobial activity of investigating bacterial strains. The bioactive components in the crude extract were also identified using GC–MS. This research will aid in the creation of new drugs to combat multidrug-resistant bacterial strains.

2. Results

2.1. Isolation and Identification

A total of n = 19 bacteria strains were isolated and identified based on colonial morphology, microscopy, biochemical characteristics, and sugar fermentation. Among all, Gram-positive, rod-shaped, mycelial, and spore-forming bacterial strains were selected for further confirmatory tests. The molecular analysis further validated the bacterial strains (BSS11, BSS17, and BSS19) as Bacillus subtilis O-3, Bacillus subtilis Md1-42, and Bacillus subtilis Khozestan2.

2.2. Morphological Characterization

The morphology of each colony from the different bacterial isolates showed regular, irregular, slightly raised, flat, white, and cream-colored colonies. A motility test determined that the bacterial isolates were motile and possessed terminal and subterminal spores (Table 1).

Table 1.

Colony morphology and microscopic presentation of the isolated bacterial species.

Bacterial Species Media Colony Color and Texture Microscopic Presentation
Bacillus subtilis O-3 (BSS11) bacillus medium white, irregular, flat gram positive, spore-forming, rod.
Bacillus subtilis Md1-42 (BSS17) bacillus medium white, irregular, flat gram positive, spore-forming, rod.
Bacillus subtilis Khozestan2 (BSS19) bacillus medium white, irregular, flat gram positive, spore-forming, rod.
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2.3. Antimicrobial Activity Assessment

All tested isolates of BSS11, BSS17, and BSS19 showed antagonistic activity against most bacterial pathogens, such as Klebsiella aerogenes ATCC 13048, Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis ATCC 12228, Candida krusei ATCC 14243, and Candida albicans ATCC 2091, and had less activity against other pathogens, such as Proteus vulgaris ATCC 6380, Klebsiella pneumoniae ATCC 13883, Shigella sonnei ATCC 25931, and Salmonella enterica ATCC 35664 (Figure 1).

Figure 1.

Figure 1

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Antagonistic activity of the bacteria of the genus Bacillus against pathogens. Antagonistic efficacy of all three isolates was examined against pathogenic bacteria, such as Salmonella enterica ATCC 35664, Serratia marcescens ATCC 13880, Klebsiella aerogenes ATCC 13048, Candida krusei ATCC 14243, Shigella sonnei ATCC 25931, Streptococcus mutans ATCC 25175, Klebseiella pneumoniae ATCC 13883, Group B Streptococcus, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 29213, Candida albicans ATCC 2091, Enterococcus hirae ATCC 10541, Proteus vulgaris ATCC 6380, and Staphylococcus epidermidis ATCC 12228. (A–A2)—BSS11, (B–B2)—BSS17, (C–C2)—BSS19.

The nine extracts showed antibacterial activity against all the bacterial pathogens except Shigella sonnei ATCC 25931, Klebsiella pneumonia ATCC 13883, Salmonella enterica ATCC 35664, and Enterococcus hirae ATCC 10541 (Table 2). Additionally, three extracts such as EAE (C), EAE (BC), and EAE (SC) from Bacillus subtilis Khozestan2 (BSS19) did not also demonstrate antibacterial activity. The nine extracts showed antibacterial activity against all the bacterial pathogens except Shigella sonnei ATCC 25931, Klebsiella pneumonia ATCC 13883, Salmonella enterica ATCC 35664, and Enterococcus hirae ATCC 10541. Additionally, three extracts such as EAE (C), EAE (BC), and EAE (SC) from Bacillus subtilis Khozestan2 (BSS19) have not demonstrated any antibacterial activity, too. The EAE (SC) preparation of Bacillus subtilis O-3 (BSS11), showed a better zone of inhibition for Staphylococcus epidermidis ATCC 12228 (25 ± 1.20 mm), Streptococcus group B (19 ± 1.20 mm), Candida krusei ATCC 14243 (29 ± 2.35 mm), Klebsiella aerogenes ATCC 13048 (17 ± 1.82 mm), and Proteus Vulgaris ATCC 6380 (18 ± 1.34 mm) when compared with the other pathogens, while the EAE (SC) preparation of Bacillus subtilis Md1-42 (BSS17) was effective against the pathogens Staphylococcus aureus ATCC 29213 (30 ± 2.50 mm) and Serratia marcescens ATCC 13880 (18 ± 1.64 mm). Additionally, the EAE (SC) preparation of Bacillus subtilis Khozestan2 (BSS19) was effective against the pathogens Streptococcus mutans ATCC 25175 (20 ± 1.32 mm), Candida albicans ATCC 2091 (40 ± 1.22 mm), and Pseudomonas aeruginosa ATCC 9027 (22 ± 1.81 mm). It was found assumingly the same potency meaning of the EAE (SC) preparations of Bacillus subtilis Md1-42 (BSS17) and Bacillus subtilis Khozestan2 (BSS19) against the pathogen Escherichia coli ATCC 25922.

Table 2.

Antibacterial activity of the bacterial culture extracts against pathogenic strains.

Species of Microorganism BSS11
(C), mm		 BSS11 (BC), mm BSS11 (SC), mm BSS17
(C), mm		 BSS17
(BC), mm		 BSS17
(SC), mm		 BSS19
(C), mm		 BSS19
(BC), mm		 BSS19
(SC), mm		 Control (Streptomycin)
Staphylococcus aureus
ATCC 29213		 19 ± 1.33 * 21 ± 1.33 * 25 ± 1.53 * 22 ± 0.50 * 26 ± 0.44 * 30 ± 2.50 * 22 ± 1.55 26 ± 1.24 29 ± 1.54 29 ± 0.33 ***
Staphylococcus epidermidis
ATCC 12228		 18 ± 1.20 * 20 ± 1.33 * 25 ± 1.20 * 19 ± 1.00 20 ± 1.26 22 ± 1.50 18 ± 1.42 * 18 ± 1.44 * 19 ± 1.54 * 22 ± 0.33 ***
Streptococcus group B 16 ± 1.20 15 ± 1.00 19 ± 1.20 13 ± 1.16 * 15 ± 1.14 * 17 ± 1.15 * 15 ± 1.33 * 14 ± 1.17 * 16 ± 1.33 * 22 ± 0.33 ***
Streptococcus mutans
ATCC 25175		 16 ± 1.22 * 17 ± 1.22 * 17 ± 1.82 * 14 ± 1.33 16 ± 1.33 19 ± 1.33 18 ± 0.31 * 19 ± 1.00 * 20 ± 1.32 * 16 ± 0.33 ***
Candida albicans
ATCC 2091		 27 ± 2.00 * 25 ± 1.50 * 30 ± 2.50 * 29 ± 1.00 31 ± 1.22 35 ± 1.26 38 ± 1.49 35 ± 1.62 40 ± 1.22 39 ± 0.33 ***
Candida krusei
ATCC 14243		 25 ± 2.33 * 27 ± 2.33 * 29 ± 2.35 * 23 ± 1.38 25 ± 0.33 25 ± 1.34 23 ± 2.00 * 26 ± 1.66 * 27 ± 2.00 * 35 ± 0.33 ***
Pseudomonas aeruginosa
ATCC 9027		 14 ± 1.22 17 ± 0.33 17 ± 1.51 13 ± 0.44 12 ± 1.44 13 ± 1.10 20 ± 1.27 * 20 ± 1.53 * 22 ± 1.81 * 23 ***
Shigella sonnei
ATCC 25931		 0 0 0 0 0 0 0 0 0 19 ± 0.33 ***
Klebsiella pneumonia
ATCC 13883		 0 0 0 0 0 0 0 0 0 15 ± 0.33 ***
Salmonella enterica
ATCC 35664		 0 0 0 0 0 0 0 0 0 19 ± 0.33 ***
Klebsiella aerogenes
ATCC 13048		 14 ± 1.82 * 17 ± 1.57 * 17 ± 1.82 * 9 ± 1.33 * 12 ± 1.22 * 13 ± 1.63 * 16 ± 0.53 * 13 ± 1.53 * 16 ± 1.53 * 17 ± 0.33 ***
Enterococcus hirae
ATCC 10541		 0 0 0 0 0 0 0 0 0 20 ± 0.33 ***
Escherichia coli
ATCC 25922		 18 ± 1.22 * 19 ± 1.57 * 20 ± 1.24 * 22 ± 1.44 * 20 ± 1.31 * 22 ± 1.54 * 19 ± 0.49 22 ± 0.46 22 ± 1.17 23 ± 0.33 ***
Serratia marcescens
ATCC 13880		 13 ± 1.22 * 15 ± 0.33 * 15 ± 1.56 * 17 ± 1.22 18 ± 1.55 18 ± 1.64 0 0 0 24 ± 0.33 ***
Proteus Vulgaris
ATCC 6380		 17 ± 1.33 * 16 ± 0.33 * 18 ± 1.34 * 11 ± 1.33 * 11 ± 1.54 * 13 ± 1.53 * 0 0 0 22 ± 0.33 ***
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* Data are represented as the means ± SE (n = 3). Values with same superscript symbols are not statistically different. Significance level * < ***.

2.4. Antibiotic Susceptibility Profile of the Isolates

The study of the antibiogram revealed that all three tested Bacillus subtilis subspecies were resistant to all antibiotics except for bacitromycin (B, 10), polymyxin (PB, 300), and cloxacillin (CX, 5) (Table 3). BSS11, BSS17, and BSS17 strains showed the highest vulnerability to gentamicin (CN, 120) with 40 ± 0.28 mm, 40 ± 0.28 mm, and 38 ± 0.28 sensitivity diameters, respectively, while strain BSS17 showed the lowest sensitivity to carbenicillin (10 ± 0.28) and to amoxycillin (12 ± 0.29) with high significant differences (p < 0.0001).

Table 3.

Antibiotic resistance profile of the Bacillus strains.

Antibiotic (AB, Charge in μg) Used Bacillus Strains
BSS11 BSS17 BSS19
Diameter (mm) S/R Diameter (mm) S/R Diameter (mm) S/R
Penicillins Penicillin G (PEN, 10) 30 ± 0.98 bc S 24 ± 0.56 ab S 23 ± 0.29 a S
Ampicillin (AMP, 10) 30 ± 1.43 ab S 27 ± 1.34 ab S 27± 0.38 a S
Amoxycillin (AMOX, 30) 32 ± 0.98 abc S 30 ± 1.30 abc S 12 ± 0.29 ab S
Amoxycillin-clavulanic acid (AMC, 30) 28 ± 1.05 bcd S 23 ± 0.33 abc S 0 ± 0.00 b S
Carbenicillin (CAR, 100) 38 ± 0.28 abc S 28 ± 0.35 ab S 10 ± 0.28 abc S
Cloxacillin (CX, 5) 0 ± 0.00 b R 0 ± 0.00 b R 0 ± 0.00 b R
Macrolides Erythromycin (ERO, 15) 35 ± 0.31 ab S 32 ± 1.41 abc S 30 ± 0.23 a S
Azithromycin (AZM, 15) 36 ± 0.28 a S 35 ± 1.43 ab S 27 ± 1.33 ab S
Cephalosporins Cefepime (FEP, 30) 35 ± 0.51 ab S 25 ± 0.57 a S 38 ± 0.86 ab S
Cefepime/clavulanic acid FEC-40 36 ± 0.98 a S 30 ± 0.36 a S 39 ± 0.67 a S
Cephalatin (KF, 30) 32 ± 0.33 ab S 40 ± 0.37 ab S 26 ± 1.32 abc S
Cefotaxime (CTX, 30) 26 ± 0.98 a S 40 ± 0.52 a S 24 ± 0.98 ab S
Aminoglycosides Gentamicin (CN, 120) 40 ± 0.28 ab S 40 ± 0.27 ab S 38 ± 0.28 ab S
Streptomycin (STR, 10) 26 ± 0.19 ab S 25 ± 0.48 ab S 22 ± 0.20 ab S
Tobramycin (TOB, 10) 33 ± 0.98 a S 39± 1.18 a S 22 ± 0.23 abc S
Tetracyclines Tetracycline (TET, 30) 35 ± 0.28 a S 30 ± 0.33 a S 20 ± 1.18 ab S
Polypeptides Polymyxin (PB, 300) 0 ± 0.00 b R 0 ± 0.00 b R 0 ± 0.00 b R
Bacitromycin (B, 10) 0 ± 0.00 b R 0 ± 0.00 b R 0 ± 0.00 b R
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The Newmann–Keuls test shows that the averages affected by the different superscript letters in the row and column are significantly different at the 5% level. Values are the means ± standard error; legend: D = dimension, S/R = sensible/resistant.

2.5. Analysis of the Isolates Using GC–MS

The crude extracts from several Bacillus bacterium species contained a number of chemicals, according to the results of the GC–MS analysis. Table 4, Table 5 and Table 6 explain the most significant and abundant components found in the crude extracts that were subjected to the GC–MS analysis, as well as information about where the chemicals found in this study had previously been identified. These substances exhibited similarities to natural products of bacterial and plant origin. According to the study of the GC–MS data, the majority derived from volatile substances, such as alkaloids, esters, ethers, and phenolic chemicals.

Table 4.

The main constituents of bacterial extract BSS11 identified through a GC–MS analysis.

Bacillus subtilis O-3 (BSS11)
No Name Molecular Formula Molecular Mass, g/mol Retention Time (min) Pubchem
Compound CID		 Similarities Area, %
1 2-Butanone C4H8O 72.11 2.111 6569 75 9.68622
2 Acetic acid ethenyl ester C4H6O2 86.09 2.63 7904 63 3.553507
3 2-Pentanone, 3-methyl- C6H12O 100.16 2.993 11262 80 3.452664
4 Disulfide, dimethyl C2H6S2 94.2 3.578 12232 81 2.579829
5 2-Heptanone, 6-methyl- C8H16O 128.21 5.602 13572 85 2.012434
6 Pyrazine, methyl- C5H6N2 94.11 5.95 7976 93 4.364266
7 Pyrazine, 2,5-dimethyl- C6H8N2 108.14 6.722 31252 92 4.600645
8 1-Hexanol C6H14O 102.17 7.055 8103 74 1.171664
9 Pyrazine, trimethyl- C7H10N2 122.17 7.824 26808 75 1.113828
10 Pyrazine, 3-ethyl-2,5-dimethyl- C8H12N2 136.19 8.355 25916 80 2.579326
11 1-Hexanol, 2-ethyl- C8H18O 130.229 8.864 7720 84 0.5058
12 Pyrrole C4H5N 67.09 9.166 8027 90 0.639997
13 Benzaldehyde C7H6O 106.12 9.374 240 82 7.155098
14 Acetic acid, trifluoro-, nonyl ester C11H19F3O2 240.26 10.344 6428483 73 0.416699
15 (S)-(+)-6-Methyl-1-octanol C9H20O 144.25 10.605 13548104 79 0.560066
16 Oxime-, methoxy-phenyl-_ C8H9NO2 151.16 12.025 9602988 70 2.008104
17 Acetamide C2H5NO 59.07 12.1 178 96 11.58042
18 Propanamide C3H7NO 73.09 12.612 6578 71 0.730287
19 2,4-Decadienal, (E,E)- C10H16O 152.23 12.825 5283349 68 0.893933
20 Hexanoic acid C6H12O2 116.16 13.043 8892 60 0.301542
21 2-Tetradecanone C14H28O 212.37 13.441 75364 86 1.671165
22 (R)-(−)-4-Methylhexanoic acid C7H14O2 130.18 13.913 12600623 70 0.496031
23 Hexanoic acid, 2-ethyl- C8H16O2 144.21 14.172 8697 66 0.587733
24 Phenol C6H6O 94.11 14.738 996 96 4.246424
25 Octanoic acid C8H16O2 144.21 15.283 379 67 0.803864
26 2,4,7,9-Tetramethyl-5-decyn-4,7-diol C14H26O2 226.35 15.658 31362 69 0.481144
27 Nonanoic acid C9H18O2 158.24 16.331 8158 82 1.003712
28 2-Octyl benzoate C15H22O2 234.33 17.33 243800 66 0.967435
29 Phenol, 2,4-bis(1,1-dimethylethyl)- C14H22O 206.32 17.784 7311 87 1.242429
30 Benzoic acid, pentyl ester C12H16O2 192.25 18.345 16296 68 0.712256
31 Benzoic acid C7H6O2 122.12 18.705 243 85 0.94121
32 1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester C16H22O4 278.34 19.822 6782 93 2.287847
33 Dibutyl phthalate C16H22O4 278.34 21.072 3026 74 2.75918
34 Hexadecanoic acid C16H32O2 256.42 22.573 985 71 6.166636
35 Oleic Acid C18H34O2 282.5 24.497 445639 82 8.522779
36 9,12-Octadecadienoic acid (Z,Z)- C18H32O2 280.4 25.011 5280450 71 3.023523
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Table 5.

The main constituents of bacterial extract BSS17 identified through a GC–MS analysis.

Bacillus subtilis Md1-42 (BSS17)
No Name Molecular Formula Molecular Mass, g/mol Retention Time (min) Pubchem
Compound CID		 Similarities Area, %
1 (2-Aziridinylethyl)amine C4H10N2 86.14 1.157 97697 78 0.440368
2 1-Propen-2-ol, acetate C5H8O2 100.12 1.664 7916 76 0.91485
3 2,3-Butanedione C4H6O2 86.09 2.649 650 93 17.04656
4 3-Penten-1-ol C5H10O 86.13 5.699 510370 83 0.269452
5 Acetoin C4H8O2 88.11 6.247 179 76 37.17943
6 3-Pentanol, 2-methyl- C6H14O 102.17 7.009 11264 80 1.154491
7 2-Nonen-1-ol C9H18O 142.24 7.108 61896 68 0.420964
8 2-Hydroxy-3-pentanone C5H10O2 102.13 7.215 521790 81 1.137636
9 Ethane-1,1-diol dibutanoate C10H18O4 202.25 8.244 551339 77 0.624527
10 Acetic acid C2H4O2 60.05 8.355 176 93 0.854423
11 1-Hexanol, 2-ethyl- C8H18O 130.229 8.889 7720 84 0.206645
12 Benzaldehyde C7H6O 106.12 9.399 240 92 2.022607
13 2,3-Butanediol C4H10O2 90.12 9.483 262 78 3.407863
14 1,6-Octadien-3-ol, 3,7-dimethyl- C10H18O 154.25 9.632 6549 83 0.530564
15 Propanoic acid, 2-methyl- C4H8O2 88.11 9.833 6590 82 3.007214
16 2,3-Butanediol, [R-(R*,R*)]- C4H10O2 90.12 9.927 225936 75 0.516676
17 1-Nonanol C9H20O 144.25 10.367 8914 79 0.235951
18 (S)-(+)-6-Methyl-1-octanol C9H20O 144.25 10.63 13548104 86 0.668355
19 Butanoic acid, 2-methyl- C5H10O2 102.13 11.076 8314 81 2.487502
20 Oxime-, methoxy-phenyl-_ C8H9NO2 151.16 12.051 151.16 70 0.59943
21 2,4-Decadienal C10H16O 152.23 12.855 5283349 70 0.310428
22 2,2,4-Trimethyl-1,3-pentanediol diisobutyrate C16H30O4 286.41 13.615 23284 73 0.259067
23 (R)-(−)-4-Methylhexanoic acid C7H14O2 130.18 13.942 12600623 74 0.173808
24 Phenol C6H6O 94.11 14.772 996 94 0.427543
25 Octanoic acid C8H16O2 144.21 15.313 379 68 0.235055
26 Nonanoic acid C9H18O2 158.24 16.358 8158 87 0.268447
27 Hexadecanoic acid, methyl ester C17H34O2 270.5 17.011 8181 89 0.296456
28 2-Octyl benzoate C15H22O2 234.33 17.353 243800 67 0.301277
29 Benzoic acid, heptyl ester C14H20O2 220.31 18.076 81591 75 0.189386
30 Benzoic acid, undecyl ester C18H28O2 276.4 18.368 229159 70 0.218367
31 Benzoic acid C7H6O2 122.12 18.538 243 84 0.270263
32 1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester C16H22O4 C16H22O4 19.238 6782 91 0.666223
33 Phenol, 2,4-bis(1,1-dimethylethyl)- C14H22O 206.32 19.849 7311 86 0.746427
34 Oleic Acid C18H34O2 282.5 20.98 445639 68 0.295063
35 Dibutyl phthalate C16H22O4 278.34 21.113 3026 82 1.029155
36 Hexadecanoic acid C16H32O2 256.42 22.592 985 85 3.51103
37 Octadecanoic acid C18H36O2 284.5 24.242 5281 68 3.107501
38 Oleic Acid C18H34O2 282.5 24.525 445639 84 5.557709
39 9,12-Octadecadienoic acid (Z,Z)- C18H32O2 280.4 25.047 5280450 84 9.157715
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Table 6.

The main constituents of bacterial extract BSS19 identified through a GC–MS analysis.

Bacillus subtilis Khozestan2 (BSS19)
No Name Molecular Formula Molecular Mass, g/mol Retention Time (min) Pubchem
Compound CID		 Similarities Area, %
1 Carbamic acid, monoammonium salt CH6N2O2 78.071 1.134 517232 88 1.700997
2 Ethanol C2H6O 46.07 2.234 702 81 0.46569
3 1-Butanol C4H10O 74.12 4.321 263 91 6.044015
4 2-Heptanone, 6-methyl- C8H16O 128.21 5.599 13572 85 1.508914
5 2-Heptanone, 5-methyl- C8H16O 128.21 5.839 28965 87 0.899369
6 Pyrazine, 2,5-dimethyl- C6H8N2 108.14 6.717 31252 94 6.575729
7 Acetic acid C2H4O2 60.05 8.318 176 93 4.27265
8 2-Decanone C10H20O 156.26 8.399 12741 76 3.555438
9 1-Hexanol, 2-ethyl- C8H18O 130.229 8.859 7720 85 1.019178
10 Benzaldehyde C7H6O 106.12 9.361 240 91 3.066211
11 1-Octene, 6-methyl- C9H18 126.24 9.587 518716 77 0.767838
12 Propanoic acid, 2-methyl- C4H8O2 88.11 9.796 6590 85 8.705907
13 1-Octanol, 2-methyl- C9H20O 144.25 9.896 102495 82 0.837655
14 1-Nonanol C9H20O 144.25 10.342 8914 81 1.748035
15 (S)-(+)-6-Methyl-1-octanol C9H20O 144.25 10.604 13548104 90 3.260679
16 Benzeneacetaldehyde C8H8O 120.15 10.821 998 69 2.227017
17 2-Furanmethanol C5H6O2 98.1 10.918 7361 87 1.948174
18 Hexanoic acid, 2-methyl- C7H14O2 130.18 11.036 20653 80 25.50356
19 2-Dodecanone C12H24O 184.32 11.198 22556 77 1.361537
20 Oxime-, methoxy-phenyl-_ C8H9NO2 151.16 12.018 9602988 65 1.54511
21 2(5H)-Furanone C4H4O2 84.07 12.114 10341 77 2.40381
22 Formamide CH3NO 45.041 12.32 713 77 0.972164
23 2-Tetradecanone C14H28O 212.37 13.44 75364 80 1.097307
24 Maltol C6H6O3 126.11 14.375 8369 93 4.442068
25 Ethanone, 1-(1H-pyrrol-2-yl)- C6H7NO 109.13 14.429 14079 67 1.456046
26 Phenol C6H6O 94.11 14.739 996 96 6.642077
27 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- C6H8O4 144.12 17.293 119838 88 1.650456
28 Benzoic acid, hept-2-yl ester C14H20O2 220.31 18.053 243678 75 0.421796
29 Benzoic acid C7H6O2 122.12 18.698 243 91 1.317335
30 5-Hydroxymethyldihydrofuran-2-one C5H8O3 116.11 19.196 98431 73 0.384416
31 1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester C16H22O4 C16H22O4 19.356 6782 91 0.986222
32 Phenol, 2,4-bis(1,1-dimethylethyl)- C14H22O 206.32 19.786 7311 85 1.085735
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The GC–MS analysis of the ethyl acetate extracts of the three isolates detected a total of 106 compounds. Based on the analysis of bacterial isolates, BSS11 and BSS17 were found to share a similar composition of volatile organic components, while bacterial isolate BSS19 was found with fewer quantities of them. For isolate BSS11, the solvent with metabolites was ethyl acetate with 36 compounds (Table 4). Phenol, benzoic acid, phenol, 2,4-bis(1,1-dimethylethyl), 1,2-benzenedicarboxylic acid, methoxyphenyl-oxime, and benzaldehyde (Figure 2) were identified in the BSS11 extract with important concentrations of 4.246424%, 0.94121%, 1.242429%, 2.287847%, 2.008104%, and 7.155098%, respectively. In the extract, the major compounds were acetamide at 11.58042% and 2-butanone at 9.68622%, while minor compounds were fatty acids and their derivatives. In strain BSS17, ethyl acetate extraction showed the presence of 39 compounds (Table 5) in comparison to 32 compounds arising out with the same extraction strain BSS19 (Table 6). GC–MS analysis for two bacterial (BSS17 and BSS19) analyses also confirmed the presence of the same volatile organic compounds but with fewer amounts compared with BSS11. The BSS19 ethyl acetate extract did not reveal the presence of some fatty acids (octanoic acid, nonanoic acid, hexadecanoic acid, octadecanoic acid, oleic acid, and 9,12-octadecanoic acid (z,z)), although they are present in the extracts of BSS11 and BSS17.

Figure 2.

Figure 2

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Structure of the components identified from Bacillus spp. isolates.

2.6. Molecular Characterization

From various samples, three bacterial isolates with enhanced antibacterial activity were discovered. Phylogenetic analysis of the 16S rRNA gene sequences revealed that all three candidate bacterial isolates, BSS11, BSS17, and BSS19, belong to three different Bacillus species, respectively (Figure 3), as they group together in the evolutionary tree with the aforementioned bacterial species.

Figure 3.

Figure 3

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The phylogenetic tree using the neighbor-joining model was constructed based on 16S rRNA gene sequences representing different Bacillus subtilis subspecies, i.e., Bacillus subtilis O-3, Bacillus subtilis Md1-42, and Bacillus subtilis Khozestan2, respectively. As an outgroup, E. coli JCM 1649 (AB242910) was used.

Bacillus subtilis O-3, Bacillus subtilis Md1-42, and Bacillus subtilis Khozestan2 were identified as having the highest hit sequence similarity for these bacterial isolates (Table 7). High bootstrap values were obtained following a phylogenetic analysis and tree topology both served to confirm the presumably described taxonomy.

Table 7.

Identification of the bacterial species based on the sequence similarities.

No Isolates 16S rRNA Amplified Region Length % Similarity NCBI Accession No
1 BSS11 1443 bp 99% with Bacillus subtilis O-3 GQ870259
2 BSS17 1454 bp 99% with Bacillus subtilis Md1-42 MF581448
3 BSS19 1450 bp 99% with Bacillus subtilis Khozestan2 MH036316
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3. Discussion

Extreme microbial diversity, abundance, and structure are also correlated with a wide range of metabolic processes, which generate a large number of metabolites with a variety of functions, including antimicrobial, anti-parasitic, anti-cancerous, and anti-pesticidal functions. The current study sought to examine the potential for specific vegetable microbial populations to exhibit antibacterial activities. Nineteen distinct bacterial isolates were discovered, as a result of a number of isolation stages, the identification of different general purposes with the selection of bacterial growth media, and biochemical studies. In recent years, a rise in the likelihood of discovering new antibiotics to combat or control untreated infectious diseases has been seen, thanks to a number of microorganisms that can produce antibiotics when grown in proper cultures [20,21]. Indeed, the development of resistance genes in bacteria through the use of mobile genetic elements or their inherent characteristics (natural phenotypic traits) are the two main causes of their antibiotic resistance [22]. All three Bacillus spp. strains were susceptible to almost all antibiotics, except for bacitracin, polymyxin, and cloxacillin, for which they were all resistant (Table 3). Similar findings on the susceptibility of various antibiotic-resistant Bacillus species were observed [23,24,25,26]. Our results also agree with those on the resistance of Bacillus strains to bacitracin, published by Adimpong et al. (2012) [27]. According to Adimpong et al. (2012) and Compaoré et al. (2013) [28], the resistance of specific Bacillus strains to particular antibiotics may be inherent or acquired and associated with the existence of resistance genes implicated in the production of resistance enzymes to these antibiotics. Conversely, the probability of passing on resistance genes to other dangerous bacteria is lower because of a natural resistance, rather than an acquired resistance. Since resistant bacteria can spread from the food chain to humans, antibiotic resistance has really become a serious global concern [29]. Although isolated Bacillus strains do not seem to harbor antibiotic resistance genes that can be passed on to dangerous germs, it is possible that they do not respond to a wide variety of antibiotics. Further research into these strains as prospective probiotic starter cultures could improve and maximize the production of high-quality, medicinal, and functional or health-promoting substances.

The antibacterial characteristic plays a crucial role in therapeutic activities. In the present study, the perpendicular streak method was used to determine the antibacterial properties of the bacterial isolates (BSS11, BSS17, and BSS19) against the selected human bacterial pathogens. This approach is regarded as a first-pass qualitative screening technique for the antimicrobial activity. The research demonstrated the strongest antagonistic action against human pathogen and as a result all tested isolates of BSS11, BSS17, and BSS19 showed antagonistic activity against most bacterial pathogens, such as Klebsiella aerogenes ATCC 13048, Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis ATCC 12228, Candida krusei ATCC 14243, and Candida albicans ATCC 2091.

Our findings demonstrated that the growth of multidrug-resistant bacterial strains is inhibited by Bacillus subtilis O-3, Bacillus subtilis Md1-42, and Bacillus subtilis Khozestan2, which has previously been published [30,31]. Previous research found that the pH of the growing medium or the generation of volatile chemicals are what cause Bacillus to have an inhibitory impact. Bacillus is known to produce polypeptide antibiotic substances, such as bacitracin, polymyxin, gramicidin S., and tyrothricin, according to a number of other investigations. These substances work well against a variety of bacteria, including Gram-positive and Gram-negative bacteria [32].

GC-MS made it possible to detect markers in the studied samples of biological material—components of a microbial cell and its metabolites (fatty acids, aldehydes, phenolic compounds). Additionally, using GC-MS was beneficial in the case of both endogenous and exogenous microflora, without preliminary isolation of a pure culture of microorganisms, which is especially important when considering the difficulties in cultivating anaerobes. The distinctive advantages of the method were the speed of analysis and the ability to quantify the content of the marker. According to the GC–MS analysis, the Bacillus species produce a variety of antifungal chemicals. The synthesis of antibiotic substances by Bacillus subtilis O-3, Bacillus subtilis Md1-42, and Bacillus subtilis Khozestan2 strains was determined by the GC–MS analysis of their crude metabolites. The most important antifungal compounds detected from all three strains were phenol, benzoic acid, phenol, 2,4-bis(1,1-dimethylethyl), 1,2-benzenedicarboxylic acid, and bis(2-methylpropyl) (Table 4, Table 5 and Table 6 and Figure 2). Additionally, the GC–MS analysis showed that all three bacterial isolates contained methoxyphenyl-oxime and benzaldehyde. Previously, methoxyphenyl-oxime was reported as a true specific antibacterial agent that controlled some bacteria [33]. Another study proved that benzaldehyde generated by Photorhabdus temperata has insecticidal, great antibacterial, and antioxidant properties [34]. Two bacterial isolates of Bacillus subtilis O-3 and Bacillus subtilis Md1-42 share common fatty acids, such as octanoic acid, nonanoic acid, hexadecanoic acid, octadecanoic acid, oleic acid, and 9,12-octadecanoic acid (z,z). It is well known that fatty acids and their derivatives have powerful antibacterial and antifungal activities [35]. Because of their great biodegradability, low toxicity, and strong resistance to extremes in pH, salinity, and temperature, they are more environmentally friendly. As food additives, they are accepted. Antifungal fatty acids are less likely to make pathogenic fungi resistant to them [36]. Most of the chemicals identified from three different Bacillus species were derived from volatile compounds, such as esters, alkaloids, ethers, and phenolics, and shared structural similarities with natural products of bacterial and plant origin. Many volatile organic compounds are major constituents of the bacterial strain and have been shown to have properties against phytopathogens [37,38]. Our results prove that Bacillus spp. share common volatile compounds and complements previous findings related to the study of chemical composition of bacterial strains using GC–MS [39,40,41,42]. Moreover, bacterial strains of Bacillus subtilis O-3 and Bacillus subtilis Md1-42 containing mentioned above volatile organic compounds (phenol, benzoic acid, phenol, 2,4-bis(1,1-dimethylethyl), 1,2-benzenedicarboxylic acid, methoxyphenyl-oxime, and benzaldehyde) and fatty acid derivatives (octanoic acid, nonanoic acid, hexadecanoic acid, octadecanoic acid, oleic acid, and 9,12-octadecanoic acid (z,z)) showed high anti-microbial activity on different pathogens. However, the bacterial strain Bacillus subtilis Khozestan2 demonstrated low antimicrobial potency, and it may be caused by due to the absenteeism of fatty acid derivatives and comparingly low concentrations of volatile organic compounds (phenol (1.456046%), benzoic acid (1.3117335%), phenol, 2,4-bis(1,1-dimethylethyl) (1.085735%), 1,2-benzenedicarboxylic acid (0.986222%), methoxyphenyl-oxime (1.54511%), and benzaldehyde (3.066211%)).

There are many purposes for bacterial extract investigation. For instance, Bacillus spp. produce a variety of compounds involved in the biocontrol of plant pathogens and the promotion of plant growth, which makes them potential candidates for most agricultural and biotechnological applications. Moreover, the Bacillus strains as a form of probiotics are not generally pathogenic to mammals and appear to have significant potential for clinical use. Nevertheless, Bacillus probiotics may also generate toxins and biogenic amines; consequently, their safety is a concern.

A molecular investigation revealed the taxonomy of three different isolated species belonging to three Bacillus spp., such as Bacillus subtilis O-3, Bacillus subtilis Md1-42, and Bacillus subtilis Khozestan2. It was tentatively determined that the three most viable candidates of bacterial isolates BSS11, BSS17, and BSS19 belong to Bacillus subtilis O-3 (99%), Bacillus subtilis Md1-42 (99%), and Bacillus subtilis Khozestan2 (99%), respectively, based on a phylogenetic analysis and top hit sequence similarity results, which were supported by a high bootstrap value. In the future, the microbial screening and the isolation of active metabolites against multidrug-resistant strains could be carried out more easily by the identification of the three separate bacterial strains and their antibacterial activity.

4. Materials and Methods

4.1. Isolation of Potential Strains of the Genus Bacillus spp.

Bacillus spp. strains have been isolated from different vegetable sources (potato, carrot, and tomato). A 15 g vegetable sample was homogenized in 100 mL of 0.85% NaCl by shaking at 150 rpm for 15 min. Then, the sample was diluted step-wise and incubated in a water bath for 10 min at 90 °C. The sample was cooled to room temperature and then 0.1 mL samples were loaded onto nutrient agar/meat peptone agar (NA/MPA) plates, which is a nutrient medium for the cultivation of non-fastidious microorganisms. NA/MPA plates were composed of gelatin peptone (5 g/L), bacteriological agar (15 g/L), and meat extract (3 g/L). The plates were incubated for 48 h at 37 °C. The isolated pure strains were refrigerated at −20 °C in nutrient broth (NB) media supplemented with 20% (v/v) glycerin. Then, the fresh culture was subjected to morphological identification and the slightly raised, flat, white, and cream-colored colonies were selected for further identification. Strain isolates in NB media were useful in further studies, particularly in the preparation of ethyl acetate extract for subjecting GC–MS analysis.

4.2. Screening for Antagonistic Activity of Isolated Bacteria against Potent Bacterial Pathogens

A preliminary antibacterial analysis of the isolates was conducted on Mueller–Hinton agar (MHA) plates using the perpendicular streak method against powerful human pathogens. Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis ATCC 12228, Streptococcus group B, Streptococcus mutans ATCC 25175, Candida albicans ATCC 2091, Candida krusei ATCC 14243, Pseudomonas aeruginosa ATCC 9027, Shigella sonnei ATCC 25931, Klebsiella pneumoniae ATCC 13883, Salmonella enterica ATCC 35664, Klebsiella aerogenes ATCC 13048, Enterococcus hirae ATCC 10541, Escherichia coli ATCC 25922, Serratia marcescens ATCC 13880, and Proteus vulgaris ATCC 6380 were employed in this investigation as bacterial pathogens. According to the widely-used method of perpendicular streaks, an exponential culture of the studied antagonist strains was streaked on the surface of an agar medium and incubated at 30 ± 4 °C for 24 h [43]. Then, an exponential culture of the test strain was inoculated perpendicularly from the edge of the cup to the stroke of the grown culture of the antagonist with a stroke by slightly touching the stroke of the antagonist strain. The plate was again incubated under conditions favorable for the growth of the test culture.

The cellular preparations were held relying on the procedure elaborated by Beiranvand et al. (2017) [44]. The chosen isolates underwent 14 days of incubation at 30 ± 4 °C while being cultivated in a nutritional broth. Then, three different techniques were used to extract the cultures. The cultures were used further in preparation of the ethyl acetate extract of culture EAE (C), the ethyl acetate extract of boiled and cooled culture EAE (BC), and the ethyl acetate extract of the sonicated culture EAE (SC). For the creation of the three different extracts, the culture broth was divided into three equal portions. In a separate flask, one part of the culture broth was combined with an equivalent amount of ethyl acetate to create EAE (C). Once separated, the ethyl acetate extract was centrifuged at 8000 rpm for 10 min. The ethyl acetate supernatant was poured into a spotless flask and heated to 50 °C for drying. Two (2) ml of DMSO were used to dissolve the dry extract. Another portion of the media was incubated in boiling water for 5 min, and then cooled for 5 min, to prepare the EAE (BC). This mixture was then diluted 1:1 with ethyl acetate. The samples were then processed using the first technique. The culture was sonicated for three minutes at 130 W to prepare EAE (SC), and extraction was then carried out as instructed in the first procedure. Three extracts were examined for their antibacterial efficacy using the well diffusion method against pathogenic bacteria, including Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis ATCC 12228, Streptococcus group B, Streptococcus mutans ATCC 25175, Candida albicans ATCC 2091, Candida krusei ATCC 14243, Pseudomonas aeruginosa ATCC 9027, Shigella sonnei ATCC 25931, Klebsiella pneumoniae ATCC 13883, Salmonella enterica ATCC 35664, Klebsiella aerogenes ATCC 13048, Enterococcus hirae ATCC 10541, Escherichia coli ATCC 25922, Serratia marcescens ATCC 13880, and Proteus vulgaris ATCC 6380. Sterile saline (0.85% NaCl) with an optical density of 0.5 McFarland standard scale (5 × 106 CFU/mL (CFU—Colony Forming Units/mL) for yeasts and 1.5 × 108 CFU/mL for bacteria) was used to prepare microbial suspensions. Each bacterial pathogen’s zone of inhibition was evaluated and control wells containing 20 μL of streptomycin (1 mg/mL) were used.

4.3. Antibiotic Susceptibility of the Bacillus Strains

Using the disk diffusion method, the antibiotic susceptibility of isolated Bacillus strains (BSS11, BSS17, and BSS19) was assessed in accordance with the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2019). Bacillus strains were spread-plated using sterile beads on Mueller–Hinton (MH) agar using an aliquot of 1 mL each, at a concentration of 106 CFU/mL (0.5 McFarland, Hi-media, India). The plates were then left to dry for an hour. Then, antibiotic disks were inserted into the agar plates containing an inoculated Bacillus strain.

The widths of the inhibition zones surrounding the antibiotic disks were measured using an electronic digital vernier caliper micrometer measuring tool caliber digital ruler (ZHHRHC LCD) following a 24 h incubation period at 37 °C (Hardened, China). This made it possible to identify the strain’s antibiotic susceptibility (S), intermediate resistance (I), or resistance (R) according to the CLSI guidelines (2012) [44,45]. Eighteen antibiotic disks contained a sample each of penicillin G (PEN, 10), ampicillin (AMP, 10), amoxycillin (AMOX, 30), amoxycillin-clavulanic acid (AMC, 30), carbenicillin (CAR, 100), cloxacillin (CX, 5), erythromycin (ERO, 15), azithromycin (AZM, 15), cefepime (FEP, 30), cefepime/clavulanic acid FEC-40, cephalatin (KF, 30), cefotaxime (CTX, 30), gentamicin (CN, 120), streptomycin (STR, 10), tobramycin (TOB, 10), tetracycline (TET, 30), polymyxin (PB, 300), and bacitromycin (B, 10).

4.4. Gas Chromatography–Mass Spectrum Analysis of the Metabolites

The volatile compounds extraction for each bacterial strain (Bacillus subtilis O-3, Bacillus subtilis Md1-42, and Bacillus subtilis Khozestan2) was done separately two times from 50 mL of the culture broth with 25 mL ethyl acetate (Sigma-Aldrich, Germany) for 30 min and two extracts were combined. Thereafter, the extract with a volume of 1.5 mL was taken into plastic vials with a volume of 2 mL and placed onto the autosampler tray for analysis using GC–MS. Thermo Scientific GC Focus Series DSQ was used to perform a GC–MS analysis on bacterial secondary metabolites. A steady flow of 1 mL of helium gas per minute was employed as the carrier gas, and an infection volume of 1 L was used. The injector and hot oven were kept at 250 °C and 110 °C, respectively, with the temperature increasing by 10 °C per minute up to 200 °C, 5 °C per minute up to 280 °C, and shutting down after 9 min at a temperature of 280 °C. The GC column was used to elute peaks of various chemicals, and the retention times of these peaks were noted. The database was searched for compounds with similar molecular masses and retention times after the data were matched with the compounds’ mass spectra. The bioactivities of previously investigated natural substances were also investigated, and the current study found a comparable correlation between the bioactivities of the bacterial extracts and their constituent parts.

4.5. Molecular Characterization of the Bacterial Isolates

Based on 16S rRNA conserved gene sequences and universal bacterial primers, isolated bacterial strains were molecularly characterized. The targeted gene sequence was amplified using the standard PCR procedure, and the final product was run through 1% gel electrophoresis to examine the size of the amplified fragments. The amplified samples and the relevant sequencing fragments were sent for sequencing, and MEGA software was used to phylogenetically analyze the nucleotide sequences that were recovered (MEGA-11). Using GenBank NCBI’s BLAST search, the bacterial isolates were further verified and classified at the species level (National Center for Biotechnology Information). With the accession numbers GQ870259, MF581448, and MH036316, 16S rRNA gene sequences for these probiotic strains were uploaded to the GenBank database (www.ncbi.nlm.nih.gov/projects/genome/clone/, accessed on 9 December 2022).

4.6. Statistical Analyses

The XLSAT software version 2016.02.27444 was used to conduct the analysis of variance (one-factor ANOVA) at the significance level (α = 0.05). The Newman–Keuls test was used to rank the means when there was a significant difference between the studied parameters.

5. Conclusions

The growth of multidrug-resistant bacterial strains could be inhibited by vegetable bacterial isolates from three different species of Bacillus, according to the current study. When examined using well diffusion and the perpendicular streak method, the crude extracts from three isolated bacterial strains were effective against bacterial strains. The potent isolates BSS11, BSS17, and BSS19 with broad-spectrum antibacterial activities were identified through this screening. The metabolic diversity within isolates was highlighted by a comparative GC–MS analysis, despite the fact that they are all members of the same Bacillus subspecies. In particular, it is evident in the case of Bacillus subtilis O-3 and Bacillus subtilis Md1-42. This study discovered a number of volatile inhibitory substances, including esters, phenolics, and ethers that may be involved in antimicrobial activity. It was found how they differ in chemical composition and how it may influence antimicrobial activity and antibiotic potency. For decades, it has been known that strains of the B. subtilis group are capable of producing a variety of secondary metabolites that mediate their antimicrobial characteristics. Along with volatile organic compounds, the presence of bacteriocins, polyketides, peptides, etc., were known. Hence, it can be concluded that the discovered organic volatile substances enhance the antimicrobial properties of Bacillus spp. together with the above substances. Bacterial strains Bacillus subtilis O-3, Bacillus subtilis Md1-42, and Bacillus subtilis Khozestan2 exhibit a tremendous metabolic capacity and adaptive biochemistry that could be employed in a variety of commercial and biotechnological activities by generating a wide range of bioactive chemical substances. Additionally, bacterial extracts, including chemicals, could be employed as antimicrobial agents to target different multidrug-resistant bacterial strains. It is anticipated that a thorough investigation of a similar kind could investigate new microbiological possibilities with undiscovered substances or metabolites that have a strong antibacterial potential. As a result, it might be a viable strategy for lowering the burden and danger posed by bacterial strains that are resistant to many drugs.

Author Contributions

Conceptualization: M.K., A.A. and A.T.; methodology: M.K., Z.S., M.A., A.A., G.Y. and E.K.; software: M.K., Z.S. and A.A.; validation: M.K., Z.S. and M.A.; formal analysis: S.A., M.A. and K.R.; investigation: M.K. and Z.S.; resources: M.K. and A.A.; data curation: M.K. and A.A.; writing—original draft: M.K. and Z.S.; writing—review and editing: M.K. and Z.S.; visualization: S.A. and K.M.; supervision: G.U.; project administration: G.U.; funding acquisition: G.U. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The authors confirm that the data supporting the findings of this study are available within the article.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds are not available from the authors.

Funding Statement

This research received no external funding.

Footnotes

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Data Availability Statement

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