Entomotherapy: An alternative treatment for diseases due to Gram-negative bacteria in Burkina Faso

doi:10.21203/rs.3.rs-3661075/v1

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Entomotherapy: An alternative treatment for diseases due to Gram-negative bacteria in Burkina Faso

https://doi.org/10.21203/rs.3.rs-3661075/v1

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Insects are known for their harmful effects. However, they also have beneficial effects on humans, animals, plants, and ecosystems. Its beneficial uses include entomophagy and entomotherapy. The objective of this study was 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 antibiotic for positive control. Extract yields varied among the insect species. All 13 extracts acted differently against certain Gram-negative bacteria. Largest inhibition diameter was 30.0 ± 0.0 mm for extracts of Cirina butyrospermi and Mylabris variabilis against Pseudomonas aeruginosa ATCC27853 and Salmonella enteritidis ATCC13076, respectively. Diameters of inhibition obtained using imipenem against these same bacterial strains were 13.0 ± 0.0mm and 22 ± 1.0 mm, respectively. The smallest inhibition diameter was 7.5 ± 0.0mm obtained using Anopheles gambiae extract against Salmonella Typhimurium ATCC14028. Imipenem used as a positive control was active on all strains tested. The highest multi-resistance index (0.85) was reported for Pseudomonas aeruginosa ATCC9027 and Serratia odorifera 652411. 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 pathologies caused by gram-negative bacteria.

Bacteriology

Entomology

Infectious Diseases

Entomotherapy

Gram negative bacteria

Insect extract

Inhibition diameter

Insects are the most numerous groups of living beings, with over 1.3 million described species¹. To colonise different ecosystems, these insects develop a wide range of peptides that enable them to defend themselves against invaders. On the basis of this observation, humans have become interested in using them to treat certain diseases ^2–6. The therapeutic potential of these insects is believed to be linked to the secretion of antimicrobial peptides. These antimicrobial peptides, which are used by humans, have antibacterial, antifungal, antiviral, and antiparasitic activities ^7–10. Research on antimicrobial peptides began in the 1980s with the discovery of drosomycin as the first antimicrobial peptide discovered in insects ¹¹. Research into antimicrobial peptides has recently increased to address bacterial multidrug resistance to commonly used antibiotics. Bacterial resistance to antibiotics is a major public health concern. According to the WHO, if nothing is done to find a palliative solution for antimicrobial resistance, it will cause 10 million deaths per year by 2050 ¹².

However, Gram-negative bacteria are the most implicated in bacterial multidrug resistance due to the production of beta-lactamases (penicillinase, cephalosporinases, and carbapenemases). These Gram-negative bacteria are responsible for many serious infections such as pneumonia, peritonitis, urinary tract infections, sepsis, kidney sepsis, wound infections, and meningitis. These infections are caused by Acinetobacter, Enterobacter, Klebsiella, Proteus, Pseudomonas, E. coli, and Serratia ¹³. Thus, apart from infections, some are responsible for food poisoning and typhoid fevers, such as strains of Salmonella, Shigella, E. coli, and Pseudomonas.

Despite their pathogenicity, some Gram-negative bacteria are exploited in food technology, medicine, and agriculture. Bacteria are responsible for the production of antimicrobial peptides (Nisin, Colicin, natamycin), exopolysaccharides (dextrans, β-glucans, fructans and levans) and enzymes (hydrolases, proteases, peptidase, lipases) ^14–16. These compounds have biotechnological applications such as texturing agents to modify the viscosity and elasticity of food products ¹⁵. In recent years, these bacteria have been the center of interest of researchers because of their potential application in food biopreservatives and their probiotic properties ^14,15. Certain Gram-negative bacteria such as Gluconoacetobacter diazotrophicus, Acetobacter nitrogenifigens, Gluconoacetobacter sacchari, Gluconoacetobacter kombuchae, Acetobacter aceti, and Gluconobacter oxydans areexploited specifically for the production of alcohols and acetic acid. In medicine, to improve the clinical effectiveness of immunotherapy and allergens, certain strains of Salmonella and Escherichia coli are used as candidate adjuvants for sublingual allergy treatment vaccines. However, the use of antibiotics as growth factors in the form of food additives favors the rapid development of bacterial resistance to antibiotics.

Certain Gram-negative bacteria are resistant to several available antibiotics and easily acquire antibiotic resistance genes. Gram-negative bacteria are more difficult to eliminate. This means that Gram-positive and Gram-negative bacteria require different treatments. The presence of these carbapenemase and beta-lactamase-producing bacteria is common in West Africa ¹⁷. However, the spread of Gram-negative bacteria producing carbapenemases and extended-spectrum beta-lactamases constitutes an “urgent” threat. Studies carried out by Sanou et al. ¹⁸ have reported the presence of these in patients in health centers in Burkina Faso. Indeed, medicinal insects produce interesting antimicrobial peptides. In addition, when faced with an increase in bacterial resistance to antibiotics. Therefore, it is imperative to identify novel bioactive molecules. In Burkina Faso, Apis mellifera, Cirina butyrospermi, Macrotermes belliccosus, Sceliphron sp., Periplaneta americana, and many other insects have been used empirically in traditional medicine as medicinal insects ¹⁹. The objective of this study was to evaluate the antimicrobial activity of medicinal insects collected from three phytogeographical areas of Burkina Faso against Gram-negative bacteria.

Sites of collection insects’

The insects were collected in three provinces Kadiogo (12°21' 56.4''N; 1°32'2''W), Houët (11°7' 55.36''N; 4°14'0.01'' W) and Séno (14°1' 48''N; 0°1'48'' W), which belong to three different phytogeographic zones. We conducted a survey among traditional health practitioners at these sites to determine their knowledge of medicinal insects. These sites were chosen because of the cosmopolitan nature of traditional health practitioners.

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 jars containing alcohol to preserve them ^20–22. Thirteen medicinal insects were collected on the basis of information provided by traditional health practitioners. These insects were listed 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.

Preparation 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 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, 4g 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, 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.

$$\text{E}\text{x}\text{t}\text{r}\text{a}\text{c}\text{t}\text{i}\text{o}\text{n} \text{y}\text{i}\text{e}\text{l}\text{d}=\frac{\text{M}\text{a}\text{s}\text{s} \text{o}\text{f} \text{d}\text{r}\text{y} \text{e}\text{x}\text{t}\text{r}\text{a}\text{c}\text{t}}{\text{I}\text{n}\text{i}\text{t}\text{i}\text{a}\text{l} \text{m}\text{a}\text{s}\text{s} \text{o}\text{f} \text{t}\text{e}\text{s}\text{t} \text{p}\text{o}\text{r}\text{t}\text{i}\text{o}\text{n}}\text{X} 100$$

Assessment of antimicrobial activity of crude extracts from insects collected

Gram-negative bacteria used for antimicrobial testing

Antimicrobial activity tests were carried out against twenty-two microbial germs including twelve Gram-negative bacteria in Table 2. Microbial strains used in this study are based on several criteria: these strains are commonly of 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.

Table 2

Information on the Gram-negative bacteria tested.
Microorganism	Species	Reference
Gram-negative bacteria	Escherichia coli	652654
	Escherichia coli	ATCC25922
	Escherichia coli	ATCC8739
	Klebsiella pneumoniae	203
	Klebsiella pneumoniae	ATCC13883
	Providencia rettgeri	652655
	Pseudomonas aeruginosa	ATCC9027
	Pseudomonas aeruginosa	ATCC27853
	Salmonella abony	NCTC6017
	Salmonella enteritidis	ATCC13076
	Salmonella Typhimurium	ATCC14028
	Serratia odorifera	652411

Antimicrobial activity testing of crude extracts from insects

Antimicrobial activity of crude extracts from insects was tested according to agar diffusion method described by Kirby-Bauer following the guidelines of the Clinical Laboratory Standards Institute. The preparation of microbial inocula was carried out from young colonies aged from 16 h to 18 h diluted in test tubes containing physiological saline. All microbial suspensions obtained were adjusted to a turbidity of 0.5 MacFarlant. The value of this standard turbidity of 0.5 McFarland corresponds approximately to a culture density of 1.5x10⁸ cells/mL. For the preparation of discs containing the crude extracts from insects, blank and sterile test antibiogram discs (MASTDISCS® AST) of 6 mm of diameter were used. These discs were impregnated solutions of crude extracts from insects contained in Eppendorf tubes during 10 min. As for carrying out the antibiogram, Mueller-Hinton agar was used. Petri dishes containing the agars were inoculated by swabbing with the different bacterial strains. The inoculated agars were dried near Bunsen burner during 5 min before to receiving discs impregnated with crude extracts from insects. Imipenem was used as positive control and blank and sterile test antibiogram discs impregnated in DMSO without extract were used as negative control. After depositing discs, Petri dishes were left at room temperature during 15 min to allow the diffusion of extracts and incubated at 37°C during 24 h. The inhibition diameters materialized by a clear halo around the disc were measured using a BioNumerical ruler (MICROBIAL DATA ANALYSIS SOFT WARE).

Determination of index multi-resistance to crude extracts from insects

Index of multi-resistance to extracts (IMRE) of crude extracts from insects was determined according to Das et al. ²⁴. IMRE was calculated using the following formula:

$$\text{I}\text{M}\text{R}\text{E}=\frac{\text{N}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{i}\text{n}\text{e}\text{f}\text{f}\text{e}\text{c}\text{t}\text{i}\text{v}\text{e} \text{e}\text{x}\text{t}\text{r}\text{a}\text{c}\text{t}\text{s} \text{o}\text{n} \text{m}\text{i}\text{c}\text{r}\text{o}\text{b}\text{i}\text{a}\text{l} \text{s}\text{t}\text{r}\text{a}\text{i}\text{n}}{\text{T}\text{o}\text{t}\text{a}\text{l} \text{n}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{e}\text{x}\text{t}\text{r}\text{a}\text{c}\text{t}\text{s} \text{t}\text{e}\text{s}\text{t}\text{e}\text{d} \text{o}\text{n} \text{m}\text{i}\text{c}\text{r}\text{o}\text{b}\text{i}\text{a}\text{l} \text{s}\text{t}\text{r}\text{a}\text{i}\text{n}}$$

Data processing and statistical analyses

Results were expressed as 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 Periplaneta americana, Pachychondyla sp., and Acrida bicolor and weak yields with Anopheles gambiae and 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, the mass-volume ratio of the ground’s solvent (m/v), maceration time, and stirring speed.

Antimicrobial activity to crude extracts from insects

The crude extracts from insects tested were inhibited the growth of the bacterial strains, as shown in Fig. 2. The antibacterial activities to crude extracts from insects against twelve Gram-negative bacteria are shown in 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 18.50 ± 01.50 mm against Klebsiella pneumoniae ATCC13883, whereas the lowest inhibition diameter was 09.00 ± 00.00 mm against Escherichia coli 652654. The crude extract of Acheta domesticus had no effect on Escherichia coli ATCC8739, Providencia rettgeri 652655, and Serratia odorifera 652411. In Latin America, Acheta domesticus has been used in the treatment of tract infections in Brazil but also as an antidiuretic and against urinary retention in Mexico ^25–27. The bioactive molecule contained in Acheta domesticus is phenoloxidase ²⁸.

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 15.00 ± 01.0 mm and lowest 07.50 ± 00.00 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 proteins ²⁹. 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 22.50 ± 0.50 mm (Providencia rettgeri 652655) and the lowest inhibition diameter of 08.0 ± 0.00 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 ³⁰ and Marques et al. ³¹ 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 a strong inhibitory activity against Salmonella enteritidis ATCC13076 (20.50 ± 0.5 mm) and weak inhibitory activity against Klebsiella pneumoniae ATCC13883 and Salmonella abony NCTC6017 (10.00 ± 00.00mm). The extract had no effect on 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 contains ^32,33.

Thus, a 75% inhibition rate of the bacterial strains tested (9/12) was observed with Cirina butyrospermum extract. The smallest and largest diameters of inhibition were respectively 08.50 ± 00.50 mm (Providencia rettgeri 652655) and 30.00 ± 00.50 mm (Pseudomonas aeruginosa ATCC 9027), respectively. Shea caterpillars are known to be rich in proteins, accounting for more than 60% of the total ^34,35. 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 (13.50 ± 01.50 mm) was recorded against Providencia rettgeri 652655 and the lowest inhibition diameter (09.00 ± 01.00 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 ³⁰.

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 (13.50 ± 0.50 mm), and Salmonella Typhimurium ATCC14028 ATCC14028 was the least sensitive strain (08.50 ± 00.50). This extract was found to be effective against the different Salmonella strains tested. This result is consistent with that reported by Afolejan et al. ³⁶. These authors revealed the inhibitory action of Macrotermes bellicosus soldier extract on different Salmonella strains. Hydroquinone and acid gluconic acid are the two molecules with antibacterial activity contained 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, P. 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 ± 00.00 mm), unlike Escherichia coli 652654 (10.00 ± 00.00 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 genus ^30,37.

The extract of Odontotermes sp. was only active against the three bacterial strains (25% inhibition). The largest inhibition diameter (19.50 ± 00.50 mm) was reported for Klebsiella pneumoniae ATCC13883 and the smallest inhibition diameter (09.00 ± 00.00 mm) was reported for Pseudomonas aeruginosa ATCC27853. Gram-negative bacteria resistant to this extract are the three Escherichia coli strains tested were 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 ³⁸.

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 17.00 ± 00.00 mm(08.00 ± 00.00mm). 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. ³⁹ made it possible to isolate twelve AMPs with strong antibacterial activity. Basserie et al. ⁴⁰ and Ali et al. ⁴¹ had 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 (15.00 ± 00.00 mm), largest diameter. However, the diameter of inhibition reported against Pseudomonas aeruginosa ATCC27853 was the lowest (10.00 ± 00.00 mm). Santos et al. ⁴² 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 largest diameter of inhibition (20.00 ± 00.00 mm) against Salmonella Typhimurium ATCC14028 and the lowest diameter of inhibition (08.00 ± 00.00 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 ^43–45.

Acrida bicolor extract inhibited the growth of 6 bacterial strains (50% inhibition). The highest inhibition diameter (23.00 ± 01.00 mm) has been reported against Salmonella Typhimurium ATCC 4028. The smallest inhibition diameter of 14.50 ± 01.50 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 ²⁶. 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 ⁴⁶. 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 ³⁰.

Table 3

Inhibition diameters of insect extracts against Gram-negative bacteria. Values in same column with different superscript letters are significantly different (p < 0.05) to Student's t-test.
Variables	Acheta domesticus	Anopheles gambiae	Apis melifera	Bunaea alcinoe	Cirina butyrospermi	Lytta sp.	Macrotermes bellicosus	Imipenem
Escherichia coli 652654	9.0 ± 0.0^b	10.5 ± 0.5^d	0.0 ± 0.0^a	0.0 ± 0.0^a	9.5 ± 0.05^b	12.0 ± 0.0^de	0.0 ± 0.0^a	20.0 ± 0.0^d
Escherichia coli ATCC25922	16.5 ± 0.5^e	10 ± 0.0^d	9.0 ± 0.0^b	0.0 ± 0.0^a	11.0 ± 0.0^c	10.0 ± 0.0^bc	0.0 ± 0.0^a	22.0 ± 0.0^e
Escherichia coli ATCC8739	0.0 ± 0.0^a	0.0 ± 0.0^a	0.0 ± 0.0^a	0.0 ± 0.0^a	0.0 ± 0.0^a	13.0 ± 0.0^e	9.5 ± 0.5^b	17.0 ± 0.0^c
Klebsiella pneumoniae 203	15.5 ± 0.5^de	0.0 ± 0.0^a	15.0 ± 1.0^cd	0.0 ± 0.0^a	0.0 ± 0.0^a	11.0 ± 0.0^cd	0.0 ± 0.0^a	15.0 ± 0.0^b
Klebsiella pneumoniae ATCC13883	18.5 ± 1.5^f	14.0 ± 0.0^e	0.0 ± 0.0^a	10.0 ± 0.0^b	12 ± 1.0^cd	0.0 ± 0.0^a	12.0 ± 0.0^c	12.0 ± 0.0^a
Providencia rettgeri 652655	0.0 ± 0.0^a	10.5 ± 0.5^d	22.5 ± 0.5^e	19.5 ± 0.5^c	8.5 ± 0.5^b	13.5 ± 1.5^e	0.0 ± 0.0^a	15.0 ± 0.0^b
Pseudomonas aeruginosa ATCC9027	0.0 ± 0.0^a	0.0 ± 0.0^a	0.0 ± 0.0^a	20.0 ± 0.0^cd	26.0 ± 0.5^f	0.0 ± 0.0 ^a	0.0 ± 0.0^a	15.5 ± 0.5^e
Pseudomonas aeruginosa ATCC27853	12.5 ± 0.0^c	9.0 ± 0.0^c	14.5 ± 0.5^c	0.0 ± 0.0^a	30.0 ± 0.0^h	0.0 ± 0.0^a	13.5 ± 0.5^d	13.0 ± 0.0^d
Salmonella abony NCTC6017	14.0 ± 1.0^d	15.0 ± 0.0^e	8.0 ± 0.0^b	10.0 ± 0.0^b	14.0 ± 0.0^f	12.0 ± 0.0^de	9.0 ± 0.0^b	28.0 ± 1.0^g
Salmonella enteritidis ATCC13076	14.0 ± 1.0^d	0.0 ± 0.0^a	16.0 ± 0.0^d	20.5 ± 0.5^d	18.5 ± 0.5^g	10.0 ± 0.0^bc	11.0 ± 1.0^c	22.0 ± 1.0^e
Serratia odorifera 652411	0.0 ± 0.0^a	15.0 ± 1.0^e	0.0 ± 0.0^a	0.0 ± 0.0^a	0.0 ± 0.0^a	12.5 ± 0.5^de	0.0 ± 0.0^a	13.0 ± 0.0^a
Salmonella Typhimurium ATCC14028	10.0 ± 0.0^b	7.5 ± 0.0^b	0.0 ± 0.0^a	0.0 ± 0.0^a	12.5 ± 0.5^d	9.0 ± 1.0^b	8.5 ± 0.5^b	19.5 ± 0.5^d

Table 3

Inhibition diameters of insect extracts against Gram-negative bacteria. Values in same column with different superscript letters are significantly different (p < 0.05) to Student's t-test. **(continued)**
Variables	Myrabris variabilis	Odontotermes sp.	Periplaneta americana	Pachycondyla sp.	Kraussaria angulifera	Acridia bicolor	Imipenem
Escherichia coli 652654	10.0 ± 0.0^b	0.0 ± 0.0^a	9.0 ± 0.0^b	14.5 ± 0.5^e	0.0 ± 0.0^a	0.0 ± 0.0^a	20.0 ± 0.0^d
Escherichia coli ATCC25922	0.0 ± 0.0^a	0.0 ± 0.0^a	12.0 ± 0.0^d	14.0 ± 0.0^de	0.0 ± 0.0^a	0.0 ± 0.0^a	22.0 ± 0.0^e
Escherichia coli ATCC8739	14.5 ± 0.5^e	0.0 ± 0.0^a	13.0 ± 1.0^d	13.0 ± 0.0^cd	8.0 ± 0.0^b	17.5 ± 1.5^c	17.0 ± 0.0^c
Klebsiella pneumoniae 203	0.0 ± 0.0^a	0.0 ± 0.0^a	8.0 ± 0.0^b	0.0 ± 0.0^a	0.0 ± 0.0^a	0.0 ± 0.0^a	15.0 ± 0.0^b
Klebsiella pneumoniae ATCC13883	0.0 ± 0.0^a	19.5 ± 0.5^d	15.0 ± 1.0^e	15.0 ± 0.0^e	0.0 ± 0.0^a	18.5 ± 0.5^c	12.0 ± 0.0^a
Providencia rettgeri 652655	15.0 ± 0.0^f	0.0 ± 0.0^a	0.0 ± 0.0^a	12.5 ± 0.5^c	0.0 ± 0.0^a	0.0 ± 0.0^a	15 ± 0.0^b
Pseudomonas aeruginosa ATCC 9027	0.0 ± 0.0^a	0.0 ± 0.0^a	0.0 ± 0.0^a	0.0 ± 0.0^a	0.0 ± 0.0^a	0.0 ± 0.0^a	15.5 ± 0.5^e
Pseudomonas aeruginosa ATCC27853	14.5 ± 0.5^e	9.0 ± 0.0^b	10.5 ± 0.5^c	10.0 ± 0.0^b	15.0 ± 1.0^c	16.5 ± 1.5^bc	13.0 ± 0.0^d
Salmonella abony NCTC6017	12.0 ± 0.0^d	0.0 ± 0.0^a	0.0 ± 0.0^a	11.0 ± 1.0^b	12.5 ± 0.5^c	15.0 ± 0.0^b	28.0 ± 1.0^g
Salmonella enteritidis ATCC13076	30.0 ± 0.0^g	18.0 ± 0.0^c	0.0 ± 0.0^a	13.0 ± 1.0^cd	16.0 ± 1.0^d	14.5 ± 1.5^b	22.0 ± 1.0^e
Serratia odorifera 652411	0.0 ± 0.0^a	0.0 ± 0.0^a	0.0 ± 0.0^a	0.0 ± 0.0^a	0.0 ± 0.0^a	0.0 ± 0.0^a	13.0 ± 0.0^a
Salmonella Typhimurium ATCC14028	11.0 ± 0.0^c	0.0 ± 0.0^a	17.0 ± 0.0^f	13.0 ± 0.0^cd	20.0 ± 0.0^e	23.0 ± 1.0^d	19.5 ± 0.5^d

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). These were tree strains 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 smallest 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 largest difference in inhibition diameter was 17 ± 00 mm, as reported for Cirina butyrospermi against Pseudomonas aeruginosa ATCC 27853. 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.
Bacterial strains	Insect extracts with a diameter larger than that of imipenem
Escherichia coli ATCC8739	Acridia bicolor.
Klebsiella pneumoniae 203	Acheta domesticus
Klebsiella pneumoniae ATCC13883	Acheta domesticus, Anopheles gambiae, Odontotermes sp., Periplaneta americana, Pachycondyla sp, and Acridia bicolor.
Providencia rettgeri 652655	Apis melifera, and Bunaea alcinoe
Pseudomonas aeruginosa ATCC9027	Bunaea alcinoe, and Cirina butyrospermi
Pseudomonas aeruginosa ATCC27853	Apis melifera, Cirina butyrospermi, Macrotermes bellicosus, Myrabris variabilis, Kraussaria angulifera, and Acridia bicolor
Serratia odolifera 652411	Anopheles gambiae
Salmonella enteritidis ATCC13076	Myrabris variabilis
Salmonella Typhimurium ATCC14028	Kraussaria angulifera, and Acridia bicolor

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 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

Index of multi-resistance and activity coefficient of crude extracts from insects.
Gram-negative bacteria Insects	Pseudomonas aeruginosa ATCC9027	Pseudomonas aeruginosa ATCC27853	Salmonella abony NCTC6017	Salmonella enteritidis ATCC13076	Serratia odorifera 652411	Salmonella Typhimurium ATCC14028
Gram-negative bacteria Insects	Activity coefficient (A) of insect extracts against bacterial strains in cm²/µL
Acheta domesticus	0	0.06	0.08	0.08	0	0.04
Anopheles gambiae	0	0.03	0.09	0	0.09	0.02
Apis melifera	0	0.08	0.03	0.1	0	0
Bunaea alcinoe	0.16	0	0.04	0.16	0	0
Cirina butyrospermi	0.27	0.35	0.08	0.13	0	0.06
Lytta sp.	0	0	0.06	0.04	0.06	0.03
Macrotermes bellicosus	0	0.07	0.03	0.05	0	0.03
Myrabris variabilis	0	0.08	0.06	0.35	0	0.05
Odontotermes sp.	0	0.03	0	0.13	0	0
Periplaneta americana	0	0.04	0	0	0	0.11
Pachycondyla sp.	0	0.04	0.05	0.07	0	0.07
Kraussaria angulifera	0	0.09	0.06	0.1	0	0.16
Acrida bicolor	0	0.11	0.09	0.08	0	0.21
Imipenem	0.09	0.07	0.31	0.19	0.07	0.15
	Index of multi-resistance to insects extracts (IMRE)
Values of IMRE	0.85	0.15	0.15	0.15	0.85	0.23

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. Therefore, these extracts are indicated for inhibiting the proliferation of the aforementioned bacteria. However, it should be noted that Providencia rettgeri 652655 is very sensitive to Apis mellifera extracts. Four insects, Bunaea alcinoe, Apis melifera, Myrabris variabilis, and Cirina butyrospermi, also have a positive correlation with Salmonella enteritidis ATCC13076. These extracts could be used to prevent the proliferation of these bacteria and to avoid nuisances due to the last debt. The extracts of Bunaea alcinoe and Myrabris variabilis were more correlated with Salmonella enteritidis ATCC13076. Therefore, they would be the best inhibitors of this bacterium. From the above, Bunaea alcinoe and Apis mellifera extracts could be used for both Salmonella enteritidis ATCC13076 and Providencia rettgeri 652655 inhibitions. In contrast, the extract of Periplaneta americana represents an extract that can be effective simultaneously against the growth of Klebsiella pneumoniae ATCC13883, and Salmonella Typhimurium ATCC14028.

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.

Author contribution statement

All authors listed have significantly contributed to the development and the writing of this article.

Funding statement

This research was funded by CEA-CFOREM of Université Joseph KI-ZERBO in the form of a doctoral scholarship provided to Mamadou Ouango.

Data availability statement

Data included in article/referenced in article.

Declaration of interest statement

The authors declare no conflicts of interest.

Additional information

No additional information is available for this paper.

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Entomotherapy: An alternative treatment for diseases due to Gram-negative bacteria in Burkina Faso

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Abstract

Figures

Introduction

Materials and Methods

Sites of collection insects’

Technical collection and information on the insect collected in this study

Preparation the insects for extraction

Extraction of crude extracts from insects collected

Extraction yield

Assessment of antimicrobial activity of crude extracts from insects collected

Gram-negative bacteria used for antimicrobial testing

Antimicrobial activity testing of crude extracts from insects

Determination of index multi-resistance to crude extracts from insects

Data processing and statistical analyses

Results

Yield values of extractions of crude extracts from insects

Antimicrobial activity to crude extracts from insects

Effectiveness of crude extracts insects compared to imipenem

Index of multi-resistance to crude extracts from insects and their activity coefficient

Correlations between bacteria and insect extracts

Conclusion

Declarations

References

Tables

Supplementary Files

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