Entomotherapy as an alternative treatment for diseases due to Gram-negative bacteria in Burkina Faso

Insects are known for their harmful effects. However, they also benefit humans, animals, plants, and ecosystems. Its beneficial uses include entomophagy and entomotherapy. This study aimed to evaluate the antibacterial activity of insect extracts against Gram-negative bacteria. Antibacterial activities of thirteen crude extracts of medicinal insects were tested against twelve Gram-negative bacteria by diffusion on agar. Imipenem was used as an antibiotic for positive control. The thirteen extracts acted differently against certain Gram-negative bacteria. The largest inhibition diameter was for extracts of Cirina butyrospermi and Mylabris variabilis against Pseudomonas aeruginosa ATCC27853 and Salmonella enteritidis ATCC13076, respectively. The diameters of inhibition obtained using imipenem against these same bacterial strains were 13.0 ± 0.0 mm and 22 ± 1.0 mm, respectively. The lowest inhibition diameter (7.5 ± 0.0 mm) was obtained using Anopheles gambiae extract against Salmonella Typhimurium ATCC14028. Imipenem was active on all strains tested. The highest values of the index multi-resistance to insect’s extracts were reported for Pseudomonas aeruginosa ATCC9027 and Serratia odorifera 652411. Overall, the results of this study confirmed the antibacterial activities of insects used by traditional health practitioners to treat different pathologies. Entomotherapy could be an alternative treatment for certain infectious pathologies caused by gram-negative bacteria.


Technical collection and information on the insect collected in this study
Insects were collected mainly using threshing methods, swath nets, and mechanical sampling with laboratory tweezers in insect nests.The insects were then placed in alcohol jars to preserve them [20][21][22] .Thirteen medicinal insects were collected based on information provided by traditional health practitioners.These insects are listed and illustrated in Table 1.Once arrived at the laboratory, the collected insects were dried in an oven at a constant temperature of 25 °C until completely dry.This allows the collected samples to maintain their complete integrity while preventing their decomposition.The collection of these insects was conducted from July 2022 to September 2023, a period of 15 months.The number of each insect species collected varied significantly from one species to another.Indeed, we had for each type of insect specimen a quantity in dry weight of 20 g.Thus, for large insects such as Periplanneta americana, Mylabris variabilis, Lytta sp., Kraussaria anguilifera, Acrida bicolor, Cirina butyrospermi, and Bunaea alcinoe, the number of specimens was 45.On the other hand, for small insects such as Macrotermes bellicosus, Odontotermes sp., Pachycondyla sp., Acheta domesticus, and Anopheles gambiae, the number of individuals collected per specimen varied from a hundred to several hundred.

Preparation of the insects for extraction
After the drying step, the insects were finely ground using a ceramic laboratory mortar.The insect powder was packaged in Falcon tubes (CONICAL BOTTOM CELLSTAR® STERILE) and stored in the oven at 37 °C for future use.

Extraction of crude extracts from insects collected
The extraction of crude extracts from insects was performed according to the method described by Dah-Nouvlessounon et al. 23 and readapted.Thus, aqueous extraction (crude extracts) was performed using sterile ultrapure Milli-Q water.For the realization, 4 g of each insect powder was macerated in 10 mL of the extraction solution for 12 h at 25 °C under magnetic stirring.The macerates were centrifuged at 3,000 rpm for 10 min at 4 °C using a JOUAN BR4 refrigerated centrifuge.After centrifugation, the supernatants were collected in Eppendorf tubes and kept cool at 4 °C.After collecting the supernatants, and the extraction solvents were evaporated to dryness in an oven at 45 °C until a dry extract of constant mass was obtained for the evaluation of extraction yield.The residues obtained were kept at 4 °C until the antimicrobial tests were performed.

Extraction yield
The extraction yield was determined by the ratio between the mass of the powdered insect after extraction and the mass of their ground material at the start according to the following formula.

Gram-negative bacteria used for antimicrobial testing
Antimicrobial activity tests were carried out against twenty-two microbial germs including twelve Gram-negative bacteria (Table 2).Microbial strains used in this study are based on several criteria: these strains are commonly of www.nature.com/scientificreports/hospital and food origin, for their high incriminations in pathologies in animals and humans, and these strains are chosen regarding their natural resistance to various types of antimicrobial agents.

Antimicrobial activity testing of crude extracts from insects
The antimicrobial activity of crude extracts from insects was tested according to the agar diffusion method described by Kirby-Bauer following the guidelines of the Clinical Laboratory Standards Institute 24 .The microbial inocula was prepared from young colonies aged from 16 to 18 h diluted in test tubes containing physiological saline.All microbial suspensions obtained were adjusted to a turbidity of 0.5 MacFarlant.This standard turbidity of 0.

Determination of index multi-resistance to crude extracts from insects
The index of multi-resistance to extracts (IMRE) of crude extracts from insects was determined according to Das et al. 25 .IMRE was calculated using the following formula: Table 2. Information on the Gram-negative bacteria tested.

Microorganism Species Reference
Gram-negative bacteria

Activity coefficient of crude extracts from insects
The activity coefficient (A) of crude extracts from insects collected against the bacterial strains tested was calculated using the following formula: Q: quantity of insect extract (μL); Z: inhibition diameter including the diameter of the disc (cm).

Data processing and statistical analyses
Results were expressed as a mean number followed by standard deviation (M ± SD) and subject to Student's t-test using R.The significance threshold was 5%.XLSTAT-2019 software was used for principal component analysis (PCA).PCA was used to explore the correlation between the activities of crude extracts from insects collected and the different Gram-negative bacteria.

Yield values of extractions of crude extracts from insects
The different yields of the compounds in the crude extracts obtained after extraction are recorded in Fig. 1.These yields vary with the species of insects.The highest yields were obtained with (36.0%)Pachychondyla sp., (28.0%)Periplaneta americana, and (20.0%)Acrida bicolor and weak yields with (8.0%) Anopheles gambiae and (7.4%) Odontotermes sp.The former contains a more water-soluble matter than the latter.Thus, several factors could strongly influence the extraction yield.Among these factors are drying time, particle size of the ground material, nature of the solvent used, mass-volume ratio of the ground's solvent (m/v), maceration time, and stirring speed.

Antimicrobial activity of crude insects extracts
The crude extracts from insects tested inhibited the growth of the bacterial strains, as shown in Fig. 2 and Table 3.
The inhibition diameters varied depending on the strain and the extract used.All thirteen crude extracts inhibited the growth of some bacterial.
The crude extract of Acheta domesticus was active on eight and twelve Gram-negative bacteria.The highest inhibition diameter was 18.5 ± 1.5 mm against Klebsiella pneumoniae ATCC13883, whereas the lowest was 09.0 ± 0.0 mm against Escherichia coli 652654.The crude extract of Acheta domesticus did not affect Escherichia coli ATCC8739, Providencia rettgeri 652655, and Serratia odorifera 652411.In Latin America, Acheta domesticus has been used in treating tract infections in Brazil and as an antidiuretic against urinary retention in Mexico [26][27][28] .The bioactive molecule contained in Acheta domesticus is phenoloxidase 29 .
The crude extract of Anopheles gambiae was active on eight Gram-positive bacteria, with an inhibition percentage of 66.66%.The highest inhibition diameter was 15.0 ± 1.0 mm and the lowest was 07.www.nature.com/scientificreports/had no inhibitory effect on Escherichia coli ATCC8739, Klebsiella pneumoniae 203, Pseudomonas aeruginosa ATCC27853, and Salmonella enteritidis ATCC13076.The inhibitory effect of Anopheles gambiae extract may be linked to its richness in different proteins 30 .Indeed, AMPs such as cecropins are found in this insect 11 .
Apis mellifera crude extract was active on six of the bacterial strains (50% inhibition), with the greatest inhibition diameter of 22.5 ± 0.5 mm (Providencia rettgeri 652655) and the lowest inhibition diameter of 08.0 ± 0.0 mm (Salmonella abony NCTC6017).This crude extract had no inhibitory effect on Escherichia coli 652654, Escherichia coli ATCC8739, Klebsiella pneumoniae ATCC13883, Pseudomonas aeruginosa ATCC9027, Serratia odorifera 652411, and Salmonella Typhimurium ATCC14028.The recorded antimicrobial activity may be due to the presence of melittin in the bee venom.Lupoli 31 and Marques et al. 32 reported the presence of inhibitory molecules, such as melittin, the main peptide in bee venom.
Thus, a 75% inhibition rate of the bacterial strains tested (9/12) was observed with Cirina butyrospermum extract.The lowest and highest diameters of inhibition were respectively 08.5 ± 0.5 mm (Providencia rettgeri 652655) and 30.0 ± 0.5 mm (Pseudomonas aeruginosa ATCC9027).Shea caterpillars are known to be rich in proteins, accounting for more than 60% of the total 35,36 .Some of these proteins have antibacterial activity.However, this extract was not active against Escherichia coli ATCC8739, Klebsiella pneumoniae ATCC13883, and Serratia odorifera 652411.
The Lytta sp.extract also inhibited 75% of the bacterial strains tested.Its maximum inhibition diameter (13.5 ± 1.5 mm) was recorded against Providencia rettgeri 652655 and the lowest inhibition diameter (09.0 ± 1.0 mm) with Salmonella Typhimurium ATCC14028.This meloid did not inhibit the growth of Klebsiella pneumoniae and the two strains of Pseudomonas aeruginosa tested.This inhibitory activity was attributed to cantharidin.Cantharidin is a bioactive molecule concentrated on the genital glands of insects in the genus Lytta, which belongs to the meloid family 31 .
Six of twelve bacterial strains were inhibited by the Macrotermes bellicosus extract (i.e., an inhibition rate of 50%).Pseudomonas aeruginosa ATCC27853 strain was the most sensitive (13.5 ± 0.5 mm), and Salmonella Typhimurium ATCC14028 was the least sensitive strain (08.5 ± 0.5 mm).This extract was found to be effective against the different Salmonella strains tested.This result is consistent with that reported by Afolejan et al. 37 .These authors revealed the inhibitory action of Macrotermes bellicosus soldier extracts on different Salmonella strains.Hydroquinone and acid gluconic acid are the two molecules with antibacterial activity in Macrotermes extracts 6 .However, some strains showed resistance to the crude extracts of this insect.These were Escherichia coli 652654, Escherichia coli ATCC25922, Klebsiella pneumoniae 203, Providencia rettgeri 652655, Pseudomonas aeruginosa ATCC9027, and Serratia odorifera 652411.
The extract of Odontotermes sp. was only active against the three bacterial strains (25% inhibition).The highest inhibition diameter (19.5 ± 0.5 mm) was reported for Klebsiella pneumoniae ATCC13883, and the lowest www.nature.com/scientificreports/inhibition diameter (09.0 ± 0.0 mm) was reported for Pseudomonas aeruginosa ATCC27853.Gram-negative bacteria resistant to this extract are the three Escherichia coli strains tested Klebsiella pneumoniae 203, Providencia rettgeri 652655, Pseudomonas aeruginosa ATCC9027, Salmonella abony NCTC6017, Serratia odorifera 652411, and Salmonella Typhimurium ATCC14028.The antimicrobial activity could be due to the bioactive molecules produced by the actinomycetes that these insects harbor 39 .
Table 3. Inhibition diameters of insect extracts against Gram-negative bacteria.Values in the same column with different superscript letters are significantly different (p < 0.05) to the Student's t-test.
The extract of Pachycondyla sp.revealed inhibitory activity against nine of the twelve strains tested (75% inhibition).The diameter of inhibition against Klebsiella pneumoniae ATCC13883 was the highest (15.0 ± 0.0 mm).However, the diameter of inhibition reported against Pseudomonas aeruginosa ATCC27853 was the lowest (10.0 ± 0.0 mm).Santos et al. 43 reported that extracts of the Pachychodyla genus contained broad-spectrum inhibitory molecules that act against Gram-positive and Gram-negative bacteria.
For the test with Kraussaria angulifera extract, antibacterial activity was reported on five of the twelve bacterial strains tested, with the highest diameter of inhibition (20.0 ± 0.0 mm) against Salmonella Typhimurium ATCC14028 and the lowest diameter of inhibition (08.0 ± 0.0 mm) against Escherichia coli ATCC8739.The strains that were not sensitive to the Orthoptera extract were Escherichia coli 652654, Escherichia coli ATCC25922, and two strains of Klebsiella pneumoniae ATCC13883, Providencia rettgeri 652655, Pseudomonas aeruginosa ATCC9027, and Serratia odorifera 652411.Locusts contain excessive amounts of protein, fats, and essential fatty acids.Some of these proteins have inhibitory activities against certain bacteria [44][45][46] .
Acrida bicolor extract inhibited the growth of 6 bacterial strains (50% inhibition).The highest inhibition diameter (23.0 ± 1.0 mm) has been reported against Salmonella Typhimurium ATCC 4028.The lowest inhibition diameter of 14.5 ± 1.5 mm was obtained against Salmonella enteritidis ATCC13076.This insect extract did not inhibit the growth of Escherichia coli 652654, Escherichia coli ATCC25922, Klebsiella pneumoniae 203, Providencia rettgeri 652655, Pseudomonas aeruginosa ATCC 9027, and Serratia odorifera 652411.Bioactive molecules from grasshoppers are little known 27 .However, in Sudan, locusts and grasshoppers are used to treat stomach problems and jaundice, these potentialities could come from the plants that these insects consume 47 .Indeed, substances from plants of the carnolidae family, calotropin and calactin, have been found in certain locusts, such as Poekilocerus bufonius of the Pyrgomorphidae family 31 .

Effectiveness of crude extracts insects compared to imipenem
For the three bacterial strains, the inhibition diameters reported with imipenem were greater than those of the insect extracts (Table 4), Escherichia coli 652654; Escherichia coli ATCC25922 and Salmonella abony NCTC6017.For the other nine bacterial strains, the inhibition diameters reported with imipenem were smaller than those of some insect extracts.The lowest difference in inhibition diameter (diameter reported with insect extractdiameter reported with imipenem) reported in the latter case was 0.5 mm reported with Acridia bicolor against Escherichia coli ATCC8739 and with Acheta domesticus against Klebsiella pneumoniae 203.The highest difference in inhibition diameter was 17 mm, as reported for Cirina butyrospermi against Pseudomonas aeruginosa ATCC27853.Table 4 shows the insect extracts for which the inhibition diameters were greater than those of imipenem against the bacterial strains tested.

Index of multi-resistance to crude extracts from insects and their activity coefficient
The different indices of the Gram-negative bacteria tested with insect extracts are given in Table 5.Thus, 3 (25%) of the bacterial strains had an index of multi-resistance to insect's extracts (IMRE) < 0.2 and 9(75%) have an IMRE > 0.2.For bacterial strains with an IMRE < 0.2, i.e., Pseudomonas aeruginosa ATCC27853, Salmonella abony NCTC6017, and Salmonella enteritidis ATCC13076, insect extracts could be used to effectively inhibit their development.As a result, these extracts can be used as.For the different extracts in which bacterial growth was inhibited, the activity coefficient was between 0.02 cm 2 /µL and 0.35 cm 2 /µL (Table 5).The best inhibitory actions were recorded with extracts of Cirina butyrospermi and Myrabris variabilis against Pseudomonas aeruginosa ATCC27853 and Salmonella enteritidis ATCC13076, respectively.

Correlations between bacteria and insect extracts
Principal Compound Analysis (PCA) was performed to understand the interaction between the extracts of the different insects and the Gram-bacteria used in the different tests (Fig. 3).According to the first two axes (F1 and F2), which account for 53.63% of the dispersion of the results, a strong positive correlation appears between the two insect extracts and Providencia rettgeri 652655.These extracts are from Bunaea alcinoe and Apis mellifera.

Conclusion
This study made it possible to highlight the antibacterial activity of insect extracts against gram-negative bacteria.
The bacteria tested in this study are responsible for several pathologies constituting a major health problem in Burkina Faso.Entomotherapy can be an alternative treatment for certain pathologies in Burkina Faso.However, this opportunity is rarely exploited in Burkina Faso, which is full of a wide variety of insects. https://doi.org/10.1038/s41598-023-50622-2

Figure 3 .
Figure 3. PCA of correlation between growth inhibitor extracts and bacteria.

Table 1 .
Presentation of insects collected in this study.

Table 4 .
Insect extracts with higher inhibition than imipenem. Gram-

Table 5 .
Activity coefficient and indes of multi-resistance of crude extracts from insects.