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

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.

Figure 1
figure 1

Extraction yields of the insects used.

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.

Figure 2
figure 2

Antimicrobial activity of insect extracts on Salmonella enteritidis and Providentia rettgeri. B.A: Bunea alcinoe; P.sp.: Pachychondyla sp.; A.D: Acheta domesticus ; M.V : Mylabris variabilis ; IMP: Imipenem ; A.G: Anopheles gambiae; A.M : Apis mellifera ; O.T : Odontotermes sp.

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 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 Mexico26,27,28. The bioactive molecule contained in Acheta domesticus is phenoloxidase29.

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 07.5 ± 0.0 mm against Salmonella abony NCTC6017 and Salmonella Typhimurium ATCC14028, respectively. Anopheles gambiae extract 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 proteins30. Indeed, AMPs such as cecropins are found in this insect11.

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. Lupoli31 and Marques et al.32 reported the presence of inhibitory molecules, such as melittin, the main peptide in bee venom.

Active on four of the twelve Gram-negative bacteria (that is, 33.33% inhibition rate), the extract of Bunaea alcinoe had strong inhibitory activity against Salmonella enteritidis ATCC13076 (20.5 ± 0.5 mm) and weak inhibitory activity against Klebsiella pneumoniae ATCC13883 and Salmonella abony NCTC6017 (10.0 ± 0.0 mm). The extract did not affect the three Gram-negative bacteria strains tested as Klebsiella pneumoniae ATCC13883, Pseudomonas aeruginosa ATCC27853, Serratia odorifera 652411, and Salmonella Typhimurium ATCC14028. Bunaea alcinoe extract has antibacterial, antitumor, and antidiuretic effects because tannin contains33,34.

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 total35,36. 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 family31.

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 extracts6. 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.

Mylabris variabilis extract inhibited 58.33% of the bacterial strains tested. The inhibitory activity was more remarkable against Salmonella enteritidis ATCC13076 (30 ± 0.0 mm), unlike Escherichia coli 652654 (10.0 ± 0.0 mm). In contrast, for Escherichia coli ATCC25922, the two strains of Klebsiella tested, Pseudomonas aeruginosa ATCC9027, and Serratia odorifera 652411, the extract had no inhibitory effect. Mylabris extract contains inhibitory molecules such as cantharidin, which is strongly produced by the Mylabris genus31,38.

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 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 harbor39.

The inhibition rate assigned to Periplaneta americana was 58.33% (seven of twelve bacteria tested). Salmonella Typhimurium ATCC14028 was the most sensitive strain to extracts of Periplaneta americana, and Klebsiella pneumoniae 203 and the least sensitive strain with inhibition diameters of 17.0 ± 0.0 mm, and 08.0 ± 0.0 mm, respectively. However, five strains were resistant to the Periplaneta americana extract. These include Providencia rettgeri 652655, Pseudomonas aeruginosa, Salmonella abony NCTC6017; Salmonella enteritidis ATCC13076, and Serratia odorifera 652411. The antibacterial activity could be attributed to the AMPs in this insect. Indeed, a study conducted in 2016 by Kim et al.40 made it possible to isolate twelve AMPs with strong antibacterial activity. Basserie et al.41 and Ali et al.42 were also identified AMPs of Periplaneta americana.

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 bacteria44,45,46.

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 known27. However, in Sudan, locusts and grasshoppers are used to treat stomach problems and jaundice, these potentialities could come from the plants that these insects consume47. Indeed, substances from plants of the carnolidae family, calotropin and calactin, have been found in certain locusts, such as Poekilocerus bufonius of the Pyrgomorphidae family31.

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 extract—diameter 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.

Table 4 Insect extracts with higher inhibition than imipenem.

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 cm2/µL and 0.35 cm2/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.

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

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