**4. Discussion**

post-application intervals when compared to control, but the colony count increased with the

The bacterial strain SP-03 isolated from soil was a rod-shaped, Gram-positive bacterium, facultatively anaerobic, grows at 5–40°C, at pH 6–7; produce subterminal ellipsoidal endo‐ spores; white-colored colonies; positive for catalase activity, Voges-Proskauer, starch hydrol‐ ysis and oxidase; and negative for methyl red, gelatin liquefaction, production of indole and citrate. The 16S rDNA gene of SP-03 was isolated and sequenced. This 16S rDNA gene sequence was then compared with previously published 16S rDNA gene sequences and based on matches the strain was classified as a member of the genus Bacillus. The sequence of strain SP-03 displayed the highest identity (100%) with the 16S rDNA gene of Bacillus weihenste‐ phanensis KBAB4 (GenBank Accession Number: HG 486214.1) (Figure 1, 2). The Bacillus

**Graph 1.** Effect of imidacloprid on bacterial populations in the soil under laboratory conditions

weihenstephanensis showed highest growth at 22°C and at pH of 7.0.

**3.4. Effect of imidacloprid on antioxidant enzymes in** *Bacillus weihenstephanensis*

On exposure of *Bacillus weihenstephanensis* to various molar concentrations (10–3 to 10–7) of imidacloprid for 24, 48, 72 and 96 h, there was a significant (*P* ≤ 0.05) increase in the activity of antioxidant enzymes studied. There was a significant increase (*P* ≤ 0.05) in the activity of superoxide dismutase (Graph 3), catalase (Graph 4) and peroxidase (Graph 5) in all the treated groups. The antioxidant enzyme activity increased with an increase in the concentration of

**3.5. Gene isolation and sequencing of the stress enzymes of** *Bacillus weihenstephanensis* **on**

In the present gene sequencing study, different markers of 400, 600, 1,000 and 1,200 bp (Figure) 6) were run along with our test sample. The superoxide dismutase gene was corresponding to 624 bp. This gene was isolated, eluted and sent for sequencing. The sequence was received and

**3.3. Identification of soil isolate**

imidacloprid.

**exposure to imidacloprid**

time.

280 Insecticides Resistance

#### **4.1. Effect of imidacloprid on soil bacterial populations in laboratory and field studies**

Results obtained in laboratory studies showed significant (*P* < 0.05) decrease in bacterial count when compared to that of control. A gradual decrease in bacterial count is observed with increase in concentration of imidacloprid, with minimal count reported at 1,000 ppm. The results obtained were similar to results reported earlier in a study involving five other pesticides [14].

The results indicate toxic effect of imidacloprid on bacterial populations. Results obtained from bacterial enumeration of imidacloprid-treated soils at recommended rate showed significant (*P* < 0.05) decrease in bacterial numbers, proving negative effect of imidacloprid on bacteria. This negative effect reduced after 14 days of treatment. The negative effect of imidacloprid was vanished by 28th day of application, indicated by bacterial count, which was almost similar to pre-treatment count. Similar results were reported in a study involving imidacloprid and five other pesticides; in the study the toxic effect was vanished by 21st day of imidacloprid application [14].

The different studies have shown that the impact of pesticides application microorganisms present in soil is variable. The impact depends on interaction between microorganisms and active substances and formulation. It also depends on surfacing of specific group of microor‐ ganisms [15]. The microorganisms can develop the ability to use an applied pesticide as a source of energy and growth [16].

The initial decrease in bacterial count is expected as pesticides are known to affect the microbial populations by controlling the survival and reproduction of individual species. Initial reduction in microbial count is also reported in studies involving different pesticides such as endosulphan, cypermirithin thiodan, etc. [12, 17–18], and herbicides like glyphosate, atrazine, simazin and alachlor [19–22] when applied at recommended rates. It has been observed in many studies that pesticides stimulated the mineralization rate of organic carbon in compar‐ ison with control samples [23–24]. Microorganisms susceptible to toxic effects of pesticides are removed from the population of soil microflora. The pesticides kill the bacterial cells by penetration and disturbing the cell metabolism.

**Graph 2.** Effect of imidacloprid on bacterial populations in soil under field conditions

The reduction in the number of sensitive microorganisms and increase in resistant microor‐ ganisms lead to reduced soil microbial biodiversity. The increase in bacterial numbers after 14th day may be due to the ability of bacteria to degrade toxic compounds like pesticides [25]. The growth of pesticide-resistant microorganisms may compensate the loss of pesticidesensitive microorganisms in the population [24]. The addition of fungicide leads to increase in bacterial populations due to no competition with fungi or antagonistic inhibition by fungi [26]. Bacteria are known to become resistant to toxic compound with production of specific degrading enzymes [27].

The application of 1.5× of imidacloprid showed significant (*P* < 0.05) decrease in bacterial number. The results were similar to recommended rates, but bacterial numbers increased slowly. These results were comparable to the results reported in similar studies with pesticides like metoalchlor, atrazine, dimethoate and endosulfan [20, 22, 28–29].

**Figure 1.** *Bacillus weihenstephanensis*

#### **4.2. Identification of soil isolate**

removed from the population of soil microflora. The pesticides kill the bacterial cells by

The reduction in the number of sensitive microorganisms and increase in resistant microor‐ ganisms lead to reduced soil microbial biodiversity. The increase in bacterial numbers after 14th day may be due to the ability of bacteria to degrade toxic compounds like pesticides [25]. The growth of pesticide-resistant microorganisms may compensate the loss of pesticidesensitive microorganisms in the population [24]. The addition of fungicide leads to increase in bacterial populations due to no competition with fungi or antagonistic inhibition by fungi [26]. Bacteria are known to become resistant to toxic compound with production of specific

The application of 1.5× of imidacloprid showed significant (*P* < 0.05) decrease in bacterial number. The results were similar to recommended rates, but bacterial numbers increased slowly. These results were comparable to the results reported in similar studies with pesticides

penetration and disturbing the cell metabolism.

282 Insecticides Resistance

**Graph 2.** Effect of imidacloprid on bacterial populations in soil under field conditions

like metoalchlor, atrazine, dimethoate and endosulfan [20, 22, 28–29].

degrading enzymes [27].

**Figure 1.** *Bacillus weihenstephanensis*

The bacterial strain SP-03 isolated from soil was a rod-shaped, Gram-positive bacterium, facultatively anaerobic, grows at 5–40°C, at pH 6–7; produce subterminal ellipsoidal endo‐ spores ; white-colored colonies; positive for catalase activity, Voges-Proskauer, starch hydrol‐ ysis and oxidase; and negative for methyl red, gelatin liquefaction, production of indole and citrate. The 16S rDNA gene of SP-03 was isolated and sequenced. This 16S rDNA gene sequence was then compared with previously published 16S rDNA gene sequences and based on matches the strain was classified as a member of the genus Bacillus. The sequence of strain SP-03 displayed the highest identity (100%) with the 16S rDNA gene of *Bacillus weihenstepha‐ nensis* KBAB4 (GenBank Accession Number: HG 486214.1). The Bacillus weihenstephanensis showed highest growth at 22°C and at pH of 7.0.

**Figure 2.** Phylogenetic tree of Bacillus weihenstephanensis

#### **4.3. Antioxidant enzymes**

Partial reduction of oxygen to water during microbial respiration gives rise to reactive oxygen intermediates, e.g. superoxide radicals, hydrogen peroxide and hydroxyl radicals. Microor‐ ganisms have developed efficient enzymatic and nonenzymatic mechanisms to eliminate these toxic and mutagenic reactive oxygen species. Superoxide is eliminated by dismutation to H2O2 catalyzed by superoxide dismutase and accumulation of H2O2 is prevented by the action of catalases and peroxidases [30].

Numerous pesticides such as paraquat, DDT, PCB, Arochlor, etc. have been used as model factors inducing oxidative stress both in vivo and in vitro [31]. The tissue damage occurs due to conversion of pesticides to free radicals or superoxide radical during their metabolism. Organisms exposed to different concentrations of xenobiotics have the risk of carcinogenic effect, neurological actions and brain damage [32]. The organisms have developed some mechanisms to control the amount of hydroxyl and superoxide radicals generated to overcome the toxic effects of xenobiotics. Antioxidants quickly scavenge the hydroxyl and superoxide radicals generated. The antioxidants can be enzymatic or nonenzymatic which safely interact with free radicals and terminate the chain reactions before vital molecules are damaged. The antioxidant enzymes include catalase, superoxide dismutase (SOD), glutathione reductase, glutathion-S-transferase and glutathione peroxidase [33].

**Graph 3.** Effect of imidacloprid on SOD activity in *Bacillus weihenstephanensis*

Significant increase in activity of SOD compared with the control may be due to the toxic effects of imidacloprid. ROS depends on the oxidative metabolism of xenobiotics or endogenous compounds. The antioxidant defense systems work by lowering the concentrations of xeno‐ biotics rather than complete elimination. When the ROS generated exceeds the antioxidants' capability of that cell, it results in oxidative stress [34].

The stress-mediated cytotoxicity results due to oxidative processes and loss of key antioxidant enzymes. *Escherichia coli*, *Salmonella typhimurium* and mammalian cells induce antioxidant proteins in response to oxidative stress [35]. It is suggested that an increase in SOD and CAT might be in response to increased oxidative stress or might be due to compensatory response to oxidative stress induced by this xenobiotic. Superoxide dismutase, catalase and peroxidase are the enzymes that participate in the protection against reactive oxygen species.

Catalase is one of the most efficient antioxidants known so far. It is present in peroxisomes of nearly all aerobic cells and protects the cells from the toxic hydrogen peroxide effects by catalyzing its decomposition into molecular oxygen and water without the production of free radicals. In addition, catalase is known to act on toxic compounds by per oxidative reactions. It is demonstrated that acetamiprid-induced oxidative stress on *Escherichia coli*, *Pseudomonas* sp and *Bacillus subtilis* resulted in elevated superoxide dismutase and catalase activities to antagonize oxidative stress [5].

**Graph 4.** Effect of imidacloprid on catalase activity in *Bacillus weihenstephanensis*

H2O2 catalyzed by superoxide dismutase and accumulation of H2O2 is prevented by the action

Numerous pesticides such as paraquat, DDT, PCB, Arochlor, etc. have been used as model factors inducing oxidative stress both in vivo and in vitro [31]. The tissue damage occurs due to conversion of pesticides to free radicals or superoxide radical during their metabolism. Organisms exposed to different concentrations of xenobiotics have the risk of carcinogenic effect, neurological actions and brain damage [32]. The organisms have developed some mechanisms to control the amount of hydroxyl and superoxide radicals generated to overcome the toxic effects of xenobiotics. Antioxidants quickly scavenge the hydroxyl and superoxide radicals generated. The antioxidants can be enzymatic or nonenzymatic which safely interact with free radicals and terminate the chain reactions before vital molecules are damaged. The antioxidant enzymes include catalase, superoxide dismutase (SOD), glutathione reductase,

Significant increase in activity of SOD compared with the control may be due to the toxic effects of imidacloprid. ROS depends on the oxidative metabolism of xenobiotics or endogenous compounds. The antioxidant defense systems work by lowering the concentrations of xeno‐ biotics rather than complete elimination. When the ROS generated exceeds the antioxidants'

The stress-mediated cytotoxicity results due to oxidative processes and loss of key antioxidant enzymes. *Escherichia coli*, *Salmonella typhimurium* and mammalian cells induce antioxidant proteins in response to oxidative stress [35]. It is suggested that an increase in SOD and CAT might be in response to increased oxidative stress or might be due to compensatory response to oxidative stress induced by this xenobiotic. Superoxide dismutase, catalase and peroxidase

Catalase is one of the most efficient antioxidants known so far. It is present in peroxisomes of nearly all aerobic cells and protects the cells from the toxic hydrogen peroxide effects by catalyzing its decomposition into molecular oxygen and water without the production of free radicals. In addition, catalase is known to act on toxic compounds by per oxidative reactions.

are the enzymes that participate in the protection against reactive oxygen species.

of catalases and peroxidases [30].

284 Insecticides Resistance

glutathion-S-transferase and glutathione peroxidase [33].

**Graph 3.** Effect of imidacloprid on SOD activity in *Bacillus weihenstephanensis*

capability of that cell, it results in oxidative stress [34].

The present study revealed that the catalase activity was significantly increased in all the groups with increase in the dose and durational exposure of imidacloprid to *Bacillus weihen‐ stephanensis*. Similarly, it has been reported that induction of major antioxidant enzymes, such as superoxide dismutase and catalase, were observed after their exposure to a single oxygen generating system in *Escherichia coli.* It is suggested that response to low concentrations of hydrogen peroxide induces catalase in *Escherichia coli* during logarithmic growth [36].

Peroxidase is found among animals, plants and microorganisms, where they perform essential roles in the metabolism. To prevent the lethal effects of such metal-ion-catalyzed oxidation (MCO), bacterial cells have evolved protective mechanisms to neutralize the formation of toxic oxygen radicals. For instance, small molecule antioxidants, such as catalases and peroxidases, have been reported to play protective roles in the enteric bacteria in *Pseudomonas* sp. and in *Bacteroides* sp. [37].

The enzyme peroxidase is an important antioxidant enzyme, which plays a pivotal role in plant growth and development. The presence of phenol substances leads to enhanced activity of peroxidase (POD). The POD helps in providing resistance to stress and self-defense by increasing the rate of respiration under stress conditions [38].

The present study revealed that the peroxidase activity in the treated groups increased significantly in higher dose (10–5, 10–4 and 10–3 M) of exposure and there was no significant increase observed in the lower dose (10–7 and 10–6 M) of imidacloprid in *Bacillus weihenstepha‐ nensis*. Similar results were reported in other organisms which suggest that a gradual increase of catalase or peroxidase production in aging cultures is not surprising since catalase and/or CP is one of the radical-scavenging enzymes in cells in response to oxidative stress [39]. On the other hand, several organisms produce two or more catalase peroxidase, whereby one

**Graph 5.** Effect of imidacloprid on peroxidase activity in *Bacillus weihenstephanensis*

enzyme was expressed at the end of exponential growth and during the stationary phase. This behavior was observed in *Escherichia coli, Pseudomonas putida, Streptomyces coelicolor* and *Arcobacter nitrofigilis* [40]. It is also reported that superoxide dismutase and peroxidase form the first line of defense against reactive oxygen species [41].

The significant increase in the antioxidant enzymes activity observed in the present study may be due to synthesis of these enzymes as a response to chemical stress induced by imidacloprid or due to inhibition of the membrane-bound enzymes by affecting the enzyme complex, oxidative stress-mediated cytotoxicity enzymes, induction of antioxidant proteins in response to oxidative stress [42].

#### **4.4. Gene sequencing of stress enzymes of** *Bacillus weihenstephanensis* **on exposure to imidacloprid**

The research in life sciences is affected significantly by the mapping of the genes and genomes of organisms. The related mapping technology is changing the current understanding of biological systems [43]. The application of life science areas of toxicology, genetics, molecular biology and environmental health to describe the response of organisms to environmental stimuli is called toxicogenomics. The toxicogenomics is developed in the past 15 years and will help in advancing the scientific basis of risk assessments for the environmental contaminants [44].

In the present study, the exposure of *Bacillus weihenstephanensis* to imidacloprid resulted in the expression of manganese containing superoxide dismutase (*sod A*) gene. MnSOD and FeSOD have an extremely broad phylogenetic distribution, being expressed in both prokaryotic (eubacterial and archaeal) and eukaryotic cells and are quite homologous [45]. Expression of *sod A* gene has also been reported for other bacterial species. A strain of *Sulfolobus sulfatari‐ cus* produced Fe-Mn SOD with half-life of 2 h at 100°C [46]. In a study, superoxide dismutase producing *Bacillus* sp. was isolated from Bulgarian thermal spring [47]. In another study, *Thiobacillus denitrificans* strain "RT" Fe-superoxide dismutase has been purified with a molecular weight of 43,000, and is composed of two identical subunits. Aerobically and anaerobically grown *Thiobacillus denitrificans* cells contain the same Fe-enzyme with similar Effect of Imidacloprid on Bacterial Soil Isolate *Bacillus weihenstephanensis* http://dx.doi.org/10.5772/61503 287


**Figure 3.** Gel image of stress enzymes amplicon

enzyme was expressed at the end of exponential growth and during the stationary phase. This behavior was observed in *Escherichia coli, Pseudomonas putida, Streptomyces coelicolor* and *Arcobacter nitrofigilis* [40]. It is also reported that superoxide dismutase and peroxidase form

The significant increase in the antioxidant enzymes activity observed in the present study may be due to synthesis of these enzymes as a response to chemical stress induced by imidacloprid or due to inhibition of the membrane-bound enzymes by affecting the enzyme complex, oxidative stress-mediated cytotoxicity enzymes, induction of antioxidant proteins in response

**4.4. Gene sequencing of stress enzymes of** *Bacillus weihenstephanensis* **on exposure to**

The research in life sciences is affected significantly by the mapping of the genes and genomes of organisms. The related mapping technology is changing the current understanding of biological systems [43]. The application of life science areas of toxicology, genetics, molecular biology and environmental health to describe the response of organisms to environmental stimuli is called toxicogenomics. The toxicogenomics is developed in the past 15 years and will help in advancing the scientific basis of risk assessments for the environmental contaminants

In the present study, the exposure of *Bacillus weihenstephanensis* to imidacloprid resulted in the expression of manganese containing superoxide dismutase (*sod A*) gene. MnSOD and FeSOD have an extremely broad phylogenetic distribution, being expressed in both prokaryotic (eubacterial and archaeal) and eukaryotic cells and are quite homologous [45]. Expression of *sod A* gene has also been reported for other bacterial species. A strain of *Sulfolobus sulfatari‐ cus* produced Fe-Mn SOD with half-life of 2 h at 100°C [46]. In a study, superoxide dismutase producing *Bacillus* sp. was isolated from Bulgarian thermal spring [47]. In another study, *Thiobacillus denitrificans* strain "RT" Fe-superoxide dismutase has been purified with a molecular weight of 43,000, and is composed of two identical subunits. Aerobically and anaerobically grown *Thiobacillus denitrificans* cells contain the same Fe-enzyme with similar

the first line of defense against reactive oxygen species [41].

**Graph 5.** Effect of imidacloprid on peroxidase activity in *Bacillus weihenstephanensis*

to oxidative stress [42].

**imidacloprid**

286 Insecticides Resistance

[44].

levels of activity. Manometric sulfite oxidation measurements suggest for the enzyme a protective function of sulfite against the auto-oxidation initiated by superoxide free radicals [48]. *Escherichia coli* when grown under anaerobic conditions contained only Fe-SOD, but exposure to oxygen induced the synthesis of Mn-SOD and New-SOD [49].

**Figure 4.** Phylogenetic tree of SOD

SOD of *B. subtilis* is manganese associated as indicated by a high similarity of the putative amino acid sequence of *B. subtilis* SodA to those of Mn-Sod from *B. caldotenax* and *B. stearo‐ thermophilus*, and presence of four conserved metal-binding sites. This SOD was found in vegetative cells and in spores [50]. A new, thermostable superoxide dismutase (SOD) from *Bacillus licheniformis* M20, is isolated from Bulgarian mineral springs. It is reported that *B.* *subtilis* contains a cytosolic Mn-superoxide dismutase [51]. Xenobiotic degrading bacteria experience oxidative stress, both as directly from the pollutants themselves and from inter‐ mediates generated during biodegradation processes [52]. Depending on the type of oxidative stress, not only different amounts of proteins can be modified but also different species may appear. It was shown that the set of oxidized proteins depended on the method of induction of oxidative stress.

The present study reveals that the genome of catalase encoded in our *Bacillus weihenstepha‐ nensis* culture on exposure to imidacloprid was *Kat E* (HPII). HPII and catalase-2 monofunc‐ tional catalases of *E. coli* and *B. subtilis*, expressed in the stationary phase, have D-isomer prosthetic groups with six haem. Catalase from *E. coli* HPI *R*., *Halobacterizlm halobitlm* and facultative alkalophilic *Bacillus* species have bifunctional catalase-peroxidases [53–56]. Several organisms produce two or more catalase/peroxidases, whereby one enzyme was expressed at the end of exponential growth and during the stationary phase. This behavior was observed in *Escherichia coli, Pseudomonas putida, Streptomyces coelicolor* and *Arcobacter nitrofigilis* [40]. It has been reported that various bacteria such as *Citrobacter freundii, Edwardsiella tarda, Entero‐ baacter aaerogenes, Klebsiella pneumonia* and *Salmonella typhimurium* exhibited patterns of catalase activity similar to that of HPI and HPII bands of *Escherichia coli*.

**Figure 5.** Phylogenetic tree of catalase

Bacterial monofunctional catalases of *E. coli* HPII [53] and *B. subtilis* catalase-2, both of which are expressed in the stationary phase, contain six haem D-isomer prosthetic groups in a hexameric structure of larger subunits. From the present study of exposure of *Bacillus weihen‐* *stephanensis* to imidacloprid, it can be concluded that the catalase enzyme can be encoded by the *Kat E* (HPII) gene.

The present sequence analysis of the peroxidase gene suggests that *Bacillus weihenstephanen‐ sis* subjected to imidacloprid expressed the glutathione peroxidase (Tpx) gene. The findings of study also support a thiol-dependent antioxidant activity for thiol peroxidase in *Streptococ‐ cus parasanguis*, which protects the organism from stress [57]. It is reported that the bacterial thiol peroxidases include a pair of cysteine residues and comprise part of the functional group for the peroxidase activity [58]. *Escherichia coli* thiol peroxidase is part of an oxidative stress defense system that uses reducing equivalents from thioredoxin (Trx1) and thioredoxin reductase to reduce alkyl hydroperoxides [59]. The specific mechanism(s) by which thiol peroxidase protects *Streptococus parasanguis* from the toxicant may be similar to those described for other thiol-specific antioxidants of *Escherichia coli*. The *Mycobacterium* sp. strain PYR-1 degrades polycyclic aromatic hydrocarbons, environmental pollutants. It was shown that inducible catalase-peroxidase of katG gene of this culture is involved in molecular mechanisms of degradation of these pollutants [60].

**Figure 6.** Phylogenetic tree of glutathione peroxidase

## **5. Conclusion**

*subtilis* contains a cytosolic Mn-superoxide dismutase [51]. Xenobiotic degrading bacteria experience oxidative stress, both as directly from the pollutants themselves and from inter‐ mediates generated during biodegradation processes [52]. Depending on the type of oxidative stress, not only different amounts of proteins can be modified but also different species may appear. It was shown that the set of oxidized proteins depended on the method of induction

The present study reveals that the genome of catalase encoded in our *Bacillus weihenstepha‐ nensis* culture on exposure to imidacloprid was *Kat E* (HPII). HPII and catalase-2 monofunc‐ tional catalases of *E. coli* and *B. subtilis*, expressed in the stationary phase, have D-isomer prosthetic groups with six haem. Catalase from *E. coli* HPI *R*., *Halobacterizlm halobitlm* and facultative alkalophilic *Bacillus* species have bifunctional catalase-peroxidases [53–56]. Several organisms produce two or more catalase/peroxidases, whereby one enzyme was expressed at the end of exponential growth and during the stationary phase. This behavior was observed in *Escherichia coli, Pseudomonas putida, Streptomyces coelicolor* and *Arcobacter nitrofigilis* [40]. It has been reported that various bacteria such as *Citrobacter freundii, Edwardsiella tarda, Entero‐ baacter aaerogenes, Klebsiella pneumonia* and *Salmonella typhimurium* exhibited patterns of

Bacterial monofunctional catalases of *E. coli* HPII [53] and *B. subtilis* catalase-2, both of which are expressed in the stationary phase, contain six haem D-isomer prosthetic groups in a hexameric structure of larger subunits. From the present study of exposure of *Bacillus weihen‐*

catalase activity similar to that of HPI and HPII bands of *Escherichia coli*.

of oxidative stress.

288 Insecticides Resistance

**Figure 5.** Phylogenetic tree of catalase

Present investigation was carried out to analyze the effect of imidacloprid on antioxidant enzymes superoxide dismutase, catalase and peroxidase in soil isolate *Bacillus weihenstepha‐ nensis*, isolated after field studies on the effect of imidacloprid at recommended and 1.5× rates, Which showed that there was an increase in the activity of all the three antioxidant enzymes. The enzyme activity increased with an increase in the concentration of insecticide proving that the inhibitory effect is dose-dependent. Further, sequencing revealed that Fe/MnSOD (sod A), hydroxyperoxidase HP(II) (Kat E) and glutathione peroxidase genes were expressed in response to stress induced by imidacloprid treatment in *Bacillus weihenstephanensis*. The present investigation indicates that imidacloprid induces stress, which results in the expres‐ sion of antioxidant enzymes in the soil isolate *Bacillus weihenstephanensis* to protect the cellular components from oxidative damage. Study also reveals that the soil isolate *Bacillus weihenste‐ phanensis* has developed the resistance to imdacloprid toxicity by synthesis of antioxidant enzymes. Further research can be performed to use it in the field for pollution monitoring and risk assessment due to imidacloprid contamination in soil, thereby exploring the possibility of using soil isolate imidacloprid-resistant *Bacillus weihenstephanensis* in the study of complex biological processes and to clean the fields with imdacloprid contamination.
